HK1154795B - Pharmaceutical compositions of paclitaxel, paclitaxel analogs or paclitaxel conjugates and related methods of preparation and use - Google Patents

Description

Pharmaceutical composition of paclitaxel, paclitaxel analogue or paclitaxel conjugate and related preparation method and application

Technical Field

The present invention relates to formulations of paclitaxel and paclitaxel analogs or combinations thereof, as well as other hydrophobic agents.

Background

Hydrophobic agents, such as paclitaxel and paclitaxel analogs, are typically either solubilized in a non-aqueous or surfactant buffer or attached to a hydrophilic moiety to increase solubility in aqueous solutions prior to administration to a patient because they are insoluble in aqueous solutions. In a commercially available paclitaxel dosage form, 6mg paclitaxel per ml, 527mg purified Cremophor (Cremophor)EL (polyoxyethylated castor oil) and 49.7% (v/v) dehydrated alcohol, USP and ethanol. Prior to administration, formulated paclitaxel was diluted in Ringer's solution (Ringer's solution) of sodium chloride/glucose or glucose. Since Cremophor can cause hypersensitivity (e.g., allergic) reactions, patients receiving paclitaxel require the administration of dexamethasone (dexamethasone) prior to surgery in order to reduce the incidence of these reactions. Because of these reactions, paclitaxel was administered 4 hours later, thus inducing hypersensitivityAnd is minimized.

Since the inclusion of Cremophor in standard paclitaxel formulations can cause a high rate of side effects, an alternative formulation is created. These formulations rely on the association of paclitaxel with soluble compounds. Abraxane (trade name) is a paclitaxel formulation with paclitaxel bound to albumin. Liposomal formulations of paclitaxel have also been proposed.

Since existing formulations of hydrophobic agents such as paclitaxel either contain undesirable excipients or are difficult to manufacture, new formulations of such agents are needed.

Disclosure of Invention

In one aspect, the invention features a composition comprising (a) a hydrophobic agent, paclitaxel, a paclitaxel analog, or a conjugate (e.g., ANG1005), or any of the hydrophobic agents described herein, the conjugate comprising (i) a polypeptide carrier and (ii) a therapeutic agent selected from the group consisting of paclitaxel and a paclitaxel analog, wherein the therapeutic agent is conjugated to a polypeptide; the composition further comprises (b) an optional tonicity agent (e.g., sodium chloride or any tonicity agent described herein); (c) a buffer (e.g., glycine, lactic acid, or citric acid, or any buffer described herein); (d) bulking agents (e.g., mannitol, sorbitol, or any bulking agent described herein); and (e) a solubilizing agent (e.g., a fatty acid polyoxyethylene ester, such as Solutol HS15 (polyethylene glycol-12-hydroxystearate-15), or any solubilizing agent described herein), e.g., wherein the solubilizing agent is not Cremophor. The polypeptide vector may comprise an amino acid sequence that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1-105 and 107-116 (e.g., AngioPep) -1(SEQ ID NO: 67), AngioPep-2 (SEQ ID NO: 97), or AngioPep-7 (SEQ ID NO: 112)) are substantially identical (e.g., at least 70%, 80%, 90%, 95%, or 100% identical). In certain embodiments, the buffer maintains the solution pH at less than 6 (e.g., pH 4-6). In certain embodiments, the composition further comprises 0.01-10% (e.g., less than 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.2%, or 0.1%) DMSO. In certain embodiments, the composition is substantially free of Cremophor (e.g., free of Cremophor). The composition is soluble in water.

In certain embodiments, the composition comprises an agent in an amount as set forth in any one of tables 1-4.

TABLE 1

Compound (I)	Percentages (by weight not water)
		ANG1005	0.1-5％
Tonicity agent	0-15％
		Buffering agent	1-10％
Expanding agent	0-15％
		Solutol HS 15	40-75％
DMSO	0.01-10％

TABLE 2

Compound (I)	Percentages (by weight not water)
		ANG1005	1.8-2.3％
Tonicity agent	9-11％
		Buffer solution	4.5-6％
Expanding agent	8-10％
		Solutol HS 15	69-75％
DMSO	0.2-1％

TABLE 3

Compound (I)	Percentages (by weight not water)
		ANG1005	1.8-4.0％
Buffer solution	0.1-6％
		Expanding agent	2-10％
Solutol HS 15	80-95％
		DMSO	0.2-1％

TABLE 4

Compound (I)	Percentages (by weight not water)
		ANG1005	2.0-3.0％

Buffer solution	0.5-6％
		Expanding agent	4-7％
Solutol HS 15	85-95％
		DMSO	0.2-0.6％

In these compositions, if a tonicity agent is present, it may be sodium chloride, the buffering agent may be glycine, lactic acid, or citric acid, and/or the bulking agent may be mannitol. The composition may be comprised of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 3.0%, 3.2%, 3.5%, 4.0%, or 5.0% ANG1005, or any range between any of these values of ANG 1005. The ANGl005 can be dissolved in a sufficient amount of Solutol HS15, and/or DMSO, which can be further diluted in an aqueous solution.

The above composition may be present in a container that can be sealed. The container can be part of a kit that further includes instructions for use (e.g., administration of the composition to treat, for example, any of the diseases described herein).

In another aspect, the invention features a method of administering the composition of several aspects above to a patient suffering from a disease, e.g., any of the diseases described herein, such as cancer (e.g., ovarian, brain, lung, liver, spleen, or kidney cancer). The method comprises administering to the patient a composition in an amount sufficient to therapeutically or prophylactically treat the disease. In certain embodiments, the cancer is a brain cancer selected from the group consisting of glioblastoma (glioblastomas), astrocytoma, glioma, medulloblastoma, and oligodendroglioma, glioma, ependymoma, and meningioma.

In another aspect, the invention features a method for preparing a pharmaceutical composition. The method comprises (a) dissolving a hydrophobic agent in a first solubilizing agent (e.g., DMSO or any such agent described herein) to form a mixture; (b) adding a second solubilizing agent (e.g., a fatty acid polyoxyethylene ester, such as Solutol HS15, or any such agent described herein) to the mixture of step (a); (c) optionally adding water and a buffer to the mixture; (d) lyophilizing the mixture of step (c); wherein the lyophilizing results in a reduction in the amount of the first solubilizing agent by at least 5% (e.g., 10%, 20%, 30%, 50%, 75%, 90%, 95%, or 99%) (e.g., to a final ratio of less than 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.5%, 2%, 3%, 4%, 5%, 8% of the total weight of the lyophilized product). In certain embodiments, the lyophilization does not significantly reduce the amount of the second solubilizing agent. In certain embodiments, the hydrophobic agent comprises paclitaxel or a paclitaxel analog. The hydrophobic agent may include or may be a combination including: (a) a polypeptide carrier and (b) an agent described herein (e.g., paclitaxel and analogs thereof), wherein the agent is bound to the carrier. The polypeptide vector may be linked to a polypeptide selected from the group consisting of SEQ id nos: 1-105 and 107-116 (e.g., angiopep-1 (SEQ ID NO: 67), angiopep-2 (SEQ ID NO: 97), or angiopep-7 (SEQ ID NO: 112)). In a particular embodiment, the conjugate is ANG 1005. In certain embodiments, water and buffer are added in step (c), and the lyophilizing of step (d) comprises (i) freezing the mixture, (ii) drying the frozen product at a first temperature and pressure sufficient to remove at least a portion (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.9%, or 99.99%) of the water, and (iii) drying the product at a second temperature and pressure sufficient to remove at least a portion (e.g., at least 5%, e.g., 10%, 20%, 30%, 50%, 75%, 90%, 95%, or 99%) of the first solvent. The mixture of step (b) may be filtered prior to lyophilization of step (d), or the mixture of step (b) may be placed in a vial or container prior to lyophilization of step (d). The method may further comprise (e) resuspending the lyophilized product.

In another aspect, the invention features a method for producing a pharmaceutical composition, comprising the steps of (a) dissolving a conjugate comprising paclitaxel or a paclitaxel analog conjugated to a polypeptide carrier in DMSO, thereby forming a mixture; (b) adding Solutol HS15 to the mixture; (c) adding water, a buffering agent and optionally a salt or a swelling agent to the mixture; and (d) lyophilizing the mixture under conditions that remove the water and DMSO from the mixture. Prior to addition to the mixture, Solutol HS15 may be mixed with water, a buffer, and optionally a tonicity agent or bulking agent, wherein the water, buffer, and optional tonicity agent are added in amounts to maintain the solubility of the conjugate in the mixture. The buffer may maintain the solution pH between 4and 6. The DMSO may be acidified to a pH between 3.5 and 4.5 prior to dissolution in step (a). In certain embodiments, the lyophilization does not significantly reduce the amount of Solutol HS15 in the mixture. The conjugate can include any of the polypeptides described herein (e.g., angiopep-2). In a particular embodiment, the paclitaxel-polypeptide conjugate is ANG 1005.

In another aspect, the invention features a pharmaceutical composition produced by any of the above methods.

"buffer" refers to any compound or group of compounds capable of maintaining the solution pH within a specified range (e.g., between any of pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, and 13.5) after addition of an agent that can otherwise alter the pH. Exemplary buffers are described herein.

"tonicity agent" refers to any agent that can alter the osmolality of an aqueous solution (e.g., any of 10, 20, 50, 75, 100, 150, 200, 250, 300, 400, 500, 750, 1000, 1500, or 2000mM or any range therebetween). Ionic salts, such as sodium chloride, can be used to adjust tonicity. Additional tonicity agents are described herein.

"bulking agent" refers to a compound that can alter the physical form of a chemical composition after a dehydration or lyophilization step. Exemplary bulking agents (extenders) are described herein.

By "solubilizer" is meant any solvent capable of dissolving a particular compound (e.g., a hydrophobic compound, such as a compound or conjugate containing paclitaxel or a paclitaxel analog). Exemplary solubilizing agents suitable for hydrophobic compounds are described herein.

"vector" refers to a compound or molecule, such as a polypeptide, that can be transported into a particular cell type (e.g., liver, lung, kidney, spleen, or muscle) or across the BBB. The carrier may be attached (covalently or non-covalently) or bound to the agent and thereby may transport the agent into a particular cell type or across the BBB. In certain embodiments, the vector may bind to a receptor located on a cancer cell or brain endothelial cell and thereby be transported into the cancer cell by transcytosis (transcytosis) or across the BBB. The vector may be a molecule for which high levels of transendothelial transport are obtained without affecting the integrity of the cell or BBB. The vector may be a polypeptide or peptidomimetic (peptidomimetic) and may be naturally occurring or produced by chemical synthesis or recombinant genetic techniques.

A "conjugate" is a carrier that is linked to an agent. Binding can be chemical, e.g., via a linker, or genetic, e.g., by recombinant genetic techniques, such as in the form of a fusion protein with, e.g., a reporter molecule (e.g., green fluorescent protein, β -galactosidase, histidine tag, etc.).

A vector or conjugate that is "efficiently delivered to a particular cell type" refers to a vector or conjugate that is capable of accumulating in that cell type (e.g., due to an increase in delivery into the cell, a decrease in outflow from the cell, or a combination thereof) to an extent that is at least 10% greater (e.g., 25%, 50%, 100%, 200%, 500%, 1,000%, 5,000%, or 10,000%) than the control substance, or if a conjugate, than the unconjugated agent.

"substantially pure" or "isolated" refers to a compound (e.g., a polypeptide or conjugate) that is separated from other chemical components. Typically, the compound is substantially pure when at least 30% by weight of the compound is free of other components. In certain embodiments, the formulation is at least 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by weight free of other components. Purified polypeptides can be obtained, for example, by expression of a recombinant polynucleotide encoding such a polypeptide or by chemical synthesis of the polypeptide. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

A pharmaceutical composition that is "substantially free" of a substance means that the amount of the substance in the composition is less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05% or 0.01% of the dry weight of the composition.

"substantially identical" refers to a polypeptide or nucleic acid that exhibits at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or even 99% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of the comparison sequence (comparison sequence) is typically at least 4 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, or 100) amino acids. For nucleic acids, the length of the comparison sequences is generally at least 60 nucleotides, preferably at least 90 nucleotides, more preferably at least 120 nucleotides, or full length. It is understood herein that gaps (gaps) may exist between amino acids of analogs that are the same as or similar to the original polypeptide amino acid. The gap may include no amino acids, one or more amino acids that are not identical or similar to the original polypeptide. Biologically active analogs of the vectors (polypeptides) of the invention may thus be included. Percent identity can be determined using a default GAP weight (gapweight), for example, using the n algorithm GAP, BESTFIT, or FASTA in Wisconsin Genetics Software Package Release7.0.

"fragment" refers to a polypeptide derived from a portion of an original or parent sequence or from an analog of said parent sequence. Fragments include polypeptides having one or more amino acid truncations (truncations), wherein the truncations may be derived from the amino terminus (N-terminus), the carboxy terminus (C-terminus), or from within the protein. Fragments may comprise sequences identical to the corresponding parts of the original sequence. The invention includes functional fragments of the vectors (polypeptides) described herein. A fragment can be at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 28, 30, 35, 40, 45, 50, 60, 75, 100, or 150) amino acids. Fragments of the invention may include, for example, polypeptides of 7, 8, 9, or 10 amino acids to 18 amino acids. Fragments may contain any of the modifications described herein (e.g., acetylation, amidation, amino acid substitutions).

"non-naturally occurring amino acid" refers to an amino acid that is not naturally occurring or found in a mammal.

"agent" refers to any compound, for example, an antibody, or a therapeutic, marker, tracer, or imaging compound.

"therapeutic agent" refers to an agent having biological activity. In some cases, the therapeutic agent is used to treat a disease, a physiological or psychological condition, a symptom of trauma or infection, and it includes anti-cancer agents, antibiotics, anti-angiogenic agents (anti-angiogenic agents) and molecules active at the central nervous system level.

"Small molecule drug" refers to a drug having a molecular weight of 1000g/mol or less (e.g., less than 800, 600, 500, 400, or 200 g/mol).

By "subject" is meant a human or non-human animal (e.g., a mammal).

"treating" a disease, disorder, or condition in a subject refers to alleviating at least one symptom of the disease, disorder, or condition by administering a therapeutic agent to the subject.

"prophylactically treating" a disease, disorder, or condition in a subject refers to reducing the frequency of occurrence (e.g., preventing) of the disease, disorder, or condition by administering a therapeutic agent to the subject.

"cancer" refers to the proliferation of any cell, the unique feature of which is the loss of normal control, resulting in uncontrolled growth, lack of differentiation, or the ability to invade tissues and metastasize. Cancer can occur in any tissue or any organ. Cancer is intended to include, but is not limited to, brain, liver, lung, kidney or spleen cancer. Additional cancers are described herein.

"administration" refers to modes of delivery, including, but not limited to, oral (oral), intra-arterial, intranasal, intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, or oral (peros). The daily dose may be divided into one, two or more doses in a suitable form, so that it is administered once, twice or more times over the whole period of time.

"therapeutically effective" or "effective amount" refers to an amount of a therapeutic agent sufficient to ameliorate, reduce, prevent, delay, inhibit or arrest any symptom of the disease or condition being treated. A therapeutically effective amount of an agent need not cure a disease or condition, but will provide treatment to a disease or condition so as to delay, block or prevent the onset of the disease or condition, or to alleviate the symptoms of the disease or condition, or to change the duration of the disease or condition or, for example, make it less severe, or to accelerate recovery in an individual.

If a "range" or "group of substances" is mentioned for a particular characteristic (e.g., temperature, concentration, time, etc.), the invention relates to and specifically incorporates herein each and every specific member and sub-range or sub-group thereof in combination. Thus, for example, for a length of 9 to 18 amino acids, it should be understood that each and every single length is specifically incorporated herein, e.g., a length of 18, 17, 15, 10, 9 and any number therebetween. Thus, unless otherwise indicated, each range recited herein is to be understood as being inclusive of the endpoints. For example, an expression of 5 to 19 amino acids in length is understood to include 5 and 19. Similarly, this also applies to other parameters, such as sequence, length, concentration, elements, etc.

The sequences, regions and portions defined herein each include each and every individual sequence, region and portion and each and every possible subsequence, sub-region and sub-portion described therein, whether such subsequence, sub-region and sub-portion is defined as positively including a particular possibility, as exclusively including a particular possibility, or as a combination of both. For example, exclusivity for a region is defined as follows: provided that the polypeptide is not shorter than 4, 5, 6, 7, 8 or 9 amino acids. Further examples of negative limitations are as follows: comprises SEQ ID.: x, wherein the sequence of SEQ ID. is excluded: a polypeptide of Y; and the like. Further examples of negative limitations are as follows: as long as the polypeptide is not (comprises or consists of) SEQ ID No.: and Z.

Other features and advantages of the invention will be apparent from the following detailed description, the accompanying drawings, and the claims.

Drawings

Fig. 1 is a schematic of an exemplary process for preparing a pharmaceutical composition comprising ANG 1005.

Figure 2 is an HPLC profile showing reconstituted ANG for injection 1005 over time, the ANG1005 diluted with D5W to a concentration of 1.0mg/ml under clinical use conditions.

FIG. 3 is an HPLC chromatogram showing a centrifuged sediment (precipitate) collected from a 2.0mg/mL sample stored for 6h and dissolved in DMSO.

Detailed Description

We have developed pharmaceutical formulations useful for hydrophobic therapeutic agents including paclitaxel and paclitaxel analogs, or conjugates thereof (e.g., ANG1005), and we have also developed methods for preparing and administering pharmaceutical compositions containing such formulations. Hydrophobic therapeutic agents (e.g., paclitaxel) are typically dissolved in hydrophobic solvents (and, in fact, are typically required to do so). Common solvents for paclitaxel include Cremophor and DMSO, which patients are poorly tolerated. In particular Cremophor can cause allergic reactions and thus require prior treatment with agents such as corticosteroids. To avoid the use of such poorly tolerated solvents, we developed a new formulation of an exemplary polypeptide-paclitaxel conjugate, ANG 1005. The formulations described herein are highly advantageous in that they can be manufactured without the use of Cremophor, can be formulated to contain extremely low DMSO concentrations, provide low degradation of the active agent and high activity, and can be manufactured using conventional methods. Compositions that do not contain a poorly tolerated excipient may be administered to a patient in higher doses, may be administered more rapidly (e.g., in the case of intravenous administration), may be administered more frequently, or may avoid the need for prior treatment with a drug (e.g., a corticosteroid) to increase tolerance to such excipients.

Development of ANG1005 formulations

In developing a novel formulation of an exemplary hydrophobic agent, ANG1005, we first tested its solubility in various solvents and solvent combinations. As stated in example 1, ANG1005, like paclitaxel, has low solubility in aqueous solution, but is highly soluble (120mg/ml) in DMSO. ANG1005 is also soluble in Solutol HS15 (BASF, Parsippany, n.j.) with ethanol at 75 ℃ (6 mg/ml). Solutol HS15 was chosen as a solubilizer due to its low toxicity and compatibility with drugs. But dissolution with Solutol HS15 alone will result in significant degradation of ANG 1005. To dissolve ANG1005, Solutol HS15 was heated to at least 65 ℃. In addition, we note that when unbuffered Solutol is heated from 25 ℃ to 50 ℃, its pH increases from 6.0 to 9.0. Thus, the combination of high temperature and high pH is likely to result in instability of ANG1005 observed under these conditions.

To avoid excessive degradation, ANG1005 was first dissolved in acidified DMSO (pH 3.5-4.0) before adding Solutol at 50 ℃ (see example 2). To further stabilize ANG1005, we acidified Solutol HS by pre-mixing Solutol HS15 with a glycine buffer of H5.0, which can maintain the solubility of ANGl 005. This minimizes degradation of the ANG 1005. Up to 20% (e.g., 1%, 5%, 10%, or 15%) of buffer can be added to the Solutol prior to adding ANG1005 without affecting the solubility of ANG1005, but mixing a greater amount of buffer with Solutol will cause incomplete dissolution.

Since we observed that ANG1005 gradually became unstable at pH above 6, we diluted the formulation in glycine buffered water at pH 5 in order to ensure stability of ANG 1005. Other buffers in this pH range were also evaluated, including acetate buffers and phosphate buffers, but these buffers are less compatible with the formulation. We also attempted to stabilize ANG1005 by lowering the final pH to 4, but the resulting lyophilized cake could not be reconstituted into a clear solution.

Exemplary ANG1005 compositions are given in table 5 below.

Table 5: component functions in ANG1005 for injection

Components	Use of	Target volume/batch	Target volume/vial
				ANG1005	API	60g	120mg
Sodium chloride, USP	Amount of seepage	290g	580mg
				Glycine	Buffer solution	150g	300mg
Mannitol	Expanding agent	260g	520mg
				Solutol HS 15	API solubilization	2000ml	4ml
DMSO	API solubilization prior to lyophilization	500ml1	1ml1
				HCl	pH adjustment	Adjusting the pH
Water for injection, USP	Solvent(s)	QS to 10L1	To 20ml1

¹Removing by freeze-drying

A bulking agent is also added to make the lyophilized product easier to reconstitute. Formulations containing mannitol and sorbitol were evaluated. Mannitol produced a better cake.

Freeze-drying

Since the DMSO/Solutol/buffer formulation contains undesirably high levels of DMSO and is not stable enough, a lyophilization protocol designed to reduce DMSO and improve ANG1005 stability was developed. A number of alternative (alternate) lyophilization cycles were evaluated to minimize DMSO content (i.e., increase temperature and increase the length of secondary drying (length); see example 3). The lyophilization conditions will be described in detail below. We tried the first lyophilization protocol, which resulted in DMSO concentrations greater than 1%. Details of this scheme are shown in table 6.

TABLE 6

We have been able to further reduce the DMSO concentration to less than 1% by using an optimized two-step drying procedure. Briefly, after freezing the product, it is lyophilized at shelf temperature (storage temperature) for a time sufficient to remove most of the water in the product. The shelf temperature is raised and the product is dried at a temperature suitable for removing DMSO. The specific conditions (precision conditions) may vary with the volume of sample being dried, the pressure and temperature used, and the formulation and buffer used. Based on the procedures described herein, one skilled in the art can determine appropriate drying conditions to produce the compositions described herein.

In one example procedure, the formulation (component) is loaded between-70 and +25 ℃ (e.g., -40 ℃). The temperature is then ramped up (ramp) to a set temperature sufficient to freeze the solution (any temperature between 0 ℃ and-70 ℃, e.g. -40 ℃) and held at that temperature for a time sufficient to freeze the product, preferably for a time sufficient to ensure that the lyophilized cake does not collapse. We determined that at-40 ℃, a lyophilization time of at least 12 hours (e.g., at least 15, 18, 20, 24, 36, or 48 hours) is required to ensure that the cake does not collapse. After freezing, the vacuum is set to a pressure (e.g., 10-500mT, such as 20, 50, 100, 200, or 500mT) and temperature (e.g., -15 to-35 ℃, such as-25 ℃) sufficient to remove water from the product for one drying cycle. For this purpose, pressures between 10-100mT were tested with minimal variation in the results. The drying time can be a time (e.g., at least 6 hours, 12 hours, 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, or 14 days) sufficient to remove a majority (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%) of the water present in the product. After the first drying cycle, a second drying cycle was performed to remove DMSO. The product is ramped up to a higher temperature (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 ℃) between 10-30 ℃ to remove the DMSO. In a preferred embodiment, the shelf temperature is ramped up to 27 ℃ over 2 hours, and then held at 27 ℃ for 1 hour. The ramp then increases (or maintains) the shelf temperature to between 23 and 27 ℃ over 30 minutes, and then at that temperature for an additional at least 10 hours (e.g., at least 15, 20, 25, 30, 40, 48, 60, or 72 hours). To prevent the residual DMSO from melting, the product may be stored below 25 ℃. An exemplary scheme of this method is shown in table 7. Lyophilization was performed using a Hull Freezer Dryer, Model 72FS 100-SS 20C.

TABLE 7

Reconstitution of products

The lyophilized product may be reconstituted prior to injection into a patient or experimental analysis of the product. Any buffer, solvent, or combination of buffer(s) and solvent(s) suitable for reconstitution can be used, with the particular buffer not being critical. However, it is generally desirable that the active agent is sufficiently stable in this solution and that the patient is sufficiently well-tolerated by the buffer(s) or solvent(s) used in the solution to which he is administered. In the case of ANG1005, it is generally desirable to use a reconstituted solvent/buffer system that can maintain a pH below 6.0, since the product is less stable above pH 6.0. For ANG1005, one preferred solvent system is ethanol in combination with ringer's lactate/5% dextrose. In this system, ethanol is added to the vial containing the product, gently mixed, and then ringer's lactate/5% dextrose is added to dissolve the product. Using conventional water for injection (WFI) or saline as a diluent can produce high pH levels, leading to degradation of ANG 1005. After dissolution, the mixture may be further diluted in water or other buffer system. Exemplary conditions for reconstitution of the lyophilized product are further described in example 4 below.

Formulation compositions (formulations compositions)

As noted above, we have developed a formulation of an exemplary hydrophobic agent suitable for administration to a patient, ANG 1005. Prior to lyophilization, in certain embodiments, the formulation may contain a substantial proportion of DMSO. Such compositions may have the following components (e.g., dry weight) given in tables 8A and 8B. Table 8C shows exemplary concentrations of the components in the aqueous solution prior to lyophilization.

Table 8A: percentages by weight not including water

Table 8B: percentage in aqueous solution (before lyophilization)

Table 8C: concentration in aqueous solution (mg/ml) before lyophilization

Prior to lyophilization, the composition is typically diluted in water (see below for lyophilization conditions). For most clinical applications, the solution is divided into amounts suitable for single dose administration of ANG1005 (e.g., about 10, 20, 30, 60, 90, 120, 150, 200, 240, 300, 400, or 500 mg). After lyophilization (e.g., under the conditions described herein), the DMSO concentration can be significantly reduced. After lyophilization, the ANG1005 compositions of the present invention can have the following characteristics (e.g., dry weight) shown in table 9.

TABLE 9

Hydrophobic agents

Any hydrophobic agent may be used in the compositions and methods of the present invention. Exemplary compounds are described below.

Taxol and related compounds

Although the present invention is exemplified with ANG1005, angio-peptide 2-paclitaxel conjugates, the formulations described herein may also be used with paclitaxel, paclitaxel analogs, or combinations thereof. Paclitaxel has the formula:

structural analogs of paclitaxel are described in U.S. patent 6,911,549 and can be represented by the formula:

wherein R is₁Selected from the group consisting of, i.e., -CH₃；-C₆H₅Or by 1, 2 or 3C₁-C₄Alkyl radical, C₁-C₃Alkoxy, halogen, C₁-C₃Alkylthio, trifluoromethyl, C₂-C₆Dialkylamino, hydroxy, or nitro substituted phenyl; and-2-furyl, 2-thienyl, 1-naphthyl, 2-naphthyl or 3, 4-methylenedioxyphenyl; r₂Selected from the group consisting of-H, -NHC (O) C₁-C₁₀Alkyl (preferably-NHC (O) C)₄-C₆Alkyl), -NHC (O) phenyl, substituted by 1, 2 or 3C₁-C₄Alkyl radical, C₁-C₃Alkoxy, halogen, C₁-C₃Alkylthio, trifluoromethyl, C₂-C₆Dialkylamino, hydroxy or nitro substituted-NHC (O) phenyl, -NHC (O) C (CH)₃)＝CHCH₃、-NHC(O)OC(CH₃)₃、-NHC(O)OCH₂Phenyl, -NH₂、-NHSO₂-4-tolyl, -NHC (O) (CH)₂)₃COOH、-NHC(O)-4-(SO₃H) Phenyl, -OH, -NHC (O) -1-adamantyl, -NHC (O) O-3-tetrahydrofuranyl, -NHC (O) O-4-tetrahydropyranyl, -NHC (O) CH₂C(CH₃)₃、-NHC(O)C(CH₃)₃、-NHC(O)OC₁-C₁₀Alkyl, -NHC (O) NHC₁-C₁₀Alkyl, -NHC (O) NHPh, substituted by 1, 2 or 3C₁-C₄Alkyl radical, C₁-C₃Alkoxy, halogen, C₁-C₃Alkylthio, trifluoromethyl, C₂-C₆Dialkylamino, or nitro substituted-NHC (O) NHPh, -NHC (O) C₃-C₈Cycloalkyl, -NHC (O) C (CH)₂CH₃)₂CH₃、-NHC(O)C(CH₃)₂CH₂Cl、-NHC(O)C(CH₃)₂CH₂CH₃Phthalimido, -NHC (O) -1-phenyl-1-cyclopentyl, -NHC (O) -1-methyl-1-cyclohexyl, -NHC (S) NHC (CH)₃)₃、-NHC(O)NHCC(CH₃)₃or-NHC (O) NHPh; r₃Selected from the group consisting of-H, -NHC (O) phenyl or-NHC (O) OC (CH)₃)₃With the proviso that R₂And R₃One is-H, but R₂And R₃Are not all-H; r₄is-H, or is selected from the group consisting of-OH, -OAc (-OC (O) CH₃)、-OC(O)OCH₂C(C1)₃、-OCOCH₂CH₂NH₃ ⁺HCOO^--NHC (O) phenyl, -NHC (O) OC (CH)₃)₃、-OCOCH₂CH₂COOH and pharmaceutically acceptable salts thereof, -OCO (CH)₂)₃COOH and pharmaceutically acceptable salts thereof, and-OC (O) -Z-C (O) -R' [ wherein Z is ethylene (-CH)₂CH₂-) propylene (-CH)₂CH₂CH₂-, -CH-, 1, 2-cyclohexyl or 1, 2-phenylene, R 'is-OH, -OH base, -NR'₂R’₃、-OR’₃、-SR’₃、-OCH₂C(O)NR’₄R’₅Wherein R'₂is-H or-CH₃，R’₃Is- (CH)₂)_nNR’₆R’₇Or (CH)₂)_nN⁺R’₆R’₇R’₈X-, wherein n is 1-3, R'₄is-H or-C₁-C₄Alkyl, R'₅is-H, -C₁-C₄Alkyl, benzyl, hydroxyethyl, -CH₂CO₂H or dimethylaminoethyl, R'₆And R'₇is-CH₃、-CH₂CH₃Benzyl, or R'₆And R'₇And NR'₆R’₇Together the nitrogen in (a) forms pyrrolidinyl, piperidinyl, morpholinyl (morpholino), or N-methylpiperazinyl (piperazino); r'₈is-CH₃、-CH₂CH₃Or benzyl, X^-Is a halide ion (halide) and the base is NH₃、(HOC₂H₄)₃N、N(CH₃)₃、CH₃N(C₂H₄)₂NH、NH₂(CH₂)₆NH₂N-methyl grapeSugar amine, NaOH or KOH]、-OC(O)(CH₂)_nNR²R³[ wherein n is 1-3, R²is-H or-C₁-C₃Alkyl and R³is-H or-C₁-C₃Alkyl radical]、-OC(O)CH(R”)NH₂[ wherein R' is selected from the group consisting of-H, -CH₃、-CH₂CH(CH₃)₂、-CH(CH₃)CH₂CH₃、-CH(CH₃)₂、-CH₂Phenyl, - (CH)₂)₄NH₂、-CH₂CH₂COOH、-(CH₂)₃NHC(＝NH)NH₂Group of]Residue of the amino acid proline, -OC (O) CH ═ CH₂、-C(O)CH₂CH₂C(O)NHCH₂CH₂SO₃ ^-Y⁺、-OC(O)CH₂CH₂C(O)NHCH₂CH₂CH₂SO₃ ^-Y⁺Wherein Y is⁺Is Na⁺Or N⁺(Bu)₄、-OC(O)CH₂CH₂C(O)OCH₂CH₂OH；R₅is-H or-OH, with the proviso that when R is₅When it is-OH, R₄is-H, with the further proviso that when R is₅When is-H, R₄Is not-H; when R is₇Is alpha-R₇₁：β-R₇₂And R is₈is-CH₃When R is₆is-H: -H, wherein R₇₁And R₇₂One of (A) is-H, R₇₁And R₇₂is-X, wherein X is halogen; when R is₇Is alpha-H: beta-R₇₄When R is₆is-H: -H, wherein R₇₄And R₈Together form a cyclopropyl ring; r₁₀is-H or-C (O) CH₃(ii) a And pharmaceutically acceptable salts thereof when the compound contains an acidic or basic functional group.

Particular paclitaxel analogs include (azidophenyl) ureido) taxane (taxoid), (2 α,5 α,7 β,9 α, 10 β, 13 α) -5, 10, 13, 20-tetraacetoxycetaxel-11-ene-2, 7, 9-triol, (2 α,5 α,9 α, 10 β) -2, 9, 10-triacetoxy-5- ((β -D-glucopyranosyl) oxy) -3, 11-cyclotax-11-ene-13-one, 1 β -hydroxybaccatin (catbacin) I, 1, 7-dihydroxytaxol (pinocembrin, taxine), 1-acetyl-5, 7, 10-deacetyl-baccatin I, 1-dehydroxybaccatin VI, 1-hydroxy-2-deacetyloxy-5-deacetyl-cinnamoyl-taxol VI j. 1-hydroxy-7, 9-dideacetyl baccatin I, 1-hydroxy baccatin I, 10-acetyl-4-deacetyltaxotere (taxotere), 10-deacetoxy paclitaxel, 10-deacetylbaccatin III dimethyl sulfoxide desolvate (discolvate), 10-deacetyl-10- (3-aminobenzoyl) paclitaxel, 10-deacetyl-10- (7- (diethylamino) coumarin-3-carbonyl) paclitaxel, 10-deacetyl-9-dihydrotaxol (taxol ), 10-deacetylbaccatin III, 10-deacetyltaxol, 10-deoxyl-10-C-morpholinoethyl taxane (docetaxel, docetaxel), 10-O-acetyl-2-O- (cyclohexylcarbonyl) -2-debenzoyltaxotere, 10-O-sec-aminoethyl-taxotere, 11-desmethylsultaine, 13-deoxy-13-acetoxy-7, 9-diacetyl-1, 2-dideoxy-taxine (taxine), 13-deoxybaccatin III, 14-hydroxy-10-deacetyl-2-O-debenzoylbaccatin III, 14-hydroxy-10-deacetylbaccatin III, 14 beta-benzoyloxy-13-deacetylbaccatin IV, 14 beta-benzoyloxy-2-deacetylbaccatin VI, 14 beta-benzoyloxy baccatin IV, 19-Hydroxybaccatin III, 2 ', 2 "-Methylenetaxerpene, 2', 2" -MethyleneTaxol, 2 '- (valyl-leucyl-lysyl-PABC) Taxol, 2' -acetyltaxol, 2 '-O-acetyl-7-O- (N- (4' -Fluorescentocarbonyl) alanyl) taxol, 2, 10, 13-triacetoxy-Taxa-4 (20), 11-diene-5, 7, 9-triol, 2, 20-O-diacetylditerpene taxane (taxumarol) N, 2- (4-azidobenzoyl) taxol, 2-deacetoxytaxol J, 2-debenzoyl-2-m-methoxybenzoyl-7-triethylsilyl-13-oxo-14-Hydroxybaccatin Carting III1, 14-carbonate, 2-O- (cyclohexylcarbonyl) -2-debenzoylbarbutin III13-O- (N- (cyclohexylcarbonyl) -3-cyclohexylisoserinate), 2 α,7 β,9 α, 10 β, 13 α -pentaacetoxytaxus-4 (20), 11-dien-5-ol, 2 α,5 α,7 β,9 α, 13 α -pentahydroxy-10 β -acetoxytaxus-4 (20), 11-dien, 2 α,7 β,9 α, 10 β, 13-pentaacetoxy-11 β -hydroxy-5 α - (3 '-N, N-dimethylamino-3' -phenyl) -propionyloxytaxus-4 (20), 12-diene, 2 α,7 β -diacetoxy-5 α, 10 β, 13 β -trihydroxy-2 (3-20) rosin (abeo) -taxus-4 (20), 11-dien-9-one, 2 α,9 α -dihydroxy-10 β, 13 α -diacetoxy-5 α - (3 ' -methylamino-3 ' -phenyl) -propionyloxytaxus-4 (20), 11-diene, 2 α -hydroxy-7 β,9 α, 10 β, 13 α -tetraacetoxy-5 α - (2 ' -hydroxy-3 ' -N, N-dimethylamino-3 ' -phenyl) -propionyloxytaxus-4 (20), 11-diene, 2 α -diacetoxy-5 α, 10 β, 13 β -trihydroxy-2 (3-20), 3 '- (4-azidobenzoylamino) taxol, 3' -N- (4-benzoyldihydrocinnamoyl) -3 '-N-debenzoylpaclitaxel, 3' -N-m-aminobenzoylamino-3 '-debenzoylpaclitaxel, 3' -p-hydroxypaclitaxel, 3, 11-cyclotaxol NN-2, 4-deacetyltaxol, 5, 13-diacetoxy-taxol-4 (20), 11-diene-9, 10-diol, 5-O-benzoyltaxol K, 5-O-phenylpropionyloxytaxol A, 5 alpha, 13 alpha-diacetoxy-10 beta-cinnamoyloxy-4 (20), 11-taxadiene-9 alpha-ol, 11-benzoyltaxol, 6,3 ' -dihydroxytaxol, 6-alpha-hydroxy-7-deoxy-10-deacetylbaccatin-III, 6-fluoro-10-acetyltaxol, 6-hydroxytryptadine, 7, 13-diacetoxy-5-cinnamoyloxy-2 (3-20) -rosin-taxol-4 (20), 11-diene-2, 10-diol, 7, 9-dideacetyl baccatin VI, 7- (5 ' -biotinylaminopropionyl) taxol, 7- (5 ' -biotinylamidopropanoyltaxol), 7-acetyltaxol, 7-deoxy-10-deacetylbaccatin-III, 7-deoxy-9-dihydrotaxol, 7-epitaxol, 7-methylthiotaxoid, 7-O- (4-benzoyldihydrocinnamoyl) taxol, 7-O- (N- (4 ' -fluorescein carbonyl) alanyl) taxol, 7-xyloside-10-deacetyltaxol, 8, 9-mono-epoxy brevifolin (8, 9-single-epoxy brevifolin), 9-dihydrobaccatin III, 9-dihydrotaxol, 9 alpha-hydroxy-2 alpha, 10 beta, 13 alpha-triacetoxy-5 alpha- (3 ' -N, N-dimethylamino-3 ' -phenyl) -propionyloxytaxol-4 (20), 11-diene, baccatin III13-O- (N-benzoyl-3-cyclohexylisoserine ester), BAY59, benzoyltaxol, BMS 181339, BMS 185660, BMS 188797, Brevlin (brevifoliol), butitaxel, cephalomannine (cephalomannine), taxane diterpene (dantaxusin) A, taxane diterpene B, taxane diterpene C, taxane diterpene D, dibromo-10-deacetylcephalomannine (cephalomannine), DJ927, taxane terpene, Flutax 2, glutaryl taxol 6-aminohexanol glucuronide, IDN 5109, IDN5111, IDN 5127, IDN 5390, isolaulimide, laulimalitide, MST 997, N- (taxol-2 '-O- (2-amino) phenylpropionate) -O- (beta-glucoside) carbamate, N- (taxol-2' -O-3, 3-dimethylbutyrate) -O- (beta-glucoside) carbamate, N-debenzoyl-N- (3- (dimethylamino) benzoyl) paclitaxel, nonataxel (nonataxel), octreotide (octreotide) -conjugated paclitaxel, paclitaxel-transferrin, PNU 166945, poly (ethylene glycol) -conjugated paclitaxel-2' -glycinate, polyglutamic acid-paclitaxel, protax, paclitaxel precursor (protaxel), RPR 109881A, SBT-101187, SBT-1102, SBT-1213, SBT-1214, SBT-1250, SBT-12843, tasumatrol E (taxanes), tasumatrol F, tasumatrol G, paclitaxel-4 (20), 11(12) -diene-5-yl acetate, paclitaxel-4 (20), 11(12) -diene-5-ol, taxane (taxanes), taxchi N, taxotere N, paclitaxel-2-beta-hydroxy-E, and paclitaxel, Paclitaxel D (taxcundine), taxoterine (taxzopidine) M, taxoterine N, taxine, taxol A, taxol M, taxol NN-1, taxol NN-7, taxol C-7-xylose, taxol-sialyl (taxol-sialyl) conjugate, diterpene taxane (taxumarol) A, diterpene taxane B, diterpene taxane G, diterpene taxane H, diterpene taxane I, diterpene taxane K, diterpene taxane M, diterpene taxane N, diterpene taxane O, diterpene taxane U, diterpene taxane V, diterpene taxane W, diterpene taxane-X, diterpene taxane-Y, diterpene taxane-Z, taxol (taxusin), taxusinane A, taxusinane B, taxol (taxusine) C, taxol-N, diterpene taxane O, diterpene taxane U, diterpene taxane V, diterpene W, diterpene taxane-X, diterpene C, taxane-X, taxane-X, yunnan Taxus chinensis T, Yunnan Taxus chinensis U, Yunnan Taxus chinensis V, tRA-96023, and wallifoliol. Other paclitaxel analogs include 1-deoxytaxel, 10-deacetoxy-7-deoxytaxel, 10-O-deacetyltaxol 10-succinyl monoester, 10-succinyl taxol, 12 b-acetoxy-2 a, 3,4, 4a, 5, 6,9, 10, 11, 12, 12a, 12 b-dodecahydro-4, 11-dihydroxy-12- (2, 5-dimethoxybenzyloxy) -4a, 8, 13, 13-tetramethyl-5-oxo-7, 11-methano (methano) -1H-cyclodecano (3, 4) benzo (1, 2-b) oxet-9-yl 3- (tert-butoxycarbonyl) amino-2-hydroxy-5-methyl-4-hexenoate, 130-nm albumin-bound paclitaxel, 2 ' -paclitaxel methyl 2-glucopyranosyl succinate, 3 ' - (4-azidophenyl) -3 ' -debenzylpaclitaxel, 4-fluoropaclitaxel, 6, 8-trimethyl-4, 4a, 5, 6, 7, 7a, 8, 9-octahydrocyclopenta (4, 5) cyclohepta (1, 2-c) -furan-4, 8-diol 4- (N-acetyl-3-phenylisoserine ester), 6, 8-trimethyl-4, 4a, 5, 6, 7, 7a, 8, 9-octahydrocyclopenta (4, 5) cyclohepta (1, 2-c) -furan-4, 8-diol 4- (N-tert-butoxycarbonyl-3-phenylisoserine ester), 7- (3-methyl-3-nitrosobutyryl) paclitaxel, 7-deoxypaclitaxel, 7-succinyl paclitaxel, A-Z-CINN310, AI-850, albumin bound paclitaxel, AZ 10992, isotaxel, MAC321, MBT-0206, NK105, paclitaxel, docetaxel, paclitaxel-EC-1 conjugate, paclitaxel and TXD 258. Other paclitaxel analogs are described in U.S. Pat. nos. 4,814,470, 4,857,653, 4,942,184, 4,924,011, 4,924,012, 4,960,790; 5,015,744; 5,157,049, respectively; 5,059,699, respectively; 5,136,060, respectively; 4,876,399, respectively; and 5,227,400.

Other hydrophobic agents

Other hydrophobic agents include analgesics and anti-inflammatory agents (e.g., aloprine, auranofin (auranofin), apazone, benorilate, diflunisal, etodolac, fenbufen, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac), anthelmintics (e.g., albendazole, benzofenapyr hydroxyfenamate, canabendazole, diclofenac, ivermectin, mebendazole, oxaniliquinone, oxfendazole, metaphenamidine pamoate, praziquantel, thiabendazole), antiarrhythmics (e.g., iodofurone hydrochloride, disopropionamide, flufenib acetate), quinuclidine sulfate (e.g., benezole), antibacterial agents (e.g., benezole, mefenaminoxidine, thiabendazole), antiarrhythmics (e.g., iodofurone hydrochloride, disopropioglitazone, flufenil, flufenib, fluquinacrine, flu, Cinoxacin, ciprofloxacin hydrochloride, clarithromycin, clofazimine, cloxacillin, demeclocycline, doxycycline, erythromycin, ethionamide, imipenem, nalidixic acid, nitrofurantoin, rifampin, spiramycin, sulfonamides (sulfobenzamide), sulfadoxine, sulfamethazine, sulfacetamide, sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfapyridine, tetracycline, trimethoprim), anticoagulants (e.g., dicoumarole, dipyridamole, nitrocoumarin (nicoumalone), phenindione), antidepressants (e.g., amoxapine, maprotiline hydrochloride, miserelin hydrochloride, nortriptyline hydrochloride, trazodone hydrochloride, trimethopremazoline maleate), antidiabetic agents (e.g., hexylurea acetate, chlorpropamide, glibenclamide), ziamide (glibenclamide), zicide (gliclazide), glipizide (glipizide), glibenclamide (glibenclamide), glibenclamide (glibenclamide), gli, Tolazamide (tolazamide), tolbutamide), antiepileptics (e.g., becloramide, carbamazepine, clonazepam, ethionine, mephenytoin, ethosuximide, tolbarbital, oxcarbazepine, metiprone, phenylacetamide, phenobarbital, phenytoin, phensunamide, pamipronide, sultamide, valproic acid), antifungals (e.g., amphotericin, butoconazole nitrate, clotrimazole, econazole nitrate, fluconazole, flucytosine, griseofulvin, itraconazole, ketoconazole, miconazole, natamycin, nystatin, sulconazole nitrate, terbinafine hydrochloride, terconazole, tioconazole, undecylenic acid), antigout agents (e.g., allopurinol, probenecosulfamide, metrazone (sulphin-pyrazone)), antihypertensive agents (e.g., amlodipine, bezazepine, dil hydrochloride, diltiazepine hydrochloride (HCl)), thiodil hydrochloride (sultiazem hydrochloride), sulindazone-sulindac, and other, Diazoxide, felodipine, guanabenz acetate, isradipine, minoxidil, nicardipine hydrochloride, nifedipine, nimodipine, phenoxybenzamine hydrochloride, prazosin hydrochloride, reserpine, terazosin hydrochloride, antimalarials (e.g., amodiaquine, chloroquine, chloropropuanidine hydrochloride, halofantrine hydrochloride, mefloquine hydrochloride, proguanil hydrochloride, pyrimethamine, quinine sulfate), antimigraine drugs (e.g., dihydroergotamine mesylate, ergotamine tartrate, dimethylacrogonine maleate, benzothidine maleate, sumatriptan succinate), antimuscarinics (e.g., atropine), benzhexol hydrochloride (benzhexol HCl), biperiden, propaferazine hydrochloride (ethoprozine), HCl, erucidine, bromphencyclamate, oxyphenamine hydrochloride (HCl), phencyclamine hydrochloride), immunosuppressants (e), such as immunosuppressants (e), aminoglutethimide, amsacrine, azathioprine, busulfan, chlorambucil, cyclosporine, dacarbazine, estramustine, etoposide, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone (mitozantrone), procarbazine hydrochloride, tamoxifen citrate, testosterone), antiprotozoal agents (e.g., benznidazole, clioquinol, decoquine, diiodohydroxyquinoline, furoic acid dicholoride, dinitramide, furazolone, metronidazole, nimorazole, nitrofural, ornidazole, sulfoniomidazole), antithyroid agents (e.g., hyperthyroid, propylthiouracil), anxiolytic agents, sedatives, hypnotics and tranquilizers (e.g., alprazolam, amobarbiturate, barbiturate, phencyclam, bromazepine, loprazine, tipepidol, tolamine, teton, tetar, barbiturate, bromhexedron, bromhexine, butoxib, Carbromurea, methotrexate, clomethiazole, chlorpromazine, clobazam, chlordiazepam, clozapine, diazepam, dapipramine, fluazinam (flunaniSone), flunitrazepam, trifluoroperazine, triflutal decanoate, fluphenazine decanoate, fluazinam, haloperidol, chlorohydroxazepam, chlordiazepam, diazepam, meprobamate, hypnone, midazolam, nitro diazepam, nordiazepam, pentobarbital, hydroxypiperidazine chlorpromazine, meprobrazine, sulpiride, hydroxydiazepam, methidaw, triazabepilone, piperazinopirox), beta-blockers (e.g., acebutolol, alprenolol, atenolol, labetalol, metoprolol, hydroxyamphetamine, pindol, pindolol, propranolol), hadrons, doxolols (e.g., amiloride, digoxin glycoside, homoeosin, clobazamide, pennogenol, clozaprinone, clobetamethamphetamol, clozaprinol, caprenolol, clozap, Eriocitrin C, methyl digoxin), corticosteroids (e.g., beclomethasone, betamethasone, budesonide, cortisone acetate, desoximetasone, dexamethasone, fludrocortisone acetate, flunisolide, fluocortolone (flucortolone), fluticasone propionate, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), diuretics: acetazolamide, amiloride, benflumethiazide, butoxyfenac, chlorothiazide, chlorthalidone, diuretic acid, furosemide, metolazone, spironolactone, triamterene, antiparkinson drugs (e.g., bromocriptine mesylate, ergometridine maleate), gastrointestinal drugs (e.g., bisacetoxyphenylmethylpyridine, cimetidine, cisapride, cyanophenamide hydrochloride, pimozide, famotidine, cloperamide, mesalazine, nizatidine, omeprazole, ondansetron hydrochloride, ranitidine hydrochloride, azapirodine), histamine H-receptor antagonists (e.g., atorvastatin, astemizole, cinnarizine, cyclizine hydrochloride, dibenzosuberidine hydrochloride, dimenhydrimine, flutolrizine hydrochloride, loratadine hydrochloride, clevidine hydrochloride, oxamide, oxfenadine, terfenadine), lipid-regulating agents (e.g., bezafibrate, triamcinolone acetonide, flufenadine, flufenide, fluazinafenadine hydrochloride, and mixtures thereof), Clofibrate, fenofibrate, gemfibrozil, probucol), nitrates and other anti-angina drugs (e.g., amyl nitrate, glyceryl trinitrate, isosorbide dinitrate, isosorbide mononitrate, pentaerythritol tetranitrate), opioid analgesics (e.g., codeine, dextropropoxyphene, diacetylmorphine, dihydrocodeine, meptazinol, methadone, morphine, naltrexone, analgesic), sex hormones (e.g., clostilbenamine citrate, norgestrel, ethinylestradiol, medroxyprogesterone, norethindrone, medroxyprogesterone, methyltestosterone, norethindrone, estradiol, conjugated estrogens, progesterone, conolone, diethylstilbestrol, testosterone, tibolone) and stimulants (e.g., amphetamine, dextroamphetamine, dextroflufen-propylamine, dextroflurprimidine, clomazone).

Polypeptide conjugates

Conjugates comprising an active agent and a polypeptide may be used in the formulations described herein. We have developed polypeptide-agent conjugates as described in U.S. patent application publication 2006/0182684, and 2006/0189515, and U.S. provisional application 61/008,880, filed 12/20/2007. Such conjugates can include any of the polypeptides, hydrophobic agents, such as paclitaxel or paclitaxel analogs (e.g., those described herein), and linkers (e.g., those described herein) described herein. The paclitaxel conjugate can be exemplified by ANG1005, which comprises a angiopep-2 peptide (SEQ ID NO: 97) bound to three paclitaxel molecules via an N-terminal ester bond, and via lysines at positions 10 and 15. The structure of ANG1005 is:

the conjugate, in certain embodiments, can cross the Blood Brain Barrier (BBB) or can preferably target certain cell types, such as hepatocytes, lung cells, kidney cells, muscle cells, or can target tumor cells (of any of the cell types described herein). These agents that bind to these peptides may exhibit an increase in the amount taken up by target cells, for example, by receptor-mediated endocytosis (e.g., by the LRP receptor). The binding agent may, alternatively or additionally, exhibit increased stability or reduced eviction by the cell (e.g., due to P-glycoprotein mediated efflux).

Polypeptides

The compositions and methods of the invention can include any of the polypeptides described herein, such as any of the polypeptides described in Table 10 (e.g., any of SEQ ID NOS: 1-105 and 107-112, such as the polypeptides defined by SEQ ID NOS: 1-97, 99, 100, 101, or 107-112), or any fragment, analog, derivative, or variant thereof. In certain embodiments, the polypeptide can have at least 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100% identity to a polypeptide described herein. The polypeptide may have one or more (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) substitutions with respect to one of the sequences described herein. Other modifications will be described in more detail below.

The invention also features fragments (e.g., functional fragments) of these polypeptides. In certain embodiments, the fragment is capable of entering or accumulating in a particular cell type (e.g., liver, lung, kidney, spleen, or muscle) or is capable of crossing the BBB. Truncations of the polypeptide may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids derived from the N-terminus of the polypeptide, the C-terminus of the polypeptide, or a combination thereof. Other fragments include sequences that are deleted internally in the polypeptide.

Other polypeptides may be identified using one of the assays or methods described in U.S. patent application publication 2006/0189515, which is incorporated herein by reference, or using any method known in the art. For example, candidate vectors can be produced by conventional polypeptide synthesis methods, can be combined with taxol, and can be administered to experimental animals. The biologically active carrier can be identified, for example, based on its potency, which can increase the survival of animals injected with tumor cells and treated with the conjugate, as compared to controls not treated with the conjugate (e.g., treated with the unconjugated agent).

In another example, a biologically active polypeptide can be identified based on its location in parenchyma (parenchyma) in an in situ brain perfusion assay. In vitro BBB assays, e.g. from CELLIAL^TMTechnically developed models can be used to identify such vectors.

Experiments can also be performed to determine its accumulation in other tissues. A labeled conjugate of the polypeptide can be administered to an animal and its accumulation in different organs can then be measured. For example, polypeptides bound to a detectable label (e.g., a near IR fluorescence spectroscopy label, such as cy5.5) allow for in vivo visualization in real time (live in vivo visualization). Administering such a polypeptide to an animal and detecting the presence of the polypeptide in an organ, thereby allowing the rate and amount of accumulation of the polypeptide in the desired organ to be determined. In other embodiments, the radioisotope (e.g.,¹²⁵I) a marker polypeptide. The polypeptide is then administered to the animal. After a period of time, the animal is sacrificed and organs of the animal are removed. The amount of radioisotope in each organ can then be measured using any means known in the art. By comparing the amount of labeled candidate polypeptide in a particular organ with the amount of labeled control, the ability of the candidate polypeptide to accumulate in a particular tissue, as well as the rate and amount of accumulation, can be determined. Suitable negative controls include any polypeptide known to be incapable of being effectively transported into a particular cell type.

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SEQ ID

NO：

1 T F V Y G G C R A K R N N F K S A E D

2 T F Q Y G G C M G N G N N F V T E K E

3 P F F Y G G C G G N R N N F D T E E Y

4 S F Y Y G G C L G N K N N Y L R E E E

5 T F F Y G G C R A K R N N F K R A K Y

6 T F F Y G G C R G K R N N F K R A K Y

7 T F F Y G G C R A K K N N Y K R A K Y

8 T F F Y G G C R G K K N N F K R A K Y

9 T F Q Y G G C R A K R N N F K R A K Y

10 T F Q Y G G C R G K K N N F K R A K Y

11 T F F Y G G C L G K R N N F K R A K Y

12 T F F Y G G S L G K R N N F K R A K Y

13 P F F Y G G C G G K K N N F K R A K Y

14 T F F Y G G C R G K G N N Y K R A K Y

15 P F F Y G G C R G K R N N F L R A K Y

16 T F F Y G G C R G K R N N F K R E K Y

17 P F F Y G G C R A K K N N F K R A K E

18 T F F Y G G C R G K R N N F K R A K D

19 T F F Y G G C R A K R N N F D R A K Y

20 T F F Y G G C R G K K N N F K R A E Y

21 P F F Y G G C G A N R N N F K R A K Y

22 T F F Y G G C G G K K N N F K T A K Y

23 T F F Y G G C R G N R N N F L R A K Y

24 T F F Y G G C R G N R N N F K T A K Y

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25 T F F Y G G S R G N R N N F K T A K Y

26 T F F Y G G C L G N G N N F K R A K Y

27 T F F Y G G C L G N R N N F L R A K Y

28 T F F Y G G C L G N R N N F K T A K Y

29 T F F Y G G C R G N G N N F K S A K Y

30 T F F Y G G C R G K K N N F D R E K Y

31 T F F Y G G C R G K R N N F L R E K E

32 T F F Y G G C R G K G N N F D R A K Y

33 T F F Y G G S R G K G N N F D R A K Y

34 T F F Y G G C R G N G N N F V T A K Y

35 P F F Y G G C G G K G N N Y V T A K Y

36 T F F Y G G C L G K G N N F L T A K Y

37 S F F Y G G C L G N K N N F L T A K Y

38 T F F Y G G C G G N K N N F V R E K Y

39 T F F Y G G C M G N K N N F V R E K Y

40 T F F Y G G S M G N K N N F V R E K Y

41 P F F Y G G C L G N R N N Y V R E K Y

42 T F F Y G G C L G N R N N F V R E K Y

43 T F F Y G G C L G N K N N Y V R E K Y

44 T F F Y G G C G G N G N N F L T A K Y

45 T F F Y G G C R G N R N N F L T A E Y

46 T F F Y G G C R G N G N N F K S A E Y

47 P F F Y G G C L G N K N N F K T A E Y

48 T F F Y G G C R G N R N N F K T E E Y

49 T F F Y G G C R G K R N N F K T E E D

50 P F F Y G G C G G N G N N F V R E K Y

51 S F F Y G G C M G N G N N F V R E K Y

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52 P F F Y G G C G G N G N N F L R E K Y

53 T F F Y G G C L G N G N N F V R E K Y

54 S F F Y G G C L G N G N N Y L R E K Y

55 T F F Y G G S L G N G N N F V R E K Y

56 T F F Y G G C R G N G N N F V T A E Y

57 T F F Y G G C L G K G N N F V S A E Y

58 T F F Y G G C L G N R N N F D R A E Y

59 T F F Y G G C L G N R N N F L R E E Y

60 T F F Y G G C L G N K N N Y L R E E Y

61 P F F Y G G C G G N R N N Y L R E E Y

62 P F F Y G G S G G N R N N Y L R E E Y

63 M R P D F C L E P P Y T G P C V A R I

64 A R I I R Y F Y N A K A G L C Q T F V Y G

65 Y G G C R A K R N N Y K S A E D C M R T C G

66 P D F C L E P P Y T G P C V A R I I R Y F Y

67 T F F Y G G C R G K R N N F K T E E Y

68 K F F Y G G C R G K R N N F K T E E Y

69 T F Y Y G G C R G K R N N Y K T E E Y

70 T F F Y G G S R G K R N N F K T E E Y

71 C T F F Y G C C R G K R N N F K T E E Y

72 T F F Y G G C R G K R N N F K T E E Y C

73 C T F F Y G S C R G K R N N F K T E E Y

74 T F F Y G G S R G K R N N F K T E E Y C

75 P F F Y G G C R G K R N N F K T E E Y

76 T F F Y G G C R G K R N N F K T K E Y

77 T F F Y G G K R G K R N N F K T E E Y

78 T F F Y G G C R G K R N N F K T K R Y

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79 T F F Y G G K R G K R N N F K T A E Y

80 T F F Y G G K R G K R N N F K T A G Y

81 T F F Y G G K R G K R N N F K R E K Y

82 T F F Y G G K R G K R N N F K R A K Y

83 T F F Y G G C L G N R N N F K T E E Y

84 T F F Y G C G R G K R N N F K T E E Y

85 T F F Y G G R C G K R N N F K T E E Y

86 T F F Y G G C L G N G N N F D T E E E

87 T F Q Y G G C R G K R N N F K T E E Y

88 Y N K E F G T F N T K G C E R G Y R F

89 R F K Y G G C L G N M N N F E T L E E

90 R F K Y G G C L G N K N N F L R L K Y

91 R F K Y G G C L G N K N N Y L R L K Y

92 K T K R K R K K Q R V K I A Y E E I F K N Y

93 K T K R K R K K Q R V K I A Y

94 R G G R L S Y S R R F S T S T G R

95 R R L S Y S R R R F

96 R Q I K I W F Q N R R M K W K K

97 T F F Y G G S R G K R N N F K T E E Y

98 M R P D F C L E P P Y T G P C V A R I

I R Y F Y N A K A G L C Q T F V Y G G

C R A K R N N F K S A E D C M R T C G G A

99 T F F Y G G C R G K R N N F K T K E Y

100 R F K Y G G C L G N K N N Y L R L K Y

101 T F F Y G G C R A K R N N F K R A K Y

102 N A K A G L C Q T F V Y G G C L A K R N N F

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E S A E D C M R T C G G A

103 Y G G C R A K R N N F K S A E D C M R T C G

G A

104 G L C Q T F V Y G G C R A K R N N F K S A E

105 L C Q T F V Y G G C E A K R N N F K S A

107 T F F Y G G S R G K R N N F K T E E Y

108 R F F Y G G S R G K R N N F K T E E Y

109 R F F Y G G S R G K R N N F K T E E Y

110 R F F Y G G S R G K R N N F R T E E Y

111 T F F Y G G S R G K R N N F R T E E Y

112 T F F Y G G S R G R R N N F R T E E Y

113 C T F F Y G G S R G K R N N F K T E E Y

114 T F F Y G G S R G K R N N F K T E E Y C

115 C T F F Y G G S R G R R N N F R T E E Y

116 T F F Y G G S R G R R N N F R T E E Y C

Peptide number 5 includes SED ID NO: 5 sequence and amidation at its C-terminus

Peptide number 67 includes SED ID NO: 67 sequence and amidation at its C-terminus

Peptide number 76 includes SED ID NO: 76 sequence and amidation at its C-terminus

Peptide number 91 includes SED ID NO: 91 sequence and amidation at its C-terminus

Peptide number 107 includes SED ID NO: 97 and acetylated at its N-terminus

Peptide number 109 includes SED ID NO: 109 sequence and acetylated at its N-terminus

Peptide number 110 includes SED ID NO: 110 sequence and acetylated at its N-terminus

The amino groups of angiopep-1 (SED ID NO: 67) and angiopep-2 (SED ID NO: 97) have been used as sites for binding of agents. To investigate the role of amino groups in binding and their effect on the total transport capacity of these vectors, new vectors based on the sequences of angiopep-1 and angiopep-2 were designed with variable reactive amino groups and variable total charge. These polypeptides are shown in table 11.

Table 11: vectors with variable amino targets

Angiopep-3 is an acetylated form of angiopep-2.

¹Ac represents acetylation.

Modified polypeptides

The compositions and methods of the invention may also include polypeptides having amino acid sequence modifications described herein (e.g., polypeptides having a sequence described in any of SEQ ID NOS: 1-105 and 107-116, e.g., angiopeptins-3, -4a, -4b, -5, -6, or-7). In certain embodiments, the modification does not significantly disrupt the desired biological activity. In some embodiments, the modification can decrease biological activity (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%). In other embodiments, the modification has no effect on biological activity, or can increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the biological activity of the original polypeptide. The modified polypeptide may have or may optimize one or more of the properties of the polypeptide of the invention, which properties may be desirable or desirable in some circumstances. Such properties include in vivo stability, bioavailability, toxicity, immunological activity and immunological properties.

Polypeptides for use in the present invention may include amino acids or sequences that have been modified by natural processes, such as post-translational processing or by chemical modification techniques known in the art. Modifications can occur anywhere in the polypeptide, including the polypeptide backbone, the amino acid side chains, and the amino-or carboxy-terminus. The same type of modification may be present to the same or different degrees at several sites in a given polypeptide, and a polypeptide may contain more than one type of modification. Polypeptides may be branched by ubiquitination (ubiquitination) and may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from post-translational natural processes or may be synthetically produced. Other modifications include pegylation, acetylation, acylation, addition of an acetamidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalently linking flavin, covalently linking a heme moiety, covalently linking a nucleotide or nucleotide derivative, covalently linking a drug, covalently linking a label (e.g., fluorescent or radioactive), covalently linking a lipid or lipid derivative, covalently linking phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-linking, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation (anchorination), hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation (prenylation), Racemization, selenoylation (selenoylation), sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation (arginylation) and ubiquitination.

Modified polypeptides may further include amino acid insertions, deletions (deletions), or conservative or non-conservative substitutions (e.g., D-amino acids, desamino acids) in the polypeptide sequence (e.g., where such changes do not substantially alter the biological activity of the polypeptide).

Substitutions may be conservative (i.e., where a residue is replaced by another residue belonging to the same general type or group) or non-conservative (i.e., where a residue is replaced by an amino acid of another type). In addition, a non-naturally occurring amino acid can be substituted for a naturally occurring amino acid (i.e., a non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution).

Synthetically made polypeptides may include substitutions of amino acids that are not naturally encoded by DNA (e.g., non-naturally occurring or non-natural amino acids). Examples of non-naturally occurring amino acids include D-amino acids, amino acids having an acetamidomethyl group attached to a cysteine sulfur atom, pegylated amino acids, amino acids having the formula NH₂(CH₂)_nOmega amino acids of COOH (where N is 2-6), neutral nonpolar amino acids such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine and norleucine. Phenylglycine may replace Trp, Tyr or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline can be replaced with hydroxyproline and remain in the conformation that confers performance.

Analogs can be generated by substitutional mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions identified as "conservative substitutions" are given in table 12. If such substitutions result in undesired changes, other types of substitutions, referred to in Table 12 as "exemplary substitutions" or as further described herein, for the class of amino acids are introduced and the products screened.

Substantial modification of functional or immunological properties can be achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of the substitution, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Naturally occurring residues can be divided into several groups based on common side chain properties:

(1) hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), histidine (His), tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe),

(2) neutral hydrophilic: cysteine (Cys), serine (Ser), threonine (Thr)

(3) Acidic/negatively charged: aspartic acid (Asp), glutamic acid (Glu)

(4) Basic: asparagine (Asn), glutamine (Gln), histidine (His), lysine (Lys), arginine (Arg)

(5) Residues that influence chain orientation: glycine (Gly), proline (Pro);

(6) aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), histidine (His),

(7) polar: ser, Thr, Asn, Gln

(8) Basic, positively charged: arg, Lys, His, and;

(9) charged: asp, Glu, Arg, Lys, His

Other conservative amino acid substitutions are listed in table 12.

Table 12: other amino acid substitutions

Original residues	Exemplary permutations	Conservative substitutions
			Ala(A)	Val、Leu、Ile	Val
Arg(R)	Lys、Gln、Asn	Lys
			Asn(N)	Gln、His、Lys、Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro	Pro
			His(H)	Asn、Gln、Lys、Arg	Arg
Ile(I)	Leu, Val, Met, Ala, Phe, norleucine	Leu
			Leu(L)	Norleucine, Ile, Val、Met、Ala、Phe	Ile
Lys(K)	Arg、Gln、Asn	Arg
			Met(M)	Leu、Phe、Ile	Leu
Phe(F)	Leu、Val、Ile、Ala	Leu
			Pro(P)	Gly	Gly
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr	Tyr
			Tyr(Y)	Trp、Phe、Thr、Ser	Phe
Val(V)	Ile, Leu, Met, Phe, Ala, norleucine	Leu

Additional analogs

The polypeptides and conjugates used in the present invention may include analogs of aprotinin polypeptides known in the art. For example, U.S. patent 5,807,980 describes bovine trypsin inhibitor (aprotinin) -derived inhibitors, their preparation and therapeutic use, which include SEQ ID NO: 102. These polypeptides have been used to treat conditions characterized by aberrant expression or amount of tissue factor and/or factor VIIIa, such as aberrant thrombosis. Us patent 5,780,265 describes serine protease inhibitors capable of inhibiting plasma kallikrein, comprising SEQ ID NO: 103. U.S. patent No.5,118,668 describes bovine trypsin inhibitor variants including SEQ ID NO: 105. aprotinin amino acid sequence (SEQ ID NO: 98), angiopep-1 amino acid sequence (SEQ ID NO: 67), and SEQ ID NO: 64, and some biologically active analogs can be found in International application publication WO 2004/060403.

Exemplary nucleotide sequences encoding aprotinin analogs are set forth in SEQ ID NO: 106(atgagaccag atttctgcct cgagccgccg tacactgggc cctgcaaagc tcgtatcatccgttacttct acaatgcaaa ggcaggcctg tgtcagacct tcgtatacgg cggctgcagagctaagcgta acaacttcaa atccgcggaa gactgcatgc gtacttgcgg tggtgcttag; Genbank accession number X04666). This sequence encodes a lysine, but not a valine, at position 16, as shown in SEQ ID NO: 98, respectively. SEQ ID NO: 106 can be introduced by methods known in the art to alter the amino acid sequence of SEQ ID NO: 98. Other mutations or fragments may be obtained using any technique known in the art.

Further examples of aprotinin analogs can be found by protein BLAST (Genbank: www.ncbi.nlm.nih.gov/BLAST /), using the synthetic aprotinin sequence (or part thereof) disclosed in International application PCT/CA 2004/000011. Exemplary aprotinin analogs are found according to accession numbers CAA37967 (GI: 58005) and 1405218C (GI: 3604747).

Preparation of polypeptide derivatives and mimetics

In addition to polypeptides consisting solely of naturally occurring amino acids, mimetic peptides or polypeptide analogs can also be used in the present invention. Polypeptide analogs are commonly used in the pharmaceutical industry as non-polypeptide drugs that have properties similar to template polypeptides. Non-polypeptide compounds are referred to as "peptide mimetics" or "peptidomimetics" (Fauchere et al, infection. Immun.54: 283-287, 1986; Evans et al, J.Med. chem.30: 1229-1239, 1987). Peptidomimetics that are structurally related to therapeutically useful polypeptides can be used to produce equivalent or enhanced therapeutic or prophylactic effects. In general, the mimetic peptides are structurally similar to the exemplary polypeptide (i.e., a polypeptide having biological or pharmaceutical activity), such as a naturally occurring receptor binding polypeptide, but having one or more Peptide bonds (peptidelinkage), by methods well known in the art (Spatola, Peptide Back bone modifications, Vega Data, 1 (3): 267, 1983); spatola et al, (Life Sci.38: 1243-1249, 1986); hudson et al, (int.J.Pept.Res.14: 177-185, 1979); and Weinstein.B., 1983, Chemistry and Biochemistry of Aminoacids, Peptides and Proteins, Weinstein eds, Marcel Dekker, New-York), optionally with a peptide bond such as-CH₂NH-、-CH₂S-、-CH₂-CH₂-, -CH-CH- (cis and trans) -, -CH₂SO-、-CH(OH)CH₂-、-COCH₂-isokey substitutions. Such peptidomimetic polypeptides can have significant advantages over naturally occurring polypeptides, including more economical production, greater chemical stability, enhanced pharmacological properties (e.g., half-life, absorption, potency, effectiveness), reduced antigenicity, and others.

Although the polypeptides used in the present invention may be effective to enter a particular cell type (e.g., those described herein), their utility may be reduced by the presence of proteases. Serum proteases have special substrate requirements. The substrate must have L-amino acids and peptide bonds for cleavage. Furthermore, exopeptidases, which represent the most prominent component of protease activity in serum, usually act on the first peptide bond of the polypeptide and require a free N-terminus (Powell et al, pharm. Res. 10: 1268-1273, 1993). In view of this, it is often advantageous to use modified forms of the polypeptides. The modified polypeptide retains the structural properties of the original L-amino acid polypeptide that confer biological activity on IGF-1, but is advantageously not susceptible to cleavage by proteases and/or exopeptidases.

Systematic substitution of one or more amino acids in the consensus sequence with the same type of D-amino acid (e.g., D-lysine instead of L-lysine) can be used to produce more stable polypeptides. Thus, the polypeptide derivatives or peptidomimetics used in the present invention may be all L-type, all D-type, or a mixture of D, L-type polypeptides. The presence of an N-terminal or C-terminal D-amino acid increases the in vivo stability of the polypeptide, since peptidases cannot utilize D-amino acids as substrates (Powell et al, pharm. Res.10: 1268-. A reverse-D polypeptide is a polypeptide containing D-amino acids arranged in a reverse sequence relative to a polypeptide containing L-amino acids. Thus, the C-terminal residue of the L-amino acid polypeptide becomes the N-terminus of the D-amino acid polypeptide, and so on. The reverse D-polypeptide retains the same tertiary conformation and thus the same activity as the L-amino acid polypeptide, but is more stable against enzymatic degradation in vivo and in vitro and thus has a stronger therapeutic efficacy than the original polypeptide (Brady and Dodson, Nature 368: 692 693, 1994; Jameson et al, Nature 368: 744-746, 1994). In addition to the reverse-D-polypeptides, restriction polypeptides (constrained polypeptides) comprising a consensus sequence or substantially identical consensus sequence variations may be generated by methods well known in the art (Rizo and Gierasch, Ann. Rev. biochem. 61: 387-418, 1992). For example, a restricted polypeptide may be produced by adding cysteine residues capable of forming disulfide bridges and thereby giving a cyclic polypeptide. The cyclic polypeptide has no free N-or C-terminus (termini). Thus, they are not easily proteolyzed by exopeptidases, although they are indeed susceptible to endopeptidases, which do not cleave at the polypeptide end. The amino acid sequences of polypeptides with an N-terminal or C-terminal D-amino acid and cyclic polypeptides, respectively, are generally identical to their corresponding polypeptide sequences, except for the presence of an N-terminal or C-terminal D-amino acid residue, or their cyclic structures.

Cyclic derivatives containing intramolecular disulfide bonds can be prepared by conventional solid phase synthesis, with the incorporation of appropriate S-protected cysteine or homocysteine residues at the positions selected for cyclization, e.g., at the amino and carboxy termini (Sah et al, J.Pharm. Pharmacol.48: 197, 1996). After completion of chain assembly, cyclization was performed as follows: (1) by selective removal of the S-protecting groups, with consequent supported oxidation (on-support oxidation) of the corresponding two free SH-functions, thereby forming S-S bonds, followed by conventional removal of the product from the support and appropriate purification procedures; or (2) by removing the polypeptide from the support and completing the side chain deprotection, followed by oxidation of the free SH-functions in a highly diluted aqueous solution.

Cyclic derivatives containing intramolecular amide bonds can be prepared by conventional solid phase synthesis, while incorporating the appropriate amino and carboxy side chain protected amino acid derivatives at the positions selected for cyclization. Cyclic derivatives containing intramolecular-S-alkyl bonds can be prepared by conventional solid phase chemistry, incorporating both an amino acid residue with a suitable amino-protected side chain and a suitable S-protected cysteine or homocysteine residue at the positions selected for cyclisation.

Another effective method of conferring resistance (resistance) to peptidases acting on the N-terminal or C-terminal residue of a polypeptide is to add a chemical group at the polypeptide end so that the modified polypeptide is no longer a substrate for the peptidase. One such chemical modification is glycosylation of the polypeptide at either or both ends. Certain chemical modifications, particularly N-terminal glycosylation, have been shown to increase the stability of polypeptides in human serum (Powell et al, pharm. Res. 10: 1268-. Other chemical modifications that enhance serum stability include, but are not limited to, the addition of an N-terminal alkyl group consisting of lower alkyl groups of 1 to 20 carbon atoms, such as acetyl, and/or the addition of a C-terminal amide or substituted amide group. In particular, the compositions and methods of the invention may include modified polypeptides consisting of polypeptides having an N-terminal acetyl group and/or a C-terminal amide group.

The invention also includes other types of polypeptide derivatives that contain additional chemical moieties that are not common to polypeptides, provided that the derivative retains the desired functional activity of the polypeptide. Examples of such derivatives include (1) N-acyl derivatives of the amino terminus or of another free amino group, wherein the acyl group may be an alkanoyl group (e.g. acetyl, hexanoyl, octanoyl), an aroyl group (e.g. benzoyl) or a blocking group, such as F-moc (fluorenylmethyl-O-CO-); (2) ester of the carboxyl terminus or another free carboxyl or hydroxyl group; (3) carboxyl-terminated or another amide of free carboxyl groups prepared by reaction with ammonia or with a suitable amine; (4) a phosphorylated derivative; (5) derivatives that bind to antibodies or other biological ligands, and other types of derivatives.

Also included are longer polypeptide sequences used in the present invention formed by the addition of additional amino acid residues to the polypeptide. Such longer polypeptide sequences are expected to have the same biological activity (e.g., into a particular cell type) as the polypeptides described above. Although polypeptides with a large number of additional amino acids are not excluded, it is recognized that some large polypeptides may assume a conformation that masks the effective sequence, thereby preventing binding to the target (e.g., a member of the LRP receptor family, such as LRP or LRP 2). These derivatives may act as competitive antagonists. Thus, while the invention includes a polypeptide or polypeptide derivative described herein having an extension (extension), it is desirable that the extension does not disrupt the cell targeting activity of the polypeptide or derivative thereof.

Other derivatives that can be used in the present invention are dual polypeptides (dual polypeptides) consisting of two identical, or two different polypeptides as described herein, wherein the two polypeptides are covalently linked to each other directly or via a spacer, for example via a short chain of alanine residues (short stretch) or via a putative site of proteolysis (for example via a cathepsin, see U.S. Pat. No.5,126,249 and european patent 495049). The polypeptide multimers used in the present invention consist of molecular polymers of the same or different polypeptides or derivatives thereof.

The invention also includes polypeptide derivatives which are chimeric or fusion proteins or fragments thereof containing a polypeptide described herein, linked at their amino or carboxy terminus, or both, to the amino acid sequence of a different protein. Such chimeric or fusion proteins can be produced by recombinant expression of a nucleic acid encoding the protein. For example, a chimeric or fusion protein may contain at least 6 amino acids of a polypeptide used in the present invention and desirably has comparable or higher functional activity than the polypeptide used in the present invention.

The polypeptide derivatives used in the present invention may be prepared by altering the amino acid sequence by substitution, addition or deletion or by altering the amino acid residues to provide functionally equivalent molecules, or functionally enhanced or reduced molecules, as desired. Derivatives for use in the present invention include, but are not limited to, those polypeptide derivatives which contain as the primary amino acid sequence all or part of the amino acid sequence of the polypeptides described herein (SEQ ID NOS: 1-105 and 107-116), including altered sequences which contain functionally equivalent amino acid residue substitutions. For example, one or more amino acid residues in the sequence may be substituted with another amino acid of similar polarity that acts as a functional equivalent, resulting in a silent change. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, positively charged (basic) amino acids include arginine, lysine and histidine. Non-polar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan, and methionine. Uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Negatively charged (acidic) amino acids include glutamic acid and aspartic acid. The amino acid glycine may be included in the family of non-polar amino acids or in the family of uncharged (neutral) polar amino acids. Substitutions made in amino acid families are generally understood to be conservative substitutions.

Assays for identifying mimetic peptides

As described above, non-peptidyl compounds (peptidomimetics) that are generated to replicate the backbone structure and pharmacophore display of the polypeptides identified by this method may have properties of higher metabolic stability, higher potency, longer duration of action, and better bioavailability.

The peptidomimetic compounds used in the present invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, including: biological libraries, spatially addressable parallel (spatially addressable) solid-phase or liquid-phase libraries, synthetic libraries requiring deconvolution, the "one-bead-compound" library approach, and synthetic library approaches using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to small molecule libraries of polypeptides, non-peptide oligomers or compounds (Lam, Anticancer Drug Des. 12: 145, 1997). Examples of methods for synthesizing libraries of molecules can be found in the art, for example, in: DeWitt et al, (Proc. Natl Acad. Sci. USA 90: 6909, 1993); erb et al, (Proc. Natl Acad. Sci. USA 91: 11422, 1994); zuckermann et al, (J.Med.chem.37: 2678, 1994); cho et al, (Science 261: 1303, 1993); carell et al, (Angew. chem, int.Ed. Engl. 33: 2059, 1994and ibid 2061); and in Gallop et al (Med. chem.37: 1233, 1994). The compound library may be present in solution (e.g.Houghten, Biotechniques 13: 412-.

Once a polypeptide useful in the present invention is identified, it can be isolated and purified by a number of standard methods, including, but not limited to, differential solubility methods (e.g., precipitation), centrifugation, chromatography (e.g., affinity, ion exchange, size exclusion, etc.), or by any other standard technique for purifying polypeptides, peptidomimetics, or proteins. The functional properties of the identified polypeptide of interest can be assessed using any functional assay known in the art. Desirably, assays for assessing downstream receptor function (e.g., cell proliferation) in intracellular signals are used.

For example, peptidomimetic compounds for use in the present invention can be obtained using the following three-stage process: (1) scanning a polypeptide for use in the invention to identify regions of secondary structure required to target a particular cell type as described herein; (2) refining (refine) the backbone structure using conformationally constrained dipeptide substitutes (surfates) and providing an organic platform (organic platform) corresponding to these substitutes; and (3) use of the optimal organic platform to show organic pharmacophores in candidate libraries designed to mimic the desired activity of the native polypeptide. These three stages are explained in more detail below. In stage 1, the leading (primary, lead) candidate polypeptide is scanned and its structure is simplified to identify the requirements for its activity. A series of polypeptide analogs of the original polypeptide are synthesized. In stage 2, the best polypeptide analogs were investigated using conformationally constrained dipeptide substitutes. Indolizidin-2-one (indolizidin-2-one), indolizidin-9-one and quinolizidine (quinolizidine) amino acids (I respectively)²aa、I⁹aa and Qaa) can be used as a platform for studying the backbone structure of an optimal polypeptide candidate. These and related platforms (reviewed in Halab et al Biopolymers 55: 10l-122, 2000, and Hanessian et al Tetrahedron 53: 12789-. Biological evaluation of these analogs can identify improved leading polypeptides (lead polypeptides) that mimic the structural requirements for activity. In stage 3, the platform from the most active leader polypeptide mayOrganic substitutes for pharmacophores designed to display the activity of the native polypeptide. The pharmacophore and the scaffold can be combined into a parallel synthetic pattern. The derivatization of the polypeptides and the above stages may also be accomplished in other ways using methods known in the art.

The structural functional relationships defined from the polypeptides, polypeptide derivatives, peptidomimetics, or other small molecules used in the present invention can be used to refine and prepare similar molecular structures with similar or better activity. Thus, compounds for use in the present invention also include molecules having common structural, polar, charge and side chain properties with the polypeptides described herein.

In summary, based on the disclosure herein, one of skill in the art can develop polypeptide or peptidomimetic screening assays useful for identifying compounds that target agents to specific cell types (e.g., those described herein). The assay can be developed for low throughput, high throughput or ultra high throughput screening formats. The tests of the present invention include tests suitable for automation.

Conjugates

The polypeptides described herein or derivatives thereof may be linked to a pharmaceutical agent. For example, a polypeptide (e.g., any of those described herein) can be attached to a therapeutic agent, a diagnostic agent, or a tag. In certain embodiments, the polypeptide is linked to or labeled with a detectable label, such as a radioimaging agent, in order to diagnose a disease or condition. Examples of such agents include a radiographic agent-antibody-carrier conjugate, wherein the antibody binds to a disease or condition-specific antigen (e.g., for diagnosis or treatment). Other binding molecules are also contemplated by the present invention. In other cases, the polypeptide or derivative is linked to a therapeutic agent, thereby treating a disease or condition, or may be linked to or labeled with a mixture thereof. The disease or condition can be treated by administering the carrier-agent conjugate to the individual under conditions that allow transport of the agent across the BBB or into a particular cell type. Each polypeptide may include at least 1, 2, 3,4, 5, 6, or 7 agents. In other embodiments, each agent has at least 1, 2, 3,4, 5, 6, 7, 10, 15, 20, or more polypeptides attached thereto. The conjugates of the invention can promote accumulation of the agent (e.g., due to increased absorption or decreased efflux) in a particular cell type or tissue of the subject, such as the liver, lung, kidney, spleen, or muscle.

The agent may be released from the carrier after transport into a particular cell type or across the BBB. The agent may be released, for example, by enzymatic cleavage or other disruption of the chemical bond between the carrier and the agent. The agent released in the absence of the carrier may then function in its intended capacity.

A therapeutic agent. The therapeutic agent can be any biologically active agent. For example, the therapeutic agent can be a drug, a radiation-emitting agent, a cytotoxin (e.g., a chemotherapeutic agent), a biologically active fragment thereof, or a mixture thereof that treats a disease (e.g., kills cancer cells), or it can be an agent that treats a disease or condition in an individual. The therapeutic agent may be a synthetic product or a product of fungal, bacterial or other microbial (e.g. mycoplasma or viral), animal, such as reptile, or plant origin. The therapeutic agent and/or biologically active fragment thereof can be an enzymatically active agent and/or fragment thereof, or can act by inhibiting or blocking an important and/or essential cellular pathway or by competing with an important and/or essential native cellular component. Other therapeutic agents include antibodies and antibody fragments.

An anticancer agent. Any anti-cancer agent known in the art may be part of the conjugates used in the present invention. In certain embodiments, the agent is paclitaxel or a paclitaxel analog (e.g., those described herein). Brain cancer can be treated with conjugates containing a vector (angiopep-1, angiopep-2, angiopep-3, angiopep-4 a, angiopep-4 b, angiopep-5 or angiopep-6) that can be efficiently transported across the BBB. Liver, lung, kidney or spleen cancer can be treated with an anti-cancer agent conjugated to a carrier that can be efficiently transported into the appropriate cell type (e.g., angiopep-7). Exemplary agents include abarelix (abarelix), aldesleukin (aldesleukin), alemtuzumab (alemtuzumab), alitretinoin (alitretinin), allopurinol (allopurinol), hexamethylmelamine (altretamine), amifostine (amifostine), anakinra (anakinra), anastrozole (anatrozole), arsenic trioxide, asparaginase, azacitidine (azacitidine), BCG Live vaccine (BCG Live), bevacizumab (bevacizumab), bexarotene (bexarotene), bleomycin (eomycin), bleomycin, bortezomib (bortezombi), bortezomib (bortezomib), busulfan (butufan), cisplatin, carthamycin (testosterone), perinone (clavulancine), carboplatin (clavulancine), carboplatin), cytosine (clavulanate), capram (capram), capram (capreomycin), capreomycin (clavulane), capreomycin (capram (capreomycin), carboplatin (clavulane (clavine), capram (clavine), capicine (clavulanine (capicine), capicine (capicine), capicine (capram), capram (capicine (capreomycin), capicine (capram), capram (capicine (capram), capram (capram), capram (capram), cap, Dactinomycin (dactinomycin), actinomycin D (actinomycin D), dalteparin (e.g. sodium), dalteparin alpha (darbepoetin alfa), dasatinib (dasatinib), daunorubicin (daunorubicin), daunomycin (daunomycin), decitabine (decitabine), dinebin (denikin), dinukin 2(denileukin difitox), dexrazoxane (dexrazoxane), docetaxel (doxorubin), doxorubin (doxorubin), dromostanone propionate (dromostanolone propiponate), kukumulumab (ecolizumab), epirubicin (epirubicin) (e.g. HCl, alfawetomidine (efavirin alfa), alfafurtinib (loxacin), estramustin (estrin), estramustine (estramustine), etofluridomycin (5) (e.g. fluvastatin (flufenine), flunixin (e), flunixin (flunixin), flunixin (, Gefitinib (gefitinib), gemcitabine (gemcitabine) (e.g. HCl), gemtuzumab ozogamicin (ozogamicin), goserelin (goserelin) (e.g. acetate), histrelin (histrelin) (e.g. acetate), hydroxyurea (hydroxyurea), temozolomide (ibrituitan), idarubicin (idarubicin), ifosfamide (ifosfamide), imatinib (imatinib) (e.g. deferoxamine (mesylate)), Interferon alpha-2 b (Interferon alfa-2b), irinotecan (irinotecan), lapatinib ditosylate (lapatinib), lenalidomide (letrozole), letrozole (letrozole), leucovorin (leucovorin), leuprolerin (leuprolivorelin) (e.g. levorphanol), leuprolide (leuprolide), melphalan (leuprolide) (e.g. acetate), melphalan (melphalan), melphalan (mebendazole), leuprolinol (leucin) (e), melphalan (e), melphalan (e) (e), melphalan (e), melphalan (e) (e), melphalan (e, Mesna (mesna), methotrexate (methotrexate), methoxsalen (methoxsalen), mitomycin c (mitomycin c), mitotane (mitotane), mitoxantrone (mitoxantrone), nandrolone phenproprione, nelarabine (nellabine), norfebuxomab (nofetumomab), opprill (oprevekinase), oxaliplatin (oxaliplatin), paclitaxel (paclitaxel), palifermin (palifermin), pamidronate (pamidronate), panitumumab (pemanide), pegagapetase (pegamumase), pegaspartase (pegamurenase), pegafenib (pegamurenase), peginterferon alpha-2 b (pegigenfa-2 b), mestranil (metrexate), pegrexate (e.g. disodium), peganufactin (paprizine), peganufactin (e.g. paprizine (paprizine), peganum (papriine), peganum (papriine (e.g. sodium), peganufactin (papriine), peganufactin (papriine (e.g. papriine), peganufactin (papriine), paprikamuramicine (papriine), papriine (papriine), paprikamikamuramikamuramikamurami, Sargramostim (sargramostim), sorafenib (sorafenib), streptozotocin (streptozocin), sunitinib (sunitinib) (e.g., maleate), talc, tamoxifen (tamoxifen), temozolomide (temozolomide), teniposide (teniposide) (VM-26), testolactone (testolactone), thalidomide (thalidomide), thioguanine (thioguanine) (6-TG), thiotepa (thiotepa), thiotepa, topotecan (topotecan) (e.g., HCl), toremifene (toremifene), Tositumomab/I-131 (Tositumomab), trastuzumab (trastuzumab), trastuzumab (ATRA), uracil azalide (mustard), penthizine (vinblastine), vinblastine (vincristine), vinblastine (vinblastine), and vinblastine (vincristine).

The label may be detected. The conjugates used in the present invention may be labeled for detection or diagnostic purposes. The detectable label or label may be a radioactive label, a fluorescent label, a nuclear magnetic resonance active label, a luminescent label, a chromophore label (chromophore label), a positron emitting isotope for a PET scanner (positron emitting isotope), a chemiluminescent label, or an enzyme label. Exemplary radioimaging agents (detectable radio-labels) that emit radiation include indium-111, technetium-99, or low-dose iodine-131. The gamma and beta emitting radionuclides include⁶⁷Cu、⁶⁷Ga、⁹⁰Y、¹¹¹In、^99mTC and²⁰¹t1. Positron emitting radionuclides include¹⁸F、⁵⁵Co、⁶⁰Cu、⁶²Cu、⁶⁴Cu、⁶⁶Ga、⁶⁸Ga、⁸²Rb and⁸⁶and Y. Fluorescent tags include Cy5.5, Alexa 488, Green Fluorescent Protein (GFP), fluorescein, and rhodamine. Chemiluminescent tags include luciferase and beta-galactosidase. The enzyme label comprises catalase and phosphatase. The histidine tag may also be a detectable tag. For example, the conjugate may comprise a carrier moiety and an antibody moiety (antibody or antibody fragment), which may further comprise a tag. In this case, the tag may be attached to a carrier or an antibody.

An antibody. The antibody may also be part of a conjugate for use in the present invention. The binding may also be achieved by any means known in the art (e.g., using the binding strategies described herein). Any diagnostic or therapeutic antibody can be bound to one or more (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, or more) of the vectors of the invention. In addition, antibody fragments (e.g., capable of binding to an antigen) can also be bound to the vectors of the invention. Antibody fragments include Fab and Fc regions, heavy chains and light chains of antibodies (e.g., any of the antibodies described herein). Exemplary antibodies for use in diagnosing and treating cancer include ABX-EGF (Panitumumab), ovaRex (Ore govemab), Theragyn (PEM yttrium monoclonal antibody) -90), Therex, Bivatuzumab, Panorex (Edbecolomab), ReoPro (AoPro)Anti (Abciximab), Bexxar (Tositumomab), Mab, idiotype 105AD7, anti-EpCAM (Rituximab), Mab lung cancer (from Cytoclonal), herceptin (Hereptin) (Trastuzumab), Rituxan (Rituximab), Avastin (Avastin) (Bevacizumab), AMD Fab (Ranibizumab), E-26 (2)^ndIgE) (Omalizumab), Horbutrin (Zevalin) (Rituxan + Yttrium-90) (ibritumomab (Ibritumomabatixetan)), Cetuximab (Cetuximab), BEC2 (Mitumomab), IMC-1C11, nuC242-DM1, Lymphocide (Epratuzumab), Lymphocide Y-90, CEA-Cide (RADBE (Labetuzumab)), CEA-CideY-90, CEA-Scan (Tc-99 m-labeled Acimomab (Artumomab)), Lempossian (Tc-99 m-labeled Thiosumab (Sulemomab)), LymphoScan (Tc-99 m-labeled pertuzumab (Actumomab)), Humata (Tc-99 m-labeled Ab-99 m-labeled Humatab (Acetuzumab)), Lymphoman (Tc-99 m-labeled Ab-99 m-X (Acerbitumomab-210)), Hutuzumab (Acetuzumab), Hutuzumab-99 m-labeled Hutuzumab (Acetuzumab)), Hutuzumab-99 m-210), Hutuzumab (Acetuzumab (Acetx-11 (Acetuzumab)), and Hittumx-III (Acetuzumab)), or Hittuy-III), or Hittuy-I), or Hitt, MDX-210/MAK, Vitaxin, Mab425, IS-IL-2, Campath (alemtuzumab), CD20 streptavidin, Avidicin, (albumin + NRLU13), Oncolom (+ iodine-131) Cotara (+ iodine-131), C215(+ Staphylococcus aureus enterotoxin, Mab lung/renal cancer (from Pharmacia), Tanacotatab (Nacolomaabtamato) enterotoxin (C242 Staphylococcus aureus enterotoxin), Nuvion (Visilizumab), SMART M195, SMART 1D10, CEAVac, TriGem, TriGeAb, radiolabeled NovoMAb-HarG 2, Monopomm C, GlioMAb-H (+ gelonin toxin (gelotuzin), Rixan (Rituxan)), and other therapeutic antibodies including Pemetuzumab (Ab-5. C), and Pemetuzumab (Ecremox-5. Ecremox-5) ABX-IL8, Antegren (Natalizumab), anti-CD 11a (Efalizumab), anti-CD 18 (from Genetech), anti-LFA 1, Antova, BTI-322, CDP571, CDP850, Corsevin M, D2E7 (Adalimumab), Humira (Adalimumab), Hu23F2G (Rovelizumab)), IC14, IDEC-114, IDEC-131, IDEC-151, IDEC-152, infliximab)(Infliximab) (Remicade), LDP-01, LDP-02, MAK-195F (Afimomab (Afelimomab)), MDX-33, MDX-CD4, MEDI-507 (Siplizumab), OKT4A, OKT3 (Muromonab) -CD3), and ReoPro (Abciximab)).

Binding attachments

Conjugates (e.g., polypeptide-agent conjugates) can be obtained using any cross-linking (binding) reagent or protocol known in the art, many of which are commercially available. Such protocols and reagents include cross-linking agents that react with amino, carboxyl, sulfhydryl, carbonyl, carbohydrate and/or phenolic groups. The amount, number and conditions of such protocols can be varied to optimize binding. Crosslinkers contain at least two reactive groups and are generally divided into homofunctional crosslinkers (containing the same reactive groups) and heterofunctional crosslinkers (containing different reactive groups). The crosslinking agents of the present invention may be homobifunctional and/or heterobifunctional. Further, the crosslinker may incorporate a "spacer" between the reactive moieties, or two reactive moieties in the crosslinker may be directly linked. The linkage may comprise an ester linkage.

Exemplary linkers include BS³[ bis (thiosuccinimidyl) suberate), NHS/EDC (N-hydroxysuccinimide and N-ethyl- (dimethylaminopropyl) carbodiimide), thio-EMCS ([ N-e-maleimidocaproic acid)]Hydrazide), SATA (N-succinimidyl-S-acetylthioacetate), and hydrazide. BS³Is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines. NHS/EDC allows the binding of primary amine groups to carboxyl groups. thio-EMCS is a heterobifunctional reactive group (maleimide and NHS-ester) that is reactive towards thiol and amino groups. Amine ligation activated with thio-NHS/EDC can be used to crosslink therapeutic antibodies with polypeptides of the invention. This is a fast, simple and repeatable joining technique. The resulting conjugate is stable and retains the biological activity of the antibody. Furthermore, it has a high binding capacity with reliable control and low non-specific interactions in the ligation procedure. SATA is reactive with amines and is added to a protectedProtected mercapto groups. The NHS-ester reacts with the primary amine to form a stable amide bond. The mercapto group can be deprotected using hydroxylamine. Hydrazides can be used to link carboxyl groups to primary amines and thus can be used to link glycoproteins.

Small molecules such as therapeutic agents can be conjugated to polypeptides (e.g., those described herein). An exemplary small molecule, paclitaxel, has two important positions (positions C2' and C7) that are useful for binding. Conjugation of the vector or vectors of the invention to paclitaxel may be performed as follows. Briefly, paclitaxel was reacted with the anhydride pyridine succinate for 3 hours at room temperature to attach the succinyl group at position 2'. 2 '-succinyl paclitaxel has a cleavable ester bond at position 2' and can simply release succinic acid. Such cleavable ester bonds can be further used for various modifications using linkers, if desired. The resulting 2 '-O-succinyl-paclitaxel was then reacted with EDC/NHS in DMSO for 9 hours at room temperature, followed by addition of the vehicle or vehicle in ringer's solution/DMSO at room temperature for an additional 4 hours. The binding reaction can be monitored by HPLC. Each intermediate such as paclitaxel, 2 '-O-succinyl-paclitaxel and 2' -O-NHS-succinyl-paclitaxel can be purified and confirmed by different methods, such as HPLC, thin layer liquid chromatography, NMR: (A-N-methyl-N-ethyl-N-methyl-L-propyl-paclitaxel)¹³C or¹H exchange), melting point, mass spectrum. The final conjugates can be analyzed by mass spectrometry and SDS-polyacrylamide gel electrophoresis. This allows the number of paclitaxel molecules bound per carrier to be determined.

Pharmaceutical composition

Since hydrophobic agents often exhibit limited solubility in aqueous solutions, the pharmaceutical compositions of the present invention may include a solubilizing agent. Exemplary formulations of ANG1005 include DMSO and Solutol HS15, although other solubilizing agents in place of or in addition to these agents may be used in the compositions of the present invention. The composition can further include a buffer, a tonicity agent, and a lyophilizate (e.g., a bulking agent or a cryoprotectant).

Solubilizer

The compositions and methods of the present invention may include any solubilizing agent known in the art. Such agents may constitute at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or 70% of the mass of the composition. Exemplary solubilizing agents include water-soluble organic solvents (e.g., polyethylene glycol 300, polyethylene glycol 400, ethanol, propylene glycol, glycerol, N-methyl-2-pyrrolidone, dimethylacetamide, and dimethylsulfoxide), nonionic surfactants (e.g., Cremophor EL, Cremophor RH 40, Cremophor RH 60, D-alpha-tocopheryl polyethylene glycol 1000 succinate, polysorbate 20, polysorbate 80, Solutol HS15 (polyethylene glycol 15-hydroxystearate), sorbitan monooleate, poloxamer (poloxamer)407, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, gerafil 44/14, Softigen 767, and mono and di-fatty acid esters of PEG 300, 400, or 1750), water-soluble lipids (e.g., castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, castor oil, corn oil, cotton seed oil, olive oil, and water, Safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and the medium chain triglycerides of coconut oil and palm kernel oil), organic liquids/semisolids (beeswax, d-alpha-tocopherol, oleic acid, medium chain mono-and diglycerides), cyclodextrins (e.g., alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, and sulfobutyl ether-beta-cyclodextrin), and phospholipids (e.g., hydrogenated soybean phosphatidylcholine, distearoylphosphatidylglycerol, 1-alpha-dimyristoylphosphatidylcholine, 1-alpha-dimyristoylphosphatidylglycerol).

Buffering agent

The compositions and methods of the present invention may also include one or more buffering agents. Depending on the hydrophobic agent, it may be desirable to maintain the pH or tonicity of the pharmaceutical composition (e.g., to minimize degradation of the active agent, or to maximize the safety or efficacy of the agent when used in therapy). Buffering to any particular pH or pH range (e.g., to pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, any range between these values) can be accomplished using an appropriate buffer. The buffer can be any concentration required to achieve the desired buffering effect (e.g., 1mM, 10mM, 20mM, 50mM, 100mM, 200mM, 500mM, 1.0M, 1.5M, or any range in these values). Exemplary buffers include citric acid/g acid salt, acetate salt, barbital (barbital), borate, boer-luodian buffer (Britton-Robinson), cacodylate salt, citrate salt, collidine (collidine), formate salt, maleate salt (maleat), Mclvaine, phosphate salt, Prideaux-Ward, succinate salt, citrate-phosphate-borate (Teorell-Stanhagen), Florana (veronal) acetate salt, MES (2- (N-morpholino) ethanesulfonic acid), BIS-TRIS (BIS (2-hydroxyethyl) iminotris (hydroxymethyl) methane), ADA (N- (2-acetamido) -2-iminodiacetic acid), ACES (N- (carbamoylmethyl) -2-aminoethanesulfonic acid), PIPES (piperazine-N, N' -BIS (2-ethanesulfonic acid)), (Britton-Robinson), Dimethylarsoni-citrate salt, Florin-phosphate-Veronal), MES (2- (N-morpholino) ethanesulfonic acid), PIPES (piperazine, MOPSO (3- (N-morpholino) -2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE (1, 3-BIS (TRIS (hydroxy-methyl) methylamino) PROPANE), BES (N, N-BIS (2-hydroxyethyl) -2-amino-ethanesulfonic acid), MOPS (3- (N-morpholino) propanesulfonic acid), TES (N-TRIS (hydroxymethyl) methyl-2-aminoethanesulfonic acid), HEPES (N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid), DIPSO (3- (N, N-BIS (2-hydroxyethyl) amino) -2-hydroxypropanesulfonic acid), MOBS (4- (N-morpholino) butanesulfonic acid), TAPSO (3- (N-TRIS (hydroxymethyl) methyl-amino) -2-hydroxypropanesulfonic acid), TRIZMA (tris (hydroxymethyl-aminomethane), HEPPSO (N- (2-hydroxyethyl) piperazine-N '- (2-hydroxy-propanesulfonic acid), POPSO (piperazine-N, N' -bis (2-hydroxypropanesulfonic acid)), TEA (triethanolamine), EPPS (N- (2-hydroxyethyl) -piperazine-N '- (3-propanesulfonic acid), TRICINE (N-tris (hydroxy-methyl) methylglycine), GLY-GLY (diglycine), BICINE (N, N-bis (2-hydroxyethyl) glycine), HEPBS (N- (2-hydroxyethyl) piperazine-N' - (4-butanesulfonic acid)), TAPS (N-tris (hydroxymethyl) methyl-3-amino-propanesulfonic acid), AMPD (2-amino-2-methyl-1, 3-propanediol), and/or any other buffer known in the art.

In addition to, or instead of, a buffer, any pharmaceutically acceptable salt known in the art may be used to maintain tonicity. Exemplary salts include sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. Such salts, either alone (along) or in combination with a buffer, are present in an amount sufficient to maintain the desired tonicity (e.g., 1mM, 10mM, 20mM, 50mM, 100mM, 200mM, 500mM, 1.0M, 1.5M, or any range between these values).

Other excipients

In certain embodiments, the compositions and methods of the invention include other excipients (e.g., bulking agents or cryoprotectants). Bulking agents are particularly desirable where the pharmaceutical composition is provided in a dehydrated (e.g., lyophilized) form. The lyophilized composition can contain less than 10% (e.g., less than 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%) by weight of water or other solvent. Because dehydrated compositions administered by parenteral routes are typically dissolved in an aqueous solution prior to administration to a patient, it is important that the dehydration process be performed in a manner that allows for re-dissolution (resolubilization). A bulking agent is added to ensure that the lyophilized product can be redissolved more easily. Such agents are known in the art and include polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, dextran (dextran); sugars such as glucose, mannitol, sucrose, lactose, trehalose, and sorbitol; amino acids, such as glycine, arginine, aspartic acid; and soluble proteins such as collagen, gelatin or serum albumin.

The composition may further comprise a preservative (e.g., thimerosal, benzyl alcohol, parabens), a polymer such as polyethylene glycol covalently attached to the protein, complexed with metal ions, or incorporated into or onto a particulate formulation of a polymeric compound such as polylactic acid, polyglycolic acid, a hydrogel, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroids (sphenoplasts). Such compositions will affect physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulations in the form of lipophilic depots (e.g., fatty acids, waxes, oils). The present invention also contemplates particulate compositions coated with polymers, such as poloxamers or poloxamines. Other embodiments of the compositions of the present invention incorporate protective coatings, protease inhibitors or permeation enhancers in particulate form for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment, the pharmaceutical composition is administered via the following route: parenteral, peri-cancerous, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricular, intracranial, and intratumoral.

Solid dosage form for oral use

Formulations for oral use include tablets containing the active ingredient(s) in admixture with non-toxic pharmaceutically acceptable excipients, and such formulations are known to those skilled in the art (e.g., U.S. Pat. nos. 5,817,307, 5,824,300, 5,830,456, 5,846,526, 5,882,640, 5,910,304, 6,036,949, 6,372,218, which are incorporated herein by reference). These excipients may, for example, be inert diluents or fillers (e.g. sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate); granulating or disintegrating agents (e.g., cellulose derivatives, including microcrystalline cellulose, starch including potato starch, croscarmellose sodium, sodium alginate, or alginic acid); a binding agent (e.g., sucrose, glucose, sorbitol, acacia (acacia), alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, glidants, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silicates, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients may be colorants, flavors, plasticizers, humectants, buffering agents, and the like.

The tablet may be uncoated or it may be coated by known techniques to optionally delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. The coating may be adapted to release the pharmaceutical agent(s) in a predetermined pattern (e.g. in order to obtain a controlled release formulation) or may be adapted to release the pharmaceutical agent(s) only after passage through the stomach (enteric coating). The coating may be a sugar coating (sugar coating), a film coating (e.g., based on hydroxypropyl methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycol, and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Additionally, a time delay material such as glycerol monostearate or glycerol distearate may be employed.

The solid tablet composition may include a coating adapted to prevent undesirable chemical changes to the composition (e.g., chemical degradation that occurs prior to release of the active). The coating may be applied to the solid dosage form in a similar manner as described in Encyclopedia of Pharmaceutical Technology (supra).

Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders and granules can be prepared in a conventional manner using the above-described ingredients in tablets and capsules using, for example, a mixer, a fluidized bed apparatus, or a spray drying equipment.

Method of treatment

The invention also features methods of treatment using the agents described herein. The anti-cancer agents and conjugates described herein (ANG1005) can be used to treat any cancer known in the art. The conjugates of the invention, including the peptides described herein, are capable of crossing the BBB (e.g., angiopep-1 through angiopep-6) and thus can be used to treat any brain or central nervous system disorder (e.g., brain cancers such as glioblastoma, astrocytoma, glioma, medulloblastoma, and oligodendroglioma, glioma, ependymoma, and meningioma). These conjugates can also be delivered efficiently to the liver, lung, kidney, spleen or muscle (e.g., angiopep-1 through angiopep-7), and thus can also be used in combination with appropriate therapeutic agents to treat diseases associated with these tissues (e.g., cancers such as hepatocellular carcinoma, liver cancer, small cell cancer (e.g., oat cell cancer), mixed small/large cell cancer, combined small cell cancer, and metastatic tumors can originate from any tissue of cancer, including breast cancer, colon cancer, prostate cancer, sarcoma, bladder cancer, neuroblastoma, Wilm tumor, lymphoma, non-Hodgkin's lymphoma, and certain T-cell lymphomas). Additional exemplary cancers that can be treated with the compositions of the invention include hepatocellular carcinoma, breast cancer, cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-hodgkin's lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, retinal carcinoma, esophageal carcinoma, multiple myeloma, ovarian cancer, uterine cancer, melanoma, colorectal cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung cancer), pancreatic cancer, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial cancer, renal cell cancer, gallbladder adenocarcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrug resistant cancers, and proliferative diseases and conditions such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet/dry AMD), corneal neovascularization, diabetic retinopathy, neoglaucoma, myopic degeneration and other proliferative diseases and conditions such as restenosis and polycystic kidney disease. Brain cancers that can be treated with vectors that are effectively transported across the BBB include astrocytomas, fibroastrocytomas, embryonic dysplastic neuroepitheliomas, oligodendrogliomas, ependymomas, glioblastoma multiforme, mixed gliomas, oligoastrocytomas, medulloblastomas, retinoblastoma, neuroblastoma, germ cell tumors, and teratomas.

The conjugate or composition of the invention may be administered to a subject by any means known in the art, for example, orally, intraarterially, intranasally, intraperitoneally, intravenously, intramuscularly, subcutaneously, transdermally, or orally. The agent may, for example, be an anti-angiogenic compound.

Dosage form

The dosage of any conjugate or composition described herein or identified using the methods described herein depends on a number of factors, including: the method of administration, the disease to be treated (e.g., cancer), the severity of the disease, whether the cancer is to be treated or prevented, and the age, weight, and health of the subject to be treated.

With respect to the therapeutic methods of the present invention, it is not intended that the administration of the carrier, conjugate or composition to a subject be limited to a particular mode of administration, dose or frequency of administration, and the present invention contemplates all modes of administration. The conjugate or composition may be administered to the subject in a single dose or in multiple doses. For example, a compound described herein or identified using a screening method of the invention may be administered once a week for, e.g., 2, 3,4, 5, 6, 7, 8, 10, 15, 20 or more weeks. It will be appreciated that for any particular subject, the particular dosage regimen will be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition. For example, the dosage of a composition can be increased if a lower dose does not provide sufficient activity in treating a disease or condition described herein (e.g., cancer). Conversely, if the disease (e.g., cancer) is reduced or eliminated, the dosage of the composition can be reduced.

While the attending physician will ultimately determine the appropriate amount and dosage regimen, a therapeutically effective amount of a carrier, conjugate or composition described herein can, for example, be in the range of 0.0035 μ g to 20 μ g/kg body weight/day or 0.010 μ g to 140 μ g/kg body weight/week. Desirably, the therapeutically effective amount is in the range of 0.025 μ g to 10 μ g/kgFor example, at least 0.025, 0.035, 0.05, 0.075, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0 or 9.0 μ g/kg body weight is administered daily, every other day or twice a week. In addition, a therapeutically effective amount may be in the range of 0.05 μ g to 20 μ g/kg, for example at least 0.05, 0.7, 0.15, 0.2, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 14.0, 16.0 or 18.0 μ g/kg body weight administered weekly, every other week, every three weeks, monthly. In addition, a therapeutically effective amount of a compound may be 0.1mg/m administered, for example, every other day, once a week, every other week, or every three weeks²～2,000mg/m²Within the range of (1). For example, the ANG1005 may be administered 50, 100, 200, 300, 400, 420, 500, 600, 700, 800, or 1,000mg/m every one, two, three, four weeks, or monthly or every other month². In a particular embodiment, ANG1005 is administered 300mg/m every three weeks²Or 420mg/m². In another embodiment, the therapeutically effective amount is 1000 μ g/m²～20,000μg/m²E.g., at least 1000, 1500, 4000 or 14,000 μ g/m administered daily, every other day, twice weekly, or every other week²A compound is provided.

The following examples are intended to illustrate, but not limit, the present invention.

Example 1

Solubility of ANG1005

ANG1005 was tested for solubility in some solvents and surfactants. The results for the single agents are given in table 13 below.

ANG1005 was also tested for solubility in a solvent/surfactant combination (combination). These results are given in table 14.

Example 2

ANG1005 dissolution conditions

In the formulation for lyophilization, ANG1005 undergoes several dissolution conditions. These results are summarized below. ANG1005 was first dissolved in DMSO as described above. To this mixture is added hot Solutol or a combination of Solutol buffers. Finally, glycine buffer was added to the ANG1005/DMSO/Solutol mixture. The dissolution conditions in table 15 were therefore tested.

Table 15: dissolution of ANG1005

The results of these tests are given in table 16.

TABLE 16

Test of	Target pH	Purity (%)	DMSO(ppm)＊	Remarks for note
					1	4.50	95.9	5855	Rapid, clear reconstitution followed by turbidity
2	4.75	94.0	6488	Fast, clear reconstitution liquid, good
					3	5.00	95.0	6256	Fast, clear reconstitution liquid, good
4	5.25	95.4	6382	Fast, clear reconstitution liquid, good
					5	4.50	96.0	6818	Rapid, clear reconstitution followed by turbidity
6	4.75	94.4	6330	Fast, clear reconstitution liquid, good
					7	5.00	94.6	6806	Fast, clear reconstitution liquid, good
8	5.25	94.0	6930	Fast, clear reconstitution liquid, good
					9	4.50	95.2	6235	Fast, clear reconstitution liquid, good
10	4.75	93.8	6932	Fast, clear reconstitution liquid, good
					11	5.00	95.1	6302	The method is quick in speed and has the advantages of high speed,clear reconstitution liquid, good
12 (ref.)	5.25	93.9	7846	Fast, clear reconstitution liquid, good
					13	4.50	97.6	7035	Turbid reconstitution fluid (+ +)

14	4.75	97.4	7071	A fast, clear reconstitution liquid, followed by turbidity and precipitation
					15	5.00	95.4-94.3	6818	Fast and clearClear reconstitution liquid, good
16	5.25	95.9-96.2	7155	Fast, clear reconstitution liquid, good
					17	5.00	96.5	6163	Turbid reconstitution fluid (+ +)

On the basis of these results, we have determined that the formulation can be processed at temperatures between 40 and 50 ℃. The pH of the solution should be above 4.5 to enable micelle (micells) formation of Solutol HS15, since pH 4.5 reconstitution can form a cloudy solution. Solutol HS15 was acidified to minimize degradation prior to the addition of ANG 1005.

Example 3

Conditions of lyophilization

After solubilization, the ANG1005 mixture is diluted in an aqueous buffer (e.g., glycine buffer, pH adjusted to 5 with HCl, mannitol, and sodium chloride), and the mixture is frozen and lyophilized. Exemplary conditions are given in table 4 above.

The loading temperature of segment (segment)1 was tested between-70 ℃ and 25 ℃. The ramp time for zone 2 may vary depending on the difference between the temperatures in zones 1 and 3 and may be as long as 6 hours. We determined that zone 3 had to be performed for at least 12 hours, as the shorter time frame (time frame) resulted in the freeze-dried cake collapsing. Sections 8 and 9 can be adjusted within the temperature ranges shown above to ensure that the product temperature is between 18 ℃ and 21 ℃ during secondary drying. The product should be kept below 25 ℃ to avoid melting. Using the solubilization/lyophilization protocol described herein, we were able to make products with greater than 96% purity and less than 1% residual DMSO in some cases, as given in table 17.

TABLE 17

1007135 further characteristics of the batches, as well as other batches, are given in Table 18 below.

Watch 18

Batch number	C1007135	C0108002	C0308011	C0608030	C1108062
						Purity of	96.5％	96.9％	95.5％	95.6％	96.9％
2: 1 conjugates	2.2％	1.4％	2.0％	2.7％	2.3％
						Measurement of	100.4％	97.2％	100.7％	102.6％	105.7％
Total related substances	3.5％	3.1％	5.0％	4.4％	3.1％
						Unbound angiopep-2	ND	ND	ND	ND	ND
Unbound paclitaxel	0.9％	0.6％	1.0％	0.7％	0.5％
						1: 1 conjugates	ND	ND	ND	ND	ND
Is unknown	0.5％	1.0％	1.0％	1.0％	0.4％
						Water content	0.05％	0.04％	0.05％	0.12％	0.03％
DMSO	0.65％	0.54％	0.37％	0.54％	0.64％

ND is not detected

Example 4

Resuspension of lyophilized ANG1005

The following procedure was developed for dissolving and suspending ANG1005 lyophilized formulations in aqueous solution. The procedure set forth is suitable for a single vial containing 120mg ANG 1005.

ANG1005 vials were equilibrated at room temperature. The vial was then vented. 4ml of absolute ethanol was slowly injected (e.g., over 30 seconds) down the side of the vial using a20 cc syringe fitted with an 18G 11/2 "needle. The vial was then placed on a spiral mixer (nutating mixer) for 10 minutes, allowing the ethanol to slowly wet the cake, providing a milky suspension.

The vial was then removed from the mixer and 12ml of ringer's lactate solution containing 5% glucose was injected down the side of the vial using a20 cc plastic syringe fitted to an 18G 1.5 "needle. The vial was then placed on the spiral mixer for 5 minutes. The vial was then rotated 180 degrees and mixing continued on the spiral mixer for an additional 5 minutes. At this point, the suspension was clear and contained very little foam. The vial is then left on the bench for 5 minutes before proceeding to the next step (e.g., dilution for injection, analysis).

Alternative diluents were also tested (table 19). While the use of these diluents resulted in clear solutions that were completely dissolved, they resulted in greater degradation of ANG1005 at room temperature relative to the mixture of ringer's lactate and ethanol, which contained 5% glucose.

Table 19: alternative diluents for resuspension:

	measurement of	Condition
			Water for injection	16ml	Heating to 40-50 deg.C
Water/ethanol for injection	12ml+4ml	RT
			D5W	16ml	Heating to 40-50 deg.C
D5W/ethanol	12ml+4ml	RT
			Lactic acid ringer's solution D5W	16ml	Heating to 40-50 deg.C

Example 5

Additional buffer and swelling agent testing

We made further efforts to reduce residual DMSO (0.5%) and shorten the lyophilization cycle (5 days).

We believe that the various excipients of the formulation (glycine and sodium chloride in particular) contribute to a reduction in the efficiency of DMSO removal during the second drying of the cycle. Formulations prepared in the absence of NaCl, glycine, mannitol or water resulted in much lower DMSO contents (of the order of 0.01%). But in the absence of mannitol, the cake is waxy (consisting primarily of Solutol). None of these low DMSO formulations was reconstituted with ethanol and D5W/ringer's lactate. In addition, when glycine is not present, the pH is not controlled. This causes degradation of ANG 1005.

Thus, in further tests, mannitol was retained as a bulking agent, glycine was replaced with a buffer comprising citric acid and lactic acid, and NaCl was removed. These formulations, using a shorter lyophilization cycle, still resulted in the cake containing 0.05% DMSO residue. At this level of DMSO, the cake was insoluble. In another formulation, soy lecithin was used in place of mannitol. This resulted in 0.2% DMSO residue. At 0.2% residual DMSO, the cake was soluble in ethanol and D5W/LR. Therefore, we considered 0.2-0.4% DMSO to be necessary for reconstitution of the vial and further dilution into infusion bags. The lyophilization time can be adjusted accordingly so that the DMSO concentration is within this range.

The details of the compositions used in these tests are as follows (tables 20-22).

Table 20: pre-lyophilization composition

％w/w	Suppliers of goods	F-37	F-38	F-39	F-40
						ANG1005		0.72	0.72	0.72	0.72
Solutol HS15		24.92	24.88	24.83	24.88
						DMSO，USP	Gaylord	13.39	13.36	13.32	13.36
1N HCl		0.311	0.311	0.306	0.311
						Citric acid		0.04
Lactic acid			0.21	0.42	0.21
						Mannitol		1.54	1.53	1.53
Soybean lecithin	PL90G				1.53
						Water for injection		59.08	59.00	58.86	59.00
Total of		100.00	100.00	100.00	100.00

85% in water

Solutol/API ratios identical to those recorded for ANG batches

DMSO/HCl ratio was the same as recorded for ANG batches

The citric acid concentration is chosen to provide a pH of 5 (based on F-34)

Calculating the lactic acid concentration to provide a pH of 5(F38) and lower (F39)

The concentration of mannitol in water was the same as that recorded for the ANG batch

Soybean lecithin is a "solubilizing" bulking agent and thus replaces mannitol

Table 21: ingredient table (composing table)

Mg/tube	Grade&Batch number	F-37	F-38	F-39	F-40
						ANG1005		184	184	184	184
Solutol HS15		6328	6328	6328	6328
						DMSO，USP		3399	3399	3395	3399
1N HCl		79	79	78	79
						Citric acid		10
Lactic acid			54	108	54
						Mannitol		390	390	390
Soybean lecithin					390
						Water for injection		15000	15000	15000	15000
Total of		25390	25434	25483	25434
						Total dry weight		6991	7035	7088	7035

85% in water

Table 22: composition table after freeze-drying

％w/w	Suppliers of goods	F-37	F-38	F-39	F-40
						ANG1005		2.64	2.62	2.60	2.62
Solutol HS15		90.65	89.95	89.28	89.95
						DMSO，USP	Gaylord
1N HCl		1.13	1.12	1.10	1.12
						Citric acid		0.14
Lactic acid			0.77	1.52	0.77
						Mannitol		5.59	5.54	5.50
Soybean lecithin					5.54
						Water for injection
Total of		100.0	100.0	100.0	100.0

85% in water

The composition was prepared as follows: stock DMSO/HCl was prepared by weighing 0.5g of 1N HCl standard solution into a 50ml falcon tube. 21.5g of DMSO was added and mixed well to obtain a stock DMSO +1N HCl solution.

For each composition, mannitol, Solutol, citric acid or lactic acid and WFI were weighed and placed in a 50ml Falcon tube. These ingredients were mixed well to dissolve. The tube was capped and heated to 51 ℃ ("buffer mixture"). The ANG1005 was weighed out using another 50ml (Falcon tube). The DMSO/HCl stock solution was then added and mixed well by vortexing until the solution became clear. The heat buffered mixture was slowly added to the DMSO/ANG1005 mixture while vortexing. The mixture was then cooled to RT. 1700-1730mg of solution was placed into each vial. The solution was lyophilized as described herein. The mixture (compound) was stored at-20 ℃.

The solution was then tested for its reconstitution capability. To reconstitute the solution, ethanol was added and mixed. Then adding the ringer's lactate solution. The amounts are given in table 23.

Table 23: reconstitution volume

Weight in each vial (wt)	Reference (ref) product	F-37～F-40
			API(mg)	125	12.5
Ethanol (mg)	3234	323.4
			Ringer's lactate solution (mg) containing D5W	12186	1218.6

If the reconstitution was successful, the sample appearance (color, crystalline or solid PPT under microscope, etc.) was recorded. Aliquots are then analyzed (e.g., determined and purity) by HPLC. The pH will also be measured.

Example 6

Stability testing of lyophilized ANG1005 products

The ANG1005 product was tested for stability over time. The activity, purity, appearance, pH and degradation of the lyophilized product stored at about-15 ℃ were monitored. The results of these tests are given in table 24 below.

Example 7

Stability of ANG1005 products after reconstitution

Several experiments were performed to evaluate the stability of ANG1005 after reconstitution into a solution. These experiments are described below.

Experiment 1

The ANG1005 injection lot C0108002 product was reconstituted to a concentration range of 1.0-2.0 mg/ml as described herein. It was previously established that ANG1005 at a concentration of 2.0mg/ml is the optimum dose for clinical use, as previously present in IND. These preliminary experiments were performed in small volumes in glass vials. Samples were stored at room temperature and visually inspected at different time points. The selected samples were filtered prior to HPLC analysis.

Table 25 shows the concentrations tested over time versus visual clarity of the solutions. The apparent turbidity (cloudiness) appears to be related to both the increase in concentration and time. HPLC analysis of the selected samples revealed a single ANG1005 peak with no significant change in purity at the different time points. No change was observed in the profile of the relevant substances (table 26). Two peaks of related material were noted, identified as 2: 1 binder (RRT 0.88) and unbound paclitaxel (RRT 0.95). A peak with an RRT of 1.15 was observed (8.1min), which is an impurity from the HPLC column because it is also present in the blank chromatogram.

Table 25: visual appearance of reconstituted ANG1005 diluted in D5W

0h

1h

2h

3h

4h

6h

1.0mg/ml

Clarification

Clarification

Clarification

Clarification

Clarification

X

1.5mg/ml

Clarification

Clarification

X

X

XX

XXX

2.0mg/ml

Clarification

X

XX

XX

XXX

XXX

X: slight turbidity

XX: turbidity (haze)

XXX: very turbid

Table 26: purity of reconstituted ANG for injection 1005 diluted in D5W

NT is not detected

Related substances: a single peak greater than 0.5% is reported, with relative retention times in parentheses

Related substances at RRT 0.88 represent 2: 1 conjugates

Related substances at RRT 0.95 represent unbound paclitaxel

Note that: a peak with a relative retention time of 1.15(8.1min) was observed, which is an impurity in the HPLC column, as it is also present in the blank chromatogram.

Experiment 2

To confirm the stability results at 1.0mg/ml obtained in experiment 1, additional studies were performed under clinical use conditions. ANG1005 for injection, lot C0108002, was reconstituted as described to make a final concentration of 1.0mg/ml in a 500ml D5W infusion bag. The solution was still visually clear with no significant change in purity or spectrum of related substances over an observation period of 6 hours at room temperature (see table 27 and figure 2).

Table 27: reconstitution of ANG1005 for injection diluted to 1.0mg/ml with D5W under clinical conditions of use

Related substances: a single peak greater than 0.5% is reported, with relative retention times in parentheses

Related substances at RRT 0.88 represent 2: 1 conjugates

Related substances at RRT 0.95 represent unbound paclitaxel

Note that: a peak with a relative retention time of 1.15(8.1min) was observed, which is an impurity in the HPLC column, as it is also present in the blank chromatogram.

Experiment 3

ANG for injection 1005, lot C0108002 was reconstituted and diluted with D5W in a glass vial to a final concentration of 2.0 mg/ml. The sample was stored at room temperature for 6 h. The solution became turbid and was centrifuged. The resulting precipitate was collected by slowly decanting the supernatant, allowed to dissolve in DMSO, and analyzed by HPLC. The major peak of the redissolved sediment was identified as ANG1005 with a purity of 97.2%. No change was observed in the related species spectra, with only two additional peaks expected (1.3% at RRT 0.88 and 1.5% at RRT 0.95). The HPLC chromatogram for this sample is shown in fig. 3. A peak with an RRT of 1.15 was observed (8.1min), which was derived from impurities in the HPLC column, as it was also present in the blank chromatogram.

The combined data indicate that the turbidity/cloudiness observed was the result of the intact ANG1005 precipitating out (going out) of solution without any degradation, possibly due to interaction between the drug product components and D5W. This phenomenon appears to be concentration and time dependent.

To reduce turbidity, it is recommended to receive ANG1005 at a dose > 300mg/m²To a final concentration of ≦ 1mg/ml for all patients.

There are several data that can indicate that this result is mostly caused by a reduction in the amount of residual DMSO in the drug product. The first batch of the drug product, batch number C0807121, had a residual DMSO content of 8.2%, while the more recent batch, batch number C0108002, had a residual DMSO content of 0.54%. It appears that this change has affected the solubility of the substance.

Additional experiments are being conducted to modify the dilution procedure to improve the stability of the reconstituted drug. The test was performed on a solution prepared with ringer's lactate as diluent instead of 5% glucose injection in the last step of the reconstitution process: each vial of ANG1005 for injection was first reconstituted as before with 4ml of absolute ethanol and 12ml of ringer's lactate/5% glucose injection to a concentration of 6mg/ml, and then further diluted with ringer's lactate. Preliminary data given in Table 28 reveal that ringer's lactate, in the same concentration range (up to 2.0mg/ml), in place of D5W, prevented the observed infusion clouding. All solutions remained clear throughout the observation period without affecting the purity of ANG 1005.

Table 28: purity of reconstituted ANG1005 diluted with lactated ringer's injection

Related substances: a single peak greater than 0.5% is reported, with relative retention times in parentheses

Related substances at RRT 0.88 represent 2: 1 conjugates

Related substances at RRT 0.95 represent unbound paclitaxel

Other embodiments

All patents, patent applications, and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent, patent application, or publication was specifically and individually indicated to be incorporated by reference.

Claims (34)

1. A composition, comprising:

（a）ANG1005；

(b) optionally a tonicity agent;

(c) a buffer to maintain a solution pH of 4 to 6;

(d) a swelling agent;

(e) solutol HS 15; and

(f) 0.2% to less than 8% DMSO.

2. The composition of claim 1, wherein the tonicity agent is sodium chloride.

3. The composition of claim 1 or 2, wherein the buffering agent is glycine.

4. The composition of claim 1 or 2, wherein the buffer is citric acid or lactic acid.

5. The composition of claim 1, wherein the bulking agent is mannitol or sorbitol.

6. The composition of claim 1, wherein the amount of DMSO is 0.2-5%.

7. The composition of claim 1, wherein the amount of DMSO is 0.2-2%.

8. The composition according to claim 7, wherein the amount of DMSO is between 0.2% and 1.0%.

9. The composition of claim 1, wherein the amount of Cremophor in the composition is less than 5%.

10. The composition of claim 9, wherein the composition is free of Cremophor.

11. The composition of claim 1, wherein the composition is dissolved in water.

12. A composition, comprising:

compound (I) Percentages by weight other than water ANG1005 0.1-5% Tonicity agent 1-15% Buffer for maintaining pH at 4-6 1-10% Expanding agent 0-15% Solutol HS15 40-75% DMSO 0.2 to less than 8 percent

13. The composition of claim 12, wherein the tonicity agent is sodium chloride, the buffering agent is glycine, and the bulking agent is mannitol.

14. The composition of claim 12, comprising:

compound (I) Percentages by weight other than water ANG1005 1.8-2.3% Tonicity agent 9-11% Buffering agent 4.5-6% Expanding agent 8-10% Solutol HS15 69-75% DMSO 0.2-2%

15. The composition of claim 14, wherein the tonicity agent is sodium chloride, the buffering agent is glycine, and the bulking agent is mannitol.

16. A composition, comprising:

compound (I) Percentages by weight other than water ANG1005 1.8-4.0% Buffer for maintaining pH at 4-6 0.1-6% Expanding agent 2-10% Solutol HS15 80-95% DMSO 0.2-1%

17. The composition of claim 16, wherein the buffer is lactic acid or citric acid and the bulking agent is mannitol.

18. Use of the composition of any one of claims 1-17 in the manufacture of a medicament for treating a patient suffering from cancer.

19. The use of claim 18, wherein the cancer is brain, ovarian, lung, liver, spleen or kidney cancer.

20. The use of claim 19, wherein the brain cancer is selected from the group consisting of glioma, ependymoma, and meningioma.

21. The use of claim 20, wherein the glioma is a glioblastoma, astrocytoma, medulloblastoma, and oligodendroglioma.

22. A sealed container containing the composition of claim 15.

23. A kit, comprising:

(a) the sealed container of claim 22; and

(b) and (5) instructions for use.

24. A process for preparing a pharmaceutical composition, the process comprising:

(a) dissolving ANG1005 in DMSO to form a mixture;

(b) adding Solutol HS15 to the mixture of step (a);

(c) optionally adding water and a buffer to the mixture, the buffer maintaining the solution pH at 4-6;

(d) lyophilizing the mixture of step (c); wherein the lyophilization results in at least a 20% reduction in the amount of DMSO, but not a significant reduction in the amount of Solutol HS 15;

wherein the final concentration of DMSO is 0.2% to less than 8%.

25. The method of claim 24, wherein water and buffer are added in step (c), and said step (d) lyophilizing comprises:

(i) freezing the mixture;

(ii) drying the frozen product at a first temperature and pressure sufficient to remove at least a portion of the water; and

(iii) drying the product at a second temperature and pressure sufficient to remove at least a portion of the DMSO.

26. The method of claim 24 or 25, wherein the mixture of step (b) is filtered prior to lyophilization of step (d).

27. The method of claim 24, wherein the mixture is placed in a vial prior to lyophilization in step (d).

28. The method of claim 24, further comprising the steps of:

(e) resuspending the lyophilized product.

29. A pharmaceutical composition produced by the method of any one of claims 24-28.

30. A process for producing a pharmaceutical composition comprising the steps of:

(a) dissolving ANG1005 in DMSO, thereby forming a mixture;

(b) adding Solutol HS15 to the mixture;

(c) adding water, a buffer, and optionally a tonicity agent or a bulking agent to the mixture, the buffer maintaining the solution pH at 4 to 6; and

(d) lyophilizing said mixture under conditions that remove said water and said DMSO from said mixture, wherein the final concentration of DMSO is between 0.2% and less than 8%.

31. The method of claim 30, wherein said Solutol HS15 is mixed with water, a buffering agent, and optionally a tonicity or bulking agent prior to addition to said mixture, wherein the water, buffering agent, and optional tonicity or bulking agent are added in an amount to maintain the solubility of said conjugate in said mixture.

32. The method of claim 30 or 31, wherein the DMSO is acidified to a ph of between 3.5 and 4.5 prior to the dissolving of step (a).

33. The method of claim 30, wherein said lyophilizing does not reduce the amount of Solutol HS15 in the mixture.

34. A pharmaceutical composition produced by the method of any one of claims 30-33.