RU2778811C2 - Complement activity modulators - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to polypeptide modulators of complement activity, including cyclic polypeptide modulators. A method of treating a complement-related disorder in a subject who has not previously received eculizumab treatment includes: introduction of a polypeptide inhibiting C5, having a core sequence SEQ ID NO: 1, to a subject in an initial loading dose of 0.1 to 0.6 mg/kg; and administering a C5 inhibiting polypeptide having the core sequence SEQ ID NO: 1, to a subject in an initial therapeutic dose of 0.1 to 0.3 mg/kg, wherein the initial therapeutic dose is administered at least twice at regular intervals after the initial loading dose, wherein lactate dehydrogenase (LDH) levels in the subject are monitored during therapy, and wherein the initial therapeutic dose is replaced with a modified therapeutic dose of 0.3 to 0.6 mg/kg after at least two weeks of administering the initial therapeutic dose or when the LDH levels in the subject do not exceed 1.5 times the normal upper limit. Also disclosed is a version of a method of treating a complement-related disorder in a subject for another group of patients.

EFFECT: group of inventions provides selective blocking of complement-mediated cell destruction.

19 cl, 34 dwg, 11 tbl, 23 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority over U.S. Provisional Application No. 62/430,959, filed December 7, 2016 under the title Modulators of Complement Activity, U.S. Provisional Application No. 62/491,702 filed April 28, 2017 under the title Modulators of Complement Activity, U.S. Provisional Application No. 62/525,284, filed June 27, 2017, titled Modulators of Complement Activity, and U.S. Provisional Application No. 62/555,711, filed September 8, 2017, titled Modulators of Complement Activity, the contents of each of which are in their entirety incorporated into this application by reference.

SEQUENCE LIST

[0002] This application contains a sequence listing that is filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The referenced ASCII copy, created on November 16, 2017, is named 2011_1022PCT_SL.txt and is 1,193 bytes in size.

BACKGROUND OF THE INVENTION

[0003] The immune response of vertebrates consists of adaptive and innate components of immunity. While the adaptive immune response is selective for specific pathogens and responds slowly, components of the innate immune response recognize a wide range of pathogens and respond quickly to infection. One such component of the innate immune response is the complement system.

[0004] The complement system includes about 20 circulating complement system proteins synthesized primarily by the liver. The components of this specific immune response were first called "complement" because of the observation that they complemented the humoral immune response in killing bacteria. These proteins remain in an inactive form until activated in response to infection. Activation occurs via a proteolytic cleavage pathway initiated by pathogen recognition and leading to destruction of the pathogen. Three such pathways are known in the complement system, which are called the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is activated when an IgG or IgM molecule binds to the surface of a pathogen. The lectin pathway is initiated by the mannan-binding protein lectin, which recognizes sugar residues in the bacterial cell wall. The alternative pathway remains active at low levels in the absence of any specific incentives. Although all three pathways differ in initiating factors, all three pathways converge on cleavage of the C3 component of the complement system. C3 is split into two products called C3a and C3b. Of these, C3b becomes covalently bound to the surface of the pathogen, while C3a acts as a diffusing signal to stimulate inflammation and attract immune cells circulating in the peripheral blood. Surface-bound C3b forms a complex with other components, initiating a cascade of reactions among the later components of the complement system. Due to the need for surface attachment, complement activity remains localized and minimizes the destruction of non-target cells.

[0005] The pathogen-associated C3b component contributes to the destruction of pathogens in two ways. In one way, C3b is directly recognized by phagocytes and results in the ingestion of the pathogen. In the second way, pathogen-associated C3b initiates the formation of a membrane attack complex (MAC). In the first step, C3b forms a complex with other complement components to form the C5-convertase complex. Depending on the initial pathway of complement activation, the components of this complex may differ. The C5 convertase derived from the classical complement pathway includes C4B and C2a in addition to C3b. When formed via an alternative pathway, C5 convertase consists of two C3b subunits, as well as one Bb component.

[0006] The C5 component of complement is cleaved by any C5 convertase complex into C5a and C5b. C5a, like C3a, diffuses into the bloodstream and stimulates inflammation, acting as a chemoattractant for inflammatory cells. C5b remains attached to the cell surface where it induces MAC formation through interaction with C6, C7, C8 and C9. The MAC is a hydrophilic pore that passes through the membrane and facilitates the free flow of fluid into and out of the cell, thereby destroying it.

[0007] An important component of all immune activity is the ability of the immune system to distinguish between self and non-self cells. Pathology occurs when the immune system is unable to make this distinction. In the case of the complement system, vertebrate cells express proteins that protect them from the effects of the complement cascade. This ensures that the targets of the complement system are restricted to pathogenic cells. Many complement-related disorders and diseases are associated with the abnormal destruction of their cells by the complement cascade. In one example, patients suffering from paroxysmal nocturnal hemoglobinuria (PNH) are unable to synthesize functional versions of the complement regulatory proteins, CD55 and CD59, on hematopoietic stem cells. This leads to complement-mediated hemolysis and various subsequent complications. Other complement related disorders and diseases include, but are not limited to, autoimmune diseases and disorders, neurological diseases and disorders, hematological diseases and disorders, and infectious diseases and disorders. Experimental evidence suggests that many complement-related disorders are attenuated by inhibition of complement activity. Therefore, there is a need to provide compositions and methods for selectively blocking complement-mediated cell destruction in order to treat associated symptoms. The present invention solves this problem by providing appropriate compositions and methods.

SUMMARY OF THE INVENTION

[0008] In some embodiments, the present invention provides a method of treating paroxysmal nocturnal hemoglobinuria (PNH) in a subject when the subject has not previously been treated with eculizumab, the method comprising: administering R5000 to the subject at an initial loading dose and administering R5000 to the subject at an initial therapeutic dose, wherein the initial the therapeutic dose is administered at least twice at regular intervals after the initial loading dose. During therapy, lactate dehydrogenase (LDH) levels in a subject can be monitored. The initial therapeutic dose may be replaced with a modified therapeutic dose of R5000 after two or more weeks of administration with the initial therapeutic dose. The modified therapeutic dose of R5000 may include an increased dose of R5000. The initial therapeutic dose may be substituted with a modified therapeutic dose when the subject's LDH levels do not exceed 1.5 times the normal upper limit. The initial loading dose may be in the range of about 0.1 mg/kg to about 0.6 mg/kg of R5000. The initial loading dose may be in the range of about 0.1 mg/kg to about 0.3 mg/kg R5000. The initial therapeutic dose may be substituted with a modified therapeutic dose of about 0.3 mg/kg to about 0.6 mg/kg R5000. Hemolysis in the subject's serum may be reduced. Negative phenomena may not be observed. At least one administration of R5000 may include self-administration. Self-administration can be controlled. Self-administration can be controlled remotely. Self-administration can be controlled with a smart device. Equal intervals of time can be from about every 12 hours to about every 168 hours.

[0009] The methods of the present invention may include a method of treating PNH in a subject, wherein the subject is currently being treated with eculizumab or has previously been treated with eculizumab, and the method comprises administering R5000 to the subject. Residual hemolytic activity in a subject may be reduced or eliminated. Communication between C5 and C5 convertase alternative pathway can be inhibited. R5000 can be co-administered with eculizumab. The subject may be a carrier of the C5 polymorphism. The polymorphism may include p.Arg885His.

[0010] In some embodiments, the present invention provides a pharmaceutical composition comprising R5001 and at least one pharmaceutically acceptable excipient. The at least one pharmaceutically acceptable excipient may include at least one of sodium chloride and sodium phosphate. The pharmaceutical composition may contain from about 25 mm to about 100 mm sodium chloride. The pharmaceutical composition may contain from about 10 mm to about 100 mm sodium phosphate. R5001 may be present at a concentration of from about 1 mg/mL to about 400 mg/mL.

[0011] In some aspects, the present invention provides a method for treating PNH in a subject, the method comprising administering a pharmaceutical composition as described herein. The subject may have previously been treated with an antibody-based drug. The subject may be resistant to or refractory to the antibody drug. The antibody drug may be eculizumab. The pharmaceutical composition may be administered at a dose of about 0.01 mg/kg to about 10 mg/kg. The pharmaceutical composition may be administered at a dose of about 0.1 mg/kg to about 0.3 mg/kg. The pharmaceutical composition can be administered every day for at least two days. The pharmaceutical composition can be administered every day for about 12 weeks. The pharmaceutical composition can be administered every day for at least 1 year. The pharmaceutical composition can be administered subcutaneously or intravenously.

[0012] In some embodiments, the present invention provides a method for inhibiting C5 binding to an alternative pathway C5 convertase by contacting C5 with R5001. C5 may be from a subject with a C5 polymorphism. The C5 polymorphism may include p.Arg885His.

[0013] In some aspects, the present invention provides a method for inhibiting residual C5 activity in a subject with PNH, the subject currently being treated with eculizumab or having previously been treated with eculizumab, the method comprising administering R5000 to the subject. Communication between C5 and C5 convertase alternative pathway can be inhibited. R5000 can be co-administered with eculizumab. The subject may be a carrier of the C5 polymorphism. The polymorphism may include p.Arg885His.

[0014] In some embodiments, the present invention provides a method for measuring C5 levels in a sample, the method comprising: immobilizing a capture agent on a substrate, wherein the capture agent binds to a site on C5 that is different from the eculizumab binding site; bringing the substrate into contact with the sample, wherein the immobilized capture agent binds to C5 in the sample; bringing the substrate into contact with a detecting agent, wherein the detecting agent binds to C5 associated with the immobilized capture agent; and measuring the level of bound detecting agent as an indication of C5 levels in the sample. The detecting agent may include a detectable label. The detection agent can be detected using a secondary detection agent. The detecting agent may include an anti-C5 antibody. Both free C5 levels and eculizumab-bound C5 levels can be measured. The substrate can be contacted with a second detecting agent, wherein the second detecting agent binds to eculizumab, and the method includes measuring the level of bound second detecting agent as an indicator of C5 levels associated with eculizumab. The second detecting agent may include a detectable label. The second detecting agent can be detected using a secondary detecting agent. The capture agent may include an R5000 variant. The R5000 variant may include an N-terminal biotinylated PEG fragment and replacement of the C-terminal lysine in R5000 with norvaline in the R5000 variant.

[0015] The methods of the present invention may include a method for measuring free eculizumab levels in a sample, the method comprising: immobilizing a capture agent on a substrate, wherein the capture agent binds to a site on C5 that is different from the eculizumab binding site; contacting the substrate with an excess of C5 to form a C5-capture agent complex, wherein the C5-capture agent complex contains immobilized C5; bringing the substrate into contact with the sample, wherein the immobilized C5 binds to eculizumab in the sample; bringing the substrate into contact with a detecting agent, wherein the detecting agent binds to eculizumab; and measuring the level of bound detecting agent as an indication of the levels of free eculizumab in the sample. The capture agent may include an R5000 variant. The R5000 variant may include an N-terminal biotinylated PEG fragment and replacement of the C-terminal lysine in R5000 with norvaline in the R5000 variant. The detecting agent may be an antibody. The detecting agent may include a detectable label. The detection agent can be detected using a secondary detection agent.

BRIEF DESCRIPTION OF THE FIGURES

[0016] The above and other objects, features and advantages will be apparent from the following description of specific embodiments of the invention, as well as the accompanying drawings explaining the principles of various embodiments of the invention.

[0017] FIG. 1 is a scatterplot showing R5000-dependent inhibition of C5a production.

[0018] FIG. 2 is a scatterplot showing R5000-dependent inhibition of membrane attack complex formation.

[0019] FIG. 3 is a scatterplot showing the activity of the R5000 inhibitor in the cynomolgus monkey model system.

[0020] FIG. 4 is a scatterplot showing the correlation of pharmacokinetics and pharmacodynamics of R5000 in male cynomolgus monkeys after several subcutaneous injections at a dose of 0.21 mg/kg.

[0021] FIG. 5 is a scatterplot showing the correlation of pharmacokinetics and pharmacodynamics of R5000 in male cynomolgus monkeys after several subcutaneous injections at a dose of 4.2 mg/kg.

[0022] FIG. 6 is a line graph showing R5000 concentrations in cynomolgus monkey after the first dose during a repeated dose toxicity study.

[0023] FIG. 7 is a line graph showing R5000 concentrations in cynomolgus monkey after the last dose during a repeated dose toxicity study.

[0024] FIG. 8 is a line graph showing predicted human plasma concentrations of R5000 when R5000 is administered daily.

[0025] FIG. 9A is a graph showing dose dependent peak plasma levels of R5000 during a human single escalating dose clinical study.

[0026] FIG. 9B is a graph showing the change in plasma concentrations over time after a single dose of R5000.

[0027] FIG. 10A is a graph showing percent hemolysis versus time after a single dose of R5000 over 4 days in humans.

[0028] FIG. 10B is a graph showing CH ₅₀ as a percentage versus time after a single dose of R5000 in humans.

[0029] FIG. 10C is a graph showing percent hemolysis at various doses over 28 days in humans.

[0030] FIG. 11 is a graph showing percent complement activity versus time after a single dose of R5000 in humans.

[0031] FIG. 12A is a graph showing percent change in hemolysis with R5000 concentration in a multiple dose human study.

[0032] FIG. 12B is a graph showing changes in plasma R5000 concentrations over time in a multiple dose study in humans.

[0033] FIG. 13A is a graph showing changes in complement activity with time during treatment with R5000 during a multiple dose human study.

[0034] FIG. 13B is a graph showing changes in complement activity over an extended period of treatment with R5000 in a multiple dose human study.

[0035] FIG. 14 is a graph showing predicted plasma drug concentration and hemolysis inhibition when R5000 is used in humans.

[0036] FIG. 15 is a graph showing the predicted pharmacokinetics and pharmacodynamics of R5000 in humans.

[0037] FIG. 16A is a graph showing hemolysis in groups of voluntary subjects receiving a single dose of R5000.

[0038] FIG. 16B is a graph showing predicted percentage trough plasma concentrations with and without a loading dose of R5000.

[0039] FIG. 16C is a graph showing the predicted inhibition of hemolysis using R5000 at 0.1 mg/kg in humans.

[0040] FIG. 17 is a graph showing the reduction in hemolysis when using mR5000 in patient samples.

[0041] FIG. 18A is a graph showing percent hemolysis in patient samples during six weeks of treatment with R5000.

[0042] FIG. 18B is a graph showing changes in hemoglobin levels in patient samples over six weeks of treatment with R5000.

[0043] FIG. 18C are a pair of graphs showing changes in lactate dehydrogenase (LDH) levels (individual and combined means) during seven weeks of treatment with R5000.

[0044] FIG. 18D is a graph showing R5000 levels in patient samples during six weeks of treatment with R5000.

[0045] FIG. 19 is a graph showing evoked response in response units versus time during a C5 inhibitor binding assay using surface plasmon resonance.

[0046] FIG. 20 is a graph showing hemolysis as a percentage of erythrocytes from PNH patients in the presence of R5000 or eculizumab.

[0047] FIG. 21 is a graph showing hemolysis as a percentage of erythrocytes from PNH patients in the presence of R5000 or eculizumab.

[0048] FIG. 22 is a graph showing percentage hemolysis of RBCs carrying recombinant C5 containing the known p.Arg885His polymorphism after exposure to various concentrations of eculizumab or R5000.

[0049] FIG. 23 is a graph showing the serum concentration of C5, C5:eculizumab complex, and eculizumab in the serum of a patient with PNH.

[0050] FIG. 24 is a graph showing percent residual hemolysis in plasma of a patient with PNH treated with eculizumab in the presence of R5000 or additional eculizumab.

DETAILED DESCRIPTION

I. Compounds and compositions

[0051] In some embodiments, the present invention provides compounds and compositions that function to modulate complement activity. These compounds and compositions may include inhibitors that block complement activation. In the context of this application, "complement activity" includes activation of the complement cascade, formation of cleavage products from a complement component such as C3 or C5, assembly of subsequent complexes occurring after a cleavage event, or any process or event accompanying or resulting from the cleavage of a component complement, such as C3 or C5. Complement inhibitors may include C5 inhibitors that block complement activation at the level of the C5 component of complement. C5 inhibitors can bind C5 and prevent it from being cleaved by C5 convertase into C5a and C5b cleavage products. In the context of the present application, "complement C5 component" or "C5" is defined as a complex that is cleaved by C5 convertase into at least the cleavage products, C5a and C5b. "C5 inhibitors", in accordance with the invention, contain any compound or composition that/th inhibits the processing or cleavage of the pre-cleaved complex of the C5 component of complement or the cleavage products of the C5 component of complement.

[0052] It should be understood that inhibition of C5 cleavage prevents the assembly and activity of the cytolytic membrane attack complex (MAC) on glycosylphosphatidylinositol (GPI)-associated protein-deficient erythrocytes. In some cases, the C5 inhibitors provided herein can also bind C5b, preventing C6 binding and subsequent assembly of the C5b-9 MAC.

Peptide compounds

[0053] In some embodiments, the C5 inhibitors of the present invention are polypeptides. In accordance with the present invention, any amino acid-based molecule (natural or synthesized) may be referred to as a "polypeptide" and the term encompasses "peptides", "peptidomimetics" and "proteins". Traditionally, "peptides" are considered to have a size ranging from about 4 to about 50 amino acids. Polypeptides of more than about 50 amino acids are commonly referred to as "proteins".

[0054] C5 polypeptide inhibitors can be linear or cyclic. Cyclic polypeptides include any polypeptides that contain as part of their structure one or more cyclic structures, such as a loop and/or an internal link. In some embodiments, cyclic polypeptides are formed when a molecule acts as a bridging moiety to link two or more regions of a polypeptide. In the context of this application, the term "bridge fragment" refers to one or more components of the bridge formed between two adjacent or non-contiguous amino acids, non-natural amino acids or non-amino acid groups in a polypeptide. Bridge fragments can be of any size and composition. In some embodiments, bridging fragments may contain one or more chemical bonds between two adjacent or non-adjacent amino acids, non-natural amino acids, non-amino acid residues, or combinations thereof. In some embodiments, such chemical bonds may be between one or more functional groups on contiguous or non-contiguous amino acids, non-natural amino acids, non-amino acid residues, or combinations thereof. Bridge moieties may contain one or more of an amide bond (lactam), a disulfide bond, a thioether bond, an aromatic ring, a triazole ring, and a hydrocarbon chain. In some embodiments, the bridging moieties comprise an amide bond between an amino group and a carboxy group that are present on the side chain of an amino acid, non-natural amino acid, or non-amino acid residue. In some embodiments, the amino or carboxy groups are part of a non-amino acid residue or a non-natural amino acid residue.

[0055] C5 polypeptide inhibitors can be cyclized through the carboxy-terminus, the amino-terminus, or through any other convenient point of attachment, for example, through the sulfur in cysteine (for example, by forming disulfide bonds between two cysteine residues in the sequence) or any side chain of the amino acid residue. Additional bonds forming cyclic loops may include, but are not limited to, maleimide bonds, amide bonds, ester bonds, ether bonds, thioether bonds, hydrazone bonds, or acetamide bonds.

[0056] In some embodiments, the C5 cyclic polypeptide inhibitors of the invention are formed using a lactam moiety. These cyclic polypeptides can be generated, for example, by synthesis on solid Wang resin using standard Fmoc chemistry. In some cases, Fmoc-ASP(allyl)-OH and Fmoc-LYS(Alloc)-OH are incorporated into polypeptides as precursor monomers for lactam bridge formation.

[0057] The C5 polypeptide inhibitors of the invention may be peptidomimetics. A "peptidomimetic" or "polypeptidomimetic" is a polypeptide in which the molecule contains structural elements that are not found in natural polypeptides (ie, polypeptides consisting of only 20 proteinogenic amino acids). In some embodiments, peptidomimetics are capable of replicating or mimicking the biological action of a natural peptide. The peptidomimetic may differ from natural polypeptides in many ways, for example, changes in the backbone structure and the presence of amino acids that do not occur naturally. In some cases, peptidomimetics may include amino acids with side chains that are not found among the known 20 proteinogenic amino acids; non-polypeptide bridging fragments used to carry out cyclization between the ends or internal regions of the molecule; replacement of the hydrogen fragment of the amide bond with methyl groups (N-methylation) or other alkyl groups; replacing a peptide bond with a chemical group or bond that is resistant to chemical or enzymatic treatment; N- and C-terminal modifications; and/or conjugation to a non-peptide extension segment (eg, polyethylene glycol, lipids, carbohydrates, nucleosides, nucleotides, nucleoside bases, various small molecule compounds, or phosphate or sulfate groups).

[0058] As used herein, the term "amino acid" includes natural amino acid residues as well as non-natural amino acids. 20 natural proteinogenic amino acids have been identified, which are designated in the present description by one-letter or three-letter following designations: aspartic acid (Asp: D), isoleucine (Ile: I), threonine (Thr: T), leucine (Leu: L), serine (Ser: S), tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys: K), alanine (Ala: A), arginine (Arg: R), cysteine (Cys, C), tryptophan (Trp: W), valine (Val: V), glutamine (Gln: Q) methionine (Met: M) , asparagine (Asn: N). Naturally occurring amino acids exist in their levorotatory (L) stereoisomeric form. Amino acids mentioned in the present description are L-stereoisomers, unless otherwise indicated. The term "amino acid" also includes amino acids bearing a conventional amino protecting group (e.g., acetyl or benzyloxycarbonyl), as well as natural and non-natural amino acids protected at the carboxy terminus (e.g., as a (C1-C6)alkyl, phenyl, or benzyl ester or amide, or as alpha-methylbenzylamide). Those skilled in the art (see, for example, Greene, T. W.; Wutz, P. G. M., Protecting Groups In Organic Synthesis; second edition, 1991, New York, John Wiley & sons, Inc., and the documents cited therein, the contents of each which are hereby incorporated by reference in their entirety) other suitable amino and carboxy protecting groups are known. The polypeptides and/or polypeptide compositions of the present invention may also include modified amino acids.

[0059] "Non-natural" amino acids have side chains or other features not present in the 20 naturally occurring amino acids listed above and include, but are not limited to: N-methyl amino acids, N-alkyl amino acids MINO acids, alpha, alpha- substituted amino acids, beta-amino acids, alpha-hydroxyamino acids, D-amino acids, and other non-natural amino acids known in the art (see, for example, Josephson et al., (2005) J. Am. Chem. Soc. 127: 11727-11735 ; Forster, A. C. et al. (2003) Proc. Natl. Acad. Sci. USA 100: 6353-6357; Subtelny et al., (2008) J. Am. Chem. Soc. 130: 6131-6136; Hartman, M. C. T. et al (2007) PLoS ONE 2:e972 and Hartman et al (2006) Proc Natl Acad Sci USA 103:4356-4361). Other non-natural amino acids useful in optimizing the polypeptides and/or polypeptide compositions of the present invention include, but are not limited to, 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid, 1-amino-2,3-hydro- 1H-indene-1-carboxylic acid, homolysin, homoarginine, homoserine, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 5-aminovaleric acid, 5-aminocaproic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, desmosine, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo- hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylpentylglycine, naphthylalanine, ornithine, pentylglycine, thioproline, norvaline, tert-butylglycine, phenylglycine, azatryptophan, 5-azatryptophan, 7-azatryptophan, 4-fluorophenylalanine, ne nicillamine, sarcosine, homocysteine, 1-aminocyclopropanecarboxylic acid, 1-aminocyclobutanecarboxylic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclohexanecarboxylic acid, 4-aminotetrahydro-2H-pyran-4-carboxylic acid, (S)-2-amino-3-( 1H-tetrazol-5-yl)propionic acid, cyclopentylglycine, cyclohexylglycine, cyclopropylglycine, η-ω-methylarginine, 4-chlorophenylalanine, 3-chlorotyrosine, 3-fluorotyrosine, 5-fluorotryptophan, 5-chlorotryptophan, citrulline, 4-chlorohomophenylalanine , homophenylalanine, 4-aminomethyl-phenylalanine, 3-aminomethyl-phenylalanine, octylglycine, norleucine, tranexamic acid, 2-aminovaleric acid, 2-aminocaproic acid, 2-aminoheptanoic acid, 2-aminocaprylic acid, 2-aminopelargonic acid, 2-aminocaprinic acid, 2-aminoundecanoic acid, 2-aminolauric acid, aminovaleric acid and 2-(2-aminoethoxy)acetic acid, pipecolic acid, 2-carboxyazetidine, hexafluoroleucine, 3-fluorovaline, 2-amino-4,4-difluoro-3- methylbutanecarboxylic acid oto, 3-fluoroisoleucine, 4-fluoroisoleucine, 5-fluoroisoleucine, 4-methylphenylglycine, 4-ethylphenylglycine, 4-isopropylphenylglycine, (S)-2-amino-5-azidovaleric acid (also referred to herein as “X02”), ( S)-2-aminohept-6-enoic acid (also referred to herein as “X30”), (S)-2-aminopent-4-oic acid (also referred to herein as “X31”), (S)-2 -aminopent-4-enoic acid (also referred to herein as "X12"), (S)-2-amino-5-(3-methylguanidino)valeric acid, (S)-2-amino-3-(4-( aminomethyl)phenyl)propionic acid, (S)-2-amino-3-(3-(aminomethyl)phenyl)propionic acid, (S)-2-amino-4-(2-aminobenzo[d]oxazol-5-yl )butyric acid, (S)-leucinol, (S)-valinol, (S)-tert-leucinol, (R)-3-methylbutan-2-amine, (S)-2-methyl-1-phenylpropan-1- amine and (S)-N,2-dimethyl-1-(pyridin-2-yl)propan-1-amine, (S)-2-amino-3-(oxazol-2-yl)propionic acid, (S) -2-amino-3-(oxazol-5-yl)propionic acid, (S)-2-amino-3-(1,3,4-oxadiazol-2-yl)propionic acid, (S)-2-amino -3-(1,2 ,4-oxadiazol-3-yl)propionic acid, (S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)propionic acid and (S)-2-amino-3-(1H -indazol-3-yl)propionic acid, (S)-2-amino-3-(oxazol-2-yl)butyric acid, (S)-2-amino-3-(oxazol-5-yl)butyric acid, (S)-2-amino-3-(1,3,4-oxadiazol-2-yl)butyric acid, (S)-2-amino-3-(1,2,4-oxadiazol-3-yl)butyric acid, (S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)butyric acid and (S)-2-amino-3-(1H-indazol-3-yl)butyric acid, 2-(2'-methoxyphenyl)-2-aminoacetic acid, tetrahydro-3-isoquinolinecarboxylic acid and their stereoisomers (including but not limited to D and L isomers).

[0060] Additional non-natural amino acids that are useful in optimizing the polypeptides or polypeptide compositions of the invention include, but are not limited to, fluorinated amino acids in which one or more carbon-bonded hydrogen atoms have been replaced by fluorine. The number of fluorine atoms included may range from 1 to all hydrogen atoms, inclusive. Examples of such amino acids include, but are not limited to, 3-fluoroproline, 3,3-difluoroproline, 4-fluoroproline, 4,4-difluoroproline, 3,4-difluoroproline, 3,3,4,4-tetrafluoroproline, 4-fluorotryptophan , 5-fluorotryptophan, 6-fluorotryptophan, 7-fluorotryptophan and their stereoisomers.

[0061] Additional non-natural amino acids that are useful in optimizing the polypeptides of the invention include, but are not limited to, amino acids that are disubstituted at the α-carbon. These include amino acids in which two substituents on the α-carbon are the same, such as α-aminoisobutyric acid and 2-amino-2-ethylbutyric acid, as well as those in which the substituents are different, such as α-methylphenylglycine and α-methylproline . In addition, substituents on the α-carbon can be combined to form a ring, e.g. , 4-aminotetrahydropyran-4-carboxylic acid, 3-aminopyrrolidine-3-carboxylic acid, 3-aminopiperidine-3-carboxylic acid, 4-aminopiperidine-4-carboxylic acid, and their stereoisomers.

[0062] Additional non-natural amino acids that are useful in optimizing the polypeptides or polypeptide compositions of the invention include, but are not limited to, tryptophan analogs in which the indole ring system is replaced by another 9- or 10-membered bicyclic ring system containing 0, 1 , 2, 3, or 4 heteroatoms independently selected from N, O, or S. Each ring system may be saturated, partially unsaturated, or fully unsaturated. The ring system may be substituted with 0, 1, 2, 3 or 4 substituents on any substitutable atom. Each substituent may be independently selected from H, F, Cl, Br, CN, COOR, CONRR', oxo, OR, NRR'. Each of R and R' may be independently selected from H, C1-C20 alkyl, or C1-C20 alkyl-O-C1-20 alkyl.

[0063] In some embodiments, tryptophan analogs (also referred to herein as "tryptophan analogs") may be useful in optimizing polypeptides or polypeptide compositions of the invention. Tryptophan analogs may include, but are not limited to, 5-fluorotryptophan [(5-F)W], 5-methyl-O-tryptophan [(5-MeO)W], 1-methyltryptophan [(1-Me-W) or (1-Me),W], D-tryptophan (D-Trp), azatryptophan (including but not limited to 4-azatryptophan, 7-azatryptophan and 5-azatryptophan) 5-chlorotryptophan, 4-fluorotryptophan, 6-fluorotryptophan , 7-fluorotryptophan and their stereoisomers. Except where otherwise indicated, the term "azatryptophan" and its abbreviation "azaTrp" as used herein refer to 7-azatryptophan.

[0064] Modified amino acid residues useful in optimizing the polypeptides and/or polypeptide compositions of the present invention include, but are not limited to, those residues that are chemically blocked, reversibly or irreversibly; chemically modified at their N-terminal amino group or their side chain groups; chemically modified in the amide backbone, for example, N-methylated stereoisomers D (non-natural amino acids) and L (natural amino acids); or residues in which the functional groups of the side chain are chemically modified to another functional group. In some embodiments, the modified amino acids include, without limitation, methionine sulfoxide; methionine sulfone; beta-methyl ester of aspartic acid, a modified amino acid of aspartic acid; N-ethylglycine modified with the amino acid glycine; alanine carboxamide; and/or a modified amino acid alanine. Non-natural amino acids can be purchased from Sigma-Aldrich (St. Louis, MO), Bachem (Torrance, CA) or other suppliers. Non-natural amino acids may further include any of those listed in Table 2 of US Patent Publication US 2011/0172126, the contents of which are hereby incorporated by reference in their entirety.

[0065] The present invention provides variants and derivatives of the polypeptides presented in this application. These include substituted, insertion, deletion and covalent variants and derivatives. In the context of this application, the term "derivative" is used as a synonym for the term "variant" and refers to a molecule that has been modified or changed in any way relative to the reference molecule or the original molecule.

[0066] The polypeptides of the invention may include the following components, portions or fragments, for which the following abbreviations are used in the present description: "Ac" and "NH2" denote the acetyl and amidated ends, respectively; "Nvl" means norvaline; "Phg" refers to phenylglycine; "Tbg" refers to tert-butylglycine; "Chg" means cyclohexylglycine; "(N-Me)X" means the N-methylated form of an amino acid indicated by a one-letter or three-letter amino acid code instead of the variable "X", written as N-methyl-X [e.g., (N-Me)D or (N-Me )Asp is the N-methylated form of aspartic acid or N-methyl-aspartic acid]; "azaTrp" means azatryptophan; "(4-F)Phe" means 4-fluorophenylalanine; "Tyr(OMe)" means O-methyl-tyrosine, "Aib" means aminoisobutyric acid; "(homo)F" or "(homo)Phe" means homophenylalanine; "(2-OMe)Phg" means 2-O-methylphenylglycine; "(5-F)W" means 5-fluorotryptophan; "D-X" refers to the D-stereoisomer of a given amino acid "X" [eg, (D-Chg) is D-cyclohexylglycine]; "(5-MeO)W" means 5-methyl-O-tryptophan; "homoC" means homocysteine; "(1-Me-W)" or "(1-Me)W" means 1-methyltryptophan; "Nle" means norleucine; "Tiq" denotes a tetrahydroisoquinoline residue; "Asp(T)" refers to (S)-2-amino-3-(1H-tetrazol-5-yl)propionic acid; "(3-Cl-Phe)" means 3-chlorophenylalanine; "[(N-Me-4-F)Phe]" or "(N-Me-4-F)Phe" means N-methyl-4-fluorophenylalanine; "(m-Cl-homo)Phe" means meta-chlorohomophenylalanine; "(desamino)C" means 3-thiopropionic acid; "(alpha-methyl)D" refers to alpha-methyl-L-aspartic acid; "2Nal" means 2-naphthylalanine; "(3-aminomethyl)Phe" means 3-aminomethyl-L-phenylalanine; "Cle" means cycloleucine; "A-pyran" means 4-amino-tetrahydropyran-4-carboxylic acid; "(Lys-C16)" refers to N-ε-palmitoyllysin; "(Lys-C12)" refers to N-ε-lauryllysin; "(Lys-C10)" refers to N-ε-capryllisin; "(Lys-C8)" refers to N-ε-capryllisin; "[x-xylyl(y,z)]" denotes a xylyl bridging moiety between two thiol-containing amino acids, where x can be m, n, or o, indicating the use of meta-, para-, or ortho-dibromoxylenes (respectively) to create bridging moieties , and the numerical identifiers, y and z, indicate the amino acid positions in the polypeptide for the amino acids involved in the cyclization process; "[cyclo(y,z)]" refers to the formation of a bond between two amino acid residues, where the numerical identifiers, y and z, indicate the positions of the residues involved in the formation of the bond; "[cyclo-olefinyl(y,z)]" refers to the formation of a bond between two amino acid residues using an olefin exchange reaction, where the numerical identifiers, y and z, indicate the positions of the residues involved in the formation of the bond; "[cyclo-thioalkyl(y,z)]" refers to the formation of a thioether bond between two amino acid residues, where the numerical identifiers, y and z, indicate the positions of the residues involved in the formation of the bond; "[cyclo-triazolyl(y,z)]" refers to the formation of a triazole ring between two amino acid residues, where the numerical identifiers, y and z, indicate the positions of the residues involved in the bond formation. "B20" refers to N-ε-(PEG2-γ-glutamic acid-N-α-octodecanedioic acid) lysine [also known as (1S,28S)-1-amino-7,16,25,30-tetraoxo-9, 12,18,21-tetraoxa-6,15,24,29-tetraazahexatetracontane-1,28,46-tricarboxylic acid].

B20

[0067] B28" is N-ε-(PEG24-γ-glutamic acid-N-α-hexadecanoyl)lysine.

B28

[0068] "K14" is N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine. All other characters refer to the standard single letter amino acid code.

[0069] Some C5 polypeptide inhibitors contain from about 5 amino acids to about 10 amino acids, from about 6 amino acids to about 12 amino acids, from about 7 amino acids to about 14 amino acids, from about 8 amino acids to about 16 amino acids, from about 10 amino acids to about 18 amino acids, from about 12 amino acids to about 24 amino acids, or from about 15 amino acids to about 30 amino acids. In some cases, C5 polypeptide inhibitors contain at least 30 amino acids.

[0070] Some C5 inhibitors of the invention include a C-terminal lipid moiety. Such lipid moieties may include fatty acid acyl groups (eg, saturated or unsaturated fatty acid acyl groups). In some cases, the fatty acid acyl group may be a palmitoyl group.

[0071] C5 inhibitors containing fatty acid acyl groups may include one or more molecular crosslinkers that attach fatty acids to a peptide. These molecular crosslinkers may include amino acid residues. In some cases, L-γ-glutamic acid residues can be used as molecular crosslinkers. In some cases, molecular crosslinkers may include one or more polyethylene glycol (PEG) crosslinkers. PEG crosslinkers of the invention may include from about 1 to about 5, from about 2 to about 10, from about 4 to about 20, from about 6 to about 24, from about 6 to about 32, or at least 32 PEG units.

[0072] The C5 inhibitors of the invention may have molecular weights from about 200 g/mol to about 600 g/mol, from about 500 g/mol to about 2000 g/mol, from about 1000 g/mol to about 5000 g/mol, from about 3000 g/mol to about 4000 g/mol, from about 2500 g/mol to about 7500 g/mol, from about 5000 g/mol to about 10000 g/mol, or at least 10000 g/mol.

[0073] In some embodiments, the C5 polypeptide inhibitors of the invention include R5000. The core amino acid sequence for R5000 ([cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg-K; SEQ ID NO: 1) contains 15 amino acids (all L- amino acid forms), including 4 non-natural amino acids [N-methyl-aspartic acid or "(N-Me)D", t-butylglycine or "Tbg", 7-azatryptophan or "azaTrp" and cyclohexylglycine or "Chg"]; lactam bridge between K1 and D6 in the polypeptide sequence; and a C-terminal lysine residue with a side chain modified to form an N-ε-(PEG24-γ-glutamic acid-N-α-hexadecanoyl)lysine residue (also referred to as "B28" herein). The side chain modification of the C-terminal lysine includes a polyethylene glycol (PEG) spacer (PEG24) where PEG24 is attached to an L-γ-glutamic acid residue that is derivatized with a palmitoyl group.

[0074] In some embodiments, the present invention includes R5000 variants. In some embodiments of R5000, the C-terminal lysine side chain fragment may be altered. In some cases, the PEG24 spacer (containing 24 PEG subunits) of the C-terminal lysine side chain fragment may include fewer or more PEG subunits. In other cases, the palmitoyl group of the C-terminal lysine side chain fragment may be substituted with another saturated or unsaturated fatty acid. In additional instances, the L-γ-glutamic acid crosslinker of the C-terminal lysine side chain fragment (between PEG and acyl groups) may be substituted with an alternative amino acid or non-amino acid crosslinker.

[0075] In some embodiments, C5 inhibitors may include active metabolites or variants of R5000. Metabolites may include R5001. In the context of this application, the term "R5001" refers to the R5000 variant with ω-hydroxylation of the palmitoyl tail. R5001 can be synthesized or can be formed by hydroxylation of the precursor R5000. R5001 can bind to C5 at an equilibrium dissociation constant (KD) equal to or similar to the given R5000 to C5 binding constant (eg, deviating about ±20% from the KD of R5000 to C5 binding). R5001 can inhibit C5 cleavage and/or block one or more subsequent events associated with C5 cleavage at a half-maximal inhibitory concentration (IC ₅₀ ) equal to or similar to a given concentration of R5000 (e.g., with a deviation of about ±20% from the IC ₅₀ for R5000 for inhibition of C5 cleavage). R5001 can inhibit complement-dependent hemolysis with an IC ₅₀ equal to or similar to a given concentration of R5000 (eg, with a deviation of about ±20% from the IC ₅₀ for R5000 for hemolysis inhibition).

[0076] In some embodiments, R5000 variants may include modifications to the core sequence of a polypeptide in R5000 that can be used in combination with one or more cyclic or C-terminal lysine side chain fragment elements in R5000. Such variants may contain sequences with at least 50%, at least 55%, at least 65%, at least 70%, at least 80%, at least 85% identity, at least 90% or at least 95% relative to the core sequence of the polypeptide for (SEQ ID NO: 1).

[0077] In some cases, R5000 variants can be cyclized by forming lactam bridges between amino acids other than those used in R5000.

[0078] The C5 inhibitors of the invention can be designed or modified to achieve specific binding characteristics. Inhibitor binding can be assessed by determining the rates of association and/or dissociation with a specific target. In some cases, the compounds exhibit strong and rapid association with the target, coupled with a low dissociation rate. In some embodiments, the C5 inhibitors of the invention exhibit strong and rapid association with C5. Such inhibitors may further exhibit low rates of dissociation from C5.

[0079] The C5 inhibitors of the invention that bind to the C5 protein of the complement system can bind to the C5 protein of the complement system at an equilibrium dissociation constant (KD) of from about 0.001 nM to about 0.01 nM, from about 0.005 nM to about 0.05 nM, from about 0.01 nM to about 0.1 nM, from about 0.05 nM to about 0.5 nM, from about 0.1 nM to about 1.0 nM, from about 0.5 nM to about 5 .0 nM, from about 2 nM to about 10 nM, from about 8 nM to about 20 nM, from about 15 nM to about 45 nM, from about 30 nM to about 60 nM, from about 40 nM to about 80 nM, from about 50 nM to about 100 nM, about 75 nM to about 150 nM, about 100 nM to about 500 nM, about 200 nM to about 800 nM, about 400 nM to about 1000 nM, or at least 1000 nM.

[0080] In some embodiments, the C5 inhibitors of the invention block the formation or formation of C5a from C5. In some case, the formation or formation of C5a is blocked after the activation of an alternative pathway of complement activation. In some cases, the C5 inhibitors of the invention block the formation of a membrane attack complex (MAC). This inhibition of MAC formation may be due to the binding of the C5 inhibitor to the C5b subunits. Binding of a C5 inhibitor to C5b subunits can prevent C6 binding, resulting in blocking of MAC formation. In some embodiments, this inhibition of MAC formation occurs after activation of the classical, alternative, or lectin pathways.

[0081] The C5 inhibitors of the invention can be synthesized using chemical processes. In some cases, such synthesis eliminates the risks associated with the manufacture of biological preparations on mammalian cell lines. In some cases, chemical synthesis can be simpler and cheaper than biological manufacturing processes.

[0082] In some embodiments, the C5 inhibitor compositions (eg, R5000 and/or its active metabolite or variant) can be pharmaceutical compositions containing at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient may include at least one of a salt and a buffer. The salt may be sodium chloride. The buffering agent may be sodium phosphate. Sodium chloride may be present at a concentration of from about 0.1 mM to about 1000 mM. In some instances, sodium chloride may be present at a concentration of from about 25 mM to about 100 mM. Sodium phosphate may be present at a concentration of from about 0.1 mM to about 1000 mM. In some cases, sodium phosphate is present at a concentration of from about 10 mM to about 100 mM.

[0083] In some embodiments, C5 inhibitor compositions (eg, R5000 and/or its active metabolite or variant) may include from about 0.01 mg/ml to about 4000 mg/ml C5 inhibitor. In some cases, C5 inhibitors are present at a concentration of from about 1 mg/mL to about 400 mg/mL.

Isotopic variations

[0084] The polypeptides of the present invention may contain one or more atoms that are isotopes. In this application, the term "isotope" is used to refer to a chemical element that has one or more additional neutrons. In one embodiment, the polypeptides of the present invention may be deuterated. In this application, the term "deuterated" is used to refer to a substance in which one or more hydrogen atoms are replaced by isotopes of deuterium. Isotopes of deuterium are isotopes of hydrogen. A hydrogen nucleus contains one proton, while deuterium nuclei contain both a proton and a neutron. Compounds and pharmaceutical compositions of the present invention can be deuterated to change such a physical property as stability, or to enable their use for diagnostic and experimental tasks.

II. Methods of application

[0085] This application provides methods for modulating complement activity using the compounds and/or compositions of the invention.

Indications for use

[0086] An important component of the activity of the entire immune system (innate and adaptive) is the ability of the immune system to distinguish between autologous and non-autologous cells. Pathology occurs when the immune system is unable to make this distinction. In the case of the complement system, vertebrate cells express inhibitory proteins that protect them from the effects of the complement cascade, and this ensures that the complement system is directed against microbial pathogens. Many complement disorders and diseases are associated with abnormal destruction of autologous cells by the complement cascade.

[0087] The methods of the invention include methods for treating complement-related disorders using the compounds and compositions of the invention. A "complement related disorder" as used herein may include any condition relating to a functional disorder of the complement system, such as splitting or processing of a complement component, such as C5.

[0088] In some embodiments, the methods of the invention include methods for inhibiting complement activity in a subject. In some cases, the percentage of complement activity inhibited in a subject may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In some instances, this degree of inhibition and/or maximum inhibition of complement activity may be achieved over a period of time from about 1 hour post-administration to about 3 hours post-administration, from about 2 hours post-administration to about 4 hours post-administration, from about 3 hours post-administration. up to about 10 hours after administration, from about 5 hours after administration to about 20 hours after administration, or from about 12 hours after administration to about 24 hours after administration. Inhibition of complement activity can last for a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days , at least 7 days, at least 2 weeks, at least 3 weeks, or at least 4 weeks. In some cases, this degree of inhibition can be achieved by daily administration. Such daily administration may include administration over at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 2 months, at least 4 months, at least 6 months, at least 1 year or at least 5 years. In some instances, compounds or compositions of the present invention may be administered to subjects during the lifetime of such subjects.

[0089] In some embodiments, the methods of the invention include methods for inhibiting C5 activity in a subject. "C5 dependent complement activity" or "C5 activity" as used herein refers to activation of the complement cascade by C5 cleavage, assembly of subsequent C5 cleavage products, or any other process or event concomitant with or resulting from C5 cleavage. In some cases, the percentage of C5 activity inhibited in a subject may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%.

[0090] In some embodiments, the methods of the invention may include methods of inhibiting hemolysis by administering one or more compounds or compositions of the invention to a subject or patient in need thereof. According to some such methods, hemolysis can be reduced by about 25% to about 99%. In other embodiments, hemolysis is reduced by about 10% to about 40%, about 25% to about 75%, about 30% to about 60%, about 50% to about 90%, about 75% to about 95%, from about 90% to about 99%, or from about 97% to about 99.5%. In some cases, hemolysis is reduced by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0091] In accordance with some methods, the percentage of hemolysis inhibition is from about ≥ 90% to about ≥ 99% (for example, ≥ 91%, (92%, (93%, (94%, (95%, 96%, (97%, (98%). In some instances, the reported degree of inhibition and/or maximum hemolysis inhibition over a period of time from about 1 hour post-administration to about 3 hours post-administration, from about 2 hours post-administration to about 4 hours post-administration , from about 3 hours after administration to about 10 hours after administration, from about 5 hours after administration to about 20 hours after administration, or from about 12 hours after administration to about 24 hours after administration. at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days,at least 2 weeks, at least 3 weeks, at least 4 weeks. In some cases, this degree of inhibition can be achieved by daily administration. Such daily administration may include administration over at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 2 months, at least 4 months, at least 6 months, at least 1 year or at least 5 years. In some instances, compounds or compositions of the present invention may be administered to subjects during the lifetime of such subjects.

[0092] The C5 inhibitors of the invention can be used to treat one or more indications, with virtually no side effects resulting from treatment with the C5 inhibitor. In some cases, no adverse effects on the cardiovascular, respiratory and/or central nervous system (CNS) occur. In some cases, there is no change in heart rate and/or blood pressure. In some cases, no change in respiratory rate, tidal volume, and/or minute volume occurs.

[0093] The expression "reduce" or "decrease" in the context of a marker or symptom of a disease means a significant reduction in a given level, often statistically significant. The decrease may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably occur to a level considered to be within the normal range. range for an individual not suffering from the disorder.

[0094] The expressions "increase" or "increase" in the context of a marker or symptom of a disease means a significant increase in a given level, often statistically significant. The increase may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably occur to a level considered to be within the normal range. range for an individual not suffering from the disorder.

[0095] A cure or prophylactic effect is evident when there is a significant improvement, often statistically significant, in one or more parameters of the severity of the disease, or when the deterioration or development of symptoms ceases when it would otherwise be expected. For example, a favorable change in a measurable disease parameter of at least 10% and preferably at least 20%, 30%, 40%, 50% or more may indicate an effective treatment. The efficacy of a given compound or composition can also be evaluated using an experimental animal model for a given disease known in the art. When using an experimental model animal, the effectiveness of the treatment is confirmed when there is a statistically significant modulation of the marker or symptom.

Paroxysmal nocturnal hemoglobinuria

[0096] In some embodiments, provided herein are methods for treating paroxysmal nocturnal hemoglobinuria (PNH) using compounds or compositions, eg, pharmaceutical compositions, of the invention. PNH is a rare complement-related disorder caused by an acquired mutation in the glycan anchor biosynthesis class A (PIG-A) gene that is derived from a multipotent hematopoietic stem cell (Pu, J.J. et al., Clin Transl Sci. 2011 Jun; 4 (3):219-24). PNH is characterized by bone marrow dysfunction, hemolytic anemia, and thrombosis. The PIG-A gene product is required to produce the glycolipid anchor, glycosylphosphatidylinositol (GPI), used to anchor proteins to the plasma membrane. Two complement regulatory proteins responsible for protecting cells from the lytic activity of the terminal complement cascade, CD55 (hemolysis promoter) and CD59 (membrane reactive lysis inhibitor), become non-functional in the absence of GPI. This leads to activation of C5 and accumulation of specific proteins of the complement system on the surface of erythrocytes (RBCs), leading to complement-mediated destruction of these cells.

[0097] The PNH patient initially suffers from hemoglobinuria, abdominal pain, smooth muscle dystonia, and fatigue, such as symptoms or disorders associated with PNH. PNH is also characterized by intravascular hemolysis (the main clinical manifestation of this disease) and venous thrombosis. Venous thrombosis can occur in unusual locations including, but not limited to, hepatic, mesenteric, cerebral, and cutaneous veins. (Parker, C. et al., 2005. Blood. 106: 3699-709 and Parker, C. J., 2007. Exp Hematol. 35: 523-33). Currently, the only approved treatment for PNH is eculizumab (SOLIRIS®, Alexion Pharmaceuticals, Cheshire, CT), an anti-C5 monoclonal antibody.

[0098] Treatment with eculizumab results in adequate containment of intravascular hemolysis in most patients with PNH (Schrezenmeier, H. et al., 2014. Haematologica. 99: 922-9). However, Nishimura et al described 11 patients in Japan (3.2% of PNH patients) who had mutations in the C5 gene that prevented eculizumab from binding to C5 and did not respond to antibody treatment (Nishimura, J-I. et al., 2014 N Engl J Med. 370: 632-9). In addition, eculizumab is administered once every 2 weeks by intravenous (IV-) infusion under the supervision of a medical specialist, which is inconvenient and difficult for patients.

[0099] Long-term IV administration can lead to serious complications, such as infections, local thrombosis, hematomas, and gradual deterioration of venous access. In addition, eculizumab is a large molecular weight protein and is associated with a risk of immunogenicity and hypersensitivity. Finally, although eculizumab binds C5 and prevents C5b formation, any C5b formed due to incomplete inhibition can initiate MAC formation and cause hemolysis.

[00100] the Peripheral blood of patients with PNH can vary in proportion to the content of normal and abnormal cells. The disease is subdivided according to the PNH Community International Group based on clinical signs, bone marrow characteristics, and percentage of GPI-AP-deficient polymorphonuclear leukocytes (PMN). Since GPI-AP-deficient erythrocytes are more susceptible to destruction in patients with PNH, flow cytometry analysis of PMN is considered more informative (Parker, C.J., 2012. Curr Opin Hematol. 19: 141-8). Analysis by flow cytometry shows that, in classical PNH, the content of GPI-AP-deficient PMN is 50-100%.

[00101] Hemolytic anemia in PNH is independent of autoimmune antibodies (Coombs-negative) and results from uncontrolled activation of the complement system via the alternative pathway (AP).

[00102] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the present invention are particularly useful in the treatment of PNH. Such compounds and compositions may include C5 inhibitors (eg, R5000 and/or its active metabolite or variant). The C5 inhibitors of the invention useful in the treatment of PNH may, in some cases, block the cleavage of C5 into C5a and C5b. In some cases, the C5 inhibitors of the invention can be used as an alternative to PNH therapy using eculizumab. Unlike eculizumab, the C5 inhibitors of the invention can bind C5b, preventing C6 binding and subsequent assembly of the C5b-9 MAC.

[00103] In some cases, R5000 and/or its active metabolites or variants, alone or in compositions, can be used to treat PNH in subjects. These subjects may include subjects who have experienced side effects, have been unresponsive, have shown no response, or have shown resistance to other treatments (eg, using eculizumab). In some embodiments, treatment with the compounds and compositions of the present invention may inhibit hemolysis of PNH erythrocytes in a dose dependent manner.

[00104] In some embodiments, R5000 and/or its active metabolite or variant is administered in combination with eculizumab in a regimen that may involve concurrent or sequential treatment.

[00105] Based on sequence and structure data, R5000 and/or its active metabolites or variants may be particularly useful for the treatment of PNH in a limited number of patients with polymorphisms in the C5 gene that prevent eculizumab from binding to C5. An example of such patients are patients with unidirectional heterozygous missense mutation C5, c. 2654G->A, which predicts the p.Arg885His (R885H) polymorphism; this and other polymorphisms at position 885 are described in Nishimura, J. et al., N Engl J Med. 2014. 370(7):632-9, content which is incorporated herein by reference in its entirety). This mutation deprives eculizumab of the ability to bind to C5 in carriers of the mutation. However, R5000 and R5001 are able to bind C5 carrying the R885H substitution. Accordingly, in some embodiments, the methods of the present invention comprise inhibition of C5 activity and/or treatment of PNH in subjects carrying the p.Arg885His polymorphism.

[00106] Like eculizumab, R5000 and R5001 block the proteolytic cleavage of C5 into C5a and C5b. Unlike eculizumab, R5000 and R5001 can also bind to C5b and block association with C6, which prevents subsequent MAC assembly. Therefore, in a preferred embodiment, any C5b that arises from incomplete inhibition by R5000 and/or R5001 is blocked from binding C6 and completing MAC assembly.

[00107] In some cases, R5000 and/or its active metabolites or variants may be used as an alternative drug to eculizumab for patients with PNH and may offer increased efficacy without the inconvenience and burden associated with IV administration and the known risks of immunogenicity and hypersensitivity associated with monoclonal antibodies. In addition, serious complications of long-term IV administration, such as infections, loss of venous access, local thrombosis, and hematomas, can be eliminated with R5000 and/or R5001 administered by subcutaneous (SC-) injection.

[00108] In some embodiments, the implementation of the methods of the present invention include the treatment of PNH in subjects who have not previously been treated with eculizumab. These methods may include administering C5 inhibitors to a subject. C5 inhibitors may include R5000 and/or its metabolites or variants. In some aspects, C5 inhibitors are administered at least twice at regular intervals. Intervals can be from about every hour to about every 12 hours, from about every 2 hours to about every 24 hours, from about every 4 hours to about every 36 hours, from about every 8 hours to about every 48 hours, from about every 12 hours to approximately every 60 hours, approximately every 18 hours to approximately every 72 hours, approximately every 30 hours to approximately every 84 hours, approximately every 40 hours to approximately every 96 hours, approximately every 50 hours to approximately every 108 hours , from about every 60 hours to about every 120 hours, from about every 70 hours to about every 132 hours, from about every 80 hours to about every 168 hours, from about every day to about every week, from about every week to about every month or longer than every m month. Administration of a C5 inhibitor may include administration of C5 inhibitors at an initial loading dose. The initial loading dose may be from about 0.01 mg/kg to about 1 mg/kg, from about 0.05 mg/kg to about 2 mg/kg, from about 0.1 mg/kg to about 3 mg/kg, from about 0.2 mg/kg to about 4 mg/kg, from about 0.3 mg/kg to about 5 mg/kg, from about 0.6 mg/kg to about 6 mg/kg, from about 1.5 mg/kg to about 10 mg/kg, or from about 5 mg/kg to about 50 mg/kg. Administration of a C5 inhibitor may include administration of C5 inhibitors at the initial therapeutic dose. The initial therapeutic dose may include the administration of C5 inhibitors at least twice at regular intervals after the initial loading dose. The initial therapeutic dose may be from about 0.01 mg/kg to about 1 mg/kg, from about 0.05 mg/kg to about 2 mg/kg, from about 0.1 mg/kg to about 3 mg/kg, from about 0.2 mg/kg to about 4 mg/kg, from about 0.3 mg/kg to about 5 mg/kg, from about 0.6 mg/kg to about 6 mg/kg, from about 1.5 mg/kg to about 10 mg/kg, or from about 5 mg/kg to about 50 mg/kg. Initial therapeutic doses may be replaced with modified therapeutic doses after the initial therapeutic dose period. The period can be from about 1 day to about 10 days, from about 1 week to about 3 weeks, from about 2 weeks to about 4 weeks, or more than 4 weeks. The modified therapeutic dose may be from about 0.01 mg/kg to about 1 mg/kg, from about 0.05 mg/kg to about 2 mg/kg, from about 0.1 mg/kg to about 3 mg/kg, from about 0.2 mg/kg to about 4 mg/kg, from about 0.3 mg/kg to about 5 mg/kg, from about 0.6 mg/kg to about 6 mg/kg, from about 1.5 mg/kg to about 10 mg/kg, or from about 5 mg/kg to about 50 mg/kg. The modified therapeutic dose may include increasing the C5 inhibitor levels administered. The levels of lactate dehydrogenase (LDH) in a subject can be monitored during therapy. Initial therapeutic doses may be substituted with modified therapeutic doses based on changes in observed LDH levels. In some aspects, subjects are switched to a modified therapeutic dose after LDH levels are found not to exceed 1.5 times the normal upper limit. In some embodiments, hemolysis in the subject's serum is reduced. In some embodiments, no adverse effects (eg, injection reactions or systemic infections) are observed in response to treatment. Administration of the C5 inhibitor may include self-administration (eg, using an automatic medical syringe). Self-administration can be controlled, for example, by a healthcare professional. In some aspects, self-administration can be controlled remotely. The control can be carried out using an intelligent device.

[00109] Eculizumab has been reported to not completely suppress C5 activity in vitro under conditions that mimic vigorous activation and potentially leave patients susceptible to poor disease control (see Brodsky et al., 2017. Blood 129; 922-923 and Harder et al., 2017. Blood 129:970-980). This phenomenon is called residual C5 activity. Residual C5 activity may be due to the inability of eculizumab to prevent binding of C5 to the alternative pathway C5 convertase (containing two C3b subunits as well as one Bb component). In some embodiments, R5000 and/or its active metabolites or variants can be used to inhibit binding between C5 and C5 alternative pathway convertase.

[00110] Residual C5 activity may also exist when vigorous complement activation results in C5 cleavage before eculizumab can bind. Like eculizumab, R5000 and its active metabolites or variants bind to C5 and inhibit C5 cleavage and activation of the terminal complement cascade. However, R5000 and R5001 bind C5 at a site different from that of eculizumab and therefore have a different molecular mechanism of inhibition. In addition, R5000 and R5001 can bind to C5b after cleavage, preventing subsequent hemolysis. In some embodiments, R5000 and/or its active metabolites or variants can be used to improve complement inhibition under conditions in which some hemolytic activity persists during or after treatment with eculizumab. Accordingly, in some embodiments, the present invention provides methods for inhibiting residual C5 activity by contacting C5 with R5000 and/or its active metabolite. C5 may be the C5 of a subject with PNH. C5 may be the C5 of a subject with a C5 polymorphism (eg, pArg885His). In some embodiments, the methods of the present invention include treating subjects with PNH when residual C5 activity persists after previous or current eculizumab treatment, by administering R5000 and/or its active metabolites or variants.

Inflammatory diseases

[00111] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to treat subjects with diseases, disorders, and/or conditions associated with inflammation. Inflammation may increase during the development of the proteolytic cascade of the complement system. While inflammation may have beneficial effects, excessive inflammation can lead to various pathologies (Markiewski et al. 2007. Am J Pathol. 17: 715-27). Accordingly, the compounds and compositions of the present invention can be used to reduce or eliminate inflammation associated with complement activation.

Sterile inflammation

[00112] In some embodiments, the compounds and compositions, for example, pharmaceutical compositions, of the present invention can be used to treat, prevent, or slow the development of sterile inflammation. Sterile inflammation is inflammation that occurs in response to any stimulus other than infection. Sterile inflammation can be a common response to stress such as genomic stress, hypoxic stress, nutritional stress, or endoplasmic reticulum stress caused by physical, chemical, or metabolic noxious stimuli. Sterile inflammation can contribute to the pathogenesis of many diseases, such as, but not limited to, ischemia-induced injury, rheumatoid arthritis, acute lung injury, drug-induced liver injury, inflammatory bowel disease, and/or other diseases, disorders, or conditions. The mechanism of sterile inflammation and methods and compositions for treating, preventing and/or delaying the onset of symptoms of sterile inflammation may include any of those recited in Rubartelli et al., Frontiers in Immunology, 2013, 4:398-99, Rock et al., Annu Rev Immunol. 2010, 28:321-342 or US Pat. No. 8,101,586, the contents of which are incorporated herein by reference in their entirety.

Systemic inflammatory response syndrome (SIRS) and sepsis

[00113] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to treat and/or prevent systemic inflammatory response syndrome (SIRS). SIRS is an inflammation that affects the entire body. If SIRS is caused by an infection, then it is called sepsis. SIRS can also be caused by non-infectious events such as trauma, injury, burns, ischemia, hemorrhage, and/or other conditions. Other negative outcomes associated with SIRS and/or sepsis include multiple organ failure (MOF). Inhibition of the complement system at the C3 level in gram-negative sepsis largely protects organs from progressive MOF caused by Escherichia coli (E. coli), but also interferes with bacterial clearance. Compounds and compositions of the present invention include complement C5 component inhibitors that can be administered to a subject with sepsis to provide organ protective benefits without adversely altering bacterial clearance.

[00114] In some embodiments, the present invention provides methods for treating sepsis. Sepsis can be caused by a microbial infection. The microbial infection may include at least one gram-negative infectious agent. In the context of this application, the term "causative agent" refers to any object that invades or otherwise infects a cell, tissue, organ, channel or fluid of a sample or subject. In some cases, infectious agents may be bacteria, viruses, or other pathogenic organisms. Gram-negative infectious agents are gram-negative bacteria. Gram-negative infectious agents may include, but are not limited to, E. coli.

[00115] Methods for treating sepsis may include administering one or more C5 inhibitors to a subject. The C5 inhibitor may be R5000. According to some methods, complement activation can be attenuated or prevented. Attenuation or prevention of complement activity can be determined by detecting one or more complement activity products in a sample from a subject. These products may include C5 cleavage products (eg C5a and C5b) or subsequent complexes formed from C5 cleavage (eg C5b-9). In some embodiments, the present invention provides methods for treating sepsis with R5000, wherein the levels of C5a and/or C5b-9 in a subject and/or in at least one sample obtained from the subject are reduced or completely suppressed. For example, in subjects (or in samples obtained from such subjects) administered R5000, C5a and/or C5b-9 levels may decrease by about 0% to about 0.05%, from about 0.01% to about 1 %, from about 0.05% to about 2%, from about 0.1% to about 5%, from about 0.5% to about 10%, from about 1% to about 15%, from about 5% to about 25%, about 10% to about 50%, about 20% to about 60%, about 25% to about 75%, about 50% to about 100%, compared to subjects (or samples from subjects), untreated with R5000 (including subjects treated with other complement inhibitors) or compared with the same subject (or samples from the same subjects) prior to treatment or prior to treatment.

[00116] In some embodiments, the C5b-9 levels reduced by R5000 treatment are C5b-9 levels associated with one or more of the classical complement activation pathway, the alternative complement pathway, and the lectin complement pathway.

[00117] In some embodiments, the presence, absence and/or levels of one or more factors associated with sepsis can be modulated by administering R5000 to a subject with sepsis. The presence or absence of such factors can be determined using assays to determine them. Changes in factor levels can be determined by determining the level of such factors in a subject with sepsis after treatment with R5000 and comparing such levels with prior levels in the same subject (either prior to treatment with R5000 or during one or more previous treatment periods) or with levels in subjects who are not receiving treatment with R5000 (including subjects with sepsis who are not receiving treatment, or subjects who are receiving some other form of treatment). Comparisons can be represented by the percentage differences between factor levels in subjects treated with R5000 and subjects not treated with R5000.

[00118] C5 cleavage products may include any proteins or complexes that may result from C5 cleavage. In some cases, C5 cleavage products may include, but are not limited to, C5a and C5b. The C5b cleavage product can proceed to form a complex with the complement proteins C6, C7, C8 and C9 (referred to herein as "C5b-9"). Accordingly, C5 cleavage products that include C5b-9 can be detected and/or quantified to determine if complement activity has been reduced or prevented. The determination of C5b-9 deposition can be performed, for example, using the WIESLAB® ELISA kit for enzyme immunoassay (Euro Diagnostica, Malmo, Sweden). Quantification of cleavage products can be measured in "Complement Conditional Units" (CAU) as described by others (e.g. see Bergseth G et al., 2013. Mol Immunol. 56:232-9, the contents of which are incorporated herein in their entirety). application by referral).

[00119] In some embodiments, treatment of sepsis with a C5 inhibitor (eg, R5000) may reduce or prevent C5b-9 production.

[00120] In accordance with the present invention, administration of R5000 to a subject may result in modulation of bacterial clearance in the subject and/or in at least one sample obtained from the subject. The clearance of bacteria, as set forth in this application, means partial or complete removal/reduction of content from/from a subject or in a sample. Clearance may occur by killing or otherwise rendering the bacteria incapable of growth and/or reproduction. In some cases, bacterial clearance may occur by lysis and/or immune injury of the bacteria (eg, phagocytosis, bacterial cell lysis, opsonization, etc.). According to some methods, bacterial clearance in subjects treated with C5 inhibitors (eg, R5000) may have no or beneficial effect on bacterial clearance. This may be due to a lack or reduced effect on C3b levels when C5 is inhibited. In some embodiments, methods of treating sepsis with R5000 may eliminate interference with C3b-dependent opsonization or enhance C3b-dependent opsonization.

[00121] In some instances, bacterial clearance may be enhanced with R5000 treatment compared to bacterial clearance in patients prior to treatment or in patients treated with another form of complement inhibitor, such as a C3 inhibitor. In some embodiments, subjects with sepsis who are treated with R5000 may show 0% to at least 100% increased bacterial clearance compared to bacterial clearance in subjects not treated with R5000 (including subjects treated with other complement inhibitors) or compared to previous levels of bacterial clearance in the same subject before treatment with R5000 or in a previous period of treatment with R5000. For example, bacterial clearance in subjects treated with R5000 and/or in at least one sample obtained from such subjects may be increased by about 0% to about 0.05%, from about 0.01% to about 1%, from about 0.05% to about 2%, from about 0.1% to about 5%, from about 0.5% to about 10%, from about 1% to about 15%, from about 5 % to about 25%, about 10% to about 50%, about 20% to about 60%, about 25% to about 75%, about 50% to about 100%, compared to untreated subjects R5000, (including subjects treated with other complement inhibitors) and/or compared with samples obtained from such subjects, or compared with the same subjects in the period before treatment or in the previous period of treatment, and/or compared with samples , received s from the same subject in the period before treatment or in the previous period of treatment.

[00122] Bacterial clearance in a subject can be measured by directly measuring bacterial levels in a patient and/or in a patient sample, or by measuring one or more measures of bacterial clearance (eg, levels of bacterial components released after bacterial lysis). In such a case, bacterial clearance rates can be determined by comparison with previous measured bacterial levels/indicators or with bacterial levels/indicators in an untreated or otherwise treated subject. In some cases, the number of colony forming units (CFU) from the collected blood is studied to determine the levels of bacteria (for example, to obtain CFU/ml of blood).

[00123] In some embodiments, treatment of sepsis with R5000 may be performed without affecting phagocytosis or without significant impairment of phagocytosis. Phagocytosis may include neutrophilic and/or monocytic phagocytosis. Treatment with R5000 without or essentially no impairment of phagocytosis can be explained by the limited or no change in C3b levels associated with R5000 treatment.

[00124] The oxidative response is a C5a-dependent process characterized by the production of peroxide by certain cells, particularly macrophages and neutrophils, due to the threat of a pathogen (see Mollnes T. E. et al., 2002. Blood 100, 1869-1877, which incorporated in this application by reference in its entirety).

[00125] In some embodiments, the implementation of the oxidative response in subjects with sepsis can be attenuated or prevented after treatment with R5000. This may be due to a decrease in C5a levels with R5000-dependent inhibition of C5. The oxidant response in subjects administered R5000 may be attenuated by about 0% to about 0.05%, about 0.01% to about 1%, about 0.05% to about 2%, from about 0. 1% to about 5%, about 0.5% to about 10%, about 1% to about 15%, about 5% to about 25%, about 10% to about 50%, about 20% to about 60%, about 25% to about 75%, about 50% to about 100%, compared to subjects not treated with R5000 (including subjects treated with other complement inhibitors) or compared to the same subject in the period before treatment or in the previous period of treatment.

[00126] Lipopolysaccharide (LPS) is a component of bacterial cell walls that is known as an immunostimulant. Complement-dependent bacteriolysis can lead to the release of LPS, which contributes to inflammatory reactions, for example, reactions that characterize sepsis. In some embodiments, treatment of sepsis with R5000 may reduce LPS levels. This effect may be due to the weakening of complement-mediated bacteriolysis, with inhibition of C5-dependent complement activity. In some embodiments, LPS levels in subjects administered R5000 (or in samples obtained from such subjects) can be reduced or reduced by about 0% to about 0.05%, from about 0.01% to about 1% , from about 0.05% to about 2%, from about 0.1% to about 5%, from about 0.5% to about 10%, from about 1% to about 15%, from about 5% to about 25%, from about 10% to about 50%, about 20% to about 60%, about 25% to about 75%, about 50% to about 100%, compared to subjects (or samples from subjects) not treated with R5000 , (including subjects treated with other complement inhibitors) or compared with the same subject (or samples from the same subjects) prior to treatment or prior to treatment.

[00127] In some embodiments, LPS levels in subjects (or samples from subjects) with sepsis who are treated with R5000 can be reduced by 100% compared to subjects (or samples from subjects) with sepsis who are not treated. drug R5000, (including subjects receiving treatment in one or more other forms) or compared with the same subject (or sample from the subject) in the period before treatment or in the previous period of treatment.

[00128] In some embodiments of the present invention, sepsis-induced levels of one or more cytokines can be reduced by treatment with R5000. Cytokines include a number of cell signaling molecules that stimulate immune responses to infection. "Cytokine storm" is an abrupt over-activation of at least four cytokines, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor alpha (TNFα), which can occur as a result of bacterial infection and contribute to sepsis. C5a is known to induce the synthesis and activity of these cytokines. Therefore, C5 inhibitors can reduce cytokine levels by lowering C5a levels. To evaluate the ability of C5 inhibitors to reduce levels of one or more inflammatory cytokines overactivated during sepsis, cytokine levels can be assessed in subjects or in samples from subjects. IL-6, IL-8, MCP-1 and/or TNFα levels can be reduced in subjects administered R5000 by about 0% to about 0.05%, from about 0.01% to about 1%, from about 0.05% to about 2%, about 0.1% to about 5%, about 0.5% to about 10%, about 1% to about 15%, about 5% to about 25%, about 10% to about 50%, about 20% to about 60%, about 25% to about 75%, about 50% to about 100%, compared to subjects not treated with R5000 (including subjects treated with treatment with other complement inhibitors) or compared with the same subject in the period before treatment or in the previous period of treatment. In some embodiments, levels of IL-6, IL-8, MCP-1, and/or TNFα in subjects with sepsis who are treated with R5000 may be reduced by 100% compared to subjects with sepsis who are not treated with R5000. R5000, (including subjects receiving one or more other forms of treatment) or compared to the same subject in the pre-treatment or prior treatment period.

[00129] One of the complications associated with sepsis is dysregulation of coagulation and/or fibrinolysis pathways (Levi M., et al., 2013. Seminars in thrombosis and hemostasis 39, 559-66; Rittirsch D., et al., 2008 (Nature Reviews Immunology 8, 776-87; and Dempfle C., 2004. A Thromb Haemost 91(2):213-24, the contents of which are hereby incorporated by reference in their entirety). While controlled local activation of these pathways is important for pathogen defense, uncontrolled systemic activation can be dangerous. Complement activity associated with bacterial infection may stimulate dysregulation of coagulation and/or fibrinolysis due to increased host cell and tissue injury associated with MAC formation. In some embodiments, treatment of sepsis with R5000 may normalize coagulation and/or fibrinolysis pathways.

[00130] Dysregulation of coagulation and/or fibrinolysis associated with sepsis may include disseminated intravascular coagulation (DIC) syndrome. The DIC syndrome is a condition that results in tissue and organ damage due to the activation of coagulation and the formation of blood clots in small blood vessels. This activity reduces blood flow to tissues and organs and depletes the clotting factors needed for coagulation in the rest of the body. The absence of these clotting factors in the bloodstream can lead to uncontrolled bleeding in other parts of the body. In some embodiments, treatment of sepsis with R5000 may ameliorate or eliminate the DIC syndrome.

[00131] Coagulation dysfunction associated with sepsis can be detected by measuring thromboplastin formation and activity time (APTT) and/or prothrombin time (PT). Test data is performed on plasma samples to determine if levels of clotting factors are low. In subjects with DIC syndrome, APTT and/or PT values are increased due to decreased levels of clotting factors. In some embodiments, treatment of sepsis in subjects with R5000 may reduce and/or normalize APTT and/or PT in samples obtained from treated subjects.

[00132] Sepsis-associated coagulation dysfunction can be further assessed by analysis of thrombin-antithrombin (TAT) complex levels and/or expression of tissue factor (TF) mRNA by leukocytes. Elevated levels of the TAT complex and expression of TF mRNA by leukocytes are associated with coagulation dysfunction and consistent with DIC. In some embodiments, treatment of sepsis with R5000 may result in a decrease in TAT levels and/or levels of leukocyte-expressed TF mRNA by from about 0.005% to about 0.05%, from about 0.01% to about 1%, from about 0 05% to about 2%, about 0.1% to about 5%, about 0.5% to about 10%, about 1% to about 15%, about 5% to about 25%, from about 10% to about 50%, about 20% to about 60%, about 25% to about 75%, about 50% to about 100%, compared to subjects not treated with R5000 (including subjects treated with treatment with other complement inhibitors) or compared with the same subject in the period before treatment or in the previous period of treatment. In some embodiments, TAT levels and/or leukocyte-expressed TF mRNA levels in septic subjects who are treated with R5000 may be reduced by 100% compared to septic subjects who are not treated with R5000, (including subjects receiving treatment in one or more other forms) or compared with the same subject in the period before treatment or in the previous period of treatment.

[00133] Factor XII is a factor important for normal plasma coagulation. Factor XII levels may decrease in plasma samples from subjects with coagulation dysfunction (eg, DIC) due to depletion of factor XII associated with coagulation in small blood vessels. In some embodiments, treatment of sepsis with R5000 may reduce factor XII depletion. Accordingly, factor XII levels in plasma samples taken from subjects with sepsis after treatment with R5000 may be elevated. Factor XII levels in plasma samples may be elevated by about 0.005% to about 0.05%, about 0.01% to about 1%, about 0.05% to about 2%, about 0.1% to about 5%, about 0.5% to about 10%, about 1% to about 15%, about 5% to about 25%, about 10% to about 50%, about 20% to about 60% , from about 25% to about 75%, from about 50% to about 100%, compared with subjects not treated with R5000 (including subjects treated with other complement inhibitors) or compared with plasma samples taken from that the same subject in the period before treatment or in the previous period of treatment. In some embodiments, factor XII levels in plasma samples from septic subjects who are receiving R5000 treatment may be increased by 100% compared to plasma samples from septic subjects who are not receiving R5000 treatment (including subjects receiving R5000 treatment). in one or more other forms) or compared with plasma samples taken from the same subject in the period before treatment or in the previous period of treatment.

[00134] Fibrinolysis is the degradation of fibrin due to enzymatic activity, i. a process that is critical in the formation of blood clots. Dysregulation of fibrinolysis can occur in severe sepsis and has been reported to disrupt normal blood coagulation in baboons infected with E. coli bacteria (P. de Boer J.P., et al., 1993. Circulatory shock. 39, 59-67, which is fully scope incorporated into this application by reference). Plasma indicators for sepsis-related fibrinolysis dysfunction (including, but not limited to, DIC-associated fibrinolysis dysfunction) may include, but are not limited to, decreased fibrinogen levels (indicative of reduced ability to form fibrin clots), elevated levels of tissue activator plasminogen (tPA), elevated levels of plasminogen activator inhibitor type 1 (PAI-1), elevated levels of plasmin-antiplasmin (PAP), elevated levels of fibrinogen/fibrin degradation products, and elevated levels of D-dimer. In some embodiments, treatment of sepsis with R5000 may result in a decrease in plasma fibrinogen levels and/or an increase in plasma levels of tPA, PAI-1, PAP, fibrinogen/fibrin degradation products, and/or D-dimer by about 0.005% to about 0.05%, from about 0.01% to about 1%, from about 0.05% to about 2%, from about 0.1% to about 5%, from about 0.5% to about 10%, from about 1% to about 15%, about 5% to about 25%, about 10% to about 50%, about 20% to about 60%, about 25% to about 75%, about 50% to about 100%, compared with levels in plasma samples from subjects not treated with R5000 (including subjects treated with other complement inhibitors) or compared with levels in plasma samples taken from the same subject during the period before treatment, or during the previous treatment period. In some embodiments, the sepsis-associated decrease in plasma fibrinogen levels and/or the sepsis-associated increase in plasma levels of tPA, PAI-1, PAP, fibrinogen/fibrin degradation products, and/or D-dimer may differ by at least 10,000%, compared to levels in plasma samples from septic subjects treated with R5000.

[00135] Another consequence of complement hyperactivity associated with sepsis is a decrease in erythrocytes due to complement-dependent hemolysis and/or C3b-dependent opsonization. Methods for treating sepsis with R5000 of the present invention may include reducing complement-dependent hemolysis. One way to evaluate complement dependent hemolysis associated with sepsis is to obtain a complete clinical blood count. CBCs can be obtained using automated processes that count the types of cells present in blood samples. CBC results usually include hematocrit, red blood cell, white blood cell (WBC) and platelet counts. Hematocrit levels are used to determine the percentage of blood (by volume) that is made up of red blood cells. Hematocrit levels, platelet levels, erythrocyte levels, and leukocyte levels in sepsis may decrease due to hemolysis. In some embodiments, treatment of sepsis with R5000 increases hematocrit levels, platelet levels, erythrocyte levels, and/or leukocyte levels. The increase may be immediate or may occur over time as a result of treatment (eg, single dose or multiple dose treatment).

[00136] In some embodiments, treatment of a subject with R5000 may reduce the activation of leukocytes (eg, neutrophils and macrophages) associated with sepsis. The term "activation" in this application in the context of leukocytes refers to the mobilization and/or maturation of these cells to perform associated immune functions. The reduction in leukocyte activation during treatment with R5000 can be determined by evaluation of the treated subject or a sample obtained from a treated subject.

[00137] In some embodiments, treatment of sepsis with R5000 may improve one or more vital signs in treated subjects. These vital signs may include, but are not limited to, heart rate, mean systemic arterial pressure (MSAP), respiratory rate, oxygen saturation, and body temperature.

[00138] In some embodiments, treatment of sepsis with R5000 may stabilize or reduce capillary leakage and/or endothelial barrier dysfunction associated with sepsis (i.e., to maintain or correct capillary leakage and/or endothelial barrier dysfunction). Stabilization or reduction of capillary leakage and/or dysfunction of the endothelial barrier can be determined by measuring levels of total plasma proteins and/or levels of plasma albumin. An increase in both levels compared to plasma levels associated with sepsis may indicate a decrease in capillary leakage. Accordingly, treatment of sepsis with R5000 may increase levels of total plasma proteins and/or plasma albumin.

[00139] The methods of the present invention may include methods for treating sepsis with R5000, wherein the levels of one or more acute phase proteins are reduced. Acute phase proteins are proteins produced by the liver in a state of inflammation. Treatment with R5000 may reduce inflammation associated with sepsis and lead to a decrease in acute phase protein production by the liver.

[00140] In accordance with some methods of the invention, sepsis-induced organ damage and/or organ dysfunction can be reduced, reverted to, or prevented by treatment with R5000. Measures that may decrease as organ function improves may include, but are not limited to, plasma lactate (showing improved vascular perfusion and clearance), creatinine, blood urea nitrogen (each of which shows increased kidney function), and hepatic transaminases (showing increased functions). In some embodiments, the implementation of the febrile reaction, the risk of secondary infection and/or the risk of recurrence of sepsis are reduced in patients treated with R5000.

[00141] The methods of the present invention may include preventing sepsis-related death and/or increasing time to post-vaccination immunity in septic subjects by treatment with R5000. The increase in time of post-vaccination immunity can be determined by comparing the time of post-vaccination immunity in subjects treated with R5000 with the time of post-vaccination immunity in untreated subjects (including subjects treated with one or more other forms). In some embodiments, post-vaccination immunity times are increased by at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days. days, at least 7 days, at least 2 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 1 year , at least 2 years, at least 5 years, or at least 10 years.

[00142] In some embodiments, administration of R5000 is performed as a single dose. In some embodiments, R5000 is administered in multiple doses. For example, administering R5000 may include administering an initial dose followed by one or more repeat doses. Repeat doses can be administered after about 1 hour to about 24 hours, from about 2 hours to about 48 hours, from about 4 hours to about 72 hours, from about 8 hours to about 96 hours, from about 12 hours to about 36 hours or from about 18 hours to about 60 hours after the previous dose. In some cases, repeat doses may be given 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 4 weeks, 2 months, 4 months, 6 months, or more than 6 months after the previous dose. doses. In some instances, repeated doses may be administered as needed to stabilize or reduce sepsis, or to stabilize or reduce one or more of the effects associated with a subject's sepsis. Repeat doses may include the same amount of R5000 or may include a different amount.

[00143] Compounds and compositions of the invention can be used to control and/or neutralize complement activation to prevent and treat SIRS, sepsis and/or MOF. Methods of using complement inhibitors for the treatment of SIRS and sepsis may include those described in US Publication No. US 2013/0053302 or US Patent No. 8,329,169, the contents of which are hereby incorporated by reference in their entirety.

Acute Respiratory Distress Syndrome (ARDS)

[00144] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to treat and/or prevent acute respiratory distress syndrome (ARDS). ARDS is a widespread inflammation of the lungs and can be caused by trauma, infection (eg, sepsis), severe pneumonia, and/or inhalation of harmful substances. ARDS is usually a severe, life-threatening complication. Studies show that neutrophils may contribute to the development of ARDS by influencing the accumulation of polymorphic cells in the affected lung alveoli and interstitial lung tissue. Accordingly, the compounds and compositions of the present invention may be administered to reduce and/or prevent neutrophil production of tissue factor in the alveoli. The compounds and compositions of the present invention may further be used to treat, prevent and/or delay the onset of ARDS, in some cases in accordance with any of the methods set forth in International Publication No. WO 2009/014633, the contents of which are hereby incorporated by reference in their entirety.

Periodontitis

[00145] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, and/or methods of the invention can be used to treat or prevent the development of periodontitis and/or associated conditions. Periodontitis is a widespread chronic inflammation resulting in the destruction of periodontal tissue, which is the tissue that supports and surrounds teeth. The condition also includes loss of alveolar bone (the bone that holds the teeth). Periodontitis can be caused by poor oral hygiene leading to a buildup of bacteria at the gum line, also known as plaque. Certain health conditions, such as diabetes or malnutrition, and/or habits such as smoking, may increase the risk of periodontitis. Periodontitis can increase the risk of stroke, myocardial infarction, atherosclerosis, diabetes, osteoporosis, premature birth, and other health problems. Studies show a correlation between periodontitis and local complement activity. Periodontitis bacteria can either inhibit or activate some components of the complement cascade. Accordingly, the compounds and compositions of the present invention can be used to prevent and/or treat periodontitis and associated diseases and conditions. Complement activation inhibitors and treatments may include any of those described in Hajishengallis and Lambris, Biochem Pharmacol. 2010, 15; 80(12): 1, or US Publication No. US 2013/0344082, the contents of each of which are hereby incorporated by reference in their entirety.

Dermatomyositis

[00146] In some embodiments, the implementation of the compounds and compositions, for example, pharmaceutical compositions, and/or methods according to the invention can be used to treat dermatomyositis. Dermatomyositis is an inflammatory myopathy characterized by muscle weakness and chronic inflammation of muscle tissue. Dermatomyositis often begins with a skin rash that is associated with or precedes muscle weakness. The compounds, compositions and/or methods of the invention can be used to relieve or prevent dermatomyositis.

Wounds and injuries

[00147] Compounds and compositions, for example, pharmaceutical compositions, according to the invention can be used to treat and/or accelerate the healing of wounds and/or lesions of various types. As used herein, the term "damage" generally refers to physical injury, but may include localized infectious or disease processes. Injury can be characterized as harm, injury or destruction caused by external events affecting parts and/or organs of the body. Wounds are associated with cuts, blows, burns, and/or other exposures to the skin that leave the skin torn or damaged. Wounds and injuries are often early, but if not properly healed, they can lead to chronic complications and/or inflammation.

Wounds and burn wounds

[00148] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to treat and/or promote wound healing. Healthy skin provides a waterproof, protective barrier against pathogens and other environmental influences. The skin also regulates body temperature and fluid evaporation. When the skin is affected, these functions are impaired, making it difficult for the skin to heal. Injury initiates a number of physiological processes associated with the immune system, which heals and regenerates tissues. Complement activation is one of these processes. Complement activation studies have identified several complement components involved in wound healing, as described in van de Goot and et al., J Burn Care Res 2009, 30:274-280 and Cazander et al., Clin Dev Immunol, 2012, 2012: 534291, the contents of which are incorporated herein by reference in their entirety. In some cases, complement activation can be excessive, causing cell death and increased inflammation (resulting in impaired wound healing and chronic wounds). In some cases, the compounds and compositions of the present invention can be used to reduce or eliminate such complement activation to promote wound healing. Treatment with the compounds and compositions of the invention may be carried out in accordance with any of the wound treatment methods disclosed in International Publication No. WO 2012/174055, the contents of which are hereby incorporated by reference in their entirety.

Head injury

[00149] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to treat and/or promote healing of a head injury. Head injuries include injuries to the scalp, skull, or brain. Examples of head injuries include, but are not limited to, concussions, bruises, skull fractures, traumatic brain injuries, and/or other injuries. Head injuries can be mild or severe. In some cases, head trauma can lead to long-term physical and/or mental complications or death. Studies show that head trauma can induce abnormal intracranial complement cascade activation, which can lead to local inflammatory responses that promote secondary brain injury through brain edema and/or neuronal death (Stahel et al., Brain Research Reviews, 1998, 27 : 243-56, the contents of which are incorporated herein by reference in their entirety). The compounds and compositions of the invention can be used to treat head trauma and/or to reduce or prevent associated secondary complications. Methods for using the compounds and compositions of the invention to control complement cascade activation in head trauma may include any of those described in US Pat. No. 8,911,733 to Holers et al., the contents of which are hereby incorporated by reference in their entirety.

Tissue crush injury

[00150] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to treat and/or promote the healing of crush injuries. Crush injuries are injuries caused by force or pressure acting on the body and causing bleeding, bruising, fractures, nerve damage, wounds, and/or other damage to the body. Compounds and compositions of the invention can be used to reduce complement activation following crush injury to thereby promote healing from crush injury (e.g., by promoting nerve regeneration, promoting fracture healing, preventing or treating inflammation, and/or other related complications with them). The compounds and compositions of the invention can be used to promote healing by any of the methods described in US Pat. No. 8,703,136; International Publication Nos. WO 2012/162215, WO 2012/174055 or US Publication No. US 2006/0270590, the contents of which are incorporated herein by reference in their entirety.

Ischemia/reperfusion injury

[00151] In some embodiments, the compounds, compositions, for example, pharmaceutical compositions, and/or methods of the present invention can be used to treat injuries associated with ischemia and/or reperfusion. These injuries may be associated with surgery (eg, transplantation). Accordingly, the compounds, compositions and/or methods of the present invention can be used to reduce or prevent ischemia and/or reperfusion injury.

Autoimmune diseases

[00152] Compounds and compositions, for example, pharmaceutical compositions, according to the invention can be used to treat subjects with autoimmune diseases and/or disorders. The immune system can be divided into innate and adaptive systems, referring respectively to non-specific immediate defense mechanisms and more complex antigen-specific systems. The complement system is part of the innate immune system that recognizes and eliminates pathogens. In addition, proteins of the complement system can modulate adaptive immunity, with a combination of innate and adaptive responses. Autoimmune diseases and disorders are immunological abnormalities that cause the system to attack its own tissues and substances. Autoimmune diseases can affect certain tissues or organs in the body. Compounds and compositions of the invention can be used to modulate complement in the treatment and/or prevention of autoimmune diseases. In some cases, such compounds and compositions can be used in accordance with the methods presented in the publication of Ballanti et al. Immunol Res (2013) 56:477-491, the contents of which are hereby incorporated by reference in their entirety.

Antiphospholipid syndrome (APS) and catastrophic antiphospholipid syndrome (CAPS)

[00153] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention may be used to prevent and/or treat antiphospholipid syndrome (APS) by controlling complement activation. APS is an autoimmune condition caused by antiphospholipid antibodies that cause blood to clot. APS can lead to recurrence of venous or arterial thrombosis in organs and complications in the placental circulation causing pregnancy complications such as miscarriage, stillbirth, preeclampsia, preterm birth and/or other complications. Catastrophic antiphospholipid syndrome (CAPS) is a severe and acute variant of a similar condition, leading to blockage of veins in several organs at the same time. Research suggests that complement activation may contribute to complications associated with APS, including pregnancy complications, thrombotic complications (formation of blood clots), and vascular complications. The compound and compositions of the invention can be used to treat conditions associated with APS by reducing or eliminating complement activation. In some cases, the compounds and compositions of the invention can be used to treat APS and/or complications associated with APS, in accordance with the methods described in Salmon et al., Ann Rheum Dis 2002;61(Suppl II):ii46-ii50 and Mackworth-Young, Clin Exp Immunol 2004, 136:393-401, the contents of which are hereby incorporated by reference in their entirety.

Cold agglutinin disease

[00154] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to treat cold agglutinin disease (CAD), also referred to as cold agglutinin-mediated hemolysis. CAD is an autoimmune disease resulting from a high concentration of IgM antibodies interacting with red blood cells at low body temperatures (Engelhardt et al. Blood, 2002, 100(5):1922-23). CAD can lead to conditions such as anemia, fatigue, dyspnea, hemoglobinuria, and/or acrocyanosis. CAD is associated with strong complement activation, and studies have shown that CAD can be treated with complement inhibitor drugs. Accordingly, the present invention provides methods for the treatment of CAD using the compounds and compositions of the invention. In some cases, the compounds and compositions of the invention can be used to treat CAD according to the methods described in Roth et al., Blood, 2009, 113:3885-86 or in International Publication No. volume is incorporated into this application by reference.

Myasthenia gravis

[00155] In some embodiments, the compounds, compositions, eg, pharmaceutical compositions, and/or methods of the invention can be used to treat myasthenia gravis. Myasthenia gravis (MG) is a rare complement-mediated autoimmune disease characterized by the production of antibodies that target proteins that are critical for normal transmission of electrical signals from nerves to muscles. Although the prognosis of MG is generally favorable, control of the disease in 10% to 15% of patients is either impossible to achieve with existing therapies or results in severe side effects of immunosuppressive therapy. This severe form of MG is known as treatment-resistant MG (rMG) and occurs in approximately 9000 individuals in the US.

[00156] Patients suffer from muscle weakness, which usually worsens with repeated use, and recovers with rest. Muscle weakness may be localized to specific muscles, such as those responsible for eye movements, but often develops into more diffuse muscle weakness. rMG can even become life-threatening when muscle weakness extends to the diaphragm and other chest wall muscles responsible for breathing. This case is the most threatening complication of rMG, known as myasthenic crisis, and requires hospitalization, intubation and mechanical ventilation. Approximately 15% to 20% of patients experience a myasthenic crisis within two years of diagnosis.

[00157] The most common target of antibodies in MG is the acetylcholine receptor, or AchR, located at the neuromuscular junction, the point at which a motor neuron transmits signals to skeletal muscle fibers. Binding of anti-AchR antibodies to the neuromuscular end plate causes activation of the classical complement cascade and MAC deposition on postsynaptic muscle fibers, resulting in local damage to the muscle membrane and impaired muscle response to neuronal stimulation.

[00158] Inhibition of the activity of the terminal complement cascade can be used to block complement-mediated damage resulting from MG and/or rMG. In some embodiments, the compounds and/or compositions of the present invention can be used to treat MG and/or rMG. These methods can be used to inhibit C5 activity to alleviate or prevent neuromuscular problems associated with MG and/or rMG.

Acute idiopathic polyneuritis

[00159] In some embodiments, the compounds, compositions, eg, pharmaceutical compositions, and methods of the invention can be used to treat acute idiopathic polyneuritis (GBS). GBS is an autoimmune disease involving an autoimmune attack on the peripheral nervous system. Compounds, compositions and/or methods of the invention can be used to alleviate or prevent problems of the peripheral nervous system associated with GBS.

Vascular diseases

[00160] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to treat vascular diseases affecting blood vessels (eg, arteries, veins, and capillaries). Such diseases can affect circulation, blood pressure, blood flow, organ function, and/or other bodily functions.

Thrombotic microangiopathy (TMA)

[00161] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to treat and/or prevent thrombotic microangiopathy (TMA) and associated diseases. Microangiopathies affect the small blood vessels (capillaries) of the body and cause capillary walls to become thick, weak, and prone to bleeding and slowing blood circulation. As a rule, TMA leads to the development of vascular thrombosis, damage to endothelial cells, thrombocytopenia and hemolysis. Organs such as the brain, kidneys, muscles, gastrointestinal tract, skin and lungs may be affected. TMAs can result from medical operations and/or conditions that include, but are not limited to, hematopoietic stem cell transplantation (HSCT), kidney disease, diabetes, and/or other conditions. TMA may be caused by underlying dysfunction of the complement system as described in Meri et all., European Journal of Internal Medicine, 2013, 24: 496-502, the contents of which are hereby incorporated by reference in their entirety. In general, TMA may result from elevated levels of certain complement components, leading to thrombosis. In some cases, this may be caused by mutations in the proteins of the complement system or related enzymes. The resulting complement dysfunction can lead to complement being targeted by endothelial cells and platelets, resulting in increased thrombosis. In some embodiments, TMA can be prevented and/or treated with the compounds and compositions of the invention. In some instances, the methods for treating TMA with the compounds and compositions of the invention can be performed as described in US Publication Nos. US 2012/0225056 or US 2013/0246083, the contents of which are incorporated herein by reference in their entirety.

Disseminated intravascular coagulation (DIC) syndrome

[00162] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent and/or treat disseminated intravascular coagulation (DIC) syndrome by controlling complement activation. The DIC syndrome is a pathological condition in which the coagulation cascade in the blood is highly activated and leads to the formation of blood clots, especially in the capillaries. The DIC syndrome can lead to obstruction of blood flow in tissues and, ultimately, can lead to organ damage. In addition, DIC syndrome interferes with the normal blood clotting process, which can lead to heavy bleeding. Compounds and compositions of the present invention can be used to treat, prevent or reduce the severity of the DIC syndrome by modulating complement activity. In some instances, the compounds and compositions of the invention may be used in accordance with any of the treatments for DIC described in US Pat. No. 8,652,477, the contents of which are hereby incorporated by reference in their entirety.

Vasculitis

[00163] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent and/or treat vasculitis. In general, vasculitis is a disorder associated with inflammation of blood vessels, including veins and arteries, characterized by leukocytes attacking tissue and causing blood vessels to swell. Vasculitis may be associated with an infection such as Rocky Mountain spotted fever or an autoimmune disease. An example of vasculitis associated with autoimmune processes is vasculitis with antineutrophil cytoplasmic autoantibodies (ANCA). ANCA vasculitis is caused by abnormal antibodies attacking the body's own cells and tissues. ANCA antibodies attack the cytoplasm of certain leukocytes and neutrophils, causing them to attack the walls of blood vessels in certain organs and tissues of the body. ANCA vasculitis may involve the skin, lungs, eyes, and/or kidneys. Studies show that in ANCA disease, the alternative complement pathway is activated and several complement components are generated that create a cycle of increased inflammation, leading to vascular damage (Jennette et al. 2013, Semin Nephrol. 33(6): 557-64, which contains incorporated in this application by reference in its entirety). In some cases, the compounds and compositions of the invention can be used to prevent and/or treat ANCA vasculitis by inhibiting complement activation.

Atypical hemolytic uremic syndrome

[00164] In some embodiments, the compounds, compositions, eg, pharmaceutical compositions, and/or methods of the present invention can be used to treat atypical hemolytic uremic syndrome (aHUS). aHUS is a rare disease caused by uncontrolled complement activation, characterized by the formation of blood clots in small blood vessels. The compositions and methods of the invention can be used to reduce or prevent complement activation associated with aHUS.

Neurological diseases

[00165] Compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent, treat, and/or alleviate symptoms of neurological diseases, including, but not limited to, neurodegenerative diseases and related disorders. Neurodegeneration generally refers to the loss of neuronal structure or function, including neuronal death. These disorders can be treated by inhibiting the action of complement on neuronal cells using the compounds and compositions of the present invention. Neurodegeneration-associated disorders include, but are not limited to, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson's disease, and Alzheimer's disease.

Amyotrophic lateral sclerosis (ALS)

[00166] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent, treat, and/or alleviate the symptoms of ALS. ALS is a fatal motor neuron disease characterized by degeneration of neurons in the spinal cord, brainstem, and motor cortex. ALS results in a loss of muscle strength, eventually leading to respiratory failure. Complement dysfunction can contribute to ALS and therefore ALS can be prevented, treated and/or symptomatically alleviated by therapy with compounds and compositions of the invention that target complement activity. In some instances, the compounds and compositions of the invention may be used to promote nerve regeneration. In some instances, the compounds and compositions of the invention may be used as complement inhibitors in accordance with any of the methods described in US Publication Nos. US 2014/0234275 or US 2010/0143344, the contents of which are incorporated herein by reference in their entirety.

Alzheimer's disease

[00167] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent and/or treat Alzheimer's disease by controlling complement activity. Alzheimer's disease is a chronic neurodegenerative disease, with symptoms that can include confusion, memory loss, mood swings, behavioral problems, and eventually loss of bodily functions. Alzheimer's disease is thought to be caused by extracellular deposits of amyloid in the brain associated with inflammatory proteins such as complement proteins (Sjoberg et al. 2009. Trends in Immunology. 30(2): 83-90, which is incorporated in this application by reference in its entirety). In some instances, the compounds and compositions of the present invention may be used as complement inhibitors in accordance with any of the treatments for Alzheimer's disease described in US Publication No. US 2014/0234275, the contents of which are hereby incorporated by reference in their entirety.

kidney disease

[00168] Compounds and compositions, for example, pharmaceutical compositions, according to the invention can be used to treat certain diseases, disorders and/or conditions associated with the kidneys, in some cases through inhibition of complement activity. The kidneys are the organs responsible for removing metabolic end products from the bloodstream. The kidneys regulate blood pressure, the urinary system, and homeostatic functions and are therefore vital to various bodily functions. The kidneys can be more susceptible to inflammation (compared to other organs) due to their unique structural features and exposure to blood. The kidneys also produce their own complement system proteins, which can be activated during infection, kidney disease, and kidney transplants. In some cases, the compounds and compositions of the invention can be used as complement inhibitors in the treatment of certain renal diseases, conditions and/or disorders in accordance with the methods described in Quigg, J Immunol 2003; 171:3319-24, the contents of which are incorporated herein by reference in their entirety.

Lupus nephritis

[00169] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent and/or treat lupus nephritis via complement inhibition. Lupus nephritis is an inflammation of the kidneys caused by an autoimmune disease called systemic lupus erythematosus (SLE). Symptoms of lupus nephritis include high blood pressure; foamy urine; swelling of the legs, feet, hands, or face; pain in the joints; muscle pain; fever and rash. Lupus nephritis can be treated with inhibitors that control the activity of the complement system, including the compounds and compositions of the present invention. Methods and compositions for preventing and/or treating lupus nephritis by complement inhibition may include any of the methods described in US Publication No. US 2013/0345257 or US Patent No. 8,377,437, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the compounds and/or compositions of the present invention can be used to prevent and/or treat lupus nephritis by binding to C5 and prevent progression of kidney disease to lupus nephritis. Binding to C5 may prevent and/or treat lupus nephritis by preventing C5 activity and blocking complement-mediated damage to kidney cells.

Membrane glomerulonephritis (MGN)

[00170] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent and/or treat membranous glomerulonephritis (MGN) by inhibiting the activation of certain complement components. MGN is a kidney dysfunction that can lead to inflammation and structural changes. MGN is caused by antibodies that bind to soluble antigen in the glomerular capillaries (glomeruli). MGN can affect kidney functions such as filtering fluids and can lead to kidney failure. The compounds and compositions of the invention may be used in accordance with the methods for preventing and/or treating MGN by complement inhibition as described in US Publication No. US 2010/0015139 or International Patent Publication No. WO 2000/021559, the contents of which are incorporated herein in their entirety. by sending.

Complications of hemodialysis

[00171] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent and/or treat complications associated with hemodialysis by inhibiting complement activation. Hemodialysis is a medical procedure used to support kidney function in subjects with kidney failure. In hemodialysis, the removal of waste products such as creatinine, urea and osmotically free water from the blood is performed extracorporeally. A common complication of the hemodialysis procedure is chronic inflammation caused by contact between the blood and the dialysis membrane. Another common complication is thrombosis, which is the formation of blood clots that impede circulation. Studies have shown that these complications are related to complement activation. Hemodialysis can be performed in combination with complement inhibitor therapy to provide a means of controlling inflammatory responses and pathologies, and/or to prevent or treat thrombosis in patients undergoing hemodialysis due to renal insufficiency. Methods for using the compounds and compositions of the invention to treat complications of hemodialysis can be carried out in accordance with any of the methods described in DeAngelis et al., Immunobiology, 2012, 217(11): 1097-1105 or in Kourtzelis et al., Blood, 2010 , 116(4):631-639, the contents of which are incorporated herein by reference in their entirety.

eye diseases

[00172] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent and/or treat certain ocular diseases, disorders and/or conditions. In a healthy eye, the complement system is activated at a low level and is constantly regulated by membrane-bound and soluble intraocular proteins that protect against pathogens. Therefore, complement activation plays an important role in several ocular complications, and control of complement activation can be used to treat such diseases. Compounds and compositions of the invention can be used as complement inhibitors in the treatment of ophthalmic disorders according to any of the methods described in Jha et al., Mol Immunol. 2007; 44 (16): 3901-3908, or US Pat. No. 8,753,625, the contents of which are hereby incorporated by reference in their entirety.

Age-Related Macular Degeneration (AMD)

[00173] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent and/or treat age-related macular degeneration (AMD) by inhibiting complement activation in the eye. AMD is a chronic eye disease that causes blurred central vision, blind spots in central vision, and/or permanent loss of central vision. Central vision affects the ability to read, drive and/or recognize faces. AMD is generally classified into two types, namely non-exudative (dry) and exudative (wet). Dry AMD refers to the deterioration of the macula, which is the tissue in the center of the retina. Wet AMD refers to damage to the blood vessels under the retina, leading to leakage of blood and fluid. Several human and animal studies have identified complement system proteins that are associated with AMD, and new therapeutic strategies have included control of complement activation pathways as described in Jha et al., Mol Immunol. 2007; 44(16): 3901-8. The methods of the invention, including the use of the compounds and compositions of the invention for the prevention and/or treatment of AMD, may include any of the methods described in US Publication No. US 2011/0269807 or US 2008/0269318, the contents of which are in full incorporated into this application by reference.

Corneal diseases

[00174] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention can be used to prevent and/or treat corneal diseases by inhibiting complement activation in the eye. The complement system plays an important role in protecting the cornea from pathogenic particles and/or inflammatory antigens. The cornea is the outer anterior part of the eye, covering and protecting the iris, pupil, and anterior chamber, and is therefore exposed to external factors. Corneal diseases include, but are not limited to, keratoconus, keratitis, ocular herpes, and/or other diseases. Corneal diseases can cause pain, blurred vision, tearing, redness, sensitivity to light, and/or scarring of the cornea. The complement system is critical for protecting the cornea, but complement activation can cause damage to corneal tissue after the infection has cleared, as some compounds of the complement system are overexpressed. Methods of the present invention for modulating complement activity in the treatment of corneal diseases may include any of the methods described in Jha et al., Mol Immunol. 2007; 44 (16): 3901-8, the contents of which are incorporated herein by reference in their entirety.

Autoimmune uveitis

[00175] In some embodiments, the compounds and compositions, eg, pharmaceutical compositions, of the invention may be used to prevent and/or treat uveitis, which is inflammation of the choroid layer of the eye. The choroid is the pigmented region of the eye that contains the choroid, iris, and ciliary body of the eye. Uveitis causes redness, blurred vision, pain, adhesions, and can eventually lead to blindness. Studies have shown that complement activation products are present in the eyes of patients with autoimmune uveitis, and complement plays an important role in the development of the disease. In some instances, the compounds and compositions of the invention may be used to treat and/or prevent uveitis according to any of the methods described in Jha et al., Mol Immunol. 2007. 44 (16): 3901-8, the contents of which are incorporated herein by reference in their entirety.

diabetic retinopathy

[00176] In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the invention can be used to prevent and/or treat diabetic retinopathy, which is a disease caused by changes in the retinal blood vessels in diabetic patients. Retinopathy can cause blood vessels to swell and leak fluid and/or abnormal blood vessels to grow. Diabetic retinopathy affects vision and can eventually lead to blindness. Studies have shown that complement activation plays an important role in the development of diabetic retinopathy. In some instances, the compounds and compositions of the invention may be used in accordance with the methods for treating diabetic retinopathy described in Jha et al. Mol Immunol. 2007; 44(16): 3901-8, the contents of which are incorporated herein by reference in their entirety.

Neuromyelitis optica (NMO)

[00177] In some embodiments, the compounds, compositions, eg, pharmaceutical compositions, and/or methods of the invention can be used to treat neuromyelitis optica (NMO). NMO is an autoimmune disease that leads to the destruction of the optic nerve. The compounds and/or methods of the invention can be used to prevent nerve damage in subjects with NMO.

Sjögren's syndrome

[00178] In some embodiments, the compounds, compositions, eg, pharmaceutical compositions, and/or methods of the invention can be used to treat Sjögren's syndrome. Sjögren's syndrome is an eye disorder characterized by xerosis ophthalmosus that can cause burning and/or itching. This disease is an autoimmune disorder in which the immune system targets the glands in the eyes and mouth that are responsible for moisturizing these areas. The compounds, compositions and/or methods of the invention may be used to treat and/or relieve symptoms of Sjögren's syndrome.

Preeclampsia and the HELLP Syndrome

[00179] In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the invention can be used to prevent and/or treat pre-eclampsia and/or HELLP syndrome (abbreviation for symptoms of the syndrome: 1) hemolysis, 2) elevated liver enzymes, and 3 ) low platelet count) with complement inhibitor therapy. Pre-eclampsia is a pregnancy disorder with symptoms including high blood pressure, edema, shortness of breath, impaired kidney function, impaired liver function, and/or low blood platelets. Preeclampsia is usually diagnosed by high levels of protein in the urine and high blood pressure. The HELLP syndrome is a combination of conditions of hemolysis, elevated liver enzymes, and low platelets. Hemolysis is a disease involving the destruction of red blood cells, leading to the release of hemoglobin from red blood cells. Elevated liver enzymes may indicate pregnancy-induced liver disease. Low platelet levels lead to a decrease in the ability to clot the blood, which raises the risk of excessive bleeding. HELLP is associated with preeclampsia and liver disease. HELLP syndrome usually occurs late in pregnancy or after childbirth. It is usually diagnosed by a blood test indicating the presence of the three components that make up it. Typically, HELLP syndrome is treated by induction of labor.

[00180] Research suggests that complement activation occurs during HELLP syndrome and preeclampsia, and that during HELLP syndrome and preeclampsia some complement components are present at elevated concentrations. Complement inhibitors can be used as drugs to prevent and/or treat these conditions. The compounds and compositions of the invention may be used in accordance with the methods for preventing and/or treating HELLP syndrome and preeclampsia as described in Heager et al., Obstetrics & Gynecology, 1992, 79(1):19-26 or International Patent Publication No. WO 201/078622, the contents of which are incorporated herein by reference in their entirety.

Preparations

[00181] In some embodiments, the compounds or compositions, eg, pharmaceutical compositions, of the invention are formulated in aqueous solutions. In some cases, aqueous solutions further include one or more salts and/or one or more buffer substances. Salts may include sodium chloride, which may be present in concentrations of from about 0.05 mM to about 50 mM, from about 1 mM to about 100 mM, from about 20 mM to about 200 mM, or from about 50 mM to about 500 mM. . Additional solutions may contain at least 500 mm sodium chloride. In some cases, aqueous solutions include sodium phosphate. Sodium phosphate may be included in aqueous solutions at a concentration of from about 0.005 mM to about 5 mM, from about 0.01 mM to about 10 mM, from about 0.1 mM to about 50 mM, from about 1 mM to about 100 mM, from about 5 mM to about 150 mM, or from about 10 mM to about 250 mM. In some cases, at least 250 mM sodium phosphate concentrations are used.

[00182] Compositions of the invention may include C5 inhibitors at a concentration of from about 0.001 mg/mL to about 0.2 mg/mL, from about 0.01 mg/mL to about 2 mg/mL, from about 0.1 mg /ml to about 10 mg/ml, from about 0.5 mg/ml to about 5 mg/ml, from about 1 mg/ml to about 20 mg/ml, from about 15 mg/ml to about 40 mg/ml, from about 25 mg/ml to about 75 mg/ml, from about 50 mg/ml to about 200 mg/ml, or from about 100 mg/ml to about 400 mg/ml. In some cases, compositions of the invention include C5 inhibitors at a concentration of at least 400 mg/ml.

[00183] The compositions of the invention may contain C5 inhibitors at a concentration having approximately, approximately or exactly any of the following values: 0.001 mg/ml, 0.2 mg/ml, 0.01 mg/ml, 2 mg/ml, 0, 1 mg/ml, 10 mg/ml, 0.5 mg/ml, 5 mg/ml, 1 mg/ml, 20 mg/ml, 15 mg/ml, 40 mg/ml, 25 mg/ml, 75 mg/ml ml, 50 mg/ml, 200 mg/ml, 100 mg/ml or 400 mg/ml. In some cases, compositions of the invention include C5 inhibitors at a concentration of at least 40 mg/ml.

[00184] In some embodiments, the compositions of the invention include aqueous compositions containing at least water and a C5 inhibitor (eg, a cyclic C5 inhibitor polypeptide). Aqueous compositions of the C5 inhibitor according to the invention may further include one or more salts and/or one or more buffer substances. In some instances, the aqueous compositions of the invention include water, a cyclic C5 inhibitor polypeptide, a salt, and a buffering agent.

[00185] Aqueous C5 inhibitor formulations of the invention may have pH levels from about 2.0 to about 3.0, from about 2.5 to about 3.5, from about 3.0 to about 4.0, from about 3, 5 to about 4.5, about 4.0 to about 5.0, about 4.5 to about 5.5, about 5.0 to about 6.0, about 5.5 to about 6.5 , from about 6.0 to about 7.0, from about 6.5 to about 7.5, from about 7.0 to about 8.0, from about 7.5 to about 8.5, from about 8.0 to about 9.0, from about 8.5 to about 9.5, or from about 9.0 to about 10.0.

[00186] In some cases, the compounds and compositions of the invention are prepared in accordance with good manufacturing practice (GMP) and/or current GMP (cGMP). Regulations applicable for the implementation of GMP and/or cGMP can be obtained from at least one of the organizations, the US Food and Drug Administration (FDA), the World Health Organization (WHO), and the International Council for Harmonization (ICH).

Dosage and Administration

[00187] For human treatment, C5 inhibitors (eg, R5000/or its active metabolites or variants) can be formulated into pharmaceutical compositions. Depending on the subject being treated, the route of administration, and the type of treatment required (eg, prevention, prophylaxis, or therapy), C5 inhibitors are formulated using methods consistent with the parameters mentioned. A summary of such methods is provided in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, (2005); and in Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York, which are hereby incorporated by reference.

[00188] The C5 inhibitors of the present invention (eg, R5000 and/or its active metabolites or variants) can be delivered in a therapeutically effective amount. In some instances, a therapeutically effective amount of a C5 inhibitor of the invention can be achieved by administering a dose of about 0.1 mg to about 1 mg, about 0.5 mg to about 5 mg, about 1 mg to about 20 mg, about 5 mg up to about 50 mg, from about 10 mg to about 100 mg, from about 20 mg to about 200 mg, or at least 200 mg of one or more C5 inhibitors.

[00189] In some embodiments, a therapeutic amount of a C5 inhibitor (eg, R5000 and/or its active metabolites or variants) may be administered to subjects based on the weight of such subjects. In some cases, C5 inhibitors are administered at a dose of from about 0.001 mg/kg to about 1.0 mg/kg, from about 0.01 mg/kg to about 2.0 mg/kg, from about 0.05 mg/kg to about 5.0 mg/kg, about 0.03 mg/kg to about 3.0 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 2.0 mg/kg, from about 0.2 mg/kg to about 3.0 mg/kg, from about 0.4 mg/kg to about 4.0 mg/kg, from about 1.0 mg/kg to about 5.0 mg/kg, from about 2.0 mg/kg to about 4.0 mg/kg, from about 1.5 mg/kg to about 7.5 mg/kg, from about 5.0 mg/kg up to about 15 mg/kg, from about 7.5 mg/kg to about 12.5 mg/kg, from about 10 mg/kg to about 20 mg/kg, from about 15 mg/kg to about 30 mg/kg, about 20 mg/kg to about 40 mg/kg, about 30 mg/kg to about flax 60 mg/kg, from about 40 mg/kg to about 80 mg/kg, from about 50 mg/kg to about 100 mg/kg, or at least 100 mg/kg. The given ranges may include ranges suitable for administration to humans. Dosage levels may be highly dependent on the nature of the disease; the effectiveness of the drug; the patient's condition; practitioner's decisions; and frequencies and regimens of administration. In some embodiments, R5000 and/or its active metabolites or variants may be administered at a dose of about 0.01 mg/kg to about 10 mg/kg. In some instances, R5000 and/or its active metabolites or variants may be administered at a dose of about 0.1 mg/kg to about 3 mg/kg.

[00190] In some instances, the C5 inhibitors of the invention (e.g., R5000 and/or active metabolites or variants thereof) are delivered at concentrations adjusted to achieve the desired C5 inhibitor level in the sample, biological system, or subject (e.g., the subject's plasma level) . In some instances, the desired concentrations of C5 inhibitors in a sample, biological system, or subject may include concentrations of about 0.001 µM to about 0.01 µM, about 0.005 µM to about 0.05 µM, about 0.02 µM to about 0.2 µM, from about 0.03 µM to about 0.3 µM, from about 0.05 µM to about 0.5 µM, from about 0.01 µM to about 2.0 µM, from about 0.1 µM to about 50 µM, from about 0.1 µM to about 10 µM, from about 0.1 µM to about 5 µM, from about 0.2 µM to about 20 µM, from about 5 µM to about 100 µM, or from about 15 µM to about 200 µM. In some cases, the required plasma concentrations of C5 inhibitors in a subject's plasma may be from about 0.1 µg/ml to about 1000 µg/ml. The desired plasma concentration of C5 inhibitors in a subject's plasma may be from about 0.01 µg/mL to about 2 µg/mL, from about 0.02 µg/mL to about 4 µg/mL, from about 0.05 µg/mL to about 5 µg/ml, about 0.1 µg/ml to about 1.0 µg/ml, about 0.2 µg/ml to about 2.0 µg/ml, about 0.5 µg/ml to about 5 µg /ml, from about 1 µg/ml to about 5 µg/ml, from about 2 µg/ml to about 10 µg/ml, from about 3 µg/ml to about 9 µg/ml, from about 5 µg/ml to about 20 µg/mL, from about 10 µg/mL to about 40 µg/mL, from about 30 µg/mL to about 60 µg/mL, from about 40 µg/mL to about 80 µg/mL, from about 50 µg/mL up to about 100 µg/ml, from about 75 µg/ml to about 150 µg/ml, or at least 150 µg/ml. In other embodiments, the C5 inhibitors are administered at a dose sufficient to produce a maximum serum concentration (C _max ) of at least 0.1 µg/mL, at least 0.5 µg/mL, at least 1 µg /ml, at least 5 µg/ml, at least 10 µg/ml, at least 50 µg/ml, at least 100 µg/ml or at least 1000 µg/ml.

[00191] In some embodiments, sufficient doses are delivered to maintain C5 inhibitor levels from about 0.1 μg/ml to about 30 μg/ml to reduce hemolysis in a subject by about 25% to about 99%.

[00192] In some embodiments, C5 inhibitors (e.g., R5000 and/or its active metabolites or variants) are administered every day at a dose sufficient to deliver from about 0.1 mg/day to about 60 mg/day per kg of subject weight . In some cases, C _max achieved with each dose is from about 0.1 µg/ml to about 1000 µg/ml. In such cases, the area under the curve (AUC) between doses can be from about 200 mg×hr/mL to about 10,000 mg×hr/mL.

[00193] According to some methods of the invention, C5 inhibitors of the invention (eg, R5000 and/or active metabolites or variants thereof) are delivered at concentrations necessary to achieve the desired effect. In some cases, the compounds and compositions of the invention are delivered in an amount necessary to halve the intensity of a given reaction or process. The concentration required to achieve this reduction in intensity is referred to herein as the half-maximal inhibitory concentration or "IC ₅₀ ". Alternatively, the compounds and compositions of the invention may be delivered in an amount sufficient to double the intensity of a given reaction, activity or process. The concentration required for such an increase is referred to herein as the half maximum effective concentration or "EC ₅₀ ".

[00194] The C5 inhibitors of the invention (eg, R5000 and/or its active metabolites or variants) may be provided in amounts totaling 0.1-95% by weight of the total weight of the composition. In some cases, C5 inhibitors are delivered by intravenous (IV-) administration. In some cases, C5 inhibitors are delivered by subcutaneous (SC-) administration.

[00195] SC administration of the C5 inhibitors of the invention (eg, R5000 and/or its active metabolites or variants) may, in some cases, provide advantages over IV administration. SC administration may allow self-treatment of patients. Such treatment may be beneficial in that patients can provide their own care at home, eliminating the need to visit a caregiver or health facility. In addition, SC administration may allow patients to avoid long-term complications associated with IV administration, such as infections, loss of venous access, local thrombosis, and hematomas. In some embodiments, SC administration may provide patients with greater convenience, increased satisfaction and quality of life, reduced treatment costs and/or drug requirements.

[00196] In some cases, daily SC administration provides stable C5 inhibitor concentrations that are achieved over 1-3 doses, 2-3 doses, 3-5 doses, or 5-10 doses. In some cases, daily SC doses of 0.1 mg/kg can provide stable C5 inhibitor levels of at least 2.5 µg/ml and/or inhibition of complement activity above 90%.

[00197] C5 inhibitors of the invention (e.g., R5000 and/or its active metabolites or variants) may have slow absorption kinetics (time to peak observed concentration greater than 4-8 hours) and high bioavailability (from about 75% to about 100% ) after SC injection.

[00198] In some embodiments, the dosage and/or administration is altered to modulate the half-life (t _1/2 ) of C5 inhibitor levels in the subject or in the subject's bodily fluids (eg, plasma). In some cases, t _1/2 is at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours at least 72 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or at least 16 weeks.

[00199] In some embodiments, C5 inhibitors of the invention (eg, R5000 and/or active metabolites or variants thereof) may have a long terminal t _1/2 . The prolonged final t _1/2 may be due to extensive target binding and/or additional plasma protein binding. In some cases, the C5 inhibitors of the invention have t _1/2 values greater than 24 hours in both plasma and whole blood. In some cases, C5 inhibitors do not lose functional activity after incubation in human whole blood at 37°C for 16 hours.

[00200] In some embodiments, the dosage and/or administration is changed to modulate a stable volume of distribution of C5 inhibitors. In some cases, the stable volume of distribution of C5 inhibitors is from about 0.1 ml/kg to about 1 ml/kg, from about 0.5 ml/kg to about 5 ml/kg, from about 1 ml/kg to about 10 ml /kg, from about 5 ml/kg to about 20 ml/kg, from about 15 ml/kg to about 30 ml/kg, from about 10 ml/kg to about 200 ml/kg, from about 20 ml/kg to about 60 ml/kg, from about 30 ml/kg to about 70 ml/kg, from about 50 ml/kg to about 200 ml/kg, from about 100 ml/kg to about 500 ml/kg, or at least 500 ml/kg. In some cases, the dosage and/or administration of C5 inhibitors is adjusted to provide a stable volume of distribution equal to at least 50% of the total blood volume. In some embodiments, distribution of the C5 inhibitor may be restricted to the plasma compartment.

[00201] In some embodiments, the C5 inhibitors of the invention (e.g., R5000 and/or its active metabolites or variants) have a total clearance of about 0.001 ml/hour/kg to about 0.01 ml/hour/kg, from about 0.005 ml /hour/kg to about 0.05 ml/hour/kg, from about 0.01 ml/hour/kg to about 0.1 ml/hour/kg, from about 0.05 ml/hour/kg to about 0, 5 ml/hour/kg, from about 0.1 ml/hour/kg to about 1 ml/hour/kg, from about 0.5 ml/hour/kg to about 5 ml/hour/kg, from about 0.04 ml/hour/kg to about 4 ml/hour/kg, from about 1 ml/hour/kg to about 10 ml/hour/kg, from about 5 ml/hour/kg to about 20 ml/hour/kg, from about 15 ml/hour/kg to about 30 ml/hour/kg or at least 30 ml/hour/kg.

[00202] The time periods during which the maximum concentration of C5 inhibitors is maintained in subjects (eg, in serum from the subject) (T _max values) can be adjusted by changing the dosage and/or administration (eg, subcutaneous administration). In some cases, C5 inhibitors have T _max values from about 1 minute to about 10 minutes, from about 5 minutes to about 20 minutes, from about 15 minutes to about 45 minutes, from about 30 minutes to about 60 minutes, from about 45 minutes up to about 90 minutes, from about 1 hour to about 48 hours, from about 2 hours to about 10 hours, from about 5 hours to about 20 hours, from about 10 hours to about 60 hours, from about 1 day to about 4 days, from about 2 days to about 10 days, or at least 10 days.

[00203] In some embodiments, C5 inhibitors of the invention (eg, R5000 and/or active metabolites or variants thereof) can be administered without side effects. In some cases, the C5 inhibitors of the invention do not inhibit the hERG gene (human heart specific potassium channel gene), even at concentrations as low as 300 μM. SC injection of the C5 inhibitors of the invention, at dose levels up to 10 mg/kg, can be well tolerated and not result in any cardiovascular (e.g., increased risk of prolonged ventricular repolarization) and/or respiratory side effects.

[00204] Doses of C5 inhibitors can be set using the maximum no side effect level (NOAEL) observed in other species. Such species may include, but are not limited to, monkeys, rats, rabbits, and mice. In some cases, human equivalent doses (HED) can be determined by allometric scaling of NOAEL doses observed in other species. In some instances, doses of HED result in therapeutic ranges from about 2-fold to about 5-fold, from about 4-fold to about 12-fold, from about 5-fold to about 15-fold, from about 10-fold up to about 30-fold, or at least 30-fold. In some cases, therapeutic range values are determined using exposure in primates and estimated _Cmax levels in humans.

[00205] In some embodiments, the C5 inhibitors of the invention provide for a short elimination period in cases of infection where prolonged inhibition of the complement system is unfavorable.

[00206] Administration of a C5 inhibitor according to the invention may be modified to reduce potential clinical risks to subjects. Meningococcal infection is a known risk with C5 inhibitors, including eculizumab. In some cases, the risk of meningococcal infection is minimized by the establishment of one or more prophylactic steps. Such steps may include exclusion of subjects that may already have been invaded by the bacteria in question. In some cases, prophylactic steps may include co-administration with one or more antibiotics. In some cases, co-administration with ciprofloxation is possible. In some cases, ciprofloxacion can be taken orally at a dose of about 100 mg to about 1000 mg (eg, 500 mg) at the same time.

[00207] In some embodiments, administration of the C5 inhibitor can be done using an automatic medical syringe. Such devices may provide for self-administration (eg, daily administration).

[00208] In some embodiments, R5000 and/or its active metabolites or variants can be co-administered with eculizumab. Co-administration can be performed to reduce residual C5 activity associated with eculizumab treatment alone (eg, due to incomplete inhibition).

Multiplicity of application

[00209] In some embodiments, the C5 inhibitors of the invention (e.g., R5000 and/or its active metabolites or variants) are administered every hour, every 2 hours, every 4 hours, every 6 hours, every 12 hours, every 18 hours, every 24 hours, every 36 hours, every 72 hours, every 84 hours, every 96 hours, every 5 days, every 7 days, every 10 days, every 14 days, every week, every two weeks, every 3 weeks, every 4 weeks , every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every year, or at least every year. In some instances, C5 inhibitors are administered once daily or administered as two, three or more sub-doses at appropriate intervals throughout the day.

[00210] In some embodiments, C5 inhibitors are administered in multiple daily doses. In some cases, C5 inhibitors are administered daily for 7 days. In some cases, C5 inhibitors are administered daily for 7-100 days. In some cases, C5 inhibitors are administered daily for at least 100 days. In some cases, C5 inhibitors are administered daily for an indefinite period.

[00211] Intravenously delivered C5 inhibitors may be infused over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. Administration can be repeated, for example, intermittently, for example, hourly, daily, weekly, twice a week (ie, every two weeks), for one month, two months, three months, four months, or longer than four months. After the initial treatment regimen, the drug can be administered less frequently. For example, after three months of administration every two weeks, the introduction can be repeated once a month for six months or at least one year. Administration of a C5 inhibitor may decrease, decrease, increase, or alter binding or any physiologically detrimental process (e.g., in a cell, tissue, blood, urine, or other patient compartment) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least at least 80% or at least 90% or more.

[00212] Before administering a full dose of a C5 inhibitor and/or a C5 inhibitor composition, patients may be given a reduced dose, such as 5% of the full dose, and monitor for adverse effects, such as an allergic or infusion reaction, or elevated lipid levels, or blood pressure. In another example, the patient can be monitored for undesirable immunostimulatory effects, such as increased levels of cytokines (eg, TNF-alpha, IL-1, IL-6, or IL-10).

[00213] Genetic predisposition plays an important role in the development of certain diseases or disorders. Therefore, a patient in need of a C5 inhibitor can be identified by examining the family history or, for example, testing for one or more markers or variants. A healthcare provider, such as a physician, nurse, or family member, may review a family history prior to prescribing or administering a drug composition of the present invention.

Analyzes for C5

[00214] In some embodiments, the present invention provides compounds and methods for detecting C5 and/or C5 associated factors in samples. In some ways, the total content of C5 is detected. The total content of C5 may include both free C5 and C5 associated with one or more C5-associated factors. C5-associated factors may include antibodies (eg, eculizumab or a similar antibody that binds to a similar epitope). According to such embodiments, probes can be added to a substrate, wherein the substrate includes a capture agent that binds to C5. The capture agent can bind to C5 on a region of C5 that is not aligned with a portion of the C5 protein recognized by C5-associated factors. Both free C5 and C5 associated with one or more C5-associated factors can be captured by capture agents, which facilitates detection using one or more detecting agents. In the context of the present application, a "detecting agent" is any factor that facilitates the recognition of an analyte, interaction, reaction, process, or event. In some embodiments, the detection agents are detection antibodies. Detecting agents may bind to the analyte to aid in the recognition of the presence or absence of the analyte. Detection agents used to detect capture of free C5 may bind to C5 in an area that is not aligned with the region bound by the capture agent or C5-associated factor. Detecting agents used to detect C5 uptake associated with one or more C5-associated factors may bind to the C5-associated factor. Detecting agents may include a detectable label.

[00215] In the context of this application, the term "detectable label" refers to one or more markers, signals or fragments that are connected, combined or associated with another object, whose markers, signals or fragments are easily detected by methods known in this field, including , but without limitation, radiography, fluorescence, chemiluminescence, enzymatic activity, absorption, and the like. Detectable labels may include, but are not limited to, radioisotopes, luminophores, chromophores, enzymes, dyes, metal ions, ligands (eg, vitamin H, avidin, streptavidin, and haptens, eg, digoxigenin), quantum dots, and the like. Detectable labels can be located in any position of the object with which they are connected, combined or associated. For example, when connected, combined or linked to a peptide or protein, the detectable labels may be within the amino acids, peptides or proteins, or at the N- or C-terminus.

[00216] In some embodiments, detection agents can be detected using a secondary detection agent that binds to the detection agent or a detectable label associated with the detection agent. The secondary detecting agent may include an antibody. A secondary detecting agent may be associated with a detectable label to aid detection (eg, by fluorescence, radiography, colorimetry, enzyme analysis, or other methods). In one embodiment, the detection agent includes digoxigenin. Digoxigenin can be detected using an anti-digoxigenin antibody.

[00217] Test substrates may include surfaces capable of retaining capture agents. Capture agents can be immobilized to substrates, for example, by covalent or non-covalent interactions. In some embodiments, the capture agents are biotinylated to promote immobilization to substrates, wherein the substrates contain a biotin-reactive compound (eg, avidin, neutravidin, or strepavidin). Substrates may include, but are not limited to, assay plates. In some embodiments, the implementation of the substrates are microspheres.

[00218] C5 capture agents may include any compound capable of binding to C5. In some embodiments, capture agents include peptides or peptide compounds. Capture agents may include R5000 or variants thereof. Some assays use the R5000 variant, the variant including an N-terminal biotinylated PEG moiety to promote immobilization to the substrate. Some variants of R5000 may further include replacing the C-terminal lysine with another compound. The compound may be another amino acid, for example, any naturally occurring or synthesized amino acid. Some R5000 variants have C-terminal lysine substitutions with norvaline.

[00219] In some embodiments, the present invention provides a method for measuring C5 or C5 levels in a sample by immobilizing a capture agent on a substrate, wherein the capture agent binds to a site on C5 that is different from the eculizumab binding site; bringing the substrate into contact with the sample, wherein the immobilized capture agent binds to C5 in the sample; bringing the substrate into contact with a detecting agent (eg, antibody), and measuring the presence or level of the detecting agent as an indication of C5 levels in the sample. The detecting agent may include a detectable label. The detection agent may be detected using a secondary detection agent. The detecting agent may be an anti-C5 antibody (eg, an anti-C5 eculizumab or non-eculizumab antibody). The method may include bringing the substrate into contact with a second detection agent (eg, an antibody), wherein the second detection agent binds to a C5-associated factor (eg, eculizumab). Measurement of the level of the second detection antibody associated with the substrate, you can perform as an indicator of the levels of C5-associated factor in the sample. In some embodiments, both free C5 levels and eculizumab-bound C5 levels are measured using the assays disclosed herein. Levels may be combined levels representing total C5 levels in samples containing both forms. In some embodiments, free C5 levels and eculizumab-bound C5 levels are different.

[00220] In some embodiments, the present invention provides methods for measuring free eculizumab levels in a sample by immobilizing a capture agent on a substrate, wherein the capture agent binds to a site on C5 that is different from the eculizumab binding site; bringing the substrate into contact with excess C5 to form a C5-capture agent complex, wherein the C5-capture agent complex contains immobilized C5; bringing the substrate into contact with the sample, wherein the immobilized C5 binds to eculizumab in the sample; bringing the substrate into contact with a detecting agent, wherein the detecting agent binds to eculizumab; and measuring the level of bound detecting agent as an indication of the levels of free eculizumab in the sample. Capture agents may include R5000 or variants thereof. Some assays use the R5000 variant, which variant includes an N-terminal biotinylated PEG moiety to promote immobilization to the substrate. The R5000 variant may include replacing the C-terminal lysine with another compound. The compound may be another amino acid, for example, any naturally occurring or synthesized amino acid. The R5000 variant may have a C-terminal substitution with norvaline. The detecting agent may be an antibody. The detecting agent may include a detectable label or be detected using a secondary detecting agent.

III. Sets

[00221] Any of the C5 inhibitors as described herein (eg, R5000 and/or its active metabolites or variants) can be supplied as part of a kit. In a non-limiting example, C5 inhibitors may be contained in a kit for treating a disease. The kit may include a vial containing a sterile dry powder of the C5 inhibitor, a sterile solution for dissolving the dry powder, and a syringe for an infusion set for administering the C5 inhibitor.

[00222] When C5 inhibitors are supplied in dry powder form, kits of the invention are expected to supply 10 micrograms to 1000 micrograms of C5 inhibitor, or at least or at most the amounts recited.

[00223] Exemplary kits may include at least one vial, vial, flask, bottle, syringe, and/or other container or device in which C5 inhibitor preparations are placed, preferably with a suitable purpose. The kits may also include one or more auxiliary containers with a sterile, pharmaceutically acceptable buffer and/or other diluent.

[00224] In some embodiments, the compounds or compositions of the invention are supplied in borosilicate glass vials. Such vials may include a cap (eg, a rubber cap). In some cases, caps include FLUROTEC® coated rubber stoppers. The caps can be secured in place with a crimp cap, including but not limited to an aluminum tear-off crimp cap.

[00225] The kits may further include instructions for using the components of the kit, as well as using any other reagent not included in the kit. The instructions may include options that may be implemented.

IV. Definitions

[00226] Bioavailability: As used herein, the term "bioavailability" refers to the systemic availability of a given amount of a compound (eg, C5 inhibitor) administered to a subject. Bioavailability can be assessed by measuring the area under the curve (AUC) or maximum serum or plasma concentration (C _max ) of the unchanged form of a compound after administration of the compound to a subject. AUC is a measure of the area under the curve plotting the concentration of a compound in serum or plasma plotted along the ordinate (Y-axis) versus time plotted along the abscissa (X-axis). In general, the AUC for a particular compound can be calculated using methods known to those of ordinary skill in the art and/or from the description in GS Banker, Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, New York, Inc., 1996, the contents of which are hereby incorporated by reference in their entirety.

[00227] Biological system: As used herein, the term "biological system" refers to a cell, group of cells, tissue, organ, group of organs, organelle, body fluid, biological signaling pathway (e.g., receptor-activated signaling pathway, charge-activated signaling pathway, metabolic pathway, cell signaling pathway, etc.), a group of proteins, a group of nucleic acids, or a group of molecules (including, but not limited to, biomolecules) that perform at least one biological function or biological task within cell membranes, cell compartments , cells, cell cultures, tissues, organs, organ systems, organisms, multicellular organisms, biological fluids, or any biological entity. In some embodiments, the biological systems are cell signaling pathways containing intracellular and/or extracellular signaling biomolecules. In some embodiments, biological systems include proteolytic cascades (eg, complement cascade).

[00228] Buffer: As used herein, the term "buffer" refers to a compound used in a solution to resist changes in pH. Such compounds may include, but are not limited to, acetic acid, adipic acid, sodium acetate, benzoic acid, citric acid, sodium benzoate, maleic acid, sodium phosphate, tartaric acid, lactic acid, potassium metaphosphate, glycine, sodium bicarbonate, phosphate potassium, sodium citrate and sodium tartrate.

[00229] Clearance rate: As used herein, the term "clearance rate" refers to the rate at which a particular compound is cleared from a biological system or fluid.

[00230] Connection: In the context of this application, the term "compound" refers to a single chemical structural unit. In some embodiments, a particular compound may exist in one or more isomeric or isotopic forms (including, without limitation, stereoisomers, geometric isomers, and isotopes). In some embodiments, the connection is provided or used in only one such form. In some embodiments, the compound is provided or used as a mixture of two or more of these forms (including, but not limited to, a racemic mixture of stereoisomers). Those skilled in the art will appreciate that some compounds exist in different forms, exhibit different properties and/or activities (including, but not limited to, biological activities). In such cases, those of ordinary skill in the art should select or exclude specific forms of the compound for use in accordance with the present invention. For example, compounds that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms.

[00231] Cyclic or cyclized: In the context of this application, the term "cyclic" refers to the presence of a continuous loop. Cyclic molecules do not have to be circular, but only connected to form an unbroken chain of subunits. Cyclic polypeptides may include a "cyclic loop" formed when two amino acids are connected by a bridge fragment. The cyclic loop contains amino acids along the polypeptide present between the bridged amino acids. Cyclic loops may contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[00232] Subsequent event: In the context of this application, the term "subsequent" or "subsequent event" refers to any event occurring after and/or as a result of another event. In some cases, subsequent events are events occurring after and as a result of C5 cleavage and/or complement activation. Such events may include, but are not limited to, C5 cleavage product formation, MAC activation, hemolysis, and hemolysis-associated disease (eg, PNH).

[00233] Equilibrium dissociation constant: As used herein, the term "equilibrium dissociation constant" or "KD" refers to a value representing the tendency of at least two agents (eg, two proteins) to reversibly separate. In some cases, KD indicates the concentration of the primary agent at which half of the total levels of the secondary agent bind to the primary agent.

[00234] Half-life: As used herein, the term "half-cycle" or "t _1/2 " refers to the time it takes for a given process or compound concentration to reach half of the final value. The term "final elimination half-life" or "final t _1/2 " refers to the time required for the plasma concentration of a factor to decrease by half after the factor concentration has reached pseudo-equilibrium.

[00235] Hemolysis: In the context of this application, the term "hemolysis" refers to the destruction of red blood cells.

[00236] Identity: In the context of this application, the term "identity", when referring to polypeptides or nucleic acids, refers to the relative relative position of the sequences. The term is used to describe the degree of sequence similarity between polymer sequences and may include the percentage of consistent monomer components with gap alignments (if any) inserted by a particular mathematical model or computer program (i.e., "algorithms"). The identity of related polypeptides can be easily calculated by known methods. Such methods include, but are not limited to, those previously described by others (Lesk, A.M., ed., Computational Molecular Biology, Oxford University Press, New York, 1988; Smith, D.W., ed., Biocomputing: Informatics and Genome Projects , Academic Press, New York, 1993; Griffin, A. M. et al., ed., Computer Analysis of Sequence Data, Part 1, Humana Press, New Jersey, 1994; von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press , 1987; Gribskov, M. et al., ed., Sequence Analysis Primer, M. Stockton Press, New York, 1991; and Carillo et al., Applied Math, SIAM J, 1988, 48, 1073).

[00237] Inhibitor: As used herein, the term "inhibitor" refers to any agent that blocks or reduces the occurrence of a particular event; cell signal; chemical way; enzymatic reaction; cellular process; interactions between at least objects; biological event; diseases; disorders; or states.

[00238] Initial loading dose: In the context of this application, "initial loading dose" refers to the first dose of a drug, which may differ from one or more subsequent doses. Initial loading doses can be used to achieve an initial drug concentration or activity level before subsequent doses are administered.

[00239] Intravenous: As used herein, the term "intravenous" refers to the area within a blood vessel. Intravenous administration generally refers to delivery of a compound into a vessel by injection into a blood vessel (eg, vein).

[00240] In vitro: In the context of this application, the term "in vitro" refers to events that occur in an artificial environment (for example, in a test tube or reaction vessel, in a cell culture, in a Petri dish, etc.), and not in organism (for example, animal, plant or microbe).

[00241] In vivo: As used herein, the term "in vivo" refers to events that occur within an organism (eg, animal, plant, or microbe, or cell or tissue thereof).

[00242] Lactam bridge: As used herein, the term "lactam bridge" refers to an amide bond that forms a bridge between chemical groups in a molecule. In some cases, lactam bridges form between amino acids in a peptide.

[00243] Cross-linking agent: The term "cross-linking agent" in the context of this application refers to a group of atoms (for example, 10-1000 atoms), molecule(s), or other compounds used to connect two or more objects. Crosslinkers can connect such objects by covalent or non-covalent (eg, ionic or hydrophobic) interactions. Crosslinkers may include chains of two or more polyethylene glycol (PEG) units. In some cases, cross-linking agents may be cleavable.

[00244] Minute volume: In the context of this application, the term "minute volume" refers to the volume of air inhaled or exhaled into/from the lungs(their) of a subject per minute.

[00245] Non-proteinogenic: In the context of this application, the term "non-proteinogenic" refers to any non-natural proteins, for example, proteins with non-natural components such as non-natural amino acids.

[00246] Patient: In the context of this application, a "patient" refers to a subject who may request or need treatment, require treatment, receive treatment, receive treatment, or a subject who is under the care of a trained professional in connection with a particular disease or condition. .

[00247] Pharmaceutical composition: In the context of this application, the term "pharmaceutical composition" refers to a composition containing at least one active ingredient (for example, a C5 inhibitor) in a form and amount that allows the active ingredient to be therapeutically effective.

[00248] Pharmaceutically acceptable: The expression "pharmaceutically acceptable" is used in this application to refer to those compounds, materials, compositions and/or dosage forms that are, within the scope of an informed medical decision, suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit/risk ratio.

[00249] Pharmaceutically acceptable excipients: The expression "pharmaceutically acceptable excipient" in the context of this application refers to any ingredient, other than active substances (for example, the active substance R5000 and / or its active metabolites or variants thereof), present in the pharmaceutical composition and having properties that are essentially non-toxic and non-inflammatory in the patient. In some embodiments, the pharmaceutically acceptable excipient is a carrier capable of suspending or dissolving the active ingredient. Excipients may include, for example: release agents, antioxidants, binders, coatings, compression aids, leavening agents, colorants (pigments), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, flavoring agents, fragrances, lubricants agents (anti-caking agents), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, phosphate (secondary acid), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose , lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinol palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol , starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C and xylitol.

[00250] Plasma compartment: As used herein, the term "plasma compartment" refers to the intravascular space occupied by blood plasma.

[00251] Salt: In the context of this application, the term "salt" refers to a compound composed of a cation with an associated anion. Such compounds may include sodium chloride (NaCl) or other classes of salts including, but not limited to, acetates, chlorides, carbonates, cyanides, nitrites, nitrates, sulfates, and phosphates.

[00252] Sample: In the context of this application, the term "sample" refers to an aliquot or portion taken from a source and/or provided for analysis or processing. In some embodiments, the sample is from a biological source such as tissue, cells, or a constituent (e.g., bodily fluid, including, but not limited to, blood, mucus, lymph fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal discharge and semen). In some embodiments, the sample may be or contain a homogenate, lysate, or extract prepared from the whole organism or a subset of its tissues, cells, or constituents, or fractions or fractions thereof, including, but not limited to, plasma, serum, cerebrospinal fluid, lymphatic fluid, external skin, respiratory, gastrointestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors or organs. In some embodiments, the sample is or contains a medium such as a nutrient broth or gel, which may contain cellular components such as proteins. In some embodiments, the "primary" sample is an aliquot of the source. In some embodiments, the primary sample is passed through one or more processing steps (eg, separation, purification, etc.) to prepare the sample for analysis or other use.

[00253] Subcutaneous: In the context of this application, the term "subcutaneous" refers to the space under the skin. Subcutaneous administration is the delivery of a compound under the skin.

[00254] Subject: As used herein, the term "subject" refers to any organism into which a compound of the invention may be administered, for example, for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (eg, mammals such as mice, rats, rabbits, pigs, non-human primates, and humans).

[00255] Substantially: In the context of this application, the term "substantially" refers to the quantitative state of demonstrating the full or nearly full magnitude or degree of a characteristic or property of interest. One of ordinary skill in the field of biology will appreciate that biological and chemical phenomena rarely, if ever, come to an end and/or come to an end, or reach or avoid an absolute result. Therefore, the term "essentially" is used in this application to characterize the potential lack of completeness inherent in many biological and chemical phenomena.

[00256] A therapeutically effective amount: As used herein, a "therapeutically effective amount" means an amount of an agent to be delivered (e.g., a C5 inhibitor) that is sufficient when administered to a subject suffering from or susceptible to a disease/s, disorder/s, and/or a condition /s to treat, improve symptoms, diagnose, prevent and/or delay the onset of a disease, disorder and/or condition.

[00257] Tidal volume: In the context of this application, the term "tidal volume" refers to the normal lung volume of air expelled between two breaths (in the absence of any external effort).

[00258] T _max : In the context of this application, the term "T _max " refers to the period of time during which the maximum concentration of the compound in the subject or in the liquid is maintained.

[00259] Treatment: In the context of this application, the term "treatment" refers to partial or complete mitigation, reduction, improvement, alleviation, delay onset, inhibition of development, reduction in severity and / or reduction in the frequency of one or more symptoms of signs of a particular disease, disorder and /or states. Treatment may be provided to a subject who is not showing signs of a disease, disorder, and/or condition and/or to a subject who is only showing early signs of a disease, disorder, and/or condition to reduce the risk of developing the pathology associated with the disease, disorder, and/or condition. .

[00260] Therapeutic dose: As used herein, "therapeutic dose" refers to one or more doses of a drug administered in the course of solving a problem or mitigating a therapeutic indication. Therapeutic doses can be adjusted to maintain the desired concentration or activity level of the drug in the bodily fluid or biological system.

[00261] Volume of distribution: As used herein, the term "volume of distribution" or " _Vdist " refers to the volume of fluid required to hold the total volume of a compound in the body at the same concentration as blood or plasma. The volume of distribution may reflect the degree of presence of the compound in the extravascular tissue. The large volume of distribution reflects the tendency of the compound to bind to tissue constituents as compared to plasma protein constituents. In a clinical setting, V _dist can be used to determine the loading dose of a compound to ensure a stable concentration of that compound.

V. Equivalents and Volume

[00262] Various embodiments of the invention have been specifically shown and described above, however, it will be apparent to those skilled in the art that various changes in form and detail may be made to the embodiments given, without departing from the spirit and scope of the invention as defined by the appended claims.

[00263] Those skilled in the art will discover, or be able to ascertain using just standard experimental testing, many equivalents to specific embodiments of the invention as described herein. The scope of the present invention is not to be limited by the above description, but is limited by the appended claims.

[00264] In the claims, the signs of the singular in the form of indefinite and definite articles can mean one or more, unless otherwise indicated, or otherwise follows from the context. Claims or descriptions that include "or" between one or more elements of a group are deemed to be satisfied if one, more or all of the elements of the group are present in, used in, or otherwise associated with a given product or process, unless otherwise stated. , or otherwise is not clear from the context. The invention includes embodiments in which one member of the group is exactly present in, used in, or otherwise associated with a given product or process. The invention includes embodiments in which two or more or all of the elements of the group are present in, used in, or otherwise associated with a given product or process.

[00265] It should also be noted that the expression "comprising" is open-ended and allows, but does not require, the inclusion of additional elements or steps. When the expression "comprising" is used in this application, the expressions "consisting of" and "or including" are also covered and disclosed.

[00266] When ranges are given, endpoints are included. In addition, it should be understood that, unless otherwise indicated, or otherwise implied by the context and understanding of one of ordinary skill in the art, values expressed as ranges may take on any particular value or sub-range within the ranges given in different embodiments of the invention, with an accuracy of one tenth unit of the lower limit of the range, unless the context clearly follows otherwise.

[00267] In addition, it should be understood that any particular embodiment of the present invention that relates to the prior art can be expressly excluded from any one or more claims. Since such embodiments are intended to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not expressly stated in this application. Any particular embodiment of a composition of the invention (e.g., any nucleic acid or protein encoded by it; any method of manufacture; any method of use; etc.) may be excluded from any one or more claims, for any reason, regardless of the relationship with known state of the art.

[00268] All listed sources, such as references, publications, databases, database elements and technologies, mentioned in this application, are included in this application by reference, even if not expressly listed. In the event of conflicting statements between the cited sources and this application, the statement in this application shall prevail.

[00269] Section and table headings are not intended to limit the invention.

EXAMPLES

Example 1. Preparation of an aqueous solution of R5000

[00270] Polypeptides were synthesized using standard Fmoc/tBu solid phase methods. Synthesis was performed on a Liberty automated microwave peptide synthesizer (CEM, Matthews NC) using standard Rink amide resin protocols, although other automated synthesizers without the microwave option can also be used. All amino acids are from commercial sources. The coupling reagent used was 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3,-tetramethylaminium hexafluorophosphate (HCTU) and the base was diisopropylethylamine (DIEA). The polypeptides were cleaved from the resin using 95% TFA, 2.5% TIS and 2.5% water for 3 hours and isolated by ether precipitation. The crude polypeptides were purified by reverse phase preparative HPLC (high performance liquid chromatography) using a C18 column in acetonitrile/water, 0.1% TFA with a 20%-50% gradient over 30 minutes. Fractions containing pure polypeptides were collected and lyophilized, and all polypeptides were analyzed by LC-MS (liquid chromatography - mass spectroscopy).

[00271] The R5000 preparation (SEQ ID NO: 1) was prepared as a cyclic peptide containing 15 amino acids (4 of which are non-natural amino acids), an acetylated N-terminus, and a C-terminal carboxylic acid. The C-terminal lysine of the core peptide has a modified side chain forming an N-ε-(PEG24-γ-glutamic acid-N-α-hexadecanoyl)lysine residue. This modified side chain includes a polyethylene glycol (PEG24) spacer attached to an L-γ-glutamic acid residue that is derivatized with a palmitoyl group. The cyclization of R5000 is carried out by a lactam bridge between the side chains of L-Lys1 and L-Asp6. All amino acids in R5000 are L-amino acids. R5000 has a molecular weight of 3562.23 g/mol and the chemical formula C172H278N24O55.

[00272] Like eculizumab, R5000 blocks the proteolytic cleavage of C5 into C5a and C5b. Unlike eculizumab, R5000 can also bind to C5b and block C6 binding, which prevents subsequent MAC assembly.

[00273] R5000 was formulated as an aqueous solution for injection containing 40 mg/ml R5000 formulated with 50 mM sodium phosphate and 76 mM sodium chloride at pH 7.0. The resulting composition was used to formulate a medicament, in accordance with current Good Manufacturing Practice (cGMP), wherein the medicament comprises a 1 ml syringe with a 1/2-inch (12.7 mm) non-removable 29 G (0 .33×12.7 mm) as part of a device for self-administration.

Example 2 Introduction and storage of R5000

[00274] R5000 is administered by subcutaneous (SC) or intravenous (IV) injection, and the dose administered (dose volume) is adjusted according to the subject's weight on a mg/kg basis. This problem is solved using a set of fixed doses, consistent with a set of weight groups. In sum, human dosing covers a wide range of weight changes from 43 to 109 kg. Subjects who have a higher body weight (>109 kg) are treated on a case by case basis in consultation with a medical advisor.

[00275] R5000 is stored at 2°C to 8°C [36°F to 46°F]. After release to subjects, R5000 is stored at controlled room temperature (20°C to 25°C [68°F to 77°F]) for up to 30 days and protected from sources of excessive temperature fluctuations such as extreme heat or exposure to light. It is advisable to avoid storing R5000 at temperatures outside the room temperature range. R5000 can be stored for up to 30 days under the conditions indicated.

Example 3 Stability Test

[00276] The stability test was performed in accordance with Rule Q1A "Stability of New Drug Substances and Products" of the International Council for Harmonization. Samples from an aqueous solution according to example 1 was kept at 3 temperatures: -20°C, 5°C and 25°C. Testing intervals are once every 1, 2 and 3 months, and then once every 3 months up to 24 months. Samples were tested for appearance (eg, clarity, color, presence of sediment), pH, osmolality, concentration, purity, target activity (eg, erythrocyte lysis assay), microparticle levels, endotoxin levels, and sterility. Samples were considered stable if, at each of the temperature conditions tested, the samples had a clear, colorless appearance, with no visually observable particles; pH level 7±0.3; osmolality 260-340 mOsm/kg; purity ≥ 95% (and without a single impurity > 3%); target activity, which is comparable with the standard sample; microparticle levels ≤ 6,000 microparticles per vial for microparticles ≥ 10 µm and levels ≤ 600 microparticles per vial for microparticles ≥ 25 µm; endotoxin levels ≤ 100 EU (endotoxin units)/ml; and lack of growth of microorganisms.

Example 4 Freeze Stability

[00277] A study was conducted to test the stability of the aqueous solution of example 1, with repeated cyclic freezing and thawing. R5000 showed no degradation or other changes after 5 freeze and thaw cycles.

Example 5 Surface Plasmon Resonance (SPR) Binding Evaluation

[00278] The binding interaction between R5000 and C5 was measured using surface plasmon resonance. R5000 bound C5 with an equilibrium dissociation constant (KD) of 0.42 nM at 25°C (n=3) and a KD of 0.78 nM at 37°C (n=3). The summary surface plasmon resonance data, combined with high resolution co-crystal structure analysis, shows that R5000 is characterized by a specific, strong and fast bonding to C5, as well as a low dissociation rate.

Example 6 Evaluation of Inhibition of C5 Cleavage

[00279] R5000 was evaluated for inhibition of C5 cleavage to C5a and C5b by ELISA (enzyme-linked immunosorbent assay (ELISA)) of the C5a cleavage product and formation of the C5b-9 membrane attack complex (MAC). The inhibitory activity of R5000 with host C5 is an important consideration in selecting appropriate animal models for drug safety. Inhibition of C5 cleavage is the basis for the clinical efficacy of eculizumab, currently the only approved drug for the treatment of PNH. R5000 demonstrated dose-dependent inhibition of C5a formation after classical pathway activation (IC ₅₀ =4.8 nM; FIG. 1) and dose-dependent inhibition of C5b (measured by C5b-9 or MAC formation) after activation of classical or alternative complement pathways (IC ₅₀ =5 .1 nM, Fig. 2).

Example 7 Inhibition of complement-induced hemolysis of erythrocytes

[00280] The erythrocyte lysis assay is a reliable way to isolate serum/plasma complement inhibitors from different species and compare the relative activities of a test drug. An in vitro functional assay was used to evaluate the inhibitory activity of peptides, including R5000, against complement function in several species. This assay tests the functional ability of classical pathway complement components to lyse ovine erythrocytes pre-coated with rabbit anti-sheep erythrocyte antibodies. When antibody-coated erythrocytes are incubated with test serum, the classical complement pathway is activated, resulting in hemolysis controlled by the release of hemoglobin. Antibody-sensitized ovine erythrocytes were used as the lysis vehicle in the present assay, and sera and/or plasma from various species were used at their preset 50% complement hemolytic activity (CH ₅₀ ).

[00281] R5000 demonstrated potent inhibition of complement-induced hemolysis of erythrocytes in serum and/or plasma of humans, non-human primates and pigs (see Table 1).

Table 1. Inhibition of erythrocyte hemolysis by R5000 in several forms

Kinds IC ₅₀ (nM) Human 6.6 Non-human primate (4 species) 3.5-17.6 Dog >4700 Rabbit >67000 Pig (2 types) 51.9-118.6 Rodents (3 species) from 591 to>100000

[00282] Weak activity was observed in rat plasma (>100 times lower than cynomolgus macaque), and little or no activity was observed in other rodents, dog or rabbit. Structural data obtained from the co-crystallization of human C5 with a molecule closely related to R5000 provided an explanation for this species selectivity by careful analysis of the primary amino acid sequence at the drug-binding site of the target protein. Although primate sequences are 100% conserved in the residues responsible for R5000 interactions, there were significant differences in these residues in rodents and, especially, dogs, which lack identical regions of the protein. These differences in amino acids were sufficient to explain the activity profiles of R5000 in different species.

[00283] The ability of R5000 to inhibit complement-mediated erythrocyte lysis via the classical and alternative pathways of complement activation was also tested. The classical pathway was evaluated using two different assays using antibody-sensitized ovine erythrocytes. In one method, hemolysis was assessed using 1% normal human serum, while a second assay was performed with 1.5% C5-poor human sera containing 0.5 nM human C5. Inhibition of the alternative pathway of complement activation was assessed using rabbit erythrocytes in 6% normal human serum in the absence of calcium (see Table 2).

Table 2. Inhibition of hemolysis by R5000 in complement activation pathways

Path _IC50 (nM) classic way 4.9 The classic way with C5-poor sera 2.4 Alternative path 59.2

[00284] R5000 showed complement mediated lysis in both classical and alternative pathway analyses.

Example 8 Pharmacodynamics in cynomolgus monkeys

[00285] R5000 is a potent complement inhibitor in primates, so cynomolgus monkeys were selected for multi-dose studies to evaluate the inhibitory activity of R5000 in an animal model. Plasma drug concentrations were determined by LC-MS and complement activity was analyzed using the previously described erythrocyte lysis assay method. The overall results of these studies indicated that monkey plasma drug levels should not be less than 2.5 μg/ml to provide >90% inhibition of complement activity (see Figure 3).

[00286] R5000 was administered to cynomolgus monkeys in multiple daily doses by subcutaneous (SC) injection during a 7-day study. Blood samples were analyzed for hemolysis as an indicator of complement activity at designated time points (during days 1, 4 and 7, data reported as the number of days after the first dose but before dosing on the respective days), using an ex vivo ovine erythrocyte lysis assay, at 1% plasma in the assay. Drug levels were determined for the same sample using an LC-MS method specific for R5000. As shown in Table 3 and FIG. 4 and FIG. 5, when R5000 was administered daily for 7 days at doses of either 0.21 or 4.2 mg/kg, minimal (<3% of pre-dose level) complement activity was observed during the dosing period.

Table 3. Mean pharmacodynamic values

Number of animals dosed Daily dose (mg/kg) Route Last date of collection (days) % Hemolysis Day 1 Day 4 Day 7 Day 8 Day 12 Day 14 Day 18 2 0.21 SC eighteen 2.9 1.9 2.4 5.4 75.4 >95 >95 2 4.2 SC eighteen <1 <1 <1 1.1 8.7 45.7 87.3

[00287] Hemolysis in the ex vivo assay was maintained below 90% of baseline after the first dose in the 0.21 mg/kg group, throughout the dosing period, and up to 24 hours after the last dose. An increase in hemolysis levels was observed after treatment was discontinued. By day 4 (264 hours in Figure 4) after the last dose was administered, hemolysis was >75% of baseline. This result correlates well with measured plasma levels for the compound during and after dosing (dashed line in FIG. 4). The second group of animals in a multi-dose study was administered a daily 4.2 mg/kg dose of R5000. In this group, hemolysis was essentially completely inhibited (was <1%) during dosing and remained below 3% about 48 hours after the last dose (day 9; 216 hours in Fig. 5). Four days after the final dose (264 hours in Fig. 5) hemolysis reached approximately 10% of baseline. This result again showed a decrease in complement activity during the dosing period (compared to pre-dosing results), which correlated with plasma drug concentrations and proved a high correlation between pharmacokinetic and pharmacodynamic values.

[00288] The complement inhibitory activity of R5000 was assessed in a 28-day, repeated-dose study in cynomolgus monkeys using an ex vivo erythrocyte hemolysis assay. R5000 was administered daily by subcutaneous injection for 28 days at doses of 0, 1, 2 or 4 mg/kg/day (day 1: Fig. 6 and day 28: Fig. 7). The results showed complete inhibition of hemolysis 2 hours after the first dose and for 28 days of dosing, with hemolysis percentages <5% in the 1, 2 and 4 mg/kg/day groups, compared to >90% in the control group. After a 28 day recovery period, sample values returned to near baseline hemolysis levels and there was little or no inhibition of the complement system. The absence of complement inhibition activity at the end of the recovery period indicated clearance of the drug from the animals.

[00289] Complement inhibition was also tested in a 13-week, multiple-dose study in cynomolgus monkeys. Monkey samples were analyzed using an ex vivo erythrocyte hemolysis assay. R5000 was administered daily by subcutaneous injection for 13 weeks at doses of 0, 0.25, 1, 2, or 10 mg/kg/day. Similar to the 28-day study, the results of the 13-week study showed complete ex vivo inhibition of hemolysis 2 hours after the first dose and for 13 weeks of dosing, with hemolysis percentages <5% in the 0.25, 1, 2, and 10 mg groups. /kg/day, compared with>90% in the control group. After a 28 day recovery period, sample values returned to near baseline hemolysis levels and there was little or no inhibition of the complement system. The absence of complement inhibition activity at the end of the recovery period indicated clearance of the drug from the animals.

[00290] During the study, oxidative metabolism led to the formation and detection of the hydroxylated metabolite R5000 with ω-hydroxylation of the palmitoyl tail (referred to in this application as "R5001").

[00291] The ability of R5001 to inhibit complement-mediated erythrocyte lysis via classical complement activation pathways was tested using antibody-sensitized ovine erythrocytes in 1% normal human serum. R5001 showed potent inhibition of complement-induced hemolysis with an IC _{50 of} 4.5 nM.

Example 9 Pharmacokinetic/Pharmacodynamic Modeling and Human Pharmacokinetic Modeling

[00292] A computer model of PK/PD was constructed using in vivo data from cynomolgus monkeys. The fit and accuracy of the model was assessed by comparing the simulated results with newly obtained experimental data. After validation in monkeys, the final model was used to predict human pharmacokinetics by allometric scaling according to human parameters. Simulation results support the target dosing interval of once daily or less for humans, with daily doses of 0.1 mg/kg providing nearly 90% of the target inhibition at steady state (see FIG. 8). Due to the long half-life of R5000, multiple doses are required to reach the final peak and trough levels of the drug. According to calculations, _Cmax in plasma after one week of daily dosing should be approximately 3 times higher than after the first dose, as drug levels reach a steady state.

Example 10 Clinical study of the effect of single incremental doses of R5000, phase 1

[00293] To evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of R5000, following subcutaneous (SC) injection, a clinical pharmacological study of the effect of single incremental doses in phase 1 was performed in healthy volunteers. The dose volume was determined by the dose requirements in the group and the weight of the subject. Subjects who were pregnant or nursing mothers, as well as any subjects with systemic infection or meningococcal infection, were excluded. The study was randomized, double-blind, placebo (PBO)-controlled, in 4 groups with SC administration of single incremental doses, who were in the department of clinical pharmacology for 3 days. All subjects received ciprofloxacin prophylaxis, and subjects in the maximum single dose group (ie, 0.4 mg/kg) were vaccinated against meningococcus at least 14 days prior to the study. All subjects received 1 dose of R5000 on day 1. Four subjects were dosed (2 subjects were treated with R5000 and 2 subjects were treated with PBO) of the smallest value (0.05 mg/kg), and 6 subjects in each group were sequentially dosed (4 subjects were treated with R5000 and 2 subjects were treated with PBO) 3 doses of the greater values (0.1, 0.2 and 0.4 mg/kg). The demographics of the subjects are shown in Table 4.

Table 4. Demographic data of subjects

placebo,
n=8 R5000,
0.05 mg/kg, n=2 R5000,
0.1 mg/kg, n=4 R5000,
0.2 mg/kg, n=4 R5000,
0.4 mg/kg, n=4 For all, n=22 Attitude
men: women 2:6 0:2 0:4 0:4 13 3:19 Average age,
years (min, max) 39
(20, 59) 23
(22, 23) 27
(20, 37) 34
(22, 65) 32
(21, 58) 33
(20, 65) Average body mass index, kg / m ² 24 twenty 21 26 27 24 white:black:asian 7:1:0 2:0:0 2:1:1 3:0:1 4:0:0 18:2:2

[00294] Safety was assessed by intensive clinical monitoring, and blood samples were taken at high frequency to determine R5000 concentrations by liquid chromatography/high-performance mass spectroscopy and the ability to inhibit complement-mediated erythrocyte lysis by ex vivo hemolysis assay of antibody-sensitized sheep erythrocytes.

[00295] Pharmacokinetic (PK) parameters measured in this study include clearance (CL), C _max (maximum plasma drug concentration, Figure 9A), T _max (time to peak plasma concentration after drug administration) ), t _1/2 (half-cycle), AUC _0-24 (area under the plasma concentration-time curve from zero time to 24 hours; see Fig. 9B for changes in plasma concentration over time), AUC _0-inf (area under the plasma concentration-time curve from zero time to infinity, see Fig. 9B for the change in plasma concentration over time, Vz (apparent volume of distribution in the terminal phase), K _el (elimination rate) and F (fractions). The results for each parameter are presented in Table 5.

Table 5. Pharmacokinetic parameters

PK parameter Statistical characteristics R5000
(0.05 mg/kg)
N=2 R5000
(0.10 mg/kg)
N=4 R5000
(0.20 mg/kg)
N=4 R5000
(0.40 mg/kg)
N=4 _Cmax (ng/ml) Mean value (SD, standard deviation) 1010 (14.142) 1550 (197.82) 2970 (317.80) 5873 (440.71) T _max (h) Median value (min, max) 4.5 (3, 6) 3.0 (3.24) 4.5 (3.48) 4.6(3.6) AUC _0-24 (ng×h/ml) Mean value (SD) 21440 (1020.9) 33230 (4605.6) 60350 (4624.8) 112300 (8623.2) AUC _0-last (ng×h/ml) Mean value (SD) 179800 (3214.7) 375400 (47513) 655100 (113710) 822600 (120760) AUC _0-inf (ng×h/ml) Mean value (SD) 190700 (3081.0) 408600 (52716) 702900 (143630) 863200 (134870) t _1/2 (h) Mean value (SD) 163.5 (10.9) 185.4 (6.4) 172.0 (24.8) 155.6 (14.3) K _el (1/h) Mean value (SD) 0.004248 (0.000283) 0.003743 (0.000128) 0.004092 (0.00058001) 0.004482 (0.00041984) CL/F (ml/h/kg) Mean value (SD) 0.2622 (0.0042) 0.2481 (0.0353) 0.2933 (0.0574) 0.4711 (0.0660) _VZ /F (ml/kg) Mean value (SD) 61.89 (5.13) 66.41 (10.20) 71.43 (7.52) 105.10 (11.68)

[00296] All groups achieved C _max levels consistent with values predicted by a PK computer model generated using data from non-human primate (NHP) studies. Plasma concentrations from a single SC injection showed a linear relationship between C _max and dose level (FIG. 9A), and dose-dependent exposure was confirmed at all dose levels (FIG. 9B). The mean maximum plasma concentration (C _max ) ranged from 1010 to 5873 ng/ml over the dose range. The mean area under the concentration-time curve from time 0 to 24 hours post-dose (AUC _0-24 ) ranged from 21440 to 112300 ng×h/ml over the dose range. These results indicate that with increasing dose of R5000 there is an approximately proportional increase in plasma concentration (C _max ) and exposure (AUC _0-24 ). The median time to maximum observed plasma concentration (T _max ) varied from 3.0 to 4.6 hours over the dose range, indicating an intermediate absorption rate of R5000 from the SC space into the central compartment (blood). The mean apparent total clearance (CL/F) after administration of R5000 was low and ranged from 0.2481 to 0.4711 ml/h/kg. The mean half-life (t _1/2 ) was consistent over a range of dose levels and ranged from 155.6 to 185.4 hours. The mean apparent total volume of distribution (Vz/F) in the terminal phase after extravascular administration ranged from 61.89 to 105.1 ml/kg, indicating that R5000 is located primarily in the circulating blood compartment with minimal extravascular distribution. Approximate t _1/2 for all groups is determined to be 7 days.

[00297] R5000 was also characterized by rapid dose-dependent inhibition of hemolysis [direct hemolysis (FIG. 10A) and %CH ₅₀ (FIG. 10B) and erythrocyte lysis in 1% plasma over time (FIG. 10C)] and attenuation of complement activity (both established by ELISA with the WIESLAB® ELISA kit for all subjects after a single dose, see Fig. 11). The maximum pharmacodynamic effect was observed approximately 3 hours after dosing. The results showed that, at the maximum plasma concentration, the maximum percent hemolysis inhibition from baseline was >90% for the 0.1, 0.2, and 0.4 mg/kg dose groups and 60% for the lowest dose group. (0.05 mg/kg). Dose-dependent inhibition of hemolysis up to 4 days was observed in groups with doses of 0.1, 0.2 and 0.4 mg/kg. That is, the mean hemolysis remained above baseline for up to 2 days in the 0.05-mg/kg group, up to 4 days in the 0.1-mg/kg group, and up to 7 days in the 0.2- and 0.4- mg/kg groups.

[00298] Similarly, analysis of complement activity showed that inhibition of complement activity remained strong for 4 days after 0.4-mg/kg injection (see Fig. 11). Human plasma samples taken from subjects receiving a 0.4-mg/kg injection were analyzed with the WIESLAB® ELISA kit (Euro Diagnostica, Malmo, Sweden). This assay measures an alternative pathway for complement activity. As shown by this analysis, complement activity was suppressed to 3% after 3 hours after dosing and remained below 13% for 96 hours after receiving R5000.

[00299] Single SC doses of R5000 were safe and well tolerated by healthy volunteers. There were no clinically significant changes in vital signs, clinical laboratory parameters, physical examination and ECG results.

[00300] This study concludes that low daily doses can provide stable levels suitable for >80% attenuation of hemolysis, and that dosing once a week may be sufficient. In particular, 0.2 mg/kg can lead to complete attenuation of complement activity and complete inhibition of hemolysis.

Example 11 Clinical Study of Multiple Doses of R5000

[00301] To evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of R5000 after single daily subcutaneous (SC) injections for 7 days, a phase 1 multiple dose clinical pharmacology study was performed in healthy volunteers. The study was single-center, randomized, double-blind and placebo (PBO) controlled. Subjects received daily SC doses of 0.2 mg/kg R5000 or consistent PBO for 7 days while in the clinical pharmacology department. The dose volume was determined by the dose requirements in the group and the weight of the subject. Subjects who were pregnant or nursing mothers, as well as any subjects with systemic infection or meningococcal infection, were excluded. All subjects received ciprofloxacin prophylaxis, and subjects in the multiple-dose group were vaccinated against meningococcus at least 14 days prior to the study. Safety was assessed by enhanced clinical monitoring, and daily blood samples were taken immediately prior to dosing, as well as 3 hours and 6 hours after each daily dose to determine R5000 concentrations by liquid chromatography/high-performance mass spectroscopy and the ability to inhibit complement-mediated erythrocyte lysis by ex vivo analysis of hemolysis of antibody-sensitized sheep erythrocytes.

[00302] A total of 6 subjects were enrolled in the study (4 received R5000 and 2 received PBO). The demographics of the subjects are shown in Table 6.

Table 6. Demographic data of subjects

placebo,
n=2 R5000,
n=4 Attitude of men: women 0:2 13 Average age, years (min, max) 27 (25, 29) 24 (22, 26) Average body mass index, kg / m ² 21 23 Whites: Asians twenty 3:1

[00303] As can be seen from Table 7 and its associated FIG. 12A (representing percent hemolysis and plasma concentration over 7 days), plasma concentrations showed a strongly increasing exposure over 7 days of dosing. According to the given data, the R5000 half-life was determined to be 7 days. Plasma levels returned to approximately 2000 ng/mL by day 15 and approximately 1000 ng/mL by day 21 (FIG. 12B).

Table 7. Plasma concentrations of R5000

Point in time (hours) R5000 concentration (ng/ml) Subject 1 Subject 2 Subject 3 Subject 4 0 0 0 0 0 3 2510 2410 2560 2520 6 2300 2390 2410 2650 24 1890 1750 1810 2220 27 3870 4050 4280 4110 thirty 3650 3730 4310 4000 48 2910 2650 3370 3330 51 5200 4910 5330 5500 54 4820 4220 5100 4900 72 3680 3340 3780 4460 75 6310 5240 5280 6110 78 5720 5570 5880 6140 96 4650 3790 4840 4540 99 6660 5320 6860 6770 102 7000 5440 6550 6820 120 4840 4430 5200 5280 123 7210 6410 7210 7700 126 7290 5850 6880 7020 144 5170 4210 4920 5430 147 7430 6320 7490 7780 150 6920 6130 7630 7110 168 5750 4940 5730 5670

[00304] R5000 PK parameters after repeated SC dosing (0.2 mg/kg/day) for 7 days are shown in Table 8. Measured pharmacokinetic (PK) parameters include clearance (CL), C _max (maximum concentration drugs in plasma), T _max (time to reach maximum plasma concentration after drug administration), t _1/2 (half-cycle), AUC _tau (area under the plasma concentration-time curve from zero time to 24 hours, AUC _0-inf (area under the plasma concentration-time curve from time zero to infinity, Vz/F (apparent volume of distribution), K _el (elimination rate) and F (fractions).

Table 8. Summary of PK parameters

PK parameter Statistical characteristics R5000 (0.20 mg/kg)
(N=4) 1 day 7 days _Cmax (ng/ml) Mean value (SD, standard deviation) 2533 (100.1) 7290 (662.4) T _max (h) Median value (min, max) 3 (3, 6) 3 (3, 6) AUC _tau (ng×h/ml) Mean value (SD) 50010 (3334.0) 151300 (12042) AUC _0-inf (ng×h/ml) Mean value (SD) Does not match 1101000 (108220) t _1/2 (h) Mean value (SD) Does not match 161.9 (14.8) K _el (1/h) Mean value (SD) Does not match 0.004309
(0.00041325) CL/F (ml/h/kg) Mean value (SD) Does not match 1.330 (0.114) _VZ /F (ml/kg) Mean value (SD) Does not match 311.6 (51.4)

[00305] Mean C _max and AUC _tau on day 1 were 2533 ng/mL and 50010 ng h/mL, respectively, consistent with results from the 0.2 mg/kg single dose group over the same period after dose administration. Following daily SC administration for 7 days, C _max and AUC _tau increased approximately 2.9 times (mean C _max = 7290 ng/ml on day 7) and 3.0 times (mean AUC _tau =151300 ng×h/ml on the 7th day), respectively. The median time to reach the maximum observed plasma concentration (T _max ) on day 7 was 3.0 hours, which is consistent with T _max after a single SC dose (median time on day 1 T _max = 3.0-4 ,6 hours). This indicates a stable absorption rate of R5000 with multiple dosing. Day 7 mean apparent total clearance of R5000 (CL/F=1.3 ml/h/kg on day 7) increased slightly relative to total clearance after a single SC dose of 0.2 mg/kg [CL/F= 0.29 ml/h/kg at a single incremental dose (SAD) of 0.2 mg/kg]. However, the elimination rate constants (K _el ) for R5000 after single and multiple dosing (mean K _el =0.0041 h ^-1 at 0.2-mg/kg SAD; mean K _el =0.0043h ^-1 at 0.2-mg/kg MD on day 7) are consistent, indicating that, with multiple doses, the clearance of R5000 does not change significantly. The apparent volume of distribution of R5000 (V _z /F) showed some increase with multiple doses of R5000 (mean V _z /F=71.4 ml/kg at 0.2-mg/kg SAD; mean V _z /F=311 .6 ml/kg at 0.2-mg/kg MD on day 7). However, V _z /F on day 7 for R5000 was still less than total body water, implying that R5000 is not distributed into the extravascular space after repeated SC administration.

[00306] To determine if low daily doses are suitable to achieve stable levels and complete and stable inhibition of complement and attenuation of hemolysis, the average percent inhibition of hemolysis in the subject's serum samples was measured (see Table 9).

Table 9. Hemolysis assay

Point in time (hours) % Hemolysis (with treatment) % Hemolysis (placebo) Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 0 100.0 100.0 100.0 100.0 100.0 100.0 3 5.8 1.9 5.0 2.9 99.4 102.7 6 6.0 1.6 5.2 2.3 90.8 99.4 24 9.3 2.4 8.9 2.9 105.8 100.7 27 4.9 1.2 4.0 1.0 112.3 96.1 thirty 4.6 1.5 4.6 2.2 110.5 100.6 48 6.6 2.0 6.9 2.1 120.6 107.2 51 4.0 1.4 3.6 1.9 118.4 97.3 54 3.7 1.4 3.6 1.8 133.5 98.0 72 5.0 1.6 3.8 1.7 114.4 97.9 75 2.9 0.8 2.7 1.3 121.4 98.2 78 3.0 0.9 4.7 1.5 119.4 94.6 96 3.9 1.1 4.0 6.5 111.5 95.2 99 2.9 0.9 2.7 1.0 122.0 101.1 102 3.6 1.4 3.8 1.7 135.1 101.4 120 4.0 2.1 3.8 2.3 115.4 95.7 123 2.7 1.3 2.7 1.6 114.1 92.2 126 3.3 1.3 3.2 1.3 113.5 96.9 144 3.4 1.2 3.8 1.4 108.8 95.7 147 2.5 1.2 2.6 1.2 121.7 103.7 150 3.4 1.3 3.0 1.4 121.3 98.3 168 3.4 1.5 3.1 1.8 119.1 102.0

[00307] Mean percent inhibition of hemolysis from baseline was ≥ 95% starting at the first time point post-dose 3 hours post-dose on Day 1 and continued for 7 days of dosing. All subjects individually showed a ≥90% reduction in hemolysis at all time points. Hemolysis observed on day 8 (24 hours after the last dose) was ≤ 3% in all subjects. Hemolysis recovered to pre-dosing levels within two weeks of the last dose.

[00308] The study concludes that low daily doses will provide stable levels suitable for complete and stable complement inhibition and attenuation of hemolysis. The study also concludes that one-week dosing may be sufficient to inhibit complement activity and reduce hemolysis in humans.

[00309] Complement activity in the subject's plasma samples was determined by analysis with the WIESLAB® ELISA kit (Euro Diagnostica, Malmo, Sweden). This assay measures an alternative pathway for complement activity. As shown by this analysis, the weakening of complement activity was rapid, complete and stable throughout the dosing period in all subjects (see Fig. 13A and table 10). The abbreviation SEM in the table stands for the standard error of the mean.

Table 10. % Complement Activity in the Multiple Dose Study

Hours after first treatment 3 48 96 Minimum % Complement Activity (SEM) 1.8 (0.8) 6.9 (0.3) 2.1 (0.1) Mean % Complement Activity (SEM) 3.1 (0.6) 8.2 (1.1) 3.1 (0.8)

[00310] The observed complement activity on day 8 (24 hours after the last dose) was ≤ 5% in all subjects. Complement activity recovered to pre-dosing levels within two weeks of the last dose (FIG. 13B).

[00311] R5000 was safe and well tolerated by healthy volunteers. There were no clinically significant changes in vital signs, clinical laboratory parameters (complete blood count, biochemical blood test, results, coagulation and urinalysis), medical examination and ECG.

[00312] R5000 and the metabolite R5001 were measured in the 0.20-mg/kg dose group in the multiple dose study and both compounds were detected.

Example 12 Pharmacokinetic/Pharmacodynamic Modeling and Human Pharmacokinetic Modeling in a Phase II Study

[00313] The PK/PD model has been developed to describe plasma concentrations of R5000 and its metabolites and their activity in an ex vivo erythrocyte lysis assay. The model is based on the quasi-stationary approximation of the target-mediated drug distribution model. In the multiple dose portion of the R5000 Phase I study, all subjects achieved drug levels consistent with values predicted by a PK computer model generated using single dose human PK and PD data. PK/PD modeling for R5000 in humans confirms the intended dosing regimen with a 0.3-mg/kg loading dose followed by daily doses of 0.1 mg/kg, resulting in ≥90% target inhibition after the first dose and at steady state (Fig. fourteen).

Example 13 Clinical Trial Plan for a Phase II Study

[00314] Based on predicted stable drug levels for nominal as well as maximum dose (based on weight grouping), a dose of 0.1 mg/kg/day R5000 was chosen. The results are shown in Table 11. In this table, C _max means the maximum plasma concentration of the drug, AUC _0-24 means the area under the plasma concentration-time curve from time 0 to 24 hours, SD means a single dose, QD means a single daily dose , and * with maximum dose indicates the potential maximum dose that patients will receive as a result of dosing based on weight grouping.

Table 11. Experimental and predicted PK values

Pharmacokinetic parameter Statistical characteristics R5000
(0.2 mg/kg) QD×7; day 7 (SD) for humans R5000 (0.1 mg/kg) prediction of steady state in humans Rated (minimum)
0.1 mg/kg once a day Maximum dose*
0.14 mg/kg once a day _Cmax (µg/ml) Mean 7.29 (0.66) 6.57 7.12 AUC _0-24 (h × mcg / ml) Mean 151.3 (12) 141.5 152.8

[00315] Based on phase II studies, hemolysis inhibition was expected to be consistently maintained ≥90% at a daily dose of 0.1 mg/kg R5000 and a loading dose of 0.3 mg/kg R5000 given on the first day of dosing. This prediction was confirmed by the overlap of expected steady state plasma concentrations of R5000 with the PK/PD relationship obtained in phase 1 studies in healthy volunteers (Fig. 15). Proper and continuous control of hemolysis is a critical component of both efficacy and safety in the treatment of paroxysmal nocturnal hemoglobinuria (PNH). In this disease, even a slight short-term weakening of complement blockade can lead to immediate "reversible" intravascular hemolysis and exacerbation of severe symptoms of PNH. This consideration was especially important in relation to the potential missed doses.

[00316] In order to effectively control hemolysis, phase 2 studies were designed to include a single loading dose of 0.3 mg/kg to properly attenuate hemolysis after the first dose. Well-tolerated single doses up to 0.4 mg/kg administered by subcutaneous injection in the single-dose groups of the phase 1 study in healthy volunteers (Fig. 16A) provided a rationale for using a loading dose of 0.3 mg/kg after superimposing the expected minimum plasma concentrations of R5000 following the first dose, with a PK/PD relationship obtained in phase 1 studies in healthy volunteers. The loading dose was not expected to affect expected exposures at steady state. The loading dose of 0.3 mg/kg was expected to provide the targeted pharmacodynamic effect (>90% inhibition of hemolysis) at minimal levels to promote immediate control of hemolysis after the first dose (see PK/PD model shown in Fig. 16B). Based on the PK/PD model, it was expected that if, after reaching steady state, the subject misses a dose, then the proposed starting dose regimen should maintain hemolysis inhibition of ≥ 90% for at least 72 hours (see model presented in Fig. 16C).

[00317] From the 2nd week visit onwards, for subjects not achieving an appropriate response (as measured by lactate dehydrogenase levels that are 1.5 times lower than the normal upper limit), and after evaluation of safety and tolerability data by the investigator and medical adviser, the dose was planned to be increased to 0.3 mg/kg per day. The dose was planned to be increased to 0.3 mg/kg per day if there was an obvious case of recurrent hemolysis (eg, hemoglobinuria).

Example 14 Dose, Administration, Contraindication in Phase II Studies

[00318] In Phase II clinical trials, R5000 is administered once daily (every 24 hours) by SC self-administration at a dose of 0.1 mg/kg following an initial loading dose of 0.3 mg/kg on the first day of dosing (1 day of the study). Some subjects may receive a higher dose of 0.3 mg/kg to optimize hemolysis control. The dose (dose volume) for each injection is determined by the prescribed dose and the body weight of the patient. An overdose of R5000 is not expected to result in acute or specific adverse systemic side effects. In the event of an overdose, clinically acceptable support measures are established, determined by the clinical scenario and in consultation with a medical advisor.

[00319] Daily dosing of R5000 allows for consistent and stable complement inhibition and therefore reduces the risk of recurrent hemolysis. The dosing regimen of R5000 is chosen to eliminate the large ratio between maximum and minimum plasma concentrations that is observed with the introduction of multiclonal antibodies once every two weeks. With an approximate 7-day plasma elimination half-life, R5000 administered daily is expected to remain within a narrow range of plasma concentrations, effectively "fixing" terminal complement activity with persistent inhibition and avoiding the "return hemolysis" phenomenon.

[00320] This action is contrary to the current standard of care for eculizumab. A proportion of patients receiving eculizumab have been reported to have inadequate control of their disease, leading to recurrent hemolysis occurring near the end of the 2-week dosing interval using the approved 900 mg biweekly dose (Hillmen P, et al. Long- term safety and efficacy of sustained eculizumab treatment in patients with paroxysmal nocturnal haemoglobinuria Br J Haematol 2013; 162:62-73 and Brodsky RA Paroxysmal nocturnal hemoglobinuria Blood 2014; 124:2804-2811). The clinically approved course of treatment with eculizumab is two weeks, i.e. one injection every 12-16 days. Although hemolysis is well controlled on the first day of a treatment cycle, efficacy often declines as plasma levels of eculizumab decrease in the last few days before reintroduction. This loss of disease control with eculizumab resulted in the need to increase doses above the approved dose, for which no safety or efficacy clinical trials have been performed. Reports from the UK National PNH Service indicate that about 22% of patients treated with eculizumab require non-standard doses due to recurrent hemolysis at the standard dose.

[00321] Subjects are instructed to self-administer SC doses daily, every 24 hours, at approximately the same time each day. Subjects are instructed to inject into the abdomen, however, injections into the thigh or upper arm may be used. During the course of the study, subjects were required to document daily confirmation of dose administration and the location of the injection. The current existing drug, eculizumab, needs to be administered by intravenous infusion every 2 weeks 2 under the supervision of a healthcare professional. In contrast, R5000 is designed for subcutaneous self-administration at home and is therefore a convenient drug for patients.

[00322] Subjects who are hypersensitive to R5000 or any of its excipients are not treated with R5000. Subjects with colonization or infection with meningococcus causing diagnostic difficulties, or suspected of having such an infection, are also excluded from treatment with R5000.

Example 15 Safety Evaluation in a Phase II Clinical Trial of R5000

[00323] The clinical study includes safety assessments to monitor side effects (including reactions to injection and systemic infection), clinical laboratory tests (including liver and pancreatic markers), ECG, vital signs, and physical examination. Phase II studies include frequent follow-up visits, with clinic visits on a weekly schedule for the first 4 weeks, followed by visits every 2 weeks. A long-term follow-up study includes continuous follow-up, with monthly clinic visits for the first 3 months and every 3 months thereafter, with continued follow-up and clinic visits expected to collect long-term safety and tolerability data.

Example 16 Inhibition of hemolysis using R5000 related peptides when erythrocytes fail to respond to eculizumab due to C5 mutation

[00324] Blood/plasma samples were analyzed from two Japanese patients with PNH who did not respond well to eculizumab. Previously, both patients were shown to have a unidirectional heterozygous C5 missense mutation, c. 2654G->A, which predicts the p.Arg885His polymorphism (a description of this polymorphism is given in Nishimura, J. et al., N Engl J Med. 2014. 370(7):632-9, the contents of which are fully incorporated into this application by referral). During the study, mR5000 was tested on patient serum samples. The mR5000 formulation is identical to R5000 except that the C-terminal lysine is not modified. The mR5000 compound has similar antihemolytic activity to R5000 but a shorter elimination half-life when administered to a patient. Patient serum was acidified and mixed with erythrocytes in the presence (+) or absence (-) of mR5000. Complete inhibition of hemolysis was observed in the presence of mR5000 (FIG. 17).

Example 17 Treatment with R5000 in Patients Who Do Not Respond to Eculizumab

[00325] Given the separate R5000 binding site, R5000 is used to treat PNH in patients with mutations in the C5 gene that prevent eculizumab from binding to C5. R5000 binds at a site on C5 that is different from the eculizumab binding site and therefore may prevent C5 activation in patients who do not respond to eculizumab due to mutations near its C5 binding site.

Example 18 Comparison between R5000 and Antibody Based C5 Inhibitors

[00326] Inhibition of complement activity was assessed by hemolysis of rabbit or PNH patient erythrocytes and by measuring C5a and C5b-9 production by ELISA. Levels of C5 and C5-eculizumab complex were measured in PNH patient samples by solid-phase ELISA using a C5 binding reagent to capture C5 or the complex, and detection by either an anti-C5 antibody followed by a secondary antibody conjugated to horseradish peroxidase (total C5), or with a horseradish peroxidase-conjugated secondary antibody to detect eculizumab (C5-eculizumab complex). Inhibition of C5 binding to alternative pathway C5 convertase (AP) was measured by the SPR method.

[00327] Residual hemolytic activity has been shown to exist after treatment with eculizumab (see Brodsky et al., 2017. Blood 129; 922-923 and Harder et al., 2017. Blood. 129:970-980). R5000 completely inhibited complement activity in several in vitro assays when residual hemolysis was evident with eculizumab treatment (Alexion Pharmaceuticals, New Haven, CT), including hemolytic activity and production of C5a and C5b-9. In serum from a PNH patient treated with eculizumab, approximately 30% of the hemolytic activity of normal human serum was retained in the rabbit erythrocyte lysis assay, even with the addition of excess eculizumab (up to 12.5 μM). The introduction of R5000 completely suppressed this residual hemolytic activity. Finally, R5000 completely inhibited C5 binding to AP C5 convertase as determined by SPR, while eculizumab only partially inhibited C5 binding. In general, these results lead to the conclusion that the mechanism of inhibition of R5000 contributes to the complete inhibition of hemolysis under conditions in which eculizumab performs partial inhibition.

Example 19 Group A Results in Two Patients at Week 7

[00328] A dose selection study to evaluate the safety, tolerability, preliminary efficacy, pharmacokinetics and pharmacodynamics of R5000 was performed in patients with PNH. The study was an open-label 12-week follow-up study. The study program was carried out with a broad coverage and was intended to cover 3 PNH groups: (group A) subjects not treated with eculizumab; (group B) subjects transitioning from eculizumab to R5000; and (group C) subjects with an inappropriate response to eculizumab. Patients received R5000 by subcutaneous injection with a loading dose of 0.3 mg/kg on day 1, followed by a daily dose of 0.1 mg/kg for the first two weeks. From the week 2 visit onwards, when the lactate dehydrogenase (LDH) level was at least 1.5 times the normal upper limit, the daily dose could be increased to 0.3 mg/kg. The primary end point of the study was achieving a change in LDH from baseline to mean levels from week 6 to week 12 of the study.

[00329] Almost complete inhibition of hemolytic activity at week 7 was obtained in two patients (patient 001 and patient 002) in group A with 100% compliance (no dosing interruptions, remote control using a remote monitoring device) of the treatment regimen. Percent hemolysis and hemoglobin levels at each time point are shown in FIG. 18A and FIG. 18B, respectively. Patient 001 was a 56-year-old female diagnosed with PNH in April 2007. Patient 001 had a granulocyte clone value of 80% and an erythrocyte clone value of 25% and had received no blood transfusions prior to enrollment in the study. Patient 002 was a 65-year-old woman diagnosed with PNH in April 2017. Patient 002 had a granulocyte clone size of 99% and an erythrocyte clone size of 47%. Patient 002 required a blood transfusion prior to enrollment in the study and received a blood transfusion at weeks 4 and 6 of the study. Rapid decreases in LDH were observed in both patients, and the mean LDH at week 7 was 1.55 times the normal upper limit (FIG. 18C). R5000 levels remained relatively stable up until week 6 (FIG. 18D). There was one short-term case of recurrent hemolysis associated with intercurrent disease. No safety or tolerability issues have been identified. No serious or related adverse changes were observed, and no reactions were observed at the injection site.

Example 20 Surface Plasmon Resonance (SPR) Measurement of C5 Inhibitor Binding

[00330] The binding of R5000 and eculizumab to alternative pathway C5 convertase was studied by surface plasmon resonance (SPR) analysis. SPR measurements were performed at 25° C. on a ProteOn XPR36 SPR setup (Bio-Rad, Hercules, CA). Eculizumab was immobilized at pH 4.5 with 10 mM sodium acetate on a Bio-Rad GLH sensor chip by amino group immobilization (NHS/EDC binding kit available from Bio-Rad) and blocked with ethanolamine (Bio-Rad) at 25°C. Biotinylated R5000 was separately fixed to a Bio-Rad NLC sensor chip. C5 binding kinetics/affinity data for eculizumab and R5000 were obtained separately by streaming serially diluted C5 (Complement Technologies, TX) onto the surface of eculizumab or R5000. The same running/assay buffer (10 mM potassium phosphate, pH 7.4 and 6.0, 150 mM NaCl, 1% DMSO and 0.01% P-20 surfactant) was used for all steps. Surface regeneration was achieved using a single injection of 10 mM glycine (pH 1.5) followed by a flow of running buffer. Data analysis (affinity and kinetic parameters) was performed using the 1:1 Langmuir method.

[00331] Biotilinated human complement component C3b (Complement Technologies, TX) was prepared by mixing EZ-link maleimide-PEG2-biotin (Thermo Fisher Scientific, Waltham, MA) with C3b starting material according to the provider's protocol. Unused biotin was separated from biotilinated C3b on a desalting column. Biotilinated C3b was captured on a Bio-Rad NLC sensor chip according to a standard protocol. To assemble the alternative pathway C5 convertase (AP) on the surface with captured C3b, sequential injections of human complement components (purchased from Complement Technologies, Tyler, TX) were performed using the co-injection method. The co-administration method included a first injection of premixed factor B (fB; 150 nM) and factor D (fD; 10 nM) (25 µl/min for 60 s), followed by 500 nM C3 (25 µl/min in for 120 s), then C5 (400 nM) alone or mixed with serially diluted R5000 or eculizumab (25 µl/min for 60 s). The dissociation time was 360 s. All assays were performed at 37° C. in a solution of 10 mM HEPES pH 7.4, 150 mM NaCl, 1% DMSO, and 0.01% P-20 surfactant. To quantify C5 binding at different concentrations of inhibitors, individual SPR sensorgrams after C5 injection were normalized to the time of C5 injection, and SPR signal fractions were calculated. The results showed a decrease in C5 binding to AP C5 convertase in the presence of R5000 and eculizumab (indicative of a reduced response to C5, see Figure 19), with 2-μM R5000 being more potent.

Example 21 Inhibition of Hemolysis of RBC Patients with PNH

[00332] Inhibition of hemolysis of erythrocytes from PNH patients was assessed in the presence or absence of R5000 and eculizumab. Erythrocytes were collected from blood samples from PNH patients by centrifugation at 1000g for 3 minutes to pellet and washed 3 times with Olswer's solution. The cell pellet was resuspended 1:2 in GVB++ buffer (Complement Technology, Tyler, TX). Donor-matched normal human serum was acidified to pH 6.4 with HCl. PNH patient erythrocytes (2.5% v/v) were incubated in a clear 96-well assay plate with 50% acidified donor-matched serum and compound concentrations were measured for 18 hours at 37°C. The cells were then centrifuged at 1000g for 3 minutes and 100 μl of the supernatant was transferred to a separate transparent 96-well assay plate to detect absorbance at 412 nm.

[00333] Results from two patients shown in FIG. 20 and FIG. 21, respectively, show that R5000 completely blocks hemolysis of erythrocytes from PNH patients, while eculizumab partially inhibits hemolysis. Recombinant C5 containing the known p.Arg885His polymorphism that confers eculizumab resistance was used as the sole source of C5 in an alternative pathway hemolysis assay using C5-poor serum and rabbit erythrocytes. During the assay, increasing concentrations of R5000 or eculizumab were tested to evaluate their ability to inhibit hemolysis (see FIG. 22). The results show that R5000 is able to bind and inhibit p.Arg885His-mediated hemolysis of complement C5, while eculizumab is not effective.

Example 22 Measurement of Plasma Levels of C5, Eculizumab and C5/Eculizumab Complex in PNH Patients

[00334] Plasma levels of C5, eculizumab, and C5/eculizumab complex in PNH patients were measured by one of three solid phase ELISA formats, using R5000 as the capture agent in each variant. The R5000 analytical variant included an N-terminal biotinylated PEG fragment and replacement of the modified C-terminal lysine residue with norvaline. 5 pmol per well of the R5000 variant was immobilized on a 96-well NeutrAvidin coated microplate (Thermo Fisher Scientific, Waltham, MA) to be used as a capture agent for C5. Immobilization was performed in assay buffer (25 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 and 1 mg/ml BSA) for 2 hours at room temperature, then washed 3 times with assay buffer.

[00335] For detection of total C5, plasma was diluted 1:10,000 in assay buffer, and 100 μl were added to assay plate wells. C5 standards were prepared (3-fold serial dilution in assay buffer starting at 100 ng/mL) and added to assay plate wells. The plate was incubated at room temperature for 1 hour and washed 5 times with assay buffer. Eculizumab (1 μg/ml in assay buffer) was then added to each well and incubated for 1 hour at room temperature to bind any C5 in the sample that had not yet complexed with eculizumab. Goat anti-human IgG horseradish peroxidase (HRP) conjugated (Abcam, Cambridge, UK; 1:5000 dilution) was injected as a helper antibody for the detection of C5 bound eculizumab and incubated for 30 minutes at room temperature. Then TMB (tetramethylbenzidine) substrate (UltraTMB ELISA, Thermo Fisher Scientific) was injected onto the plate and incubated for 30 minutes. The reaction was stopped with 2-N sulfuric acid and absorbance read on a SPECTRAMAX® M3 plate reader (Molecular Devices, Sunnyvale, CA) at 450 nm. The amount of total C5 was calculated relative to the standard C5 curve.

[00336] Detection of the level of the C5/eculizumab complex was performed using the same method as described above, but without adding excess eculizumab. This assay used a diluted, premixed C5/eculizumab complex (1:1 molar ratio, 3-fold serial dilution starting at 100 ng/mL) as a standard.

[00337] To measure free eculizumab, 100 μl of a 1-μg/ml solution of C5 in assay buffer was added to a C5-binding peptide-coated assay plate and incubated for 1 hour at room temperature. Patient plasma diluted 1:10,000 in assay buffer was added to the test wells and diluted eculizumab (3-fold serial dilution starting at 100 ng/mL) was used as a standard.

[00338] The concentration of total C5, eculizumab-bound C5, and free eculizumab was determined from samples from eculizumab-treated PNH patients or from normal human serum as shown in FIG. 23. These results indicate the presence of sufficient eculizumab in PNH patient samples to saturate C5 and form C5:eculizumab complexes.

Examples 23 Effect of R5000 on Residual Hemolysis in Plasma of PNH Patients Treated with Eculizumab

[00339] Plasma hemolytic activity from a patient treated with eculizumab was determined in the presence of additional eculizumab and a C5 inhibitory peptide (R5000) by an assay designed to detect alternative pathway activation. Rabbit erythrocytes (Ers; #B302; Complement Technology Inc., Tyler, TX) were incubated with 25% plasma from an eculizumab-treated patient in the presence of MgEGTA (B106; Complement Technology Inc., Tyler, TX), complement inhibitors at 12.5 μM, 4, 2 μM, and 1.4 μM, and assay buffer (GVB; #B101; Complement Technology Inc., Tyler, TX) for 30 minutes at 37°C in a 96-well transparent assay plate. After incubation, the plate was centrifuged at 1000 g and 100 μl of the supernatant was transferred to a new 96-well transparent assay plate and the absorbance at 412 nm was measured. Absorption values were normalized to a control plate with 25% (final concentration) normal human serum (#NHS; Complement Technology Inc., Tyler, TX), which was provided for 100% hemolysis.

[00340] The results shown in FIG. 24 lead to the conclusion that even in the presence of sufficient eculizumab to bind to all of the available C5 in the patient sample, in this case there is still sufficient activation of the alternative complement pathway to cause hemolysis of rabbit erythrocytes. The addition of eculizumab for treatment provided only slight inhibition of hemolysis, confirming that the hemolytic activity was not due to a lack of eculizumab in the sample. However, the addition of R5000 completely abolished the residual hemolytic activity, demonstrating that the residual activity is C5 dependent and leading to the conclusion that R5000 can further inhibit hemolysis in the presence of excess eculizumab.

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SEQUENCE LIST

<110> RA PHARMACEUTICALS, INC.

<120> COMPLEMENT ACTIVITY MODULATORS

<130> 2011.1022PCT

<140> PCT/US2017/065005

<141> 2017-12-07

<150> 62/555.711

<151> 2017-09-08

<150> 62/525.284

<151> 2017-06-27

<150> 62/491.702

<151> 2017-04-28

<150> 62/430.959

<151> 2016-12-07

<160> 1

<170> Version 3.5 of the patent

<210> 1

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<220>

<223> N-terminus acetylated

<220>

<221> OTHER_SIGNIFICANTS

<222> (1)..(6)

<223> /note="Lactam bridge between residues"

<220>

<221> MODIFIED_REMAIN

<222>(7)..(7)

<223> N-methyl-aspartic acid

<220>

<221> MODIFIED_REMAIN

<222>(8)..(8)

<223> Tert-butylglycine

<220>

<221> MODIFIED_REMAIN

<222> (10)..(10)

<223> 7-Azatryptophan

<220>

<221> MODIFIED_REMAIN

<222> (14)..(14)

<223> Cyclohexylglycine

<220>

<221> MODIFIED_REMAIN

<222> (15)..(15)

<223> N-epsilon-(PEG24-gamma-glutamic acid-N-alpha-

hexadecanoyl)Lys

<400> 1

Lys Val Glu Arg Phe Asp Xaa Xaa Tyr Xaa Glu Tyr Pro Xaa Lys

1 5 10 15

<---

Claims (32)

1. A method of treating a complement related disorder in a subject, wherein the subject has not previously received eculizumab treatment, the method comprising: administering a C5 inhibitory polypeptide having the core sequence of SEQ ID NO: 1 to a subject at an initial loading dose of 0.1 to 0.6 mg/kg; and administering a C5 inhibitory polypeptide having the core sequence of SEQ ID NO: 1 to a subject at an initial therapeutic dose of about 0.1 mg/kg to about 0.3 mg/kg, moreover, the initial therapeutic dose is administered at least twice at regular intervals after the initial loading dose, wherein the subject's lactate dehydrogenase (LDH) levels are monitored during therapy, and wherein the initial therapeutic dose is replaced with a modified therapeutic dose of about 0.3 mg/kg to about 0.6 mg/kg at least two weeks after the initial therapeutic dose, or when the subject's LDH levels do not exceed 1.5 times the normal upper limit. 2. The method of claim 1, wherein the modified therapeutic dose of the C5 inhibitory polypeptide having the core sequence of SEQ ID NO: 1 contains an increased dose thereof. 3. The method of claim 1 or 2, wherein hemolysis in the subject's serum is reduced. 4. The method according to any one of paragraphs. 1-3, in which no adverse events are observed. 5. The method according to any one of paragraphs. 1-4, wherein at least one administration of a C5 inhibitory polypeptide having the core sequence of SEQ ID NO: 1 comprises a self-administration. 6. The method of claim 5 wherein self-administration is controlled. 7. The method of claim 6 wherein the self-administration is controlled remotely. 8. The method according to claim 6 or 7, wherein the self-administration is controlled using an intelligent device. 9. The method according to any one of paragraphs. 1-8, in which equal intervals of time are from every 12 hours to approximately every 168 hours. 10. The method of any one of claims 1-9, wherein residual hemolytic activity in the subject is reduced or eliminated. 11. The method of claim 1 wherein binding between C5 and C5 alternative pathway convertase is inhibited. 12. The method according to any one of paragraphs. 1-9, wherein the C5 inhibitory polypeptide having the core sequence of SEQ ID NO: 1 is co-administered with eculizumab. 13. The method according to any one of paragraphs. 1-12, wherein the complement disorder includes hemolysis and/or paroxysmal nocturnal hemoglobinuria. 14. The method of claim 11, wherein the subject is a carrier of a C5 polymorphism, wherein the polymorphism contains p.Arg885His. 15. A method of treating a complement related disorder in a subject in which the subject is currently being treated with eculizumab or has previously been treated with eculizumab, the method comprising administering a C5 inhibitory polypeptide having the core sequence of SEQ ID NO: 1 to the subject at baseline. loading dose, which is from 0.1 to 0.6 mg/kg; and administering a C5 inhibitory polypeptide having the core sequence of SEQ ID NO: 1 to a subject at an initial therapeutic dose of about 0.1 mg/kg to about 0.3 mg/kg, moreover, the initial therapeutic dose is administered at least twice at regular intervals after the initial loading dose, wherein the subject's lactate dehydrogenase (LDH) levels are monitored during therapy, and wherein the initial therapeutic dose is replaced with a modified therapeutic dose of about 0.3 mg/kg to about 0.6 mg/kg at least two weeks after the initial therapeutic dose, or when the subject's LDH levels do not exceed 1.5 times the normal upper limit. 16. The method of claim 15, wherein residual hemolytic activity in the subject is reduced or eliminated. 17. The method of claim 15 wherein binding between C5 and C5 alternative pathway convertase is inhibited. 18. The method according to any one of paragraphs. 15-17, wherein the C5 inhibitory polypeptide having the core sequence of SEQ ID NO: 1 is co-administered with eculizumab. 19. The method according to any one of paragraphs. 15-18, wherein the complement disorder includes hemolysis and/or paroxysmal nocturnal hemoglobinuria.

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