HK40013212B - Compstatin peptides with improved pharmacokinetic properties - Google Patents

Description

Compstatin peptides with improved pharmacokinetic properties

The present application is a divisional application of chinese patent application No.201280050936.4, filed on 9/7 of 2012, entitled "compstatin analog with improved pharmacokinetic properties".

Government funding

In accordance with 35u.s.c. ≡202 (c), it is acknowledged that the U.S. government may have certain rights in the invention described herein, in part with funds from the national institutes of health under the foundation numbers GM 62134, AI30040, AI068730, GM097747 and EY 020633.

Technical Field

The present invention relates to activation of the complement cascade in the body. In particular, the present invention provides peptides and peptidomimetics (pepidomimetics) that bind C3 protein with nanomolar affinity and inhibit complement activation, exhibit strong aqueous solubility, plasma stability, and in vivo residence, and can have bioavailability through a variety of routes of administration.

Background

Throughout the specification various publications are referenced, including patents, published applications, technical papers, and academic papers. Each of these cited publications is incorporated by reference in its entirety.

The human complement system is a powerful participant in the defense against pathogenic organisms and in the mediation of immune responses. Complement can be activated by 3 different pathways: classical pathway, lectin pathway and alternative pathway. The main activation event common to the 3 different pathways is proteolytic cleavage of the centroprotein C3 of the complement system by the C3 convertase into its activation products C3a and C3b. The generation of these fragments results in the regulatory action of C3b and iC3b on pathogenic cells, which render them susceptible to phagocytosis or clearance, and immune cell activation by interaction with complement receptors (Markiewski & Lambris,2007,Am J Pathol 171:715-727). Deposition of C3b on target cells also induces the formation of new invertase complexes, thereby initiating a self-amplification loop.

Together, plasma and cell surface binding proteins carefully regulate complement activation, preventing host cells from self-attack by the complement cascade. However, excessive activation or inappropriate regulation of complement can lead to a number of pathogenic conditions, including autoimmune to inflammatory diseases (Holers, 2003,Clin Immunol 107:140-51; markiewski & Lambris,2007, supra; ricklin & Lambris,2007,Nat Biotechnol 25:1265-75; sahu et al, 2000,J Immunol 165:2491-9). It is therefore highly desirable to develop therapeutic complement inhibitors. In this specification, C3 and C3b have been shown to be promising targets because their central role in the cascade allows simultaneous inhibition of complement initiation, amplification and downstream activation (Ricklin & Lambris,2007, supra).

Compstatin (compstatin) is the first complement inhibitor of non-host origin shown to block all 3 activation pathways (Sahu et al 1996,J Immunol 157:884-91;U.S.Patent 6,319,897). The cyclic tridecapeptide binds both C3 and C3b and prevents cleavage of native C3 by the C3 convertase. Its high inhibitory potency has been confirmed by a series of studies using experimental models indicating its potential as a therapeutic agent (Fine et al, 1999a,Xenotransplantation 6:52-65; fine et al, 1999b,Transplant Proc 31:934-935; nilsson et al, 1998Blood 92:1661-1667;Ricklin&Lambris,2008,Adv Exp Med Biol 632:273-292; schmidt et al, 2003,J Biomed Mater Res A66:491-499; soulika et al, 2000,Clin Immunol 96:212-221). The gradual optimization of compstatin has resulted in analogs with improved activity (Ricklin & Lambris,2008, supra; WO2004/026328; WO 2007/062249). One of these analogs is currently being tested in clinical trials for the treatment of age-related macular degeneration (AMD) (the leading cause of blindness in elderly patients in industrialized countries) (Coleman et al 2008,Lancet 372:1835-1845; ricklin & lambris,2008, supra). In view of its therapeutic potential in AMD and other diseases, it is important to further optimize compstatin for even greater efficacy.

Early structure-activity studies have identified the cyclic nature of compstatin peptides as well as the presence of β -turns and hydrophobic clusters as the main features of the molecule (Morikis et al 1998,Protein Sci 7:619-627; WO99/13899; morikis et al 2002,J Biol Chem 277:14942-14953; ricklin & lambris,2008, supra). The hydrophobic residues at positions 4 and 7 were found to be particularly important, and modification of them with unnatural amino acids resulted in analogs with 24-fold improvement in activity over the original compstatin peptide (Katragadda et al 2006,J Med Chem 49:4616-4622; WO 2007/062249).

While the previous optimization steps were based on combinatorial screening studies, solution structures and computer models (Chiu et al, 2008,Chem Biol Drug Des 72:249-256; mulakala et al, 2007,Bioorg Med Chem 15:1638-1644; ricklin & lambris,2008, supra), the publication of the compstatin co-crystal structure in complex with complement fragment C3C (Janssen et al, 2007,J Biol Chem 282:29241-29247; WO 2008/153963) represented important milestones to initiate rational optimization. The crystal structure shows a shallow binding site at the interface of the Macroglobulin (MG) domains 4 and 5 of C3C and 9 of the 13 amino acids are shown to directly participate in binding by hydrogen bonding or hydrophobic interactions. Compared to the structure of the compstatin peptide in solution (Morikis et al, 1998, supra), the conjugated form of compstatin undergoes a conformational change, shifting the position of the β -turn from residues 5-8 to 8-11 (Janssen et al, 2007, supra; WO 2008/153963).

The present inventors have recently developed a series of compstatin analogs with improved potency based on methylation at the peptide backbone (particularly at position 8 of the peptide) and substitution at flanking position 13 (Qu et al 2011,Molec Immunol 48:481-489, wo 2010/127336). These modifications are reported to produce compstatin analogs with binding affinities superior to most of the active analogs reported to date.

Compstatin and its analogs have significant potential for clinical use. Recent examples include reducing filter-induced adverse effects during hemodialysis and preservation of organs in sepsis. Importantly, intravitreal use of compstatin analogs has shown promising results in the treatment of age-related macular degeneration (AMD) both in non-human primate (NHP) studies and in phase I clinical trials. The low molecular weight of compstatin and its analogs, their high specificity and potency, and their ability to inhibit all complement activation and amplification pathways simultaneously, facilitate a beneficial drug modality. However, extended clinical applications (e.g., systemic administration via multiple routes) place additional demands on the molecular properties of compstatin derivatives. For example, adverse pharmacokinetic profiles resulting from rapid elimination from plasma still pose major limitations for peptide drugs. Furthermore, while oral delivery is the most convenient and popular route of drug administration, most peptide drugs show little or no oral activity. This is believed to be due primarily to degradation in the gastrointestinal tract by enzymes and extreme conditions and poor permeability of the intestinal mucosa. Thus, most protein-based therapeutic agents are administered by frequent injection (via parenteral routes, e.g., by intravenous, intramuscular, and subcutaneous injection). These forms of administration are very expensive and may require medical professionals, all of which can lead to poor patient acceptance and compliance. In view of the foregoing, it is apparent that the development of modified compstatin peptides or mimics with greater activity, in vivo stability, plasma residence time, and bioavailability would constitute a significant advance in the art.

Summary of The Invention

The present invention provides analogs of the complement inhibitory peptide compstatin that have improved complement inhibitory activity compared to compstatin and also have improved solubility and stability as well as pharmacokinetic properties, including bioavailability through a variety of routes of administration.

One aspect of the invention is a compound comprising a modified compstatin peptide (ICVVQDWGHHRCT (loop C2-C12; SEQ ID NO: 1) or an analog thereof, wherein the modification comprises an added or substituted N-terminal composition that improves (1) the C3, C3b or C3C binding affinity of the peptide, (2) the solubility of the peptide in an aqueous liquid, and/or (3) the plasma stability and/or plasma residence time of the peptide compared to an equivalent condition of the unmodified compstatin peptide.

Components that may be added to the N-terminus of a peptide include amino acid residues other than L-Gly or peptide or non-peptide analogs of such amino acids. In certain embodiments, the added component is a D-amino acid, and/or the component may include at least one aromatic ring. In one embodiment, the added component is D-Tyr. In other embodiments, the added component includes an N-methylated amino acid. In one embodiment, the N-methylated amino acid is N-methylated L-Gly, also referred to herein as Sar. Thus, in various embodiments, the component added is D-Tyr, D-Phe, tyr (Me), D-Trp, tyr, D-Cha, cha, phe, sar, arg, mPhe, mVal, trp, mIle, D-Ala, mAla, thr, or Tyr.

In other embodiments, the modified compstatin peptide comprises a substituted N-terminal component wherein Ile at position 1 is replaced with Ac-Trp or the dipeptide Tyr-Gly.

The compounds may also include other modifications. For example, his at position 9 (based on the number of compstatin) may be replaced by Ala. In addition, val at position 4 may be replaced by Trp or an analog of Trp. Specific analogs of Trp at position 4 include 1-methyl Trp or 1-formyl Trp. The Trp at position 7 may also be replaced with an analog of Trp (including but not limited to halogenated Trp). Other modifications include modifications of Gly at position 8 to constrain backbone conformation at this disposal. Specifically, the backbone can be constrained by replacing Gly (Gly 8) at position 8 with Nα -methyl Gly (Sar). Other modifications include substitution of Thr at position 13 with Ile, leu, nle, N-methyl Thr or N-methyl Ile. Still other modifications include the substitution of disulfide bonds between C2 and C12 with thioether bonds to form cystathionine or lanthionine (lantithinine). Another modification includes replacing Arg at position 11 with Orn and/or replacing Asp at position 6 with Asn.

In a specific embodiment, the compstatin analog comprises a peptide having the sequence of SEQ ID No. 29:

Xaa1-Xaa2-Cys-Val-Xaa3-Gln-Xaa4-Xaa5-Gly-Xaa6-His-Xaa7-Cys-Xaa8, wherein Gly between Xaa5 and Xaa6 (position 8 of compstatin) is optionallyIs modified to constrain the backbone conformation, and wherein: xaa1 is absent or Tyr, D-Tyr or Sar; xaa2 is Ile, gly or Ac-Trp; xaa3 is Trp or a Trp analog, wherein said Trp analog has enhanced hydrophobic character compared to Trp; xaa4 is Asp or Asn; xaa5 is Trp or a Trp analog comprising a chemical modification to an indole ring thereof, wherein the chemical modification enhances the hydrogen bonding potential of the indole ring; xaa6 is His, ala, phe or Trp; xaa7 is Arg or Orn; and Xaa8 is Thr, ile, leu, nle, N-methyltr or N-methylile, wherein the carboxyl terminus-OH of either Thr, ile, leu, nle, N-methyltr or N-methylile is optionally replaced by-NH ₂ Instead, and wherein the peptide is cyclic through a Cys-Cys or thioether bond.

Some embodiments of the analog include the following features: gly at position 8 is N-methylated; xaa1 is D-Tyr or Sar; xaa2 is Ile; xaa3 is Trp, 1-methyl-Trp or 1-formyl-Trp; xaa5 is Trp; xaa6 is Ala; and Xaa8 is Thr, ile, leu, nle, N-methyltr or N-methylile, optionally with the carboxyl terminus-OH being-NH ₂ Instead of this. More specifically Xaa8 can be Ile, N-methyl Thr or N-methyl Ile, optionally with the carboxyl terminal-OH being-NH ₂ Instead of this. Exemplary analogs include SEQ ID NO. 7 and SEQ ID NO. 18. Another aspect of the invention relates to a compound that inhibits complement activation comprising a non-peptide or partial peptide mimetic of SEQ ID NO. 7 or SEQ ID NO. 18, wherein the compound binds C3 and inhibits complement activation at least 500-fold more activity than the activity of a peptide comprising SEQ ID NO. 1 under equivalent assay conditions.

Another aspect of the invention relates to the above compounds, which include additional components that extend the in vivo residence (i.e., residence time) of the compounds. In one embodiment, the additional component is polyethylene glycol (PEG). In other embodiments, the additional component is an albumin binding small molecule or albumin binding peptide. In some particular embodiments, an albumin binding small molecule or albumin binding peptide is attached to the peptide at the N or C terminus. The connection may be direct or through a linker or spacer.

Another aspect of the invention relates to a pharmaceutical composition comprising any of the above compounds and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition is formulated for oral administration. In another embodiment, it is formulated for topical administration. In another embodiment, it is formulated for pulmonary administration. In another embodiment, the pharmaceutical composition is formulated for subcutaneous or intramuscular injection. In another embodiment, it is formulated for intravenous injection or infusion.

In another aspect the invention provides the use of any of the above compounds for inhibiting complement activation in vivo, ex vivo, in situ or in vitro and for the manufacture of a medicament for inhibiting complement activation.

The various features and advantageous aspects of the invention will be understood by reference to the following detailed description, figures and examples.

Brief description of the drawings

FIG. 1 interaction of compstatin analogs with C3 b. (A) Kinetic profiles of compstatin lead compound 4 (1 MeW) (lowest, narrowest group of peaks), cp20 (middle group of peaks) and peptide 14 (Cp 40 (SEQ ID NO: 18)) (highest widest group of peaks), as determined by single cycle kinetic analysis using surface plasmon resonance. (B) Peptides 1-20 with an isoperistic line (isoaffinic line) and a Rate plot (Rate plot) of reference compound 4 (1 MeW) and Cp20 (SEQ ID NO: 3), as shown by the dashed lines. A baseline of the velocity constant and affinity of Cp20 (SEQ ID NO: 3) is shown.

FIG. 2 correlation between free energy value (ΔG) (y-axis) calculated from calculated docking experiments (computational docking experiment) between compstatin analogs and C3C and free energy value (x-axis) calculated from experimentally determined affinity values of the same analogs of C3 b. Peptide numbers are shown alongside each tag on the figure. The correlation over the whole data set is shown as a solid line, while the dotted line represents the correlation after excluding peptides 1, 5 and 7.

FIG. 3 docking of compstatin analogs within the binding site of C3C. (A) The docking conformation of peptide 14 (Cp 40 (SEQ ID NO: 18), blue-green in the color chart) and peptide 4 (gray in the color chart) (note that in the achromatic chart the aromatic ring of the dY side chain (CP 40 (SEQ ID NO: 18)) is superimposed before the ring of Y (peptide 4) and can be distinguished in this way). Other D-amino acids have a similar conformation to peptide 14 in the docking model. The side chains of other residues in peptide 4 are omitted for clarity. (B) the docking conformation of peptide 19.

FIG. 4 stability of peptide 3 (Cp 30 (SEQ ID NO: 7)) and peptide 14 (Cp 40 (SEQ ID NO: 18)) at 37℃in human plasma. Cp30 (SEQ ID NO: 7), cp40 (SEQ ID NO: 18) and positive control peptide 2B were incorporated into human plasma to reach a final concentration of 20. Mu.M. Plasma was incubated at 37℃and 100. Mu.L of samples were taken at different time points. Peptides were extracted from plasma using solid phase extraction and analyzed using UPLC-MS. 3A: the area of each sample at the various time points was plotted over time (square: cp40 (SEQ ID NO: 18), circle: cp30 (SEQ ID NO: 7), triangle: control peptide 2B). 3B: chromatograms of samples from time 0 (upper), 24h (middle) and 120h (lower).

Fig. 5 pharmacokinetic assessment of compstatin analogs in non-human primates. (A) A linear plot of peptide levels over time following a single fast intravenous injection of 2mg/kg in cynomolgus monkeys shows a biphasic model with a fast initial elimination phase followed by a slow log-linear end phase. Cp20 (SEQ ID NO: 3) -the bottom two lines; cp30 (SEQ ID NO: 7) (peptide 3) -the middle two lines; cp40 (SEQ ID NO: 18) (peptide 14) -upper two lines. (B) Calculation of plasma elimination half-life (t) from the terminal phase (1-24 h) _1/2 ). Cp20 (SEQ ID NO: 3) -two lines below; cp30 (SEQ ID NO: 7) (peptide 3) -the middle two lines; cp40 (SEQ ID NO: 18) (peptide 14) -upper two lines. The dashed lines mark the range of measured plasma levels of target protein C3 in panels a and B. (C) Superposition of the kinetic binding profile of analog Cp20 (SEQ ID NO: 3) with immobilized C3 from human, baboon, cynomolgus and macaque, as assessed by SPR.

FIG. 6 plasma concentrations of the analog post-compstatin analog Cp40 (SEQ ID NO: 18) in cynomolgus monkeys by two different routes for single administration. Plasma concentrations were measured by mass spectrometry at various time points after subcutaneous injection (top panel) or oral administration (bottom panel).

FIG. 7 plasma concentration and complement inhibitory activity of the compstatin analog Cp40 (SEQ ID NO: 18) after a single administration of the analog by intramuscular injection in baboons. Plasma concentrations were measured by mass spectrometry at various time points after intramuscular injection (circles). Inhibition of complement activation via the alternative pathway was measured by erythrocyte hemolysis assay (triangle).

Detailed description of illustrative embodiments

Definition:

various terms relating to methods and other aspects of the present invention are used throughout the specification and claims. Unless otherwise indicated, such terms will have their ordinary meaning in the art. Other well-defined terms are to be construed in a manner consistent with the definitions provided herein.

The following abbreviations may be used herein: ac, acetyl; DCM, dichloromethane; DIC,1, 3-diisoendocarbodiimide; DIPEA, N-diisopropylethylamine; DPBS, dulbecco's phosphate buffered saline; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; fmoc, 9-fluorenylmethoxycarbonyl; HOAt, 1-hydroxy-7-azabenzotriazole; ITC, isothermal titration calorimetry; MALDI, matrix-assisted laser desorption ionization; MBHA, 4-methylbenzylamine (4-methylbenzylamine); NMP, N-methylpyrrolidone; sar, N-methylglycine; SPR, surface plasmon resonance; TIPS, triisopropylsilane; trt, trityl; WFI, water for injection.

As used herein, the term "about" when referring to a measurable value, e.g., amount, length of time, etc., is intended to include a ± 20% or ± 10% change from the specified value, in some embodiments a ± 5% change, in some embodiments a ± 1% change, in some embodiments a ± 0.1% change, as such changes are suitable for making and using the disclosed compounds and compositions.

As used herein, the term "compstatin" refers to peptide ICVVQDWGHHRCT (cyclic C2-C12 formed by disulfide bonds) comprising SEQ ID No. 1. The term "compstatin analog" refers to a modified compstatin comprising substitutions of natural and/or unnatural amino acids or amino acid analogs, modified within or between different amino acids (as described in more detail herein and as known in the art). When referring to the positions of particular amino acids or analogs within compstatin or compstatin analogs, these positions are sometimes referred to as "positions" within the peptide, where the positions are numbered from 1 (Ile in compstatin) to 13 (Thr in compstatin). For example, gly residues occupy "position 8".

The terms "pharmaceutically active" and "biological activity" refer to the ability of a compound of the invention to bind to C3 or a fragment thereof and inhibit complement activation. The biological activity may be measured by one or more assays recognized in the art (as described in more detail herein).

As used herein, "alkyl" refers to an optionally substituted straight, branched, or cyclic hydrocarbon having from about 1 to 10 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 1 to about 7 carbon atoms being preferred. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl, cyclooctyl, adamantyl, 3-methylpentyl, 2-dimethylbutyl, and 2, 3-dimethylbutyl. The term "lower alkyl" refers to an optionally substituted saturated straight, branched or cyclic hydrocarbon having from about 1 to about 5 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein). Lower alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl, isopentyl, and neopentyl.

As used herein, "halogen" refers to F, cl, br or I.

As used herein, "alkanoyl" is used interchangeably with "acyl" and refers to an optionally substituted straight or branched aliphatic acyclic residue having from about 1 to about 10 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 1 to about 7 carbon atoms being preferred. Alkanoyl includes, but is not limited to, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, isopentanoyl, 2-methyl-butyryl, 2-dimethylpropionyl, hexanoyl, heptanoyl, octanoyl, and the like. The term "lower alkanoyl" refers to an optionally substituted straight or branched aliphatic acyclic residue having from about 1 to about 5 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein). Lower alkanoyl includes, but is not limited to, formyl, acetyl, n-propionyl, iso-propionyl, butyryl, iso-butyryl, pentanoyl, iso-pentanoyl and the like.

As used herein, "aryl" refers to an optionally substituted mono-or bi-cyclic aromatic ring system having from about 5 to about 14 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 6 to about 10 carbons being preferred. Non-limiting examples include, for example, phenyl and naphthyl.

As used herein, "aralkyl" refers to an alkyl group as defined above having an aryl substituent and having from about 6 to about 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 6 to about 12 carbon atoms being preferred. Aralkyl groups may be optionally substituted. Non-limiting examples include, for example, benzyl, naphthylmethyl, benzhydryl, trityl, phenethyl, and diphenylethyl.

As used herein, the terms "alkoxy" and "alkoxy" refer to an optionally substituted alkyl-O-group, wherein alkyl is as previously defined. Exemplary alkoxy and alkoxy groups include methoxy, ethoxy, n-propylamino, i-propoxy, n-butoxy, heptyloxy and the like.

As used herein, "carboxyl" refers to a-C (=o) OH group.

As used herein, "alkoxycarbonyl" refers to a-C (=o) O-alkyl group, wherein alkyl is as previously defined.

As used herein, "aroyl" refers to a-C (=o) -aryl group, wherein aryl is as previously defined. Exemplary aroyl groups include benzoyl and naphthoyl.

Typically, the substituted chemical moiety comprises one or more substituents replacing hydrogen at selected positions on the molecule. Exemplary substituents include, for example, halogen, alkyl, Cycloalkyl, aralkyl, aryl, mercapto, hydroxy (-OH), alkoxy, cyano (-CN), carboxyl (-COOH), acyl (alkanoyl: -C (=o) R); -C (=o) O-alkyl, aminocarbonyl (-C (=o) NH ₂ ) -N-substituted aminocarbonyl (-C (=o) NHR "), CF ₃ 、CF ₂ CF ₃ Etc. In connection with the substituents described above, each moiety "R" may independently be, for example, any of H, alkyl, cycloalkyl, aryl, or aralkyl.

As used herein, "L-amino acid" refers to the natural L-amino acid or any of the alkyl esters of these alpha-amino acids that are commonly found in proteins. The term D-amino acid refers to a dextrorotatory alpha-amino acid. All amino acids mentioned herein are L-amino acids unless explicitly indicated otherwise.

"hydrophobic" or "nonpolar" may be used synonymously herein to refer to any intermolecular or intramolecular interaction that is not characterized by dipoles.

"PEGylation" refers to a reaction in which at least one polyethylene glycol (PEG) moiety, regardless of size, is chemically attached to a protein or peptide to form a PEG-peptide conjugate. By "pegylated" is meant that at least one PEG moiety (regardless of size) is chemically attached to a peptide or protein. The term PEG is generally accompanied by a numerical suffix indicating the approximate average molecular weight of the PEG polymer; for example, PEG-8,000 refers to polyethylene glycol having an average molecular weight of about 8,000 daltons (or g/mol).

As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by the formation of an acid or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; basic or organic salts of acidic residues such as carboxylic acids; etc. Thus, the term "acid addition salt" refers to the corresponding salt derivative of the parent compound that has been prepared by the addition of an acid. Pharmaceutically acceptable salts include, for example, conventional salts or quaternary ammonium salts of the parent compound formed from inorganic or organic acids. For example, such conventional salts include, but are not limited to, salts derived from mineral acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like. Certain acidic or basic compounds of the present invention may exist in zwitterionic forms. All forms of the compounds (including free acid, free base and zwitterion) are included within the scope of the invention.

Description of:

the present invention is derived in part from the inventors' development of compstatin analogs with improved inhibition potency and pharmacokinetic parameters. Selective modification of the N-terminus of compstatin using non-protein derived amino acids and/or other molecular entities results in certain binding affinities (K) _D =0.5 nM) and other similarly potent derivatives with improved solubility in clinically relevant solvents. Pharmacokinetic evaluations in non-human primates showed plasma half-life values beyond that expected for peptide drugs. Bioavailability evaluations in two non-human primate models showed that certain analogs have subcutaneous, intramuscular, and oral bioavailability.

One modification according to the invention comprises adding a component to the N-terminus of compstatin (Ile- [ Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys ] -Thr; SEQ ID NO: 1) which improves peptide solubility and plasma stability while maintaining or improving C3 binding affinity and complement inhibitory activity. In particular embodiments, the added component is an amino acid residue, particularly a residue that is resistant to proteolytic cleavage, such as an N-methylated amino acid (e.g., N-methyl Gly (Sar) or D-amino acid ((e.g., D-Tyr)) as such, the D configuration of the N-terminal residue may better configure the free amino group to interact with the C3 polarity as described in more detail below.

With reference to the exemplary analogs shown below, the analogs show advantages over compstatin and even the highly potent analogs Ac-Ile- [ Cys-Val-Trp (Me) -Gln-Asp-Trp-Gly-Ala-His-Arg-Cys]-Thr-NH ₂ (SEQ ID NO: 2) (Katragadda et al, 2006, supra, WO 2007/062249; sometimes referred to herein as "4 (1 MeW)") and several other advantageous features described in detail below.

"compstatin 30" (Cp 30):

Sar-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH ₂

(SEQ ID NO:7; also referred to as "peptide 3" in the examples)

"compstatin 40" (Cp 40):

dTyr-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH ₂

(SEQ ID NO:18; also referred to as "peptide 14" in the examples)

Without wishing to be bound or limited by theory, it is believed that the improved C3 binding affinity of the analogs described herein is due, at least in part, to higher affinity interactions mediated by the N-terminus. For example, SPR and ELISA data show that D-amino acids or amino acids with hydrophobic side chains improve C3 binding, while a combination of features (i.e., D-amino acids with aromatic side chains, such as D-Tyr) is most advantageous. In general, D-amino acids with aromatic side chains show advantages over D or L configuration amino acids with shorter side chains. Furthermore, docking studies indicate that improved affinity results from additional polar and nonpolar interactions involving localization of free amino groups, as well as nature and localization of side chains on the N-terminal residues. For example, the increased affinity of Cp40 (SEQ ID NO: 18) was determined to be due at least in part to a combination of interactions with C3 at the N-terminus of the analog; (1) The D configuration of the N-terminal Tyr better provides free amino groups for polar interactions with C3, a property that generally explains the advantageous aspects of the D configuration; and (2) bulky hydrophobic side chains are capable of fitting the hydrophobic pocket on C3C and also provide hydroxyl groups to hydrogen bond with C3C. Furthermore, docking studies predicted that analogs comprising Ac-Trp (example 2) at the N-terminus bind C3 with high affinity. SPR analysis of peptide 1 did show high binding affinity comparable to that of Cp40 (SEQ ID NO: 18) (peptide 14). Both peptides have been determined to utilize a hydrophobic binding pocket on C3, C3b or C3C near the N-terminus of compstatin.

N-methylation can affect peptides in several ways. First, the potential hydrogen bond donors are replaced with methyl groups, which are unable to form hydrogen bonds. Second, N-methyl is a weak electron donor, meaning that it can slightly increase the basicity of adjacent carbonyl groups. Third, the size of the N-methyl group can cause steric constraints depending on the nature of the adjacent residues. Finally, N-methylation can alter the trans/cis population of amide linkages, thereby altering the local peptide conformation in a similar manner to proline. In the case of Cp30 (SEQ ID NO: 7), the SPR data indicated a slightly faster association rate and a slightly slower dissociation rate than Cp20 (SEQ ID NO: 3), which indicates that Cp30 (SEQ ID NO: 7) has a more favourable free solution conformation for binding C3/C3b/C3C and binds more strongly. Given the lack of an Ac group at the N-terminal position and the presence of a methyl group, it can be reasonably generalized that the modification allows the N-terminal to participate in a stronger polar interaction with residues S388/S437/D349 of C3C. This is made possible by the localization of the free N-terminus to a favorable position (by N-methylation in a manner that enhances polar interactions with the binding site on C3/C3C).

In addition to improved C3 binding affinity, the analogs of the invention also have improved solubility characteristics compared to previously available analogs such as Cp20 (SEQ ID NO: 3). For systemic drug administration, analogues with high solubility in water for injection (WFI) and Phosphate Buffered Saline (PBS) should preferably minimize the required injection volume. By comparison, analogs having high solubility in WFI and lower solubility in PBS can produce longer-acting gels, precipitates, or suspensions for topical administration or local injection, such as intraocular injection (e.g., for treatment of AMD). Cp30 (SEQ ID NO: 7) was determined to be soluble in WFI and PBS, however Cp40 (SEQ ID NO: 18) was less soluble in PBS than in WFI.

Peptide analogs of the invention also exhibit favorable plasma stability characteristics, which are believed to be due at least in part to the presence of one or more N-terminal components (e.g., D-amino acid residues or N-methyl groups) or albumin binding molecules that resist protease attack. In addition, the analogs specifically and strongly bind to C3, C3b and C3C in plasma. Importantly, the stability provided by the N-terminal and/or other modifications described herein contributes to improved bioavailability for oral, subcutaneous, or intramuscular administration, as shown in the mouse and two non-human primate model systems, as well as improved (i.e., slower) in vivo plasma elimination half-life values like shown in the primate model systems.

The above-described N-terminal modifications can be combined with other modifications of compstatin previously shown to improve activity, thereby producing peptides with significantly improved complement inhibitory activity. For example, acetylation of the N-terminus generally enhances the complement inhibitory activity of compstatin and its analogs. Thus, the addition of an acyl group at the amino terminus of a peptide (including but not limited to N-acetylation) is one embodiment of the invention, although it may not be necessary if the N-terminus of the peptide is already stable or if solubility becomes an issue.

As another example, substitution of His at position 9 by Ala is known to improve the activity of compstatin and is also a preferred modification of the peptides of the invention. Substitution of Tyr for Val at position 4 has also been determined to result in a modest improvement in activity (Klepeis et al 2003,J Am Chem Soc 125:8422-8423).

WO2004/026328 and WO2007/0622249 disclose that Trp and certain Trp analogues at position 4 and certain Trp analogues at position 7 (in particular in combination with Ala at position 9) give many times higher activity than compstatin. These modifications are likewise used to obtain advantageous aspects in the present invention.

Specifically, peptides comprising 5-fluoro-tryptophan or 5-methoxy-, 5-methyl-or 1-methyl-tryptophan or 1-formyl-tryptophan at position 4 have been shown to have 31-264 fold higher activity than compstatin. Particular preference is given to 1-methyl-and 1-formyltryptophan. It is believed that the indole 'N' -mediated hydrogen bond at position 4 is not necessary for compstatin binding and activity. The absence of this hydrogen bond or the reduction of the polar character enhances the binding and activity of compstatin by replacing the hydrogen with a lower alkyl, alkanoyl or indole nitrogen at position 4. Without wishing to be bound by any particular theory or mechanism of action, it is believed that the hydrophobic interaction or effect at position 4 enhances the interaction of compstatin with C3. Thus, modification of Trp at position 4 (e.g., altering the structure of the side chain according to methods well known in the art), or substitution of Trp analogs that maintain or enhance the above hydrophobic interactions at position 4 or position 7, are considered advantageous modifications in the present invention, combining the modifications at positions 8 and 13 described above. Such analogs are well known in the art and include, but are not limited to, the analogs illustrated herein, as well as unsubstituted or otherwise substituted derivatives thereof. Examples of suitable analogues can be found by reference to the following list of publications and many other publications: beene, et al, 2002,Biochemistry 41:10262-10269 (describing mono-and poly-halogenated Trp analogs, etc.); babitzky & Yanofsky,1995, J.biol. Chem.270:12452-12456 (describing methylation and halogenated Trp and other Trp and indole analogs, etc.); and U.S. patent nos. 6,214,790, 6,169,057, 5,776,970, 4,870,097, 4,576,750 and 4,299,838. As known in the art, trp analogs may be introduced into compstatin peptides by in vitro or in vivo expression, or by peptide synthesis.

In certain embodiments, a substituted alkyl group is substituted with a substituent comprising a 1-alkyl group, more particularly a lower alkyl group (e.g., C ₁ -C ₅ ) Analogs of substituents (as defined above) replace Trp at position 4 of compstatin. These analogs include, but are not limited to, N (alpha) methyl tryptophan and 5-methyl tryptophan. In other embodiments, a compound comprising a 1-alkanoyl, more specifically a lower alkanoyl (e.g., C ₁ -C ₅ ) Analogs of substituents (as defined below) such as N (. Alpha.) formyl tryptophan, 1-acetyl-L-tryptophan and L-. Beta. -homotryptophan replace Trp at position 4 of compstatin.

WO2007/0622249 discloses that the incorporation of 5-fluoro-tryptophan at position 7 of compstatin increases the enthalpy of interaction between the resulting compstatin analogue and C3 relative to compstatin, whereas the incorporation of 5-fluoro-tryptophan at position 4 decreases the enthalpy of interaction. Thus, modification of Trp at position 7, as described in WO2007/0622249, in combination with the above described N-terminal modification is considered a useful modification in the present invention.

Other modifications are described in WO 2010/127336. One modification disclosed in this document includes constraints on the peptide backbone at position 8 of the peptide. In one embodiment, the amino acid sequence is obtained by substituting glycine (Gly) at position 8 with N-methylglycine ⁸ ) To constrain the backbone. Another modification disclosed in this document includes substitution of Thr at position 13 with Ile, leu, nle (norleucine), N-methyl Thr or N-methyl Ile.

Still other modifications are described in co-pending application Ser. No.61/385,711. One such modification includes by adding CH ₂ To replace the C2-C12 disulfide bond to form homocysteine on C2 or C12, and to introduce a thioether bond to form a cystathionine, such as γ -cystathionine or δ -cystathionine. Another modification includes the addition of a thioether bond rather than CH ₂ To replace the C2-C12 disulfide bond, thereby forming lanthionine. Analogs containing thioether linkages exhibit substantially the same activity as that of certain disulfide-bonded analogs and also have equivalent or improved stability characteristics.

Other internal modifications are described in this application. For example, substitution of ornithine for arginine at position 11, and/or asparagine at position 6 of certain compstatin analogs (e.g., cp20, SEQ ID NO:3,Cp40,SEQ ID NO:18) results in analogs having similar binding and complement inhibitory activity as the parent compound. Furthermore, one or both of these substitutions are expected to make the analog less susceptible to metabolism by certain physiological enzymes found in the gut, liver or plasma.

The modified compstatin peptides of the invention may be prepared by condensation of one or more amino acid residues according to conventional peptide synthesis methods using various peptide synthesis methods. For example, peptides are synthesized according to standard solid phase methods. Other methods of synthesizing peptides or peptide mimetics by solid phase methods or in liquid phase are well known to those skilled in the art. During peptide synthesis, branched amino and carboxyl groups may be protected/deprotected as desired using known protecting groups. An example of a suitable peptide synthesis is shown in example 1. Modifications to peptides and peptide derivatives using alternative protecting groups will be apparent to those skilled in the art.

Alternatively, certain peptides of the invention may be produced by expression in a suitable prokaryotic or eukaryotic system. For example, the DNA construct may be inserted into a plasmid vector suitable for expression in a bacterial cell, such as e.coli (e.coli), or a yeast cell, such as s.cerevisiae, or into a baculovirus vector for expression in an insect cell or a viral vector for expression in a mammalian cell. Such vectors contain regulatory elements necessary for expression of the DNA in the host cell in a localized manner that permits expression of the DNA in the host cell. Regulatory elements required for such expression include promoter sequences, transcription initiation sequences, and optionally enhancer sequences.

Peptides may also be produced by expressing a nucleic acid molecule in vitro or in vivo. DNA constructs encoding concatemers of peptides (the upper limit of the concatemers depends on the expression system used) can be introduced into an in vivo expression system. After concatamers are generated, cleavage between the C-terminal Asn and then the N-terminal G is achieved by exposing the polypeptide to hydrazine.

Peptides produced by gene expression in recombinant prokaryotic or eukaryotic systems can be purified according to methods known in the art. Combinations of gene expression and synthesis may also be used to produce compstatin analogs. For example, analogs can be produced by gene expression and subsequently subjected to one or more post-translational synthetic processes, e.g., to modify the N-or C-terminal or cyclized molecules.

Advantageously, peptides incorporating unnatural amino acids, e.g., methylated amino acids, can be produced by in vivo expression in a suitable prokaryotic or eukaryotic system. For example, N-methylated amino acids or other unnatural amino acids can be introduced at selected positions of compstatin using methods such as those described by Katragadda & Lambris (2006,Protein Expression and Purification 47:289-295) for introducing unnatural Trp analogs into compstatin by expression in E.coli auxotrophs.

The structure of compstatin is known in the art and the structure of the foregoing analogs is determined by similar methods. After confirming a particularly desirable conformation of a short peptide, methods for designing peptides or peptide mimics that fit the conformation are well known in the art. Of particular relevance to the present invention, the design of peptide analogs can be further improved by considering the contributions of the individual side chains of the amino acid residues, as discussed above (i.e., with respect to the effect of the functional groups or with respect to stereochemistry).

It will be appreciated by those skilled in the art that peptide mimetics can equally well be used as peptides to provide the specific backbone conformation and side chain functionality required for binding to C3 and inhibiting complement activation. Thus, it is within the scope of the invention to generate compounds that bind C3, inhibit complement, by using naturally occurring amino acids, amino acid derivatives, analogs, or non-amino acid molecules that can be linked to form an appropriate backbone conformation. The non-peptide analogs or analogs comprising a peptide and a non-peptide component are sometimes referred to herein as "peptide mimetics" or "isomimetics (isosteric mimetic)", to denote substitution or derivatization of the peptides of the invention that have the same backbone conformational characteristics and/or other functionalities so as to be sufficiently similar to the exemplified peptides to inhibit complement activation.

The use of peptide mimetics for the development of high affinity peptide analogs is well known in the art (see, e.g., vagner et al, 2008, curr. Opin. Chem. Biol.12:292-296; robinson et al, 2008,Drug Disc.Today 13:944-951). Given that rotation constraints similar to those of amino acid residues in peptides, analogs containing non-amino acid moieties, can be analyzed, their conformational motifs can be verified using any computerized technique known in the art.

The modified compstatin peptides of the invention may be modified by adding a polyethylene glycol (PEG) component to the peptide. As is well known in the art, pegylation can increase the in vivo half-life of therapeutic peptides and proteins. In one embodiment, the PEG has an average molecular weight of about 1,000 to about 50,000. In another embodiment, the PEG has an average molecular weight of about 1,000 to about 20,000. In another embodiment, the PEG has an average molecular weight of about 1,000 to about 10,000. In an exemplary embodiment, the PEG has an average molecular weight of about 5,000. The polyethylene glycol may be branched or linear, preferably linear.

The compstatin analog of the invention may be covalently bound to PEG through a linking group. Such methods are well known in the art. (reviewed in Kozlowski A. Et al 2001,BioDrugs 15:419-29; see also Harris JM and Zalipsky S, eds. Poly (ethylene glycol), chemistry and Biological Applications, ACS Symposium Series 680 (1997)). Non-limiting examples of acceptable linking groups include ester groups, amide groups, imide groups, carbamate groups, carboxyl groups, hydroxyl groups, carbohydrates, succinimide groups (including but not limited to Succinimide Succinate (SS), succinimide Propionate (SPA), succinimide Carboxymethyl (SCM), succinimide Succinamide (SSA), and N-hydroxysuccinimide (NHS)), epoxide groups, oxycarbonyl imidazole groups (including but not limited to Carbonyldiimidazole (CDI)), nitrophenyl groups (including but not limited to nitrophenyl carbonate (NPC) or trichlorophenyl carbonate (TPC)), tosylate (trysilate) groups, aldehyde groups, isocyanate groups, vinyl sulfone groups, tyrosine groups, cysteine groups, histidine groups, or primary amines. In certain embodiments, the linking group is a succinimide group. In one embodiment, the linking group is NHS.

Alternatively, the compstatin analogs of the invention may be directly coupled to PEG (i.e., no linking group) via an amino, thiol, hydroxyl, or carboxyl group. In one embodiment, PEG may be coupled to a lysine residue added to the C-terminus of compstatin.

Alternatively to pegylation, in vivo clearance of the peptide may also be reduced by attaching the peptide to some other molecule or peptide. For example, certain Albumin Binding Peptides (ABPs) exhibit a significantly long half-life of 2.3h when injected into rabbits by intravenous bolus injection (Dennis et al, 2002,J Biol Chem.277:35035-35043). This type of peptide fused to D3H44 against tissue factor Fab enables Fab to bind albumin while retaining the ability of Fab to bind tissue factor (Nguyen et al, 2006,Protein Eng Des Sel.19:291-297.). This interaction with albumin results in significantly reduced in vivo clearance and prolonged half-life in mice and rabbits when compared to wild-type D3H44Fab, comparable to that seen for pegylated Fab molecules, immunoadhesins and albumin fusions. WO2010/127336 proposes a suitable synthetic strategy using ABP and albumin binding small molecules (ABM), and optionally spacers or linkers between components. These methods result in the production of ABP-and ABM-compstatin analog conjugates that are capable of inhibiting complement activation and also exhibit prolonged survival in vivo. Example 1 herein describes the use of these and other methods for using higher affinity albumin to bind small molecule ABM2 for the production of compstatin analog-ABM 2C-terminal conjugates using linker molecules. Example 1 further describes the generation of N-terminal conjugates of certain compstatin analogs with 3 different albumin binding small molecules ABM, ABM0, and ABM2 using direct ligation without a linker. Such conjugates, whether conjugated directly via the C-terminus, the N-terminus, or via a spacer or linker, exhibit C3 binding and complement inhibitory activity comparable to or exceeding that of the unconjugated analog, as well as favorable in vivo retention.

The complement activation inhibitory activity of compstatin analogs, peptidomimetics and conjugates can be tested by a variety of assays known in the art. In certain embodiments, the assays described in the examples are utilized. A non-exhaustive list of other assays is shown in U.S. Pat. No. 6,319,897, WO99/13899, WO2004/026328, WO2007/062249 and WO2010/127336, including but not limited to (1) peptide binding of C3 and C3 fragments; (2) various hemolysis assays; (3) measurement of C3 convertase mediated C3 cleavage; and (4) measurement of factor D to factor B cleavage.

The peptides and peptidomimetics described herein have practical utility for any purpose known in the art that utilizes compstatin itself. Such uses include, but are not limited to: (1) Inhibiting complement activation in serum and on cells, tissues or organs of a patient (human or animal), which may be beneficial in the treatment of certain diseases or conditions including, but not limited to, age-related macular degeneration, rheumatoid arthritis, spinal cord injury, parkinson's disease, alzheimer's disease, cancer, sepsis, paroxysmal nocturnal hemoglobinuria, psoriasis, and respiratory disorders such as asthma, chronic Obstructive Pulmonary Disease (COPD), allergic inflammation, emphysema, bronchitis, bronchiectasis, cystic fibrosis, tuberculosis, pneumonia, respiratory distress syndrome (RDS-newborns and adults), rhinitis, and sinusitis; (2) Inhibiting complement activation that occurs during cell or organ transplantation or during use of the artificial organ or implant (e.g., by systemic administration prior to, during and/or after performing the method, or by coating or otherwise treating the cell, organ, artificial organ or implant with a peptide of the invention); (3) Inhibiting complement activation that occurs during in vitro diversion of physiological solutions (blood, urine) (e.g., by systemic administration at a time limit before, during, and/or after performing the method, or by coating a conduit through which the fluid is diverted with a peptide of the invention); and (4) screening the small molecule library to identify other inhibitors of compstatin activation (e.g., liquid or solid phase high throughput assays designed to measure the ability of a test compound to compete with compstatin analogs for binding to C3 or C3 fragments).

To perform one or more of the above functions, another aspect of the present invention features a pharmaceutical composition comprising a compstatin analog or conjugate described and illustrated herein. Such pharmaceutical compositions may consist of the active ingredient alone (in a form suitable for administration to a subject), or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these agents. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt (e.g., in combination with a physiologically acceptable cation or anion), as is well known in the art.

Based on its solubility characteristics, the particular compstatin analog of the invention may be selected for a particular formulation. As mentioned above, analogs that are highly soluble in water or buffered saline may be particularly suitable for systemic injection because the injection volume may be minimized. By comparison, analogs having high solubility in water and lower solubility in buffered saline may yield longer lasting gels, suspensions, or precipitates for topical administration or local injection, such as intraocular injection. Thus, for illustrative purposes and not intended to be limiting, cp30 (SEQ ID NO: 7) may be selected for pharmaceutical formulations to be administered by systemic injection, while Cp40 (SEQ ID NO: 18) may be selected for formulations for intravitreal injection. Notably, cp40 (SEQ ID NO: 18) has been shown to be obtainable orally and by subcutaneous or intramuscular injection, which provides an important additional delivery route, as discussed below.

The formulation of the pharmaceutical composition may be prepared by any method known in the art of pharmaceutical technology or developed in the future. Generally, such preparative methods include the steps of bringing the active ingredient into association with the carrier or one or more other auxiliary agents and then, if necessary or desired, shaping or packaging the product into the desired dosage unit or units.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical component that can be combined with a compstatin analog and, upon combination, can be used to administer the compstatin analog to a subject.

As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient that is compatible with any other ingredients in the pharmaceutical composition, and is not deleterious to the subject to which the composition is to be administered.

The pharmaceutical compositions useful in practicing the present invention may be administered in a single bolus mode or in a repeated regimen or in a combination readily determinable by one of skill in the art to deliver doses of 1ng/kg to 100mg/kg body weight. In certain embodiments, the dosage comprises at least 0.1mg/kg, or at least 0.2mg/kg, or at least 0.3mg/kg, or at least 0.4mg/kg, or at least 0.5mg/kg, or at least 0.6mg/kg, or at least 0.7mg/kg, or at least 0.8mg/kg, or at least 0.9mg/kg, or at least 1mg/kg, or at least 2mg/kg, or at least 3mg/kg, or at least 4mg/kg, or at least 5mg/kg, or at least 6mg/kg, or at least 7mg/kg, or at least 8mg/kg, or at least 9mg/kg, or at least 10mg/kg, or at least 15mg/kg, or at least 20mg/kg, or at least 25mg/kg, or at least 30mg/kg, or at least 35mg/kg, or at least 40mg/kg, or at least 45mg/kg, or at least 50mg/kg, or at least 55mg/kg, or at least 60mg, or at least 6mg/kg, or at least 80mg, or at least 75mg, or at least 5mg/kg on a daily basis, or another suitable periodic regimen. In a specific embodiment, the dosage is from about 0.5mg/kg to about 20mg/kg, or from about 1mg/kg to about 10mg/kg, or from about 2mg/kg to about 6mg/kg.

In one embodiment, the invention includes administering a dose that results in a serum concentration of the compstatin analog in the subject in the range of about 0.01 μm to about 30 μm. In certain embodiments, the combined dosages and regimens may result in a serum concentration of the compstatin analog or an average serum concentration over time of at least about 0.01 μm, or at least about 0.02 μm, or at least about 0.03 μm, or at least about 0.04 μm, or at least about 0.05 μm, or at least about 0.06 μm, or at least about 0.07 μm, or at least about 0.08 μm, or at least about 0.09 μm, or at least about 0.1 μm,0.11 μm, or at least about 0.12 μm, or at least about 0.13 μm, or at least about 0.14 μm, or at least about 0.15 μm, or at least about 0.16 μm, or at least about 0.17 μm, or at least about 0.18 μm, or at least about 0.19 μm, or at least about 0.2 μm, or at least about 0.3 μm, or at least about 0.4 μm, or at least about 0.5 μm, or at least about 0.6 μm, or at least about 0.7 μm or at least about 0.8 μm, or at least about 1. Mu.M or at least about 1.5. Mu.M, or at least about 2. Mu.M, or at least about 2.5. Mu.M, or at least about 3.5. Mu.M, or at least about 4.5. Mu.M, or at least about 5. Mu.M, or at least about 5.5. Mu.M, or at least about 6. Mu.M, or at least about 6.5. Mu.M, or at least about 7. Mu.M, or at least about 7.5. Mu.M, or at least about 8. Mu.M, or at least about 8.5. Mu.M, or at least about 9. Mu.M, or at least about 9.5. Mu.M, or at least about 10. Mu.M, or at least about 10.5. Mu.M, or at least about 11. Mu.M, or at least about 11.5. Mu.M, or at least about 12. Mu.M, or at least about 12.5. Mu.M, or at least about 13. Mu.M, or at least about 13.5. Mu.M, or at least about 14.5. Mu.M, or at least about 15.M, or at least about 15.5. Mu.M, or at least about 16.M or at least about 17. Mu.M, or at least about 17.5 μm, or at least about 18 μm, or at least about 18.5 μm, or at least about 19 μm, or at least about 19.5 μm, or at least about 20 μm, or at least about 20.5 μm, or at least about 21 μm, or at least about 21.5 μm, or at least about 22 μm, or at least about 22.5 μm, or at least about 23 μm, or at least about 23.5 μm, or at least about 24 μm, or at least about 24.5 μm, or at least about 25 μm, or at least about 25.5 μm, or at least about 26 μm, or at least about 26.5 μm, or at least about 27 μm, or at least about 27.5 μm, or at least about 28 μm, or at least about 28.5 μm, or at least about 29 μm, or at least about 29.5 μm, or at least about 30 μm. In certain embodiments, the combined dosages and regimens may result in a serum concentration or average serum concentration over time of the compstatin analog of: no more than about 0.1 μm, or no more than about 0.11 μm, or no more than about 0.12 μm, or no more than about 0.13 μm, or no more than about 0.14 μm, or no more than about 0.15 μm, or no more than about 0.16 μm, or no more than about 0.17 μm, or no more than about 0.18 μm, or no more than about 0.19 μm, or no more than about 0.2 μm, or no more than about 0.3 μm, or no more than about 0.4 μm, or no more than about 0.5 μm, or no more than about 0.6 μm, or no more than about 0.7 μm, or no more than about 0.8 μm, or no more than about 0.9 μm, or no more than about 1 μm, or no more than about 1.5 μm, or no more than about 2.5 μm, or no more than about 3 μm, or no more than about 3.5 μm, or no more than about 4.5 μm, or no more than about 5 μm, or no more than about 6.5. Mu.M, or no more than about 7.5. Mu.M, or no more than about 8. Mu.M, or no more than about 8.5. Mu.M, or no more than about 9. Mu.M, or no more than about 9.5. Mu.M, or no more than about 10. Mu.M, or no more than about 10.5. Mu.M, or no more than about 11. Mu.M, or no more than about 11.5. Mu.M, or no more than about 12. Mu.M, or no more than about 12.5. Mu.M, or no more than about 13. Mu.M, or no more than about 13.5. Mu.M, or no more than about 14. Mu.M, or no more than about 14.5. Mu.M, or no more than about 15. Mu.M, or no more than about 15.5. Mu.M, or no more than about 16. Mu.M, or no more than about 16.5. Mu.M, or no more than about 17. Mu.M, or no more than about 17.5. Mu.M, or no more than about 18. Mu.M, or no more than about 18.5. Mu.M, or no more than about 19.5. M, or no more than about 19.5. Mu.M, or no more than about 20.5 μm or no more than about 21.5 μm or no more than about 22 μm or no more than about 22.5 μm or no more than about 23 μm or no more than about 23.5 μm or no more than about 24 μm or no more than about 24.5 μm or no more than about 25 μm or no more than about 25.5 μm or no more than about 26 μm or no more than about 26.5 μm or no more than about 27 μm or no more than about 27.5 μm or no more than about 28 μm or no more than about 28.5 μm or no more than about 29 μm or no more than about 29.5 μm or no more than about 20 μm.

Suitable ranges include from about 0.1 to about 30. Mu.M, or from about 1 to about 29. Mu.M, or from about 2 to about 28. Mu.M, or from about 3 to about 27. Mu.M, or from about 4 to about 26. Mu.M, or from about 5 to about 25. Mu.M, or from about 6 to about 24. Mu.M, or from about 7 to about 23. Mu.M, or from about 8 to about 22. Mu.M, or from about 9 to about 21. Mu.M, or from about 10 to about 20. Mu.M, or from about 11 to about 19. Mu.M, or from about 12 to about 18. Mu.M, or from about 13 to about 17. Mu.M, or from about 1 to about 5. Mu.M, or from about 5 to about 10. Mu.M, or from about 10 to about 15. Mu.M, or from about 15 to about 20. Mu.M, or from about 20 to about 25. Mu.M, or from about 25 to about 30. Mu.M. While the precise dosage to be administered varies depending on many factors, including but not limited to the type of patient and the type of disease state to be treated, the age of the patient and the route of administration, such dosages can be readily determined by one of skill in the art.

The pharmaceutical composition may be administered to the patient as frequently as several times per day, or may be administered less frequently, such as once per day, once per week, once per two weeks, once per month, or even less frequently (e.g., once per month or even once per year or less frequently). The frequency of administration will be apparent to those skilled in the art and will depend on many factors such as, but not limited to, the type and severity of the disease to be treated, the type and age of the patient, as described above.

The pharmaceutical compositions useful in the methods of the invention may be administered systemically in the form of oral formulations, parenteral formulations, ophthalmic formulations (including intravitreal formulations), suppositories, aerosols, topical formulations, transdermal formulations or other similar formulations. Such pharmaceutical compositions may comprise a pharmaceutically acceptable carrier and other ingredients known to enhance and facilitate administration of the drug. Other formulations such as nanoparticles, liposomes, re-encapsulated erythrocytes, and immune-based systems can also be used to administer compstatin analogs according to the methods of the invention.

As used herein, "oral administration" or "enteral administration" of a pharmaceutical composition includes any route of administration characterized by introduction into the gastrointestinal tract. Such administration includes feeding through the mouth and oral or intragastric administration. Such administration may also include sublingual, buccal, intranasal, pulmonary or rectal administration, and the like, as known in the art.

Formulations of pharmaceutical compositions suitable for oral administration comprise the active ingredient in combination with a pharmaceutically acceptable carrier, in a variety of dosage forms including, but not limited to, pills, tablets, granules, powders, capsules, dispersions, suspensions, solutions, emulsions, microemulsions, gels and films, to name a few. Such dosage forms typically include carriers and excipients to facilitate formulation and delivery of the active ingredient.

The pharmaceutically acceptable carrier is selected from the group consisting of proteins, carbohydrates, lipids, organic and inorganic molecules, and combinations thereof. The active ingredient may be combined with the carrier in suitable diluents to form a solution or suspension. Such liquid formulations may be viscous or non-viscous, depending on the amount and carrier used. The liquid formulation may be used directly or may be further formulated into a suitable capsule, gel capsule or solid by methods known to those skilled in the art. Alternatively, the solid formulation may be prepared by combining the solid components. Such solid formulations may be used as powders or formulated as granules, capsules, tablets or films, any of which may be prepared as a time release formulation (time release formulation).

Suitable proteins for use as carriers in oral dosage forms include milk proteins such as casein, sodium caseinate, whey, low lactose whey, whey protein concentrate, gelatin, soy protein (isolated), brown algae protein, red algae protein, baker's yeast extract and albumin. Suitable carbohydrates include cellulose such as methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, cellulose acetate and ethyl cellulose, starches such as corn starch, potato starch, tapioca starch, wheat starch, acid modified starches, pregelatinized starch and non-modified starches, alginates such as ammonium alginate, sodium alginate and calcium alginate, gluten such as corn gluten (corn gluten) and wheat gluten, gums such as gum arabic (gum arabic), gum ghatti, guar gum, karaya gum (karaya gum) and gum (tragacanth gum), insoluble glucose isomerase preparations, sugars such as corn sugar, invert sugar, corn syrup, high fructose corn syrup and sodium gluconate. Suitable lipids include tocopherols such as alpha-tocopherol acetate, short-, medium-and long-chain fatty acids and esters thereof, fatty alcohols and ethers thereof, oils such as coconut oil (refined), soybean oil (hydrogenated) and rapeseed oil, aluminum palmitate, didodecyl thiodipropionate, enzyme modified lecithin, calcium stearate, enzyme modified fats, glyceryl palmitostearate, lecithin, mono-and diglycerides, glycerol and waxes such as beeswax (yellow and white), candelilla wax and carnauba wax, and vegetable oils. Suitable organic and inorganic materials include methyl and vinyl pyrrolidones such as polyvinylpyrrolidone, methylsulfonylmethane, dimethyl sulfoxide and related compounds, hydroxy acids and polyhydroxy acids such as polylactic acid and the like.

In some embodiments, controlled release forms may be prepared to achieve sustained or position-specific release of compstatin analogs within the digestive tract to improve absorption and prevent metabolism of certain forms. For example, acid resistant coatings or acid resistant capsule materials of tablets may be used to prevent the release of compstatin analogs in the stomach and to protect the compounds from gastric enzyme metabolism. Suitable materials and coatings to achieve controlled release after passing through the stomach are composed primarily of fatty acids, waxes, shellac, plastics and vegetable fibers including, but not limited to, methacrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate, sodium alginate or stearic acid. Sustained release in the gastrointestinal tract may be achieved, for example, by embedding compstatin analogs in a matrix of insoluble materials such as various acrylates, chitin, and the like. Methods of preparing such formulations are known to those skilled in the art.

Compstatin can be formulated as a suppository or enema for rectal, vaginal or urethral administration. For this purpose, the compstatin analog may be dissolved or suspended in a oleaginous base carrier such as cocoa butter, which is solid or semi-solid at room temperature but melts at body temperature, or suspended in a water-soluble solid matrix such as polyethylene glycol or glycerol (prepared from glycerol and gelatin). Other excipients may be added to improve the formulation and suppositories may be molded into a form that is convenient for administration. In other embodiments, liquid suppositories composed of compstatin analogs dissolved or suspended in a liquid carrier suitable for rectal delivery for administration with a small syringe may be used.

For the treatment of chronic or acute lung conditions in which complement activation is involved, a preferred route of administration of the pharmaceutical composition is pulmonary administration. Thus, the pharmaceutical compositions of the present invention may be prepared, packaged or marketed in a formulation suitable for pulmonary administration through the oral cavity. Such formulations may include dry particles comprising the active ingredient and having a diameter in the range of about 0.5 to about 7 nanometers, preferably about 1 to about 6 nanometers. Such compositions are conveniently presented in dry powder form for administration using a dry powder reservoir containing a spray of propellant that can be directed to it to disperse the powder or using a device that self-distributes the container of the propellant/powder (e.g., a device containing the active ingredient dissolved or suspended in a low boiling point propellant in a closed container). Preferably, such powders include particles wherein at least 98% by weight of the particles have a diameter greater than 0.5 nanometers and at least 95% by number of the particles have a diameter less than 7 nanometers. More preferably, at least 95% by weight of the particles have a diameter greater than 1 nanometer, and at least 90% by number of the particles have a diameter less than 6 nanometers. The dry powder composition preferably includes a solid fine powder diluent such as sugar and is conveniently provided in unit dosage form.

Low boiling point propellants typically include liquid propellants having a boiling point of less than 65°f at atmospheric pressure. In general, the propellant may constitute 50 to 99.9% (w/w) of the composition and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may also contain additional ingredients such as liquid nonionic or solid anionic surfactants or solid diluents (preferably having the same particle size scale as the particles containing the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of drops in solution or suspension. Such formulations may be prepared, packaged, or sold, optionally sterile, in the form of aqueous or diluted alcoholic solutions or suspensions containing the active ingredient, and may be conveniently applied using any of the atomizing or spraying devices. Such formulations may also contain one or more additional ingredients including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, including in place of lung surfactants or preservatives such as methylparaben. Drops provided by this route of administration preferably have an average diameter in the range of about 0.1 to about 200 nanometers.

Formulations described herein as useful for pulmonary delivery may also be used for intranasal delivery of the pharmaceutical compositions of the invention.

Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of about 0.2 to 500 microns. Such formulations are administered in a manner wherein snuff is employed (i.e., by rapid inhalation through the nasal passages from a powder container held close to the nostrils).

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) up to 100% (w/w) of the active ingredient, and may also comprise one or more additional ingredients described herein.

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical damage to the tissue of a subject and administration of the pharmaceutical composition through a wound of the tissue. Parenteral administration thus includes, but is not limited to, administration of pharmaceutical compositions by injection of the composition, by administration of the composition through a surgical incision, by administration of the composition through a tissue penetrating non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intramuscular, intra-articular, intravitreal, intrasternal and renal dialysis infusion techniques.

Formulations of pharmaceutical compositions suitable for parenteral administration comprise the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline solution. Such formulations may be prepared, packaged or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged or sold in unit dosage forms (e.g., in ampoules or in multi-dose containers containing a preservative). Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, pastes, and implantable sustained release or biodegradable formulations in oily or aqueous vehicles. Such formulations may also contain one or more additional ingredients including, but not limited to, suspending, stabilizing or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granule) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged or sold in the form of sterile injectable aqueous or oleaginous suspensions or solutions. The suspensions or solutions may be formulated according to known techniques and may contain, in addition to the active ingredient, further ingredients such as dispersing agents, wetting agents or suspending agents as described herein. Such sterile injectable preparations may be prepared, for example, using non-toxic parenterally-acceptable diluents or solvents, such as water or 1, 3-butanediol. Other acceptable excipients and solvents include, but are not limited to, ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or diglycerides. Other parenterally administrable formulations useful include those in which the active ingredient is in the form of microcrystalline cellulose, in the form of liposomal formulations, for use in microbubbles for delivery by ultrasound release or as a component in a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: an excipient; surfactants, including substituted lung surfactants; a dispersing agent; an inert diluent; granulating agents and disintegrating agents; an adhesive; a lubricant; a sweetener; a flavoring agent; a colorant; a preservative; physiologically degradable compositions such as gelatin; an aqueous vehicle and a solvent; an oily vehicle and a solvent; a suspending agent; a dispersant or wetting agent; emulsifying agent and demulcent; a buffering agent; a salt; a thickener; a filler; an emulsifying agent; an antioxidant; an antibiotic; an antifungal agent; a stabilizer; and a pharmaceutically acceptable polymeric or hydrophobic material. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed.,1985,Remington's Pharmaceutical Sciences,Mack Publishing Co, easton, PA.

The method comprises the following steps:

another aspect of the invention relates to methods of modulating complement activation. Generally, the methods comprise contacting a medium in which modulation of complement activation is desired with a compstatin analog of the invention, wherein the contacting results in modulation of complement activation in the medium. The medium may be any medium in which modulation of complement activation is desired. In certain embodiments, the medium comprises cells or tissue of an organism, including (1) cultured cells or tissue, (2) cells or tissue within the body of a subject or patient; and (3) cells or tissues that have been removed from the body of one subject and are to be placed in the body of the same patient (e.g., extracorporeal flow of blood or autograft) or transferred to another patient. In the latter embodiment, the medium may also comprise biological material, such as a line, filter or membrane that contacts cells or tissue during in vitro bypass. Alternatively, the medium may comprise a biological material that is placed into the subject.

In certain embodiments, methods of modulating complement activation are suitable for use in a living patient or subject, and include part or all of a method of treating a patient for a pathological condition associated with complement activation, particularly AP-mediated complement activation. Many such pathological conditions are known in the art (see, e.g., holers,2008, supra), including, but not limited to, conditions such as atypical hemolytic uremic syndrome (aHUS), dense deposit disease, age-related macular degeneration (AMD), paroxysmal sleep hemoglobinuria (PNH), cold-set disease (CAD), rheumatoid Arthritis (RA), systemic Lupus Erythematosus (SLE), several autoimmune and autoimmune renal diseases, autoimmune myocarditis, multiple sclerosis, traumatic brain and spinal cord injuries, intestinal and renal ischemia-reperfusion (IR) injuries, spontaneous and recurrent pregnancy failure, antiphospholipid syndrome (APS), alzheimer's disease, asthma, anti-cytoplasmatic related oligovasculitis (anti-nuclear cytoplasmic antigen-associated pauci-immune vasculitis) (Wegener's syndrome), non-lupus autoimmune skin such as pemphigus, bullous pemphigoid and bullous epidermolysis, post-traumatic injury (post-traumatic shock), certain forms of atherosclerosis, and certain forms of atherosclerosis. In some specific embodiments, the pathological condition is associated with mutations and polymorphisms in the genes encoding FH and/or CD46, but is not limited to: AMD, aHUS and membranoproliferative glomerulonephritis type II (MPGN-II, also known as Dense Deposit Disease (DDD)). In other embodiments, compstatin analogs of the invention are suitable for use as alternatives to eculizumab or TT30 for the treatment of diseases in which these agents are currently prescribed, or in the treatment of diseases in which preclinical and clinical research and development is underway. These diseases include, but are not limited to aHUS, PNH, CAD and AMD.

The methods of treatment generally comprise (1) identifying a subject having a disease or condition treatable by modulation of complement activation (as described above), and (2) administering to the subject an effective amount of a compstatin analog of the invention using a treatment regimen and duration appropriate for the condition to be treated. Development of appropriate dosages and treatment regimens will vary depending on many factors, including, but not limited to, the type of patient and the type of disease state to be treated, the age of the patient, and the route of administration. Those skilled in the art are familiar with the design of dosing regimens that take such variables into account. For example, it will be apparent to those skilled in the art that oral administration of the compstatin analogs of the invention requires a higher initial formulation due to less bioavailability than, for example, intravenous injection.

The following examples are provided to describe the invention in more detail. They are intended to illustrate the invention but not to limit it.

Example 1

This example describes the synthesis of compstatin analogs with N-terminal modifications and conjugates of certain analogs with albumin binding small molecules.

Chemical. Rink amide MBHA resin, oxyma and the following Fmoc-amino acids were obtained from Novabiochem (San Diego, CA): ile, cys (Trt), val, tyr (tBu), gln (Trt), asp (OtBu), trp (Boc), gly, sar, ala, meAla, his (Trt), arg (Pbf), meIle, phe, mePhe and D-Cha. DIC and Fmoc-Trp (Me) -OH were purchased from AnaSpec (San Jose, calif.). HOAt is available from Advanced ChemTech (Louisville, KY). NMP and DCM were obtained from Fisher Scientific (Pittsburgh, pa.). All other chemicals used for the synthesis were purchased from Sigma-Aldrich (st.louis, MO) and used directly without further purification.

Peptide synthesis and purification. All peptides were synthesized by Fmoc solid phase method using DIC and Oxyma as coupling agents. The following procedure was used to synthesize linear peptides: rink amide MBHA resin (0.59 mmol/g) was placed in a peptide synthesis glass vessel equipped with frit at the bottom and swollen in DCM for 30min. After removal of the Fmoc protecting group (25% piperidine in NMP, 5 and 10 min), the resin was washed 7 times with NMP, 2 times with DCM, and each amino acid was coupled to the resin. For each coupling, 3 equivalents of amino acid, HOAt and DIC were used, preactivated in NMP for 10 min. All couplings were carried out for 1h and monitored by the Kaiser test or the chloranil test. In the case of a positive test result, the coupling is repeated until a negative test result is observed. After coupling Cys at position 1, synthesis was terminated. The resin was then separated from the bottom frit in an HSW polypropylene injection (Torviq, niles, MI) and additional amino acids were coupled using the previously reported method.

After completion of the solid phase synthesis, the resin was washed 4 times with NMP, DCM and DCM/diethyl ether (1:1) and dried under high vacuum for 4h. The peptides were cleaved from the resin using 94% TFA,2.5% water, and a mixture of 2.5% EDT and 1% TIPS for 2h. After evaporation of TFA under vacuum, the peptide was precipitated and washed 3 times with cold diethyl ether. The liquid was separated from the solid by centrifugation and decanted. The crude peptide was dried in vacuo and dissolved in 30% acetonitrile. The pH of the solution was adjusted to 8-9 using concentrated aluminum hydroxide. To the solution was added diluted hydrogen peroxide (1:100, 2 eq) with vigorous stirring. Cyclization was monitored by using MALDI-TOF. When the reaction was complete, the solution was supplemented with TFA to lower the pH to 2. The solution was freeze-dried. The crude peptide was purified using RP-HPLC as previously described (Qu et al, 2011, supra). The purity of the purified peptide was >95% as determined by analytical RP-HPLC (Phenomenex 00G-4041-E0Luna 5. Mu.C 18 100A column, 250X4.60mm; phenomenex, torrance, calif.). The mass of each peptide was confirmed using Waters MALDI micro MX instrument or Synapt HDMS.

Some compstatin analogs were conjugated to albumin binding small molecules, examples of which are shown below.

/>

In one construct, ABM2 was conjugated to the C-terminus of peptide Cp30 (SEQ ID NO:7; table 1 below) via a mini PEG-3 spacer according to the method described in WO 2010/127336.

In other constructs, ABM0 or ABM2 is coupled to the N-terminus of CP20 (SEQ ID NO: 3) or CP40 (SEQ ID NO: 18) without a spacer.

Example 2

C3 binding and complement inhibitory activity of compstatin analogs synthesized by the method described in example 1 were measured.

Materials and methods:

inhibition of complement activation. The ability of compstatin analogs to inhibit activation of the classical pathway of complement was assessed by ELISA as described elsewhere (Katragadda et al, 2006, supra; mallik et al, 2005, supra). Percent inhibition was plotted against peptide concentration and the resulting data set was fitted to a log dose-response function using Origin 8.0 software. IC (integrated circuit) ₅₀ The values are obtained from the fitting parameters that yield the smallest χ2 value. Each analogue was assayed at least 3 times.

SPR analysis. The interaction of compstatin analogs with C3b was characterized using a Biacore 3000 instrument (GE Healthcare, corp., piscataway, NJ). The running buffer was PBS containing 0.005% Tween-20, pH 7.4 (10 mM sodium phosphate, 150mM NaCl). Biotinylated C3b was site-specifically captured on streptavidin chips at densities of about 3000 and 5000 RU; two untreated flow cells were used as reference surfaces. For kinetic analysis, groups of 5 increasing concentrations of the specific compounds were injected one after the other on the chip surface in a single cycle. Injection of 3-fold dilution series (0.49-40 nM) at 30 ul/min; each injection was performed for 2min, allowing the peptide to dissociate for 5min each time before starting the next injection. At the end of the last injection, a dissociation time of 40min was performed. Peptide 4 (1 MeW) was included in each experimental series as an internal control and reference. Data analysis was performed using a scanner (BioLogic Software, campbell, australia) and BiaEvaluation (GE Healthcare, corp., piscataway, NJ). Signals from untreated flow cells and buffer blank injection populations were subtracted to correct for buffer effects and injection artifacts. The processed biosensor data was globally fitted to a 1:1langmuir binding model (provided by GE Helthcare friendly) from equation K _D ＝k _d /k _a Calculating equilibrium dissociation constant (K) _D ). Each assay was performed at least 2 times.

The peptide was docked to C3C. AutoDock Vina (Trott and Olson, 2010) was used for docking studies. Except for rings that can be treated as rigid by VinaAll other parts of the peptide (terminal residues, side chains) outside the backbone of the core region were determined to be flexible in each round of docking. The C3C residues near the N-terminus of analog Cp20 (SEQ ID NO: 3) (i.e., asp349, lys386, ser388, asn390, ser437, asn452, leu454, asp491 and Leu 492) were determined to be flexible for docking experiments to allow more reasonable interactions between the extended N-terminus of these peptides and C3C. The only exception is peptide 19, which does not extend to other peptides at its N-terminus; the binding site on C3C thus remains rigid in the docking of peptide 19. The initial structure of all peptides was manually set up in PyMol based on the C3C binding structure of 4W 9A. AutoDockTools was used to define the binding pocket and to prepare the initial structure of C3C, all peptides from pdb were formatted into the input format of Vina (pdbqt). 55. In a comparative graph of binding free energy (Δg) calculated relative to the experiment, experiment Δg was calculated from affinity values determined by SPR as Δg=rtln (KD), r=1.986 cal K ^-1 mol ^-1 And t=293.15K.

Results:

structure/activity of N-terminal extension. By using a molecular modeling method, cp20 (SEQ ID NO: 3) was substituted for the early compstatin analog 4W9A in the eutectic structure with target fragment C3C. Computational analysis of this complex confirmed that the methyl group of Sar8 forms a contact (distance over) with the oxygen atom of G489 in C3C). Analysis of the binding sites also showed the presence of a hydrophobic region on C3C that can be utilized by N-terminal extension of the peptide ligand. Although not embedded in the binding pocket of C3C, the N-terminus of compstatin has previously been protected by an acetyl moiety to improve the stability of the peptide; however, such capping also has a beneficial effect on the inhibitory potency. Based on the current lead compound Cp20 (SEQ ID NO: 3), the effect of the substituted N-terminal acetyl moiety on target binding was evaluated (Table 1). For this purpose, the analogs were plotted against the quantitative kinetics of binding to C3b and compared to the clinically used analog 4 (1 MeW) and Cp20 (SEQ ID NO: 3) (Table 1, FIG. 1). In fact, substitution of the terminal acetyl group with a shorter methyl group (peptide 1) results in an affinity decrease of almost 1 order of magnitude, lower than 4 #1 MeW), confirming the advantage of N-terminal capping. Conversely, capping with glycine residue (peptide 2) improves the dissociation rate (k _d ) However, the association rate (k) is slightly reduced _a ) Resulting in only a very small net change in affinity (compared to Cp20 (SEQ ID NO: 3)). Glyn-methylation to Sar (peptide 3) restores associative properties while maintaining beneficial dissociation values resulting in significant improvement in affinity (K _D =1.6 nM; compounds of table 1).

To further exploit the benefits of N-terminal optimization, additional Cp20 (SEQ ID NO: 3) -based analogs with natural amino acids (peptides 4-8), methylated amino acids (peptides 9-13) and D-amino acids (peptides 14-18) at position Xaa0 (FIG. 1B; table 1) were screened. The set includes representative hydrophobic, hydrophilic and charged side chains. All compounds tested showed strong binding (K _D <20 nM), k in the whole panel _a Value (1-4X 10) ⁶ M ^-1 s ^-1 ) Display ratio k _d Values (1-25X 10) ^-3 s ^-1 ) There was less variability (table 1, fig. 1B). When screening for binding to C3b, all analogs followed a 1:1langmuir kinetic model, strongly supporting the presence of a single high affinity binding site. In general, D-amino acids having hydrophobic side chains appear to be more advantageous than the acetyl (Ac) moiety of Cp20 (SEQ ID NO: 3). Of these peptides, peptide 14 with Dtyr at this position was the most potent, with sub-nanomolar affinity (K _D =0.5 nM; table 1) and the slowest dissociation rates within the panel. The affinity of peptides in which Ac was replaced with other amino acids fell between the affinities of peptides 1 and 14, with most of the analogs clustered around the profile of Cp20 (SEQ ID NO: 3) (FIG. 1B). Tyrosine generally appears to be preferred because all peptides with an N-terminal Tyr, its O-methyl analog and its D-isomer belong to the best binders with an affinity of about 1nM or below. In contrast, residues with shorter side chains such as Gly, thr or Ala derivatives appear to be less advantageous than Cp20 (SEQ ID NO: 3) and do not improve affinity. Thus, substitution of the capping Xaa0 residue appears to be well tolerated for a wide range of amino acid residues with different properties (from hydrophobic to charged).

TABLE 1A series of compstatin analogs with modifications at the N-terminus (Xaa 0-Xaa1- [ Cys-Val-Trp (Me) -Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH ₂ ) (SEQ ID NO: 4) kinetic parameters and inhibition efficacy. k (k) _a Rate of association; k (k) _d Dissociation rate; k (K) _D Binding constants from SPR; IC (integrated circuit) ₅₀ Peptide concentration achieving 50% inhibition of classical pathway complement activation. ND, not determined.

^a Ac-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys]-Thr-NH ₂ (Katragadda et al, 2006, supra, WO 2007/062249; sometimes referred to herein as "4 (1 MeW)"); included as a standard in all assays, but not following the Cp20 (SEQ ID NO: 3) template.

^b A base compound for N-terminal modification; binding/potency values from previous publications (WO 2010/127336).

^c Selected for further determination: peptide 3-Cp30 (SEQ ID NO: 7); peptide 14-Cp40 (SEQ ID NO: 18)

And (5) calculating and analyzing. Extended dwell analysis was performed to provide structural evidence of the observed effect of binding affinity and to generate computational models for predicting novel analogs. Initially, the docking strategy was validated using the screened dataset from the N-terminal modified analog (peptides 1-18; table 1) of Cp20 (SEQ ID NO: 3). For this purpose, compounds were prepared on-chip, which were left in the compstatin binding pocket of human C3C (Janssen et al, 2007, supra), the binding free energy (Δg) was calculated, which was compared to the SPR affinity derivative value by determining the pearson coefficient ((R, fig. 2). Based on 5 independent docking studies over the whole data set, the overall correlation between experimental and calculated Δg values was 0.46 (fig. 2.) of the 19 analogues of the data set, 3 peptides with very short moieties (methyl; peptide 1) or aromatic natural amino acids (peptides 5 and 7) showed significantly higher bias; when these analogues were not included, the correlation increased to 0.69 (fig. 2).

More detailed analysis of the stay peptide indicated that most N-terminal modified compstatin analogs formed additional contacts with the polar region and shallow pocket on C3C. For example, the polar region of C3C involved in Asp349, ser388, and Ser437 interacted with the N-terminal amino group of Dtyr in peptide 14 (fig. 3A). In contrast, such polar interactions are detrimental to peptides having natural amino acids at that position due to the different orientations of the amino groups, as illustrated for peptide 4 (fig. 3A). Furthermore, the side chain of the extended amino acid (DTyr) in peptide 14 forms additional hydrophobic contacts with Leu454 and Leu492 in shallow extended pockets on C3C. Finally, the hydroxyl group of Dtyr forms weak hydrogen bonds with Asn452 of C3C. The combination of these effects may contribute to the observed sub-nanomolar binding affinity of peptide 14.

To further explore the unique strategy to elucidate the N-terminal pocket, two analogues were designed in which the aromatic residues were located at either position Xaa0 or Xaa1 (peptides 19 and 20; table 1). Based on the computational model developed above, the side chain of the new Trp in peptide 19 was predicted to fit well into the hydrophobic binding pocket (fig. 3B), whereas a short flexible Gly linker was chosen in peptide 20 to enable better orientation of the Tyr side chain when compared to homolog peptide 4. Although peptide 20 showed a weak binding affinity of 1/3 of that of peptide 4, peptide 19 reached a sub-molar concentration of binding affinity (K _D =0.5 nM; table 1) so that it is as potent as peptide 14. Taken together, these results demonstrate the advantage of correctly oriented hydrophobic residues adjacent to Cys at position 2.

Additional analogs were constructed based on Cp40 (peptide 14, SEQ ID NO:18, table 1). These analogs are shown in table 2 below.

Table 2. Kinetic parameters and inhibition potency of Cp40 (SEQ ID NO: 18) -based analogues with modifications inside the peptide were evaluated. The numbering in the peptide names indicates the position relative to compstatin. k (k) _a Rate of association; k (k) _d Dissociation rate; k (K) _D Binding constants from SPR; IC (integrated circuit) ₅₀ Peptide concentration achieving 50% inhibition of classical pathway complement activation.

^a Ornithine replaces arginine at position 11.

^b Asparagine replaces aspartic acid at position 6.

As described above, ABM0 or ABM2 is coupled to the N-terminus of CP20 (SEQ ID NO: 3) or CP40 (SEQ ID NO: 18) and certain variants thereof without the use of spacers. These analogs exhibit binding and complement inhibitory activity within the same range as the Cp40 analogs and derivatives thereof shown in table 2.

Example 3

The solubility of certain compstatin analogs synthesized as described in example 1 was measured in water for injection (WFI) and Dulbecco's PBS (DPBS).

Materials and methods:

about 5mg of each peptide (acetate form) was weighed into separate LoBind Eppendorf tubes and 50. Mu.L of water for injection (WFI) was added to each tube. Each sample was centrifuged at 13000rpm for 2min, followed by dilution to measure Optical Density (OD) at 280nm using a measuring NanoDrop 2000 spectrophotometer (thermo scientific, wilmington, DE). Each concentrated sample was diluted 1:20 into Dulbecco's phosphate buffered saline (DPBS, free of potassium and calcium; invitrogen, carlsbad, calif.). The samples were monitored for precipitation, each sample was vortexed for 5 minutes and centrifuged at 13000rpm for 2 minutes. OD of each DPBS supernatant was measured to determine the concentration of peptide at saturation.

Results:

although the presence of 3 acidic or basic residues (Asp 6, his10, arg 11) in most compstatin analogues contributes to the overall favourable solubility in aqueous solutions, their zwitterionic properties may adversely affect the solubility in buffers. Thus, the selected compounds were evaluated for solubility in two clinically relevant solvents, i.e., water for injection (WFI) and Dulbecco's PBS (DPBS). In addition, the ultra-high performance liquid chromatography (UPLC) residence time of these peptides on C18 column was measured to reflect their apparent relative hydrophobicity (table 3).

Table 3. Solubility of peptides in WFI (Water for injection) and DPBS, and UPLC (ultra high Performance liquid chromatography) residence time as an indicator of hydrophobicity.

^a OD at saturation (280 nm); WFI = water for injection, DPBS = dubelco's phosphate buffered saline solution

^b Measured as residence time during UPLC analysis on C18 column

^c During the ELISA study, peptide 19 could not be dissolved in PBS at a concentration of 100 μm or higher.

Solubility in WFI is excellent, with all compounds having values exceeding 50mg/mL, except Cp20 (SEQ ID NO: 3). In general, the solubility in DPBS is significantly lower for all analogs. The reduced solubility of Cp20 (SEQ ID NO: 3) in both solvents compared to 4 (1 MeW) is believed to be the result of its hydrophobicity due to the substitution of the two N-methylation (positions 8 and 13) and the C-terminal Thr to Ile. Substitution of the N-terminal acetyl moiety of 4 (1 MeW) and Cp20 (SEQ ID NO: 3) with uncapped amino acid residues induced a significant increase in the hydrophobicity of Cp30 (SEQ ID NO: 7) and Cp40 (SEQ ID NO: 18) and restored their high solubility (> 50 mg/mL) in WFI. However, incorporation of hydrophobic Dtyr at its N-terminus adversely affected the solubility of Cp40 (SEQ ID NO: 18) in DPBS (0.8 mg/mL). In contrast, the presence of a small N-terminal Sar in Cp30 (SEQ ID NO: 7) greatly improved its solubility in DPBS (6.9 mg/mL), making the peptide nearly 2-fold more soluble than the 4 (1 MeW) analog for clinical use.

Example 4

Plasma stability and plasma protein binding in human plasma were measured for certain compstatin analogs synthesized as described in example 1.

Materials and methods:

plasma stability. Fresh human plasma containing lepirudin (3.75 units/ml) was combined at 37℃with Cp30 (SEQ ID NO: 7), cp40 (SEQ ID NO: 18) or control at a final concentration of 20. Mu.M eachPeptide 2B was incubated together. 100. Mu.L of the sample was taken for solid phase extraction. A96-well plate HLB Oasis 30 μm 10mg (Waters, milford, mass.) was used for extraction. SPE materials are conditioned by adding 500. Mu.L each of methanol and ACN, followed by 500. Mu.L milli-Q water. By 4%H ₃ PO ₄ The samples were diluted 1:1. After loading the samples, the samples were washed 2 times with 500 μl of 10% acn in 0.1% formic acid. Samples were eluted with 200 μl of 65% acn in 0.1% formic acid and collected in Eppendorf LoBind collection plates. Samples for UPLC-MS were diluted 1:10 in milli-Q water containing 0.1% formic acid. Cp20 (SEQ ID NO: 3) was incorporated into each sample before SPE was used as an internal standard.

Plasma protein binding. Cp30 (SEQ ID NO: 7) was incorporated into 500. Mu.L of fresh human plasma containing lepirudin (3.75 units/mL) so that the final peptide concentration was 20. Mu.M (C3: 1.2mg/mL, 6.4. Mu.M). Control samples were prepared in the same manner using Cp30 (SEQ ID NO: 7) and milli-Q water to determine the area of peptide in UPLC-MS at 1. Mu.M. Plasma samples were equilibrated for 10min at room temperature. Subsequently, with mixing, 500 μl of 30% peg (MW 3350) in milli-Q water was slowly added to the plasma sample. The mixture was centrifuged at 14000rpm for 10min to separate the supernatant. The pellet was dissolved in 1000. Mu.L of FPLC buffer A and separated by FPLC using a Mono Q5/5 column, and fractions were collected at 1mL per tube. 0.5mL of each fraction was combined with the same volume of 4%H ₃ PO ₄ Mix for SPE and UPLC-MS analysis.

UPLC-MS analysis. UPLC-MS analysis was performed on SYNAPT HDMS (Waters, milford, mass.) equipped with an ESI source controlled by MassLynx 4.1 software (Waters). Each sample was injected in quadruplicates. The on-line ACQUITY UPLC (Waters) system was used for peptide separation by reverse phase liquid chromatography. The capillary voltage was 3.2kV, the cone voltage was 30V and the source temperature was 120 ℃. The [ Glu1] -fibrinogen peptide was used for lock-mass correction (lock-mass correction) at a sampling rate of 30 s. Mass spectra were obtained in positive mode at a scan rate of 1s in the m/z range of 200-2000 Da. The presence of analyte is confirmed by retention time and mass. The selectivity was studied by analyzing blank plasma samples and pure peptide to determine the presence of any interference co-eluting with the analyte. After injection, the analytes were separated on a 1.7 μm UPLC BEH130C18 column (Water, 2.1. Mu. m x 150mm, part number 186003556). The analytical column temperature was maintained at 40 ℃. The peptide was isolated at a flow rate of 0.3 mL/min. The gradient was linear 10-60% b (0.1% formic acid in acetonitrile) over 8 minutes.

Results:

plasma stability. To investigate the stability of novel analogues with a free N-terminus Cp30 (SEQ ID NO: 7) and Cp40 (SEQ ID NO: 18) were selected for incubation in human plasma at 37℃FIG. 4A. Control linear peptide 2B (LRFLNPFSLDGSGFW, SEQ ID NO: 28) was cleaved rapidly after contact with plasma. The 0-time samples show cleavage at Arg position. The peptide completely disappeared within 30 min. Under the same conditions, both Cp30 (SEQ ID NO: 7) and Cp40 (SEQ ID NO: 18) showed significant plasma stability. After 5 days, more than 55% of the peptide was retained. UPLC-MS chromatograms at time points 0, 24 and 120h were very similar (FIG. 4B). No major cleavage products were observed.

Plasma protein binding. To investigate the binding specificity of Cp30 (SEQ ID NO: 7), excess peptide was incubated in fresh human plasma. Plasma proteins were precipitated with PEG3350 and isolated using a small Mono Q column. The presence of Cp30 (SEQ ID NO: 7) in each 1mL fraction was measured. Fractions containing Cp30 (SEQ ID NO: 7) were further quantitatively analyzed using UPLC-MS and the identity of the co-eluted proteins was tested. It was found that 7.5% of Cp30 (SEQ ID NO: 7) was located in the flow-through, while 88.0% and 4.5% co-eluted with C3 and C3C, respectively. The identity of these proteins was identified by SDS-PAGE followed by Coomassie staining and Western blotting. Furthermore, the total amount of Cp30 (SEQ ID NO: 7) detected was equal to the amount of plasma C3.

Example 5

The in vivo retention of the compstatin analogs Cp20 (SEQ ID NO: 3), cp30 (SEQ ID NO: 7) and Cp40 (SEQ ID NO: 18) synthesized as described in example 1 in the cynomolgus monkey model was measured. Using the SPR method described above, in 4 primate species: the binding profiles of these peptides were compared in human, cynomolgus, macaque and baboon plasma.

Materials and methods:

primate studies and sample collection. Plasma half-life and production of the major metabolites were evaluated in cynomolgus monkeys (Macaca fascicu laris) at Simian Conservation Breeding and Research Center (SICONBREC, makati City, philippines). For each analog (Cp 20 (SEQ ID NO: 3), cp30 (SEQ ID NO: 7) and Cp40 (SEQ ID NO: 18)), two healthy animals were sedated and intravenous injection was performed with 2mg/kg of the compound (dissolved in saline for injection). Blood samples (1-2 mL) were collected in EDTA-coated vacuum blood collection tubes immediately prior to compound injection and at different time points (2, 5 and 30min;1, 2, 4, 6 and 24 hours) after injection to prevent clotting and complement activation, which were centrifuged at-800 x g for 10min to obtain plasma. The plasma samples were immediately frozen and stored for future analysis. All NHP studies were performed in accordance with animal welfare regulations.

Analysis of plasma samples. The compstatin analog in the plasma samples was extracted by Solid Phase Extraction (SPE) in 96 well plate format (HLB Oasis 30 μm,10mg; waters, milford, mass.) prior to analysis by UPLC-MS. The SPE material was conditioned thoroughly using acetonitrile and water. Plasma samples were diluted 1:1 with 4% phosphoric acid, and a constant concentration of Cp20 (SEQ ID NO: 3) (5 pM) was incorporated into all samples containing Cp30 (SEQ ID NO: 7) or Cp40 (SEQ ID NO: 18) as internal standard; in the case of a sample containing Cp20 (SEQ ID NO: 3), cp40 (SEQ ID NO: 18) was used as an internal control. Samples were loaded onto SPE plates and washed with 10% acetonitrile in 0.1% formic acid. The extracted peptide was eluted with 200 μl of 65% acetonitrile in 0.1% formic acid and collected in a LoBind tube (Eppendorf) to avoid peptide adsorption. Finally, 5 μl of each eluate was diluted with 45 μl of 0.1% formic acid and injected into a UPLC-MS system consisting of online ACQUITY UPLC coupled to a SYNAPT G2-S HDMS instrument equipped with an ESI source and controlled by MassLynx 4.1 software (Waters). Each sample was injected in quadruplicates. Reverse phase liquid chromatography was used for peptide separation using a 1.7 μm UPLC BEH130C18 column (2.1 μm. Times.150 mm; waters) at a column temperature of 40 ℃. Peptides were isolated using a linear gradient of 10-60% acetonitrile in water containing 0.1% formic acid at a flow rate of 0.15mL/min over 8 min. Directly analyzing the eluted peptides using HDMS; the ESI source capillary voltage was set to 3.2kV, the cone voltage was set to 30V and the source temperature was set to 120 ℃. [ Glu1] -fibrinopeptide B (Sigma) was used for locking mass correction at a sampling rate of 30 s. Mass spectra were obtained in positive mode at a scan rate of 1s in the m/z range of 50-1950 Da.

Measurement of plasma half-life. On the day of analysis, calibration curves were prepared by incorporating compstatin analogs (Cp 20 (SEQ ID NO: 3), cp30 (SEQ ID NO: 7) and Cp40 (SEQ ID NO: 18)) at final concentrations of 0.5, 1, 2, 4 and 8. Mu.M into freshly thawed plasma from untreated cynomolgus monkeys. SPE was performed on all calibration samples and measurements were performed using the UPLC-HDMS described above. The MS peak area was determined by integration and plotted against concentration to produce a calibration curve that showed good linearity with a regression coefficient (R2) greater than 0.993. For pharmacokinetic analysis, plasma concentrations (Cp) at each time point were calculated from the extracted peak areas of each peptide using the corresponding standard curve. The elimination constant (k) was determined from the slope of the final elimination period (0.5-24 h) using the following formula _e ) And plasma half-life (t) _1/2 )：ln(Cp)＝ln(Cp0)-k _e X t and t _1/2 ＝0.693/k _e . Measurement of C3 levels. The C3 plasma concentrations of each cynomolgus monkey used in this study were determined by ELISA. Briefly, 96-well plates (MaxiSorp; nunc) were coated overnight at 4.25℃with 1. Mu.g/ml of monoclonal anti-C3 antibody in PBS (clone 8E11; tosic et al, 1989,J.Immunol.Methods 120:241-249). The wells were washed with PBS/Tween 0.05% followed by blocking with PBS/BSA 1% for 1h at room temperature. Plasma (diluted 1:10,000 and 1:20,000 in PBS/BSA) or serial dilutions of purified cynomolgus monkey C3 were then incubated at room temperature for 1h, followed by washing, and peroxidase-conjugated anti-C3 (MP Biomedicals, solon, OH) diluted 1:1,000 in PBS/BSA was incubated at room temperature for 1h. The tetramethylbenzidine substrate (R &Dsystems, minneapolis, MN) and the optical density was measured using a microtiter plate reader set at a wavelength of 450 nm.

And (5) hemolysis measurement. Rabbit erythrocytes were washed with Phosphate Buffered Saline (PBS), followed by Fluona sodium buffered saline (VBS) ^Mg+ Washing with EGTA. At the position ofDilutions of 1:20 were prepared in VBS buffer. Plasma samples (1:10 in VBS-100. Mu.l) were incubated with rabbit red blood cell solution (50. Mu.l) in 96-well plates at 37℃for 1h. EDTA (0.2 mM-15. Mu.l) was added to terminate the reaction and the plates were centrifuged (2500 Xg 3 min). The supernatant (100 μl) was transferred to a new well and the optical density was measured at 405 nm. Red blood cell incubations with water or buffer were used as positive (100% lysis) and negative (0% lysis) controls, respectively.

And combining the characteristic spectrum. For NHP-specific experiments, C3 from human, cynomolgus, macaque and baboon plasma was immobilized on each flow cell of a CM5 sensor chip (GE Healthcare) using standard amine coupling methods to achieve target densities of 6,000-7,000RU. The peptides Cp20 (SEQ ID NO: 3) and Cp40 (SEQ ID NO: 18) were evaluated quantitatively using the single cycle kinetic method as described in example 2. For visual comparison of kinetic profiles independent of differences in target density or activity, each binding curve was normalized for maximum response and superimposed in the original curve.

Results:

peptide drugs are often hindered by relatively rapid elimination from plasma, which can be extremely limited in clinical applications that rely on constant systemic drug levels (e.g., PNH in the case of complement inhibitors). Comparative studies including Cp20 (SEQ ID NO: 3) and newly developed Cp30 (SEQ ID NO: 7) and Cp40 (SEQ ID NO: 18) were performed, wherein plasma levels were assessed by LC-MS over a 24 hour period using 2mg/kg of each analogue of cynomolgus monkey injected intravenously. All the tested analogues followed a similar biphasic elimination profile, in which plasma levels decreased more rapidly within the first hour after injection, followed by a much slower decrease over the entire later time point (fig. 5A). The peptide concentration at which the kinetic changes occur is very similar to the concentration of the desired physiological plasma level of the target protein C3. In fact, measurement of C3 levels in the relevant monkeys by ELISA (4.9-12.8 μm) confirmed that the initial decline in compstatin levels slowed down within the established C3 range (fig. 5A). These observations indicate a target-driven elimination model in which tight binding to abundant target C3 greatly influences peptide excretion. In fact, when plasma half-life was calculated based on the terminal log-linear portion (1-24 h), a direct correlation with binding affinity for C3 was observed, with half-life values of Cp20 (SEQ ID NO: 3), cp30 (SEQ ID NO: 7) and Cp40 (SEQ ID NO: 18) being 9.3, 10.1 and 11.8h, respectively (FIG. 5B). The half-life of the Cp30-ABM2 conjugate was observed to be 22 hours (not shown).

The concentration of compstatin analog was measured for inhibition of complement activation by alternative pathways in plasma samples using a erythrocyte hemolysis assay. The concentration of analog in the sample measured at each time point was observed following complement inhibitory activity.

In view of the strong apparent dependence of the elimination phase on binding affinity, translating these NHP-based studies into the adult system appears to be affected by the differential affinity of these compstatin analogs for human and NHP C3. Thus, the binding profile of the peptides to C3 and 3 related NHPs (cynomolgus monkey, baboon) from humans was measured using the SPR method described above. The affinity and kinetic profile of all analogs was highly comparable (fig. 5C).

Example 6

Bioavailability of the compstatin analog Cp40 (SEQ ID NO: 18) synthesized as described in example 1 from subcutaneous and oral routes of administration in the cynomolgus model was measured.

Materials and methods:

primate studies and sample collection. The bioavailability was evaluated in cynomolgus monkeys at Simian Conservation Breeding and Research Center (SICONBREC, makati City, philippines). Two healthy animals were used for each route of administration. Animals were sedated and either subcutaneously injected with 2mg/kg of the compound or orally administered 4mg/kg of the compound by intragastric administration. Blood samples (1-2 mL) were collected in EDTA-coated vacuum blood collection tubes at different time points (2, 5 and 30min;1, 2, 4, 6 and 24 hours) immediately before and after injection of the compound to prevent clotting and complement activation, which were centrifuged at-800 x g for 10min to obtain plasma. The plasma samples were immediately frozen and stored for future analysis. All NHP studies were performed in accordance with animal welfare regulations.

And (5) analyzing. Assays of plasma samples, plasma half-life and determination of complement inhibitory activity in plasma were performed as described in example 5.

Results:

in addition to being very expensive, not well tolerated by the patient, and often requiring trained professionals for intravenous administration, the bioavailability of peptide drugs when administered by any other route is often low. After subcutaneous or oral delivery, the compstatin analog Cp40 (SEQ ID NO: 18) was tested for bioavailability. The cynomolgus monkey was subcutaneously injected with 2mg/kg of the analogue or orally injected with 4mg/kg of the analogue, and plasma levels were assessed by LC-MS over a 24-hour period. The results are shown in fig. 6.

The plasma concentration of Cp40 (SEQ ID NO: 18) reached a peak of about 12.5. Mu.M within 4-5 hours after administration by subcutaneous injection (FIG. 6, top panel). Oral injection of the analog resulted in a plasma concentration of about 0.023 μm within 1 hour of injection (fig. 6, bottom frame; note that oral injection was successful in only one of the two monkeys). By comparison (fig. 5B), intravenous injection of this analog resulted in a peak plasma concentration of about 28 μm immediately after injection.

The concentration of compstatin analog was measured for inhibition of complement activation by another pathway in plasma samples from subcutaneous injections using a erythrocyte hemolysis assay. The concentration of the analog was closely followed at each time point where complement inhibitory activity was measured.

Example 7

The in vivo residence of the compstatin analog Cp30 (SEQ ID NO: 7) and Cp-30-ABM2 conjugates in the baboon model as described in example 1 was measured.

Materials and methods:

teenager baboons (P.Anubis, baboon Research Resources, university of Oklahoma) weighing 5-8kg were used. Two baboons were used for the study, one baboon for each compound. Each animal received a single dose of peptide (10 mg) by injection via the peripheral vein. Blood samples for LC-MS/MS assays were collected in 1-ml plastic tubes containing 50 μg lepirudin and centrifuged at 2000g for 20min at 4 ℃ for plasma separation. The plasma samples were stored at-70 ℃. Blood samples were collected at predetermined time intervals after injection of Cp30 (SEQ ID NO: 7) or Cp30 (SEQ ID NO: 7) -ABM2 conjugate. Samples were treated with SPE and analyzed using LC-MS/MS. Calibration curves were generated using standard peptides at different concentrations in plasma to determine peptide concentration for each sample.

The compstatin analogs were extracted from plasma samples using SPE. A96-well plate HLB Oasis 30 μm 10mg (Waters, milford, mass.) was used for extraction. SPE materials were conditioned by adding 500. Mu.l of methanol, ACN followed by 500. Mu.l of milli-Q water. By 4%H ₃ PO ₄ The samples were diluted. After loading the samples, washing was performed with 500 μl of water and 10% acn containing 0.1% formic acid. Samples were eluted with 200 μl of 65% acn (in 0.1% formic acid) and collected in a collection plate. Samples for LC-MS were diluted 1:2 to 1:11 in milli-Q water containing 10% ACN with 0.1% formic acid. CP20 (SEQ ID NO: 3) was incorporated as an internal standard into each sample prior to SPE.

LC-MS/MS analysis. LC-MS/MS analysis was performed on SYNAPT HDMS (Waters, milford, MA) equipped with ESI source controlled by MassLynx 4.1 software (Waters). Each sample was injected in triplicate. The on-line ACQUITY UPLC (Waters) system was used for peptide separation by reverse phase liquid chromatography. The capillary voltage was 3.2kV, the cone voltage was 30V and the source temperature was 120 ℃. The [ Glu1] -fibrinogen peptide was used for lock quality correction at a sampling rate of 30 s. A mass spectrum was obtained in positive mode at a scan rate of 1s in the m/z range of 500-1800 Da. The presence of analyte is confirmed by retention time and mass. After injection, the analytes were separated on a 1.7 μm UPLC BEH130C18 column (Water, 1.0. Mu. m x 100 mm). The analytical column temperature was maintained at 40 ℃. The peptide was isolated at a flow rate of 0.15 mL/min. The gradient was linear 15-55% b (0.1% formic acid in acetonitrile) over 7 minutes.

Results:

plasma concentrations of peptide Cp30 (SEQ ID NO: 7) and ABM2 conjugates were determined using LC-MS/MS after a single bolus intravenous injection into baboons. Peptide Cp30 (SEQ ID NO: 7) showed a half-life of 5 hours, cp30 (SEQ ID NO: 7) -ABM2 showed a half-life of 7.5 hours. In comparison, the compstatin analog 4 (1 MeW) and the potent analog (peptide 3) disclosed in WO2010/127336 were previously determined to have half-lives of about 60-90 minutes in the same baboon model.

Example 8

Bioavailability from intramuscular administration of the compstatin analog Cp40 (SEQ ID NO: 18) synthesized as described in example 1 was measured in a baboon model.

The method comprises the following steps:

teenager baboons were intramuscularly injected with 2mg/kg Cp40 (SEQ ID NO: 18). Blood samples for LC-MS/MS assays were collected in 1-ml plastic tubes containing 50 μg lepirudin and centrifuged at 2000g for 20min at 4 ℃ for plasma separation. The plasma samples were stored at-70 ℃. Blood samples were collected at predetermined time intervals after injection of the analog. Samples were treated with SPE and analyzed using LC-MS/MS. Calibration curves were generated using standard peptides at different concentrations in plasma to determine peptide concentration for each sample.

Compstatin analogs were extracted from plasma samples and subjected to LC-MS/MS analysis as described in example 7. The hemolysis assay was performed as described in example 5.

Results:

the results are shown in fig. 7. The plasma concentration of Cp40 (SEQ ID NO: 18) reached a peak of about 10. Mu.M within about 5-6 hours after administration by intramuscular injection. Complement inhibitory activity was observed to closely follow the concentration of analog in the samples measured at each time point.

The invention relates to the following embodiments:

1. a compound comprising a modified compstatin peptide (ICVVQDWGHHRCT (cyclo C2-C12; SEQ ID NO: 1) or an analogue thereof, wherein the modification comprises an added or substituted N-terminal component that improves (1) the C3, C3b or C3C binding affinity of the peptide, (2) the solubility of the peptide in an aqueous liquid, and/or (3) the plasma stability and/or plasma residence time of the peptide compared to an unmodified compstatin peptide under equivalent conditions.

2. A compound of embodiment 1 wherein the added component is an amino acid other than L-Gly or a non-peptide analog of an amino acid.

3. A compound of embodiment 2 wherein the added component is a D-amino acid.

4. A compound of embodiment 2 wherein the added component includes at least one aromatic ring.

5. A compound of embodiment 2 or embodiment 3 wherein the added component is D-Tyr.

6. A compound of embodiment 2 wherein the amino acid is N-methylated.

7. A compound of embodiment 6 wherein the amino acid is N-methylglycine (Sar).

8. A compound of any one of embodiments 2-6 wherein the added component is D-Tyr, D-Phe, tyr (Me), D-Trp, tyr, D-Cha, cha, phe, sar, arg, mPhe, mVal, trp, mIle, D-Ala, mAla, thr, or Tyr.

9. A compound of embodiment 1 comprising a substituted N-terminal component wherein lie at position 1 is substituted with Ac-Trp or dipeptide Tyr-Gly.

10. The compound of any one of embodiments 1-9, further comprising an substitution of Ala for His at position 9.

11. A compound of any one of embodiments 1-10 further comprising a substitution of Trp or an analog of Trp for Val at position 4.

12. A compound of embodiment 11 wherein the Trp analog at position 4 is 1-methyl Trp or 1-formyl Trp.

13. The compound of embodiment 11 further comprising a substitution of the Trp analog for Trp at position 7.

14. A compound of embodiment 13 wherein the Trp analog at position 7 is a halogenated Trp.

15. The compound of any one of embodiments 1-14, further comprising a modification of Gly at position 8 to constrain the backbone conformation at that position.

16. The compound of embodiment 15 wherein the compound is prepared by reacting a compound of embodiment 15 with N ^α Methyl Gly replaces Gly at position 8 (Gly 8) to constrain the backbone.

17. The compound of embodiment 15 further comprising replacing Thr at position 13 with Ile, leu, nle, N-methyl Thr or N-methyl Ile.

18. A compound of any one of embodiments 1-17 further comprising replacing the disulfide bond between C2 and C12 with a thioether bond to form cystathionine or lanthionine.

19. The compound of any one of embodiments 1-18, further comprising replacing Arg at position 11 with Orn.

20. A compound of any one of embodiments 1-19 further comprising replacing Asp at position 6 with Asn.

21. A compound of embodiment 1 which is a compstatin analog comprising a peptide having the sequence shown in SEQ ID No. 29:

xaa1-Xaa2-Cys-Val-Xaa3-Gln-Xaa4-Xaa5-Gly-Xaa6-His-Xaa7-Cys-Xaa8, wherein Gly between Xaa4 and Xaa5 is optionally modified to constrain the backbone conformation;

wherein:

xaa1 is absent or Tyr, D-Tyr or Sar;

xaa2 is Ile, gly or Ac-Trp;

xaa3 is Trp or a Trp analog, wherein the Trp analog has enhanced hydrophobic character compared to Trp;

xaa4 is Asp or Asn;

xaa5 is Trp or a Trp analog comprising a chemical modification to an indole ring thereof, wherein the chemical modification increases the hydrogen bonding potential of the indole ring;

Xaa6 is His, ala, phe or Trp;

xaa7 is Arg or Orn; and

xaa8 is Thr, ile, leu, nle, N-methyl Thr or N-methyl Ile, wherein the carboxy terminal-OH of either Thr, ile, leu, nle, N-methyl Thr or N-methyl Ile is optionally substituted with-NH ₂ Instead, and

the peptide is cyclic through a Cys-Cys or thioether bond.

22. A compound of embodiment 19 wherein:

gly at position 8 is N-methylated;

xaa1 is D-Tyr or Sar;

xaa2 is Ile;

xaa3 is Trp, 1-methyl-Trp or 1-formyl-Trp;

xaa5 is Trp;

xaa6 is Ala; and

xaa8 is Thr, ile, leu, nle, N-methyl Thr or N-methyl Ile, optionally with-NH ₂ Instead of the carboxyl terminal-OH.

23. A compound of embodiment 22 wherein Xaa8 is lie, N-methyltr or N-methylile, optionally with-NH ₂ Instead of the carboxyl terminal-OH.

24. A compound of embodiment 23 comprising SEQ ID No. 7 or SEQ ID No. 18.

25. A compound of any one of the preceding embodiments comprising an additional component that increases the bioavailability of the compound or extends the in vivo residence of the compound.

26. A compound of embodiment 25 wherein the additional component is polyethylene glycol (PEG).

27. The compound of embodiment 25, wherein the additional component is an albumin binding small molecule.

28. The compound of embodiment 27, wherein the albumin binding small molecule is linked to the peptide at the N-terminus or the C-terminus.

29. A compound of embodiment 25 comprising a spacer between the peptide and the albumin-binding small molecule.

30. The compound of embodiment 25, wherein the additional component is an albumin binding peptide.

31. A pharmaceutical composition comprising a compound of any of the preceding embodiments and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of embodiment 31, formulated for oral administration of the compound.

33. The pharmaceutical composition of embodiment 31, formulated for topical administration of the compound.

34. The pharmaceutical composition of embodiment 31, formulated for pulmonary administration of the compound.

35. The pharmaceutical composition of embodiment 31, formulated for subcutaneous or intramuscular injection of the compound.

36. The pharmaceutical composition of embodiment 31, formulated for intravenous injection of the compound.

37. Use of a compound of any one of the preceding embodiments for the manufacture of a medicament for inhibiting complement activation.

38. Use of a compound of any of the preceding embodiments for inhibiting complement activation.

The invention is not limited to the embodiments described and illustrated hereinabove, but is capable of modification and variation within the scope of the appended embodiments.

Claims (6)

1. Consists of SEQ ID NO:18, and a peptide consisting of 18.

2. A compound, which is coupled to the peptide of claim 1 with additional components that prolong the in vivo retention of the peptide.

3. The compound of claim 2, wherein the additional component is selected from the group consisting of:

(a) Polyethylene glycol (PEG);

(b) Albumin binds small molecules; and

(c) Albumin binding peptides.

4. The compound of claim 3, wherein the albumin binding small molecule is ABM2 of the structure:

5. the compound of claim 3, wherein the albumin binding small molecule is attached to the terminus of the peptide directly or through a spacer between the peptide and albumin binding small molecule.

6. A pharmaceutical composition comprising the peptide of claim 1 or the compound of any one of claims 2-5, and a pharmaceutically acceptable carrier.