HK1183804A

HK1183804A - Therapeutic peptide-polymer conjugates, particles, compositions, and related methods

Info

Publication number: HK1183804A
Application number: HK13111219.7A
Authority: HK
Inventors: Oliver S. Fetzer; Jungyeon Hwang; Patrick Lim Soo; Pei-Sze Ng; Sonke Svenson
Original assignee: Cerulean Pharma Inc.
Priority date: 2010-08-20
Filing date: 2011-08-18
Publication date: 2014-01-10

Description

Therapeutic peptide-polymer conjugates, particles, compositions, and related methods

Priority requirement

The present application claims priority from U.S. s.N.61/375,771 filed on 20/2010 and U.S. s.N.61/477,827 filed on 21/4/2011, which are all incorporated herein by reference.

Background

Delivery of therapeutic peptides by controlled release of the therapeutic peptide is desirable to provide optimal use and effectiveness. Controlled release polymer systems can increase the efficacy of therapeutic peptides and minimize patient compliance issues.

Summary of The Invention

Described herein are particles that can be used, for example, to deliver therapeutic peptides or proteins in the treatment of, for example, the following conditions: cancer, inflammatory disorders (e.g., inflammatory disorders including inflammatory disorders caused by, for example, infectious diseases) or autoimmune disorders, cardiovascular diseases, or other disorders (e.g., infectious diseases). Generally, the particles comprise a hydrophilic-hydrophobic polymer (e.g., a diblock or triblock copolymer) and a therapeutic peptide or protein. In some embodiments, the particles further comprise a hydrophobic polymer or surfactant. Generally, the therapeutic peptide is linked to a polymer, e.g., a hydrophilic-hydrophobic polymer, or to a hydrophobic polymer (if present). In embodiments where the therapeutic peptide or protein is charged, the particle may further comprise a counter ion for the therapeutic peptide. Also described herein are conjugates such as therapeutic peptides or protein-polymer conjugates, mixtures, compositions, and dosage forms containing particles or conjugates, methods of using particles (e.g., for treating a disorder), kits comprising therapeutic peptides or protein-polymer conjugates and particles, methods of making therapeutic peptides or protein-polymer conjugates and particles, methods of storing particles, and methods of analyzing particles.

In one aspect, the disclosure features a particle comprising:

a) a plurality of hydrophobic polymers;

b) a plurality of hydrophilic-hydrophobic polymers; and

c) a plurality of therapeutic peptides or proteins, wherein at least a portion of the plurality of therapeutic peptides or proteins are covalently attached to the hydrophobic polymer of a) or the hydrophilic-hydrophobic polymer of b).

In some embodiments, the particles further comprise a hydrophobic moiety, such as chitosan, poly (vinyl alcohol), or poloxamer (poloxamer).

In some embodiments, at least a portion of the hydrophobic polymer of a) is not covalently attached to the therapeutic peptide or protein of c). In some embodiments, at least a portion of the hydrophobic polymer of a) is covalently attached to the therapeutic peptide or protein of c), e.g., at least a portion of the hydrophobic polymer of a) is covalently attached to a single therapeutic peptide or protein of c), or at least a portion of the hydrophobic polymer of a) is covalently attached to multiple therapeutic peptides or proteins of c).

In some embodiments, at least a portion of the hydrophobic polymer of a) is covalently attached directly to the therapeutic peptide or protein of c) (e.g., at the carboxy terminus or the hydroxy terminus of the hydrophobic polymer). In some embodiments, at least a portion of the therapeutic peptide or protein of c) is covalently attached to the hydrophobic polymer via a linker. Exemplary linkers include: linkers comprising moieties formed using "click chemistry" (e.g., as described in WO 2006/115547), and linkers comprising an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, an oxime, a carbamate, a carbonate, a silyl ether, or a triazole (e.g., an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, or a triazole). In some embodiments, the linker comprises a functional group, such as a bond that is cleavable under physiological conditions. In some embodiments, the linker comprises a plurality of functional groups, such as bonds that are cleavable under physiological conditions. In some embodiments, the linker comprises a functional group, such as a bond or functional group described herein, that is not directly connected to the first or second moiety linked through the linker at the terminus of the linker, but is internal to the linker. In some embodiments, the linker is hydrolyzable under physiological conditions, the linker is enzymatically cleavable under physiological conditions, or the linker comprises a disulfide that is reducible under physiological conditions. In some embodiments, the linker is not cleaved under physiological conditions, e.g., the linker has a sufficient length that a therapeutic peptide or protein need not be cleaved to be active, e.g., the linker is at least about 20 angstroms (e.g., at least about 24 angstroms) in length.

In some embodiments, at least a portion of the hydrophobic polymer of a) is covalently attached to at least a portion of the therapeutic peptide or protein of c) through the amino terminus of the therapeutic peptide or protein; a) at least a portion of the hydrophobic polymer of (a) is covalently attached to at least a portion of the therapeutic peptide or protein of c) through the carboxy terminus of the therapeutic peptide or protein; and/or at least a portion of the hydrophobic polymer of a) is covalently attached to at least a portion of the therapeutic peptide or protein of c) through the amino acid side of the therapeutic peptide or protein.

In some embodiments, at least a portion of the hydrophobic polymer of a) is coupled with a moiety that can suppress the pH of the hydrophobic polymer or particle. Exemplary pH-inhibiting moieties include weakly basic salts, such as calcium carbonate, magnesium hydroxide, and zinc carbonate, and proton sponges (e.g., including one or more amine groups), such as polyamines.

In some embodiments, at least a portion of the hydrophilic-hydrophobic polymer of b) is covalently attached to the therapeutic peptide or protein of c). In some embodiments, at least a portion of the hydrophilic-hydrophobic polymer of b) is covalently attached to the single therapeutic peptide or protein of c). In some embodiments, at least a portion of the hydrophilic-hydrophobic polymers of b) are covalently attached to a plurality of therapeutic peptides or proteins of c).

In some embodiments, at least a portion of the hydrophilic-hydrophobic polymer of b) is directly covalently attached to the therapeutic peptide or protein of c). In some embodiments, at least a portion of the therapeutic peptide or protein of c) is covalently attached to the hydrophilic-hydrophobic polymer of b) via a linker. Exemplary linkers include: linkers comprising moieties formed using "click chemistry" (e.g., as described in WO 2006/115547), and linkers comprising an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, an oxime, a carbamate, a carbonate, a silyl ether, or a triazole (e.g., an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, or a triazole). In some embodiments, the linker comprises a functional group, such as a bond that is cleavable under physiological conditions. In some embodiments, the linker comprises a plurality of functional groups, such as bonds that are cleavable under physiological conditions. In some embodiments, the linker comprises a functional group, such as a bond or functional group described herein, that is not directly connected to the first or second moiety linked through the linker at the terminus of the linker, but is internal to the linker. In some embodiments, the linker is hydrolyzable under physiological conditions, the linker is enzymatically cleavable under physiological conditions, or the linker comprises a disulfide that is reducible under physiological conditions. In some embodiments, the linker is not cleaved under physiological conditions, e.g., the linker has a sufficient length that a therapeutic peptide or protein need not be cleaved to be active, e.g., the linker is at least about 20 angstroms (e.g., at least about 24 angstroms) in length.

In some embodiments, at least a portion of the hydrophilic-hydrophobic polymer of b) is covalently attached to the therapeutic peptide or protein of c) at the carboxy terminus or the hydroxy terminus of the hydrophobic polymer.

In some embodiments, at least a portion of the hydrophilic-hydrophobic polymer of b) is covalently attached to at least a portion of the therapeutic peptide or protein of c) through the amino terminus of the therapeutic peptide or protein. In some embodiments, at least a portion of the hydrophilic-hydrophobic polymer of b) is covalently attached to at least a portion of the therapeutic peptide or protein of c) through the carboxy terminus of the therapeutic peptide or protein. In some embodiments, at least a portion of the hydrophilic-hydrophobic polymer of b) is covalently attached to at least a portion of the therapeutic peptide or protein of c) through the amino acid side of the therapeutic peptide or protein.

In some embodiments, the particle further comprises a plurality of other therapeutic peptides or proteins, wherein the other therapeutic peptides or proteins are different from the therapeutic peptides or proteins of c), e.g., at least a portion of the plurality of other therapeutic peptides or proteins are attached to at least a portion of the hydrophobic polymer of a) and/or the hydrophilic-hydrophobic polymer of b).

In some embodiments, at least a portion of the hydrophobic polymer of a) is a copolymer of lactic acid and glycolic acid (i.e., PLGA). For example, in some embodiments, a portion of the hydrophobic polymers of a) are PLGA having a ratio of lactic acid to glycolic acid of about 15: 85 or 25: 75 to about 75: 25 or 85: 15, e.g., a ratio of lactic acid to glycolic acid of about 50: 50.

In some embodiments, the hydrophobic polymer of a) has a weight average molecular weight of about 6 to about 12kDa, e.g., about 8 to about 10 kDa. In other embodiments, the hydrophobic polymer of a) has a weight average molecular weight of about 4 to about 8 kDa. In some embodiments, the hydrophobic polymer of a) has a weight average molecular weight of about 10 to about 100 kDa.

In some embodiments, the hydrophobic polymer of a) comprises from about 35% to about 80% by weight of the particle.

In some embodiments, at least a portion of the hydrophobic polymer of a) is covalently attached to a therapeutic peptide or protein and a portion of the hydrophobic polymer of a) is attached to a plurality of therapeutic peptides or proteins.

In some embodiments, the hydrophilic-hydrophobic polymer of b) is a block copolymer. Exemplary block copolymers comprise a neutral hydrophilic block (e.g., which can enhance circulation), and a pH-responsive block (e.g., which can facilitate inclusion body escape). Exemplary pH-responsive blocks include those having cis-acetoxyl, hydrazone, or acetal linkages that can be hydrolyzed at, for example, pH4 to 6.5. In some embodiments, the polymer includes reversible peptide conjugation sites, e.g., which may provide a means of releasing the peptide from the carrier upon reaching the cytosol (e.g., sulfhydryl).

In some embodiments, the hydrophilic-hydrophobic polymer of b) is a diblock copolymer (e.g., PEG-PLGA). In some embodiments, the hydrophilic-hydrophobic polymer of b) is a triblock copolymer (e.g., PEG-PLGA-PEG). In some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymers of b) has a hydroxyl terminus. In some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymer of b) has a hydroxyl end and the hydroxyl end is capped (e.g., capped with an acyl moiety). For example, in some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymer of b) has a hydroxyl terminus and the hydroxyl terminus is terminated with an acyl moiety.

In some embodiments, the hydrophobic portion of the hydrophilic-hydrophobic polymer of b) comprises a copolymer of lactic acid and glycolic acid (i.e., PLGA). In some embodiments, the hydrophobic portion of the hydrophilic-hydrophobic polymer of b) comprises PLGA having a ratio of lactic acid to glycolic acid of about 15: 85 or 25: 75 to about 75: 25 or 85: 15, for example, a ratio of lactic acid to glycolic acid of about 50: 50.

In some embodiments, the hydrophilic portion of the hydrophilic-hydrophobic polymer of b) has a weight average molecular weight of about 1 to about 6kDa (e.g., about 2 to about 6 kDa). In some embodiments, the hydrophobic portion of the hydrophilic-hydrophobic polymer of b) has a weight average molecular weight of about 8 to about 13 kDa.

In some embodiments, the plurality of hydrophilic-hydrophobic polymers of b) comprise about 5% to about 25% (e.g., about 10% to about 25%) by weight of the particle.

In some embodiments, the hydrophilic portion of the hydrophilic-hydrophobic polymer of b) comprises PEG.

In some embodiments, the hydrophilic portion of the hydrophilic-hydrophobic polymer terminates with a methoxy group.

In some embodiments, at least a portion of the hydrophilic-hydrophobic polymers of b) are covalently attached to a therapeutic peptide or protein and a portion of the hydrophilic-hydrophobic polymers of b) are attached to a plurality of therapeutic peptides or proteins.

In some embodiments, the therapeutic peptide is a therapeutic peptide described herein. In some embodiments, the therapeutic peptide comprises from about 2 to about 50 amino acid residues, e.g., from about 2 to about 40 amino acid residues or from about 2 to about 30 amino acid residues.

In some embodiments, the protein is a protein described herein.

In some embodiments, at least a portion of the therapeutic peptide or protein is chemically modified.

In some embodiments, the plurality of therapeutic peptides comprises about 1% to about 90% (e.g., about 50% to about 90%, about 70% to about 90%, about 10% to 50%, about 10% to about 30%) by weight of the particle.

In some embodiments, the particle further comprises a surfactant. In some embodiments, the surfactant is a polymer, for example, the surfactant is PVA. In some embodiments, PVA has a weight average molecular weight of about 23 to about 26 kDa. In some embodiments, the surfactant comprises from about 15% to about 35% by weight of the particle.

In some embodiments, the particles further comprise a counter ion. For example, in embodiments where the therapeutic peptide is a charged peptide, the particles may comprise a counterion, wherein the counterion has a charge opposite to the charge on the therapeutic peptide. In some embodiments, the ratio of the charge of the therapeutic peptide to the charge of the counter ion in the particle is from about 1: 1.5 to about 1.5: 1 (e.g., from about 1.25: 1 to about 1: 1.25, or about 1: 1).

In some embodiments, the counter ion can function as a surfactant (e.g., a single moiety can function as both the counter ion and the surfactant).

In some embodiments, the particles have a diameter of less than about 200nm (e.g., less than about 150 nm).

In some embodiments, the surface of the particle is substantially coated with a polymer such as PEG.

In some embodiments, the zeta potential of the particles is from about-10 to about 10mV (e.g., from about-5 to about 5 mV).

In some embodiments, the particles are chemically stable for at least 1 day (e.g., at least 7 days, at least 14 days, at least 21 days, at least 30 days) under conditions comprising a temperature of 23 degrees celsius and a percent humidity of 60%.

In some embodiments, the particles are lyophilized particles.

In some embodiments, the particles are formulated as a pharmaceutical composition.

In some embodiments, the surface of the particle is substantially free of the targeting agent.

In some embodiments, the therapeutic peptide or protein is attached to the hydrophobic polymer of a) and the therapeutic peptide or protein-hydrophobic polymer conjugate has one or more of the following properties:

i) the hydrophobic polymer attached to the therapeutic peptide or protein may be a homopolymer or a polymer composed of more than one monomeric subunit;

ii) the hydrophobic polymer attached to the therapeutic peptide or protein has a weight average molecular weight of about 4-15 kDa;

iii) the hydrophobic polymer is composed of a first and a second type of monomeric subunit, and the ratio of the first to second type of monomeric subunit in the hydrophobic polymer attached to the therapeutic peptide or protein is from about 15: 85 or 25: 75 to about 75: 25 or 85: 15, e.g., about 50: 50;

iv) the hydrophobic polymer is PLGA; and

v) the therapeutic peptide or protein comprises from about 1% to about 100% by weight (e.g., from about 50% to about 100%, from about 70% to about 100%, from about 50% to 90%) of the particle.

In some embodiments, the hydrophobic polymer attached to the therapeutic peptide or protein has a weight average molecular weight of about 4-15kDa, such as 6-12kDa, such as 8-10 kDa.

In some embodiments, the hydrophilic-hydrophobic polymer of b) has one or more of the following properties:

i) the hydrophilic moiety has a weight average molecular weight of about 1-6kDa (e.g., 2-6 kDa);

ii) the hydrophobic polymer has a weight average molecular weight of about 4-15 kDa;

iii) the hydrophilic polymer is PEG;

iv) the hydrophobic polymer is composed of a first and a second type of monomeric subunit, and the ratio of the first and second types of monomeric subunit in the hydrophobic polymer is from about 15: 85 or 25: 75 to about 75: 25 or 85: 15, e.g., about 50: 50; and

v) the hydrophobic polymer is PLGA.

In some embodiments, if the weight average molecular weight of the hydrophilic portion of the hydrophilic-hydrophobic polymer of b) is between about 1-3kDa, such as about 2kDa, then the ratio of the weight average molecular weight of the hydrophilic portion to the weight average molecular weight of the hydrophobic portion is between 1: 3-1: 7, and if the weight average molecular weight of the hydrophilic portion is between about 4-6kDa, such as about 5kDa, then the ratio of the weight average molecular weight of the hydrophilic portion to the weight average molecular weight of the hydrophobic portion is between 1: 1-1: 4.

In some embodiments, the hydrophilic portion of the hydrophilic-hydrophobic polymer of b) has a weight average molecular weight of about 2-6kDa and the hydrophobic portion has a weight average molecular weight of between about 8-13 kDa.

In some embodiments, the hydrophilic portion of the hydrophilic-hydrophobic polymer of b) terminates with a methoxy group.

In some embodiments, the therapeutic peptide is attached to the hydrophobic polymer of a) and the therapeutic peptide-hydrophobic polymer conjugate has one or more of the following properties:

i) the hydrophobic polymer attached to the therapeutic peptide can be a homopolymer or a polymer composed of more than one monomeric subunit;

ii) the hydrophobic polymer attached to the therapeutic peptide has a weight average molecular weight of about 4-15 kDa;

iii) the hydrophobic polymer is comprised of a first and a second type of monomeric subunit, and the ratio of the first and second type of monomeric subunit in the hydrophobic polymer attached to the therapeutic peptide or protein is from about 15: 85 or 25: 75 to about 75: 25 or 85: 15, e.g., about 50: 50; and

iv) the hydrophobic polymer is PLGA.

In some embodiments, the particles further comprise a surfactant (e.g., PVA).

In another aspect, the disclosure features a particle comprising:

a) a plurality of therapeutic peptide or protein-polymer conjugates comprising a therapeutic peptide or protein linked to a hydrophobic polymer; and

b) a variety of hydrophilic-hydrophobic polymers.

In some embodiments, the particles further comprise a hydrophobic polymer (e.g., PLGA).

In some embodiments, the particles further comprise a hydrophobic moiety, such as chitosan, poly (vinyl alcohol), or poloxamer.

In some embodiments, the therapeutic peptide or protein is covalently attached to the hydrophobic polymer via a linker. Exemplary linkers include: linkers comprising moieties formed using "click chemistry" (e.g., as described in WO 2006/115547), and linkers comprising an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, an oxime, a carbamate, a carbonate, a silyl ether, or a triazole (e.g., an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, or a triazole). In some embodiments, the linker comprises a functional group, such as a bond that is cleavable under physiological conditions. In some embodiments, the linker comprises a plurality of functional groups, such as bonds that are cleavable under physiological conditions. In some embodiments, the linker comprises a functional group, such as a bond or functional group described herein, that is not directly connected to the first or second moiety linked through the linker at the terminus of the linker, but is internal to the linker. In some embodiments, the linker is hydrolyzable under physiological conditions, the linker is enzymatically cleavable under physiological conditions, or the linker comprises a disulfide that is reducible under physiological conditions. In some embodiments, the linker is not cleaved under physiological conditions, e.g., the linker has a sufficient length that a therapeutic peptide or protein need not be cleaved to be active, e.g., the linker is at least about 20 angstroms (e.g., at least about 24 angstroms) in length.

In some embodiments, the particle further comprises a plurality of other therapeutic peptides or proteins, wherein the other therapeutic peptides or proteins are different from the therapeutic peptides or proteins of a). In some embodiments, at least a portion of a plurality of other therapeutic peptides or proteins are attached to at least a portion of a hydrophobic polymer and/or a hydrophilic-hydrophobic polymer of b).

In some embodiments, the hydrophilic-hydrophobic polymer of b) is a block copolymer, e.g., the hydrophilic-hydrophobic polymer of b) is a diblock copolymer. In some embodiments, the hydrophilic-hydrophobic polymer of b) is a block copolymer. Exemplary block copolymers comprise a neutral hydrophilic block (e.g., which can enhance circulation), and a pH-responsive block (e.g., which can facilitate inclusion body escape). Exemplary pH-responsive blocks include those having cis-acetoxyl, hydrazone, or acetal linkages that can be hydrolyzed at, for example, pH4 to 6.5. In some embodiments, the polymer includes reversible peptide conjugation sites, e.g., which may provide a means of releasing the peptide from the carrier upon reaching the cytosol (e.g., sulfhydryl).

In some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymers of b) has a hydroxyl terminus. In some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymer of b) has a hydroxyl end and the hydroxyl end is capped (e.g., capped with an acyl moiety). For example, in some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymer of b) has a hydroxyl terminus and the hydroxyl terminus is terminated with an acyl moiety.

In some embodiments, the protein is a protein described herein.

In some embodiments, at least a portion of the therapeutic peptide is chemically modified.

In some embodiments, the plurality of therapeutic peptides comprises about 1% to about 50% (e.g., about 1% to about 20%) by weight of the particle.

In some embodiments, the particles further comprise a counter ion. For example, in embodiments where the therapeutic peptide is a charged peptide, the particles may comprise a counter ion, wherein the counter ion has a charge opposite to the charge on the therapeutic peptide or protein. In some embodiments, the ratio of the charge of the therapeutic peptide or protein to the charge of the counter ion in the particle is from about 1: 1.5 to about 1.5: 1 (e.g., from about 1.25: 1 to about 1: 1.25, or about 1: 1).

In some embodiments, the particles are lyophilized particles.

i) the hydrophobic polymer attached to the therapeutic peptide or protein can be a homopolymer or a polymer composed of more than one monomeric subunit;

iv) the hydrophobic polymer is PLGA; and

v) the therapeutic peptide comprises from about 1% to about 20% by weight of the particle.

iii) the hydrophilic polymer is PEG;

iv) the hydrophobic polymer is composed of a first and a second type of monomeric subunit, and the ratio of the first and second type of monomeric subunit in the hydrophobic polymer is from about 15: 85 or 25: 75 to about 75: 25 or 85: 15, e.g., about 50: 50; and

v) the hydrophobic polymer is PLGA.

In some embodiments, the hydrophilic portion of the hydrophilic-hydrophobic polymer of b) terminates in a methoxy group.

iv) the hydrophobic polymer is PLGA.

In some embodiments, the particles further comprise a surfactant (e.g., PVA).

In some embodiments, the protein is a protein described herein.

In some embodiments, the plurality of therapeutic peptides or proteins comprises from about 1% to about 100% (e.g., from about 50% to about 100%, from about 70% to about 100%, from about 50% to about 90%) by weight of the particle.

In some aspects, the disclosure features a particle comprising:

a) optionally a plurality of hydrophobic polymers; and

b) a plurality of therapeutic peptide or protein-hydrophilic-hydrophobic polymer conjugates comprising a therapeutic peptide or protein linked to a hydrophilic-hydrophobic polymer.

In some embodiments, the particles are substantially free of hydrophobic polymers. In some embodiments, the particles further comprise a hydrophobic moiety, such as chitosan, poly (vinyl alcohol), or poloxamer.

In some embodiments, the particle further comprises a plurality of hydrophilic-hydrophobic polymers, wherein each of the hydrophilic-hydrophobic polymers of the plurality of hydrophilic-hydrophobic polymers comprises a hydrophilic moiety attached to a hydrophobic moiety.

In some embodiments, the hydrophobic-hydrophilic polymer of the conjugate of b) is covalently attached to the therapeutic peptide or protein via a linker. Exemplary linkers include: linkers comprising moieties formed using "click chemistry" (e.g., as described in WO 2006/115547), and linkers comprising an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, an oxime, a carbamate, a carbonate, a silyl ether, or a triazole (e.g., an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, or a triazole). In some embodiments, the linker comprises a functional group, such as a bond that is cleavable under physiological conditions. In some embodiments, the linker comprises a plurality of functional groups, such as bonds that are cleavable under physiological conditions. In some embodiments, the linker comprises a functional group, such as a bond or functional group described herein, that is not directly connected to the first or second moiety linked through the linker at the terminus of the linker, but is internal to the linker. In some embodiments, the linker is hydrolyzable under physiological conditions, the linker is enzymatically cleavable under physiological conditions, or the linker comprises a disulfide that is reducible under physiological conditions. In some embodiments, the linker is not cleaved under physiological conditions, e.g., the linker has a sufficient length that a therapeutic peptide or protein need not be cleaved to be active, e.g., the linker is at least about 20 angstroms (e.g., at least about 24 angstroms) in length.

In some embodiments, the particle further comprises a plurality of other therapeutic peptides or proteins, wherein the other therapeutic peptides or proteins are different from the therapeutic peptides or proteins of b). In some embodiments, at least a portion of a plurality of other therapeutic peptides or proteins are attached to at least a portion of the hydrophobic polymer and/or hydrophilic-hydrophobic polymer of a). In some embodiments, at least a portion of a plurality of other therapeutic peptides or proteins are attached to at least a portion of the hydrophobic polymer of a).

In some embodiments, the particles comprise a hydrophobic polymer. In some embodiments, at least a portion of the hydrophobic polymer of a) has a carboxyl terminus. In some embodiments, at least a portion of the hydrophobic polymer of a) has hydroxyl termini. In some embodiments, at least a portion of the hydrophobic polymer of a) having hydroxyl termini has hydroxyl termini that are capped (e.g., capped with an acyl moiety).

In some embodiments, the end of the hydrophobic polymer is modified (e.g., by reaction with a functional moiety), for example, the hydroxyl end of the hydrophobic polymer is modified (e.g., by reaction with a functional moiety) and/or the carboxyl end of the hydrophobic polymer is modified (e.g., by reaction with a functional moiety). For example, the hydroxyl or carboxyl terminus is modified with a reactive moiety that can be used, for example, to attach a therapeutic peptide or protein to a polymer via a linker. In some embodiments, the reactive moiety is not reacted with the therapeutic peptide or protein and remains on the polymer or is hydrolyzed in a subsequent reaction.

In some embodiments, at least a portion of the hydrophobic polymer of a) has a carboxyl terminus and a hydroxyl terminus, e.g., at least a portion of the hydrophobic polymer of a) having a hydroxyl terminus has a hydroxyl terminus that is capped (e.g., with an acyl moiety).

In some embodiments, the hydrophilic-hydrophobic polymer of b) is a block copolymer. In some embodiments, the hydrophilic-hydrophobic polymer of b) is a block copolymer. Exemplary block copolymers comprise a neutral hydrophilic block (e.g., which can enhance circulation), and a pH-responsive block (e.g., which can facilitate inclusion body escape). Exemplary pH-responsive blocks include those having cis-acetoxyl, hydrazone, or acetal linkages that can be hydrolyzed at, for example, pH4 to 6.5. In some embodiments, the polymer includes reversible peptide conjugation sites, e.g., which may provide a means of releasing the peptide from the carrier upon reaching the cytosol (e.g., sulfhydryl). In some embodiments, the hydrophilic-hydrophobic polymer of b) is a diblock copolymer (e.g., PEG-PLGA). In some embodiments, the hydrophilic-hydrophobic polymer of b) is a triblock copolymer (e.g., PEG-PLGA-PEG).

In some embodiments, the hydrophobic polymer has one or more of the following characteristics:

i) the hydrophobic polymer may be a homopolymer or a polymer composed of more than one monomeric subunit;

iii) the hydrophobic polymer is composed of first and second types of monomeric subunits, and the ratio of first and second types of monomeric subunits in the hydrophobic polymer attached to the agent is from about 15: 85 or 25: 75 to about 75: 25 or 85: 15, e.g., about 50: 50; and

iv) the hydrophobic polymer is PLGA.

In some embodiments, the hydrophobic polymer has a weight average molecular weight of about 4-15kDa, such as 6-12kDa, such as 8-10 kDa.

i) the hydrophilic moiety has a weight average molecular weight of about 1-6kDa (e.g., 2-6kDa),

iii) the hydrophilic polymer is PEG;

iv) the hydrophobic portion of the hydrophilic-hydrophobic polymer is comprised of first and second types of monomeric subunits, and the ratio of the first and second types of monomeric subunits in the hydrophobic portion is from about 15: 85 or 25: 75 to about 75: 25 or 85: 15, e.g., about 50: 50; and

v) the hydrophobic part of the hydrophilic-hydrophobic polymer is PLGA.

In some embodiments, if the weight average molecular weight of the hydrophilic portion of the hydrophilic-hydrophobic polymer is between about 1-3kDa, such as about 2kDa, the ratio of the weight average molecular weight of the hydrophilic portion to the weight average molecular weight of the hydrophobic portion is between 1: 3-1: 7, and if the weight average molecular weight of the hydrophilic portion is between about 4-6kDa, such as about 5kDa, the ratio of the weight average molecular weight of the hydrophilic portion to the weight average molecular weight of the hydrophobic portion is between 1: 1-1: 4.

In some embodiments, the hydrophilic portion of the hydrophilic-hydrophobic polymer has a weight average molecular weight of about 2-6kDa and the hydrophobic portion has a weight average molecular weight of between about 8-13 kDa.

iii) the hydrophobic polymer is comprised of first and second types of monomeric subunits, and the ratio of the first and second types of monomeric subunits in the hydrophobic polymer is from about 15: 85 or 25: 75 to about 75: 25 or 85: 15, e.g., about 50: 50; and

iv) the hydrophobic polymer is PLGA.

In some embodiments, the protein is a protein described herein.

In some embodiments, the particles further comprise a counter ion. For example, in embodiments where the therapeutic peptide is a charged peptide, the particles can comprise a counterion, wherein the counterion has a charge opposite to the charge on the therapeutic peptide. In some embodiments, the ratio of the charge of the therapeutic peptide or protein to the charge of the counter ion in the particle is from about 1: 1.5 to about 1.5: 1 (e.g., from about 1.25: 1 to about 1: 1.25, or about 1: 1).

In some embodiments, the particles are lyophilized particles.

In some aspects, the disclosure features a particle comprising:

a) optionally, a plurality of hydrophobic polymers;

b) a plurality of hydrophilic-hydrophobic polymer-conjugates, wherein the hydrophilic-hydrophobic polymer conjugate comprises a hydrophilic-hydrophobic polymer attached to a charged peptide; and

c) a plurality of charged therapeutic peptides or proteins, wherein the charge of the therapeutic peptide or protein is opposite to the charge of the peptide conjugated to the hydrophilic-hydrophobic polymer, and wherein the charged therapeutic peptide or protein forms a non-covalent bond (e.g., an ionic bond) with the charged peptide or protein of the hydrophilic-hydrophobic polymer-conjugate.

In some embodiments, the particles further comprise a hydrophilic-hydrophobic polymer, such as a block copolymer (e.g., PEG-PLGA). Exemplary block copolymers comprise a neutral hydrophilic block (e.g., which can enhance circulation), and a pH-responsive block (e.g., which can facilitate inclusion body escape). Exemplary pH-responsive blocks include those having cis-acetoxyl, hydrazone, or acetal linkages that can be hydrolyzed at, for example, pH4 to 6.5. In some embodiments, the polymer includes reversible peptide conjugation sites, e.g., which may provide a means of releasing the peptide from the carrier upon reaching the cytosol (e.g., sulfhydryl). In some embodiments, the hydrophilic-hydrophobic polymer of b) is a diblock copolymer (e.g., PEG-PLGA). In some embodiments, the hydrophilic-hydrophobic polymer of b) is a triblock copolymer (e.g., PEG-PLGA-PEG).

In some embodiments, the block copolymer is a diblock or triblock copolymer. Exemplary block copolymers comprise a neutral hydrophilic block (e.g., which can enhance circulation), and a pH-responsive block (e.g., which can facilitate inclusion body escape). Exemplary pH-responsive blocks include those having cis-acetoxyl, hydrazone, or acetal linkages that can be hydrolyzed at, for example, pH4 to 6.5. In some embodiments, the polymer includes reversible peptide conjugation sites, e.g., which may provide a means of releasing the peptide from the carrier upon reaching the cytosol (e.g., sulfhydryl).

In some embodiments, the hydrophobic-hydrophilic polymer of the conjugate of b) is covalently attached to the therapeutic peptide or protein via a linker. Exemplary linkers include: linkers comprising moieties formed using "click chemistry" (e.g., as described in WO 2006/115547), and linkers comprising an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, an oxime, a carbamate, a carbonate, a silyl ether, or a triazole (e.g., an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, or a triazole). In some embodiments, the linker comprises a functional group, such as a bond that is cleavable under physiological conditions. In some embodiments, the linker comprises a plurality of functional groups, such as bonds that are cleavable under physiological conditions. In some embodiments, the linker comprises a functional group, such as a bond or a functional group described herein, that is not directly connected to the first or second moiety attached through the linker at the terminus of the linker, but is internal to the linker. In some embodiments, the linker is hydrolyzable under physiological conditions, the linker is enzymatically cleavable under physiological conditions, or the linker comprises a disulfide that is reducible under physiological conditions. In some embodiments, the linker is not cleaved under physiological conditions, e.g., the linker has a sufficient length that a therapeutic peptide or protein need not be cleaved to be active, e.g., the linker is at least about 20 angstroms (e.g., at least about 24 angstroms) in length.

In some embodiments, the particle further comprises a plurality of other therapeutic peptides or proteins, wherein the other therapeutic peptides or proteins are different from the therapeutic peptides or proteins of b). In some embodiments, at least a portion of a plurality of other therapeutic peptides or proteins are attached to at least a portion of the hydrophobic polymer and/or hydrophilic-hydrophobic polymer of a). In some embodiments, at least a portion of a plurality of other therapeutic peptides or proteins are attached to at least a portion of the hydrophobic polymers of a).

In some embodiments, at least a portion of the hydrophobic polymer of a) has a carboxyl terminus and a hydroxyl terminus, and for example, at least a portion of the hydrophobic polymer of a) having a hydroxyl terminus has a hydroxyl terminus that is capped (e.g., capped with an acyl moiety).

In some embodiments, at least a portion of the hydrophobic polymer of a) is covalently attached to a therapeutic peptide or protein, and a portion of the hydrophobic polymer of a) is attached to a plurality of therapeutic peptides or proteins.

In some embodiments, the hydrophilic-hydrophobic polymer of b) is a block copolymer. Exemplary block copolymers comprise a neutral hydrophilic block (e.g., which can enhance circulation), and a pH-responsive block (e.g., which can facilitate inclusion body escape). Exemplary pH-responsive blocks include those having cis-acetoxyl, hydrazone, or acetal linkages that can be hydrolyzed at, for example, pH4 to 6.5. In some embodiments, the polymer includes reversible peptide conjugation sites, e.g., which may provide a means of releasing the peptide from the carrier upon reaching the cytosol (e.g., sulfhydryl). In some embodiments, the hydrophilic-hydrophobic polymer of b) is a diblock copolymer (e.g., PEG-PLGA). In some embodiments, the hydrophilic-hydrophobic polymer of b) is a triblock copolymer (e.g., PEG-PLGA-PEG).

iii) the hydrophilic polymer is PEG;

v) the hydrophobic part of the hydrophilic-hydrophobic polymer is PLGA.

In some embodiments, the protein is a protein described herein.

In some embodiments, the plurality of therapeutic peptides or proteins comprises from about 1% to about 90% (e.g., from about 50% to about 90%, from about 70% to about 90%, from about 20% to about 70%) by weight of the particle.

In some embodiments, the particles further comprise a counter ion. For example, in embodiments where the therapeutic peptide is a charged peptide, the particles may comprise a counterion, wherein the counterion has a charge opposite to the charge on the therapeutic peptide. In some embodiments, the ratio of the charge of the therapeutic peptide or protein to the charge of the counter ion in the particle is from about 1: 1.5 to about 1.5: 1 (e.g., from about 1.25: 1 to about 1: 1.25, or about 1: 1).

In some embodiments, the particles are lyophilized particles.

In some aspects, the disclosure features a composition comprising a plurality of particles described herein. In some embodiments, the composition is a pharmaceutical composition.

In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or all of the particles have a diameter of less than about 200 nM.

In some embodiments, the particles have a Dv90 diameter of less than 200nm (e.g., less than 150 nm).

In some embodiments, the composition is substantially free of polymers having a molecular weight of less than about 500 Da.

In some embodiments, the composition is substantially free of the therapeutic peptide or protein in free form (i.e., the therapeutic peptide or protein is not embedded in or attached to the particle).

In some embodiments, the composition is chemically stable at ambient conditions for at least 1 day (e.g., at least 7 days, at least 14 days, at least 21 days, at least 30 days). In some embodiments, the composition is chemically stable for at least 1 day (e.g., at least 7 days, at least 14 days, at least 21 days, at least 30 days) under conditions comprising a temperature of 23 degrees celsius and a humidity of 60, 70, or 80 percent.

In some embodiments, the composition is a lyophilized composition.

In some embodiments, the composition, when administered to a subject, results in an increase in AUC of at least 10%, 20%, 50%, 75%, 80%, 90%, 100%, 200%, or 500% as compared to the AUC of the free form of the therapeutic peptide or protein (i.e., not in particle form) administered to the subject. In some embodiments, the composition and the administered therapeutic peptide or protein in free form are administered under similar conditions. In some embodiments, the amount of therapeutic peptide or protein in the particle composition administered to the subject, e.g., in terms of weight or number of molecules, is the same as the amount of therapeutic peptide in free form administered. In some embodiments, the curve defining AUC is selected from:

a) a plot of therapeutic peptide or protein in a selected target compartment, e.g., a selected tissue, organ, or other compartment, versus time.

In some embodiments, the curve is a plot of therapeutic peptide or protein in a selected target compartment, e.g., peripheral blood, versus time. In some embodiments, the AUC is calculated over a period of 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 2 days, or 7 days. In some embodiments, the period begins at or 1 minute, 10 minutes, 60 minutes, 2 hours, 12 hours, 24 hours, 2 days, or 7 days after the administration of a dose of the composition or the therapeutic peptide or protein in free form.

In some embodiments, the subject is any one of a mouse, rat, dog, or human.

In some embodiments, the composition results in a peak plasma concentration (C) when administered to a subject_max) Less than C of the free form of the therapeutic peptide or protein administered to a subject_max90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1%. In some embodiments, the composition and the administered therapeutic peptide or protein in free form are administered under similar conditions. In some embodiments, the amount of therapeutic peptide or protein in the particle composition administered to the subject, e.g., in terms of weight or number of molecules, is the same as the amount of free form administered. In some embodiments, C_maxMeasured by the presence of the labeled therapeutic peptide or protein in free form in plasma. In some embodiments, C is taken over a period of 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 2 days, or 7 days_maxAnd (6) measuring the values. In some embodiments, the period begins at or 1 minute, 10 minutes, 60 minutes, 2 hours, 12 hours, 24 hours, 2 days, or 7 days after the administration of a dose of the composition or therapeutic peptide or protein. In some embodiments, the subject is any one of a mouse, rat, dog, or human.

In some embodiments, the volume of distribution (V) caused by the composition when administered to a subject_z) V less than the therapeutic peptide or protein in free form administered to a subject_z90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1%.

In some embodiments, the composition and the free form of the therapeutic peptide or protein administered are inApplied under similar conditions. In some embodiments, the amount of therapeutic peptide or protein in the particle composition administered to the subject, e.g., in terms of weight or number of molecules, is the same as the amount of free form administered. In some embodiments, V_zMeasured by detecting the free form of the labeled therapeutic peptide or protein in plasma. In some embodiments, V is taken over a period of 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 2 days, or 7 days_zAnd (6) measuring the values. In some embodiments, the period begins at or 1 minute, 10 minutes, 60 minutes, 2 hours, 12 hours, 24 hours, 2 days, or 7 days after the administration of a dose of the composition or the therapeutic peptide or protein in free form. In some embodiments, the subject is any one of a mouse, rat, dog, or human.

In some aspects, the disclosure features a kit comprising a plurality of particles described herein or a composition described herein.

In some aspects, the disclosure features a single dosage unit comprising a plurality of particles described herein or a composition described herein.

In some aspects, the disclosure features a method of treating a subject having a disorder, the method comprising administering to the subject an effective amount of a particle described herein or a composition described herein.

In one embodiment, the disorder is a proliferative disorder, e.g., cancer, in a subject, e.g., a human, the method comprising: a composition comprising a conjugate or particle described herein is administered to a subject in an amount effective to treat a disorder, thereby treating a proliferative disorder. In one embodiment, the composition is administered in combination with one or more other anti-cancer agents, e.g., a chemotherapeutic agent or combination of chemotherapeutic agents described herein, and radiation.

In one embodiment, the cancer is a cancer described herein. For example, the cancer may be a cancer of the following organs: bladder (including accelerated and metastatic bladder cancer), breast (e.g., estrogen receptor positive breast cancer; estrogen receptor negative breast cancer; HER-2 positive breast cancer; HER-2 negative breast cancer; progesterone receptor positive breast cancer; progesterone receptor negative breast cancer; estrogen receptor negative, HER-2 negative and progesterone receptor negative breast cancer (i.e., triple negative breast cancer); inflammatory breast cancer), colon (including colorectal cancer), kidney (e.g., transitional cell carcinoma), liver, lung (including small and non-small cell lung cancers, adenocarcinoma of the lung, and squamous cell carcinoma), genitourinary tract, e.g., ovary (including fallopian tube and peritoneal carcinoma), cervix, prostate, testis, kidney, and ureter, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gall bladder, gallbladder, Thyroid, skin (including squamous cell carcinoma), brain (including glioblastoma multiforme), head and neck (e.g., occult primary), and soft tissue (e.g., Kaposi's sarcoma; e.g., AIDS-related Kaposi's sarcoma), leiomyosarcoma, angiosarcoma, and histiocytoma). Preferred cancers include breast cancer (e.g., metastatic or locally advanced breast cancer), prostate cancer (e.g., hormone refractory prostate cancer), renal cell carcinoma, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma, e.g., unresectable, locally advanced or metastatic non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma), pancreatic cancer, gastric cancer (e.g., metastatic gastric adenocarcinoma), colorectal cancer, rectal cancer, head and neck squamous cell carcinoma, lymphoma (Hodgkin's lymphoma) or non-Hodgkin's lymphoma), renal cell carcinoma, urothelial cancer, soft tissue sarcoma (e.g., kaposi's sarcoma (e.g., AIDS-related kaposi's sarcoma), leiomyosarcoma, angiosarcoma, and histiocytoma), glioma, myeloma (e.g., multiple myeloma), melanoma (e.g., advanced or metastatic melanoma), germ cell tumors, ovarian cancer (e.g., advanced ovarian cancer, e.g., advanced fallopian tube or peritoneal membrane cancer), and gastrointestinal cancer.

In one embodiment, the disease or disorder associated with inflammation is a disease or disorder described herein. For example, the disease or condition associated with inflammation may be, for example, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, degenerative joint disease, spondyloarthropathies (spondilobarthropathies), gouty arthritis, systemic lupus erythematosus, juvenile arthritis, rheumatoid arthritis, osteoarthritis, osteoporosis, diabetes (e.g., insulin-dependent diabetes or juvenile onset diabetes), menstrual pain, cystic fibrosis, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, mucous colitis, ulcerative colitis, gastritis, esophagitis, pancreatitis, peritonitis, Alzheimer's disease, shock, ankylosing spondylitis, gastritis, conjunctivitis, pancreatitis (pancreatitis) (acute or chronic), multiple organ injury syndrome (e.g., sepsis or secondary to trauma), myocardial infarction, or a combination thereof, Atherosclerosis, stroke, reperfusion injury (e.g., due to cardiopulmonary bypass or renal dialysis), acute glomerulonephritis, vasculitis, thermal injury (i.e., sunburn), necrotizing enterocolitis, granulocyte infusion-related syndrome, and/or Sjogren's syndrome. Exemplary inflammatory conditions of the skin include, for example, eczema, atopic dermatitis, contact dermatitis, rubella, scleroderma, psoriasis, and skin diseases with an acute inflammatory component.

In another embodiment, compositions comprising the particles or conjugates described herein may be used to treat or prevent allergies and respiratory conditions, including asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen poisoning, emphysema, chronic bronchitis, acute respiratory distress syndrome, and any Chronic Obstructive Pulmonary Disease (COPD). The particles or conjugates described herein can be used to treat chronic hepatitis infections, including hepatitis b and hepatitis c.

In addition, compositions comprising the particles or conjugates described herein can be used to treat autoimmune diseases and/or inflammation associated with autoimmune diseases, such as organ-tissue autoimmune diseases (e.g., Raynaud's syndrome), scleroderma, myasthenia gravis, transplant rejection, endotoxic shock, sepsis, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmune thyroiditis, uveitis, systemic lupus erythematosus, Addison's disease, autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome), and Grave's disease.

In one embodiment, the disorder is associated with a cardiovascular disease, such as a heart disease, in a subject, such as a human, the method comprising: a composition comprising a particle or conjugate described herein is administered to a subject in an amount effective to treat a disorder, thereby treating a cardiovascular disease.

In one embodiment, the cardiovascular disease is a disease or disorder described herein. For example, the cardiovascular disease may be cardiomyopathy or myocarditis; such as idiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholic cardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy, and hypertensive cardiomyopathy. The particles, conjugates, compositions, and methods described herein can also be used to treat or prevent atherosclerotic disorders of major blood vessels (macrovascular disease), such as the aorta, coronary arteries, carotid arteries, cerebrovascular arteries, renal arteries, iliac arteries, femoral arteries, and popliteal arteries. Other vascular diseases that may be treated or prevented include those associated with: platelet aggregation, retinal arterioles, glomerular arterioles, neurotrophic vessels, cardiac arterioles, and associated capillary beds of the eye, kidney, heart, and central and peripheral nervous systems. Other conditions that may be treated with the particles, conjugates, compositions, and methods described herein include restenosis following, for example, coronary intervention, and conditions associated with abnormal levels of high and low density cholesterol.

In one embodiment, a composition comprising a particle or conjugate described herein is administered to a subject undergoing or having undergone angioplasty. In one embodiment, a composition comprising a particle or conjugate described herein is administered to a subject undergoing or having undergone angioplasty accompanied by stent placement. In some embodiments, a composition comprising a particle or conjugate described herein can be used as a strut of a stent or a coating of a stent.

In one embodiment, the disorder is associated with the kidney of a subject, e.g., a human, e.g., a renal disorder, the method comprising: a composition comprising a particle or conjugate described herein is administered to a subject in an amount effective to treat a disorder, thereby treating a disease or disorder associated with kidney disease.

In one embodiment, the disease or disorder associated with the kidney is a disease or disorder described herein. For example, the kidney-related disease or disorder can be, for example, acute renal failure, acute renal syndrome, analgesic nephropathy, congenic infarct nephropathy, chronic renal failure, chronic nephritis, congenital nephrotic syndrome, end-stage renal disease, Goodpasture syndrome, interstitial nephritis, kidney damage, kidney infection, kidney injury, kidney stones, lupus nephritis, membranoproliferative GN I, membranoproliferative GN II, membranous nephropathy, minimal disease, necrotizing glomerulonephritis, nephroblastoma, renal calcinosis, nephrogenic diabetes insipidus, nephrotic degenerative disease (nephrotic syndrome), polycystic kidney disease, post streptococcal GN, reflux nephropathy, renal artery embolism, renal artery stenosis, renal papillary necrosis, renal tubular acidosis type I, renal tubular acidosis type II, renal hypoperfusion, renal venous thrombosis.

In some aspects, the disclosure features a therapeutic peptide or protein-hydrophobic polymer conjugate that includes a therapeutic peptide or protein covalently attached to a hydrophobic polymer, e.g., the therapeutic peptide or protein is covalently attached to the hydrophobic polymer via a carboxy terminus, the therapeutic peptide or protein is covalently attached to the hydrophobic polymer via an amino terminus, and/or the therapeutic peptide or protein is covalently attached to the hydrophobic polymer via an amino acid side chain.

In some embodiments, the therapeutic peptide or protein is covalently attached to the hydrophobic polymer at a terminus of the polymer.

In some embodiments, the therapeutic peptide or protein is covalently attached to the polymer on the backbone of the hydrophobic polymer.

In some embodiments, a single therapeutic peptide or protein is covalently attached to a single hydrophobic polymer. In other embodiments, a plurality of therapeutic peptides or proteins are covalently linked to a single hydrophobic polymer.

In some embodiments, the therapeutic peptide or protein is covalently attached directly to the hydrophobic polymer (e.g., via an amide bond). In some embodiments, the therapeutic peptide or protein is covalently attached to the hydrophobic polymer via a linker. Exemplary linkers include: linkers comprising moieties formed using "click chemistry" (e.g., as described in WO 2006/115547), and linkers comprising an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, an oxime, a carbamate, a carbonate, a silyl ether, or a triazole (e.g., an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, or a triazole). In some embodiments, the linker comprises a functional group, such as a bond that is cleavable under physiological conditions. In some embodiments, the linker comprises a plurality of functional groups, such as bonds that are cleavable under physiological conditions. In some embodiments, the linker comprises a functional group, such as a bond or functional group described herein, that is not directly connected to the first or second moiety linked through the linker at the terminus of the linker, but is internal to the linker. In some embodiments, the linker is hydrolyzable under physiological conditions, the linker is enzymatically cleavable under physiological conditions, or the linker comprises a disulfide that is reducible under physiological conditions. In some embodiments, the linker is not cleaved under physiological conditions, e.g., the linker has a sufficient length that a therapeutic peptide or protein need not be cleaved to be active, e.g., the linker is at least about 20 angstroms (e.g., at least about 24 angstroms) in length.

In some embodiments, the hydrophobic polymer has a terminal hydroxyl moiety. In some embodiments, the hydroxyl terminus of the hydrophobic polymer is modified (e.g., by reaction with a functional moiety). In some embodiments, the hydrophobic polymer has a terminal hydroxyl moiety that is capped (e.g., capped with an acyl moiety).

In some embodiments, the hydrophobic polymer has a terminal carboxyl moiety. In some embodiments, the carboxyl terminus of the hydrophobic polymer is modified (e.g., by reaction with a functional moiety).

In some embodiments, the hydrophobic polymer of the therapeutic peptide or protein-hydrophobic polymer conjugate has one or more of the following properties:

i) the hydrophobic polymer attached to the therapeutic peptide or protein is a homopolymer or a polymer composed of more than one monomeric subunit;

ii) the hydrophobic polymer attached to the therapeutic peptide or protein has a weight average molecular weight of about 4-15kDa (e.g., 6-12kDa, e.g., 8-10 kDa);

iv) the hydrophobic polymer is PLGA.

In some aspects, the disclosure features a composition comprising a plurality of therapeutic peptides or protein-hydrophobic polymer conjugates described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a reaction mixture.

In some embodiments, the composition is substantially free of unconjugated therapeutic peptides or proteins.

In some embodiments, the composition is substantially free of hydrophobic polymers having a molecular weight of less than about 500 Da.

In some aspects, the disclosure features a method of making a therapeutic peptide or protein-hydrophobic polymer conjugate described herein, the method comprising:

providing a therapeutic peptide or protein and a polymer; and

the therapeutic peptide or protein and the polymer are subjected to conditions to effect covalent attachment of the therapeutic peptide or protein to the polymer.

In some embodiments, the method is performed in a reaction mixture, e.g., a reaction mixture comprising a single solvent or a reaction mixture comprising a solvent system of multiple solvents (e.g., the multiple solvents are miscible, the solvent system comprises water and a polar solvent (e.g., DMF, DMSO, acetone, or acetonitrile), or the solvent system is biphasic (e.g., comprises an organic phase and an aqueous phase)).

In some embodiments, the polymer is attached to an insoluble matrix.

In some embodiments, the methods comprise forming a bond using "click chemistry" (e.g., as described in WO 2006/115547).

In some embodiments, the method results in the formation of an amide bond, a disulfide bond, an ester bond, and/or a triazole.

In some embodiments, the hydrophobic polymer has an aqueous solubility of less than about 1 mg/ml.

In some embodiments, the hydrophobic polymer is covalently attached to the therapeutic peptide or protein through the amino terminus of the therapeutic peptide or protein. In some embodiments, the hydrophobic polymer is covalently attached to the therapeutic peptide or protein through the carboxy terminus of the therapeutic peptide or protein. In some embodiments, the hydrophobic polymer is covalently attached to the therapeutic peptide or protein through an amino acid side chain of the therapeutic peptide or protein.

In some embodiments, the therapeutic peptide or protein is covalently attached to the polymer at a terminus of the hydrophobic polymer.

In some embodiments, the hydrophobic polymer has hydroxyl and/or carboxylic acid termini.

In some embodiments, the method produces a therapeutic peptide or protein-hydrophobic polymer conjugate having a purity of at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 99%).

In some embodiments, the method produces at least about 100mg of the therapeutic peptide or protein-hydrophobic polymer conjugate (e.g., at least about 1 g).

In some aspects, the disclosure features a therapeutic peptide or protein-hydrophobic polymer conjugate made by the methods described herein.

In some aspects, the disclosure features a therapeutic peptide or protein-hydrophilic-hydrophobic polymer conjugate comprising a therapeutic peptide or protein covalently attached to a hydrophilic-hydrophobic polymer, wherein the hydrophilic-hydrophobic polymer comprises a hydrophilic moiety attached to a hydrophobic moiety.

In some embodiments, the therapeutic peptide or protein is attached to a hydrophilic portion of a hydrophilic-hydrophobic polymer.

In some embodiments, the therapeutic peptide is attached to a hydrophobic portion of a hydrophilic-hydrophobic polymer.

In some embodiments, the hydrophilic-hydrophobic polymer is covalently attached to the therapeutic peptide or protein through the amino terminus of the therapeutic peptide or protein, the hydrophilic-hydrophobic polymer is covalently attached to the therapeutic peptide or protein through the carboxy terminus of the therapeutic peptide or protein, and/or the hydrophilic-hydrophobic polymer is covalently attached to the therapeutic peptide or protein through an amino acid side chain of the therapeutic peptide or protein.

In some embodiments, the therapeutic peptide or protein is covalently attached to the hydrophilic-hydrophobic polymer at the terminus of the polymer. In some embodiments, the therapeutic peptide or protein is covalently attached to the polymer on the backbone of the hydrophilic-hydrophobic polymer.

In some embodiments, a single therapeutic peptide or protein is covalently linked to a single hydrophilic-hydrophobic polymer.

In some embodiments, a plurality of therapeutic peptides or proteins are covalently attached to a single hydrophilic-hydrophobic polymer, e.g., a therapeutic peptide or protein is attached to a hydrophilic portion of a hydrophilic-hydrophobic polymer, and a therapeutic peptide or protein is attached to a hydrophobic portion of a hydrophilic-hydrophobic polymer.

In some embodiments, the therapeutic peptide or protein is covalently attached directly to the hydrophobic portion of the hydrophobic-hydrophobic polymer (e.g., via an amide or ester bond).

In some embodiments, the therapeutic peptide or protein is covalently attached directly to the hydrophilic portion of the hydrophilic-hydrophobic polymer (e.g., via an amide or ester bond).

In some embodiments, the therapeutic peptide or protein is covalently attached to the hydrophilic-hydrophobic polymer via a linker. Exemplary linkers include: linkers comprising moieties formed using "click chemistry" (e.g., as described in WO 2006/115547), and linkers comprising an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, an oxime, a carbamate, a carbonate, a silyl ether, or a triazole (e.g., an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, or a triazole). In some embodiments, the linker comprises a functional group, such as a bond that is cleavable under physiological conditions. In some embodiments, the linker comprises a plurality of functional groups, such as bonds that are cleavable under physiological conditions. In some embodiments, the linker comprises a functional group, such as a bond or a functional group described herein, that is not directly connected to the first or second moiety attached through the linker at the terminus of the linker, but is internal to the linker. In some embodiments, the linker is hydrolyzable under physiological conditions, the linker is enzymatically cleavable under physiological conditions, or the linker comprises a disulfide that is reducible under physiological conditions. In some embodiments, the linker is not cleaved under physiological conditions, e.g., the linker has a sufficient length that a therapeutic peptide or protein need not be cleaved to be active, e.g., the linker is at least about 20 angstroms (e.g., at least about 24 angstroms) in length.

In some embodiments, the hydrophilic-hydrophobic polymer has one or more of the following properties:

ii) the hydrophobic polymer has a weight average molecular weight of about 4-15kDa (e.g., 6-12kDa, 8-10 kDa);

iii) the hydrophilic polymer is PEG;

iv) the hydrophobic polymer is composed of a first and a second type of monomeric subunit, and the ratio of the first and second type of monomeric subunit in the hydrophobic polymer attached to the therapeutic peptide is from about 15: 85 or 25: 75 to about 75: 25 or 85: 15, e.g., about 50: 50; and

v) the hydrophobic polymer is PLGA.

In some embodiments, if the weight average molecular weight of the hydrophilic portion of the hydrophilic-hydrophobic polymer is between about 1-3kDa, such as about 2kDa, then the ratio of the weight average molecular weight of the hydrophilic portion to the weight average molecular weight of the hydrophobic portion is between 1: 3-1: 7, and if the weight average molecular weight of the hydrophilic portion is between about 4-6kDa, such as about 5kDa, then the ratio of the weight average molecular weight of the hydrophilic portion to the weight average molecular weight of the hydrophobic portion is between 1: 1-1: 4.

In some aspects, the disclosure features a composition comprising a plurality of therapeutic peptides or protein-hydrophilic-hydrophobic polymer conjugates described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a reaction mixture.

In some embodiments, the composition is substantially free of unconjugated therapeutic peptide.

In some embodiments, the composition is substantially free of hydrophilic-hydrophobic polymers having a molecular weight of less than about 500 Da.

In some aspects, the disclosure features a method of making a therapeutic peptide or protein-hydrophilic-hydrophobic polymer conjugate described herein, the method comprising:

providing a therapeutic peptide or protein and a hydrophilic-hydrophobic polymer; and

the therapeutic peptide or protein and the hydrophilic-hydrophobic polymer are subjected to conditions to effect covalent attachment of the therapeutic peptide or protein to the polymer.

In some embodiments, the method is performed in a reaction mixture, e.g., the reaction mixture comprises a single solvent or the reaction mixture comprises a solvent system comprising multiple solvents (e.g., the multiple solvents are miscible, or the solvent system is biphasic (e.g., comprises an organic phase and an aqueous phase)).

In some embodiments, at least one of a therapeutic peptide, protein, or hydrophilic-hydrophobic polymer is attached to the insoluble substrate, e.g., the hydrophilic-hydrophobic polymer is attached to the insoluble substrate.

In some embodiments, the method results in the formation of an amide bond, a disulfide bond, an ester bond, and/or a tetrazole.

In some embodiments, the hydrophilic-hydrophobic polymer is covalently attached to the therapeutic peptide through the amino terminus of the therapeutic peptide or protein, the hydrophilic-hydrophobic polymer is covalently attached to the therapeutic peptide or protein through the carboxy terminus of the therapeutic peptide or protein, and/or the hydrophilic-hydrophobic polymer is covalently attached to the therapeutic peptide or protein through an amino acid side chain of the therapeutic peptide or protein.

In some embodiments, the therapeutic peptide or protein is covalently attached to the hydrophobic-hydrophilic polymer at a terminus of the polymer.

In some embodiments, the therapeutic peptide or protein is covalently attached to the hydrophobic-hydrophilic polymer on a hydrophilic portion of the polymer. In some embodiments, the therapeutic peptide or protein is covalently attached to the hydrophobic-hydrophilic polymer on a hydrophobic portion of the polymer. In some embodiments, the therapeutic peptide or protein is covalently attached to the hydrophobic-hydrophilic polymer on the backbone of the polymer.

In some embodiments, a single therapeutic peptide or protein is covalently linked to a single hydrophobic-hydrophilic polymer.

In some embodiments, a plurality of therapeutic peptides or proteins are covalently linked to a single hydrophobic-hydrophilic polymer. In some embodiments, the therapeutic peptide or protein is covalently attached to the hydrophobic-hydrophilic polymer on a hydrophilic portion of the polymer, the therapeutic peptide or protein is covalently attached to the hydrophobic-hydrophilic polymer on a hydrophobic portion of the polymer, and/or the therapeutic peptide or protein is covalently attached to the hydrophobic-hydrophilic polymer on a backbone of the polymer.

In some embodiments, the method produces a therapeutic peptide or protein-hydrophilic-hydrophobic polymer conjugate having a purity of at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 99%).

In some aspects, the disclosure features a therapeutic peptide or protein-hydrophilic-hydrophobic polymer conjugate made by the methods described herein.

In another aspect, the invention features a method of storing a conjugate, particle, or composition, the method including:

providing the conjugate, particle, or composition disposed in a container (e.g., a gas-tight or liquid-tight container, e.g., a container described herein, e.g., a container with an inert gas, e.g., a headspace filled with argon or nitrogen);

storing the conjugate, particle, or composition, e.g., under preselected conditions, e.g., temperature, e.g., temperatures described herein;

and, moving the container to a second location or removing all or an aliquot of the conjugate, particle, or composition from the container.

In one embodiment, the conjugate, particle or composition is evaluated for physical properties such as stability or activity, e.g., color, aggregation, flow or pouring ability, or particle size or charge, of the therapeutic peptide or protein. The evaluation may be compared to a standard and, optionally, the conjugate, particle, or composition is classified in response to the standard.

In one embodiment, the conjugate, particle, or composition is stored in a reconstituted formulation (e.g., in a liquid state as a solution or suspension).

In one aspect, in any of the aspects and embodiments described above, a protein may be used instead of a therapeutic peptide. As used herein, a "protein" has more than 100 amino acids or more, e.g., a protein is at least 110 amino acids long.

Brief Description of Drawings

Fig. 1A-C depict exemplary linkers that may be used to link portions described herein.

Detailed Description

The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Described herein are particles, conjugates (e.g., therapeutic peptide-polymer conjugates, and protein-polymer conjugates), and compositions. Also disclosed are dosage forms containing the conjugates, particles, and compositions; methods of using the conjugates, particles, and compositions (e.g., for treating a disorder); kits comprising conjugates, particles, and compositions; methods of making conjugates, particles, and compositions; methods of storing conjugates, particles, and compositions; and methods of analyzing conjugates, particles, and compositions.

The headings and other identifiers (e.g., (a), (b), (i), etc.) are presented only to facilitate a reading of the specification and claims. The use of headings and other identifiers in this specification and claims does not require that the steps or elements be performed in alphabetical or numerical order or the order in which they appear.

Definition of

The term "ambient conditions" as used herein, unless otherwise indicated, refers to ambient conditions at about one atmosphere of pressure, 50% relative humidity, and about 25 ℃.

The term "anionic moiety" refers to a moiety having a pKa and/or a negative charge of less than 3, 2, 1, or 0 under at least one of the following conditions: during production of the particles described herein, upon formulation into the particles described herein, or after administration of the particles described herein to a subject, e.g., while circulating within the subject and/or while in an inclusion body. Anionic moieties include polymeric species, such as moieties having more than one charge.

The term "anionic polymer" refers, for example, to an anionic moiety having multiple negative charges (i.e., at least 2 under at least 1 condition as described above) when formulated into a particle as described herein. In some embodiments, the anionic polymer has at least 3, 4, 5, 10, 15, or 20 negative charges.

The term "linked" (e.g., a therapeutic peptide is attached to a polymer) as used herein with respect to the relationship of a first moiety to a second moiety refers to the formation of a covalent bond between the first moiety and the second moiety. In the same context, the term "linkage" refers to a covalent bond between the first and second moieties. For example, a therapeutic peptide attached to a polymer is a therapeutic peptide covalently bonded to a polymer (e.g., a hydrophobic polymer described herein). The linkage may be direct, e.g., through a direct bond of the first moiety to the second moiety, or may be through a linker (e.g., through a covalent linking chain of one or more atoms disposed between the first and second moieties). For example, when the linkage is through a linker, the first moiety (e.g., drug) is covalently bonded to the linker, which in turn is covalently bonded to the second moiety (e.g., hydrophobic polymer described herein).

The term "biodegradable" includes polymers, compositions, and formulations that are intended to degrade during use, as described herein. Biodegradable polymers are generally different from non-biodegradable polymers in that the former can degrade during use. In certain embodiments, such use relates to in vivo use, such as in vivo therapy, and in other certain embodiments, such use relates to in vitro use. In general, degradation attributable to biodegradability involves degradation of the biodegradable polymer into its constituent subunits, or digestion (e.g., by biochemical processes) of the polymer into smaller, non-polymeric subunits. In certain embodiments, two different types of biodegradation can generally be identified. For example, one type of biodegradation may involve cleavage of bonds (whether covalent or otherwise) in the polymer backbone. In such biodegradation, monomers and oligomers are typically produced, and even more typically, such biodegradation occurs through cleavage of bonds linking one or more subunits of the polymer. In contrast, another type of biodegradation may involve cleavage of the interior of the side chain or the bond (whether covalent or otherwise) connecting the side chain to the polymer backbone. In certain embodiments, one or the other or both general types of biodegradation may occur during use of the polymer.

The term "biodegradation" as used herein includes both general types of biodegradation described above. The degradation rate of biodegradable polymers often depends in part on a variety of factors, including the chemical nature (identity) of the bonds responsible for any degradation, molecular weight, crystallinity, biostability, and degree of crosslinking of such polymers, the physical characteristics (e.g., shape and size) of the polymers, the assembly of the polymers or particles, and the manner and location of administration. For example, greater molecular weight, higher crystallinity, and/or higher biostability often results in slower biodegradation.

The term "cationic moiety" refers to a moiety having a pKa5 or greater (e.g., a lewis base having a pKa of 5 or greater) and/or a positive charge under at least one of the following conditions: during preparation of the particles described herein, when formulated into the particles described herein, or after administration of the particles described herein to a subject, e.g., when circulating within the subject and/or in an endosome. Exemplary cationic moieties include amine-containing moieties (e.g., charged amine moieties such as quaternary amines), guanidine-containing moieties (e.g., charged guanidines such as guanidine cationic moieties), and heterocyclic and/or heteroaromatic moieties (e.g., charged moieties such as pyridinium or histidine moieties). Cationic moieties include polymeric species, such as moieties having more than one charge, for example, facilitated by the repeated presence of moieties, (e.g., cationic PVA and/or polyamines). Cationic moieties also include zwitterions, meaning compounds having both a positive and negative charge (e.g., amino acids such as arginine, lysine, or histidine).

The term "cationic polymer," e.g., polyamine, refers to, for example, a polymer having multiple positive charges (i.e., at least 2 under at least 1 condition as described above) when formulated into particles described herein (the term "polymer" is described below). In some embodiments, the cationic polymer, e.g., a polyamine, has at least 3, 4, 5, 10, 15, or 20 positive charges.

The phrase "cleavable under physiological conditions" refers to a bond that has a half-life of less than about 50 or less than about 100 hours when subjected to physiological conditions. For example, enzymatic degradation can occur within a period of less than about five years, one year, six months, three months, one month, fifteen days, five days, three days, or one day after exposure to physiological conditions (e.g., aqueous solutions having a pH of about 4 to 8 and a temperature of about 25 ℃ to about 37 ℃).

An "effective amount" or an "effective. The effective amount of the therapeutic peptide-polymer conjugate, particle, or composition can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. An effective amount is also an amount by which any toxic or deleterious effects of the therapeutic peptide-polymer conjugate, particle or composition are outweighed by the therapeutically beneficial effects.

The term "entrapped" as used herein refers to the disposition of a first moiety with or within a second moiety, such as a therapeutic peptide and a polymer (e.g., a therapeutic or diagnostic agent and a hydrophobic polymer), by forming a non-covalent interaction between the first moiety and the second moiety. In one embodiment, where reference is made to a moiety embedded in a particle, this moiety (e.g., a therapeutic peptide or counterion) is associated with the polymer or other component of the particle through one or more non-covalent interactions such as van der Waals interactions (van der Waals interactions), hydrophobic interactions, hydrogen bonding, dipole-dipole interactions, ionic interactions, pi stacking (nesting), and the absence of covalent bonds between the moiety and the other component of the polymer or particle. The embedded portion may be completely or partially surrounded by the polymer or particles embedding it.

The term "hydrophobic" as used herein describes a moiety that can only be dissolved in an aqueous solution to an extent of less than about 0.05mg/mL (e.g., about 0.01mg/mL or less) at physiological ionic strength.

The term "hydrophilic" as used herein describes a moiety that has a solubility in aqueous solution of at least about 0.05mg/mL or greater at physiological ionic strength.

The term "hydrophilic-hydrophobic polymer" as used herein describes a polymer comprising a hydrophilic moiety linked to a hydrophobic moiety. Exemplary hydrophilic-hydrophobic polymers include block copolymers, for example, comprising a block of a hydrophilic polymer and a block of a hydrophobic polymer.

As used herein, "hydroxy-Protecting Groups" are well known in the art and include those Protecting Groups described in detail in Protecting Groups in Organic Synthesis, t.w.greene and p.g.m.wuts, third edition, John Wiley & Sons, 1999, all of which are incorporated herein by reference. Suitable hydroxyl protecting groups include, for example, acyl (e.g., acetyl), Triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), 2, 2, 2-trichloroethoxycarbonyl (Troc), and benzyloxycarbonyl (Cbz).

As used herein, "inert atmosphere" refers to an atmosphere consisting essentially of an inert gas that is not chemically reactive with the polymer-reagent conjugates, particles, compositions, or mixtures described herein. An example of an inert gas is nitrogen (N)₂) Helium and argon.

As used herein, a "linker" is a moiety that links two or more moieties (e.g., a therapeutic peptide or counterion and a polymer, such as a hydrophobic or hydrophilic-hydrophobic, or hydrophilic polymer) together. The linking group has at least two functional groups. For example, a linker having two functional groups may have a first functional group capable of reacting with a functional group on a moiety such as: a therapeutic peptide, a counterion, a hydrophobic moiety such as a polymer, or a hydrophilic-hydrophobic polymer described herein; and a second functional group capable of reacting with a functional group on a second moiety such as: a therapeutic peptide, a counterion, a hydrophobic moiety such as a polymer, or a hydrophilic-hydrophobic polymer as described herein.

The linker may have more than two functional groups (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more functional groups) that may be used, for example, to link reagents to a polymer or to provide a biocleavable moiety within the linker. In some embodiments, for example, when the linker has more than two functional groups, e.g., and the linker includes functional groups in addition to the two functional groups that link the first moiety to the second moiety, the additional functional group (e.g., a third functional group) can be disposed between the first and second groups, and in some embodiments can be cleaved, e.g., under physiological conditions. For example, the linker may have the form:

wherein f is₁A first functional group, for example, a first functional group capable of reacting with a functional group on a moiety such as: a therapeutic peptide or protein, a counterion, a hydrophobic moiety such as a polymer (e.g., a hydrophobic polymer described herein), or a hydrophilic-hydrophobic moiety such as a hydrophilic-hydrophobic polymer described herein; f. of₂Is a second functional group, e.g., a functional group capable of reacting with a functional group on a second moiety such as: a therapeutic peptide or protein described herein or a counterion described herein; f. of ₃Is a biocleavable functional group, e.g., a biocleavable bond as described herein; and isRepresents a spacer for a linking functional group, such as an alkylene (divalent alkyl) group, wherein optionally one or more carbon atoms of the alkylene linker are replaced with one or more heteroatoms (e.g., yielding one of the group consisting of thioether, amino, ester, ether, ketone, amide, silyl ether, oxime, carbamate, carbonate, disulfide, heterocycle, or heteroaromatic). As appropriate, a linker may refer to a linker before attachment to either of the first or second moieties (e.g., therapeutic peptide or polymer), after attachment to one moiety but not before attachment to the other moietyA linker moiety prior to attachment to the second moiety, or a residue of a linker present after attachment to the first and second moieties.

The term "lyoprotectant" as used herein refers to a substance present in a lyophilized formulation. It is usually present prior to the lyophilization process and persists in the resulting lyophilized formulation. Lyoprotectants are typically added after the particles are formed. If a concentration step is present, e.g., between particle formation and lyophilization, a lyoprotectant may be added before or after the concentration step. Lyoprotectants can be used to protect particles during lyophilization, e.g., to reduce or prevent aggregation, particle breakage, and/or other types of damage. In one embodiment, the lyoprotectant is a cryoprotectant.

In one embodiment, the lyoprotectant is a carbohydrate. The term "carbohydrate" as used herein refers to and includes monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

In one embodiment, the lyoprotectant is a monosaccharide. The term "monosaccharide" as used herein refers to a single carbohydrate unit (e.g., a simple sugar) that cannot be hydrolyzed to a simpler carbohydrate unit. Exemplary monosaccharide lyoprotectants include glucose, fructose, galactose, xylose, ribose, and the like.

In one embodiment, the lyoprotectant is a disaccharide. The term "disaccharide" as used herein refers to a compound or chemical moiety formed from 2 monosaccharide units bonded together by glycosidic linkages (e.g., by 1-4 linkages or 1-6 linkages). Disaccharides can be hydrolyzed into two monosaccharides. Exemplary disaccharide lyoprotectants include sucrose, trehalose, lactose, maltose, and the like.

In one embodiment, the lyoprotectant is an oligosaccharide. The term "oligosaccharide" as used herein refers to a compound or chemical moiety formed from 3 to about 15, preferably 3 to about 10 monosaccharide units bonded together by glycosidic linkages (e.g., by 1-4 or 1-6 linkages) to form a linear, branched, or cyclic structure. Exemplary oligosaccharide lyoprotectants include cyclodextrin, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. The oligosaccharides may be oxidized or reduced.

In one embodiment, the lyoprotectant is a cyclic oligosaccharide. The term "cyclic oligosaccharide" as used herein refers to a compound or chemical moiety formed from 3 to about 15, preferably 6, 7, 8, 9 or 10 monosaccharide units bonded together by glycosidic bonds (e.g., by 1-4 or 1-6 bonds) to form a cyclic structure. Exemplary cyclic oligosaccharide lyoprotectants include cyclic oligosaccharides as discrete compounds, such as alpha cyclodextrin, beta cyclodextrin, or gamma cyclodextrin.

Other exemplary cyclic oligosaccharide lyoprotectants include compounds that include a cyclodextrin moiety in a larger molecular structure, such as a polymer comprising a cyclic oligosaccharide moiety. The cyclic oligosaccharide may be oxidized or reduced, for example, to the dicarbonyl form. The term "cyclodextrin moiety" as used herein refers to a cyclodextrin (e.g., alpha, beta, or gamma cyclodextrin) group that is incorporated into or is part of a larger molecular structure (e.g., a polymer). The cyclodextrin moiety can be bonded to one or more other moieties directly or through an optional linker. The cyclodextrin moiety can be oxidized or reduced, e.g., oxidized to the dicarbonyl form.

The carbohydrate lyoprotectant (e.g., cyclic oligosaccharide lyoprotectant) can be a derivatized carbohydrate. For example, in one embodiment, the lyoprotectant is a derivatized cyclic oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2 hydroxypropyl-beta cyclodextrin, e.g., a partially etherified cyclodextrin (e.g., a partially etherified beta cyclodextrin) disclosed in U.S. patent No. 6,407,079, the contents of which are incorporated herein by reference. Another example of a derivatized cyclodextrin is β -cyclodextrin sulfobutyl ether sodium.

An exemplary lyoprotectant is a polysaccharide. The term "polysaccharide" as used herein refers to a compound or chemical moiety formed from at least 16 monosaccharide units bonded together by glycosidic linkages (e.g., by 1-4 linkages or 1-6 linkages) to form a linear, branched, or cyclic structure, and includes polymers comprising polysaccharides as their backbone structure. The polysaccharide may be linear or cyclic in the backbone. Exemplary polysaccharide lyoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin, and the like.

The term "derivatized carbohydrate" refers to an entity having at least one atom that is different from the subject non-derivatized carbohydrate. For example, instead of-OH present on the non-derivatized carbohydrate, the derivatized carbohydrate may have-OX, wherein X is not H. Derivatives may be obtained by chemical functionalization and/or substitution or by de novo synthesis-the term "derivative" implies that there are no process-based limitations.

In some embodiments, the lyoprotectant is a reduced sugar alcohol, such as mannitol.

The term "nanoparticle" is used herein to refer to a material structure having a size in at least any one dimension (e.g., x, y, and z cartesian dimensions) of less than about 1 micrometer (micron) (e.g., less than about 500nm or less than about 200nm or less than about 100nm) and greater than about 5 nm. In embodiments, the size is less than about 70nm but greater than about 20 nm. The nanoparticles can have a variety of geometries, e.g., spherical, ellipsoidal, and the like. The term "nanoparticles" is used as a plural of the term "nanoparticles".

As used herein, "particle polydispersity index (PDI)" or "particle polydispersity" refers to the width of the particle size distribution. Can be derived from the equation PDI-2 a₂/a₁ ²Calculating the particle PDI, wherein a₁Is the first cumulative quantity or moment used to calculate the intensity weighted Z-means particle size and a₂Is the second moment used to calculate the parameter defined as the polydispersity index (PdI). The particle PDI of 1 is the theoretical maximum and will be a completely flat particle size distribution plot. Compositions of particles described herein may have particle PDIs of less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1.

As used herein, "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient with the polymer-agent conjugates, particles, or compositions described herein, and which does not destroy the pharmacological activity thereof and is non-toxic when administered at a dosage sufficient to deliver a therapeutic amount of the particles. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, mannitol, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) astragalus membranaceus gel powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) no pyrogen water; (17) isotonic saline; (18) a ringer's solution; (19) ethanol; (20) a phosphate buffer solution; and (21) other non-toxic compatible materials employed in pharmaceutical compositions.

The term "polymer" as used herein is given its ordinary meaning as used in the art, i.e., a molecular structure characterized by one or more repeating units (monomers) linked by covalent bonds. The repeat units may all be the same, or in some cases, more than one type of repeat unit may be present in the polymer. The polymer may be a natural or non-natural (synthetic) polymer. The polymer may be a homopolymer or a copolymer containing two or more monomers. The polymer may be linear or branched.

A polymer is a "copolymer" if more than one type of repeating unit is present in the polymer. It is to be understood that in any embodiment employing a polymer, the polymer employed may be a copolymer. The repeat units forming the copolymer may be arranged in any manner. For example, the repeating units may be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., containing one or more regions each containing a first repeating unit (e.g., a first block), and one or more regions each containing a second repeating unit (e.g., a second block), and the like. The block copolymer may have two (diblock copolymer), three (triblock copolymer) or more number of distinct blocks. With respect to sequence, the copolymer may be random, block, or contain a combination of random and block sequences.

In some cases, the polymer is bio-derived, i.e., a biopolymer. Non-limiting examples of biopolymers include polypeptides or proteins (i.e., polymers of different amino acids) or nucleic acids such as DNA or RNA.

As used herein, "polymer polydispersity index (PDI)" or "polymer polydispersity" refers to the distribution of molecular mass in a given polymer sample. The calculated polymer PDI is the weight average molecular weight divided by the number average molecular weight. It indicates the distribution of individual molecular masses in a batch of polymers. The polymer PDI has a value generally greater than 1, but as the polymer chains approach uniform chain lengths, the PDI approaches one (1).

As used herein, the terms "prevent" or "preventing" as used in the context of administering an agent to a subject refers to subjecting the subject to a regimen (e.g., administration of a polymer-agent conjugate, particle, or composition) such that onset of at least one symptom of the disorder is delayed compared to that observed in the absence of the regimen.

The term "protein" as used herein refers to a plurality of linked amino acids having 100 or more amino acids. For example, a protein may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500 or more amino acids in length. Proteins include, for example, adaptor proteins, antibodies, carbohydrate binding proteins, carrier proteins, cyclins, chemokines, chromosomal proteins, collagens, cytokines, fibrous proteins, growth factors, heat shock proteins, interferons, oncoproteins, proteases, ubiquitin, zinc finger proteins, and fragments thereof.

As used herein, the term "subject" is intended to include both human and non-human animals. Exemplary human subjects include human patients with a disorder (e.g., a disorder described herein) or normal subjects. The term "non-human animal" includes all vertebrates, e.g., non-mammals (e.g., chickens, amphibians, reptiles) and mammals, e.g., non-human primates, domesticated and/or agriculturally useful animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

The term "therapeutic peptide" as used herein refers to a peptide comprising two or more amino acids, but up to 100 amino acids, covalently linked together through one or more amide bonds, wherein upon administration of the peptide to a subject, the subject receives a therapeutic effect (e.g., administration of a therapeutic peptide-treated cell, or cure, alleviate, or ameliorate symptoms of a disorder), as opposed to, for example, using a peptide in the form of a linker that itself has no therapeutic effect. The therapeutic peptide may comprise, for example, more than three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen amino acids. In some embodiments, the therapeutic peptide comprises more than 15, e.g., greater than 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 amino acids. For example, in some embodiments, the therapeutic peptide is more than 9, 10, 11, or 12 amino acids long.

The therapeutic effect of the therapeutic peptide may occur through the therapeutic peptide acting as an agonist or antagonist. The term "agonist" as used herein is meant to refer to a peptide that mimics or upregulates (e.g., potentiates or complements) the activity of a protein. A direct agonist has at least one activity of the substance to be agonized. For example, a direct agonist can be a wild-type peptide or derivative thereof having at least one activity of a wild-type protein. An indirect agonist may be a peptide that increases at least one activity of a protein. Indirect agonists include peptides that increase the interaction of a polypeptide with another molecule, such as a target peptide or nucleic acid. An "antagonist" as used herein means a peptide that reduces or down-regulates (e.g., suppresses or inhibits at least one activity of a protein.

Exemplary therapeutic peptides include, for example, peptides that treat cells, or cure, reduce, alleviate, or ameliorate symptoms of metabolic disorders, such as hormones, e.g., anti-glycogenic peptides; peptides that treat cells, or cure, reduce, alleviate or ameliorate the symptoms of a proliferative disorder, such as a tumor or metastasis thereof; a peptide that treats cells, or cures, alleviates, or ameliorates symptoms of cardiovascular disorders; a peptide that treats a cell, or cures, alleviates, or ameliorates a symptom of an infectious disease; and peptides that treat cells, or cure, reduce, alleviate, or ameliorate symptoms of allergic, inflammatory, or autoimmune disorders. In some cases, the therapeutic peptide is not a hormone. For example, in some embodiments, the therapeutic peptide is a peptide other than Luteinizing Hormone Releasing Hormone (LHRH). In some embodiments, the therapeutic peptide is a peptide other than tubulin statin (tubulysin). In some embodiments, the therapeutic peptide does not interact with, e.g., does not bind to, an integrin. For example, in one embodiment, the therapeutic peptide does not have the sequence Arg-Gly-asp.

The therapeutic peptide may comprise alpha-, beta-and/or gamma-amino acids. For example, the therapeutic peptide can comprise three or more α -amino acids, e.g., three or more consecutive α -amino acids. In one embodiment, the therapeutic peptide comprises at least four, five, six, seven, eight, nine, ten, or more a-amino acids, e.g., at least four, five, six, seven, eight, nine, ten, or more consecutive a-amino acids. Typically, all of the amino acids of the therapeutic peptide are alpha-amino acids or the therapeutic peptide comprises less than 5, 4, 3, or 2 non-alpha amino acids. The therapeutic peptide can be linear, branched, cyclic, or a combination thereof.

In some cases, the therapeutic peptide is a "standard therapeutic peptide," i.e., a majority of the amino acids (i.e., greater than 50% of the amino acids, e.g., 51%, 55%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or all of the amino acids) of the therapeutic peptide are standard amino acids. Standard amino acids are Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Asx, and Glx. In other embodiments, the therapeutic peptide is a "non-standard therapeutic peptide," i.e., a majority of the amino acids (i.e., greater than 50% of the amino acids, e.g., 51%, 55%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or all of the amino acids) of the therapeutic peptide are non-standard amino acids. The term "non-standard amino acid" as used herein refers to an amino acid having the desired amino, carboxylic, and side chains, but not Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Asx, or Glx.

A "therapeutic peptide" can be a fragment of a protein, e.g., a fragment having an amino acid sequence corresponding to the sequence of a known protein. In some embodiments, the therapeutic peptide is a fragment having an amino acid sequence corresponding to the sequence of a commercially available reference protein, and the glycan structure of the fragment is different from the glycan structure of the fragment of the commercially available protein fragment. For example, the glycan structure of the therapeutic peptide may differ from the naturally occurring glycosylation pattern of the peptide by one or more glycans, e.g., two, e.g., three, e.g., four, e.g., five, e.g., six, e.g., seven, e.g., eight, e.g., nine, e.g., ten or more glycans.

In preferred embodiments, the therapeutic peptide is linked to the polymer via a linker (e.g., through a covalently-linked chain of one or more atoms disposed between the therapeutic peptide or protein and the polymer). The linker may, for example, be a linker as described herein.

In one embodiment, the therapeutic peptide has no substantial effect on the localization of the particle, e.g., it does not target the particle to a ligand, e.g., a surface protein or an extracellular matrix component, by affinity.

In some embodiments, if the conjugate comprises a targeting agent that is a peptide, then the targeting agent is a peptide or protein that is different from the therapeutic peptide or protein.

As used herein, the term "treating" or "treating" a subject having a disorder refers to subjecting the subject to a regimen (e.g., administration of a polymer-agent conjugate, particle, or composition) such that at least one symptom of the disorder is cured, healed, reduced, alleviated, altered, cured, improved, or improved. Treatment includes administration of an amount effective to alleviate, alter, cure, ameliorate, improve or affect the disorder or symptoms of the disorder. Treatment may inhibit the deterioration or worsening of symptoms of the disorder.

The term "zwitterionic moiety" refers to a moiety that has positive and negative charges under at least one of the following conditions: during production of the particles described herein, upon formulation into the particles described herein, or after administration of the particles described herein to a subject, e.g., while circulating within the subject and/or while in an inclusion body. Zwitterionic moieties include polymeric species, such as moieties having more than one charge.

The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted (e.g., by one or more substituents). Exemplary acyl groups include acetyl (CH) ₃C (O), benzoyl (C)₆H₅C (O) -) and acetylamino acids (e.g., acetylglycine, CH₃C(O)NHCH₂C(O)-)。

The term "alkoxy" refers to an alkyl group as defined below having an oxygen group attached. Representative alkoxy groups include methoxy, ethoxy, propoxy, t-butoxy, and the like.

The term "carboxy" refers to-C (O) OH or a salt thereof.

The terms "hydroxy" and "hydroxyl" are used interchangeably and refer to-OH.

The term "substituent" refers to a group that is "substituted" on any atom of an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group. Any atom may be substituted. Suitable substituents include, but are not limited to, alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12 straight or branched chain alkyl), cycloalkyl, haloalkyl (e.g., perfluoroalkyl such as CF)₃) Aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, alkoxy, haloalkoxy (e.g., perfluoroalkoxy such as OCF)₃) Halo, hydroxy, carboxy, carboxylate, cyano, nitro, amino, alkylamino, SO₃H. Sulfate ester group, phosphate ester group, methylenedioxy (-O-CH) ₂-O-wherein the oxygen is attached to an ortho atom), ethylenedioxy, oxo, thio (e.g., C ═ S), imino (alkyl, aryl, aralkyl), S (O)_nAlkyl (wherein n is 0-2), S (O)_nAryl (wherein n is 0-2), S (O)_nHeteroaryl (wherein n is 0-2), S (O)_nHeterocyclyl (where n is 0-2), amines (mono, di, alkyl, cycloalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, and combinations thereof), esters (alkyl, aralkyl, heteroaralkyl, aryl, heteroaryl), amides (mono, di, alkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, and combinations thereof), sulfonamides (mono, di, alkyl, aralkyl, heteroaralkyl, and combinations thereof). In one aspect, the substituents on the group are independently any one of the above substituents singly or in any subset. On the other hand, the substituent itself may be substituted with any of the above-mentioned substituents.

Particles

In general, the particles comprise a therapeutic peptide or protein, and a counter ion, such as at least one of a hydrophobic portion of a polymer, or a hydrophilic-hydrophobic polymer. In some embodiments, the particles comprise a therapeutic peptide or protein and a counter ion, and at least one of a hydrophobic portion, such as a polymer, or a hydrophilic-hydrophobic polymer. In some embodiments, the particles described herein comprise a hydrophobic moiety such as a hydrophobic polymer or lipid (e.g., a hydrophobic polymer), a polymer containing a hydrophilic moiety and a hydrophobic moiety, a therapeutic peptide or protein, and a counterion. In some embodiments, a therapeutic peptide or protein and/or a counterion is attached to the moiety. For example, a therapeutic peptide or protein and/or a counterion can be attached to a polymer (e.g., a hydrophobic polymer or a polymer containing both hydrophilic and hydrophobic portions). In some embodiments, the therapeutic peptide or protein is linked to a polymer (e.g., a hydrophobic polymer or a polymer containing hydrophilic and hydrophobic portions), and the counter ion is not linked to the polymer (e.g., the counter ion is embedded in the particle). In some embodiments, the therapeutic peptide or protein and the counter ion are both attached to a polymer (e.g., a hydrophobic polymer or a polymer containing hydrophilic and hydrophobic portions). In some embodiments, the counter ion is attached to a polymer (e.g., a hydrophobic polymer or a polymer containing hydrophilic and hydrophobic portions), and the therapeutic peptide or protein is not attached to the polymer (e.g., the therapeutic peptide or protein is embedded in the particle). In some embodiments, neither the therapeutic peptide or protein nor the counter ion is attached to the polymer. The therapeutic peptide or protein and/or the counterion may also be attached to other moieties. For example, a therapeutic peptide or protein may be attached to a counterion or a hydrophilic polymer such as PEG.

In addition to hydrophobic moieties such as hydrophobic polymers or lipids (e.g., hydrophobic polymers), polymers containing hydrophilic and hydrophobic moieties, therapeutic peptides or proteins, and counterions, the particles described herein can include one or more other components, such as other therapeutic peptides or proteins or other counterions. The particles described herein may also comprise compounds having at least one acidic moiety, such as a carboxylic acid group. The compound may be a small molecule or a polymer having at least one acidic moiety. In some embodiments, the compound is a polymer such as PLGA.

In some embodiments, the particles are configured such that upon administration to a subject, the therapeutic peptide or protein is preferentially released to a preselected compartment. The preselected compartment can be a target site, location, tissue type, cell type (e.g., a disease-specific cell type, such as a cancer cell), or a subcellular compartment, such as a cytosol. In one embodiment, the particles provide preferential release in the tumor as opposed to other compartments (e.g., non-tumor compartments, such as peripheral blood). In embodiments where the therapeutic peptide or protein is linked to a polymer or counter ion, the therapeutic peptide or protein is released (e.g., by reductive cleavage of the linker) to a greater extent in the tumor than in a non-tumor compartment of the subject, e.g., peripheral blood. In some embodiments, the particle is configured such that, upon administration to a subject, it delivers more therapeutic peptide or protein to a compartment of the subject, e.g., a tumor, than if the therapeutic peptide or protein in free form were administered.

In some embodiments, the particles are associated with an excipient, e.g., a carbohydrate component or a stabilizer or a lyoprotectant, e.g., a carbohydrate component, a stabilizer, or a lyoprotectant described herein. While not wishing to be bound by theory, the carbohydrate component may act as a stabilizer or lyoprotectant. In some embodiments, the carbohydrate component, stabilizer, or lyoprotectant comprises one or more carbohydrates (e.g., one or more carbohydrates described herein, such as sucrose, cyclodextrin, or a derivative of cyclodextrin (e.g., 2-hydroxypropyl-beta-cyclodextrin, sometimes referred to herein as HP-beta-CD), a salt, PEG, PVP, or a crown ether) Sugars include those oligosaccharides that include less than 10, 8, 6, or 4 monosaccharide subunits (e.g., mono-or disaccharides (e.g., sucrose, trehalose, lactose, maltose), or combinations thereof).

In one embodiment, the carbohydrate component, stabilizer, or lyoprotectant comprises first and second components, such as cyclic carbohydrates and non-cyclic carbohydrates (e.g., monosaccharides, disaccharides, or tetrasaccharides).

In one embodiment, the weight ratio of cyclic carbohydrate to non-cyclic carbohydrate associated with the particle is a weight ratio described herein, e.g., 0.5: 1.5 to 1.5: 0.5.

In one embodiment, the carbohydrate component, stabilizer, or lyoprotectant comprises the following first and second components (designated herein as a and B):

(A) comprises a cyclic carbohydrate and (B) comprises a disaccharide; (A) comprises more than one cyclic carbohydrate, e.g., beta-cyclodextrin (sometimes referred to herein as beta-CD) or a beta-CD derivative (e.g., HP-beta-CD), and (B) comprises a disaccharide;

(A) comprises a cyclic carbohydrate, e.g., β -CD or a β -CD derivative (e.g., HP- β -CD), and (B) comprises more than one disaccharide;

(A) comprises more than one cyclic carbohydrate, and (B) comprises more than one disaccharide;

(A) comprises a cyclodextrin, e.g., β -CD or a β -CD derivative (e.g., HP- β -CD), and (B) comprises a disaccharide;

(A) comprises a β -cyclodextrin, e.g., a β -CD derivative (e.g., HP- β -CD), and (B) comprises a disaccharide;

(A) Comprises a beta-cyclodextrin, e.g., a beta-CD derivative (e.g., HP-beta-CD), and (B) comprises sucrose;

(A) comprises a β -CD derivative, e.g., HP- β -CD, and (B) comprises sucrose;

(A) comprises a beta-cyclodextrin, e.g., a beta-CD derivative (e.g., HP-beta-CD), and (B) comprises trehalose;

(A) comprises a beta-cyclodextrin, e.g., a beta-CD derivative (e.g., HP-beta-CD), and (B) comprises sucrose and trehalose.

(A) Comprising HP-beta-CD, and (B) comprising sucrose and trehalose.

In one embodiment, components a and B are present in the following ratios: 0.5: 1.5 to 1.5: 0.5. In one embodiment, components a and B are present in the following ratios: 3-1: 0.4-2; 3-1: 0.4-2.5; 3-1: 0.4-2; 3-1: 0.5-1.5; 3-1: 0.5-1; 3-1: 1; 3-1: 0.6-0.9; and 3: 1: 0.7. In one embodiment, components a and B are present in the following ratios: 2-1: 0.4-2; 3-1: 0.4-2.5; 2-1: 0.4-2; 2-1: 0.5-1.5; 2-1: 0.5-1; 2-1: 1; 2-1: 0.6-0.9; and 2: 1: 0.7. In one embodiment, components a and B are present in the following ratios: 2-1.5: 0.4-2; 2-1.5: 0.4-2.5; 2-1.5: 0.4-2; 2-1.5: 0.5-1.5; 2-1.5: 0.5-1; 2-1.5: 1; 2-1.5: 0.6-0.9; 2: 1.5: 0.7. In one embodiment, components a and B are present in the following ratios: 2.5-1.5: 0.5-1.5; 2.2-1.6: 0.7-1.3; 2.0-1.7: 0.8-1.2; 1.8: 1; 1.85: 1 and 1.9: 1.

In one embodiment, component a comprises a cyclodextrin, e.g., a β -CD derivative (e.g., HP- β -CD), and (B) comprises sucrose, and they are present in the following ratios: 2.5-1.5: 0.5-1.5; 2.2-1.6: 0.7-1.3; 2.0-1.7: 0.8-1.2; 1.8: 1; 1.85: 1 and 1.9: 1.

In some embodiments, the particle comprises a plurality of hydrophobic moieties. For example, the particles may comprise a hydrophobic polymer such as PLGA and another hydrophobic moiety such as chitosan, poly (vinyl alcohol) or poloxamer.

In some embodiments, the particles comprise a pH-suppressing molecule, e.g., a compound that can act as a buffer. Exemplary pH suppressors include base salts (e.g., calcium carbonate, magnesium hydroxide, and zinc carbonate) to buffer the system and proton sponges (e.g., amine groups) that may also help buffer the system.

The particles may also comprise a counter ion, for example, to counter the charge on the therapeutic peptide or protein. For example, if the therapeutic peptide or protein-conjugate is positively charged, exemplary counterions include acetic acid, adamantanecarboxylic acid, alpha ketoglutaric acid, D-or L-aspartic acid, benzenesulfonic acid, benzoic acid, 10-camphorsulfonic acid (camphosulfucnic acid), citric acid, 1, 2-ethanedisulfonic acid, fumaric acid, D-gluconic acid, D-glucuronic acid, glucaric acid, D-or L-glutamic acid, glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, 1-hydroxy-2-naphthoic acid (1-hydroxy-2-naphthoic acid), lactobionic acid (lactobionic acid), maleic acid, L-malic acid, mandelic acid, methanesulfonic acid, mucic acid, 1, 5-naphthalenedisulfonic acid tetrahydrate, 2-naphthalenesulfonic acid, nitric acid, oleic acid, and mixtures thereof, Pamoic acid, phosphoric acid, p-toluenesulfonic acid hydrate, D-saccharic acid monopotassium salt, salicylic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, D-or L-tartaric acid. If the therapeutic peptide-conjugate is negatively charged, exemplary counterions include N-methyl D-reduced glucamine, choline, arginine, lysine, procaine, Tromethamine (TRIS), spermine, N-methyl-morpholine, glucosamine, N-bis (2-hydroxyethyl) glycine, diazabicycloundecene, creatine, arginine ethyl ester, amantadine, rimantadine, ornithine, taurine, and citrulline.

In some embodiments, the particles are nanoparticles. In some embodiments, the nanoparticle has a diameter of less than or equal to 220nm (e.g., less than or equal to about 215nm, 210nm, 205nm, 200nm, 195nm, 190nm, 185nm, 180nm, 175nm, 170nm, 165nm, 160nm, 155nm, 150nm, 145nm, 140nm, 135nm, 130nm, 125nm, 120nm, 115nm, 110nm, 105nm, 100nm, 95nm, 90nm, 85nm, 80nm, 75nm, 70nm, 65nm, 60nm, 55nm, or 50 nm). In one embodiment, the nanoparticles have a diameter of at least 10nm (e.g., at least about 20 nm).

The particles described herein may also include a targeting agent or a lipid (e.g., on the surface of the particle).

The composition of the plurality of particles described herein can have an average diameter of about 50nm to about 500nm (e.g., about 50nm to about 200 nm). The composition of the plurality of particle particles can have a median particle diameter (about 50nm to about 500nm (e.g., about 75nm to about 220 nm)) of about 50nm to about 220nm (e.g., about 75nm to about 200nm) D_v50 (50% of the particles present by volume below the particle size)). The composition of the plurality of particle particles can have a D of about 50nm to about 500nm (e.g., about 75nm to about 220nm)_v90 (90% of the particles present by volume are below this particle size). In some embodiments, the composition of the plurality of particles has a Dv90 of less than about 150 nm. A composition of the plurality of particles may have particle PDI of less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1.

The particles described herein can have a surface zeta potential in the range of about-20 mV to about 50mV when measured in water. The zeta potential is a measure of the surface potential of the particle. In some embodiments, the particles may have a surface zeta potential in the range of about-20 mV to about 20mV, about-10 mV to about 10mV, or neutral, when measured in water.

In one embodiment, a particle or composition comprising a plurality of particles described herein can retain at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of its activity when stored in an open or closed container at 25 ℃ ± 2 ℃/60% relative humidity ± 5% relative humidity for 20, 30, 40, 50, or 60 days, e.g., as determined in an in vivo model system.

In one embodiment, the particles are stable in a non-polar organic solvent (e.g., any of hexane, chloroform, or dichloromethane). For example, the particles do not substantially transform, e.g., if present, the outer layer does not internalize, or a significant amount of the surface components internalize relative to their configuration in an aqueous solvent. In embodiments, the distribution of the components in the non-polar organic solvent and in the aqueous solvent is substantially the same.

In one embodiment, the particle lacks at least one component of the micelle, e.g., it lacks a core that is substantially free of hydrophilic components.

In one embodiment, the core of the particle comprises a plurality of hydrophilic components.

In one embodiment, the core of the particle comprises a substantial amount, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% (by weight or number) of the therapeutic peptide.

In one embodiment, the core of the particle comprises a substantial amount, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% (by weight or number) of the counter-ions of the particle, e.g., polycationic moieties.

The particles described herein can include a small amount of residual solvent, for example, solvents used in making the particles such as acetone, t-butyl methyl ether, benzyl alcohol, dioxane, heptane, dichloromethane, dimethylformamide, dimethyl sulfoxide, ethyl acetate, acetonitrile, tetrahydrofuran, ethanol, methanol, isopropanol, methyl ethyl ketone, butyl acetate, or propyl acetate (e.g., isopropyl acetoacetate). In some embodiments, the particles can include less than 5000ppm of solvent (e.g., less than 4500ppm, less than 4000ppm, less than 3500ppm, less than 3000ppm, less than 2500ppm, less than 2000ppm, less than 1500ppm, less than 1000ppm, less than 500ppm, less than 250ppm, less than 100ppm, less than 50ppm, less than 25ppm, less than 10ppm, less than 5ppm, less than 2ppm, or less than 1 ppm).

In some embodiments, the particles are substantially free of class II or class III solvents as defined by the U.S. department of health and public service food and drug administration "Q3 c-table and list". In some embodiments, the particles comprise less than 5000ppm acetone. In some embodiments, the particles comprise less than 5000ppm of tert-butyl methyl ether. In some embodiments, the particles comprise less than 5000ppm heptane. In some embodiments, the particles comprise less than 600ppm methylene chloride. In some embodiments, the particles comprise less than 880ppm dimethylformamide. In some embodiments, the particles comprise less than 5000ppm of ethyl acetate. In some embodiments, the particles comprise less than 410ppm acetonitrile. In some embodiments, the particles comprise less than 720ppm tetrahydrofuran. In some embodiments, the particles comprise less than 5000ppm ethanol. In some embodiments, the particles comprise less than 3000ppm methanol. In some embodiments, the particles comprise less than 5000ppm isopropyl alcohol. In some embodiments, the particles comprise less than 5000ppm methyl ethyl ketone. In some embodiments, the particles comprise less than 5000ppm butyl acetate. In some embodiments, the particles comprise less than 5000ppm propyl acetate.

The particles described herein can include varying amounts of hydrophobic moieties such as hydrophobic polymers, for example, from about 20% to about 90% (e.g., from about 20% to about 80%, from about 25% to about 75%, or from about 30% to about 70%) by weight of the particle or of the starting material used to make the particle. The particles described herein can comprise varying amounts of the hydrophilic-hydrophobic polymer, for example, up to about 50 wt% (e.g., any of about 4 to about 50 wt%, about 5 wt%, about 8 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 23 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt%). For example, the weight percent of the hydrophilic-hydrophobic polymer of the particles is about 3% to 30%, about 5% to 25%, or about 8% to 23%.

The particles described herein can include different amounts of counter ions, for example, from about 0.1% to about 60% (e.g., from about 1% to about 60%, from about 2% to about 20%, from about 3% to about 30%, from about 5% to about 40%, from about or from about 10% to about 30%) by weight of the particle or of the starting material used to make the particle.

The particles described herein can comprise varying amounts of the therapeutic peptide, for example, from about 0.1% to about 50% (e.g., from about 1% to about 50%, from about 0.5% to about 20%, from about 2% to about 20%, from about or from about 5% to about 15%) by weight of the particle or of the starting material used to make the particle.

When the particles comprise a surfactant, the particles may comprise a different amount of surfactant, for example, up to about 40% by weight, or from about 15% to about 35% or from about 3% to about 10% of the particles or of the starting material used to make the particles. In some embodiments, the surfactant is PVA. In some embodiments, the particles may comprise from about 2% to about 5% PVA (e.g., about 4%) and from about 0.1% to about 3% cationic PVA (e.g., about 1%).

The particles described herein can be substantially free of a targeting agent (e.g., a targeting agent covalently linked to a component in the particle, e.g., a targeting agent capable of binding or otherwise associating with a target biological entity, e.g., a membrane component, a cell surface receptor, a prostate-specific membrane antigen, etc.). The particles described herein may be substantially free of a targeting agent selected from the group consisting of: nucleic acid aptamers, growth factors, hormones, cytokines, interleukins, antibodies, integrins, fibronectin receptors, p-glycoprotein receptors, peptides, and cell binding sequences. In some embodiments, no polymer within the particle is conjugated to the targeting moiety. The particles described herein may be free of moieties added for the purpose of selectively targeting the particles to a site within the body of a subject, for example, by using moieties on the particles that have a high specific affinity for a target within the body of a subject.

In some embodiments, the particles are free of lipids, e.g., free of phospholipids. The particles described herein may be substantially free of an amphiphilic layer that reduces water penetration into the nanoparticles. The particles described herein may comprise less than 5% or 10% (e.g., as determined by w/w, v/v) of a lipid, e.g., a phospholipid. The particles described herein may be substantially free of lipid layers, e.g., phospholipid layers, e.g., lipid layers that reduce water penetration into the nanoparticles. The particles described herein can be substantially free of lipids, e.g., substantially free of phospholipids.

The particles described herein can be substantially free of a radiopharmaceutical, e.g., a radiotherapeutic agent, a radiodiagnostic agent, a prophylactic agent, or other radioisotope. The particles described herein can be substantially free of an immunomodulatory agent, e.g., an immunostimulatory agent or an immunosuppressive agent. The particles described herein can be substantially free of vaccines or immunogens, e.g., peptides, sugars, lipid-based immunogens, B cell antigens, or T cell antigens.

The particles described herein can be substantially free of water-soluble hydrophobic polymers, such as PLGA, e.g., PLGA having a molecular weight of less than about 1kDa (e.g., less than about 500 Da).

Exemplary particles

Exemplary particles include particles comprising:

a) a plurality of hydrophobic polymers;

b) a plurality of hydrophilic-hydrophobic polymers; and

Another exemplary particle includes a particle comprising:

b) a variety of hydrophilic-hydrophobic polymers.

Another exemplary particle includes a particle comprising:

a) optionally a plurality of hydrophobic polymers; and

Another exemplary particle includes a particle comprising:

a) optionally, a plurality of hydrophobic polymers;

c) A plurality of charged therapeutic peptides or proteins, wherein the charge of the therapeutic peptide or protein is opposite to the charge of the peptide conjugated to the hydrophilic-hydrophobic polymer, and wherein the charged therapeutic peptide or protein forms a non-covalent bond (e.g., an ionic bond) with the charged peptide of the hydrophilic-hydrophobic polymer-conjugate.

Method for making particles and compositions

The particles described herein can be prepared using any method known in the art for preparing particles, such as nanoparticles. Exemplary methods include spray drying, emulsion (e.g., emulsion-solvent evaporation or double emulsion), precipitation (e.g., nanoprecipitation), and phase inversion.

In one embodiment, the particles described herein can be prepared by precipitation (e.g., nanoprecipitation). Such methods include dissolving the components of the particles (i.e., the one or more polymers, optionally one or more other components, and the reagent), individually or in combination, in one or more solvents to form one or more solutions. For example, a first solution containing one or more components may be poured into a second solution containing one or more components (at a suitable rate or speed). The solutions may be combined, for example, using a syringe pump, a MicroMixer (MicroMixer), or any device that allows for vigorous, controlled mixing. In some cases, the nanoparticles may be formed when the first solution contacts the second solution, e.g., precipitation of the polymer after contact causes the polymer to form nanoparticles. The control of such particle formation can be easily optimized.

In one set of embodiments, the particles are formed by: one or more solutions containing one or more polymers and other components are provided and the solutions are contacted with certain solvents to produce particles. In a non-limiting example, a hydrophobic polymer (e.g., PLGA) is conjugated to a therapeutic peptide or protein in order to form a conjugate. The therapeutic peptide or protein-polymer conjugate, a polymer comprising a hydrophilic moiety and a hydrophobic moiety (e.g., PEG-PLGA), and optionally a third polymer (e.g., a biodegradable polymer, such as PLGA) are dissolved in an organic solvent (e.g., acetone) that is miscible with the water moiety. The solution is added to an aqueous solution containing a surfactant to form the desired particles. The two solutions can be sterile filtered separately prior to mixing/precipitation.

The formed nanoparticles may be exposed to further processing techniques in order to remove the solvent or to purify the nanoparticles (e.g., dialysis). For purposes of the above process, water-miscible solvents include acetone, ethanol, methanol, and isopropanol; and the organic solvent that is partially miscible with water includes acetonitrile, tetrahydrofuran, ethyl acetate, isopropanol, isopropyl acetate, or dimethylformamide.

Another method that can be used to produce the particles described herein is as described by Johnson, b.k. et al, AlChE Journal (2003) 49: 2264. multidot. 2282 and U.S.2004/0091546 describe a process known as "flash nanoprecipitation", each of which is incorporated herein by reference in its entirety. This process enables the production of controlled size, polymer stabilized and protected nanoparticles of hydrophobic organics with high loading and yield. The rapid nanoprecipitation technique is based on the inhibited nucleation and growth of amphiphilic diblock copolymers of hydrophobic organics. Amphiphilic diblock copolymers dissolved in a suitable solvent can form micelles as the solvent quality of one block decreases. To achieve this change in solvent mass, a tangential flow mixing unit (vortex mixer) is used. The vortex mixer consists of a closed volume chamber (confined volume chamber) in which one jet containing the diblock copolymer and the active agent dissolved in a water-miscible solvent is mixed at high speed with another jet containing water, i.e. the active agent and the anti-solvent for the hydrophobic block of the copolymer. The rapid mixing and high energy dissipation involved in the process provides a time scale shorter than that of nucleation and growth of particles, resulting in the formation of nanoparticles with active loading levels and size distributions not otherwise provided by the technology. When nanoparticles are formed via rapid nanoprecipitation, mixing occurs sufficiently rapidly to allow high levels of supersaturation of all components to be reached before aggregation begins. Thus, the active agent and polymer precipitate simultaneously, and the limitations of low active agent incorporation and aggregation present with widely used techniques based on slow solvent exchange (e.g., dialysis) are overcome. The rapid nanoprecipitation process is not sensitive to the chemical specificity of the components, making it a versatile nanoparticle formation technique.

The particles described herein can also be prepared using mixer techniques, such as static mixers or micromixers (e.g., split-recombine micromixers, slit-interdigitated micromixers, star laminar interdigitated micromixers (star laminar interdigitated micromixers), super focus (superfocus) interdigitated micromixers, liquid-liquid micromixers, or impinging jet micromixers).

The split-recombine micromixer uses a mixing principle that includes: the streams are separated, superimposed/directed onto each other and recombined in each mixing step (8 to 12 such steps are included). Mixing eventually occurs via diffusion within milliseconds (excluding the residence time of the multi-step flow channel). In addition, at higher flow rates, turbulence increases this mixing effect, further improving the overall mixing quality.

The slit interdigitated micromixer combines the regular flow patterns created by the multi-laminar flow with geometric focusing to accelerate liquid mixing. Due to the two-step mixing, the slot mixer is suitable for a wide variety of processes.

The particles described herein can also be prepared using Microfluidic Reaction Technology (MRT). The core of the MRT is a continuous, colliding jet microreactor that can be expanded to at least 50 liters/min. In the reactor, high velocity liquid reactants are forced to interact inside a volume on the microliter scale. The reactants are mixed at the nanometer level when exposed to high shear stress and turbulence. MRT provides precise control over the feed rate and mixing location of the reactants. This ensures control of the nucleation and growth process, resulting in uniform crystal growth and a stable rate.

The particles described herein may also be prepared by emulsion. An exemplary emulsification process is disclosed in U.S. Pat. No. 5,407,609, which is incorporated herein by reference. The method comprises dissolving or otherwise dispersing a reagent, liquid or solid in a solvent containing dissolved wall-forming material, dispersing the reagent/polymer-solvent mixture in a treatment medium to form an emulsion, and immediately transferring all of the emulsion to a bulk treatment medium or other suitable extraction medium to immediately extract the solvent from the droplets in the emulsion to form a microencapsulated product, such as microcapsules or microspheres. The most common method for preparing polymeric delivery vehicle formulations is the solvent emulsification-evaporation method. This method involves dissolving the polymer and drug in an organic solvent that is completely immiscible with water (e.g., methylene chloride). The organic mixture is added to water containing a stabilizer, most often poly (vinyl alcohol) (PVA), and then typically sonicated.

After the particles are prepared, they may be classified by filtration, sieving, extrusion or ultracentrifugation in order to recover particles within a specific size range. One method of sorting includes extruding an aqueous suspension of particles through a series of polycarbonate membranes having a selected uniform pore size; the pore size of the membrane will correspond approximately to the maximum size of the particles produced by extrusion through such a membrane. See, for example, U.S. patent 4,737,323, which is incorporated herein by reference. Another method is continuous ultracentrifugation at specified speeds (e.g., 8,000, 10,000, 12,000, 15,000, 20,000, 22,000, and 25,000rpm) to separate fractions of specified sizes. Another method is tangential flow filtration, in which a solution containing particles is pumped tangentially along the membrane surface. The applied pressure serves to force a portion of the liquid through the membrane to the filtrate side. Particles too large to pass through the membrane pores are retained on the upstream side. The retained components do not accumulate at the membrane surface as in normal flow filtration, but are removed by tangential flow. Thus, tangential flow filtration can be used to remove excess surfactant present in the aqueous solution or to concentrate the solution via diafiltration.

After purifying the particles, they may be sterile filtered while in solution (e.g., using a 0.22 micron filter).

In certain embodiments, the particles are prepared to be approximately uniform in size within a selected size range. The maximum diameter of the particles is preferably in the range of 30nm to 300nm (e.g., about 30nm to about 250 nm). The particles may be analyzed by techniques known in the art such as dynamic light scattering and/or electron microscopy (e.g., transmission electron microscopy or scanning electron microscopy) in order to determine the size of the particles. The particles may also be tested for reagent loading and/or the presence or absence of impurities.

Lyophilization process

The particles described herein can be prepared for dry storage via a lyophilization process commonly referred to as freeze-drying. Lyophilization is the process of extracting water from a solution to form a granular solid or powder. The process is carried out by freezing the solution and then extracting any water or moisture by sublimation under vacuum. Advantages of lyophilization include maintaining the mass of the material and minimizing degradation of the therapeutic compound. Lyophilization may be particularly useful in the development of pharmaceutical products that are reconstituted and administered to a patient by injection, such as parenteral pharmaceutical products. Alternatively, lyophilization is suitable for the development of oral pharmaceutical products, particularly fast-melt or fast-dissolving formulations.

The lyophilization process can be performed in the presence of a lyoprotectant, e.g., a lyoprotectant described herein. In some embodiments, the lyoprotectant is a carbohydrate (e.g., a carbohydrate described herein, such as sucrose, cyclodextrin, or a derivative of cyclodextrin (e.g., 2-hydroxypropyl- β -cyclodextrin)), a salt, PEG, PVP, or a crown ether.

Therapeutic peptides or protein-polymer conjugates

The therapeutic peptide or protein-polymer conjugates described herein comprise a polymer (e.g., a hydrophobic polymer or a hydrophilic-hydrophobic polymer) and a therapeutic peptide or protein. The therapeutic peptides or proteins described herein can be attached, e.g., directly or through a linker, to a polymer described herein. The therapeutic peptide or protein may be linked to a hydrophobic polymer (e.g., PLGA), or a polymer having a hydrophobic portion and a hydrophilic portion (e.g., PEG-PLGA). The therapeutic peptide or protein may be attached to one end of the polymer, to both ends of the polymer, or to a point along the polymer chain. In some embodiments, multiple therapeutic peptides or proteins may be attached to multiple points along the polymer chain, or multiple therapeutic peptides or proteins may be attached to the terminus of the polymer via a multifunctional linker.

Polymer and method of making same

A wide variety of polymers and methods of forming therapeutic peptides or protein-polymer conjugates and particles thereof are known in the art of therapeutic peptide delivery. Any polymer may be used according to the present invention. The polymer may be a natural or non-natural (synthetic) polymer. The polymer may be a homopolymer or a copolymer containing two or more monomers. The polymer may be linear or branched.

A polymer is considered to be a "copolymer" if more than one type of repeating unit is present in the polymer. It is understood that in any embodiment employing a polymer, the polymer employed may be a copolymer. The repeat units forming the copolymer may be arranged in any manner. For example, the repeat units can be arranged in random order, alternating order, or in the form of a "block" copolymer, i.e., containing one or more regions each containing a first repeat unit (e.g., a first block), and one or more regions each containing a second repeat unit (e.g., a second block), and so forth. The block copolymer may have two (diblock copolymer), three (triblock copolymer), or a greater number of different blocks. In terms of sequence, the copolymer may be random, block, or contain a combination of random and block sequences.

Hydrophobic moieties

Hydrophobic polymers

The particles described herein may comprise a hydrophobic polymer. The hydrophobic polymer can be linked to a therapeutic peptide or protein and/or a counter ion to form a conjugate (e.g., a therapeutic peptide/protein-hydrophobic polymer conjugate or a counter ion-hydrophobic polymer conjugate).

In some embodiments, the hydrophobic polymer is not attached to another moiety. The particles may comprise a plurality of hydrophobic polymers, for example wherein some of the hydrophobic polymers are linked to another moiety such as a therapeutic peptide and/or a counterion, and some of the hydrophobic polymers are free.

Exemplary hydrophobic polymers include the following: acrylates including methyl acrylate, ethyl acrylate, propyl acrylate, n-Butyl Acrylate (BA), isobutyl acrylate, 2-ethyl acrylate, and t-butyl acrylate; methacrylates including ethyl methacrylate, n-butyl methacrylate, and isobutyl methacrylate; acrylonitrile; methacrylonitrile; vinyl compounds including vinyl acetate, vinyl versatate, vinyl propionate, vinyl formamide, vinyl acetamide, vinyl pyridine, and vinyl imidazole; aminoalkyl compounds including aminoalkyl acrylates, aminoalkyl methacrylates, and aminoalkyl (meth) acrylamides; styrene; cellulose acetate phthalate; cellulose acetate succinate; hydroxypropyl methylcellulose phthalate; poly (D, L-lactide); poly (D, L-lactide-co-glycolide); poly (glycolide); poly (hydroxybutyrate); poly (alkyl carbonates); poly (ortho esters); a polyester; poly (hydroxyvaleric acid); polydioxanone; poly (ethylene terephthalate); poly (malic acid); poly (tartronic acid); a polyanhydride; polyphosphazene; poly (amino acids) and their copolymers (see generally, Svenson, S (eds.), Polymeric Drug Delivery: volume I: Particulate Drug carriers.2006; ACS symposium; Amiji, M.M (eds.), Nanotechnology for Cancer therapy.2007; Taylor & Francis Group, LLP; Nair et al, prog.polymer.sci. (2007) 32: 762-798); hydrophobic peptide-based polymers and poly (L-amino acid) -based copolymers (Lavasanifar, A., et al, advanced drug Delivery Reviews (2002) 54: 169-190); poly (ethylene-vinyl acetate) ("EVA") copolymers; silicone rubber; polyethylene; polypropylene; polydienes (polybutadiene, polyisoprene, and hydrogenated versions of these polymers); maleic anhydride copolymers of vinyl methyl ether and other vinyl ethers; polyamides (nylon 6, 6); a polyurethane; poly (ester polyurethane); poly (ether polyurethanes); and poly (ester-ureas).

Hydrophobic polymers suitable for use in preparing the polymer-agent conjugates or particles described herein also include biodegradable polymers. Examples of biodegradable polymers include polylactides, polyglycolides, caprolactone-based polymers, poly (caprolactone), polydioxanones, polyanhydrides, polyamines, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyphosphoesters, polyesters, polybutylene terephthalate, polyorthocarbonates, polyphosphazenes, succinates, poly (malic acid), poly (amino acids), poly (vinylpyrrolidone), polyethylene glycols, polyhydroxycelluloses, polysaccharides, chitins, chitin and hyaluronic acid and copolymers, terpolymers and mixtures thereof. Biodegradable polymers also include copolymers (including caprolactone-based polymers, polycaprolactone) and copolymers including polybutylene terephthalate.

In some embodiments, the polymer is a polyester synthesized from monomers selected from the group consisting of: d, L-lactide, D-lactide, L-lactide, D, L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, epsilon-caprolactone, epsilon-hydroxycaproic acid, gamma-butyrolactone, gamma-hydroxybutyric acid, delta-valerolactone, delta-hydroxyvaleric acid, hydroxybutyric acid and malic acid.

Copolymers may also be used in the polymer-agent conjugates or particles described herein. In some embodiments, the polymer may be PLGA, which is a biodegradable random copolymer of lactic acid and glycolic acid. PLGA polymers can have different ratios of lactic acid: glycolic acid, for example, ranges from about 0.1: 99.9 to about 99.9: 0.1 (e.g., about 75: 25 to about 25: 75, about 60: 40 to 40: 60, or about 55: 45 to 45: 55). In some embodiments, for example, in PLGA, the ratio of lactic acid monomer to glycolic acid monomer is 50: 50, 60: 40, or 75: 25.

In particular embodiments, parameters such as water absorption, agent release (e.g., "controlled release"), and polymer degradation kinetics can be optimized by optimizing the ratio of lactic acid monomer to glycolic acid monomer in the PLGA polymer in the polymer-agent conjugate or particle. Furthermore, adjusting the ratio will also affect the hydrophobicity of the copolymer, which will in turn affect the drug loading.

In certain embodiments where the biodegradable polymer also has a therapeutic peptide, protein, or other material, such as a counterion attached thereto, the rate of biodegradation of the polymer can be characterized by the release rate of the material. In such cases, the rate of biodegradation may depend not only on the chemical and physical characteristics of the polymer, but also on the nature of the material to which it is attached. Degradation of the subject compositions includes not only cleavage of intramolecular bonds, e.g., by oxidation and/or hydrolysis, but also cleavage of intermolecular bonds, such as dissociation of host/guest complexes resulting from competitive complex formation with an inclusion host (inclusion host). In some embodiments, release may be affected by another component in the particle, e.g., a compound having at least one acidic moiety (e.g., free acid PLGA).

In certain embodiments, particles comprising one or more polymers (e.g., hydrophobic polymers) biodegrade over a period of time acceptable for the desired application. In certain embodiments, such as in vivo therapy, such degradation occurs over a period of time typically less than about five years, one year, six months, three months, one month, fifteen days, five days, three days, or even one day upon exposure to a physiological solution having a pH between 4 and 8 and a temperature between 25 ℃ and 37 ℃. In other embodiments, the polymer degrades over a period of time between about one hour and several weeks, depending on the desired application.

When the polymer is used to deliver therapeutic peptides in vivo, it is important that the polymer itself is non-toxic and that the polymer degrades into non-toxic degradation products when it is eroded by body fluids. However, many synthetic biodegradable polymers, after in vivo erosion, produce oligomers and monomers that interact adversely with surrounding tissues (d.f. williams, j.mater.sci.1233 (1982)). To minimize the toxicity of the intact polymer carrier and its degradation products, polymers have been designed based on naturally occurring metabolites. Exemplary polymers include polyesters derived from lactic acid and/or glycolic acid and polyamides derived from amino acids.

Various biodegradable polymers are known and used for controlled release of pharmaceutical agents. Such polymers are described, for example, in U.S. patent nos. 4,291,013; 4,347,234, respectively; 4,525,495, respectively; 4,570,629, respectively; 4,572,832, respectively; 4,587,268, respectively; 4,638,045, respectively; 4,675,381; 4,745,160; and 5,219,980; and PCT publication WO2006/014626, each of which is incorporated herein by reference in its entirety.

The hydrophobic polymers described herein may have a plurality of end groups. In some embodiments, the end groups of the polymer are not further modified, for example, when the end groups are carboxylic acids, hydroxyl groups, or amino groups. In some embodiments, the end groups may be further modified. For example, a polymer with a hydroxyl end group can be derivatized with an acyl group to produce an acyl-terminated polymer (e.g., an acetyl-terminated polymer or a benzoyl-terminated polymer) or with an alkyl group to produce an alkoxy-terminated polymer (e.g., a methoxy-terminated polymer) or with a benzyl group to produce a benzyl-terminated polymer. The end group may also be further reacted with a functional group, for example, to provide a bond to another moiety, such as a nucleic acid reagent, a counterion, or an insoluble matrix. In some embodiments, the particles comprise a functionalized hydrophobic polymer (e.g., a hydrophobic polymer, such as PLGA (e.g., 50: 50PLGA)) functionalized with a moiety (e.g., N- (2-aminoethyl) maleimide, 2- (2- (pyridin-2-yl) disulfide) ethylamino, or succinimidyl-N-methyl ester) that is not reactive with another moiety (e.g., a therapeutic peptide).

The hydrophobic polymer may have a weight average molecular weight in the range of about 1kDa to about 70kDa (e.g., about 4kDa to about 66kDa, about 2kDa to about 12kDa, about 6kDa to about 20kDa, about 5kDa to about 15kDa, about 6kDa to about 13kDa, about 7kDa to about 11kDa, about 5kDa to about 10kDa, about 7kDa to about 10kDa, about 5kDa to about 7kDa, about 6kDa to about 8kDa, about 6kDa, about 7kDa, about 8kDa, about 9kDa, about 10kDa, about 11kDa, about 12kDa, about 13kDa, about 14kDa, about 15kDa, about 16kDa, or about 17 kDa).

The hydrophobic polymers described herein can have a polymer polydispersity index (PDI) of less than or equal to about 2.5 (e.g., less than or equal to about 2.2, less than or equal to about 2.0, or less than or equal to about 1.5). In some embodiments, the hydrophobic polymers described herein may have a polymer PDI of about 1.0 to about 2.5, about 1.0 to about 2.0, about 1.0 to about 1.7, or about 1.0 to about 1.6.

The particles described herein can include varying amounts of hydrophobic polymer, for example, from about 10 wt% to about 90 wt% (e.g., from about 20 wt% to about 80 wt%, from about 25 wt% to about 75 wt%, or from about 30 wt% to about 70 wt%) of the particles.

The hydrophobic polymers described herein may be commercially available, for example, from commercial suppliers such as BASF, Boehringer Ingelheim, Durcet Corporation, PuracAmerica, and SurModics Pharmaceuticals. The polymers described herein may also be synthetic. Methods for synthesizing polymers are known in the art (see, e.g., Polymer Synthesis: Theroy and Practice Fundamentals, Methods, experiments. D. Braun et al, fourth edition, Springer, Berlin, 2005). Such methods include, for example, polycondensation, free radical polymerization, ionic polymerization (e.g., cationic or anionic polymerization), or ring opening metathesis polymerization.

Commercially available or synthetic polymer samples can be further purified prior to forming the polymer-reagent conjugate or incorporating the particles or polymers described herein. In some embodiments, purification can reduce the polydispersity of the polymer sample. The polymer may be purified by precipitation from solution or onto a solid such as diatomaceous earth. The polymer may also be further purified by Size Exclusion Chromatography (SEC).

Other hydrophobic moieties

Other suitable hydrophobic moieties for the particles described herein include lipids, e.g., phospholipids. Exemplary lipids include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithins (ESM), cephalins, cardiolipins, phosphatidic acid, cerebrosides, dicetyl phosphate, Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), Dioleoylphosphatidylglycerol (DOPG), Dipalmitoylphosphatidylglycerol (DPPG), Dioleoylphosphatidylethanolamine (DOPE), palmitoleoylphosphatidylcholine (POPC), palmitoleoylphosphatidylethanolamine (POPE), palmitoleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), Dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine (DEPE), stearoyl-oleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, and dilinoleoyl-phosphatidylcholine.

Other exemplary hydrophobic moieties include cholesterol and vitamin ETPGS.

In one embodiment, the hydrophobic moiety is not a lipid (e.g., is not a phospholipid) or does not comprise a lipid.

Hydrophobic-hydrophilic polymers

The particles described herein may comprise a polymer comprising a hydrophilic portion and a hydrophobic portion, e.g., a hydrophobic-hydrophilic polymer. The hydrophobic-hydrophilic polymer may be linked to another moiety such as a therapeutic peptide or protein (e.g., via a hydrophilic or hydrophobic moiety). In some embodiments, the hydrophobic-hydrophilic polymer is free (i.e., not attached to another moiety). The particles may comprise a plurality of hydrophobic-hydrophilic polymers, for example wherein some of the hydrophobic-hydrophilic polymers are linked to another moiety such as a therapeutic peptide, protein, and/or counterion, and some of the hydrophobic-hydrophilic polymers are free.

The polymer containing a hydrophilic portion and a hydrophobic portion may be a copolymer in which a hydrophilic block is coupled to a hydrophobic block. These copolymers can have a weight average molecular weight between about 5kDa and about 30kDa (e.g., about 5kDa to about 25kDa, about 10kDa to about 22kDa, about 10kDa to about 15kDa, about 12kDa to about 22kDa, about 7kDa to about 15kDa, about 15kDa to about 19kDa, or about 11kDa to about 13kDa, e.g., about 9kDa, about 10kDa, about 11kDa, about 12kDa, about 13kDa, about 14kDa, about 15kDa, about 16kDa, about 17kDa, about 18kDa, or about 19 kDa). Polymers containing hydrophilic and hydrophobic moieties may be attached to the agent.

Examples of suitable hydrophobic moieties of the polymer include those described above. The hydrophobic portion of the copolymer can have a weight average molecular weight of about 1kDa to about 20kDa (e.g., about 8kDa to about 15kDa, about 1kDa to about 18kDa, 17kDa, 16kDa, 15kDa, 14kDa, or 13kDa, about 2kDa to about 12kDa, about 6kDa to about 20kDa, about 5kDa to about 18kDa, about 7kDa to about 17kDa, about 8kDa to about 13kDa, about 9kDa to about 11kDa, about 10kDa to about 14kDa, about 6kDa to about 8kDa, about 6kDa, about 7kDa, about 8kDa, about 9kDa, about 10kDa, about 11kDa, about 12kDa, about 13kDa, about 14kDa, about 15kDa, about 16kDa, or about 17 kDa).

Examples of suitable hydrophilic moieties of the polymer include the following: carboxylic acids including acrylic acid, methacrylic acid, itaconic acid, and maleic acid; polyoxyethylene or polyethylene oxide (PEG); polyacrylamides (e.g., polyhydroxypropylmethacrylamide) and copolymers thereof with dimethylaminoethyl methacrylate, diallyldimethylammonium chloride, vinylbenzyltrimethylammonium chloride, acrylic acid, methacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid and styrenesulfonate, poly (vinylpyrrolidone), polyoxazolines, polysialic acid, starch and starch derivatives, dextran and dextran derivatives; polypeptides, such as polylysine, polyarginine, polyglutamic acid; hyaluronic acid, alginic acid, polylactic acid, polyethyleneimine, polyionolene, polyacrylic and polyiminocarboxylic acid esters, gelatin and unsaturated ethylene mono-or dicarboxylic acids. A list of suitable hydrophilic polymers can be found in Handbook of Water-solvent Gums and Resins, R.Davidson, McGraw-Hill (1980). The hydrophilic portion of the copolymer can have a weight average molecular weight of about 1kDa to about 21kDa (e.g., about 1kDa to about 8kDa, about 1kDa to about 3kDa (e.g., about 2kDa), or about 2kDa to about 6kDa (e.g., about 3.5kDa), or about 4kDa to about 6kDa (e.g., about 5 kDa)). In one embodiment, the hydrophilic moiety is PEG, and the weight average molecular weight is about 1kDa to about 21kDa (e.g., about 1kDa to about 8kDa, about 1kDa to about 3kDa (e.g., about 2kDa), or about 2kDa to about 6kDa (e.g., about 3.5kDa), or about 4kDa to about 6kDa (e.g., about 5 kDa)). In one embodiment, the hydrophilic moiety is PVA, and the weight average molecular weight is about 1kDa to about 21kDa (e.g., about 1kDa to about 8kDa, about 1kDa to about 3kDa (e.g., about 2kDa), or about 2kDa to about 6kDa (e.g., about 3.5kDa), or about 4kDa to about 6kDa (e.g., about 5 kDa)). In one embodiment, the hydrophilic moiety is a polyoxazoline, and the weight average molecular weight is about 1kDa to about 21kDa (e.g., about 1kDa to about 8kDa, about 1kDa to about 3kDa (e.g., about 2kDa), or about 2kDa to about 6kDa (e.g., about 3.5kDa), or about 4kDa to about 6kDa (e.g., about 5 kDa)). In one embodiment, the hydrophilic moiety is polyvinylpyrrolidone, and the weight average molecular weight is about 1kDa to about 21kDa (e.g., about 1kDa to about 8kDa, about 1kDa to about 3kDa (e.g., about 2kDa), or about 2kDa to about 6kDa (e.g., about 3.5kDa), or about 4kDa to about 6kDa (e.g., about 5 kDa)). In one embodiment, the hydrophilic moiety is a polyhydroxypropylmethacrylamide, and the weight average molecular weight is from about 1kDa to about 21kDa (e.g., from about 1kDa to about 8kDa, from about 1kDa to about 3kDa (e.g., about 2kDa), or from about 2kDa to about 6kDa (e.g., about 3.5kDa), or from about 4kDa to about 6kDa (e.g., about 5 kDa)). In one embodiment, the hydrophilic moiety is polysialic acid, and the weight average molecular weight is from about 1kDa to about 21kDa (e.g., from about 1kDa to about 8kDa, from about 1kDa to about 3kDa (e.g., about 2kDa), or from about 2kDa to about 6kDa (e.g., about 3.5kDa), or from about 4kDa to about 6kDa (e.g., about 5 kDa)).

The polymer containing a hydrophilic portion and a hydrophobic portion may be a block copolymer, for example, a diblock or triblock copolymer. In some embodiments, the polymer may be a diblock copolymer containing a hydrophilic block and a hydrophobic block. In some embodiments, the polymer may be a triblock copolymer containing a hydrophobic block, a hydrophilic block, and another hydrophobic block. The two hydrophobic blocks may be the same hydrophobic polymer or different hydrophobic polymers. The block copolymers used herein can have different ratios of hydrophilic moieties to hydrophobic moieties, for example, in the range of 1: 1 to 1: 40 by weight (e.g., about 1: 1 to about 1: 10 by weight, about 1: 1 to about 1: 2 by weight, or about 1: 3 to about 1: 6 by weight).

The polymer containing hydrophilic and hydrophobic moieties may have a plurality of end groups. In some embodiments, the end group can be a hydroxyl group or an alkoxy group (e.g., methoxy group). In some embodiments, the end groups of the polymer are not further modified. In some embodiments, the end groups may be further modified. For example, the end groups can be alkyl terminated to produce alkoxy terminated polymers (e.g., methoxy terminated polymers), can be derivatized with targeting agents (e.g., folic acid) or dyes (e.g., rhodamine), or can be reacted with functional groups.

The polymer containing a hydrophilic portion and a hydrophobic portion may include a linking group between the two blocks of the copolymer. For example, such a linker may be an amide, ester, ether, amino, carbamate, or carbonate linkage.

Polymers containing hydrophilic and hydrophobic moieties described herein can have a polymer polydispersity index (PDI) of less than or equal to about 2.5 (e.g., less than or equal to about 2.2, less than or equal to about 2.0, or less than or equal to about 1.5). In some embodiments, the polymer PDI is from about 1.0 to about 2.5, for example, from about 1.0 to about 2.0, from about 1.0 to about 1.8, from about 1.0 to about 1.7, or from about 1.0 to about 1.6.

The particles described herein can include varying amounts of the polymer containing the hydrophilic portion and the hydrophobic portion, for example, up to about 50 wt% (e.g., about 4 wt% to about 50 wt%, about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt%) of the particle. For example, the weight percentage of the second polymer within the particles is about 3% to 30%, about 5% to 25%, or about 8% to 23%.

The polymers containing hydrophilic and hydrophobic moieties described herein may be commercially available or may be synthetic. Methods for synthesizing polymers are known in the art (see, e.g., Polymer Synthesis: Theroy and Practice Fundamentals, Methods, experiments. D. Braun et al, fourth edition, Springer, Berlin, 2005). Such methods include, for example, polycondensation, free radical polymerization, ionic polymerization (e.g., cationic or anionic polymerization), or ring opening metathesis polymerization. Block copolymers can be prepared by separately synthesizing two polymer units and then conjugating the two moieties using established methods. For example, a coupling agent such as EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) can be used to link the blocks. After conjugation, the two blocks may be linked via amide, ester, ether, amino, urethane or carbonate linkages.

Commercially available or synthetic polymer samples can be further purified prior to forming the polymer-reagent conjugate or incorporating the particles or polymers described herein. In some embodiments, purification can remove lower molecular weight polymers that may result in non-filterable polymer samples. The polymer may be purified by precipitation from solution or onto a solid such as diatomaceous earth. The polymer may also be further purified by Size Exclusion Chromatography (SEC).

Peptide-polymer conjugates

In some embodiments, a polymer, such as a hydrophilic-hydrophobic polymer, is attached to the charged peptide. The charged therapeutic peptide or protein may then form a non-covalent bond with the charged peptide. The charged peptide can be conjugated to the same polymer (e.g., hydrophobic and hydrophilic-hydrophobic polymers) as described above using the same methods described above.

Therapeutic peptides

Therapeutic peptides can be delivered to a subject using the described therapeutic peptide-polymer conjugates, particles, or compositions. In some embodiments, the therapeutic peptide is a compound having pharmaceutical activity. In another embodiment, the therapeutic peptide is a clinically used or studied drug. In another embodiment, the therapeutic peptide has been approved by the U.S. food and drug administration for use in humans or other animals. In some embodiments, the therapeutic peptide is a charged peptide (e.g., having a positive or negative charge).

Metabolic disorders

The disclosed therapeutic peptide-polymer conjugates, particles, and compositions can be useful for the prevention and treatment of metabolic disorders.

In some embodiments, the therapeutic peptide is a hormone. Examples of hormones include enkephalin, GLP-1 (e.g., GLP-1(7-37), GLP-1(7-36)), GLP-2, insulin-like growth factor-1, insulin-like growth factor-2, orexin A, orexin B, neuropeptide Y, growth hormone-releasing hormone, thyroid stimulating hormone-releasing hormone, cholecystokinin, melanocyte-stimulating hormone, corticotropin-releasing factor, melanin concentrating hormone, galanin, bombesin, calcitonin gene-related peptide, neurotensin, endorphin, dynorphin, and the C-peptide of proinsulin.

Preferably, the therapeutic peptide is an anti-glycogenic peptide. Anti-glycogenic peptides include peptides having one or more of the following activities: 1) the ability to increase insulin secretion; 2) (ii) an ability to increase insulin biosynthesis; 3) the ability to decrease glucagon secretion; 4) the ability to delay gastric emptying; 5) reducing hepatic glucose neogenesis; 6) improving insulin sensitivity; 7) improving glucose sensing in beta cells; 8) enhancing glucose treatment; 9) reducing insulin resistance; and 10) promoting beta cell function or viability. Examples of anti-glycogenic peptides include glucagon-like peptide-1 (GLP-1), insulin-like growth factor-1, insulin-like growth factor-2, exedin-4, and gastric inhibitory polypeptides and variants and derivatives thereof. Variants of some of the smaller peptides listed above are known. For example, known variants of GLP-1 include, for example, GLP-1: (7-36)、GLP-1(7-37)、Gln⁹-GLP-1(7-37)、Thr¹⁶-Lys¹⁸-GLP-1(7-37)、Lys¹⁸GLP-1(7-37) and Gly⁸-GLP-1. Derivatives include, for example, acid addition salts, carboxylic acid salts, lower alkyl esters, and amides, such as those described in PCT publication WO 91/11457.

Exemplary therapeutic peptides include:

a-71378(Abbott Laboratories), a six amino acid peptide (and variants and derivatives thereof), may be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as obesity;

PYY3-36(Amylin Pharmaceuticals), a thirty-four amino acid peptide (and variants and derivatives thereof), may be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as obesity;

AC-253(Antam, Amylin Pharmaceuticals) and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, and/or gestational diabetes) and obesity;

albiglutide (GSK-716155, Syncria, GlaxoSmithKline) and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

AKL-0707(LAB GHRH, Akela Pharma), a 29 amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as lipodystrophy and malnutrition;

AOD-9604(Metabolic Pharmaceuticals, Ltd.), a cyclic 16 amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat Metabolic disorders such as obesity;

BAY-73-7977(Bayer AG), and variants and derivatives thereof, are useful in the particles, conjugates, and compositions described herein for treating metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, and gestational diabetes);

BMS-686117(Bristol-Myers Squibb), an eleven amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, and gestational diabetes);

BIM-44002(Ipsen), a twenty-eight amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as hypercalcemia;

CVX-096(Pfizer-Covx) and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, and gestational diabetes);

davintrinide (davalintide) (AC-2307, Amylin Pharmaceuticals), a cyclic thirty amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as obesity;

AC-2993(LY-2148568、Byetta^TMAmylin Pharmaceuticals), a thirty-eight amino acid peptide and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes) and obesity;

exsulin (INGAP peptide, Exsulin), a fifteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

glucagon (Glucogen)^TMNovo Nordisk), a twenty-nine amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

ISF402(Dia-B Tech), a four amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

larazotide (AT-1001, SPD-550, Alba therapeutics corp), an eight amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

Liraglutide (Victoza)^TMNovo Nordisk), a thirty-one amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes) and obesity;

risperidone (lixisenatide) (AVE-0010, ZP-10, Sanofi Aventis), a forty-four amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

LY-2189265(EliLilly & Co.) and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

LY-548805(EliLilly & Co.) and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

NBI-6024(Neurocrine Biosciences, Inc.), a fifteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

Ornithipide (obinepitide) (7TM Pharma), a thirty-six amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating metabolic disorders such as obesity;

peptide YY (3-36) (MDRNA Inc.), a thirty-four amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as obesity;

pramlintide (Symlin)^TMAmylin Pharmaceuticals), a cyclic thirty-four amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes) and obesity;

r-7089(Roche), and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

semaglutide (NN-9535, Novo Nordisk), and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

SST analogs (Merck & co.inc.), and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

SUN-E7001(CS-872, Daiichi Sankyo), a thirty amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

taspoglutide (BIM-51077, Roche), a thirty amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

temorelin (tesamorelin) (TH-9507, theratetechnologies), a forty-four amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates and compositions described herein for the treatment of metabolic disorders such as growth hormone deficiency, muscle atrophy and lipodystrophy;

TH-0318(OctoPlus NV) and variants and derivatives thereof, which can be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

TKS-1225 (oxyntomodulin, Wyeth), a thirty-seven amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as obesity;

TM-30339(7TM Pharma) and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as obesity;

TT-223(E1-INT, Eli Lilly & Co.) and variants and derivatives thereof, may be used in the particles, conjugates and compositions described herein for the treatment of metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes);

non-acylated ghrelin (AZP-01, Alize Pharma), a twenty-eight amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat metabolic disorders such as diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes); and

urocortin II (Neurocrine Biosciences Inc.), a thirty-eight amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of metabolic disorders such as obesity.

Cancer treatment

The disclosed therapeutic peptide-polymer conjugates, particles, and compositions are useful for treating proliferative disorders, e.g., treating tumors and metastases thereof, wherein the tumors or metastases thereof are cancers described herein.

The therapeutic peptide can be, for example, a peptide inhibitor of proliferation signal transduction (e.g., an inhibitor of mitotic signal transduction or a peptide that restores the activity of a tumor suppressor protein such as p 53), a cell cycle inhibitor, or an inducer of apoptosis. For example, peptide inhibitors of proliferative signal transduction include peptide inhibitors of Ras activation, peptide inhibitors of MAP kinase, peptide inhibitors of NF-. kappa.B activation, and peptide inhibitors of c-Myc activation. See, e.g., Bidwell et al, (2009) ExpertOpin. drug Delivery6 (10): 1033-1047, the contents of which are incorporated herein by reference.

Examples of therapeutic peptides that can be used in the claimed conjugates, particles and compositions include the following:

a-6(Angstrom Pharmaceuticals Inc.), an eight amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., ovarian cancer);

PPI-149 (abarelix), Pleenaxis ^TM) A ten amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., prostate cancer);

ABT-510(Abbott Laboratories), a nine amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating proliferative disorders such as cancer (e.g., lung cancer (e.g., small cell or non-small cell lung cancer), renal cell carcinoma, sarcoma, lymphoma, solid tumors, melanoma, and malignant glioma);

ADH-1(Exherin^TMadheex Technologies), a cyclic five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., solid tumors and melanoma);

AEZS-108(AN-152, ZEN-008, AEtherna Zentaris), a ten amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating proliferative disorders such as cancer (e.g., endometrial, breast, ovarian, and prostate cancer);

Afanotide (afamelanotide) (EP-1647, CUV-1647, Melanotan^TMClinuvel Pharmaceuticals, Ltd.), a thirteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., skin cancer);

amamustine (PTT-119, Abbott Laboratories), a three amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., lymphoma (e.g., non-Hodgkin's lymphoma) and lung cancer (e.g., small cell or non-small cell lung cancer);

antagonist G (PTL-68001, Arana Therapeutics), a six amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., lung cancer (e.g., small cell or non-small cell lung cancer), pancreatic cancer, and colorectal cancer);

ATN-161 (antenuon LLC), a five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., glioma);

Avorelin (EP-23904, Meterelin)^TM、Lutrelin^TMMediiolum faceutici SpA), a nine amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., prostate and breast cancer);

buserelin (suprefect)^TM、Suprecur^TMSanofi-Aventis), a ten amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating proliferative disorders such as cancer (e.g., prostate cancer);

carfilzomib (PR-171, Proteolix Inc.), a four amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating proliferative disorders such as cancer (e.g., multiple myeloma, lymphoma, hematological tumors, and solid tumors);

CBP-501(Takeda Pharmaceuticals), a twelve amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat proliferative disorders such as cancer (e.g., lung cancer (e.g., small cell or non-small cell lung cancer) and mesothelioma);

Cimadotin (LU-103793, Abbott Laboratories), a five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer;

cetrorelix (NS-75, cetroride)^TMAEterna Zentaris), a ten amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as benign prostatic hyperplasia, fibroids (e.g., uterine fibroids), cancer (e.g., breast cancer, ovarian cancer, prostate cancer);

chlorotoxin (TM-601, TransMolecular Inc.), a thirty-six amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., glioma);

cilengitide (EMD-121974, EMD-85189), a five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., lung cancer (e.g., small cell or non-small cell lung cancer), glioblastoma, pancreatic cancer, and prostate cancer);

CTCE-9908 (chemical Therapeutics Corp.), a seventeen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer;

CVX-045(Pfizer-Covx) and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat proliferative disorders such as cancer (e.g., solid tumors);

CVX-060(Pfizer-Covx) and variants and derivatives thereof, useful in the particles, conjugates, and compositions described herein for treating proliferative disorders such as cancer;

degarelix (FE200486, Ferring Pharmaceuticals), a ten amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., prostate cancer);

deslorelin (Somagard)^TMShire) and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., lymphoma (e.g., non-hodgkin's lymphoma), brain cancer, melanoma);

A six amino acid peptide, and variants and derivatives thereof, of sphingosine (didemnin) B (NSC-325319, PharmaMar) may be used in the particles, conjugates and compositions described herein for the treatment of proliferative disorders such as cancer (e.g. lymphoma (e.g. non-hodgkin's lymphoma), brain cancer, melanoma);

DRF-7295(Dabur India Ltd.) and variants and derivatives thereof, may be used in the particles, conjugates and compositions described herein for the treatment of proliferative disorders such as cancer (e.g. breast cancer and colorectal cancer);

edotreotide (SMT-487, OctreoTherrTM, Onaita)^TMMolecular weight Pharmaceuticals), a cyclic seven amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat proliferative disorders such as cancer;

elipidosin (PM-02734, Irvalec)^TMPharmaMar), and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., lung cancer (e.g., small cell or non-small cell lung cancer));

EP-100(Esperance Pharmaceuticals Inc.), a thirty-three amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating proliferative disorders such as cancer (e.g., prostate cancer);

Ganirelix (Org-37462, RS-26306, Orgalutran)^TM、Antagon^TMSchering-Plough Corp), and variants and derivatives thereof, may be used in the particles, conjugates and compositions described herein for the treatment of proliferative disorders such as endometriosis and cancer (e.g., prostate and breast cancer);

oxidized glutathione (glutoxim) (NOV-002, Pharma Vam), a six amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating proliferative disorders such as cancer (e.g., lung cancer (e.g., small cell or non-small cell lung cancer) and ovarian cancer);

gorralide (BIM-32001, Ipsen), a four amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer;

goserelin (goserelin) (ICI-118630, AstraZeneca), a ten amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., prostate, breast, and uterine cancers);

Histrelin (Vantas)^TM，Johnson&Johnson), a nine amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., prostate cancer);

labradilil (RMP-7, Cereport)^TM，Johnson&Johnson), a nine amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., glioma and brain cancer);

leuprorelin (leuprolide) (Lupron)^TM、Pro stap^TM、Leuplin^TM、Enantone^TMTakeda Pharmaceuticals), a nine amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as fibroids (e.g., uterine fibroids) and cancer (e.g., prostate cancer);

LY-2510924(AVE-0010, Sanofi-Aventis), a cyclic amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as and cancer (e.g., breast cancer);

mifamurtide (Junnovan)^TM、Metpact^TMTakeda pharmaceuticals), a three amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., osteosarcoma);

Methionine-enkephalin (meta-enkephalin) (INNO-105, innovive pharmaceuticals Inc.), a five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., solid tumors, pancreatic cancer);

muramyl tripeptide (Novartis), a three amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer;

nafarelin (nafarelin) (RS-94991, Samynarel)^TM、Nasanyl^TM、Synarel^TM、Synareia^TMRoche), and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as endometriosis and cancer (e.g., prostate and breast cancer);

octreotide (octreotide) (SMS-201-995, Sandostatin)^TMNovartis) and variants thereofXenologies and derivatives that may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as benign prostatic hyperplasia and cancer (e.g., prostate cancer);

oxazarelix (D-63153, SPI-153, spectra pharmaceuticals), a ten amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as benign prostatic hyperplasia and cancer (e.g., prostate cancer);

POL-6326 (polypar) and variants and derivatives thereof, useful in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer;

ramorelix (ramorelix) (Hoe-013, Sanofi Aventis), a nine amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as fibroids (e.g., uterine fibroids) and cancer (e.g., prostate cancer);

RC-3095(AEterna Zentaris), a six amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., solid tumors);

Re-188-P-2045(P2045、Neotide^TMbryan Oncor), an eleven amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating proliferative disorders such as cancer (e.g., lung cancer (e.g., small cell or non-small cell lung cancer));

romurtide (Romurtide) (DJ-7041, Nopia)^TMMuroctasin (TM), Daiichi Sankyo), a two amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat proliferative disorders such as cancer;

YHI-501(TZT-1027, Yakult Honsha KK), a two amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating proliferative disorders such as cancer (e.g., solid tumors);

SPI-1620(Spectrum Pharmaceuticals), a fourteen amino acid peptide and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., solid tumors);

talobilauride (RP-56142, Sanofi Aventis), a three amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates and compositions described herein for the treatment of proliferative disorders such as cancer;

TAK-448(Takeda Pharmaceuticals) and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., prostate cancer);

TAK-683(Takeda Pharmaceuticals) and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat proliferative disorders such as cancer (e.g., prostate cancer);

Tacidotin (ILX-651, BSF-223651, Genzyme), a five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., melanoma, prostate cancer, and lung cancer (e.g., small cell or non-small cell lung cancer));

teverelix (teverelix) (EP-24332, Antarelix)^TMArdana Biosciences), a ten amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as endometriosis, benign prostatic hyperplasia, and cancer (e.g., prostate cancer);

tipepitide (PCK-3145, Kotinos Pharmaceuticals), a fifteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as endometriosis, benign prostate hyperplasia, and cancer (e.g., prostate cancer);

thymalfasin (Zadaxin)^TM、Timosa^TM、Thymalfasin^TMSciClone Pharmaceuticals), a twenty-eight amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., melanoma, lung cancer (e.g., small cell or non-small cell lung cancer), and carcinoma (e.g., hepatocellular carcinoma));

TLN-232(CAP-232, TT-232, hallusion Pharmaceuticals), a seven amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as endometriosis, benign prostatic hyperplasia, and cancer;

triptorelin (triptorelin) (WY-42462, debiophama), a ten amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as endometriosis, fibroids (e.g., uterine fibroids), benign prostatic hyperplasia, and cancer (e.g., prostate and breast cancer);

tyroselerotide (CMS-024, China Medical System), a three amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., liver cancer (e.g., hepatocellular carcinoma); and

tyroservatide (CMS-024-02, China Medical Systems), a three amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of proliferative disorders such as cancer (e.g., lung cancer (e.g., small cell or non-small cell lung cancer)).

Cardiovascular diseases

The disclosed therapeutic peptide-polymer conjugates, particles, and compositions can be useful for the prevention and treatment of cardiovascular disease.

Exemplary therapeutic peptides that can be used in the disclosed conjugates, particles, and compositions include the following:

AC-2592(Betatropin^TMamylin Pharmaceuticals), a thirty amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as heart failure;

AC-625(Amylin Pharmaceuticals), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat cardiovascular disorders such as hypertension;

anaritide (Auriculin)^TM，Johnson&Johnson), a cyclic twenty-five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as renal failure, heart failure, and hypertension;

APL-180(Novartis), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as coronary artery disorders;

Atrial natriuretic peptide (Atriopeptin) (Astellas Pharma), a twenty-five amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat cardiovascular disorders;

BGC-728(BTG plc), a cyclic peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as myocardial infarction and cerebrovascular ischemia;

carperitide (SUN-4936, HANP)^TMDaiichi Sankyo), a cyclic peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as heart failure;

CD-np (nie therapeutics), a forty-one amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as heart failure;

CG-77X56(Cardeva^TMteva Pharmaceuticals), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as heart failure;

D-4F (APP-018, Novartis), an eighteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as atherosclerosis;

Danegatide (danegatide) (ZP-1609, WAY-261134, GAP-134, Zealand Pharma), a two amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as arrhythmia;

DMP-728(DU-728, Bristol-Myers Squibb), a cyclic three amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as thrombosis (e.g., coronary thrombosis);

efegatran (Efegatran) (LY-294468, Eli Lilly and Co.), a three amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates and compositions described herein for the treatment of cardiovascular disorders such as myocardial infarction and thrombosis (e.g., coronary thrombosis);

EMD-73495(Merck kGaA), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders;

eptifibatide (C68-22, Integrin)^TM、Integrilin^TMTakeda pharmaceuticals), a cyclic six amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as acute coronary syndrome, myocardial infarction, and unstable angina;

ET-642 (RLT-peptide, Pfizer), a twenty-two amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as atherosclerosis;

FE202158(Ferring Pharmaceuticals), a cyclic nine amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat cardiovascular disorders such as vasodilatory hypotension (e.g., sepsis and intradialytic hypotension);

FX-06(Ikaria), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates and compositions described herein for the treatment of cardiovascular disorders such as reperfusion injury;

icaritin (icrocatide) (ITF-1697, italfarnco), a four amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as respiratory distress syndrome;

KAI-1455(KAI Pharmaceuticals), a twenty amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as cardiovascular surgical cytoprotection;

KAI-9803(Bristo-Myers Squibb), a twenty-three amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as myocardial infarction, reperfusion injury, and coronary artery disease;

l-346670(Merck & co.inc.), a cyclic twenty-six amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as hypertension;

l-364343(Merck & co.inc.), a cyclic twenty-nine amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as hypertension;

LSI-518P (Lipid Sciences Inc.), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders;

nesiritide (Nesiritide) (Noratak)^TM、Natrecor^TM，Johnson&Johnson), a thirty-two amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as heart failure;

A peptide chymosin inhibitor (Pfizer), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders;

PL-3994(Palatin Technologies), a fifteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as hypertension and heart failure;

rotigotine (rotigotide) (ZP-123, GAP-486, Zealand Pharma), a six amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates and compositions described herein for the treatment of cardiovascular disorders such as ventricular arrhythmia and atrial fibrillation;

saralasin (Saralasin) (P-113, Sarenin)^TM，Procter&Gamble), an eight amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders;

SKF-105494(GlaxoSmithKline), a cyclic seven amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as hypertension;

Teragilren (Terlakiren) (CP-80794, Pfizer), a two amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as hypertension;

thymalfasin (Zadaxin)^TM、Timosa^TM、Thymalfasin^TMSciclone pharmaceuticals), a twenty-eight amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat cardiovascular disorders such as angiogenic disorders;

a tricarbotide (AP-214, Action Pharma), a ten amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as reperfusion injury and renal disease;

uraritide (CDD-95-126, ESP-305, CardioBISS)^TM、Nephrobiss^TMEKR Therapeutics), a cyclic thirty-two amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat cardiovascular disorders such as heart failure and renal failure;

urocortin II (Neurocrine Biosciences Inc.), a thirty-eight amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as heart failure; and

ZP-120(Zealand Pharma), a twelve amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of cardiovascular disorders such as isolated systolic hypertension and heart failure.

Infectious diseases

The conjugates, particles, and compositions described herein can comprise peptides that treat or prevent infectious diseases. Exemplary therapeutic peptides that can be used in the disclosed conjugates, particles, and compositions include the following:

eboweitai (albivirtide) (Frontier Biotechnologies), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as HIV infection;

ALG-889(Allergene Inc.), a sixteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as HIV infection and immune disorders;

Alloferon(Allokine-alpha^TMentoparm co. ltd.), a thirteen amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as hepatitis b virus infection, hepatitis c virus infection, herpes virus infection, and cancer;

ALX-40-ac (nps pharmaceuticals), a nine amino acid peptide, and variants and derivatives thereof, are useful in the particles, conjugates, and compositions described herein for treating microbial or viral disorders such as HIV infection;

CB-182804(Cubist Pharmaceuticals), a peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as multi-drug resistant gram negative bacterial infections;

CB-183315 (cube Pharmaceuticals), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as Clostridium difficile (Clostridium difficile) -associated diarrhea;

CZEN-002(Migami), a polymeric eight amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as vulvovagaginalcanydiasi;

enfuvirtide (T-20, Fuzeon)^TMRoche), a thirty-six amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating microbial or viral disorders such as HIV infection;

Glucosaminyl muramyl tripeptide (Theramide)^TMDOR BioPharma Inc.), a three amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating microbial or viral disorders such as herpes virus infection, post-surgical infection, psoriasis, respiratory disorders (e.g., pulmonary disorders), and tuberculosis;

GMDP(Likopid^TM、Licopid^TMarana Therapeutics), a two amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as herpes virus infection, post-surgical infection, psoriasis, respiratory disorders (e.g., pulmonary disorders), and tuberculosis;

golimod (Golotimod) (SCV-07, SciClone Pharmaceuticals), a two amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as hepatitis c, viral infections, and tuberculosis;

GPG-NH2(Tripep), a three amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as HIV infection;

hLF (1-11) (AM-Pharma Holding BV), an eleven amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as bacterial infections, mycoses, bacteremia, and candidemia;

IMX-942(Inimex Pharmaceuticals), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as hospital-acquired bacterial infections;

isaccagenan (IB-367, Ardea Biosciences Inc.), a cyclic sixteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as stomatitis and nosocomial pneumonia;

moraxel butyl ester (Murabutide) (VA-101, CY-220, Sanofi-Aventis), a two amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as hepatitis virus infection and HIV infection;

Neogen(Neogen^TMimmunotech Developments), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as viral infections, bacterial infections, and hematopoietic disorders;

NP-213(Novexatin^TMNovabitics), a cyclic amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as onychomycosis;

ovofanide (IM-862, Implicit Bioscience), a two amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as hepatitis c virus infection;

omeganan (Omiganan) (CPI-226, Omigard)^TMMigenix Inc.), a twelve amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as catheter infections and rosacea;

OP-145(OctoPlus NV), a peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating microbial or viral disorders such as otitis;

p-1025(Sinclair Pharma plc), a nineteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as dental caries;

P-113(PAC-113、HistaWash^TM、Histat gingivitis gel^TM、Histatperiodontal wafer^TMPacgen biopharmaceutics Corp.), a twelve amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as candida albicans infection and gingivitis;

Pep-F (5K, Milkhaus Laboratory Inc.), a peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as herpes virus infections;

R-15-K(BlockAide/CR^TMadvntrx Pharmaceuticals Inc.), a fifteen amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as HIV infection;

a thirty-six amino acid peptide (siffuvirtide) (FusoGen Pharmaceuticals Inc.), a thirty-six amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as HIV infection;

SPC-3(Columbia Laboratories), a polymeric fifty-six amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as HIV infection;

Thymalfasin (Zadaxin)^TM、Timosa^TM、Thymalfasin^TMSciclone pharmaceuticals), a twenty-eight amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as cancer (e.g., hepatocellular carcinoma), hepatitis b virus infection, hepatitis c virus infection, HIV infection, influenza virus infection, aspergillus infection, and wound healing;

thymonoctan (FCE-25388, Pfizer), an eight amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as hepatitis virus infection and HIV infection;

thymopentin (TP-5, Timunox)^TM，Johnson&Johnson), a five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating microbial disorders or virusesDisorders such as lung infection and HIV infection;

tefuvirtide (tiffuvirtide) (R-724, T-1249, Roche), a thirty-nine amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as HIV infection;

TRI-1144(Trimeris Inc.), a thirty-eight amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of microbial or viral disorders such as HIV infection;

VIR-576(Pharis Biotec), a forty amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as HIV infection; and

XOMA-629(XOMA Ltd.), a fifteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat microbial or viral disorders such as acne, staphylococcus aureus (staphyloccocusareus) infection, and impetigo;

allergic, inflammatory and autoimmune disorders

The conjugates, particles, and compositions described herein can comprise peptides that treat or prevent allergic, inflammatory, and/or autoimmune disorders. Exemplary therapeutic peptides that can be used in the disclosed conjugates, particles, and compositions include the following;

a-623(AMG-623, Anthera Pharmaceuticals), a peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat allergy, inflammatory disorders, or immune disorders such as lupus erythematosus and chronic lymphocytic leukemia;

AG-284(AnergiX.MS^TMGlaxoSmithKline), a nineteen amino acid peptide, and variations thereofAnd derivatives, which may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders, or immune disorders such as multiple sclerosis;

AI-502(AutoImmune), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat allergy, inflammatory or immune disorders such as transplant rejection;

Allotrap2702(B-2702、Allotrap2702^TMgenzyme), a ten amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat allergies, inflammatory disorders, or immune disorders such as transplant rejection;

AZD-2315(AstraZeneca), an eight amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders or immune disorders such as rheumatoid arthritis;

Cnsnqic-Cyclic (802-2, adoona Pharmaceuticals), a Cyclic five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders or immune disorders such as factor VIII deficiency, multiple sclerosis, and graft versus host disease;

The Delmitide (RDP-58, Genzyme), a ten amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating allergies, inflammatory or immune disorders such as inflammatory bowel disease, ulcerative colitis, and crohn's disease;

direcotide (Dirucotide) (MBP-8298, Eli Lilly and Co.), a seventeen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders, or immune disorders such as multiple sclerosis;

disitetide (NAFB-001, P-144, ISDIN SA), a cyclic fourteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders, or immune disorders such as scleroderma;

dnaJP1(AT-001, Adeona Pharmaceuticals), a fifteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders, or immune disorders such as rheumatoid arthritis;

Edratide (TV-4710, Teva Pharmaceuticals), a twenty amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating allergies, inflammatory disorders, or immune disorders such as systemic lupus erythematosus;

f-991(Clinquest Inc.), a nine amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating allergy, inflammatory disorders or immune disorders such as allergic asthma and skin disorders;

FAR-404(Enkorten^TMfaracija doo), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergies, inflammatory disorders or immune disorders such as functional bowel disorders, multiple sclerosis, rheumatoid arthritis, asthma, and systemic lupus erythematosus;

glatirmod (glasipimod) (SKF-107647, GlaxoSmithKline), an eight amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergies, inflammatory disorders, or immune disorders such as leukopenia drug-induced fungal infections, immune disorders, viral infections, bacterial infections, and immune deficiencies;

Glatiramer (Glatiramer) (COP-1, Copaxone)^TMTeva pharmaceuticals), a peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat allergies, inflammatory or immune disorders such as glaucoma, Huntington's chorea, motor neuron disease, multiple sclerosis, and neurodegenerative diseases;

glucosaminyl muramyl tripeptide (Theramide)^TMDOR BioPharma Inc.), a three amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating allergies, inflammatory or immune disorders such as herpes virus infections, post-surgical infections, psoriasis, respiratory disorders (e.g., pulmonary disorders), and tuberculosis;

GMDP(Likopid^TM、Licopid^TMarana Therapeutics), a two amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of allergies, inflammatory or immune disorders such as herpes virus infections, post-surgical infections, psoriasis, respiratory disorders (e.g., pulmonary disorders), and tuberculosis;

icatibant (Icatant) (JE-049, HOE-140, Firazyr) ^TMShire), an eight amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergies, inflammatory or immune disorders such as hereditary angioedema, rhinitis, asthma, osteoarthritis, pain, and cirrhosis;

IPP-201101(Lupuzor^TMimmu pharma Ltd.), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating allergy, inflammatory disorders or immune disorders such as systemic lupus erythematosus;

an MS peptide (Briana Bio-Tech Inc.), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating allergy, inflammatory disorders, or immune disorders such as multiple sclerosis;

Org-42982(AG-4263、AnergiX.RA^TMGlaxoSmithKline), a thirteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating allergy, inflammatory disorders, or immune disorders such as rheumatoid arthritis;

pentigtide (Pentigetide) (TA-521, Pentyde)^TM，Bausch&Lomb), a five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders, or immune disorders such as allergic rhinitis and allergic conjunctivitis;

PI-0824(Genzyme), a nineteen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders or immune disorders such as pemphigus vulgaris;

PI-2301 (peptimmunee), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating allergy, inflammatory disorders, or immune disorders such as multiple sclerosis;

PLD-116(Barr Pharmaceuticals Inc.), a fifteen amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat allergies, inflammatory or immune disorders such as ulcerative colitis;

PMX-53(Arana Therapeutics), a cyclic six amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat allergies, inflammatory or immune disorders such as inflammation, rheumatoid arthritis, and psoriasis;

PTL-0901(Acambis plc), a nine amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat allergy, inflammatory disorders, or immune disorders such as allergic rhinitis;

RA peptide (Acambis plc), a four amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders, or immune disorders such as rheumatoid arthritis;

TCMP-80(Elan Corp.), a two amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for treating allergy, inflammatory disorders, or immune disorders;

thymodepressin (immunotech developments), a two amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat allergies, inflammatory or immune disorders such as relapsed autoimmune cytopenia (1, 2, 3 lineage), aplastic anemia, rheumatoid arthritis, and psoriasis;

thymopentin (TP-5, Timunox)^TM，Johnson&Johnson), a five amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat allergy, inflammatory disorders, or immune disorders such as lung infection, rheumatoid arthritis, HIV infection, and primary immunodeficiency;

Telimotide (tiplimitide) (NBI-5788, Neurocrine Biosciences Inc.), a seventeen amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders, or immune disorders such as multiple sclerosis;

uraritide (CDD-95-126, ESP-305, CardioBISS)^TM、Nephrobiss^TMEKR Therapeutics), a cyclicThirty-two amino acid peptides, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of allergy, inflammatory disorders, or immune disorders such as asthma; and

ZP-1848(Zealand Pharma), a peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat an allergy, inflammatory disorder, or immune disorder.

Renal disease

The disclosed therapeutic peptide-polymer conjugates, particles, and compositions are useful for treating renal disorders, such as those described herein.

The therapeutic peptide can be, for example, a peptide agonist of the GHRH receptor, a peptide agonist of the ANP receptor, a peptide agonist of the AVP receptor, a peptide agonist of the CALC receptor, a peptide agonist of the CRH receptor, a peptide agonist of the SST receptor, a peptide agonist of the IL-2 receptor, and a peptide agonist of the MC receptor.

AKL-0707(Aleka Pharma), a twenty-nine amino acid peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat renal disorders, such as renal dysfunction associated with lipodystrophy.

Aniritide (Johnson & Johnson), a twenty-five amino acid cyclic peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating renal disorders, such as renal failure;

BIM-44002(Ipsen), a twenty-eight amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat renal disorders, such as renal failure, e.g., hypercalcemia associated with renal failure;

human calcitonin (also known as) (Novartis), a thirty-two amino acid peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat renal disorders, such as renal failure, e.g., hypercalcemia associated with renal failure;

salmon calcitonin (also known as ) (Sanofi-Aventis), a thirty-two amino acid cyclic peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat renal disorders, such as renal failure, e.g., hypercalcemia associated with renal failure;

c-peptide (also known as SPM-933) (Cebix), a linear peptide of thirty-one amino acids, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat renal disorders, such as renal disease, e.g., diabetic nephropathy;

desmopressin (also known as desmopressin)Or) (Ferringpharmaceuticals), a nine amino acid cyclic peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat renal disorders, such as renal disease, e.g., diabetic nephropathy;

DG-3173 (also called PTR-3173 or) (DeveroGen), an eight amino acid cyclic peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for treating kidneyDisorders, such as nephropathy, e.g. diabetic nephropathy;

EA-230 (expenential Biotherapies), a four amino acid linear peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat renal disorders, such as renal failure;

Elcatonin (also known as Elcatonin)Or) (Asahi KaseiPharma), a thirty-one amino acid cyclic peptide, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein to treat renal disorders, such as renal failure, e.g., hypercalcemia associated with renal failure;

lypressin (also known as Lypressin)) (Novartis), a cyclic peptide of nine amino acids, and variants and derivatives thereof, may be used in the particles, conjugates, and compositions described herein for the treatment of renal disorders, such as diabetes insipidus;

terlipressin (also known as Terlipessin)) (Ferringpharmaceuticals), a twelve amino acid cyclic peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein for the treatment of renal disorders, such as hepatorenal syndrome;

the trekk peptide (also known as AP-214) (Action Pharma), a linear peptide of ten amino acids, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat renal disorders; and

uraritide (also known as CDD-95-126, ESP-305, Ulatride),Or) (EKR Therapeutics), a thirty-two amino acid cyclic peptide, and variants and derivatives thereof, can be used in the particles, conjugates, and compositions described herein to treat renal disorders, such as renal failure.

Renal disorders

The disclosed polymer-agent conjugates, particles, and compositions are useful for treating renal disorders, e.g., treating the renal disorders described herein. In some embodiments where the agent is a diagnostic agent, the polymer-agent conjugates, particles, and compositions described herein can be used to assess or diagnose a renal disorder.

Exemplary renal conditions include, for example, acute renal failure, acute nephrotic syndrome, analgesic nephropathy, congenic infarct nephropathy, chronic renal failure, chronic nephritis, congenital nephrotic syndrome, end-stage renal disease, goodpasture's syndrome, interstitial nephritis, renal damage, renal infection, renal injury, kidney stones, lupus nephritis, membranoproliferative GN I, membranoproliferative GNII, membranous nephropathy, minimal disease, necrotizing glomerulonephritis, nephroblastoma, nephrocalcinosis, nephrogenic diabetes insipidus, nephrotic variant disease (nephrotic syndrome), polycystic kidney disease, GN post streptococcal infection, reflux nephropathy, renal artery embolism, renal artery stenosis, renal papillary necrosis, tubular acidosis type I, tubular acidosis type II, renal hypoperfusion, and renal vein thrombosis.

In some embodiments, the agent is a derivative of a therapeutic peptide having pharmaceutical activity, such as an acetylated derivative or a pharmaceutically acceptable salt. In some embodiments, the therapeutic peptide is a prodrug, such as a hexanoate conjugate.

A therapeutic peptide can mean a combination of therapeutic peptides that have been combined and attached to a polymer and/or loaded into a particle. Any combination of therapeutic peptides may be used. In certain embodiments of treating cancer, at least two conventional chemotherapeutic therapeutic peptides are attached to a polymer and/or loaded into a particle.

In certain embodiments, a therapeutic peptide can be linked to a polymer to form a therapeutic peptide-polymer conjugate.

In certain embodiments, the therapeutic peptide in the particle is attached to the polymer of the particle. The therapeutic peptide may be attached to any polymer in the particle, for example, a hydrophobic polymer or a polymer containing both hydrophilic and hydrophobic portions.

In certain embodiments, the therapeutic peptide is embedded in the particle. The therapeutic peptide may be associated with other components of the polymer or particle by one or more non-covalent interactions such as van der waals interactions, hydrophobic interactions, hydrogen bonding, dipole-dipole interactions, ionic interactions, and pi stacking.

The therapeutic peptide can be present in varying amounts in the therapeutic peptide-polymer conjugates, particles, or compositions described herein. When present in the particles, the therapeutic peptide can be present in an amount of, for example, about 1% to about 100% by weight (e.g., about 2% to about 30%, about 4% to about 25%, about 50% to about 100%, about 70% to about 100%, about 50% to about 90%, or about 5% to about 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% by weight).

Conjugates

One or more components of the particle may be in the form of a conjugate, i.e., attached to another moiety. Exemplary conjugates include therapeutic peptide/protein-polymer conjugates (e.g., therapeutic peptide or protein-hydrophobic polymer conjugates, therapeutic peptide or protein-hydrophobic-hydrophilic polymer conjugates, or therapeutic peptide or protein-hydrophilic polymer conjugates), counterion-polymer conjugates (e.g., counterion-hydrophobic polymer conjugates or counterion-hydrophobic-hydrophilic polymer conjugates), and therapeutic peptide or protein-hydrophobic moiety conjugates.

The therapeutic peptide or protein-polymer conjugates described herein comprise a polymer (e.g., a hydrophobic polymer, a hydrophilic polymer, or a hydrophilic-hydrophobic polymer) and a therapeutic peptide or protein. The therapeutic peptides or proteins described herein can be attached to the polymers described herein, for example, directly (e.g., in the absence of an atom intervening a spacer moiety), or through a linker. The therapeutic peptide or protein may be attached to a hydrophobic polymer (e.g., PLGA), a hydrophilic polymer (e.g., PEG), or a hydrophilic-hydrophobic polymer (e.g., PEG-PLGA). The therapeutic peptide or protein may be attached to one end of the polymer, to both ends of the polymer, or to a point along the polymer chain. In some embodiments, multiple therapeutic peptides or proteins may be attached to multiple points along the polymer chain, or multiple therapeutic peptides or proteins may be attached to the terminus of the polymer via a multifunctional linker. The therapeutic peptide or protein can be attached to the polymer described herein through the amino terminus or the carboxy terminus of the therapeutic peptide or protein. Therapeutic peptides or proteins can also be attached to the polymers described herein through functional groups of the side chains of amino acids that are part of the therapeutic peptide or protein.

The counterion-polymer conjugates described herein comprise a polymer (e.g., a hydrophobic polymer or a polymer containing hydrophilic and hydrophobic portions) and a counterion. The counterions described herein can be attached to the polymers described herein, for example, directly (e.g., in the absence of an atom intervening a spacer moiety), or through a linker. The counter ion can be attached to a hydrophobic polymer (e.g., PLGA), or a polymer having a hydrophobic portion and a hydrophilic portion (e.g., PEG-PLGA). The counter ion may be attached to one end of the polymer, to both ends of the polymer, or to a point along the polymer chain. In some embodiments, multiple counterions can be attached to one point along the polymer chain, or multiple counterions can be attached to the ends of the polymer via a multifunctional linker.

Connection mode

The therapeutic peptides, proteins, or counterions described herein can be directly (e.g., in the absence of an atom intervening a spacer moiety) attached to a polymer or hydrophobic moiety (e.g., a polymer) described herein. The attachment may be at the end of the polymer or along the backbone of the polymer. In some embodiments, the therapeutic peptide or protein is modified at the point of attachment to the polymer; for example, the terminal amine or terminal carboxylic acid moiety of a therapeutic peptide or protein is converted to a functional group that reacts with the polymer (e.g., the carboxylic acid moiety is converted to a thioester moiety). The reactive functional group of the therapeutic peptide, protein, or counterion can be directly attached (e.g., in the absence of an atom intervening a spacer moiety) to a functional group on the polymer. The therapeutic peptide, protein, or counterion can be attached to the polymer via a variety of linkages, such as amide, ester, sulfide (e.g., maleimide sulfide), disulfide, succinimide, oxime, silyl ether, carbonate, or urethane linkages. For example, in one embodiment, the carboxyl group of the therapeutic peptide, protein, or counterion can react with the hydroxyl group of the polymer, thereby forming a direct ester linkage between the therapeutic peptide, protein, or counterion and the polymer. In another embodiment, the amine group of the therapeutic peptide, protein, or counterion can be attached to the carboxylic acid group of the polymer, thereby forming an amide linkage. In one embodiment, the sulfhydryl-modified therapeutic peptide or protein may be reacted with a reactive moiety on the end of a polymer (e.g., acrylate PLGA, or pyridyl-SS-activated PLGA, or maleimide activated PLGA) to form a sulfide or disulfide or thioether bond (i.e., sulfide bond). Exemplary ligation patterns include those generated by click chemistry (e.g., amide linkages, ester linkages, ketals, succinates or triazoles as well as those described in WO 2006/115547).

In certain embodiments, suitable protecting groups may be required at the terminus of other polymers or on the reactive side chain of therapeutic peptides or proteins in order to facilitate formation of a particular desired conjugate. For example, a polymer having a hydroxyl terminus can be protected, for example, with a silyl group (e.g., trimethylsilyl) or an acyl group (e.g., acetyl). A therapeutic peptide or protein having one or more reactive groups on a side chain can be protected with, for example, an acetyl group on a hydroxyl or amino group, such that the therapeutic peptide or protein can be selectively attached to the polymer, for example, through the terminus of the therapeutic peptide or protein.

In some embodiments, the process of linking a therapeutic peptide, protein, or counterion to a polymer can result in a composition comprising a mixture of conjugates having the same polymer and the same therapeutic peptide, protein, or counterion, but differing in the nature of the bond between the therapeutic peptide, protein, or counterion and the polymer. For example, when a therapeutic peptide, protein, or counterion has multiple reactive moieties that can react with a polymer, the reaction product of the therapeutic peptide, protein, or counterion with the polymer can include conjugates in which the therapeutic peptide, protein, or counterion is attached to the polymer via one reactive moiety, and conjugates in which the therapeutic peptide, protein, or counterion is attached to the polymer via another reactive moiety. For example, when a therapeutic peptide or protein is linked to a polymer, the reaction product may include a conjugate in which some of the therapeutic peptide or protein is linked to the polymer through the carboxy terminus of the therapeutic peptide or protein and some of the therapeutic peptide or protein is linked to the polymer through the amino terminus of the therapeutic peptide or protein. Likewise, when the counter ion has multiple reactive groups, such as multiple amines, the reaction product may include a conjugate in which some of the counter ion is linked to the polymer through a first reactive group and some of the counter ion is linked to the polymer through a second reactive group.

In some embodiments, the process of attaching a therapeutic peptide, protein, or counterion to a polymer can include the use of a protecting group. For example, when a therapeutic peptide, protein, or counterion has multiple reactive moieties that can react with a polymer, the therapeutic peptide, protein, or counterion can be protected at certain reactive sites so that the polymer will be attached via the designated site. In one embodiment, the therapeutic peptide or protein may be protected at the carboxy terminus or amino terminus of the therapeutic peptide or protein when attached to the polymer. In one embodiment, the therapeutic peptide or protein may be protected at the side chain of the therapeutic peptide or protein when attached to the polymer. In one embodiment, the therapeutic peptide or protein may be protected at the side chain and the terminus (e.g., amino terminus or carboxy terminus) of the therapeutic peptide or protein.

In some embodiments, selectively coupled products (such as those coupled products described above) may be combined to form a mixture of therapeutic peptide/protein-polymer conjugates. For example, PLGA linked to a therapeutic peptide or protein through the carboxy-terminus of the therapeutic peptide or protein, and PLGA linked to a therapeutic peptide or protein through the amino-terminus of the therapeutic peptide or protein may be combined to form a mixture of the two conjugates, and the mixture may be used to prepare the particle.

The polymer-agent (e.g., polymer-therapeutic peptide or polymer-protein) conjugate can comprise a single therapeutic peptide or protein or counterion attached to the polymer. The therapeutic peptide, protein, or counterion may be attached to the end of the polymer, or to a point along the polymer chain.

In some embodiments, the conjugate can comprise a plurality of therapeutic peptides, proteins, or counterions attached to the polymer (e.g., 2, 3, 4, 5, 6, or more agents can be attached to the polymer). The therapeutic peptide, protein or counterion may be the same or different. In some embodiments, a plurality of therapeutic peptides, proteins, or counterions can be linked to a multifunctional linker (e.g., a polyglutamic acid linker). In some embodiments, a plurality of therapeutic peptides, proteins, or counterions can be attached at multiple points along the polymer chain.

Linking group

The therapeutic peptides, proteins, or counterions can be linked to a moiety such as a polymer or a hydrophobic moiety such as a lipid, or to each other through a linker such as the linkers described herein. For example: the hydrophobic polymer may be linked to a counterion; the hydrophobic polymer may be linked to a therapeutic peptide or protein; the hydrophilic-hydrophobic polymer may be linked to a therapeutic peptide or protein; the hydrophilic polymer may be attached to a therapeutic peptide or protein; the hydrophilic polymer may be linked to a counterion; or a hydrophobic moiety may be attached to a counterion, or a therapeutic peptide or protein may be attached to a counterion. A therapeutic peptide or protein can be attached to a moiety, such as a polymer described herein, through a carboxylic acid position of the therapeutic peptide or protein, such as a terminal carboxylic acid position of the therapeutic peptide or protein (e.g., through a linker described herein). The therapeutic peptide or protein can be attached to a moiety, such as a polymer described herein, through an amine position of the therapeutic peptide or protein, such as a terminal amine position of the therapeutic peptide or protein (e.g., through a linker described herein). In some embodiments, the therapeutic peptide or protein is attached through the terminus of a polymer (e.g., a PLGA polymer, where attachment is at the hydroxyl terminus or the carboxyl terminus).

In certain embodiments, a plurality of linker moieties are attached to the polymer, thereby allowing for attachment of a plurality of therapeutic peptides, proteins, or counterions to the polymer through the linkers, e.g., where the linkers are attached at multiple placements on the polymer, such as along the polymer backbone. In some embodiments, the linker is configured such that multiple first moieties are linked to a second moiety by the linker, e.g., multiple therapeutic peptides or proteins can be linked to a single polymer, such as a PLGA polymer, via a branched linker, wherein the branched linker comprises multiple functional groups through which the therapeutic peptides or proteins can be linked. In some embodiments, the therapeutic peptide or protein is released from the linker under biological conditions (i.e., cleavable under physiological conditions). In another embodiment, a single linker is attached to the polymer, e.g., at a terminus of the polymer.

The linking group may comprise, for example, an alkylene group (divalent alkyl group). In some embodiments, one or more carbon atoms of the alkylene linker may be replaced with one or more heteroatoms or functional groups (e.g., thioether, amino, ether, ketone, amide, silyl ether, oxime, carbamate, carbonate, disulfide, heterocyclic, or heteroaromatic moieties). For example, an acrylate polymer (e.g., acrylate PLGA) can be reacted with a sulfhydryl-modified therapeutic peptide or protein to form a therapeutic peptide/protein-polymer conjugate that is linked by a sulfide bond. The acrylate may be attached to the end of the polymer (e.g., the hydroxyl end of a PLGA polymer such as a 50: 50PLGA polymer) by reacting acryloyl chloride (acryloyl chloride) with the hydroxyl end of the polymer.

In some embodiments, the linker has other functional groups in addition to the functional group that allows for the attachment of the first moiety to the second moiety. In some embodiments, the other functional groups can be cleaved under physiological conditions. Such a linker may be formed, for example, by: reacting a first activating moiety, such as a therapeutic peptide or protein, e.g., a therapeutic peptide or protein described herein, with a second activating moiety, such as a polymer, e.g., a polymer described herein, so as to generate a linker comprising a functional group formed by conjugating the therapeutic peptide or protein to the polymer. Optionally, other functional groups may provide sites for other linkages or allow cleavage under physiological conditions. For example, other functional groups may include sulfide, disulfide, ester, oxime, carbonate, carbamate, or amide linkages, which are cleavable under physiological conditions. In some embodiments, one or both of the functional groups that attach the linker to the first or second moiety, such as an ester, amide, or disulfide, can be cleaved under physiological conditions.

In some embodiments, the other functional group is a heterocyclic or heteroaromatic moiety.

The therapeutic peptide or protein can be attached to the portion of the polymer as described herein through a linker (e.g., a linker comprising two or three functional groups, such as a linker described herein), through a carboxylic acid or amine group of the therapeutic peptide or protein, such as a terminal carboxylic acid or amine of the therapeutic peptide or protein, or through a reactive group on a side chain of an amino acid of the therapeutic peptide or protein. In some embodiments, the therapeutic peptide or protein is linked through the terminus of the polymer (e.g., a PLGA polymer, where the linkage is at the hydroxyl terminus or the carboxyl terminus).

In some embodiments, the linker comprises a moiety that can modulate the reactivity of a functional group in the linker (e.g., another functional group or atom that can increase or decrease the reactivity of the functional group, e.g., under biological conditions).

For example, as shown in fig. 1A-C, a Therapeutic Peptide (TP) having a first reactive group can be reacted with a polymer having a second reactive group to attach the therapeutic peptide to the polymer while providing a biologically cleavable functional group. The resulting linker comprises a first spacer, such as an alkylene spacer, that links the therapeutic peptide to the functional group resulting from the linkage (i.e., by forming a covalent bond), and a second spacer, such as an alkylene spacer (e.g., about C), that links the polymer to the functional group resulting from the linkage ₁To about C₆)。

As shown in fig. 1A-C, a therapeutic peptide can be attached to a first spacer via a moiety Y that is also biocleavable. Y may beFor example, -O-, -S-, -NH-, -C (═ O) NH-, or-C (═ O) O-. In some embodiments, the second spacer may be attached to a leaving group X-, such as a halo (e.g., chloro) or N-hydroxysuccinimide (NHS). The second spacer may be attached to the polymer via other functional groups (Z) bonded to the polymer terminus, such as the terminal-OH, -CO of the polymer₂H、-NH₂or-SH, e.g. terminal-OH or-CO of PLGA₂H. The other functional group (Z) may be, for example, -O-, -OC (═ O) O-, -OC (═ O) NR-, -NRC (═ O) O-, -NRC (═ O) NR' -, -NRs (═ O)₂-、-S-、-S(＝O)-、-S(＝O)₂-, -C (═ O) O-or-C (═ O) NR-and provide further sites for reactions such as ligation or cleavage. The therapeutic peptide may be attached through a carboxylic acid or amine group of the therapeutic peptide, such as a terminal carboxylic acid or amine of the therapeutic peptide, or through a reactive group on a side chain of an amino acid of the therapeutic peptide. In some embodiments, the therapeutic peptide is linked to the terminus of the polymer (e.g., a PLGA polymer, where the linkage is at the hydroxyl terminus or the carboxyl terminus) through a spacer.

In one embodiment, such as shown in fig. 1A, a sulfhydryl-modified therapeutic peptide can be reacted with a pyridyl-SS-activated polymer (e.g., pyridyl-SS-activated PLGA, such as pyridyl-SS-activated 5050PLGA) to form a therapeutic peptide-polymer conjugate that is linked by a disulfide bond. In one embodiment, the sulfhydryl-modified therapeutic peptide can be reacted with a maleimide-activated polymer (e.g., maleimide-activated PLGA, e.g., maleimide-activated 5050PLGA) to form a therapeutic peptide-polymer conjugate that is linked by a maleimide sulfide bond. In one embodiment, the sulfhydryl-modified therapeutic peptide can be reacted with an acrylate-activated polymer (e.g., acrylate-activated PLGA, e.g., acrylate-activated 5050PLGA) to form a therapeutic peptide-polymer conjugate via a mercaptopropionic ester bond. The therapeutic peptide can be attached via a carboxylic acid or amine group of the therapeutic peptide, such as a terminal carboxylic acid or amine of the therapeutic peptide, or by a reactive group on a side chain of an amino acid of the therapeutic peptide. In some embodiments, the therapeutic peptide is linked to the terminus of the polymer (e.g., a PLGA polymer, where the linkage is at the hydroxyl terminus or the carboxyl terminus) through a spacer.

In one embodiment, for example, as shown in fig. 1B, the amine-modified therapeutic peptide can be reacted with a polymer having an activated carboxylic acid or ester (e.g., activated carboxylic PLGA, e.g., activated carboxylic 5050PLGA, e.g., SPA-activated carboxylic 5050PLGA) to form a therapeutic peptide-polymer conjugate linked by an amide bond. In one embodiment, the amine-modified therapeutic peptide can be reacted with an activated polymer (e.g., activated PLGA, e.g., -activated 5050PLGA) to form a therapeutic peptide-polymer conjugate that is linked by a carbamate linkage. In one embodiment, the amine-modified therapeutic peptide can be reacted with an activated polymer (e.g., activated PLGA, e.g., activated 5050PLGA) to form a therapeutic peptide-polymer conjugate linked by a carbonyldiamine linkage (urea). In one embodiment, the amine-modified therapeutic peptide can be reacted with an activated polymer (e.g., activated PLGA, e.g., activated 5050PLGA) to form a therapeutic peptide-polymer conjugate linked by an aminoalkyl sulfonamide linkage. The therapeutic peptide may be attached through a carboxylic acid or amine group of the therapeutic peptide, such as a terminal carboxylic acid or amine of the therapeutic peptide, or through a reactive group on a side chain of an amino acid of the therapeutic peptide. In some embodiments, the therapeutic peptide is linked to the terminus of the polymer through a spacer (e.g., a PLGA polymer, where the linkage is at the hydroxyl terminus or the carboxyl terminus).

In one embodiment, for example, as shown in fig. 1C, a hydroxylamine-modified therapeutic peptide can be reacted with an aldehyde-activated polymer (e.g., aldehyde-activated PLGA, e.g., aldehyde-activated 5050PLGA, e.g., formaldehyde-activated 5050PLGA) to form a therapeutic peptide-polymer conjugate that is linked by an aldoxime bond. The therapeutic peptide may be attached through a carboxylic acid or amine group of the therapeutic peptide, such as a terminal carboxylic acid or amine of the therapeutic peptide, or through a reactive group on a side chain of an amino acid of the therapeutic peptide. In some embodiments, the therapeutic peptide is linked to the terminus of the polymer (e.g., a PLGA polymer, where the linkage is at the hydroxyl terminus or the carboxyl terminus) through a spacer.

In one embodiment, for example, as shown in fig. 1C, an alkyne (alkylyne) modified therapeutic peptide can be reacted with an azide-activated polymer (e.g., azide-activated PLGA, e.g., azide-activated 5050PLGA) to form a therapeutic peptide-polymer conjugate linked by a triazole linkage. The therapeutic peptide may be attached through a carboxylic acid or amine group of the therapeutic peptide, such as a terminal carboxylic acid or amine of the therapeutic peptide, or through a reactive group on a side chain of an amino acid of the therapeutic peptide. In some embodiments, the therapeutic peptide is linked to the terminus of the polymer (e.g., a PLGA polymer, where the linkage is at the hydroxyl terminus or the carboxyl terminus) through a spacer.

In some embodiments, the linker may have one or more of the following functional groups prior to attachment to the reagent and the polymer: amine, amide, hydroxyl, carboxylic acid, ester, halogen, mercapto, maleimide, carbonate or carbamate. In some embodiments, the functional group remains in the linker after the first and second moieties are linked by the linker. In some embodiments, the linker comprises one or more atoms or groups that modulate the reactivity of the functional group (e.g., such that the functional group is cleaved, such as by hydrolysis or reduction under physiological conditions).

In some embodiments, the linker may comprise an amino acid or peptide within the linker. Often, in these embodiments, the peptide linker may be cleaved by hydrolysis under reducing conditions, or by a particular enzyme (e.g., under physiological conditions).

When the linker is the residue of a divalent organic molecule, cleavage of the linker may be within the linker itself, or cleavage may be at one bond coupling the linker to the remainder of the conjugate, e.g., to a therapeutic peptide or polymer.

In some embodiments, the linker may be selected from or may comprise one of the following:

Wherein m is 1-10, n is 1-10, p is 1-10, and R is an amino acid side chain.

The linker may comprise a bond generated by click chemistry (e.g., an amide bond, an ester bond, a ketal, a succinate, or a triazole, and those described in WO 2006/115547). The linker can be cleaved, for example, by hydrolysis, reduction, oxidation, pH shift, photolysis, or a combination thereof; or by enzymatic reaction. The linker may also comprise a bond that may be cleaved under oxidizing or reducing conditions, or may be acid sensitive.

In some embodiments, the linker is not cleaved under physiological conditions, e.g., the linker has a sufficient length that the therapeutic peptide need not be cleaved to be active, e.g., the length of the linker is at least about 20 angstroms (e.g., at least about 30 angstroms or at least about 50 angstroms).

Methods of making therapeutic peptide-polymer conjugates and protein-polymer conjugates

Therapeutic peptide-polymer conjugates and protein-polymer conjugates can be prepared using a variety of methods known in the art, including those described herein. In some embodiments, to covalently attach the agent to the polymer, the polymer or agent may be chemically activated using any technique known in the art. The activated polymer is then mixed with the agent or the activated agent is mixed with the polymer under suitable conditions such that a covalent bond is formed between the polymer and the agent. In some embodiments, a nucleophilic group, such as a sulfhydryl, hydroxyl, or amino group, on the reagent attacks an electrophilic group (e.g., an activated carbonyl group) to create a covalent bond. The agent may be attached to the polymer via a variety of linkages such as amide, ester, succinimide, carbonate, or urethane linkages.

The coupling reaction typically occurs in a solvent system and may comprise a mixture of solvents. Exemplary water-miscible solvents include acetone, DMSO, acetonitrile, DMF, dioxane, and THF. Exemplary water-immiscible solvents include ethyl acetate, benzyl alcohol, chloroform, and dichloromethane. The solvent system may vary based on the length and type of amino acids present in the peptide or protein. In some embodiments, an aqueous buffer solution may be used, for example, with a hydrophilic peptide. In some embodiments, the following solvents are used with minimal or no amounts: acetic acid, acetonitrile, DMF, DMSO, ethanol or isopropanol.

In some embodiments, the agent may be attached to the polymer through a linker. In these embodiments, the linker may be first covalently attached to the polymer, and then to the agent. In other embodiments, the linker may be attached to the agent first, and then to the polymer.

Exemplary therapeutic peptide-polymer conjugates

Many different combinations of the components described herein can be used to make therapeutic peptide-polymer conjugates. For example, various combinations of polymers (e.g., PLGA, PLA, or PGA), linkers to link the therapeutic peptide to the polymer, and therapeutic peptides are described herein.

Exemplary therapeutic peptide-polymer conjugates include the following.

1) PLGA-ester linker-therapeutic peptides

Such conjugates typically comprise a modification of the carbonyl terminus of the peptide with an amino group that can be conjugated to a PLGA polymer. Such linkers have an ester bond to the therapeutic peptide that can be cleaved off at high pH or by enzymes such as esterases. An exemplary scheme is shown below.

2) PLGA-amide linker-therapeutic peptides

Such conjugates typically comprise a modification of the carbonyl end group of PLGA with an amine functional group. The amino group of the PLGA derivative may then react with the carbonyl end group of the therapeutic peptide or the carbonyl group on the side chain of an amino acid such as glutamic acid or aspartic acid to form a stable amide bond. An exemplary scheme is shown below.

3) PLGA-disulfide linker-therapeutic peptides

Such conjugates typically comprise a modification of the carbonyl end group of PLGA with a reactive sulfhydryl (sulfhydryl). Such groups may be reactive with therapeutic peptides containing cysteine groups that may be located at the terminal group or along the chain. It can also react with peptides derived by sulfhydryl groups. Disulfide bonds may be reduced internally to release the peptide. An exemplary scheme is shown below.

4) PLGA-disulfideCompound linker-therapeutic peptides

Such conjugates typically comprise a modification of the hydroxyl group on tyrosine with a disulfide amino group that can be conjugated to PLGA. Following disulfide bond reduction, the linker cyclizes and kicks out (kick out) the polypeptide. Tyrosine or phenolic derived amino acids may be used. Disulfide bonds may be reduced internally to release the therapeutic peptide. An exemplary scheme is shown below.

5) PLGA-thioether linker-therapeutic peptides

Such conjugates typically comprise a modification of the carbonyl end group of PLGA with a maleimide group. This group can be reactive with therapeutic peptides containing cysteines located at the terminal group or along the peptide chain. It can also react with peptides derived by sulfhydryl groups. Such conjugates have a non-releasing thioether bond. An exemplary scheme is shown below.

6) Alkyne-terminated PLGA/azide-functional therapeutic peptides

PLGA polymers terminated with ethynyl (i.e., alkyne) can be conjugated to therapeutic peptides. The terminal amino functional group (e.g., glycine) can be converted to an alkyne (alkyen) moiety via a coupling reaction with 4-pentynoic acid in the presence of N, N' -dicyclohexylcarbodiimide. The reaction can also be accomplished using click chemistry, for example, using a catalyst such as copper bromide to react an azide-terminated polymer (e.g., an azide-terminated PLGA polymer) and an alkyne-functional therapeutic peptide. The 2, 2 '-bipyridine may also be dissolved in N-methylpyrrolidone to complex the copper bromide and the 2, 2' -bipyridine, the product of which may be dialyzed against water (e.g., pure water). The reaction may be performed on a solid support, for example, to prepare an azide-functionalized therapeutic peptide. An exemplary reaction scheme is shown below.

7) Linker formed by Diels-Alder (Diels Alder) chemistry

The PLGA polymer terminates in a moiety that can be used to react a conjugated diene into an olefinic group to form a cyclohexene group to attach the therapeutic peptide to the polymer. An exemplary diels-alder reaction may use Michael's Addition (1, 4 Addition), for example, accomplished in the presence of a base (NaOH or KOH) to form an enolate. The resulting enolate may then be reacted with an α, β -unsaturated ketone. Other exemplary reactions include, for example, epoxy ring opening with amines or hydroxyl groups (nucleophilic substitution-Sn 2 reaction).

8) Linker for use in antibody drug conjugates

Exemplary linkers include acid-labile hydrazone linkers: (6-maleimidocaproyl) hydrazone linker to a cysteine residue (e.g., as used for BR 96-doxorubicin, BMS); and 4- (4' -acetylphenoxy) butanoic acid (e.g., as used in Mylotarg, Pfizer).

Other linkers include enzyme-linked conjugates. Certain advantages of such linkers include improved stability in blood circulation relative to hydrazone linkers. Exemplary enzyme-linked conjugates include valine-citrulline, valine-lysine (Seattle Genetics), and phenylalanine-lysine.

9) Linker synthesized using click chemistry

The acetylene group (e.g., acetylene) -terminated PLGA polymer may be conjugated to the therapeutic peptide having an azide group, or the azide group-terminated PLGA polymer may be conjugated to the therapeutic peptide having an acetylene group. To enable easier release of the therapeutic peptide, a cleavable linker (e.g., an ester or disulfide) may be introduced between the azide or alkyne functionality and the therapeutic peptide.

Acetylene group (alkyne) -terminated PLGA can be reacted with azide-functional therapeutic peptides. Synthesis may include the use of an insoluble matrix, for example, to functionalize the therapeutic peptide. In some embodiments, the terminal amino functional group (e.g., glycine) may be converted to an alkyne moiety via a coupling reaction with 4-pentynoic acid in the presence of N, N' -dicyclohexylcarbodiimide.

Other exemplary coupling reactions using click chemistry include michael addition (1, 4 addition) (e.g., addition of a base (NaOH or KOH) to form an enolate and allow the enolate to react with an α, β -unsaturated ketone); diels-alder reactions (e.g., reaction of a conjugated diene into an olefinic group so as to form a cyclohexene group); and epoxy ring opening with amines or hydroxyl groups (e.g., nucleophilic substitution-Sn 2 reaction).

Compositions of therapeutic peptide-polymer conjugates and protein-polymer conjugates

The composition of the therapeutic peptide/protein-polymer conjugate described above may comprise a mixture of products. For example, conjugation of a therapeutic peptide or protein to a polymer can be performed in less than 100% yield, and thus, a composition comprising a therapeutic peptide/protein-polymer conjugate can also comprise an unconjugated polymer.

The composition of the therapeutic peptide/protein-polymer conjugate may also comprise therapeutic peptide/protein-polymer conjugates having the same polymer and the same agent, and differing in the nature of the bond between the agent and the polymer. The therapeutic peptide/protein-polymer conjugate can be present in the composition in varying amounts. For example, when a therapeutic peptide or protein having multiple available attachment points is reacted with a polymer, the resulting composition may comprise more products conjugated via carboxyl groups having greater reactivity, and fewer products attached via carboxyl groups having lower reactivity.

In addition, the therapeutic peptide/protein-polymer conjugate compositions may comprise a therapeutic peptide or protein linked to more than one polymer chain.

Surface active agent

In some embodiments, the particles described herein comprise a surfactant. Exemplary surfactants include PEG, poly (vinyl alcohol) (PVA), poly (vinyl pyrrolidone) (PVP), poloxamers, polysorbates, polyoxyethylene esters, PEG-lipids (e.g., PEG-ceramide, d- α -tocopheryl polyethylene glycol 1000 succinate), 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine), or lecithin. In some embodiments, the surfactant is PVA, and the PVA is from about 3kDa to about 50kDa (e.g., from about 5kDa to about 45kDa, from about 7kDa to about 42kDa, from about 9kDa to about 30kDa, or from about 11 to about 28kDa) and up to about 98% hydrolyzed (e.g., from about 75% -95%, from about 80-90% hydrolyzed, or about 85% hydrolyzed). In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant isHS15(BASF, Florham Park, NJ). In some embodiments, the surfactant is present in an amount up to about 35% by weight of the system (e.g., up to about 20% or up to about 25%, about 15% to about 35%, about 20% to about 30%, or about 23% to about 26%).

Counter ion

The particles described herein can also comprise one or more counter ions, for example, charged, cationic, anionic, or zwitterionic moieties. The counter ion can neutralize the charge associated with the therapeutic peptide or protein, allowing for improved formulation (e.g., improved stability, solubility, or transport). In some embodiments, the charged moiety is associated with the therapeutic peptide or protein (e.g., hydrogen bonded to the therapeutic peptide or protein, or a portion of the solvated layer surrounding the therapeutic peptide or protein). In some embodiments, the charged moiety is covalently attached to the polymer of the particle described herein. In some embodiments, the charged moiety is covalently attached to a polymer that is covalently attached to the therapeutic peptide or protein. In some embodiments, the charged moiety is a peptide.

In some embodiments, the charged moiety is covalently attached to the hydrophobic polymer via a linker (e.g., at the carboxy terminus or the hydroxy terminus of the hydrophobic polymer). In some embodiments, the linker comprises a bond formed using "click chemistry" (e.g., as described in WO 2006/115547). In some embodiments, the linker comprises an amide linkage, an ester linkage, a disulfide linkage, a sulfide linkage, a ketal, a succinate, or a triazole. In some embodiments, a single charged moiety is covalently attached to a single hydrophobic polymer (e.g., at a terminus of the hydrophobic polymer). In some embodiments, the charged moiety is covalently linked to the hydrophilic-hydrophobic polymer through an amide, ester, or ether linkage through the hydrophobic moiety. In some embodiments, a single hydrophobic polymer is covalently attached to a plurality of charged moieties. In some embodiments, at least a portion of the plurality of charged moieties is attached to the backbone of at least a portion of the hydrophobic polymer.

In some embodiments, the cationic moiety is a cationic polymer (e.g., PEI, cationic PVA, poly (histidine), poly (lysine), or poly (2-dimethyl (dimethylamino) amino) ethyl methacrylate). In some embodiments, the cationic moiety is an amine (e.g., a primary, secondary, tertiary, or quaternary amine). In some embodiments, at least a portion of the cationic portion comprises a plurality of amines (e.g., primary, secondary, tertiary, or quaternary amines). In some embodiments, at least one amine in the cationic portion is a secondary or tertiary amine. In some embodiments, at least a portion of the cationic moiety comprises a polymer, for example, polyethyleneimine or polylysine. The polymeric cationic moiety has a variety of molecular weights (e.g., in the range of about 500 to about 5000Da, e.g., about 1 to about 2kDa or about 2.5 kDa).

In some embodiments, the cationic moiety is, for example, a polymer having one or more secondary or tertiary amines, such as cationic PVA (e.g., as provided by Kuraray, such as CM-318 or C-506), chitosan, and polyvinylamine. Cationic PVA may be manufactured, for example, by polymerizing a vinyl acetate/N-vinylformamide (N-vinylformamide) copolymer, for example, as described in US2002/0189774, the contents of which are incorporated herein by reference. Other examples of cationic PVAs include those described in US6, 368,456 and Fatehi (Carbohydrate Polymers79(2010)423-428, the contents of which are incorporated herein by reference.) in some embodiments, at least a portion of the cationic moiety comprises a cationic PVA (e.g., as provided by Kuraray, such as CM-318 or C-506).

Other exemplary cationic moieties include poly (histidine) and poly (2-dimethyl (dimethylamino) amino) ethyl methacrylate). In some embodiments, the amine is positively charged at acidic pH. In some embodiments, the amine is positively charged at physiological pH. In some embodiments, at least a portion of the cationic moiety is selected from the group consisting of: protamine sulfate, hexyldimethylamine bromide, hexadecyltrimethylammonium bromide, spermine, and spermidine. In some embodiments, at least a portion of the cationic moiety is selected from the group consisting of: tetraalkylammonium moieties, trialkylammonium moieties, imidazolium moieties, arylammonium moieties, iminium moieties, amidinium moieties, guanidinium moieties, thiazolium moieties, pyrazolium moieties, pyrazinium moieties, pyridinium moieties, and phosphonium moieties. In some embodiments, at least a portion of the cationic moiety is a cationic lipid. In some embodiments, at least a portion of the cationic moiety is conjugated to a non-polymeric hydrophobic moiety (e.g., cholesterol or vitamin E TPGS). In some embodiments, the plurality of cationic moieties comprises from about 1% to about 60% by weight of the particle. In some embodiments, the ratio of the charge of the plurality of cationic moieties to the charge of the plurality of therapeutic peptides is from about 1: 1 to about 50: 1 (e.g., 1: 1 to about 10: 1 or 1: 1 to 5: 1).

Exemplary cationic moieties for the particles and conjugates described herein include amines, such as polyamines (e.g., Polyethyleneimine (PEI) or derivatives thereof, such as polyethyleneimine-polyethylene glycol-N-acetyl galactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethylene glycol-tri-N-acetyl galactosamine (PEI-PEG-tri-GAL) derivatives), cationic lipids (e.g., DOTIM, dimethyl dioctadecyl ammonium bromide, 1, 2 dioleyloxypropyl-3-trimethylammonium bromide, DOTAP, 1, 2-dimyristoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, edmp, ethyl-PC, DODAP, DC-cholesterol and MBOP, CLinDMA, pCLinDMA, eCLinDMA, DMOBA, and DMLBA), polyamino acids (e.g., poly (lysine), poly (histidine), and poly (arginine)), and polyvinylpyrrolidone (PVP). The cationic moiety may be positively charged at physiological pH.

Other exemplary cationic moieties include protamine sulfate, hexyldimethylamine bromide, cetyltrimethylammonium bromide, spermine, spermidine, and those described in, for example, WO2005007854, US7,641, 915, and WO2009055445, the contents of each of which are incorporated herein by reference. The cationic moiety may include N-methyl D-reduced glucamine, choline, arginine, lysine, procaine, Tromethamine (TRIS), spermine, N-methyl-morpholine, glucosamine, N-bis (2-hydroxyethyl) glycine, diazabicycloundecene, creatine, arginine ethyl ester, amantadine, rimantadine, ornithine, taurine, and citrulline. The cationic moiety may additionally include sodium, potassium, calcium, magnesium, ammonium, monoethanolamine, diethanolamine, triethanolamine, tromethamine, lysine, histidine, arginine, morpholine, methyl reduced glucamine, and glucosamine.

Anionic moieties that may be suitable for formulation with a therapeutic peptide or protein having a net positive charge include, but are not limited to, acetate, propionate, butyrate, valerate, hexanoate, heptanoate, levulinate, chloride, bromide, iodide, citrate, succinate, maleate, glycolate, glucuronate, 3-hydroxyisobutyrate, 2-hydroxyisobutyrate, lactate, malate, pyruvate, fumarate, tartrate, tartronate, nitrate, phosphate, benzenesulfonate, methanesulfonate, sulfate, sulfonate, acetate, adamantanecarboxylic acid, alpha ketoglutarate, D-or L-aspartic acid, benzenesulfonic acid, benzoic acid, 10-camphorsulfonic acid, citric acid, 1, 2-ethanedisulfonic acid, fumaric acid, D-gluconic acid, D-glucuronic acid, glucaric acid, D-or L-glutamic acid, glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, 1-hydroxy-2-naphthoic acid, lactobionic acid, maleic acid, L-malic acid, mandelic acid, methanesulfonic acid, mucic acid, 1, 5-naphthalenedisulfonic acid tetrahydrate, 2-naphthalenesulfonic acid, nitric acid, oleic acid, pamoic acid, phosphoric acid, p-toluenesulfonic acid hydrate, D-saccharic acid monopotassium salt, salicylic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, D-or L-tartaric acid.

In some embodiments, pharmaceutically acceptable salts are formed by including a counterion (e.g., a charged moiety described herein) in a particle or conjugate described herein.

Storage method

The therapeutic peptide/protein-polymer conjugates, particles, or compositions described herein can be stored in the container for at least about 1 hour (e.g., at least about 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 2 days, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, or 3 years). Thus, described herein are containers comprising the therapeutic peptide/protein-polymer conjugates, particles, or compositions described herein.

The therapeutic peptide/protein-polymer conjugate, particle, or composition can be stored under a variety of conditions, including environmental conditions. The therapeutic peptide/protein-polymer conjugate, particle, or composition can also be stored at low temperatures, e.g., a temperature of less than or equal to about 5 ℃ (e.g., less than or equal to about 4 ℃ or less than or equal to about 0 ℃). The polymer-agent conjugate, particle, or composition can also be frozen and stored at a temperature of less than about 0 ℃ (e.g., -80 ℃ and-20 ℃). The polymer-reagent conjugate, particle, or composition can also be stored under an inert atmosphere, for example, an atmosphere containing an inert gas such as nitrogen or argon. Such an atmosphere may be substantially free of atmospheric oxygen and/or other reactive gases, and/or substantially free of moisture.

The therapeutic peptide/protein-polymer conjugates, particles, or compositions described herein can be stored in a variety of containers, including light-blocking containers such as amber vials. The container may be a vial, for example, a sealed vial with a rubber or silicone enclosure (e.g., an enclosure made from polybutadiene or polyisoprene). The container may be substantially free of atmospheric oxygen and/or other reactive gases, and/or substantially free of moisture.

Method for evaluating particles

The particles described herein can be subjected to a number of analytical methods. For example, the particles described herein can be subjected to measurements to determine the presence or absence of impurities or residual solvents (e.g., via Gas Chromatography (GC)), to determine the relative amounts of one or more components (e.g., via High Performance Liquid Chromatography (HPLC)), to measure particle size (e.g., via dynamic light scattering and/or scanning electron microscopy), or to determine the presence or absence of surface components.

In some embodiments, the particles described herein can be evaluated using dynamic light scattering. The particles may be irradiated with laser light and the intensity of the scattered light fluctuates at a rate dependent on the particle size as smaller particles are further "kicked" out by the solvent molecules and move more rapidly. Analysis of these intensity fluctuations produces the velocity of brownian motion and thus uses the Stokes-Einstein relationship to produce particle size. The diameter measured in dynamic light scattering is called the hydrodynamic diameter and refers to how the particles diffuse in the liquid. The diameter obtained by this technique is the diameter of a sphere with the same translational diffusion coefficient as the measured particle.

In some embodiments, the particles described herein can be evaluated using low temperature scanning electron microscopy (Cryo-SEM). SEM is a type of electron microscopy in which the surface of a sample is imaged by scanning the sample in a raster scan pattern using a high energy electron beam. The interaction of the electrons with the atoms that make up the sample produces a signal containing information about the surface topology, composition, and other characteristics of the sample, such as electrical conductivity. For Cryo-SEM, the SEM was equipped with a cold stage for cryomicroscopy. Cryofixation may be used and cryoscanning electron microscopy may be performed on cryofixed specimens. Cryo-fixed specimens can be cryo-fractured under vacuum in special equipment to reveal internal structures, sputter coated, and transferred to a SEM cryo-stage while still frozen.

In some embodiments, the particles described herein can be evaluated using Transmission Electron Microscopy (TEM). In this technique, an electron beam is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through. Forming an image from the interaction of electrons projected through the specimen; the image is magnified and focused onto an imaging device, such as a phosphor screen, on the film layer or detected by a sensor, such as a Charge Coupled Device (CCD) camera.

Pharmaceutical composition

Provided herein are compositions, e.g., pharmaceutical compositions, comprising a plurality of particles described herein and a pharmaceutically acceptable carrier or adjuvant.

In some embodiments, the pharmaceutical composition can include a pharmaceutically acceptable salt of a compound described herein (e.g., a therapeutic peptide-polymer conjugate). Pharmaceutically acceptable salts of the compounds described herein include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, pamoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, and undecanoate. Salts derived from suitable bases include alkali metal (e.g., sodium) salts, alkaline earth metal (e.g., magnesium) salts, ammonium salts, and N- (alkyl) 4+ salts. The present invention also contemplates the quaternization of any basic nitrogen-containing group in the compounds described herein. By this quaternization, water-or oil-soluble products can be obtained.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxybenzyl ether (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The composition may comprise a liquid for suspending the polymer-agent conjugate, particle or composition, which may be any liquid solution compatible with the polymer-agent conjugate, particle or composition, and is also suitable for use in pharmaceutical compositions, such as pharmaceutically acceptable non-toxic liquids. Suitable suspending liquids include, but are not limited to, suspending liquids selected from the group consisting of: water, aqueous sucrose syrup, corn syrup, sorbitol, polyethylene glycol, propylene glycol, D5W, and mixtures thereof.

The compositions described herein may also include another component, such as an antioxidant, an antibacterial agent, a buffer, a compatibilizer, a chelating agent, an inert gas, a tonicity agent, and/or a viscosity agent.

In one embodiment, the polymer-agent conjugate, particle, or composition is provided in a lyophilized form and reconstituted prior to administration to a subject. The lyophilized polymer-reagent conjugates, particles or compositions may be administered by a dilute solution, such as a salt or saline solution, for example, sodium chloride solution at a pH between 6 and 9, Lactated Ringer's injection solution or commercially available diluent, such as PLASMA-LYTE A injection at pH(Baxter, Deerfield, IL) for reconstitution.

In one embodiment, the lyophilized formulation includes a lyoprotectant or stabilizer to maintain physical and chemical stability by protecting the particles and active agent from the crystal formation and fusion processes during lyophilization. The lyoprotectant or stabilizer may be one or more of the following: polyethylene glycol (PEG), PEG lipid conjugates (e.g., PEG-brain amide or D- α -tocopheryl polyethylene glycol 1000 succinate), poly (vinyl alcohol) (PVA), poly (vinyl pyrrolidone) (PVP), polyoxyethylene esters, poloxamers, polysorbates, polyoxyethylene esters, lecithin, sugars, oligosaccharides, polysaccharides, carbohydrates, cyclodextrins (e.g., 2-hydroxypropyl- β -cyclodextrin) and polyols (e.g., trehalose, mannitol, sorbitol, lactose, sucrose, glucose, and dextran), salts, and crown ethers.

In some embodiments, the lyophilized polymer-reagent conjugate, particle, or composition is reconstituted using: water, 5% dextrose injection, lactated ringer's and dextrose injection, or equal parts by volume of a mixture of anhydrous ethanol, USP and a non-ionic surfactant, such as the polyoxyethylene castor oil surfactant available from GAF Corporation, Mount Olive, n.j under the trade mark cremophor el. The lyophilized product and vehicle for reconstitution can be packaged separately in suitably light-shielded vials. To minimize the amount of surfactant in the reconstituted solution, only sufficient vehicle may be provided to form a solution of polymer-reagent conjugates, particles, or compositions. Once dissolution of the drug is achieved, the resulting solution is further diluted with a suitable parenteral diluent and subsequently injected. Such diluents are well known to those of ordinary skill in the art. These diluents are generally available in clinical settings. However, within the scope of the present invention, a third vial containing sufficient parenteral diluent is used to package the subject polymer-agent conjugate, particle, or composition to prepare the final concentration for administration. A typical diluent is lactated ringer's injection.

Final dilution of reconstituted polymer-reagent conjugates, particles or compositions can be performed with other formulations having similar utility, such as 5% dextrose injection, lactated ringer's and dextrose injection, sterile water for injection, and the like. However, due to the narrow pH range: pH6.0 to 7.5, lactated ringer's injection is most typical. Every 100mL of lactated ringer's injection contains sodium chloride USP0.6g, sodium lactate 0.31g, potassium chloride USP0.03g and calcium chloride 2H2O USP0.02g. The osmolality was 275mOsmol/L, which is very close to isotonicity.

The compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. The amount of active agent that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form varies depending on the host treated, the particular mode of administration. The amount of active agent that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form is generally that amount of the composition which produces a therapeutic effect.

Route of administration

The pharmaceutical compositions described herein can be administered orally, parenterally (e.g., via intravenous, subcutaneous, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intraocular, or intracranial injection), topically, mucosally (e.g., rectal or vaginal), nasally, buccally, ocularly, via an inhalation spray (e.g., delivered by atomization, propellant, or dry powder device), or via an implantable drug reservoir.

Pharmaceutical compositions suitable for parenteral administration comprise a combination of one or more polymer-agent conjugates, particles or compositions with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters, such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the action of microorganisms can be ensured by the incorporation of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

In some cases, to prolong the effect of the drug, it is desirable to slow the absorption of the agent following subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility. The rate of absorption of the polymer-agent conjugate, particle, or composition then depends on its rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered pharmaceutical form is achieved by dissolving or suspending the polymer-agent conjugate, particle or composition in an oily vehicle.

Pharmaceutical compositions suitable for oral administration may be in the form of: capsules, cachets, pills, tablets, chewing gum, lozenges (using a flavoring base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base such as gelatin and glycerin, or sucrose and acacia) and/or as mouthwash and the like, each containing a predetermined amount of the agent as an active ingredient. The compounds may also be administered in the form of a bolus, electuary or paste.

Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (for example, gelatin or hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrating agents (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agents. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.

Tablets and other solid dosage forms, such as dragees, capsules, pills, and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may also be formulated to provide sustained or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose, other polymer matrices, liposomes and/or microspheres in varying ratios to provide the desired release profile. They may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents, in the form of sterile solid compositions that can be dissolved in sterile water or some other sterile injectable medium just prior to use. These compositions may also optionally contain opacifying agents and may be such that: they release one or more active ingredients only or preferentially in certain parts of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the polymer-agent conjugates, particles, or compositions, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

In addition to the polymer-agent conjugates, particles, or compositions, the suspensions may contain suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

Pharmaceutical compositions suitable for topical administration are useful where the desired treatment involves the topical application of an easily accessible area or organ. For topical application to the skin, the pharmaceutical compositions should be formulated in a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for topical administration of the particles described herein include, but are not limited to: mineral oil, liquid paraffin, white paraffin, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active particles suspended or dissolved in a carrier using suitable emulsifiers. Suitable vectors include, but are not limited to: mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions described herein may also be administered topically to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topical transdermal patches are also included herein.

The pharmaceutical compositions described herein may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art, and may be prepared as solutions in saline.

The pharmaceutical compositions described herein may also be administered in the form of suppositories for rectal or vaginal administration. Suppositories can be prepared by mixing one or more of the polymer-agent conjugates, particles or compositions described herein with one or more suitable non-irritating excipients that are solid at room temperature but liquid at body temperature. The composition will thus melt within the rectal or vaginal cavity and release the polymer-agent conjugate, particle or composition. Such materials include, for example, cocoa butter, polyethylene glycol, suppository waxes or salicylates. Compositions of the invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Ophthalmic formulations, ophthalmic ointments, powders, solutions, and the like are also encompassed within the scope of the present invention. Ocular tissue (e.g., the deep cortex, supranuclear, or aqueous humor regions of the eye) can be contacted with an ophthalmic formulation that is allowed to distribute into the lens. Any suitable method of administering or applying the ophthalmic formulations of the present invention may be employed (e.g., topically, by injection, parenterally, by air-transmission, etc.). For example, the contacting can be via topical administration or via injection.

Dosage and dosing regimen

The therapeutic peptide/protein-polymer conjugate, particle, or composition may be formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the therapeutic peptide that is effective to achieve the desired therapeutic response, without being toxic to the subject, for the particular subject, composition, and mode of administration.

In one embodiment, the therapeutic peptide/protein-polymer conjugate, particle, or composition is from about 0.1 to 300mg/m, for example²About 5 to 275mg/m²About 10 to 250mg/m²E.g., about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290mg/m²Is administered to the subject. Administration may be at regular intervals, such as every 1, 2, 3, 4 or 5 days, or weekly, or every 2, 3, 4, 5, 6 or 7 or 8 weeks. Administration may be over a period of about 10 minutes to about 6 hours, such as about 30 minutes to about 2 hours, about 45 minutes to 90 minutes, such as about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or more. In one embodiment, the therapeutic peptide-polymer conjugate, particle, or composition is administered in a bolus injection (bolus infusion) or intravenous bolus injection, e.g., over a period of 15 minutes, 10 minutes, 5 minutes, or less. In one embodiment, a therapeutic peptide-polymer conjugate, particle or composition Such that the desired dosage amount of agent is administered. Preferably, the dosage of the therapeutic peptide/protein-polymer conjugate, particle or composition is a dosage described herein.

In one embodiment, the subject receives 1, 2, 3, to 10, to 12, to 15 treatments or more, or until the disorder or symptoms of the disorder are cured, restored, alleviated, reduced, altered, corrected, improved, alleviated, improved, or affected. For example, the subject receives an infusion every 1, 2, 3, or 4 weeks until the disorder or symptoms of the disorder are cured, restored, alleviated, reduced, altered, corrected, improved, alleviated, improved, or affected. Preferably, the dosing regimen is a dosing regimen as described herein.

The therapeutic peptide/protein-polymer, particle, or composition can be administered as a first line therapy, e.g., alone or in combination with one or more other agents. In other embodiments, the therapeutic peptide/protein-polymer conjugate, particle, or composition is administered after the subject develops resistance to first-line therapy, fails to respond to first-line therapy, or has relapsed after first-line therapy. The therapeutic peptide/protein-polymer conjugate, particle, or composition may be administered in combination with a second agent. Preferably, the therapeutic peptide/protein-polymer conjugate, particle or composition is administered in combination with a second agent described herein. The second agent may be the same as or different from the agent in the particle.

Reagent kit

The therapeutic peptide/protein-polymer conjugates, particles, or compositions described herein can be provided in a kit. The kit comprises a therapeutic peptide/protein-polymer conjugate, particle, or composition described herein and optionally a container, a pharmaceutically acceptable carrier, and/or informational material. The informational material may be descriptive, instructive, marketable, or other material that relates to the methods described herein and/or the use of the particles for the methods described herein.

The information material of the kit is not limited in its form. In one embodiment, the informational material may include information regarding: production of a therapeutic peptide/protein-polymer conjugate, particle, or composition, physical characteristics of a therapeutic peptide/protein-polymer conjugate, particle, or composition, concentration, expiration date, lot or production site information, and the like. In one embodiment, the information material relates to a method of administering a therapeutic peptide/protein-polymer conjugate, particle, or composition.

In one embodiment, the informational material may include instructions to administer a therapeutic peptide/protein-polymer conjugate, particle, or composition described herein in a suitable manner, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein) in order to perform a method described herein. In another embodiment, the informational material can include instructions to administer a therapeutic peptide/protein-polymer conjugate, particle, or composition described herein to a suitable subject, e.g., a human having or at risk of having a disorder described herein. In another embodiment, the informational material may include instructions to reconstitute a therapeutic peptide/protein-polymer conjugate or particle described herein into a pharmaceutically acceptable composition.

In one embodiment, the kit comprises instructions for using the therapeutic peptide/protein-polymer conjugate, particle, or composition, for e.g., treating a subject. The instructions may comprise methods of reconstituting or diluting the therapeutic peptide-polymer conjugate, particle, or composition for use by a particular subject or in combination with a particular chemotherapeutic agent. The instructions may also include methods of reconstituting or diluting the therapeutic peptide/protein-polymer composition for use in a particular mode of administration, such as intravenous infusion.

In another embodiment, the kit includes instructions for treating a subject having a particular indication, such as a particular cancer.

The information material of the kit is not limited in its form. In many cases, informational material (e.g., instructions) is provided in printed matter, e.g., printed text, pictures, and/or photographs, such as labels or printed sheets. However, the informational material may also be provided in other formats, such as braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is contact information, e.g., a physical address, an email address, a website, or a phone number, where a user of the kit can obtain substantial information about the particles described herein and/or their use in the methods described herein. The informational material may also be provided in any combination of formats.

In addition to the therapeutic peptide/protein-polymer conjugates, particles, or compositions described herein, the compositions of the kits can comprise other ingredients, such as surfactants, lyoprotectants or stabilizers, antioxidants, antibacterial agents, bulking agents, chelating agents, inert gases, tonicity and/or viscosity agents, solvents or buffers, stabilizers, preservatives, flavoring agents (e.g., bitter antagonists or sweeteners), fragrances, dyes, or colorants, e.g., to color or stain one or more components of the kit, or other cosmetic ingredients, pharmaceutically acceptable carriers, and/or second agents to treat the conditions or disorders described herein. Alternatively, the other ingredients may be contained in the kit, but in a different composition or container than the particles described herein. In such embodiments, the kit can comprise instructions for mixing the polymer-reagent conjugates, particles, or compositions described herein with other ingredients, or for using the therapeutic peptide/protein-polymer conjugates, particles, or compositions described herein with other ingredients.

In another embodiment, the kit includes a second therapeutic agent, such as a second chemotherapeutic agent. In one embodiment, the second reagent is in lyophilized or liquid form. In one embodiment, the therapeutic peptide/protein-polymer conjugate, particle, or composition and the second therapeutic agent are in separate containers, and in another embodiment, the therapeutic peptide/protein-polymer conjugate, particle, or composition and the second therapeutic agent are packaged in the same container.

In some embodiments, the components of the kit are stored in sealed vials, e.g., with rubber or silicone inclusions (e.g., polybutadiene or polyisoprene inclusions). In some embodiments, the components of the kit are stored under inert conditions (e.g., under nitrogen or another inert gas such as argon). In some embodiments, the components of the kit are stored under anhydrous conditions (e.g., with a desiccant). In some embodiments, the components of the kit are stored in a light-blocking container, such as an amber vial.

The therapeutic peptide/protein-polymer conjugates, particles, or compositions described herein can be provided in any form, such as a liquid, frozen, dried, or lyophilized form. Preferably, the polymer-agent conjugates, particles, or compositions described herein are substantially pure and/or sterile. In one embodiment, the therapeutic peptide/protein-polymer conjugate, particle, or composition is sterile. When the therapeutic peptide/protein-polymer conjugates, particles, or compositions described herein are provided in the form of a liquid solution, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being preferred. In one embodiment, the therapeutic peptide/protein-polymer conjugate, particle, or composition is provided in lyophilized form and optionally a diluent solution is provided to reconstitute the lyophilizate. Diluents may include, for example, saline or saline solutions, e.g., sodium chloride solution having a pH between 6 and 9, ringer's lactate injection, D5W or PLASMA-LYTEA injection (Baxter，Deerfield，IL)。

The kit may include one or more containers for a composition containing a therapeutic peptide/protein-polymer conjugate, particle, or composition described herein. In some embodiments, the kit contains separate containers, dispensers or compartments for the composition and informational material. For example, the composition may be contained in a bottle, vial, IV blending bag, IV infuser, carrier (piggyback set), or syringe, and the informational material may be contained in a plastic sleeve (sleeve) or packaging (packet). In other embodiments, the individual elements of the kit are contained in a single, non-separate container. For example, the composition is contained in a bottle, vial or syringe having the informational material in the form of a label attached thereto. In some embodiments, a kit comprises a plurality (e.g., a set) of individual containers, each container containing one or more unit dosage forms (e.g., dosage forms described herein) of a polymer-agent conjugate, particle, or composition described herein. For example, a kit comprises a plurality of syringes, ampoules, foil packets, or blister packets, each containing a single unit dose of the particles described herein. The container of the kit may be air-impermeable, water-resistant (e.g., impermeable to changes in moisture or evaporation), and/or light-impermeable.

The kit optionally includes a device suitable for administering the composition, such as a syringe, inhaler, pipette, forceps, measuring spoon, dropper (e.g., eye dropper), swab (e.g., cotton or wood swab), or any such delivery device. In one embodiment, the device is a medical implant device, e.g., packaged for surgical insertion.

Methods of using particles and compositions

The polymer-agent conjugates, particles, and compositions described herein can be administered, e.g., in vitro or ex vivo, to cultured cells, or, e.g., in vivo, to a subject, in order to treat or prevent a variety of disorders, including those described herein below. The polymer-agent conjugates, particles, and compositions can be used as part of a first-line, second-line, or adjuvant therapy, and can also be used alone or in combination with one or more other treatment regimens.

Having thus described aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Examples

Example 1.5050 purification and characterization of PLGA

Step A: 5050PLGA (300g, Mw: 7.8 kDa; Mn: 2.7kDa) and acetone (900mL) were charged to a 3-L round bottom flask equipped with a mechanical stirrer. The mixture was stirred at ambient temperature for 1 hour to form a clear yellowish solution.

And B: MTBE (9.0L, 30 volumes for 5050PLGA mass) was charged to a 22-L jacketed reactor equipped with a mechanical stirrer with a bottom discharge valve. Will be provided with(795g) Added to the solution and stirred overhead at about 200rpm to produce a suspension. The solution from step a was slowly added to this suspension over 1 hour. After addition of the polymer solution, the mixture was stirred for another hour and filtered through a polypropylene filter. The filter cake was washed with MTBE (3X 300mL), conditioned for 0.5 hour, and air dried at ambient temperature (typically 12 hours) until residual MTBE ≦ 5 wt% (e.g., from ¹H NMR analysis determined).

And C: acetone (2.1L, 7 volumes for the mass of 5050 PLGA) was charged to a 12-L jacketed reactor equipped with a mechanical stirrer with a bottom discharge valve. The polymer from step B is-The complex was charged to a reactor where overhead stirring was performed at about 200rpm to produce a suspension. The suspension was stirred at ambient temperature for a further 1 hour and filtered through a polypropylene filter. The filter cake was washed with acetone (3 × 300mL) and the combined filtrates were purified through a 0.45mM in-line filter to yield a clear solution. This solution was concentrated to about 1000 mL.

Step D: water (9.0L, 30 volumes) was charged to a 22-L jacketed reactor equipped with a mechanical stirrer with a bottom discharge valve and cooled to 0 ℃ to 5 ℃ using a chiller. The solution from step C was slowly added over 2 hours with overhead stirring at about 200 rpm. After the addition of the solution, the mixture was stirred for another hour and filtered through a polypropylene filter. The filter cake was conditioned for 1 hour, air dried at ambient temperature for 1 day, then vacuum dried for 3 days to yield purified 5050PLGA [258g, 86% yield as a white powder ]。¹H NMR analysis was consistent with the expected product range and Karl Fisher analysis showed 0.52 wt% water. The product was analyzed by HPLC (AUC, 230nm) and GPC (AUC, 230 nm). The process results in a narrower polymer polydispersity, i.e., Mw: 8.8kDa and Mn: 5.8 kDa.

Example 2.5050 purification and characterization of PLGA lauryl ester

MTBE (4L) and heptane (0.8L) were charged to a 12-L round bottom flask equipped with a mechanical stirrer. The mixture was stirred at about 300rpm and a solution of 5050PLGA lauryl ester (65g) in acetone (300mL) was added dropwise to the mixture. Over time, a gummy solid formed and eventually caked on the bottom of the flask. The supernatant was slowly decanted and the solid was dried under vacuum at 25 ℃ for 24 hours to provide 40g of purified 5050PLGA lauryl ester as a white powder [ yield: 61.5 percent]。¹H NMR(CDCl₃，300MHz)：δ5.25-5.16(m，53H)，4.86-4.68(m，93H)，4.18(m，7H)，1.69-1.50(m，179H)，1.26(bs，37H)，0.88(t，J＝6.9Hz，6H)。¹H NMR analysis was consistent with the expected product range. GPC (AUC, 230 nm): 6.02-9.9min, t_R＝7.91min。

Example 3.7525 purification and characterization of PLGA

12L of MTBE was charged to a 22-L round bottom flask equipped with a mechanical stirrer, to which was added a solution of 7525PLGA (150g, about 6.6kDa) in dichloromethane (DCM, 750mL) dropwise over one hour with stirring at about 300rpm, forming a gummy solid. The supernatant was slowly decanted and the gummy solid was dissolved in DCM (3L). The solution was transferred to a round bottom flask and concentrated to a residue which was dried under vacuum at 25 ℃ for 40 hours to provide 94g of purified 7525PLGA as a white foam [ yield: 62.7 percent ]。¹H NMR(CDCl₃，300MHz)：δ5.24-5.15(m，68H)，4.91-4.68(m，56H)，3.22(s，2.3H，MTBE)，1.60-1.55(m，206H)，1.19(s，6.6H，MTBE)。¹HNMR analysis was consistent with the expected product range. GPC (AUC, 230 nm): 6.02-9.9min, t_R＝7.37min。

Example 4 Synthesis, purification and characterization of O-acetyl-5050-PLGA

The purified 5050PLGA [220g, Mn 5700%]And DCM (660mL) was charged to a 2000-mL round bottom flask equipped with an overhead stirrer. The mixture was stirred for 10 minutes to form a clear solution. Ac is added₂O (11.0mL, 116mmol) and pyridine (9.4mL, 116mmol) were added to the solution, resulting in a minimum temperature rise of about 0.5 ℃. The reaction was stirred at ambient temperature for 3 hours and concentrated to about 600 mL. The solution was added to the top of the flask over 1 hour with overhead stirring at about 200rpm(660g) Was added to the suspension of MTBE (6.6L, 30 vol). The suspension was filtered through a polypropylene filter and the filter cake was air dried at ambient temperature for 1 day. The suspension is stirred in the topAcetone (1.6L, about 8 vol) was suspended for 1 hour. The slurry was filtered through a glass fritted funnel (coarse) and the filter cake was washed with acetone (3 × 300 mL). By passingThe pad purges the combined filtrate to provide a clear solution. The solution was concentrated to about 700mL over 2 hours with 200rpm overhead stirring and added to cold water (7.0L, 0 ℃ to 5 ℃). The suspension was filtered through a polypropylene filter. The filter cake was washed with water (3X 500mL) and adjusted for 1 hour to provide 543g of wet cake. The wet cake was transferred to two glass trays and air dried overnight at ambient temperature to provide 338g of wet product which was then vacuum dried at 25 ℃ for 2 days to constant weight to provide 201g of product as a white powder [ yield: 91 percent ]。¹H NMR analysis was consistent with the expected product range. The product was analyzed by HPLC (AUC, 230nm) and GPC (Mw: 9.0kDa and Mn: 6.3 kDa).

Example 5 Synthesis, purification and characterization of folate-PEG-PLGA-lauryl ester.

Synthesis of folate-PEG-PLGA-lauryl ester involves direct coupling of folate to PEG diamine (Sigma-Aldrich, n 75, MW3350 Da). PEG diamine was purified due to the possibility of small molecular weight amines being present in the product. 4.9g of PEG diamine was dissolved in DCM (25mL, 5 volumes) and then transferred to MTBE (250mL, 50 volumes) with vigorous stirring. The polymer precipitated as a white powder. The mixture was then filtered and the solid dried under vacuum to give 4.5g of product [ 92%]. Of solids¹H NMR analysis gave a clear spectrum; however, not all alcohol groups are converted to amines (63% diamine, 37% monoamine) based on the integration of a-methylene to amine groups.

Synthesis of folate- (. gamma.) CO-NH-PEG-NH Using purified PEG diamine₂. Folic acid (100mg, 1.0 equiv) was dissolved in hot DMSO (4.5mL, 3 volumes for PEG diamine). The solution was cooled to ambient temperature and (2- (7-aza-1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium hexafluorophosphate) (HATU, 104mg, 1.2 equiv.) and N were added, N-diisopropylethylamine (DIEA, 80. mu.L, 2.0 equiv.). The resulting yellow solution was stirred for 30 minutes and added with PEG diamine (1.5g, 2 equivalents) in DMSO (3mL, 2 volumes). An excess of PEG diamine is used to avoid the possible formation of bis-adducts of PEG diamine and to improve the conversion of folic acid. The reaction was stirred at 20 ℃ for 16 hours and passedThe reaction was directly purified using a C18 chromatography column (RediSep, 43g, C18). The product containing fractions were combined and CH was removed under vacuum₃And (C) CN. The remaining aqueous solution (about 200mL) was extracted with chloroform (200 mL. times.2). The combined chloroform phases were concentrated to about 10mL and transferred to MTBE to precipitate the product as a yellow powder. To completely remove any unreacted PEG diamine in the material, the yellow powder was washed three times with acetone (200 mL). The remaining solid was dried under vacuum to afford a yellow semi-solid product (120 mg). HPLC analysis indicated 97% purity and¹h NMR analysis showed the product to be pure.

Reacting folic acid- (gamma) CO-NH-PEG-NH₂With p-nitrophenyl-COO-PLGA-CO₂Lauryl to provide folate-PEG-PLGA-lauryl ester. For the preparation of p-nitrophenyl-COO-PLGA-CO₂Lauryl, PLGA5050 (lauryl ester) [10.0g, 1.0 equiv ]And p-nitrophenyl chloroformate (0.79g, 2.0 equivalents) was dissolved in DCM. One portion of TEA (3.0 equivalents) was added to the dissolved polymer solution. The resulting solution was stirred at 20 ℃ for 2 hours and¹h NMR analysis indicated complete conversion. The reaction solution was then transferred to a 4: 1 solvent mixture of MTBE/heptane (50 volumes). The product precipitated and gelled. The supernatant was decanted and the solid was dissolved in acetone (20 volumes). The resulting acetone suspension was filtered and the filtrate was concentrated to dryness to give the product as a white foam [7.75g, 78% based on GPCMn 4648%]。¹H NMR analysis indicated a pure product with no detectable p-nitrophenol.

Adding folic acid- (gamma) CO-NH-PEG-NH₂(120mg, 1.0 equiv.) was dissolved in DMSO (5mL) and TEA (3.0 equiv.) was added. Inverse directionThe pH of the mixture should be 8 to 9. Adding p-nitrophenyl-COO-PLGA-CO₂Lauryl (158mg, 1.0 equiv) in DMSO (1mL) and the reaction monitored by HPLC. A new peak at 16.1 min (about 40%, AUC, 280nm) was observed from the HPLC chromatogram within 1 hour. With an excess of 1, 8-diazabicyclo [5.4.0]A small sample of the reaction mixture was treated with undec-7-ene (DBU) and immediately turned dark yellow in color. HPLC analysis of this sample indicated p-nitrophenyl-COO-PLGA-CO ₂The lauryl and peak at 16.1 minutes disappeared completely. On the contrary, in folic acid- (gamma) CO-NH-PEG-NH₂Peaks appear on the right. Can conclude p-nitrophenyl-COO-PLGA-CO under the condition of strong alkali₂Lauryl and possible products are unstable. To identify the new peak at 16.1 min, the peak was determined byTo purify a reaction mixture of about 1/3. Finally using 1: 4 DMSO/CH₃CN in a solvent mixture to elute the material. This material was observed to be yellow, which may indicate folate content. This material was not separated from the solution due to the large amount of DMSO present. Adding the mixture containing unreacted folic acid- (gamma) CO-NH-PEG-NH₂Fractions of (a) were combined and concentrated to a residue. This residual ninhydrin test gave a negative result, which may mean that the end of the PEG lacks an amine group. This observation may also indicate incomplete conversion of the reaction.

By passingTo purify the remaining reaction solution. Similar to the previous purification, the column retained the suspected yellow product. The material was eluted using MeOH with 0.5% TFA. The fractions containing the possible products were combined and concentrated to dryness. Of this sample¹HNMR analysis indicated the presence of folic acid, PEG, and lauryl-PLGA, and the combination of these segments was close to the desired 1: 1 ratio for all three components. High purity was observed from both HPLC and GPC analysis. The GPC-based Mn was 8.7 kDa. Samples in DMSO were recovered by precipitation into MTBE.

Example 6 Synthesis of PLGA-PEG-PLGA therapeutic peptide conjugates

The triblock copolymer PLGA-PEG-PLGA will be synthesized using the method developed by Zentner et al, Journal of controlled Release, 72, 2001, 203-215. The molecular weight of PLGA obtained using this method is about 3 kDa. A similar method as reported by Chen et al, International Journal of pharmaceuticals, 288, 2005, 207-. The LA/GA ratio is typically, but not limited to, a 1: 1 ratio. The minimum PEG molecular weight is 2kDa, and the upper limit is 30 kDa. The preferred range of PEG is 3-12 kDa. PLGA has a molecular weight of 4kDa with a minimum and a maximum of 30 kDa. A preferred range of PLGA is 7-20 kDa. Therapeutic peptides (e.g., histrelin or thymopentin) can be conjugated to PLGA through an appropriate linker (i.e., as set forth in the examples) so as to form a polymer-therapeutic peptide conjugate. In addition, the same therapeutic peptide or a different therapeutic peptide can be linked to another PLGA to form a dual therapeutic peptide polymer conjugate having two of the same therapeutic peptide or two of the different therapeutic peptides. Nanoparticles can be formed from PLGA-PEG-PLGA alone or from single therapeutic peptide or dual therapeutic peptide polymer conjugates consisting essentially of such triblock copolymers.

Example 7 Synthesis of polycaprolactone-poly (ethylene glycol) -polycaprolactone (PCL-PEG-PCL) therapeutic peptide conjugates

Triblock PCL-PEG-PCL was synthesized using a ring-opening polymerization method in the presence of a catalyst (i.e., tin octylate) as reported in Hu et al, Journal of Controlled Release, 118, 2007, 7-17. The molecular weight of the PCL obtained from the synthesis ranges from 2 to 22 kDa. PCL-PEG-PCL was also synthesized using a non-catalytic method as shown in the Journal of Pharmaceutical Sciences, 91, 2002, 1463-1473 article by Ge et al. The molecular weight range of PCL that can be obtained from the specific synthesis is 9 to 48 kDa. Similarly, another catalyst-free method developed by Cerrai et al, Polymer, 30, 1989, 338-343 was used to synthesize triblock copolymers having a molecular weight range of PCL from 1 to 9 kDa. The minimum PEG molecular weight will be 2kDa with an upper limit of 30 kDa. A preferred range for PEG will be 3 to 12 kDa. The PCL has a molecular weight of 4kDa at the minimum and 30kDa at the maximum. The preferred range for PCL is 7-20 kDa. Therapeutic peptides (e.g., histrelin or thymopentin) can be conjugated to PCL through an appropriate linker (i.e., as set forth in the examples) so as to form a polymer-therapeutic peptide conjugate. In addition, the same therapeutic peptide or a different therapeutic peptide can be linked to another PCL to form a dual therapeutic peptide polymer conjugate having two of the same therapeutic peptide or two of the different therapeutic peptides. Nanoparticles can be formed from PCL-PEG-PCL alone or from single therapeutic peptide or dual therapeutic peptide polymer conjugates consisting essentially of such triblock copolymers.

Example 8 Synthesis of polylactide-poly (ethylene glycol) -polylactide (PLA-PEG-PLA) therapeutic peptide conjugates

Triblock PLA-PEG-PLA copolymers will be synthesized using ring opening polymerization using the catalyst reported in Chen et al, Polymers for Advanced Technologies, 14, 2003, 245-. The molecular weight of the PLA formed is in the range of 6 to 46 kDa. The lower molecular weight range (i.e., 1-8kDa) can be obtained using the method shown in Zhu et al, Journal of applied Polymer Science, 39, 1990, 1-9. The minimum PEG molecular weight is 2kDa, and the upper limit is 30 kDa. The preferred range of PEG is 3-12 kDa. PLA has a molecular weight of a minimum of 4kDa and a maximum of 30 kDa. A preferred range of PLA is 7-20 kDa. Therapeutic peptides (e.g., histrelin or thymopentin) can be conjugated to PLA through an appropriate linker (i.e., as set forth in the examples) so as to form a polymer-therapeutic peptide conjugate. In addition, the same therapeutic peptide or a different therapeutic peptide can be linked to another PLA to form a dual therapeutic peptide polymer conjugate having two of the same therapeutic peptide or two of the different therapeutic peptides. Nanoparticles can be formed from PLA-PEG-PLA alone or from a single therapeutic peptide or dual therapeutic peptide polymer conjugate consisting primarily of such triblock copolymers.

Example 9 Synthesis of p-Dioxycyclohexanone-co-lactide-poly (ethylene glycol) -p-dioxanone-co-lactide (PDO-PEG-PDO) therapeutic peptide conjugates

Triblock PDO-PEG-PDO will be synthesized in the presence of a catalyst (stannous 2-ethyl hexanoate) using a method developed by Bhattari et al, Polymer International, 52, 2003, 6-14. The molecular weight of the PDO obtained by this method is in the range of 2-19 kDa. The minimum PEG molecular weight is 2kDa, and the upper limit is 30 kDa. The preferred range of PEG is 3-12 kDa. The molecular weight of PDO is a minimum of 4kDa and a maximum of 30 kDa. The preferred range of PDO is 7-20 kDa. Therapeutic peptides (e.g., histrelin or thymopentin) can be conjugated to PDO through an appropriate linker (i.e., as set forth in the examples) to form a polymer-therapeutic peptide conjugate. In addition, the same therapeutic peptide or a different therapeutic peptide can be linked to another PDO to form a dual therapeutic peptide polymer conjugate having two of the same therapeutic peptide or two of the different therapeutic peptides. Nanoparticles can be formed from PDO-PEG-PDO alone or from single therapeutic peptide or dual therapeutic peptide polymer conjugates consisting essentially of such triblock copolymers.

Example 10 Synthesis of Multi-functionalized PLGA/PLA-based polymers

One can synthesize PLGA/PLA-related polymers with functional groups dispersed throughout the polymer chain, which are readily biodegradable and whose components are all biodegradable components (i.e., known to be safe in the human body). Specifically, PLGA/PLA-related polymers derived from 3-S- [ benzyloxycarbonyl) methyl ] -1, 4-dioxane-2, 5-dione (3-S- [ benzyloxycarbonyl) methyl ] -1, 4-dioxane-2, 5-dione) (BMD) can be synthesized (see structures below). (the following structure is intended to represent random copolymers having the monomer units shown in parentheses.) exemplary R groups include negative charges, H, alkyl, and aralkyl groups.

1. BMD-derived PLGA/PLA-related polymers

2. PLGA/PLA related polymers derived from BMD and 3, 5-dimethyl-1, 4-dioxane-2, 5-dione (bis-DL-lactic acid cyclic diester)

3. PLGA/PLA related polymers derived from BMD and 1, 4-dioxane-2, 5-dione (bis-glycolic acid cyclic diester)

In a preferred embodiment, a number of different pendant functional groups will be used to prepare PLGA/PLA polymers derived from BMD and the cyclic diester of-DL-lactic acid by varying the ratio of BMD and lactide. For ease of reference, if it is assumed that each polymer has a number average molecular weight (Mn) of 8kDa, then 100 wt% of polymers derived from BMD have about 46 pendant carboxylic acid groups (1 acid group per 0.174 kDa). Similarly, a polymer derived from BMD at 25 wt% and from 3, 5-dimethyl-1, 4-dioxane-2, 5-dione (bis-DL-lactic acid cyclic diester) at 75 wt% has about 11 pendant carboxylic acid groups (1 acid group per 0.35 kDa). In contrast, the unfunctionalized 8kDa PLGA polymer has exactly 1 acid group and if 4 sites are added during functionalization of the end groups of the linear PLGA/PLA polymer, there is 1 acid group per 2kDa or 1 acid group per 1kDa if the 4kDa molecule is linked to four functional groups.

Specifically, the method of Kimura et al, Macromolecules, 21, 1988, 3338-3340 was used to develop PLGA/PLA-related polymers derived from BMD. The polymer will have repeating units of glycolic and malic acid per unit [ RO (COCH)₂OCOCHR₁O)_nH, wherein R is H or alkyl or PEG unit, etc., and R₁Is CO₂H]Having 1 pendant carboxylic acid group thereon. For every 174 mass units, there is one pendant carboxylic acid group. The molecular weight and polymer polydispersity of the polymer may vary with different reaction conditions (i.e., initiator type, temperature, processing conditions). Mn may range from 2 to 21 kDa. Also, there will be one pendant carboxylic acid group for every two monomer components in the polymer. Based on the above reference, NMR analysis showed that transesterification or other mechanisms did not produce detectable amounts of β -malate polymer.

Another type of PLGA/PLA related Polymer derived from BMD and 3, 5-dimethyl-1, 4-dioxane-2, 5-dione (bis-DL-lactic acid cyclic diester) was synthesized using the method developed by Kimura et al, Polymer, 1993, 34, 1741-one 1748. They showed that the highest BMD ratio used was 15 mol% and this was interpreted as a polymer containing 14 mol% (16.7 wt%) BMD derived units. This level of BMD incorporation indicates about 8 carboxylic acid residues per 8kDa polymer (1 carboxylic acid residue per kDa polymer). Similar to BMD alone, no polymer derived from β -malate was detected. Furthermore, Kimura et al report the glass transition temperature (T) _g) Is in the low 20 ℃ + range, despite the use of high polymer molecular weights (36 to 67 kDa). Whether the carboxylic acid is free or benzyl, T is for these polymers_gAre all 20 ℃ to 23 ℃. Incorporation of a more hardening element (i.e., a carboxylic acid that can form strong hydrogen bonds) should increase T_g. Due to the possible lower T_gThe value, would need to be evaluated for the possible prevention of coalescence of any particles formed by the polymeric drug conjugate derived from the particular polymer.

Another method for synthesizing PLA-PEG polymers containing varying amounts of benzyl malate glycolate involved polymerizing BMD in the presence of 3, 5-dimethyl-1, 4-dioxane-2, 5-dione (bis-DL-lactic acid cyclic diester), reported by Lee et al, Journal of Controlled Release, 94, 2004, 323-. They report that synthetic polymers contain 1.3 to 3.7 carboxylic acid units in the PLA chain with about 5 to 8kDa (the total weight of the polymer is about 11 to 13kDa, where PEG is 5kDa) depending on the amount of BMD used in the polymerization. In one polymer, there are 3.7 carboxylic acid units per hydrophobic block, where BMD represents about 19 wt% of the weight of the hydrophobic block. The ratio of BMD to lactide was similar to that observed by Kimura et al, Polymer, 1993, 34, 1741-1748 and the acid residues were similar in the resulting Polymer (approximately 1 acid unit per kDa hydrophobic Polymer).

The more easily hydrolyzed polymers functionalized with BMD were prepared using the method developed by Kimura et al, International Journal of biologicalcompromotes, 25, 1999, 265- "271. They report that the rate of hydrolysis is related to the number of free acid groups present (with polymers with more acid groups hydrolyzing faster). The polymer has a BMD content of about 5 mol% or 10 mol%. Furthermore, in the Lee et al, Journal of controlled Release, 94, 2004, 323-335 reference, the rate of polymer hydrolysis is fastest (6 days for polymers containing 19.5 wt% BMD and 20 days for polymers containing 0 wt% BMD) when the concentration of pendant acid groups is highest.

A therapeutic peptide (e.g., histrelin or thymopentin) can be conjugated to a PLGA/PLA related polymer with BMD (see previous examples above). Likewise, particles can be prepared from such polymeric therapeutic peptide conjugates.

Example 11 Synthesis of Polymer prepared Using beta-lactone of benzyl malate

One can prepare polymers by polymerizing MePEGOH with malic acid RS- β -benzyl lactone (β -lactone) and DL-lactide (cyclic diester of lactic acid) to provide a polymer containing MePEG (lactic acid) (malic acid) Me (OCH) ₂CH₂O)[OCCCH(CH₃)O]_m[COCH₂CH(CO₂H)O]Such as those developed by Wang et al, Colloid Polymer Sci, 2006, 285, 273-. These polymers will likely degrade faster because they contain higher levels of acidic groups. It should be noted that the use of beta-lactone leads to the use of 3- [ (benzyloxycarbonyl) methyl group]Polymers different from the polymers obtained from (E) -1, 4-dioxane-2, 5-dione. In these polymers, no presenceIn the case of methylene spacers, the carboxylic acid groups are attached directly to the polymer chain.

Ouhib et al, Ch.Des.Monoeres.Polym, 2005, 1, 25 report another polymer that can be prepared directly from β -lactones. The resulting polymer (i.e., poly-3, 3-dimethylmalic acid) is soluble in water as the free acid, has pendant carboxylic acid groups on each unit of the polymer chain and 3, 3-dimethylmalic acid is also reported to be a non-toxic molecule.

One can polymerize 4-benzyloxycarbonyl-, 3, 3-dimethyl-2-oxetanone in the presence of 3, 5-dimethyl-1, 4-dioxane-2, 5-dione (DDD) and beta-butyrolactone to produce block copolymers with pendant carboxylic acid groups, as shown by Coulembier et al, Macromolecules, 2006, 39, 4001-. The polymerization reaction is carried out with a carbene catalyst in the presence of ethylene glycol. The catalyst used is a triazole carbene catalyst, which forms polymers with a narrow polydispersity.

Example 12 Synthesis of PLGA-histrelin conjugate

PLGA5050, PLGA75/25 or PLGA85/15 polymers (recommended MW in the range of 10-100kDa, but not exclusively limited thereto) will be conjugated to histrelin on its serine by using a glycine linker modified on the hydroxyl group. This ester linkage between glycine and the therapeutic peptide can be cleaved off at high pH or by enzymes such as esterases.¹H NMR was used to confirm the identity of the product. HPLC was used to analyze the product purity. GPC is used to determine the purity, molecular weight, and polydispersity of the product.

Example 13 Synthesis of PLGA-nesiritide conjugates

PLGA5050, PLGA75/25 or PLGA85/15 polymers (recommended MW in the range of 10-100kDa, but not exclusively limited thereto) will be modified with an alkynyl functionality at the carbonyl end. Nesiritide will be functionalized with an azide group at the carbonyl terminus of the histidine group. Then, PLGA with an alkynyl group was conjugated to nesiritide with an azide group by click chemistry to form a triazole. This ester linkage between the triazole and the therapeutic peptide can be cleaved off at high pH or by enzymes such as esterases. ¹H NMR was used to confirm the identity of the product. HPLC was used to analyze the product purity. GPC is used to determine the purity, molecular weight, and polydispersity of the product.

Example 14 Synthesis of PLGA-Thymopentin

PLGA5050, PLGA75/25 or PLGA85/15 polymers (recommended MW in the range of 10-100kDa, but not exclusively limited thereto) will be modified with azide functionality at the carbonyl end group. Thymopentin will be functionalized with an alkynyl group at the amino terminus of the arginine group. PLGA with azide groups was then conjugated to thymopentin with alkynyl groups by click chemistry to form triazoles.¹H NMR was used to confirm the identity of the product. HPLC was used to analyze the product purity. GPC is used to determine the purity, molecular weight, and polydispersity of the product.

Example 15 Synthesis of PLGA-RWJ-800088

PLGA5050, PLGA75/25 or PLGA85/15 polymers (recommended MW between 10-100 k)Da range, but not exclusively limited thereto) will be conjugated to RWJ-800088 by forming an amide bond between PLGA and the amino end group of lysine on RWJ-800088.¹H NMR was used to confirm the identity of the product. HPLC was used to analyze the product purity. GPC is used to determine the purity, molecular weight, and polydispersity of the product.

Claims

1. A particle, comprising:

a) a plurality of hydrophobic polymers;

b) a plurality of hydrophilic-hydrophobic polymers; and

c) a plurality of therapeutic peptides or proteins, wherein at least a portion of the plurality of therapeutic peptides or proteins are covalently attached to any of the hydrophobic polymers of a) or the hydrophilic-hydrophobic polymers of b).

2. The particle of claim 1, wherein at least a portion of the hydrophobic polymers of a) are not covalently attached to the therapeutic peptides or proteins of c).

3. The particle of any of claims 1-2, wherein at least a portion of the hydrophobic polymer of a) is covalently attached to a therapeutic peptide or protein of c).

4. The particle of any of claims 1-3, wherein the at least a portion of the therapeutic peptide or protein of c) is covalently attached to the hydrophobic polymer via a linker.

5. The particle of any of claims 1-4, wherein at least a portion of the hydrophobic polymer of a) is covalently attached to at least a portion of the therapeutic peptide or protein of c) through an amino acid side chain of the therapeutic peptide or protein.

6. The particle of any of claims 1-5, wherein at least a portion of the hydrophilic-hydrophobic polymers of b) are covalently attached to the therapeutic peptides or proteins of c).

7. The particle of any of claims 1-6, wherein at least a portion of the hydrophilic-hydrophobic polymers of b) are directly covalently attached to the therapeutic peptides or proteins of c).

8. The particle of any of claims 1-7, wherein at least a portion of the therapeutic peptide or protein of c) is covalently attached to the hydrophilic-hydrophobic polymer of b) via a linker.

9. The particle of any of claims 1-8, wherein at least a portion of the hydrophilic-hydrophobic polymer of b) is covalently attached to at least a portion of the therapeutic peptide or protein of c) through an amino acid side chain of the therapeutic peptide or protein.

10. The particle of any one of claims 1-9, wherein the particle further comprises a plurality of other therapeutic peptides or proteins, wherein the other therapeutic peptides or proteins are different from the therapeutic peptides or proteins of c).

11. The particle of any of claims 1-10, wherein at least a portion of the plurality of other therapeutic peptides or proteins are attached to at least a portion of the hydrophobic polymer of a) and/or the hydrophilic-hydrophobic polymer of b).

12. The particle of any one of claims 1-11, further comprising a counter ion.

13. A particle, comprising:

a) optionally a plurality of hydrophobic polymers;

b) a plurality of hydrophilic-hydrophobic polymer-conjugates, wherein the hydrophilic-hydrophobic polymer conjugates comprise a hydrophilic-hydrophobic polymer attached to a charged peptide or charged protein; and

c) a plurality of charged therapeutic peptides or charged proteins, wherein the charge of the therapeutic peptides or proteins is opposite to the charge of the peptides or proteins conjugated to the hydrophilic-hydrophobic polymer, and wherein the charged therapeutic peptides or proteins form non-covalent bonds (e.g., ionic bonds) with the charged peptides or the charged proteins of the hydrophilic-hydrophobic polymer-conjugate.

14. The particle of claim 13, wherein the particle is substantially free of hydrophobic polymers.

15. The particle of claim 13 or 14, wherein the hydrophobic-hydrophilic polymer of the conjugate of b) is covalently attached to the charged peptide via a linker.

16. The particle of any of claims 1-15, wherein at least a portion of the hydrophobic polymers of a) are copolymers of lactic and glycolic acid (i.e., PLGA).

17. The particle of claim 16, wherein a portion of the hydrophobic polymers of a) are PLGA having a ratio of about 50: 50 of lactic acid to glycolic acid.

18. The particle of any of claims 1-17, wherein the hydrophobic portion of the hydrophilic-hydrophobic polymers of b) comprises a copolymer of lactic and glycolic acid (i.e., PLGA).

19. The particle of claim 18, wherein the hydrophobic portion of the hydrophilic-hydrophobic polymers of b) comprises PLGA having a ratio of about 50: 50 of lactic acid to glycolic acid.

20. The particle of any one of claims 1-19, wherein the hydrophilic portion of the hydrophilic-hydrophobic polymer of b) comprises PEG.

21. The particle of any of claims 1-20, wherein the therapeutic peptide comprises from about 2 to about 60 amino acid residues.

22. The particle of any one of claims 1-21, wherein the therapeutic peptide or protein is selected from the therapeutic peptides or proteins described herein.

23. The particle of any one of claims 1-22, further comprising a surfactant.

24. The particle of any one of claims 1 to 23, wherein the particle has a diameter of less than about 200nm (e.g., less than about 150 nm).

25. The particle of any one of claims 1-24, wherein the zeta potential of the particle is from about-20 to about +20mV (e.g., from about-5 to about +5 mV).

26. A particle, comprising:

a) a plurality of hydrophobic polymers;

b) a plurality of hydrophilic-hydrophobic polymers; and

c) a protein, wherein the protein is covalently linked to the hydrophobic polymer of a) or the hydrophilic-hydrophobic polymer of b).

27. A composition comprising a plurality of particles of any one of the preceding claims.

28. A kit comprising a plurality of particles of any of the preceding claims or a composition of any of the preceding claims.

29. A single dosage unit comprising a plurality of particles of any one of the preceding claims or a composition of any one of the preceding claims.

30. A method of treating a subject having a disorder, comprising administering to the subject an effective amount of the particle of any one of the preceding claims or the composition of any one of the preceding claims.

31. A therapeutic peptide-hydrophobic polymer conjugate comprising a therapeutic peptide covalently attached to a hydrophobic polymer; or a protein-hydrophobic polymer conjugate comprising a protein covalently linked to a hydrophobic polymer.

32. The therapeutic peptide-hydrophobic polymer conjugate or protein-hydrophobic polymer conjugate of claim 31, wherein the therapeutic peptide or protein is covalently attached to the hydrophobic polymer via the carboxy terminus of the therapeutic peptide or protein.

33. The therapeutic peptide-hydrophobic polymer conjugate or protein-hydrophobic polymer conjugate of claim 31, wherein the therapeutic peptide or protein is covalently attached to the hydrophobic polymer via the amino terminus of the therapeutic peptide or protein.

34. The therapeutic peptide-hydrophobic polymer conjugate or protein-hydrophobic polymer conjugate of claim 31, wherein the therapeutic peptide or protein is covalently attached to the hydrophobic polymer via an amino acid side chain of the therapeutic peptide or protein.

35. The therapeutic peptide-hydrophobic polymer conjugate or protein-hydrophobic polymer conjugate of any one of claims 31-34, wherein the therapeutic peptide or protein is covalently attached to the hydrophobic polymer at a terminus of the polymer.

36. The therapeutic peptide-hydrophobic polymer conjugate or protein-hydrophobic polymer conjugate of any of claims 31-34, wherein the therapeutic peptide or protein is covalently attached to the polymer along the backbone of the hydrophobic polymer.

37. The therapeutic peptide-hydrophobic polymer conjugate or protein-hydrophobic polymer conjugate of any one of claims 31-36, wherein the therapeutic peptide or protein is covalently attached to the hydrophobic polymer via a linker.

38. A composition comprising a plurality of therapeutic peptide-hydrophobic polymer conjugates or protein-hydrophobic polymer conjugates of any one of claims 31-37.

39. A method of making a therapeutic peptide-hydrophobic polymer conjugate or protein-hydrophobic polymer conjugate of claim 31, the method comprising:

providing a therapeutic peptide or protein and a polymer; and

subjecting the therapeutic peptide or protein and polymer to conditions to effect covalent attachment of the therapeutic peptide or protein to the polymer.

40. A therapeutic peptide-hydrophilic-hydrophobic polymer conjugate or protein-hydrophilic-hydrophobic polymer conjugate comprising a therapeutic peptide or protein covalently attached to a hydrophilic-hydrophobic polymer, wherein the hydrophilic-hydrophobic polymer comprises a hydrophilic moiety attached to a hydrophobic moiety.

41. The therapeutic peptide-hydrophilic-hydrophobic polymer conjugate or protein-hydrophilic-hydrophobic polymer conjugate of claim 40, wherein the therapeutic peptide or protein is attached to the hydrophilic portion of the hydrophilic-hydrophobic polymer.

42. The therapeutic peptide-hydrophilic-hydrophobic polymer conjugate or protein-hydrophilic-hydrophobic polymer conjugate of claim 40, wherein the therapeutic peptide or protein is attached to a hydrophobic portion of the hydrophilic-hydrophobic polymer.

43. The therapeutic peptide-hydrophilic-hydrophobic polymer conjugate or protein-hydrophilic-hydrophobic polymer conjugate of any one of claims 40-42, wherein the hydrophilic-hydrophobic polymer is covalently attached to the therapeutic peptide or protein through the amino terminus of the therapeutic peptide or protein.

44. The therapeutic peptide-hydrophilic-hydrophobic polymer conjugate or protein-hydrophilic-hydrophobic polymer conjugate of any one of claims 40-42, wherein the hydrophilic-hydrophobic polymer is covalently attached to the therapeutic peptide or protein through the carboxy-terminus of the therapeutic peptide or protein.

45. The therapeutic peptide-hydrophilic-hydrophobic polymer conjugate or protein-hydrophilic-hydrophobic polymer conjugate of any one of claims 40-42, wherein the hydrophilic-hydrophobic polymer is covalently attached to the therapeutic peptide or protein through an amino acid side chain of the therapeutic peptide or protein.

46. The therapeutic peptide-hydrophilic-hydrophobic polymer conjugate or protein-hydrophilic-hydrophobic polymer conjugate of any one of claims 40-45, wherein the therapeutic peptide or protein is attached to the hydrophilic-hydrophobic polymer via a linker.

47. A composition comprising a plurality of therapeutic peptide-hydrophilic-hydrophobic polymer conjugates or protein-hydrophilic-hydrophobic polymer conjugates of any one of claims 40-46.

48. A method of making a therapeutic peptide-hydrophilic-hydrophobic polymer conjugate or protein-hydrophilic-hydrophobic polymer conjugate of claim 40, comprising:

subjecting the therapeutic peptide or protein and hydrophilic-hydrophobic polymer to conditions to effect covalent attachment of the therapeutic peptide or protein to the polymer.

49. A method of storing the conjugate of any one of claims 31 to 37 or 40 to 46, the particle of any one of claims 1 to 26, or the composition of any one of claims 27, 38, or 47, the method comprising:

(a) Providing the conjugate, particle, or composition disposed in a container;

(b) storing the conjugate, particle or composition; and

(c) moving the container to a second location or removing all or an aliquot of the conjugate, particle, or composition from the container.

50. The method of claim 49, wherein the stored conjugate, particle, or composition is a reconstituted formulation.