Modulators of Complement Activity

Abstract

To provide pharmaceutical compositions for treating complement-related disorders.SOLUTION: The present disclosure relates to polypeptide modulators of complement activity, including cyclic polypeptide modulators. A pharmaceutical composition of the present disclosure is a composition for treating a patient afflicted with complement-related disorders including paroxysmal nocturnal hemoglobinuria (PNH), and includes a polypeptide including an amino acid sequence of SEQ ID NO: 1.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to modulators of complement activity. [Background technology]

[0002] The immune response of vertebrates is composed of adaptive and innate immune components. The adaptive immune response is selective and slow to react to specific pathogens, whereas the components of the innate immune response recognize a broad range of pathogens and respond rapidly upon infection. One such component of the innate immune response is the complement system.

[0003] The complement system includes approximately 20 circulating complement component proteins that are primarily synthesized by the liver. This particular component of the immune response was first named "complement" because it was observed to complement the antibody response in the destruction of bacteria. These proteins remain in an inactive form before being activated in response to infection. Activation occurs through a pathway of proteolytic cleavage that is initiated by pathogen recognition, resulting in pathogen destruction. Three such pathways are known in the complement system, termed the classical, lectin, and alternative pathways. The classical pathway is activated when IgG or IgM molecules bind to the surface of a pathogen. The lectin pathway is initiated by mannan-binding lectin proteins that recognize sugar residues on the bacterial cell wall. The alternative pathway remains active at a low level in the absence of any specific stimulus. Although all three pathways differ with respect to the initiating event, all three pathways converge on the cleavage of complement component C3. C3 is cleaved into two products, termed C3a and C3b. Of these, C3b is covalently bound to pathogen surfaces, whereas C3a acts as a diffusible signal for promoting inflammation and recruiting circulating immune cells. Surface-associated C3b forms complexes with other components to initiate cascade reactions between later components of the complement system. The requirement for surface binding keeps complement activity localized, minimizing the destruction of non-target cells.

[0004] Pathogen-associated C3b promotes pathogen destruction in two ways. In one pathway, C3b is directly recognized by phagocytes, leading to phagocytosis of the pathogen. In the second pathway, pathogen-associated C3b initiates the formation of the membrane attack complex (MAC). In the first step, C3b complexes with other complement components to form the C5 convertase complex. Depending on the initial complement activation pathway, the components of this complex can differ. The C5 convertase formed as a result of the classical complement pathway contains C4b and C2a in addition to C3b. When formed by the alternative pathway, the C5 convertase contains two subunits of C3b as well as one Bb component.

[0005] Complement component C5 is cleaved into C5a and C5b by one of the C5 convertase complexes. C5a, much like C3a, diffuses into the circulation to promote inflammation and acts as a chemoattractant for inflammatory cells. C5b remains bound to the cell surface and triggers the formation of MAC through interactions with C6, C7, C8, and C9. MACs are hydrophilic pores present across cell membranes that disrupt cells by facilitating the free flow of fluids into and out of the cells.

[0006] A key component of all immune activity is the immune system's ability to distinguish between self and non-self cells. Pathological conditions result when the immune system fails 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 complement system is limited to targeting pathogenic cells. Many complement-related disorders and diseases involve the abnormal destruction of self cells by the complement cascade. In one example, subjects suffering from paroxysmal nocturnal hemoglobinuria (PNH) are unable to synthesize functional forms of the complement regulatory proteins CD55 and CD59 on hematopoietic stem cells. This results in complement-mediated hemolysis and a variety of downstream complications. Other complement-related disorders and diseases include, but are not limited to, autoimmune, neurological, hematological, and infectious diseases and disorders. Experimental evidence suggests that many complement-related disorders can be alleviated through inhibition of complement activity. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need for compositions and methods for selectively blocking complement-mediated cell destruction to treat associated indications. The present invention fulfills this need by providing related compositions and methods. [Means for solving the problem]

[0008] In some embodiments, the disclosure provides a method of treating paroxysmal nocturnal hemoglobinuria (PNH) in a subject not previously treated with eculizumab, comprising administering an initial loading dose of R5000 to the subject and administering an initial therapeutic dose of R5000 to the subject, wherein the initial therapeutic dose is administered two or more times at regular intervals after the initial loading dose. Lactate dehydrogenase (LDH) levels in the subject may be monitored throughout treatment. The initial therapeutic dose may be replaced with a modified therapeutic dose of R5000 after two or more weeks of administration of the initial therapeutic dose. The modified therapeutic dose of R5000 may comprise an increased dose of R5000. The initial therapeutic dose may be replaced with the modified therapeutic dose when the subject's LDH level is less than or equal to 1.5 times the upper limit of normal. The initial loading dose may comprise about 0.1 mg / kg to about 0.6 mg / kg of R5000. The initial therapeutic dose may comprise about 0.1 mg / kg to about 0.3 mg / kg of R5000. The initial therapeutic dose may be replaced with a modified therapeutic dose of about 0.3 mg / kg to about 0.6 mg / kg of R5000. Hemolysis in the subject's serum may be reduced. No adverse events may be observed. At least one administration of R5000 may comprise self-administration. The self-administration may be monitored. The self-administration may be remotely monitored. The self-administration may be monitored using a smart device. The regular interval may be about every 12 hours to about every 168 hours.

[0009] The disclosed methods may include a method of treating PNH in a subject currently being treated with eculizumab or previously treated with eculizumab, comprising administering R5000 to the subject. Residual hemolytic activity in the subject may be reduced or eliminated. The association between C5 and alternative pathway C5 convertase may be inhibited. R5000 may be co-administered with eculizumab. The subject may have a C5 polymorphism. The polymorphism may include p.Arg885His.

[0010] In some embodiments, the present disclosure provides a pharmaceutical composition comprising R5001 and at least one pharma- ceutically acceptable excipient. The at least one pharma-ceutically acceptable excipient may comprise at least one of sodium chloride and sodium phosphate. The pharmaceutical composition may comprise about 25 mM to about 100 mM sodium chloride. The pharmaceutical composition may comprise about 10 mM to about 100 mM sodium phosphate. R5001 may be present in a concentration of about 1 mg / mL to about 400 mg / mL.

[0011] In some aspects, the disclosure provides a method of treating PNH in a subject, comprising administering a pharmaceutical composition described herein. The subject may have been previously treated with an antibody-based therapeutic. The subject may be resistant or non-responsive to treatment with an antibody-based therapeutic. The antibody-based therapeutic 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 may be administered daily for at least two days. The pharmaceutical composition may be administered daily for about 12 weeks. The pharmaceutical composition may be administered daily for at least one year. The pharmaceutical composition may be administered subcutaneously or intravenously.

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

[0013] In some aspects, the disclosure provides a method of inhibiting residual C5 activity in a subject with PNH who is currently being treated with eculizumab or has previously been treated with eculizumab, comprising administering R5000 to the subject. The association between C5 and alternative pathway C5 convertase can be inhibited. R5000 can be co-administered with eculizumab. The subject can have a C5 polymorphism. The polymorphism can include p.Arg885His.

[0014] In some embodiments, the disclosure provides a method of measuring C5 levels in a sample, comprising immobilizing a capture agent on a substrate, where the capture agent binds to a site on C5 that is different from the eculizumab binding site; contacting the substrate with a sample, where the immobilized capture agent binds to C5 in the sample; contacting the substrate with a detection reagent, where the detection reagent binds to the C5 bound to the immobilized capture agent; and measuring the level of bound detection reagent as an indication of C5 levels in the sample. The detection reagent may comprise a detectable label. The detection reagent may be detected using a secondary detection reagent. The detection reagent may comprise an anti-C5 antibody. Both free C5 levels and C5 levels associated with eculizumab may be measured. The substrate may be contacted with a second detection reagent, where the second detection reagent binds to eculizumab, and the method comprises measuring the level of bound second detection reagent as an indication of C5 levels associated with eculizumab. The second detection reagent may comprise a detectable label. The second detection reagent may be detected using a secondary detection reagent. The capture agent may include a mutant of R5000. The R5000 mutant may include an N-terminal biotinylated PEG moiety and a substitution of the C-terminal lysine of R5000 in the R5000 mutant with norvaline.

[0015] The disclosed method may include a method of measuring free eculizumab levels in a sample, comprising immobilizing a capture agent on a substrate, where 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, where the C5-capture agent complex comprises immobilized C5; contacting the substrate with a sample, where the immobilized C5 binds to eculizumab in the sample; contacting the substrate with a detection reagent, where the detection reagent binds to eculizumab; and measuring the level of bound detection reagent as an indication of free eculizumab levels in the sample. The capture agent may comprise a variant of R5000. The R5000 variant may comprise an N-terminal biotinylated PEG moiety and a substitution of the C-terminal lysine of R5000 in the R5000 variant with norvaline. The detection reagent may be an antibody. The detection reagent may comprise a detectable label. The detection reagent may be detected using a secondary detection reagent.

[0016] The above and other objects, features and advantages will become apparent from the following description of specific embodiments of the invention as well as the accompanying drawings which illustrate, by way of example, the principles of various embodiments of the invention. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a scatter plot showing R5000 inhibition of C5a production. [Diagram 2] FIG. 13 is a scatter plot showing R5000 inhibition of membrane attack complex formation. [Diagram 3] FIG. 1 is a scatter plot showing R5000 inhibitor activity in a cynomolgus monkey model. [Figure 4] FIG. 1 is a scatter plot showing the pharmacokinetic and pharmacodynamic correlations of R5000 in male cynomolgus monkeys after multiple subcutaneous doses at 0.21 mg / kg. [Diagram 5] FIG. 1 is a scatter plot showing the pharmacokinetic and pharmacodynamic correlations of R5000 in male cynomolgus monkeys after multiple subcutaneous doses at 4.2 mg / kg. [Figure 6] 1 is a line graph showing the concentration of R5000 in cynomolgus monkeys after the first dose in a repeated dose toxicity study. [Figure 7] 1 is a line graph showing the concentration of R5000 in cynomolgus monkeys after the final dose in a repeated dose toxicity study. [Figure 8] 1 is a graph showing predicted plasma concentrations of R5000 in humans following daily dosing of R5000. [Figure 9A] 1 is a graph showing dose-dependent maximum plasma concentration levels of R5000 in a single ascending dose clinical trial in humans. [Figure 9B] FIG. 1 is a graph showing plasma concentrations over time following administration of a single dose of R5000. [Figure 10A] 1 is a graph showing percent hemolysis over time following administration of a single dose of R5000 over a 4 day duration in humans. [Figure 10B] 1 is a graph showing percent CH50 over time following administration of a single dose of R5000 in humans. [Figure 10C] 1 is a graph showing percent hemolysis at various doses over a 28 day duration in humans. [Figure 11] 1 is a graph showing percent complement activity over time following administration of a single dose of R5000 in humans. [Figure 12A] 1 is a graph showing the change in percent hemolysis versus R5000 concentration in a multiple dose human study. [Figure 12B] 1 is a graph showing plasma concentrations of R5000 over time in a multiple dose human study. [Figure 13A] 1 is a graph showing changes in complement activity over time with R5000 treatment in a multiple dose human study. [Figure 13B] 1 is a graph showing changes in complement activity over time with R5000 treatment in a multiple dose human study. [Figure 14] 1 is a graph showing predicted drug plasma concentrations and hemolysis inhibition by R5000 in humans. [Figure 15]1 is a graph showing the predicted pharmacokinetics and pharmacodynamics of R5000 in humans. [Figure 16A] 1 is a graph showing hemolysis in the R5000 single dose cohort of healthy human volunteers. [Figure 16B] 1 is a graph showing predicted percent trough plasma concentrations in the presence and absence of a loading dose of R5000. [Figure 16C] 1 is a graph showing predicted inhibition of hemolysis by 0.1 mg / kg R5000 in humans. [Figure 17] 1 is a graph showing reduction of hemolysis by mR5000 in patient samples. [Figure 18A] 1 is a graph showing percent hemolysis in patient samples over 6 weeks of treatment with R5000. [Figure 18B] 1 is a graph showing the change in hemoglobin levels in patient samples over 7 weeks of treatment with R5000. [Figure 18C] 1 is a set of graphs showing the change in patients' lactate dehydrogenase (LDH) levels (individual and combined averages) over 7 weeks of treatment with R5000. [Figure 18D] 1 is a graph showing levels of R5000 in patient samples over 6 weeks of treatment with R5000. [Figure 19] 1 is a graph showing response units over time generated during surface plasmon resonance analysis of C5 inhibitor binding. [Figure 20] 1 is a graph showing percent hemolysis of PNH patient red blood cells in the presence of R5000 or eculizumab. [Figure 21] 1 is a graph showing percent hemolysis of PNH patient red blood cells in the presence of R5000 or eculizumab. [Figure 22] 1 is a graph showing percent hemolysis in red blood cells carrying recombinant C5 with the known polymorphism p.Arg885His following exposure to various concentrations of eculizumab or R5000. [Diagram 23]1 is a graph showing concentrations of C5, C5:eculizumab, and eculizumab in PNH patient serum. [Figure 24] 1 is a graph showing percent residual hemolysis in eculizumab-treated PNH patient plasma in the presence of R5000 or additional eculizumab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] I. Compounds and Compositions In some embodiments, the present disclosure provides compounds and compositions that function to regulate complement activity. Such compounds and compositions may include inhibitors that block complement activation. As used herein, "complement activity" includes the activation of the complement cascade, the formation of cleavage products from complement components such as C3 or C5, the assembly of downstream complexes following a cleavage event, or any process or event that is associated with or results from the cleavage of a complement component, such as C3 or C5. Complement inhibitors may include C5 inhibitors that block complement activation at the level of complement component C5. C5 inhibitors may bind to C5 and prevent its cleavage by C5 convertase into cleavage products C5a and C5b. As used herein, "complement component C5" or "C5" is defined as the complex that is cleaved by C5 convertase into at least cleavage products C5a and C5b. "C5 inhibitors" according to the present invention include any compound or composition that inhibits the processing or cleavage of the precleaved complement component C5 complex or the cleavage products of complement component C5.

[0019] It is understood that inhibition of C5 cleavage blocks the assembly and activity of the cytolytic membrane attack complex (MAC) on glycosylphosphatidylinositol (GPI) adhesive protein-deficient erythrocytes. In some cases, the C5 inhibitors presented herein can also bind to C5b and block the binding of C6 and the subsequent assembly of the C5b-9MAC.

[0020] Peptide-Based Compounds In some embodiments, the C5 inhibitors of the present disclosure are polypeptides. According to the present invention, any amino acid-based molecule (natural or non-natural) may be referred to as a "polypeptide", which term encompasses "peptides", "peptidomimetics" and "proteins". "Peptides" are traditionally considered to range in size from about 4 to about 50 amino acids. Polypeptides larger than about 50 amino acids are commonly referred to as "proteins".

[0021] C5 inhibitor polypeptides may be linear or cyclic. Cyclic polypeptides include any polypeptides that have one or more cyclic features, such as loops and / or internal bonds, as part of their structure. In some embodiments, cyclic polypeptides are formed when a molecule acts as a bridging moiety to link two or more regions of a polypeptide. As used herein, the term "bridging moiety" refers to one or more components of a bridge formed between two adjacent or non-adjacent amino acids, non-natural amino acids, or non-amino acids in a polypeptide. A bridging moiety may be of any size composition. In some embodiments, a bridging moiety may include 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 in adjacent or non-adjacent amino acids, non-natural amino acids, non-amino acid residues, or combinations thereof. A bridging moiety may include 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, a bridging moiety includes an amide bond between an amine functionality and a carboxylic acid functionality, each present in an amino acid, non-natural amino acid, or non-amino acid residue side chain. In some embodiments, the amine or carboxylic acid functionality is part of a non-amino acid residue or a non-naturally occurring amino acid residue.

[0022] The C5 inhibitor polypeptide may be cyclized through the carboxy terminus, amino terminus, or any other convenient attachment point, such as the sulfur of a cysteine ​​(e.g., through the formation of a disulfide bond between two cysteine ​​residues in the sequence) or through any side chain of an amino acid residue. Additionally, the bonds forming the cyclic loop may include, but are not limited to, maleimide bonds, amide bonds, ester bonds, ether bonds, thiol ether bonds, hydrazone bonds, or acetamide bonds.

[0023] In some embodiments, the cyclic C5 inhibitor polypeptide of the present invention is formed with lactam moiety.Such cyclic polypeptide may be formed, for example, by synthesis on solid support Wang resin using standard Fmoc chemistry.In some cases, Fmoc-ASP(allyl)-OH and Fmoc-LYS(alloc)-OH are incorporated into polypeptide to serve as precursor monomer for lactam bridge formation.

[0024] The C5 inhibitor polypeptide of the present invention may be a peptide mimetic. A "peptide mimetic" or "polypeptide mimetic" is a polypeptide whose molecule contains structural elements that are not found in natural polypeptides (i.e., polypeptides that are composed of only the 20 proteinogenic amino acids). In some embodiments, a peptide mimetic can reproduce or mimic the biological action of natural peptides. A peptide mimetic may differ from natural polypeptides in many ways, for example, through changes in backbone structure or through the presence of non-naturally occurring amino acids. In some cases, peptidomimetics may include amino acids having side chains not found in the 20 known proteinogenic amino acids; non-polypeptide bridging moieties used to effect cyclization between the ends or interior of the molecule; replacement of amide bond hydrogen moieties with methyl groups (N-methylation) or other alkyl groups; replacement of peptide bonds with chemical groups or bonds that are resistant to chemical or enzymatic treatment; modifications of the N- and C-termini; and / or conjugation to non-peptide extensions (e.g., polyethylene glycol, lipids, carbohydrates, nucleosides, nucleotides, nucleoside bases, various small molecules, or phosphate or sulfate groups).

[0025] As used herein, the term "amino acid" includes residues of natural and non-natural amino acids. The 20 naturally occurring proteinogenic amino acids are identified and referred to herein by either their one-letter or three-letter designations, such as 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). Natural amino acids exist in their levorotatory (L) stereoisomeric form. The amino acids referred to herein are L-stereoisomers unless otherwise specified. The term "amino acid" also includes amino acids having conventional amino protecting groups (e.g., acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g., as (C1-C6) alkyl, phenyl, or benzyl esters or amides; or as α-methylbenzyl amides). Other suitable amino- and carboxy-protecting groups are known to those of skill in the art (see, e.g., Greene, TW; Wutz, PGM, Protecting Groups In Organic Synthesis, 2nd Edition, 1991, New York, John Wiley & sons, Inc., and documents cited therein, the contents of each of which are incorporated herein by reference in their entirety). The polypeptides and / or polypeptide compositions of the present invention may also include modified amino acids.

[0026] "Unnatural" 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, α,α-substituted amino acids, β-amino acids, α-hydroxy amino acids, D-amino acids, and other unnatural amino acids known in the art (see, e.g., Josephson et al., 2005, J. Am. Chem. Soc., vol. 127, 2005). (See, for example, pp. 11727-11735; Forster, AC et al., 2003, Proc. Natl. Acad. Sci. USA 100, pp. 6353-6357; Subtelny et al., 2008, J. Am. Chem. Soc. 130, pp. 6131-6136; Hartman, MCT et al., 2007, PLoS ONE 2:e972; and Hartman et al., 2006, Proc. Natl. Acad. Sci. USA 103, pp. 4356-4361). Additional unnatural amino acids useful for optimizing the polypeptides and / or polypeptide compositions of the present invention include 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid, 1-amino-2,3-hydro-1H-indene-1-carboxylic acid, homolysine, homoarginine, homoserine, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 5-aminopentanoic acid, 5-aminohexanoic 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, penicillamine, 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) Propanoic acid, cyclopentylglycine, cyclohexylglycine, cyclopropylglycine, η-ω-methyl-arginine, 4-chlorophenylalanine, 3-chlorotyrosine, 3-fluorotyrosine, 5-fluorotryptophan, 5-chlorotryptophan, citrulline, 4-chloro-homophenylalanine, homophenylalanine, 4-aminomethyl-phenylalanine, 3-aminomethyl-phenylalanine, octylglycine, norleucine, tranexamic acid, 2-aminopentanoic acid, 2-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, 2-aminodecanoic acid, 2-aminoundecanoic acid, 2-aminododecanoic acid, aminovaleric acid and 2-(2-aminoethoxy)acetic acid, pipecolic acid, 2-carboxyazetidine, hexafluoroleucine, 3-fluorovaline, 2-amino-4,4-Difluoro-3-methylbutanoic acid, 3-fluoro-isoleucine, 4-fluoroisoleucine, 5-fluoroisoleucine, 4-methyl-phenylglycine, 4-ethyl-phenylglycine, 4-isopropyl-phenylglycine, (S)-2-amino-5-azidopentanoic acid (also referred to herein as "X02"), (S)-2-aminohept-6-enoic acid (also referred to herein as "X30"), (S)-2-aminopent-4-ynoic acid (also referred to herein as "X40"), (S)-2-aminopent-4-ynoic acid (also referred to herein as "X50"), (S)-2-aminopent-4-ynoic acid (also referred to herein as "X60"), (S)-2-aminopent-4-ynoic acid (also referred to herein as "X70"), (S)-2-aminopent-4-ynoic acid (also referred to herein as "X80"), (S)-2-aminopent-4-ynoic acid (also referred to herein as "X90"), (S)-2-aminopent-4-ynoic acid (also referred to herein as "X1 ..."). (S)-2-aminopent-4-enoic acid (also referred to herein as "X12"), (S)-2-amino-5-(3-methylguanidino)pentanoic acid, (S)-2-amino-3-(4-(aminomethyl)phenyl)propanoic acid, (S)-2-amino-3-(3-(aminomethyl)phenyl)propanoic acid, (S)-2-amino-4-(2-aminobenzo[d]oxazol-5-yl)butanoic acid, (S)-leucinol, (S)-valinol, (S)- S)-tert-Leucinol, (R)-3-Hethylbutan-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)propanoic acid, (S)-2-Amino-3-(oxazol-5-yl)propanoic acid, (S)-2-Amino-3-(1,3,4-oxadiazol-2 ... 2,4-oxadiazol-3-yl)propanoic acid, (S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)propanoic acid, and (S)-2-amino-3-(1H-indazol-3-yl)propanoic acid, (S)-2-amino-3-(oxazol-2-yl)butanoic acid, (S)-2-amino-3-(oxazol-5-yl)butanoic acid, (S)-2-amino-3-(1,3,4-oxadiazol-2-yl)butanoic acid, (S)-2-amino-3-(1,2,4-oxadiazol-3-yl)butanoic acid, (S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)butanoic acid, and (S)-2-amino-3-(1H-indazol-3-yl)butanoic acid, 2-(2'MeOphenyl)-2-aminoacetic acid, tetrahydro-3-isoquinolinecarboxylic acid, and their stereoisomers (including but not limited to D and L isomers).

[0027] Other unnatural amino acids useful in optimizing a polypeptide or polypeptide composition of the invention include, but are not limited to, fluorinated amino acids in which one or more carbon-bonded hydrogen atoms have been replaced with fluorine. The number of fluorine atoms can range from one to all hydrogen atoms. Examples of such amino acids include, but are not limited to, 3-fluoroproline, 3,3-difluoroproline, 4-fluoroproline, 4,4-difluoroproline, 3,4-difluroproline, 3,3,4,4 tetrafluoroproline, 4-fluorotryptophan, 5-flurotryptophan, 6-fluorotryptophan, 7-fluorotryptophan, and stereoisomers thereof.

[0028] Additional unnatural amino acids useful for optimizing the polypeptides of the present invention include, but are not limited to, those disubstituted at the α-carbon. These include amino acids in which the two substituents on the α-carbon are the same, such as α-aminoisobutyric acid and 2-amino-2-ethylbutanoic acid, as well as those in which the substituents are different, such as α-methylphenylglycine and α-methylproline. Additionally, the substituents on the α-carbon may join together to form a ring, such as 1-aminocyclopentanecarboxylic acid, 1-aminocyclobutanecarboxylic acid, 1-aminocyclohexanecarboxylic acid, 3-aminotetrahydrofuran-3-carboxylic acid, 3-aminotetrahydropyran-3-carboxylic acid, 4-aminotetrahydropyran-4-carboxylic acid, 3-aminopyrrolidine-3-carboxylic acid, 3-aminopiperidine-3-carboxylic acid, 4-aminopiperidine-4-carboxylic acid, and stereoisomers thereof.

[0029] Other unnatural amino acids useful for optimizing a polypeptide or polypeptide composition of the invention include, but are not limited to, analogs of tryptophan 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 can be saturated, partially unsaturated, or fully unsaturated. The ring system can be substituted at any substitutable atom with 0, 1, 2, 3, or 4 substituents. Each substituent can be independently selected from H, F, Cl, Br, CN, COOR, CONRR', oxo, OR, NRR'. Each R and R' can be independently selected from H, C1-C20 alkyl, or C1-C20 alkyl-O-C1-20 alkyl.

[0030] In some embodiments, analogs of tryptophan (also referred to herein as "tryptophan analogs") may be useful in optimizing the polypeptides or polypeptide compositions of the present 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 stereoisomers thereof. Unless otherwise specified, the term "azatryptophan" and its abbreviation "azaTrp" as used herein refer to 7-azatryptophan.

[0031] Modified amino acid residues useful for optimizing the polypeptides and / or polypeptide compositions of the present invention include, but are not limited to, those that are chemically blocked (reversibly or irreversibly); those that have been chemically modified at the N-terminal amino group or at its side chain group; those that have been chemically modified at the amide backbone, e.g., N-methylated, D (unnatural amino acid) and L (natural amino acid) stereoisomers, or residues where the side chain functional group has been chemically modified to another functional group. In some embodiments, modified amino acids include, but are not limited to, methionine sulfoxide, methionine sulfone, aspartic acid-(β-methyl ester), modified amino acids of aspartic acid, N-ethylglycine, modified amino acids of glycine, alanine carboxamide, and / or modified amino acids of alanine. Unnatural amino acids may be purchased from Sigma-Aldrich (St. Louis, MO), Bachem (Torrance, CA), or other suppliers. Unnatural amino acids can further include any of those listed in Table 2 of US Patent Application Publication No. 2011 / 0172126, the contents of which are incorporated herein by reference in their entirety.

[0032] The present invention contemplates variants and derivatives of the polypeptides presented herein. These include substitution, insertion, deletion, and covalent variants and derivatives. As used herein, the term "derivative" is used synonymously with the term "variant" and refers to a molecule that is modified or altered in any way relative to a reference or starting molecule.

[0033] Polypeptides of the invention may include any of the following components, features, or moieties, where the abbreviations used herein include: "Ac" and "NH2" represent acetyl and amidated termini, respectively; "Nvl" is an abbreviation for norvaline; "Phg" is an abbreviation for phenylglycine; "Tbg" is an abbreviation for tert-butylglycine; "Chg" is an abbreviation for cyclohexylglycine; and "(N-Me)X" is an abbreviation for the N-methylated form of an amino acid represented by the one-letter or three-letter amino acid code in place of the variable "X" written as N-methyl-X [e.g., (N-Me)D or (N-Me)Asp represent aspartate, aryl ... aspartic acid or N-methyl-aspartic acid], "azaTrp" stands for azatryptophan, "(4-F)Phe" stands for 4-fluorophenylalanine, "Tyr(OMe)" stands for O-methyltyrosine, "Aib" stands for aminoisobutyric acid, "(homo)F" or "(homo)Phe" stands for homophenylalanine, "(2-OMe)Phg" stands for 2-O-methylphenylglycine, and "(5-F)W" stands for 5-fluorotryptophan. where "DX" means the D-stereoisomer of a given amino acid "X" [e.g., (D-Chg) is an abbreviation for 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" means a tetrahydroisoquinoline residue, and "Asp(T)" means (S)-2-amino-3-(1H-tetrahydro-2-phenylindole) residue. "(N-Me-4-F)Phe" or "(N-Me-4-F)Phe" means N-methyl-4-fluorophenylalanine, "(m-Cl-homo)Phe" means meta-chlorohomophenylalanine, "(des-amino)C" means 3-thiopropionic acid, "(α-methyl)D" means α-methyl-L-aspartic acid, and "2Nal" means 2-naphthylalanine;"(3-aminomethyl)Phe" refers to 3-aminomethyl-L-phenylalanine, "Cle" refers to cycloleucine, "Ac-pyran" refers to 4-amino-tetrahydro-pyran-4-carboxylic acid, "(Lys-C16)" refers to N-ε-palmitoyl lysine, "(Lys-C12)" refers to N-ε-lauryl lysine, "(Lys-C10)" refers to N-ε-caprylyl lysine, "(Lys-C8)" refers to N-ε-caprylic acid lysine, "[xxylyl(y,z)]" refers to a xylyl bridge moiety between two thiol-containing amino acids, where x can be m, p, or o and represents the use of meta-, para-, or ortho-dibromoxylene (respectively) to form the bridge moiety, and the numerical identifiers y and z represent the ring structure. determines the amino acid position within the polypeptide of the amino acid involved in the conjugation, "[cyclo(y,z)]" refers to the formation of a bond between two amino acid residues, where the numerical identifiers y and z determine the positions of the residues involved in the conjugation, "[cyclo-olefinyl(y,z)]" refers to the formation of a bond between two amino acid residues by olefin metathesis, where the numerical identifiers y and z determine the positions of the residues involved in the conjugation, "[cyclo-thioalkyl(y,z)]" refers to the formation of a thioether bond between two amino acid residues, where the numerical identifiers y and z determine the positions of the residues involved in the conjugation, and "[cyclo-triazolyl(y,z)]" refers to the formation of a triazole ring between two amino acid residues, where the numerical identifiers y and z determine the positions of the residues involved in the conjugation. "B20" means N-ε-(PEG2-γ-glutamic acid-N-α-octadecanedioic 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].

[0034] B20

[0035] [ka]

[0036] "B28" means N-ε-(PEG24-γ-glutamic acid-N-α-hexadecanoyl)lysine. B28

[0037] [ka]

[0038] "K14" refers to N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine. All other symbols refer to the standard single letter amino acid codes.

[0039] Some C5 inhibitor polypeptides include 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, the C5 inhibitor polypeptide includes at least 30 amino acids.

[0040] Some C5 inhibitors of the present invention include a C-terminal lipid moiety. Such lipid moiety may include a fatty acyl group (e.g., a saturated or unsaturated fatty acyl group). In some cases, the fatty acyl group may be a palmitoyl group.

[0041] C5 inhibitors having fatty acyl groups may include one or more molecular linkers linking the fatty acid to the peptide. Such molecular linkers may include amino acid residues. In some cases, L-gamma glutamic acid residues may be used as molecular linkers. In some cases, the molecular linkers may include one or more polyethylene glycol (PEG) linkers. The PEG linkers of the present invention may include about 1 to about 5, about 2 to about 10, about 4 to about 20, about 6 to about 24, about 8 to about 32, or at least 32 PEG units.

[0042] The C5 inhibitors of the present invention may have a molecular weight of about 200 g / mol to about 600 g / mol, about 500 g / mol to about 2000 g / mol, about 1000 g / mol to about 5000 g / mol, about 3000 g / mol to about 4000 g / mol, about 2500 g / mol to about 7500 g / mol, about 5000 g / mol to about 10000 g / mol, or at least 10000 g / mol.

[0043] In some embodiments, the C5 inhibitor polypeptide of the present invention comprises R5000. The core amino acid sequence of R5000 ([cyclo(1,6)]Ac-KVERFD-(N-Me)D-Tbg-Y-azaTrp-EYP-Chg-K; SEQ ID NO:1) comprises 15 amino acids (all L-amino acids), such as four unnatural amino acids [N-methyl-aspartic acid or "(N-Me)D", tert-butylglycine or "Tbg", 7-azatryptophan or "azaTrp", and cyclohexylglycine or "Chg"]; a lactam bridge between K1 and D6 of the polypeptide sequence; and a C-terminal lysine residue with a modified side chain, forming N-ε-(PEG24-γ-glutamic acid-N-α-hexadecanoyl)lysine residue (also referred to herein as "B28"). The C-terminal lysine side chain modification comprises a polyethylene glycol (PEG) spacer (PEG24), where PEG24 is attached to an L-gamma glutamic acid residue derivatized with a palmitoyl group.

[0044] In some embodiments, the present invention includes variants of R5000. In some R5000 variants, the C-terminal lysine side chain moiety may be modified. In some cases, the PEG24 spacer (having 24 PEG subunits) of the C-terminal lysine side chain moiety may contain fewer or additional PEG subunits. In other cases, the palmitoyl group of the C-terminal lysine side chain moiety may be replaced with another saturated or unsaturated fatty acid. In further cases, the L-gamma glutamic acid linker of the C-terminal lysine side chain moiety (between the PEG group and the acyl group) may be replaced with an alternative amino acid or non-amino acid linker.

[0045] In some embodiments, the C5 inhibitor may include an active metabolite or variant of R5000. The metabolite may include R5001. As used herein, the term "R5001" refers to a variant of R5000 with ω-hydroxylation of the palmitoyl tail. R5001 may be synthesized or formed by hydroxylation of an R5000 precursor. R5001 may have the ability to bind to C5 with an equilibrium dissociation constant (KD) comparable to or similar to that of R5000 binding to C5 (e.g., about ±20% of the KD of R5000 binding to C5). R5001 may have a half-maximal inhibitory concentration (IC) comparable to or similar to that of R5000. 50 ) (e.g., IC of R5000 to inhibit cleavage of C5 50 R5001 may have the ability to inhibit cleavage of C5 and / or block one or more downstream events associated with cleavage of C5 at an IC5 concentration of 0.01 or 0.02 or 0.05 or 0.05% relative to that of R5000. 50 (e.g., IC of R5000 to inhibit hemolysis 50 The antibody may have the ability to inhibit complement dependent hemolysis at about ±20% of the complement concentration.

[0046] In some embodiments, R5000 variants may contain modifications in the core polypeptide sequence in R5000 that may be combined with features of the cyclic or C-terminal lysine side chain moieties of R5000. Such variants may have at least 50%, at least 55%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the core polypeptide sequence of (SEQ ID NO:1).

[0047] In some cases, the R5000 variant may be cyclized by forming a lactam bridge between amino acids other than those used in R5000. The C5 inhibitor of the present invention may be developed or modified to achieve specific binding properties. Inhibitor binding may be evaluated by measuring the rate of association and / or dissociation with a specific target. In some cases, compounds show strong and rapid association with targets combined with slow dissociation. In some embodiments, the C5 inhibitor of the present invention shows strong and rapid association with C5. Such inhibitors may further show slow dissociation with C5.

[0048] The C5 inhibitor of the present invention that binds to the C5 complement protein may be about 0.001 nM to about 0.01 nM, about 0.005 nM to about 0.05 nM, about 0.01 nM to about 0.1 nM, about 0.05 nM to about 0.5 nM, about 0.1 nM to about 1.0 nM, about 0.5 nM to about 5.0 nM, about 2 nM to about 10 nM, about 8 nM to about An equilibrium dissociation constant (K D ) may bind to the C5 complement protein.

[0049] In some embodiments, the C5 inhibitor of the present invention blocks the formation or production of C5a from C5. In some cases, the formation or production of C5a is blocked after activation of the alternative pathway of complement activation. In some cases, the C5 inhibitor of the present invention blocks the formation of the membrane attack complex (MAC). Such inhibition of MAC formation may be due to the binding of the C5 inhibitor to the C5b subunit. The binding of the C5 inhibitor to the C5b subunit may prevent the binding of C6, resulting in the disruption of MAC formation. In some embodiments, this inhibition of MAC formation occurs after activation of the classical, alternative, or lectin pathway.

[0050] The C5 inhibitor of the present invention may be synthesized using chemical processes.In some cases, such synthesis removes the risks associated with the production of biological products in mammalian cell lines.In some cases, chemical synthesis may be simpler and more cost-effective than biological production processes.

[0051] In some embodiments, the C5 inhibitor (e.g., R5000 and / or its active metabolites or variants) composition may be a pharmaceutical composition comprising at least one pharma- ceutically acceptable excipient. In some embodiments, the pharma- ceutically acceptable excipient may comprise at least one of a salt and a buffering agent. The salt may be sodium chloride. The buffering agent may be sodium phosphate. The sodium chloride may be present at a concentration of about 0.1 mM to about 1000 mM. In some cases, the sodium chloride may be present at a concentration of about 25 mM to about 100 mM. The sodium phosphate may be present at a concentration of about 0.1 mM to about 1000 mM. In some cases, the sodium phosphate is present at a concentration of about 10 mM to about 100 mM.

[0052] In some embodiments, the C5 inhibitor (e.g., R5000 and / or its active metabolites or variants) composition may contain from about 0.01 mg / mL to about 4000 mg / mL of the C5 inhibitor. In some cases, the C5 inhibitor is present at a concentration of from about 1 mg / mL to about 400 mg / mL.

[0053] Isotopic Mutations The polypeptides of the present invention may contain one or more atoms that are isotopes. As used herein, the term "isotope" refers to a chemical element that has one or more additional neutrons. In one embodiment, the polypeptides of the present invention may be deuterated. As used herein, the term "deuterated" refers to a substance that has one or more hydrogen atoms replaced by a deuterium isotope. A deuterium isotope is an isotope of hydrogen. A hydrogen nucleus contains one proton, whereas a deuterium nucleus contains both a proton and a neutron. The compounds and pharmaceutical compositions of the present invention may be deuterated to alter physical properties such as stability or to allow for use in diagnostic and experimental applications.

[0054] II.How to use Provided herein are methods of modulating complement activity using the compounds and / or compositions of the invention.

[0055] treatment index A key component of all immune activity (innate and adaptive) is the ability of the immune system to distinguish between self and non-self cells. Pathology arises 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, ensuring that the complement system is specific for pathogenic microorganisms. A number of complement-related disorders and diseases are associated with the abnormal destruction of self cells by the complement cascade.

[0056] The methods of the invention include methods of treating complement-associated disorders using the compounds and compositions of the invention. "Complement-associated disorders," as referred to herein, may include any condition related to a dysfunction of the complement system, such as the cleavage or processing of a complement component, such as C5.

[0057] In some embodiments, the methods of the invention include methods of inhibiting complement activity in a subject. In some cases, the percentage of complement activity inhibited in the 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 cases, this level of inhibition and / or maximum inhibition of complement activity may be achieved from about 1 hour to about 3 hours after administration, from about 2 hours to about 4 hours after administration, from about 3 hours to about 10 hours after administration, from about 5 hours to about 20 hours after administration, or from about 12 hours to about 24 hours after administration. The inhibition of complement activity may continue throughout 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 level of inhibition may be achieved through daily administration. Such daily administration may include administration for 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 cases, a subject may be administered a compound or composition of the present disclosure for the life of such subject.

[0058] In some embodiments, the method of the present invention includes a method of inhibiting C5 activity in a subject. "C5-dependent complement activity" or "C5 activity" as used herein refers to the activation of complement cascade through cleavage of C5, assembly of downstream cleavage products of C5, or any other process or event associated with or resulting from cleavage of C5. 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%.

[0059] 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 may 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%, about 90% to about 99%, or about 97% to about 99.5%. In some cases, hemolysis is reduced by at least 50%, 60%, 70%, 80%, 90% or 95%.

[0060] According to some methods, the percent inhibition of hemolysis is about ≧90% to about ≧99% (e.g., ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%). In some cases, this level of inhibition and / or maximum inhibition of hemolysis may be achieved. It may be achieved from about 1 hour to about 3 hours after administration, from about 2 hours to about 4 hours after administration, from about 3 hours to about 10 hours after administration, from about 5 hours to about 20 hours after administration, or from about 12 hours to about 24 hours after administration. The inhibition of the hemolytic activity level may continue throughout 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 level of inhibition may be achieved through daily administration. Such daily administration may include administration for 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 cases, a subject may be administered a compound or composition of the present disclosure for the life of such subject.

[0061] The C5 inhibitors of the present invention may be used to treat one or more indications, where C5 inhibitor treatment results in little or no adverse effects. In some cases, no adverse cardiovascular, respiratory, and / or central nervous system (CNS) effects occur. In some cases, no changes occur in heart rate and / or arterial pressure. In some cases, no changes occur to respiratory rate, tidal volume, and / or minute ventilation.

[0062] "Reduce" or "lower" in the context of a disease marker or symptom means a significant, often a statistically significant, decrease in such level, which can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably to a level that is recognized as within the normal range in individuals without such disorder.

[0063] "Increase" or "elevate" in the context of a disease marker or symptom means a significant, often a statistically significant, increase in such level, which can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably to a level that is recognized as being within the normal range in individuals without such disorder.

[0064] The therapeutic or prophylactic effect is evident when there is a significant, often statistically significant, improvement in one or more parameters of the pathology, or by not worsening or developing symptoms that would normally be expected. By way of example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more in measurable parameters of the disease may indicate effective treatment. The efficacy of a given compound or composition may also be determined using experimental animal models of a given disease known in the art. When using experimental animal models, the efficacy of treatment is demonstrated when there is a statistically significant modulation in markers or symptoms.

[0065] Paroxysmal nocturnal hemoglobinuria In some embodiments, provided herein is a method of treating paroxysmal nocturnal hemoglobinuria (PNH) with a compound or composition, e.g., a pharmaceutical composition, of the present invention. PNH is a rare complement-related disorder caused by acquired mutations in the phosphatidylinositol glycan anchor biosynthesis class A (PIG-A) gene derived from pluripotent hematopoietic stem cells (Pu, JJ et al., Clin Transl Sci., June 2011, Vol. 4, No. 3, pp. 219-24). PNH is characterized by bone marrow failure, hemolytic anemia, and thrombosis. The PIG-A gene product is required for the generation of the glycolipid anchor glycosylphosphatidylinositol (GPI), which is utilized to tether proteins to the plasma membrane. Two complement regulatory proteins involved in protecting cells from the lytic activity of the terminal complement complex, CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis), become non-functional in the absence of GPI, leading to the activation of C5 and the accumulation of specific complement proteins on the surface of red blood cells (RBCs), resulting in complement-mediated destruction of these cells.

[0066] Patients with PNH initially present with hemoglobinuria, abdominal pain, smooth muscle dystonia, and fatigue, i.e., PNH-associated symptoms or disorders. PNH is also characterized by intravascular hemolysis (the primary clinical manifestation of the disease) and venous thrombosis. Venous thrombosis can occur at abnormal sites, including, but not limited to, the hepatic, mesenteric, cerebral, and cutaneous veins (Parker, C. et al., 2005, Blood 106:3699-709 and Parker, CJ, 2007, Exp Hematol. 35:523-33). Currently, the C5 inhibitor monoclonal antibody eculizumab (SOLIRIS®, Alexion Pharmaceuticals, Cheshire, Connecticut) is the only approved treatment for PNH.

[0067] Treatment with eculizumab results in sufficient control of intravascular hemolysis in most PNH patients (Schrezenmeier, H. et al., 2014, Haematologica, 99:922-9). However, Nishimura and colleagues described 11 patients in Japan (3.2% of patients with PNH) with mutations in the C5 gene that prevent eculizumab from binding to C5 and that did not respond to treatment with the antibody (Nishimura, JI. et al., 2014, N Engl J Med., 370:632-9). Furthermore, eculizumab is administered every 2 weeks as an IV infusion under the supervision of a medical professional, which is inconvenient and burdensome for the patient.

[0068] Long-term IV administration can lead to serious complications such as infection, local thrombosis, hematoma, and progressively reduced venous access. In addition, eculizumab is a large protein and is associated with the risk of immunogenicity and hypersensitivity. Finally, while eculizumab binds to C5 and blocks the production of C5b, any C5b produced due to incomplete inhibition can initiate the formation of MAC and cause hemolysis.

[0069] Peripheral blood of patients with PNH can contain variable ratios of normal and abnormal cells. The disease is classified by the International PNH Interest Group on the basis of clinical features, bone marrow characteristics, and the percentage of GPI-AP-deficient polymorphonuclear leukocytes (PMNs). Group 1 (GPI-AP-deficient PMNs) are subclassified. Flow cytometric analysis of PMNs may be more informative, as GPI-AP-deficient erythrocytes are more susceptible to destruction in PNH patients (Parker, CJ, 2012, Curr Opin Hematol, 19, 141-8). Flow cytometric analysis in classical PNH shows 50-100% GPI-AP-deficient PMNs.

[0070] The hemolytic anemia of PNH is autoantibody independent (Coombs negative) and is due to uncontrolled activation of the alternative pathway of complement (AP). In some embodiments, the compounds and compositions of the present invention, e.g., pharmaceutical compositions, are particularly useful in treating PNH. Such compounds and compositions may include a C5 inhibitor (e.g., R5000 and / or its active metabolite or variant). The C5 inhibitors of the present invention useful for treating PNH may, in some cases, block the cleavage of C5 into C5a and C5b. In some cases, the C5 inhibitors of the present invention may be used as an alternative to eculizumab treatment for PNH. Unlike eculizumab, the C5 inhibitors of the present invention may bind to C5b, preventing its binding to C6 and subsequent assembly of C5b-9MAC.

[0071] In some cases, R5000 and / or its active metabolites or variants, alone or in compositions, can be used to treat PNH in subjects. Such subjects can include subjects who have adverse effects, are non-responsive, have reduced responsiveness, or are resistant to other treatments (e.g., with eculizumab). In some embodiments, treatment with the compounds and compositions of the present disclosure can inhibit the hemolysis of PNH red blood cells in a dose-dependent manner.

[0072] In some embodiments, R5000 and / or its active metabolite or variant is administered in combination with eculizumab in a treatment regimen that may include concurrent or sequential treatment. R5000 and / or its active metabolites or variants may be particularly useful in treating PNH in a limited number of patients who, based on sequence and structural data, have a polymorphism in the C5 gene that prevents eculizumab from binding to C5. An example of such a patient is one who has a single missense C5 heterozygous mutation c.2654G->A, which predicts the p.Arg885His polymorphism (R885H; for a description of this and other polymorphisms at position 885, see Nishimura, J. et al., N Engl J Med., 2014, 370, no. 7, pp. 632-9, the contents of which are incorporated herein by reference in their entirety). This mutation destroys the ability of eculizumab to bind to C5 in carriers of the mutation. However, R5000 and R5001 are capable of binding to C5 with the R885H substitution. Thus, in some embodiments, the methods of the disclosure include inhibiting the activity of C5 and / or treating PNH in a subject with the p.Arg885His polymorphism.

[0073] Similar to eculizumab, R5000 and R5001 block the proteolytic cleavage of C5 into C5a and C5b. Unlike eculizumab, R5000 and R5001 can also bind to C5b, block its association with C6, and prevent subsequent assembly of MAC. Thus, advantageously, any C5b resulting from incomplete inhibition by R5000 and / or R5001 is prevented from binding to C6 and completing assembly of MAC.

[0074] In some cases, R5000 and / or its active metabolites or variants may be used as an alternative treatment to eculizumab in patients with PNH, and may provide additional efficacy without the inconvenience and disadvantages of intravenous administration and the known risks of immunogenicity and hypersensitivity associated with monoclonal antibodies. Furthermore, serious complications of long-term intravenous administration, such as infection, lack of venous access, local thrombosis, and hematoma, may be overcome by R5000 and / or R5001 administered by subcutaneous (SC) injection.

[0075] In some embodiments, the method of the present disclosure comprises treating PNH in a subject who has not been previously treated with eculizumab. Such a method may comprise administering a C5 inhibitor to the subject. The C5 inhibitor may comprise R5000 and / or its metabolites or variants. In some aspects, the C5 inhibitor is administered two or more times at regular intervals. The intervals may be about every hour to about every 12 hours, about every 2 hours to about every 24 hours, about every 4 hours to about every 36 hours, about every 8 hours to about every 48 hours, about every 12 hours to about every 60 hours, about every 18 hours to about every 72 hours, about every 30 hours to about every 84 hours, about every 40 hours to about every 96 hours, about every 50 hours to about every 108 hours, about every 60 hours to about every 120 hours, about every 70 hours to about every 132 hours, about every 80 hours to about every 168 hours, about every day to about every week, about every week to about every month, or longer than every month. Administration of the C5 inhibitor may include administering a loading dose of the C5 inhibitor. The initial loading dose may be about 0.01 mg / kg to about 1 mg / kg, about 0.05 mg / kg to about 2 mg / kg, about 0.1 mg / kg to about 3 mg / kg, about 0.2 mg / kg to about 4 mg / kg, about 0.3 mg / kg to about 5 mg / kg, about 0.6 mg / kg to about 6 mg / kg, about 1.5 mg / kg to about 10 mg / kg, or about 5 mg / kg to about 50 mg / kg. The administration of the C5 inhibitor may include administering the C5 inhibitor at an initial therapeutic dose. The initial therapeutic dose may include administering the C5 inhibitor two or more times at regular intervals after the initial loading dose. The initial therapeutic dose may be about 0.01 mg / kg to about 1 mg / kg, about 0.05 mg / kg to about 2 mg / kg, about 0.1 mg / kg to about 3 mg / kg, about 0.2 mg / kg to about 4 mg / kg, about 0.3 mg / kg to about 5 mg / kg, about 0.6 mg / kg to about 6 mg / kg, about 1.5 mg / kg to about 10 mg / kg, or about 5 mg / kg to about 50 mg / kg. The initial therapeutic dose may be replaced with a modified therapeutic dose after a period of administration with the initial therapeutic dose. The period may be about 1 day to about 10 days, about 1 week to about 3 weeks, about 2 weeks to about 4 weeks, or more than 4 weeks.The modified therapeutic dose may be about 0.01 mg / kg to about 1 mg / kg, about 0.05 mg / kg to about 2 mg / kg, about 0.1 mg / kg to about 3 mg / kg, about 0.2 mg / kg to about 4 mg / kg, about 0.3 mg / kg to about 5 mg / kg, about 0.6 mg / kg to about 6 mg / kg, about 1.5 mg / kg to about 10 mg / kg, or about 5 mg / kg to about 50 mg / kg. The modified therapeutic dose may include an increase in the level of the administered C5 inhibitor. Lactate dehydrogenase (LDH) levels in the subject may be monitored throughout treatment. The initial therapeutic dose may be replaced with the modified therapeutic dose based on changes in LDH levels observed. In some aspects, the subject is transitioned to the modified therapeutic dose after an LDH level of 1.5 times or less than the upper limit of normal is detected. In some embodiments, hemolysis in the serum of the subject is reduced. In some embodiments, there is no adverse event in response to treatment (e.g., injection reaction or systemic infection). Administration of the C5 inhibitor may include self-administration (e.g., using an auto-injector device). Self-administration may be monitored, for example, by a medical professional. In some embodiments, self-administration may be remotely monitored. Monitoring may be performed using a smart device.

[0076] It has been reported that eculizumab does not completely abolish C5 activity in vitro under conditions that mimic strong activation, leaving patients potentially vulnerable to inadequate disease control (see Brodsky et al., 2017, Blood 129:922-923 and Harder et al., 2017, Blood 129:970-980). This is referred to as residual C5 activity. Residual C5 activity may result from eculizumab's inability to block the association of C5 with the alternative pathway C5 convertase (which contains two subunits of C3b and one Bb component). In some embodiments, R5000 and / or its active metabolites or variants may be used to inhibit the association between C5 and the alternative pathway C5 convertase.

[0077] Residual C5 activity may also occur when strong complement activation results in cleavage of C5 before eculizumab can bind. Like eculizumab, R5000 and its active metabolites or variants bind to C5 and inhibit cleavage of C5 and activation of the terminal complement cascade. However, R5000 and R5001 bind to C5 at a different site than eculizumab, and thus have a different molecular mechanism for inhibition. Furthermore, R5000 and R5001 may associate with C5b after cleavage and block subsequent hemolysis. In some embodiments, R5000 and / or its active metabolites or variants may be used to improve complement inhibition under conditions where some hemolytic activity persists during or after treatment with eculizumab. Thus, in some embodiments, the present disclosure provides a method of inhibiting residual C5 activity by contacting C5 with R5000 and / or its active metabolites. The C5 may be C5 of a subject with PNH. The C5 may be C5 of a subject having a C5 polymorphism (e.g., pArg885His). In some embodiments, the methods of the disclosure include treating a subject having PNH by administering R5000 and / or an active metabolite or variant thereof, where residual C5 activity remains after prior or current treatment with eculizumab.

[0078] Inflammatory Indications In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat subjects with diseases, disorders, and / or conditions associated with inflammation. Inflammation may be upregulated during the proteolytic cascade of the complement system. Although inflammation may be a beneficial effect, excessive inflammation may lead to a variety of pathological conditions (Markiewski, 2007, Am J Pathol. 17:715-27). Thus, the compounds and compositions of the present invention may be used to reduce or eliminate inflammation associated with complement activation.

[0079] sterile inflammation In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention can be used to treat, prevent, or delay the onset of sterile inflammation. Sterile inflammation is inflammation that occurs in response to stimuli other than infection. Sterile inflammation can be a normal response to stress, such as genomic stress, hypoxic stress, nutritional stress, endoplasmic reticulum stress, etc., caused by physical, chemical, or metabolic noxious stimuli. Sterile inflammation can contribute to the pathogenesis of many diseases, including, 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. Methods and compositions for treating, preventing, and / or delaying the mechanisms of sterile inflammation and symptoms of sterile inflammation may include any of those taught in Rubartelli, Frontiers in Immunology, 2013, Vol. 4, pp. 398-99; Rock et al., Annu Rev Immunol., 2010, Vol. 28, pp. 321-342; or U.S. Pat. No. 8,101,586, the contents of each of which are incorporated herein by reference in their entireties.

[0080] Systemic Inflammatory Response (SIRS) and Sepsis In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used for the treatment and / or prevention of systemic inflammatory response syndrome (SIRS). SIRS is inflammation that affects the entire body. When SIRS is caused by infection, it is referred to as sepsis. SIRS can also be caused by non-infectious events, such as trauma, injury, burns, ischemia, hemorrhage, and / or other pathologies. Negative outcomes associated with SIRS and / or sepsis include multiple organ failure (MOF). Complement inhibition at the C3 level in Gram-negative sepsis not only significantly protects organs against E. coli-induced progressive MOF, but also impedes bacterial clearance. The compounds and compositions described herein include C5 complement component inhibitors that may be administered to subjects with sepsis to provide the benefit of organ protection without adversely altering bacterial clearance.

[0081] In some embodiments, the present disclosure provides a method of treating sepsis. Sepsis may be induced by a microbial infection. The microbial infection may include at least one gram-negative infectious agent. As used herein, the term "infectious agent" refers to any entity that invades or otherwise infects a cell, tissue, organ, compartment, or bodily fluid of a sample or subject. In some cases, the infectious agent may be a bacterium, a virus, or other pathogen. The gram-negative infectious agent is a gram-negative bacterium. The gram-negative infectious agent may include, but is not limited to, E. coli.

[0082] A method 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 may be reduced or prevented. The reduction or prevention of complement activity may be determined by detecting one or more products of complement activity in a subject sample. Such products may include C5 cleavage products (e.g., C5a and C5b) or downstream complexes formed as a result of C5 cleavage (e.g., C5b-9). In some embodiments, the present disclosure provides a method for treating sepsis with R5000, wherein the levels of C5a and / or C5b-9 are reduced or eliminated in the subject and / or in at least one sample obtained from the subject. For example, C5a and / or C5b-9 levels may be reduced in a subject (or in a sample obtained from such a subject) administered R5000 when compared to a subject (or subject sample) not treated with R5000 (including subjects treated with other complement inhibitors), or when compared to the same subject (or subject sample) during a pre-treatment or initial period of treatment, by about 0% 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%, about 25% to about 75%, or about 50% to about 100%.

[0083] In some embodiments, the C5b-9 levels reduced by R5000 treatment are C5b-9 levels associated with one or more of the classical pathway of complement activation, the alternative pathway of complement activation, and the lectin pathway of complement activation.

[0084] In some embodiments, the presence, absence, and / or level of one or more factors associated with sepsis may be modulated by administering R5000 to a subject with sepsis. The presence or absence of such factors may be determined using an assay for their detection. Changes in factor levels may be determined by measuring the levels of such factors in a subject with sepsis after R5000 treatment and comparing those levels to earlier levels in the same subject (either before R5000 treatment or during one or more earlier periods of treatment) or to levels in a subject not treated with R5000 (including a subject with sepsis not receiving any treatment or a subject receiving some other form of treatment). Comparisons may be shown by the percentage difference in factor levels between subjects treated with R5000 and subjects not treated with R5000.

[0085] C5 cleavage products may include any protein or complex that may result from C5 cleavage. In some cases, C5 cleavage products may include, but are not limited to, C5a and C5b. C5b cleavage products may proceed to form complexes with complement proteins C6, C7, C8, and C9 (referred to herein as "C5b-9"). Thus, C5 cleavage products, including C5b-9, may be detected and / or quantified to determine whether complement activity is reduced or inhibited. Detection of C5b-9 deposition may be performed, for example, through the use of a WIESLAB® ELISA (Euro Diagnostica, Malmö, Sweden) kit. Quantitation of cleavage products may be measured in "complement arbitrary units" (CAU) as described by others (see, e.g., Bergseth G. et al., 2013, Mol Immunol. 56:232-9, the contents of which are incorporated herein by reference in their entirety).

[0086] In some embodiments, treating sepsis with a C5 inhibitor (eg, R5000) can reduce or prevent the production of C5b-9. According to the present invention, administration of R5000 to a subject can result in the modulation of bacterial clearance in the subject and / or in at least one sample obtained from the subject. Bacterial clearance, as referred to herein, is the partial or complete removal / reduction of bacteria from a subject or sample. Clearance can occur through killing or otherwise rendering bacteria unable to grow and / or reproduce. In some cases, bacterial clearance can occur through bacteriolysis and / or immune destruction (e.g., through phagocytosis, bacterial cell lysis, opsonization, etc.). According to some methods, bacterial clearance in a subject treated with a C5 inhibitor (e.g., R5000) can have no effect or beneficial effect on bacterial clearance. This can occur due to the absence or reduction of the effect on C3b levels by C5 inhibition. In some embodiments, the method of treating sepsis with R5000 can avoid interference with or enhance C3b-dependent opsonization.

[0087] In some cases, bacterial clearance associated with R5000 treatment may be enhanced compared to bacterial clearance in untreated subjects or subjects treated with another form of complement inhibitor, such as a C3 inhibitor. In some embodiments, subjects with sepsis who are treated with R5000 may experience between 0% and at least 100% enhanced bacterial clearance when compared to bacterial clearance in subjects not treated with R5000 (including subjects treated with other complement inhibitors) or when compared to earlier bacterial clearance levels in the same subjects prior to treatment with R5000 or during earlier treatment periods with R5000. For example, bacterial clearance in a subject treated with R5000 and / or in at least one sample obtained from such a subject may be enhanced by about 0% 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%, about 25% to about 75%, about 50% to about 100% when compared to a subject not treated with R5000 (including subjects treated with other complement inhibitors) and / or when compared to a sample obtained from such a subject, or when compared to the same subject during a pre-treatment period or early period of treatment and / or when compared to a sample obtained from the same subject during a pre-treatment period or early period of treatment.

[0088] Bacterial clearance may be measured in a subject by directly measuring the bacterial level in the subject and / or in a subject sample, or by measuring one or more indicators of bacterial clearance (e.g., the level of bacterial components released after lysis).The level of bacterial clearance may then be determined by comparison with previous measurements of the bacteria / indicator level or with the bacteria / indicator level in a subject not undergoing treatment or undergoing a different treatment.In some cases, colony forming units (cfu) from collected blood are tested to determine the bacterial level (e.g., to obtain cfu / ml of blood).

[0089] In some embodiments, sepsis treatment with R5000 can be performed without effect on phagocytosis or without substantial impairment of phagocytosis. This may include neutrophil-dependent and / or monocyte-dependent phagocytosis. Unimpaired or substantially unimpaired phagocytosis with R5000 treatment may be due to limited or non-existent changes to C3b levels with R5000 treatment.

[0090] The oxidative burst is a C5a-dependent process characterized by the generation of peroxides by certain cells, particularly macrophages and neutrophils, following pathogen challenge (see Mollnes TE et al., 2002, Blood 100:1869-1877, the contents of which are incorporated herein by reference in their entirety).

[0091] In some embodiments, oxidative burst may be reduced or prevented in subjects with sepsis after treatment with R5000. This may be due to a reduction in C5a levels due to R5000-dependent C5 inhibition. Oxidative burst may be reduced by about 0% 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%, about 25% to about 75%, about 50% to about 100% in subjects administered R5000 when compared to subjects not treated with R5000 (including subjects treated with other complement inhibitors) or when compared to the same subjects during a pre-treatment period or early period of treatment.

[0092] Lipopolysaccharide (LPS) is a component of bacterial cell envelope, which is a known immune stimulator. Complement-dependent bacteriolysis can lead to the release of LPS, which contributes to inflammatory responses, such as those characteristic of sepsis. In some embodiments, treating sepsis with R5000 can reduce LPS levels. This can be attributed to the reduction in complement-mediated bacteriolysis due to the inhibition of C5-dependent complement activity. In some embodiments, LPS levels may be reduced or eliminated by about 0% 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%, about 25% to about 75%, or about 50% to about 100% in a subject administered R5000 (or in a sample obtained from such a subject) when compared to a subject (or subject sample) not treated with R5000 (including subjects treated with other complement inhibitors), or when compared to the same subject (or subject sample) during a pre-treatment or early period of treatment.

[0093] In some embodiments, LPS levels may be reduced by 100% in a subject (or subject sample) with sepsis who is treated with R5000, compared to a subject (or subject sample) with sepsis who is not treated with R5000 (including subjects receiving one or more other forms of treatment), or when compared to the same subject (or subject sample) during a pre-treatment or early period of treatment.

[0094] In some embodiments of the present disclosure, sepsis-induced levels of one or more cytokines may be reduced with R5000 treatment. Cytokines include a number of cell signaling molecules that stimulate the immune response to infection. A "cytokine storm" is a dramatic upregulation of at least four cytokines, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor alpha (TNFα), that can result from bacterial infection and contribute to sepsis. C5a is known to induce the synthesis and activity of these cytokines. Thus, inhibitors of C5 may reduce cytokine levels by reducing the levels of C5a. Cytokine levels may be assessed in a subject or subject sample to assess the ability of a C5 inhibitor to reduce the levels of one or more inflammatory cytokines that are upregulated during sepsis. IL-6, IL-8, MCP-1 and / or TNFα levels may be decreased by about 0% 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%, about 25% to about 75%, about 50% to about 100% in a subject administered R5000 when compared to a subject not treated with R5000 (including subjects treated with other complement inhibitors) or when compared to the same subject during a pre-treatment or early treatment period. In some embodiments, IL-6, IL-8, MCP-1, and / or TNFα levels may be reduced by 100% in subjects with sepsis who are treated with R5000 compared to subjects with sepsis who are not treated with R5000 (including subjects receiving one or more other forms of therapy), or when compared to the same subjects during a pre-treatment or early period of treatment.

[0095] One complication associated with sepsis is dysregulation of coagulation and / or fibrinolytic 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 each of which are incorporated herein by reference in their entirety). While controlled local activation of these pathways is important for defense against pathogens, uncontrolled systemic activation can be detrimental. Complement activity associated with bacterial infections may promote dysregulation of coagulation and / or fibrinolysis due to increased host cell and tissue damage associated with MAC formation. In some embodiments, treatment of sepsis with R5000 may normalize coagulation and / or fibrinolysis pathways.

[0096] Dysregulation of coagulation and / or fibrinolysis associated with sepsis may include disseminated intravascular coagulation (DIC). DIC is a condition that results in tissue and organ damage due to activation of coagulation and clot formation in small blood vessels. This activity reduces blood flow to tissues and organs and consumes blood factors necessary for clotting in other parts of the body. The absence of these blood factors in the bloodstream can result in uncontrolled bleeding in other parts of the body. In some embodiments, treatment of sepsis with R5000 can reduce or eliminate DIC.

[0097] Sepsis-related coagulation dysfunction may be detected by measuring the activated partial thromboplastin time (APTT) and / or prothrombin time (PT). These are tests performed on plasma samples to determine whether clotting factor levels are low. In subjects with DIC, the APTT and / or PT are prolonged due to reduced levels of clotting factors. In some embodiments, treatment of a septic subject with R5000 may reduce and / or normalize the APTT and / or PT in samples obtained from the treated subject.

[0098] Sepsis-associated coagulation dysfunction may be further assessed through analysis of thrombin-antithrombin (TAT) complex levels and / or leukocyte expression of tissue factor (TF) mRNA. Increased levels of TAT complexes and leukocyte expression of TF mRNA are associated with coagulation dysfunction and consistent with DIC. In some embodiments, treatment of sepsis with R5000 may result in a reduction in TAT levels and / or leukocyte TF mRNA levels of 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%, about 25% to about 75%, about 50% to about 100% when compared to subjects not treated with R5000 (including subjects treated with other complement inhibitors) or when compared to the same subjects during a pre-treatment or early period of treatment. In some embodiments, TAT levels and / or leukocyte TF mRNA levels may be reduced by 100% in subjects with sepsis treated with R5000 compared to subjects with sepsis not treated with R5000 (including subjects receiving one or more other forms of therapy), or when compared to the same subjects during a pre-treatment or early period of treatment.

[0099] Factor XII is an important factor for normal coagulation in plasma. Factor XII level may be reduced in plasma samples taken from subjects with coagulation dysfunction (e.g., DIC) due to consumption of factor XII associated with coagulation in small blood vessels. In some embodiments, sepsis treatment with R5000 may reduce consumption of factor XII. Thus, factor XII level may be increased in plasma samples taken from subjects with sepsis after treatment with R5000. Factor XII levels may be elevated in plasma samples 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%, about 25% to about 75%, or about 50% to about 100% when compared to subjects not treated with R5000 (including subjects treated with other complement inhibitors) or when compared to plasma samples taken from the same subjects during a pre-treatment or early period of treatment. In some embodiments, factor XII levels may be increased by 100% in plasma samples from subjects with sepsis who are treated with R5000, when compared to plasma samples from subjects with sepsis who are not treated with R5000 (including subjects receiving one or more other forms of therapy), or when compared to plasma samples taken from the same subjects during a pre-treatment or early period of treatment.

[0100] Fibrinolysis is the degradation of fibrin due to enzymatic activity, a process critical for blood clot formation. Dysregulation of fibrinolysis can occur in severe sepsis and has been reported to affect normal coagulation in baboons challenged with E. coli (P. de Boer JP et al., 1993, Circulatory shock 39:59-67, the contents of which are incorporated herein by reference in their entirety). Plasma indices of sepsis-dependent fibrinolytic dysfunction (including but not limited to fibrinolytic dysfunction associated with DIC) may include, but are not limited to, decreased fibrinogen levels (indicating decreased ability to form fibrin clots), elevated tissue plasminogen activator (tPA) levels, elevated plasminogen activator inhibitor type 1 (PAI-1) levels, elevated plasmin-antiplasmin (PAP) levels, increased fibrinogen / fibrin degradation products, and elevated D-dimer levels. In some embodiments, treatment of sepsis with R5000 may result in a reduction 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%, 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%, about 25% to about 75%, about 50% to about 100%, when compared to levels in plasma samples from subjects not treated with R5000 (including subjects treated with other complement inhibitors) or compared to levels in plasma samples taken from the same subjects during a pre-treatment period or an early period of treatment. In some embodiments, sepsis-associated decrease in plasma fibrinogen levels and / or 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% when compared to levels in plasma samples from subjects with sepsis treated with R5000.

[0101] Another consequence of overactive complement activity associated with sepsis is a reduction in red blood cells due to complement-dependent hemolysis and / or C3b-dependent opsonization. A method of treating sepsis with R5000 according to the present disclosure may include reducing complement-dependent hemolysis. One method of assessing complement-dependent hemolysis associated with sepsis includes obtaining a complete blood count. A complete blood count may be obtained through an automated process that counts the cell types present in a blood sample. Results from a complete blood count analysis typically include hematocrit, red blood cell (RBC) count, white blood cell (WBC) count, and platelet levels. Hematocrit levels are used to determine the percentage of blood (by volume) that is composed of red blood cells. Hematocrit levels, platelet levels, RBC levels, and WBC levels may be reduced in sepsis due to hemolysis. In some embodiments, treatment of sepsis with R5000 increases hematocrit levels, platelet levels, RBC levels, and / or WBC levels. The increase may be immediate or may occur over time with treatment (e.g., single or multiple dose treatment).

[0102] In some embodiments, treatment of a subject with R5000 may reduce sepsis-associated white blood cell (e.g., neutrophils and macrophages) activation. "Activation," as used herein in relation to white blood cells, refers to the recruitment and / or maturation of these cells to perform associated immune functions. Reduction of white blood cell activation by R5000 treatment may be determined by evaluating the subject or a sample obtained from the subject.

[0103] In some embodiments, treatment of sepsis with R5000 may improve one or more vital signs in the subject being treated. Such vital signs may include, but are not limited to, heart rate, mean systemic arterial pressure (MSAP), respiratory rate, oxygen saturation, and body temperature.

[0104] In some embodiments, treating sepsis with R5000 may stabilize or reduce capillary leakage and / or endothelial barrier dysfunction associated with sepsis (i.e., to maintain or improve capillary leakage and / or endothelial barrier dysfunction). Stabilizing or reducing capillary leakage and / or endothelial barrier dysfunction may be determined by measuring total plasma protein levels and / or plasma albumin levels. An increase in either level compared to the plasma levels associated with sepsis may indicate a decrease in capillary leakage. Thus, treating sepsis with R5000 may increase total plasma protein and / or plasma albumin levels.

[0105] The method of the present disclosure may include using R5000 to treat sepsis, wherein the level of one or more acute phase proteins is reduced.Acute phase proteins are proteins produced by the liver under inflammatory conditions.R5000 treatment can reduce inflammation associated with sepsis and cause the liver to reduce the production of acute phase proteins.

[0106] According to some methods of the present invention, treatment with R5000 can reduce, reverse, or prevent sepsis-induced organ damage and / or organ dysfunction. Indicators that can be reduced with improved organ function include, but are not limited to, plasma lactate (indicating improved vascular perfusion and clearance), creatinine, blood urea nitrogen (both indicating improved renal function), and liver transaminase (indicating improved liver function). In some embodiments, subjects treated for sepsis with R5000 have reduced fever response, risk of secondary infection, and / or risk of sepsis recurrence.

[0107] The method of the present disclosure may include preventing sepsis-related death and / or improving the survival time of subjects suffering from sepsis through treatment with R5000. Improved survival time may be determined through a comparison of survival time in subjects treated with R5000 to survival time in untreated subjects (including subjects treated with one or more other forms of treatment). In some embodiments, survival time is 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, 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.

[0108] In some embodiments, the administration of R5000 is performed in a single dose. In some embodiments, the administration of R5000 is performed in multiple doses. For example, the administration of R5000 may include administration of an initial dose followed by one or more repeat doses. The repeat dose may be administered about 1 hour to about 24 hours, about 2 hours to about 48 hours, about 4 hours to about 72 hours, about 8 hours to about 96 hours, about 12 hours to about 36 hours, or about 18 hours to about 60 hours after the previous administration. In some cases, the repeat dose may be administered 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 administration. In some cases, the repeat dose may be administered as required to stabilize or reduce sepsis or stabilize or reduce one or more effects associated with sepsis in a subject. Repeat doses may contain the same or different amounts of R5000.

[0109] The compounds and compositions of the present invention may be used to control and / or balance complement activation for the prevention and treatment of SIRS, sepsis, and / or MOF. Methods of application of complement inhibitors to treat SIRS and sepsis may include those described in U.S. Patent Application Publication No. 2013 / 0053302, or U.S. Patent No. 8,329,169, the contents of each of which are incorporated herein by reference in their entirety.

[0110] Acute respiratory distress syndrome (ARDS) In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat and / or prevent the onset of acute respiratory distress syndrome (ARDS). ARDS is a widespread inflammation of the lungs that can be caused by trauma, infection (e.g., sepsis), severe pneumonia, and / or inhalation of harmful substances. ARDS is typically a severe, life-threatening complication. Studies suggest that neutrophils may contribute to the onset of ARDS by affecting the accumulation of polymorphonuclear cells in injured alveolar and interstitial tissues of the lung. Thus, the compounds and compositions of the present invention may be administered to reduce and / or prevent the production of tissue factor in alveolar neutrophils. The compounds and compositions of the present invention may further be used in some cases to treat, prevent, and / or delay ARDS according to any of the methods taught in WO2009 / 014633, the contents of which are incorporated herein by reference in their entirety.

[0111] Periodontitis In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat or prevent the onset of periodontitis and / or associated conditions. Periodontitis is a widespread chronic inflammation that results in the destruction of periodontitis, the tissue that surrounds and supports the teeth. The condition also includes alveolar bone loss (the bone that holds the teeth). Periodontitis may occur due to lack of oral hygiene that results in the accumulation of bacteria at the gum line, also known as plaque. Certain health conditions, e.g., diabetes or malnutrition and / or habits such as smoking, may increase the risk of periodontitis. Periodontitis may increase the risk of stroke, myocardial infarction, atherosclerosis, diabetes, osteoporosis, premature birth, as well as other health problems. Studies have demonstrated a correlation between periodontitis and local complement activity. Periodontal bacteria may inhibit or activate certain components of the complement cascade. Thus, the compounds and compositions of the present invention may be used to prevent and / or treat periodontitis and associated diseases and conditions. Complement activation inhibitors and methods of treatment may include any of those taught by Hajishengallis, Biochem Pharmacol. 2010, 15;80(12):1 and Lambris, or U.S. Patent Application Publication No. 2013 / 0344082, the contents of each of which are incorporated herein by reference in their entireties.

[0112] dermatomyositis In some embodiments, the compounds, compositions, e.g., pharmaceutical compositions, and / or methods of the present invention may be used to treat dermatomyositis. Dermatomyositis is an inflammatory muscle disease characterized by muscle weakness and chronic myositis. Dermatomyositis often begins with an associated skin rash or precedes muscle weakness. The compounds, compositions, and / or methods of the present invention may be used to reduce or prevent dermatomyositis.

[0113] Wounds and injuries The compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat and / or promote healing of various types of wounds and / or injuries. As used herein, the term "injury" typically refers to physical trauma, but may include localized infection or disease processes. Injuries may be characterized by damage, injury, or destruction caused by an external event affecting a living body part and / or organ. Wounds are associated with cuts, bruises, burns, and / or other impacts on the skin that result in destruction or damage to the skin. Wounds and injuries are often acute, but may cause chronic complications and / or inflammation if not healed properly.

[0114] Wounds and burn wounds In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat and / or promote wound healing. Healthy skin provides a waterproof protective barrier against pathogens and other environmental agents. Skin also controls body temperature and fluid evaporation. When skin is damaged, these functions are disrupted, making it difficult for skin to heal. Injury initiates a set of physiological processes related to the immune system that repair and regenerate tissue. Complement activation is one of these processes. As taught by van de Goot et al., J Burn Care Res, 2009, 30:274-280, and Cazander et al., Clin Dev Immunol, 2012, 534-291, the contents of each of which are incorporated herein by reference in their entirety, studies of complement activation have identified several complement components involved in wound healing. In some cases, complement activation can become excessive, causing cell death and increased inflammation (leading to 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 present invention may be according to any of the methods for treating wounds disclosed in WO 2012 / 174055, the contents of which are incorporated herein by reference in their entirety.

[0115] head injury In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat and / or promote healing of head trauma. Head trauma includes injury to the scalp, skull, or brain. Examples of head trauma include, but are not limited to, concussion, contusion, skull fracture, traumatic brain injury, and / or other injuries. Head trauma can be mild or severe. In some cases, head trauma can lead to long-term physical and / or mental complications or death. Research suggests that head trauma can induce inappropriate intracranial complement cascade activation, which can lead to a local inflammatory response that can cause secondary brain damage by the development of cerebral edema and / or neuronal death (Stahel et al., Brain Research Reviews, 1998, vol. 27, pp. 243-56, the contents of which are incorporated herein by reference in their entirety). The compounds and compositions of the present invention may be used to treat head trauma and / or reduce or prevent associated secondary complications. Methods of using the compounds and compositions of the present invention to control complement cascade activation in head trauma may include any of those taught by Holers et al. in U.S. Patent No. 8,911,733, the contents of which are incorporated herein by reference in their entirety.

[0116] crush injury In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat and / or promote healing of crush injuries. Crush injuries are injuries caused by force or pressure applied to the body, resulting in bleeding, bruising, fractures, nerve injuries, wounds, and / or other damage to the body. The compounds and compositions of the present invention may be used to promote healing after crush injuries (e.g., promote nerve regeneration, promote fracture healing, prevent or treat inflammation and / or other related complications) by reducing complement activation after crush injuries. The compounds and compositions of the present invention may be used to promote healing according to any of the methods taught in U.S. Pat. No. 8,703,136, WO 2012 / 162215, WO 2012 / 174055, or U.S. Patent Publication No. 2006 / 0270590, the contents of each of which are incorporated herein by reference in their entirety.

[0117] Ischemia / reperfusion injury In some embodiments, the compounds, compositions, e.g., pharmaceutical compositions, and / or methods of the present disclosure may be used to treat injury associated with ischemia and / or reperfusion. Such injury may involve surgical intervention (e.g., transplantation). Thus, the compounds, compositions, and / or methods of the present disclosure may be used to reduce or prevent ischemia and / or reperfusion injury.

[0118] autoimmune diseases The compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat subjects with autoimmune diseases and / or disorders. The immune system may be divided into the innate and adaptive systems, which represent a non-specific immediate defense mechanism and a more complex antigen-specific system, respectively. The complement system is part of the innate immune system, which recognizes and eliminates pathogens. In addition, complement proteins may modulate adaptive immunity, linking innate and adaptive responses. Autoimmune diseases and disorders are immune disorders in which the system targets self-tissues and substances. Autoimmune diseases may involve certain tissues or organs of the body. The compounds and compositions of the present invention may be used to modulate complement in the treatment and / or prevention of autoimmune diseases. In some cases, such compounds and compositions may be used according to the methods set forth in Ballanti et al., Immunol Res, 2013, 56:477-491, the contents of which are incorporated herein by reference in their entirety.

[0119] Antiphospholipid syndrome (APS) and fulminant antiphospholipid syndrome (CAPS) In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present 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 may cause recurrent venous or arterial thrombosis in organs and complications in the placental circulation that may lead to pregnancy-related complications, e.g., miscarriage, stillbirth, preeclampsia, premature birth, and / or other complications. Fulminant antiphospholipid syndrome (CAPS) is the most acute form of a similar condition that simultaneously leads to venous blockage in several organs. Research suggests that complement activation may contribute to APS-related complications, including pregnancy-related complications, thrombotic (clotting) complications, and vascular complications. The compounds and compositions of the present invention may be used to treat APS-related conditions by reducing or eliminating complement activation. In some cases, the compounds and compositions of the present invention may be used to treat APS and / or APS-related complications according to the methods taught by Salmon et al., Ann Rheum Dis, 2002, 61(Suppl II): ii46-ii50, and Mackworth-Young, Clin Exp Immunol, 2004, 136: 393-401.

[0120] cold agglutinin disease In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat cold agglutinin disease (CAD), also referred to as cold agglutinin-mediated hemolysis. CAD is an autoimmune disease caused by high concentrations of IgM antibodies that interact with red blood cells at low body temperatures (Engelhardt et al., Blood, 2002, vol. 100, no. 5, pp. 1922-23). ​​CAD may result in conditions such as anemia, fatigue, dyspnea, hemoglobinuria, and / or acrocyanosis. CAD is associated with robust complement activation, and studies have shown that CAD may be treated with complement inhibitor therapy. Thus, the present invention provides methods of treating CAD using the compounds and compositions of the present invention. In some cases, the compounds and compositions of the present invention may be used to treat CAD according to the methods taught in Roth et al., Blood, 2009, 113:3885-86, or in WO 2012 / 139081, the contents of each of which are incorporated herein by reference in their entireties.

[0121] myasthenia gravis In some embodiments, the compounds, compositions, e.g., pharmaceutical compositions, and / or methods of the present invention may be used to treat myasthenia gravis. Myasthenia gravis (MG) is a rare complement-mediated autoimmune disease characterized by the production of autoantibodies that target proteins critical for the normal transmission of electrical signals from nerves to muscles. The prognosis for MG is generally benign, but in 10%-15% of patients, disease control cannot be achieved with current therapies or results in severe side effects of immunosuppressive treatment. A severe form of MG is known as refractory MG (rMG) and affects approximately 9,000 people in the United States.

[0122] Patients characteristically present with muscle weakness that becomes more severe with repetitive use and resolves with rest. Weakness may be localized to specific muscles, such as those involved in eye movement, but often progresses to more diffuse weakness. rMG can become even more life-threatening when the weakness involves the diaphragm and other chest wall muscles involved in breathing. This is the most feared complication of rMG, also known as myasthenic crisis, and requires hospitalization, intubation, and mechanical ventilation. Approximately 15%-20% of patients experience a myasthenic crisis within 2 years of diagnosis.

[0123] The most common target of autoantibodies in MG is the acetylcholine receptor, or AchR, located at the neuromuscular junction, the point where motor neurons transmit signals to skeletal muscle fibers. Binding of anti-AchR autoantibodies to muscle endplates leads to activation of the classical complement cascade and deposition of MAC on postsynaptic muscle fibers, resulting in local damage to the muscle membrane and reduced responsiveness of muscles to neuronal stimulation.

[0124] Inhibition of terminal complement activity may be used to block complement-mediated damage caused by MG and / or rMG. In some embodiments, the compounds and / or compositions of the present disclosure may be used to treat MG and / or rMG. Such methods may be used to inhibit C5 activity to reduce or prevent neuromuscular problems associated with MG and / or rMG.

[0125] Guillain-Barré syndrome In some embodiments, the compounds, compositions, e.g., pharmaceutical compositions, and methods of the present invention may be used to treat Guillain-Barre syndrome (GBS). GBS is an autoimmune disease involving autoimmune attacks of the peripheral nervous system. The compounds, compositions, and / or methods of the present invention may be used to reduce or prevent peripheral nerve problems associated with GBS.

[0126] Vascular Indications In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat vascular indications that affect blood vessels (e.g., arteries, veins, and capillaries) that may affect blood circulation (blood pressure), blood flow, organ function, and / or other bodily functions.

[0127] Thrombotic microangiopathy (TMA) In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat and / or prevent thrombotic microangiopathy (TMA) and related disorders. Microangiopathy affects the body's small blood vessels (capillaries), making the capillary walls thicker and weaker, making them more susceptible to bleeding and slower blood circulation. TMA tends to result in the development of vascular thrombosis, endothelial cell damage, thrombocytopenia, and hemolysis. Organs such as the brain, kidneys, muscles, gastrointestinal system, skin, and lungs may be affected. TMA may result from medical procedures and / or conditions, including, but not limited to, hematopoietic stem cell transplantation (HSCT), kidney disorders, diabetes, and / or other conditions. TMA may be caused by an underlying complement system dysfunction as described by Meri et al., European Journal of Internal Medicine, 2013, vol. 24, pp. 496-502, the contents of which are incorporated herein by reference in their entirety. In general, TMA may result from increased levels of certain complement components leading to thrombosis. In some cases, this may be caused by mutations in complement proteins or associated enzymes. The resulting complement dysfunction may cause complement targeting of endothelial cells and platelets, resulting in increased thrombosis. In some embodiments, TMA may be prevented and / or treated using the compounds and compositions of the present invention. In some cases, treatment of TMA with the compounds and compositions of the present invention may be performed as described in U.S. Patent Application Publication No. 2012 / 0225056 or U.S. Patent Application Publication No. 2013 / 0246083, the contents of each of which are incorporated by reference in their entireties.

[0128] Disseminated intravascular coagulation (DIC) In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to treat and / or prevent disseminated intravascular coagulation (DIC) by controlling complement activation. DIC is a pathological condition in which the coagulation cascade is activated extensively in the blood, resulting in the formation of blood clots, especially in capillaries. DIC may cause obstruction of blood flow in tissues and ultimately damage organs. In addition, DIC may affect the normal process of blood clotting, causing severe bleeding. The compounds and compositions of the present invention may be used to treat, prevent, or reduce the severity of DIC by modulating complement activity. In some cases, the compounds and compositions of the present invention may be used according to any of the methods of treating DIC taught in U.S. Pat. No. 8,652,477, the contents of which are incorporated herein by reference in their entirety.

[0129] vasculitis In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent and / or treat vasculitis. Generally, vasculitis is a disorder associated with inflammation of blood vessels, including veins and arteries, characterized by white blood cells attacking the tissues, causing the vessels to expand. Vasculitis may be associated with an infection, such as in Rocky Mountain spotted fever or autoimmune diseases. An example of an autoimmune-related vasculitis is antineutrophil cytoplasmic autoantibody (ANCA) vasculitis. ANCA vasculitis is caused by abnormal antibodies that attack the body's own cells and tissues. ANCA attacks the cytoplasm of certain white blood cells and neutrophils, causing the attack of blood vessel walls in certain organs and tissues of the body. ANCA vasculitis may affect the skin, lungs, eyes, and / or kidneys. Studies indicate that ANCA diseases activate the alternative complement pathway and produce certain complement components that form an inflammatory amplification loop that leads to vascular damage (Jennette et al., 2013, Semin Nephrol. 33(6):557-64, the contents of which are incorporated herein by reference in their entirety). In some cases, compounds and compositions of the present invention may be used to prevent and / or treat ANCA vasculitis by inhibiting complement activation.

[0130] Atypical hemolytic uremic syndrome In some embodiments, the compounds, compositions, e.g., pharmaceutical compositions, and / or methods of the present disclosure may be useful for the treatment of atypical hemolytic uremic syndrome (aHUS). aHUS is a rare disease caused by uncontrolled complement activation characterized by clot formation in small blood vessels. The compositions and methods of the present invention may be useful for reducing or preventing complement activation associated with aHUS.

[0131] Neurological Indications The compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent, treat, and / or alleviate symptoms of neurological indications, including, but not limited to, neurodegenerative diseases and related disorders. Neurodegeneration is generally associated with loss of neuronal structure or function, including neuronal death. Such disorders may be treated by inhibiting the action of complement on neuronal cells using the compounds and compositions of the present invention. Neurodegeneration-related disorders include, but are not limited to, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson's disease, and Alzheimer's disease.

[0132] Amyotrophic lateral sclerosis (ALS) In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent, treat, and / or alleviate symptoms of ALS. ALS is a fatal motor neuron disease characterized by degeneration of spinal cord neurons, brain stem, and motor cortex. ALS causes muscle weakness that ultimately leads to respiratory failure. Complement dysfunction may contribute to ALS, so therapy with the compounds and compositions of the present invention that target complement activity may prevent, treat, and reduce symptoms of ALS. In some cases, the compounds and compositions of the present invention may be used to promote neuronal regeneration. In some cases, the compounds and compositions of the present invention may be used as complement inhibitors according to any of the methods taught in U.S. Patent Application Publication No. 2014 / 0234275 or U.S. Patent Application Publication No. 2010 / 0143344, the contents of each of which are incorporated herein by reference in their entirety.

[0133] Alzheimer's Disease In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may 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 may include disorientation, memory loss, mood swings, behavioral problems, and ultimately loss of bodily function. Alzheimer's disease is thought to be caused by extracellular brain deposits of amyloid associated with inflammation-related proteins, such as complement proteins (Sjoberg et al., 2009, Trends in Immunology, vol. 30, no. 2, pp. 83-90, the contents of which are incorporated herein by reference in their entirety). In some cases, the compounds and compositions of the present invention may be used as complement inhibitors according to any of the methods of treating Alzheimer's disease taught in U.S. Patent Application Publication No. 2014 / 0234275, the contents of which are incorporated herein by reference in their entirety.

[0134] Kidney-related Indications The compounds and compositions, e.g., pharmaceutical compositions, of the present invention may, in some cases, be used to treat certain kidney-related diseases, disorders, and / or conditions by inhibiting complement activity. The kidney is an organ involved in removing metabolic waste products from the bloodstream. The kidney is essential for various bodily functions, as it controls blood pressure, the urinary system, and homeostatic mechanisms. The kidney may be more severely affected by inflammation (compared to other organs) due to its unique structural characteristics and exposure to blood. The kidney also produces its own complement proteins, which may be activated by infection, kidney disease, and kidney transplantation. In some cases, the compounds and compositions of the present invention may be used as complement inhibitors in the treatment of certain kidney-related diseases, disorders, and / or conditions, according to the method taught by Quigg, J Immunol, 2003, 171, pp. 3319-24, the contents of which are incorporated herein by reference in their entirety.

[0135] Lupus nephritis In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent and / or treat lupus nephritis by inhibiting complement activity. Lupus nephritis is nephritis 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; joint pain; muscle pain; fever; and rash. Lupus nephritis may be treated with inhibitors that control complement activity, 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 those taught in U.S. Patent Application Publication No. 2013 / 0345257 or U.S. Patent No. 8,377,437, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the compounds and / or compositions of the present disclosure may be used to prevent and / or treat lupus nephritis by binding to C5 and preventing the progression of kidney disease in lupus nephritis. Binding to C5 may prevent and / or treat lupus nephritis by blocking C5 activity and complement-mediated damage to renal cells.

[0136] Membranous glomerulonephritis (MGN) In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent and / or treat membranous glomerulonephritis (MGN) disorders by inhibiting the activation of certain complement components. MGN is a kidney disorder that can result in inflammation and structural changes. MGN is caused by antibodies that bind to soluble antigens in the kidney capillaries (glomeruli). MGN can affect kidney functions, such as fluid filtration, and can lead to kidney failure. The compounds and compositions of the present invention may be used according to the methods of preventing and / or treating MGN by complement inhibition taught in U.S. Patent Publication No. 2010 / 0015139 or WO 2000 / 021559, the contents of each of which are incorporated herein by reference in their entirety.

[0137] hemodialysis complications In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent and / or treat complications associated with hemodialysis by inhibiting complement activation. Hemodialysis is a medical procedure used to maintain kidney function in subjects with renal failure. In hemodialysis, removal of waste products from the blood, such as creatinine, urea, and free water, is performed externally. A common complication of hemodialysis treatment is chronic inflammation caused by contact between the blood and the dialysis membrane. Another common complication is thrombosis, which refers to the formation of blood clots that disrupt blood circulation. Studies suggest that such complications are related to complement activation. Hemodialysis may be combined with complement inhibitor therapy to provide a means of controlling inflammatory responses and pathological conditions and / or preventing or treating thrombosis in subjects undergoing hemodialysis due to renal failure. Methods of using the compounds and compositions of the present invention for the treatment of hemodialysis complications may be performed according to any of the methods taught by DeAngelis et al., Immunobiology, 2012, Vol. 217, No. 11, pp. 1097-1105, or Kourtzelis et al., Blood, 2010, Vol. 116, No. 4, pp. 631-639, the contents of each of which are incorporated herein by reference in their entirety.

[0138] eye disease In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent and / or treat certain eye-related diseases, disorders, and / or conditions. In a healthy eye, the complement system is activated at a low level and is continuously regulated by membrane-bound and soluble intraocular proteins that protect against pathogens. Thus, complement activation plays an important role in several eye-related complications, and control of complement activation may be used to treat such diseases. The compounds and compositions of the present invention may be used as complement inhibitors in the treatment of eye diseases according to any of the methods taught by Jha et al., Mol Immunol., 2007, vol. 44, no. 16, pp. 3901-3908, or U.S. Pat. No. 8,753,625, the contents of each of which are incorporated herein by reference in their entirety.

[0139] Age-related Macular Degeneration (AMD) In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent and / or treat age-related macular degeneration (AMD) by inhibiting intraocular complement activation. AMD is a chronic eye disease that causes central vision impairment, central blind spots, and / or eventual central vision loss. Central vision affects the ability to read, drive, and / or recognize faces. AMD is generally classified into two types: nonexudative (dry) and exudative (wet). Dry AMD refers to the deterioration of the macula, the central tissue of the retina. Wet AMD refers to the insufficiency of blood vessels under the retina that results in the leakage of blood and fluid. As discussed by Jha et al., Mol Immunol., 2007, Vol. 44, No. 16, pp. 3901-8, several human and animal studies have identified complement proteins associated with AMD, and novel treatment strategies involve the control of complement activation pathways. Methods of the present invention, including the use of compounds and compositions of the present invention for the prevention and / or treatment of AMD, may include any of those taught in U.S. Patent Application Publication No. 2011 / 0269807 or U.S. Patent Application Publication No. 2008 / 0269318, the contents of each of which are incorporated herein by reference in their entireties.

[0140] Corneal disorders In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent and / or treat corneal diseases by inhibiting intraocular complement activation. The complement system plays an important role in protecting the cornea from pathogenic particles and / or inflammatory antigens. The cornea is the outermost front part of the eye that covers and protects 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 complications may cause pain, blurred vision, tearing, redness, photosensitivity, and / or corneal scarring. Although the complement system is crucial for corneal protection, complement activation may cause damage to corneal tissue after the infection is cleared, since certain complement compounds are highly expressed. Methods of the present invention for modulating complement activity in the treatment of corneal disease may include any of those taught by Jha et al., Mol Immunol., 2007, Vol. 44, No. 16, pp. 3901-8.

[0141] Autoimmune uveitis In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent and / or treat uveitis, which is an inflammation of the uveal layer of the eye. The uvea is the pigmented area of ​​the eye that includes the choroid, iris, and ciliary body of the eye. Uveitis can cause redness, blurred vision, pain, adhesions, and ultimately blindness. Complement activation products are present in the eyes of patients with autoimmune uveitis, and studies suggest that complement plays an important role in disease development. In some cases, the compounds and compositions of the present invention may be used to prevent and / or treat uveitis according to any of the methods identified in Jha et al., Mol Immunol., 2007, Vol. 44, No. 16, pp. 3901-8, the contents of which are incorporated herein by reference in their entirety.

[0142] diabetic retinopathy In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent and / or treat diabetic retinopathy, a disease caused by changes in retinal blood vessels in diabetic patients. Retinopathy may cause blood vessels to expand and leak fluid and / or abnormal blood vessel growth. Diabetic retinopathy may affect vision and ultimately lead to blindness. Studies suggest that complement activation plays an important role in the progression of diabetic retinopathy. In some cases, the compounds and compositions of the present invention may be used according to the method of treating diabetic retinopathy described in Jha et al., Mol Immunol., 2007, Vol. 44, No. 16, pp. 3901-8, the contents of which are incorporated herein by reference in their entirety.

[0143] Neuromyelitis optica (NMO) In some embodiments, the compounds, compositions, e.g., pharmaceutical compositions, and / or methods of the present invention may 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 present invention may be used to prevent neurodestruction in subjects with NMO.

[0144] Sjögren's syndrome In some embodiments, the compounds, compositions, e.g., pharmaceutical compositions, and / or methods of the present invention may be used to treat Sjogren's syndrome. Sjogren's syndrome is an eye disease characterized by dry eyes that may cause burning and / or itching. It is an autoimmune disorder in which the immune system targets glands in the eyes and mouth that are responsible for moistening those areas. The compounds, compositions, and / or methods of the present disclosure may be used to treat and / or reduce the symptoms of Sjogren's syndrome.

[0145] Preeclampsia and HELLP syndrome In some embodiments, the compounds and compositions, e.g., pharmaceutical compositions, of the present invention may be used to prevent and / or treat pre-eclampsia and / or HELLP (an abbreviation that stands for syndrome features of 1) hemolysis, 2) elevated liver enzymes, and 3) low platelet count) syndrome with complement inhibitor therapy. Pre-eclampsia is a pregnancy disorder with symptoms including elevated blood pressure, swelling, shortness of breath, renal insufficiency, liver dysfunction, and / or low platelet count. Pre-eclampsia is typically diagnosed by high urinary protein levels and high blood pressure. HELLP syndrome is a combination of hemolysis, elevated liver enzymes, and low platelet pathology. Hemolysis is a disease involving the rupture of red blood cells resulting in the release of hemoglobin from the red blood cells. Elevated liver enzymes may represent pregnancy-induced liver pathology. Low platelet levels result in reduced clotting ability leading to excessive bleeding. HELLP is associated with pre-eclampsia and liver damage. HELLP syndrome typically occurs at a later stage of pregnancy or after birth. It is typically diagnosed by a blood test that indicates the presence of the three conditions involved: HELLP is typically treated by induction of labor.

[0146] Studies suggest that complement activation occurs during HELLP syndrome and pre-eclampsia, and that complement components are present at increased levels during HELLP and pre-eclampsia. Complement inhibitors may be used as therapeutic agents to prevent and / or treat these conditions. The compounds and compositions of the present invention may be used according to the methods of prevention and / or treatment of HELLP and pre-eclampsia taught by Heager et al., Obstetrics & Gynecology, 1992, Vol. 79, No. 1, pp. 19-26, or WO 201 / 078622, the contents of each of which are incorporated herein by reference in their entirety.

[0147] formulation In some embodiments, the compound or composition, e.g., pharmaceutical composition, of the present invention is formulated in an aqueous solution. In some cases, the aqueous solution further comprises one or more salts and / or one or more buffering agents. The salt may include sodium chloride, which may be included at a concentration of about 0.05 mM to about 50 mM, about 1 mM to about 100 mM, about 20 mM to about 200 mM, or about 50 mM to about 500 mM. Furthermore, the solution may include at least 500 mM sodium chloride. In some cases, the aqueous solution includes sodium phosphate. The sodium phosphate may be included in the aqueous solution at a concentration of about 0.005 mM to about 5 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 50 mM, about 1 mM to about 100 mM, about 5 mM to about 150 mM, or about 10 mM to about 250 mM. In some cases, a sodium phosphate concentration of at least 250 mM is used.

[0148] The compositions of the invention may contain a C5 inhibitor at a concentration of about 0.001 mg / mL to about 0.2 mg / mL, about 0.01 mg / mL to about 2 mg / mL, about 0.1 mg / mL to about 10 mg / mL, about 0.5 mg / mL to about 5 mg / mL, about 1 mg / mL to about 20 mg / mL, about 15 mg / mL to about 40 mg / mL, about 25 mg / mL to about 75 mg / mL, about 50 mg / mL to about 200 mg / mL, or about 100 mg / mL to about 400 mg / mL. In some cases, the compositions of the invention contain a C5 inhibitor at a concentration of at least 400 mg / mL.

[0149] The compositions of the invention may contain a C5 inhibitor at a concentration of approximately, roughly, 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, 50 mg / mL, 200 mg / mL, 100 mg / mL, or 400 mg / mL. In some cases, the compositions of the invention contain a C5 inhibitor at a concentration of at least 40 mg / mL.

[0150] In some embodiments, the composition of the present invention comprises an aqueous composition comprising at least water and a C5 inhibitor (e.g., a cyclic C5 inhibitor polypeptide). The aqueous C5 inhibitor composition of the present invention may further comprise one or more salts and / or one or more buffers. In some cases, the aqueous composition of the present invention comprises water, a cyclic C5 inhibitor polypeptide, a salt, and a buffer.

[0151] Aqueous C5 inhibitor formulations of the invention may have a pH level of about 2.0 to about 3.0, about 2.5 to about 3.5, about 3.0 to about 4.0, 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, about 6.0 to about 7.0, about 6.5 to about 7.5, about 7.0 to about 8.0, about 7.5 to about 8.5, about 8.0 to about 9.0, about 8.5 to about 9.5, or about 9.0 to about 10.0.

[0152] In some cases, the compounds and compositions of the present invention are prepared in accordance with Good Manufacturing Practice (GMP) and / or current GMP (cGMP). Guidelines used to implement GMP and / or cGMP may be obtained from one or more of the U.S. Food and Drug Administration (FDA), the World Health Organization (WHO), and the International Council on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH).

[0153] Dosage and Administration In the treatment of human subjects, the C5 inhibitor (e.g., R5000 and / or its active metabolites or variants) may be formulated as a pharmaceutical composition. Depending on the subject to be treated, the mode of administration, and the type of treatment desired (e.g., prevention, prophylaxis, or treatment), the C5 inhibitor may be formulated in a manner suitable for these parameters. Overviews of such technology can be found in Remington, The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, 2005; and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

[0154] The C5 inhibitors of the present invention (e.g., R5000 and / or its active metabolites or variants) may be provided in a therapeutically effective amount. In some cases, a therapeutically effective amount of a C5 inhibitor of the present invention may be achieved by administration of one or more doses of C5 inhibitors 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 to about 50 mg, about 10 mg to about 100 mg, about 20 mg to about 200 mg, or at least 200 mg.

[0155] In some embodiments, a therapeutic amount of a C5 inhibitor (e.g., R5000 and / or an active metabolite or variant thereof) may be administered to a subject based on the subject's body weight. In some cases, the C5 inhibitor may be administered at a dose of about 0.001 mg / kg to about 1.0 mg / kg, about 0.01 mg / kg to about 2.0 mg / kg, 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, about 0.2 mg / kg to about 3.0 mg / kg, about 0.4 mg / kg to about 4.0 mg / kg, about 1.0 mg / kg to about 5.0 mg / kg, about 2 ... The 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 embodiments, the 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 cases, 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.

[0156] In some cases, the C5 inhibitors of the present invention (e.g., R5000 and / or its active metabolites or variants) are provided at a concentration adjusted to obtain a desired level of the C5 inhibitor in a sample, biological system, or subject (e.g., plasma level in a subject). In some cases, the desired concentration of the C5 inhibitor in a sample, biological system, or subject may include a concentration 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, about 0.03 μM to about 0.3 μM, about 0.05 μM to about 0.5 μM, about 0.01 μM to about 2.0 μM, about 0.1 μM to about 50 μM, about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, about 0.2 μM to about 20 μM, about 5 μM to about 100 μM, or about 15 μM to about 200 μM. In some cases, the desired concentration of the C5 inhibitor in the subject's plasma may be about 0.1 μg / mL to about 1000 μg / mL. Desirable concentrations of C5 inhibitor in subject plasma are about 0.01 μg / mL to about 2 μg / mL, about 0.02 μg / mL to about 4 μg / mL, 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, about 1 μg / mL to about 5 μg / mL, about 2 μg / mL In other embodiments, the C5 inhibitor may have a maximum serum concentration (C5) 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 5 μg / mL, at least 10 μg / mL, at least 50 μg / mL, at least 100 μg / mL, or at least 1000 μg / mL. max ) is administered in a dose sufficient to achieve

[0157] In some embodiments, a dose sufficient to maintain a C5 inhibitor level of about 0.1 μg / mL to about 30 μg / mL is provided to reduce hemolysis in a subject by about 25% to about 99%.

[0158] In some embodiments, the C5 inhibitor (e.g., R5000 and / or its active metabolites or variants) is administered daily at a dose sufficient to deliver about 0.1 mg / day to about 60 mg / kg body weight of the subject. In some cases, the C5 inhibitor achieved with each administration is max is about 0.1 μg / mL to about 1000 μg / mL. In such a case, the area under the curve (AUC) during administration is about 200 μg / mL. * time / mL ~ approx. 10,000μg * It may be hours / mL.

[0159] According to some methods of the invention, the C5 inhibitors of the invention (e.g., R5000 and / or its active metabolites or variants) are provided at a concentration required to obtain a desired effect. In some cases, the compounds and compositions of the invention are provided in an amount required to reduce a given reaction or process by half. The concentration required to achieve such a reduction is referred to herein as the half maximal inhibitory concentration or "IC 50 Alternatively, the compounds and compositions of the invention may be provided in an amount required to enhance a given reaction, activity or process by half. The concentration required for such enhancement is referred to herein as the half maximal effective concentration, or "EC 50 "It is called "

[0160] The C5 inhibitors of the present invention (e.g., R5000 and / or its active metabolites or variants) may be present in a total amount of 0.1-95% by weight of the total weight of the composition. In some cases, the C5 inhibitors are provided by intravenous (IV) administration. In some cases, the C5 inhibitors are provided by subcutaneous (SC) administration.

[0161] SC administration of the C5 inhibitors of the present invention (e.g., R5000 and / or its active metabolites or variants) may provide advantages over IV administration in some cases. SC administration may be able to provide self-treatment to patients. Such treatment may be advantageous in that patients can provide treatment to themselves in their own homes, avoiding the need to travel to a provider or medical facility. Furthermore, SC treatment may allow patients to avoid long-term complications associated with IV administration, such as infections, reduced venous access, local thrombosis, and hematomas. In some embodiments, SC treatment may enhance patient compliance, patient satisfaction, quality of life, and reduce treatment costs and / or drug requirements.

[0162] In some cases, daily SC dosing provides steady state C5 inhibitor concentrations that are reached within 1-3 doses, within 2-3 doses, within 3-5 doses, or within 5-10 doses. In some cases, a daily SC dose of 0.1 mg / kg can achieve sustained C5 inhibitor levels of 2.5 μg / mL or greater and / or greater than 90% inhibition of complement activity.

[0163] The C5 inhibitors of the present invention (e.g., R5000 and / or its active metabolites or variants) may exhibit slow absorption kinetics (time to maximum observed concentration greater than 4-8 hours) and high bioavailability (about 75% to about 100%) following SC administration.

[0164] In some embodiments, the dose and / or administration is based on the half-life (t 1 / 2 ) is changed to adjust for t 1 / 2is 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.

[0165] In some embodiments, the C5 inhibitors of the present invention (e.g., R5000 and / or its active metabolites or variants) have a long terminal t 1 / 2 It can show extended terminal t 1 / 2 may be due to additional target binding and / or additional plasma protein binding. In some cases, the C5 inhibitors of the present invention have a t of greater than 24 hours in both plasma and whole blood. 1 / 2 Values ​​are shown in Table 1. In some cases, C5 inhibitors do not lose functional activity after incubation in human whole blood for 16 hours at 37° C.

[0166] In some embodiments, the dose and / or administration is altered to adjust the steady-state volume of distribution of the C5 inhibitor. In some cases, the steady-state volume of distribution of the C5 inhibitor is about 0.1 mL / kg to about 1 mL / kg, about 0.5 mL / kg to about 5 mL / kg, about 1 mL / kg to about 10 mL / kg, about 5 mL / kg to about 20 mL / kg, about 15 mL / kg to about 30 mL / kg, about 10 mL / kg to about 200 mL / kg, about 20 mL / kg to about 60 mL / kg, about 30 mL / kg to about 70 mL / kg, about 50 mL / kg to about 200 mL / kg, about 100 mL / kg to about 500 mL / kg, or at least 500 mL / kg. In some cases, the dose and / or administration of the C5 inhibitor is adjusted to ensure that the steady-state volume of distribution is equal to at least 50% of the total blood volume. In some embodiments, the C5 inhibitor distribution may be restricted to the plasma compartment.

[0167] In some embodiments, the C5 inhibitors of the present invention (e.g., R5000 and / or its active metabolites or variants) exhibit a total clearance rate of about 0.001 mL / hr / kg to about 0.01 mL / hr / kg, about 0.005 mL / hr / kg to about 0.05 mL / hr / kg, about 0.01 mL / hr / kg to about 0.1 mL / hr / kg, about 0.05 mL / hr / kg to about 0.5 mL / hr / kg, about 0.1 mL / hr / kg to about 1 mL / hr / kg, about 0.5 mL / hr / kg to about 5 mL / hr / kg, about 0.04 mL / hr / kg to about 4 mL / hr / kg, about 1 mL / hr / kg to about 10 mL / hr / kg, about 5 mL / hr / kg to about 20 mL / hr / kg, about 15 mL / hr / kg to about 30 mL / hr / kg, or at least 30 mL / hr / kg.

[0168] The time period during which the maximum concentration of C5 inhibitor is maintained in a subject (e.g., in the subject's serum) (T maxThe T value may be adjusted by altering the dose and / or administration (e.g., subcutaneous administration). In some cases, the C5 inhibitor has a T of about 1 minute to about 10 minutes, about 5 minutes to about 20 minutes, about 15 minutes to about 45 minutes, about 30 minutes to about 60 minutes, about 45 minutes to about 90 minutes, about 1 hour to about 48 hours, about 2 hours to about 10 hours, about 5 hours to about 20 hours, about 10 hours to about 60 hours, about 1 day to about 4 days, about 2 days to about 10 days, or at least 10 days. max It has a value.

[0169] In some embodiments, the C5 inhibitors of the present invention (e.g., R5000 and / or its active metabolites or variants) can be administered without off-target effects. In some cases, the C5 inhibitors of the present invention do not inhibit hERG (human ether-a-go-go related gene) even at concentrations of 300 μM or less. SC injection of the C5 inhibitors of the present invention at dose levels up to 10 mg / kg may be well tolerated and may not result in any adverse cardiovascular effects (e.g., increased risk of prolonged ventricular repolarization) and / or adverse respiratory effects.

[0170] The dose of the C5 inhibitor may be determined using a no observed adverse effect level (NOAEL) observed in another species. Such species may include, but are not limited to, monkeys, rats, rabbits, and mice. In some cases, the human equivalent dose (HED) may be determined by non-proportional scaling from the NOAEL observed in the other species. In some cases, the HED results in a therapeutic limit of about 2-fold to about 5-fold, about 4-fold to about 12-fold, about 5-fold to about 15-fold, about 10-fold to about 30-fold, or at least 30-fold. In some cases, the therapeutic limit is determined based on the exposure to primates and the human C5 inhibitor assessed in humans. max The level is determined by:

[0171] In some embodiments, the C5 inhibitors of the present invention allow for rapid drug holiday in cases of infection where long-term inhibition of the complement system proves detrimental. The administration of C5 inhibitors according to the present invention may be modified to reduce potential clinical risks to subjects. Infection with Neisseria meningitidis is a known risk of C5 inhibitors, including eculizumab. In some cases, the risk of infection with Neisseria meningitidis is minimized by initiating one or more preventative steps. Such steps may include the exclusion of subjects that may already be colonized by these bacteria. In some cases, preventative steps may include the co-administration of one or more antibiotics. In some cases, ciprofloxacin may be co-administered. In some cases, ciprofloxacin may be co-administered orally at a dose of about 100 mg to about 1000 mg (e.g., 500 mg).

[0172] In some embodiments, C5 inhibitor administration may be performed using an auto-injector device. Such a device may allow for self-administration (e.g., daily administration). In some embodiments, R5000 and / or its active metabolites or variants may be co-administered with eculizumab. Co-administration may be performed to reduce residual C5 activity (e.g., due to incomplete inhibition) associated with eculizumab treatment alone.

[0173] Dosage frequency In some embodiments, the C5 inhibitor of the present invention (e.g., R5000 and / or its active metabolites or variants) is administered hourly, 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 2 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 cases, the C5 inhibitor is administered once a day, or in two, three or more sub-doses at appropriate intervals throughout the day.

[0174] In some embodiments, the C5 inhibitor is administered in multiple daily doses. In some cases, the C5 inhibitor is administered daily for 7 days. In some cases, the C5 inhibitor is administered daily for 7-100 days. In some cases, the C5 inhibitor is administered daily for at least 100 days. In some cases, the C5 inhibitor is administered daily indefinitely.

[0175] Intravenously delivered C5 inhibitors may be delivered by chronic infusion, for example, over 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes. Administration may be repeated, for example, periodically, for example, hourly, daily, weekly, biweekly (i.e., every 2 weeks), one month, two months, three months, four months, or more than four months. After an initial treatment regimen, the therapeutic agent may be administered on a less frequent basis. For example, after three months of biweekly administration, administration may be repeated once a month, six months, or once a year or more. Administration of C5 inhibitors may decrease, reduce, increase, or alter binding or any physiologically deleterious process (e.g., in the patient's cells, tissues, blood, urine, or other compartments) 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 80%, or at least 90% or more.

[0176] Prior to administration of the full dose of C5 inhibitor and / or C5 inhibitor composition, the patient may be administered a smaller dose, e.g., 5% of the full dose, and monitored for adverse effects, e.g., allergic or infusion reactions, or elevated lipid levels or blood pressure. In another example, the patient may be monitored for undesired immune stimulatory effects, e.g., elevated cytokine (e.g., TNF-α, Il-1, Il-6, or Il-10) levels.

[0177] Genetic predisposition plays a role in the development of some diseases or disorders.Therefore, the patient who needs C5 inhibitor can be identified by taking family history or by screening one or more genetic markers or variants, for example.Before prescribing or administering the therapeutic composition of the present invention, medical provider, for example, doctor, nurse, or family member, can analyze family history.

[0178] C5 assay In some embodiments, the disclosure provides compounds and methods for detecting C5 and / or C5-associated factors in a sample. Total C5 is detected using several methods. Total C5 may include both free C5 and C5 associated with one or more C5-associated factors. The C5-associated factors may include antibodies (e.g., eculizumab or similar antibodies that bind similar epitopes). According to such embodiments, the sample may be added to a substrate that includes a capture agent that associates with C5. The capture agent may bind to C5 on a region of C5 that does not overlap with a portion of the C5 protein recognized by the C5-associated factors. Free C5 and C5 associated with one or more C5-associated factors may be captured by the capture agent to facilitate detection using one or more detection reagents. As used herein, a "detection reagent" is any agent that facilitates recognition of an analyte, interaction, reaction, process, or event. In some embodiments, the detection reagent is a detection antibody. The detection reagent may bind to the analyte to facilitate recognition of the presence or absence of the analyte. A detection reagent used to detect capture of free C5 may bind to C5 on a region that does not overlap with the region bound by the capture agent or C5 association factor. A detection reagent used to detect capture of C5 associated with one or more C5 association factors may bind to the C5 association factor. The detection reagent may include a detectable label.

[0179] As used herein, the term "detectable label" refers to one or more markers, signals, or moieties that are bound, incorporated, or associated with another entity, and that are easily detected by methods known in the art, including, but not limited to, radiography, fluorescence, chemiluminescence, enzyme activity, absorbance, and the like. Detectable labels may include, but are not limited to, radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands (e.g., biotin, avidin, streptavidin, and haptens such as digoxigenin), quantum dots, and the like. Detectable labels may be located at any position of the entity to which they are bound, incorporated, or associated. For example, when detectable labels are bound, incorporated, or associated with a peptide or protein, they may be present within an amino acid, peptide, or protein, or may be located at the N- or C-terminus.

[0180] In some embodiments, the detection reagent may be detected using a secondary detection reagent that binds to the detection reagent or a detectable label associated with the detection reagent. The secondary detection reagent may include an antibody. The secondary detection reagent may be associated with a detectable label to facilitate detection (e.g., through fluorescent, radiographic, colorimetric, enzymatic, or other detection methods). In one embodiment, the detection reagent includes digoxigenin. Digoxigenin may be detected using an anti-digoxigenin antibody.

[0181] The assay substrate may include a surface capable of retaining a capture agent. The capture agent may be immobilized to the substrate, for example, through covalent or non-covalent interactions. In some embodiments, the capture agent is biotinylated to facilitate substrate immobilization with a substrate having a biotin-interacting compound (e.g., avidin, neutravidin, or streptavidin). The substrate may include, but is not limited to, an assay plate. In some embodiments, the substrate is a bead.

[0182] The C5 capture agent may include any compound capable of associating with C5. In some embodiments, the capture agent includes a peptide or peptide-based compound. The capture agent may include R5000 or a variant thereof. In some assays, a variant of R5000 is used that includes an N-terminal biotinylated PEG moiety to facilitate substrate immobilization. Some R5000 variants may further include a substitution of the C-terminal lysine with another compound. The compound may be another amino acid, for example, any natural or unnatural amino acid. Some R5000 variants have a substitution of the C-terminal lysine with norvaline.

[0183] In some embodiments, the present disclosure provides a method for measuring C5 or C5 levels in a sample by immobilizing a capture agent on a substrate, where the capture agent binds to a site on C5 that is different from the eculizumab binding site; contacting the substrate with the sample, where the immobilized capture agent binds to C5 in the sample; contacting the substrate with a detection reagent (e.g., an antibody) and measuring the presence or level of the detection reagent as an indication of C5 levels in the sample. The detection reagent may include a detectable label. The detection reagent may be detected using a secondary detection reagent. The detection reagent may be an anti-C5 antibody (e.g., eculizumab or a non-eculizumab anti-C5 antibody). The method may include contacting the substrate with a second detection reagent (e.g., an antibody) that binds to a C5-associated factor (e.g., eculizumab). Measuring the level of the second detection antibody that binds to the substrate may be performed as an indication of the level of the C5-associated factor in the sample. In some embodiments, both free C5 levels and C5 levels associated with eculizumab are measured using the assays disclosed herein. The level may be a combined level resulting in a total C5 level in a sample having both forms. In some embodiments, free C5 levels and C5 levels associated with eculizumab are distinguished.

[0184] In some embodiments, the present disclosure provides a method for measuring the level of free eculizumab in a sample by immobilizing a capture agent on a substrate, where 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, where the C5-capture agent complex comprises immobilized C5; contacting the substrate with a sample, where the immobilized C5 binds to eculizumab in the sample; contacting the substrate with a detection reagent, where the detection reagent binds to eculizumab; and measuring the level of bound detection reagent as an indication of the level of free eculizumab in the sample. The capture agent may include R5000 or a variant thereof. In some assays, a variant of R5000 that includes an N-terminal biotinylated PEG moiety is used to facilitate substrate immobilization. The R5000 variant may include a substitution of the C-terminal lysine with another compound. The compound may be another amino acid, for example, any natural or unnatural amino acid. The R5000 variant may have a substitution of the C-terminal lysine with norvaline. The detection reagent may be an antibody. The detection reagent may include a detectable label or may be detected using a secondary detection reagent.

[0185] III. Kit Any of the C5 inhibitors (e.g., R5000 and / or its active metabolites or variants) described herein may be provided as part of a kit. In a non-limiting example, the C5 inhibitor may be included in a kit for treating a disease. The kit may include a vial of sterile dry C5 inhibitor powder, a sterile solution for dissolving the dry powder, and an infusion set syringe for administering the C5 inhibitor.

[0186] When the C5 inhibitor is provided as a dry powder, it is contemplated that between 10 micrograms and 1000 milligrams of the C5 inhibitor, or at least or at most these amounts, are provided in the kits of the present invention.

[0187] A typical kit may include at least one vial, test tube, flask, bottle, syringe and / or other container or device in which the C5 inhibitor formulation is provided, preferably suitably allocated. The kit may also include one or more secondary containers having sterile pharma- ceutically acceptable buffers and / or other diluents.

[0188] In some embodiments, the compounds or compositions of the present invention are provided in borosilicate vials. Such vials may include a cap (e.g., a rubber stopper). In some cases, the cap includes a rubber stopper coated with FLUROTEC®. The cap may be secured in place with an overseal, such as, but not limited to, an aluminum flip-off overseal.

[0189] The kit may further include instructions for utilizing the kit components, as well as the use of any other reagents not included in the kit. The instructions may include executable variations.

[0190] IV. Definition Bioavailability: As used herein, the term "bioavailability" refers to the systemic availability of a given amount of a compound (e.g., a C5 inhibitor) administered to a subject. Bioavailability is measured as the area under the curve (AUC) or maximum serum or plasma concentration (C) of the unaltered form of the compound following administration of the compound to a subject. maxAUC can be assessed by measuring the area under the curve when the serum or plasma concentration of a compound along the ordinate (Y-axis) is plotted against time along the abscissa (X-axis). In general, the AUC for a particular compound can be calculated using methods known to those of skill in the art and / or as described in Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, Vol. 72, by GS Banker, Marcel Dekker, New York, Inc., 1996, the contents of which are incorporated herein by reference in their entirety.

[0191] Biological system: As used herein, the term "biological system" refers to a cell, a group of cells, a tissue, an organ, a group of organs, an organelle, a biological fluid, a biological signaling pathway (e.g., a receptor-activated signaling pathway, a charge-activated signaling pathway, a metabolic pathway, a 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 a cell membrane, a cell compartment, a cell, a cell culture, a tissue, an organ, an organ system, an organism, a multicellular organism, a body fluid, or any biological entity. In some embodiments, the biological system is a cell signaling pathway that includes intracellular and / or extracellular signaling biomolecules. In some embodiments, the biological system includes a proteolytic cascade (e.g., a complement cascade).

[0192] Buffering Agent: As used herein, the term "buffering agent" refers to a compound used in a solution intended to resist changes in pH. Such compounds 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, potassium phosphate, sodium citrate, and sodium tartrate.

[0193] Clearance rate: As used herein, the term "clearance rate" refers to the rate at which a particular compound is eliminated from a biological system or fluid. Compound: As used herein, the term "compound" refers to a distinctive chemical entity. In some embodiments, a particular compound may exist in one or more isomeric or isotopic forms (including but not limited to stereoisomers, geometric isomers and isotopes). In some embodiments, a compound is provided or utilized only in a single such form. In some embodiments, a compound is provided or utilized as a mixture of two or more such forms (including but not limited to racemic mixtures of stereoisomers). One of ordinary skill in the art will appreciate that some compounds exist in different forms and exhibit different properties and / or activities (including but not limited to biological activity). In such cases, it is within the skill of the art to select or avoid a particular form of a compound for use in accordance with the present invention. For example, compounds containing asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms.

[0194] Circular or cyclized: As used herein, the term "cyclic" refers to the presence of a continuous loop. Circular molecules need not be circular, only when linked to form an unbroken chain of subunits. A cyclic polypeptide may contain a "cyclic loop" that is formed when two amino acids are connected by a bridging moiety. A cyclic loop includes the amino acids along the polypeptide that lie between the bridged amino acids. A cyclic loop may contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0195] Downstream Event: As used herein, the term "downstream" or "downstream event" refers to any event that occurs after and / or as a result of another event. In some cases, downstream events are events that occur after or as a result of C5 cleavage and / or complement activation. Such events may include, but are not limited to, production of C5 cleavage products, activation of MAC, hemolysis, and hemolysis-related diseases (e.g., PNH).

[0196] Equilibrium dissociation constant: As used herein, the term "equilibrium dissociation constant" or "K D " refers to a value that represents the tendency of two or more agents (e.g., two proteins) to reversibly separate. In some cases, K D indicates the concentration of the primary drug when half of the total level of the secondary drug is associated with the primary drug.

[0197] Half-life: As used herein, the term "half-life" or "t 1 / 2 "Terminal half-life" or "terminal t" refers to the time it takes for a given process or compound concentration to reach half of its final value. 1 / 2 " refers to the time it takes for the plasma concentration of a factor to decrease by half once the concentration of the factor has reached pseudoequilibrium.

[0198] Hemolysis: As used herein, the term "hemolysis" refers to the destruction of red blood cells. Identity: As used herein, the term "identity," when referring to a polypeptide or nucleic acid, refers to a comparative relationship between sequences. The term is used to describe the degree of sequence relatedness between polymeric sequences, and may include the percentage of monomeric components that match in a gapped alignment (if any) as processed by a particular mathematical model or computer program (i.e., an "algorithm"). The identity of related polypeptides may be readily calculated by known methods, including, but not limited to, those described by others (Lesk, AM, ed., Computational Molecular Biology, Oxford University Press, 2002). University Press, New York, 1988; Smith, DW, ed., Biocomputing: Informatics and Genome Projects, Academic Press, New York, 1993; Griffin, AM, et al., eds., Computer Analysis of Sequence Data, (including those previously described in, for example, von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, 1987; Gribskov, M. et al., eds., Sequence Analysis Primers, M. Stockton Press, New York, 1991; and Carillo et al., Applied Math, SIAM J, 1988, vol. 48, p. 1073).

[0199] Inhibitor: As used herein, the term "inhibitor" refers to any agent that blocks or causes a decrease in the occurrence of a specific event; a cell signal; a chemical pathway; an enzymatic reaction; a cellular process; an interaction between two or more entities; a biological event; a disease; a disorder; or a pathological condition.

[0200] Loading dose: As used herein, an "initial loading dose" refers to an initial dose of a therapeutic agent, which may be different from one or more subsequent doses. An initial loading dose may be used to obtain an initial concentration or level of activity of the therapeutic agent before subsequent doses are administered.

[0201] Intravenous: As used herein, the term "intravenous" refers to the area inside a blood vessel. Intravenous administration typically refers to the delivery of a compound into the blood via injection into a blood vessel (e.g., a vein).

[0202] In vitro: As used herein, the term "in vitro" refers to events that occur not inside a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment (e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc.).

[0203] In vivo: As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0204] 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 are formed between amino acids in a polypeptide.

[0205] Linker: The term "linker," as used herein, refers to a group of atoms (e.g., 10-1,000 atoms), molecule, or other compound used to link two or more entities. A linker may link such entities through covalent or non-covalent (e.g., ionic or hydrophobic) interactions. A linker may include a chain of two or more polyethylene glycol (PEG) units. In some cases, a linker may be cleavable.

[0206] Minute ventilation: As used herein, the term "minute ventilation" refers to the volume of air inhaled or exhaled from a subject's lungs every minute. Non-proteinogenic: As used herein, the term "non-proteinogenic" refers to any non-naturally occurring protein, e.g., having non-naturally occurring components, e.g., non-naturally occurring amino acids.

[0207] Patient: As used herein, "patient" refers to a subject who may seek or require treatment, who is in need of treatment, who is receiving treatment, who will receive treatment, or who is under the care of a skilled professional for a particular disease or condition.

[0208] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition containing at least one active ingredient (e.g., a C5 inhibitor) in a form and amount that allows the active ingredient to be therapeutically effective.

[0209] Pharmaceutically acceptable: The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable risk-benefit ratio, and within the scope of sound medical judgment.

[0210] Pharmaceutically acceptable excipient: The phrase "pharmaceutical acceptable excipient" as used herein refers to any component present in a pharmaceutical composition other than the active agent (e.g., the active agent R5000 and / or its active metabolites or variants) that has substantially non-toxic and non-inflammatory properties in patients. In some embodiments, the pharmaceutical acceptable excipient is a medium capable of suspending or dissolving the active agent. The excipient may include, for example, anti-adhesives, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (coloring agents), softeners, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, glidants (glidants), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), 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, retinyl 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.

[0211] Plasma compartment: As used herein, the term "plasma compartment" refers to the intravascular space occupied by plasma. Salt: As used herein, the term "salt" refers to a compound consisting of an anion combined with a cation. 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.

[0212] Sample: As used herein, the term "sample" refers to an aliquot or portion taken from a source and / or provided for analysis or processing. In some embodiments, a sample is derived from a biological source, such as a tissue, cell, or component (e.g., bodily fluids, including but not limited to, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). In some embodiments, a sample may be or include a homogenate, lysate, or extract (prepared from a whole organism or a subset of its tissues, cells, or components, or a fraction or portion thereof, such as, but not limited to, plasma, serum, cerebrospinal fluid, lymph, external sections of the skin, respiratory tract, intestinal tract, and urogenital tract, tears, saliva, milk, blood cells, tumors, or organs). In some embodiments, a sample is or includes a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins. In some embodiments, a "primary" sample is an aliquot of a source. In some embodiments, the primary sample may be subjected to one or more processing (eg, separation, purification, etc.) steps to prepare the sample for analysis or other uses.

[0213] Subcutaneous: As used herein, the term "subcutaneous" refers to the space below the skin. Subcutaneous administration is the delivery of a compound below the skin. Subject: As used herein, the term "subject" refers to any organism to which a compound according to the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Exemplary subjects include animals (e.g., mammals such as mice, rats, rabbits, pig subjects, non-human primates, and humans).

[0214] Substantially: As used herein, the term "substantially" refers to a qualitative state exhibiting a degree or extent of a total or near total amount of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or completeness, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0215] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., a C5 inhibitor) to be delivered that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition.

[0216] Tidal Volume: As used herein, the term "tidal volume" refers to the normal lung volume of air exchanged between breaths (in the absence of excessive effort). T max As used herein, the term "T max " refers to the period of time during which a maximum concentration of a compound in a subject or bodily fluid is maintained.

[0217] Treat: As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit symptoms of the disease, disorder, and / or condition and / or to subjects who exhibit only early signs of the disease, disorder, and / or condition, with the goal of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0218] Therapeutic Dose: As used herein, "therapeutic dose" refers to one or more doses of a therapeutic agent administered in the course of addressing or alleviating a therapeutic indication. Therapeutic doses may be adjusted to maintain a desired concentration or level of activity of the therapeutic agent in a bodily fluid or biological system.

[0219] Volume of distribution: As used herein, the term “volume of distribution” or “V dist " refers to the volume of body fluid required to contain the total amount of a compound in the body at the same concentration as in blood or plasma. The volume of distribution may reflect the extent to which a compound is present in extravascular tissues. A large volume of distribution reflects the tendency of a compound to bind to tissue components compared to plasma protein components. In a clinical situation, V dist can be used to determine a loading dose of the compound to achieve a steady state concentration of the compound.

[0220] V. Equivalents and Scope While various embodiments of the present invention have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

[0221] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not limited to the above description, but is instead set forth in the appended claims.

[0222] In the claims, articles such as "a," "an," and "the" may mean one or more, unless otherwise indicated to the contrary or otherwise evident from the context. A claim or description including "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are present in, utilized in, or otherwise adapted to a given product or process, unless otherwise indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of a group is present in, utilized in, or otherwise adapted to a given product or process. The invention includes embodiments in which more than one or all of the group members are present in, utilized in, or otherwise adapted to a given product or process.

[0223] It should also be noted that the term "comprising" is intended to be open, permitting but not requiring the incorporation of additional elements or steps. Thus, when the term "comprising" is used herein, the terms "consisting of" and "or including" are also encompassed and disclosed.

[0224] When ranges are given, the endpoints are included. Moreover, unless otherwise specified or otherwise evident from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to be capable of assuming any particular value or subrange within the stated range in various embodiments of the invention, to the tenth unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0225] In addition, it should be understood that any particular embodiment of the present invention that is encompassed by the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly set forth herein, since they are deemed to be known to those of skill in the art. Any particular embodiment of the composition of the present invention (e.g., any nucleic acid, any protein encoded thereby, any method of manufacture, any method of use, etc.) may be excluded from any one or more of the claims for any reason, regardless of the existence of prior art.

[0226] All cited sources, e.g., references, publications, databases, database entries, and techniques cited herein, are incorporated by reference into this application, even if not expressly stated in the citation. In the event of a conflict between the contents of a cited source and the contents of this application, the contents of this application shall control.

[0227] The sections and headings are not intended to be limiting. (Example) Example 1. Preparation of R5000 aqueous solution Polypeptides were synthesized using standard solid-phase Fmoc / tBu methods. Synthesis was performed on a Liberty automated microwave peptide synthesizer (CEM, Matthews, NC) using standard protocols with Rink amide resin, although other automated synthesizers without microwave capabilities may be used. All amino acids were obtained from commercial suppliers. 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). Polypeptides were cleaved from the resin using 95% TFA, 2.5% TIS, and 2.5% water for 3 h and isolated by precipitation with ether. Crude polypeptides were purified by reversed-phase preparative HPLC using a C18 column with a gradient of 20% to 50% acetonitrile / water. Purification was performed with 0.1% TFA for 30 min. Fractions containing pure polypeptide were collected and lyophilized, and all polypeptides were analyzed by LC-MS.

[0228] R5000 (SEQ ID NO:1) was prepared as a cyclic peptide with 15 amino acids (four of which are unnatural), 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 contains a polyethylene glycol spacer (PEG24) attached to an L-γ glutamic acid residue that has been derivatized with a palmitoyl group. Cyclization of R5000 is via a lactam bridge between the side chains of L-Lys1 and L-Asp6. All of the amino acids in R5000 are L-amino acids. R5000 has a molecular weight of 3562.23 g / mol and a C 172 H 278 N 24 O 55 It has the chemical formula:

[0229] Like eculizumab, R5000 blocks the proteolytic cleavage of C5 into C5a and C5b. Unlike eculizumab, R5000 also binds to C5b and can block its binding to C6, preventing the subsequent assembly of the MAC.

[0230] R5000 was prepared as an aqueous solution for injection containing 40 mg / mL R5000 in a formulation of 50 mM sodium phosphate and 76 mM sodium chloride at a pH of 7.0. The resulting composition was used to prepare a medical product that conforms to current Good Manufacturing Practice (cGMP), namely, a 1 ml syringe with a 29 gauge, 12.7 mm (1 / 2 inch) staked needle placed within a self-administration device.

[0231] Example 2. Administration and storage of R5000 R5000 is administered by subcutaneous (SC) or intravenous (IV) injection and the dose administered (dosage volume) is adjusted based on subject weight on a mg / kg basis. This is achieved using a set of fixed doses adjusted to a set of weight brackets. Overall, human dosing supports a broad weight range of 43-109 kg. Subjects presenting with a heavier weight (>109 kg) are addressed on a case-by-case basis with consideration of medical monitoring.

[0232] R5000 is stored at 2°C to 8°C [36° to 46°F]. Once dispensed into subjects, R5000 is stored at controlled room temperature (20°C to 25°C [68°F to 77°F]) for a maximum of 30 days, protected from sources of excessive temperature fluctuations such as high heat or exposure to light. Storage of R5000 at other than room temperature is preferably avoided. R5000 may be stored under these conditions for a maximum of 30 days.

[0233] Example 3. Stability test Stability testing is performed according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q1A "Stability of New Drug Substances and Products". Samples from the aqueous solution of Example 1 are held at three temperatures: -20°C, 5°C, and 25°C. Testing intervals are 1 month, 2 months, and 3 months, then every 3 months up to 24 months. Samples are tested for appearance (e.g., clarity, color, presence of precipitate), pH, osmolality, concentration, purity, target activity (e.g., by RBC lysis assay), particulate levels, endotoxin levels, and sterility. A sample is considered stable if, at each of the temperature conditions tested, it has a clear, colorless appearance with no visible particles; a pH of 7±0.3; an osmolality of 260-340 mOsm / kg; a purity of ≥95% (and no single impurities of >3%); target activity comparable to a reference standard; a particulate level of ≤6,000 particulates / vial for particles ≥10 μm and ≤600 particulates / vial for particles ≥25 μm; an endotoxin level of ≤100 EU / mL; and no growth of microorganisms.

[0234] Example 4. Freeze-thaw stability Studies were conducted to test the stability of the aqueous solutions of Example 1 when exposed to multiple freeze and thaw cycles. R5000 showed no degradation or other changes after five cycles of freezing and thawing.

[0235] Example 5. Surface Plasmon Resonance (SPR)-Based Binding Assessment The binding interaction between R5000 and C5 was measured using surface plasmon resonance. R5000 has an equilibrium dissociation constant (K D ) (n=3) and a K of 0.78 nM at 37 °C D (n=3) bound to C5. Overall surface plasmon resonance data, combined with analysis of the high-resolution co-crystal structure, indicate that R5000 exhibits specific, strong and rapid association with C5 and slow dissociation kinetics.

[0236] Example 6. Evaluation of C5 cleavage inhibition R5000 was evaluated for inhibition of cleavage of C5 into C5a and C5b by ELISA analysis of C5a cleavage products and C5b-9 membrane attack complex (MAC) formation. The inhibitory activity of R5000 on host C5 is an important factor in selecting an appropriate animal model for drug safety. Inhibition of C5 cleavage is the basis for the clinical efficacy of eculizumab, the only therapeutic agent currently approved for the treatment of PNH. R5000 demonstrated a dose-dependent inhibition (IC) of C5a formation following classical pathway activation. 50 = 4.8 nM; Figure 1 ), as well as dose-dependent inhibition of C5b (as measured by C5b-9 or MAC formation) upon activation of the classical and alternative complement pathways (IC 50 = 5.1 nM; Figure 2).

[0237] Example 7. Inhibition of Complement-Induced Red Blood Cell (RBC) Hemolysis The RBC lysis assay is a reliable method to screen complement inhibitors in serum / plasma from various species and compare the relative activity of test substances. An in vitro functional assay was used to evaluate the inhibitory activity of peptides, including R5000, on complement function in several species. In this assay, the classical pathway complement components are tested for their functional ability to lyse sheep RBCs pre-coated with rabbit anti-sheep RBC antibodies. When the antibody-coated RBCs are incubated with the test serum, the classical pathway of complement is activated and the hemolytic outcome is monitored by the release of hemoglobin. Antibody-sensitized sheep red blood cells are used as the medium for lysis in this assay, and serum and / or plasma from various species are tested at their predefined 50% hemolytic complement activity (CH). 50 ) was used.

[0238] R5000 demonstrated potent inhibition of complement-induced RBC hemolysis in human, non-human primate, and porcine serum and / or plasma (see Table 1).

[0239] [Table 1]

[0240] Weak activity was observed in rat plasma (>100-fold lower than in cynomolgus monkeys) and little activity was observed in other rodents, dogs, or rabbits. Structural data obtained from co-crystallization of human C5 with a molecule closely related to R5000 offer an explanation for this type of selectivity through careful analysis of the primary amino acid sequence at the drug-binding site of the target protein. While the primate sequence was 100% conserved within the residues involved in the R5000 interaction, there were significant differences within these residues in rodents, especially dogs, where no identical portion of the protein exists. These amino acid differences were sufficient to explain the activity profile of R5000 in the different species.

[0241] The ability of R5000 to inhibit complement-mediated lysis of red blood cells via the classical and alternative complement activation pathways was also tested. The classical pathway was assessed using two different assays using antibody-sensitized sheep red blood cells. In one method, hemolysis was assessed using 1% normal human serum, while in the second assay, 1.5% C5-depleted human serum containing 0.5 nM human C5 was used. Inhibition of the alternative complement activation pathway was assessed using rabbit red blood cells in 6% normal human serum in the absence of calcium (see Table 2).

[0242] [Table 2]

[0243] R5000 demonstrated complement-mediated lysis in both classical and alternative pathway assays. Example 8. Pharmacodynamics in Cynomolgus Monkeys Because R5000 is a potent inhibitor of complement in primates, cynomolgus monkeys were selected for multi-dose studies to evaluate the inhibitory activity of R5000 in an animal model. Plasma drug concentrations were measured by LC-MS, and complement activity was assayed using the RBC lysis assay described above. Overall results from these studies showed that plasma drug concentrations in monkeys needed to be 2.5 μg / mL or higher to achieve >90% inhibition of complement activity (see Figure 3).

[0244] In a 7-day study, R5000 was administered to cynomolgus monkeys via subcutaneous injection (SC) in multiple daily doses. Blood samples were analyzed for hemolysis as an indicator of complement activity at the indicated times (days 1, 4, and 7 after the first dose, but data are reported prior to dosing on each day) using an ex vivo sheep RBC lysis assay (using 1% plasma in the assay). Drug concentrations were measured from the same samples using an LC-MS method specific for R5000. As shown in Table 3 and Figures 4 and 5, minimal (<3% of pre-dose) complement activity was observed throughout the dosing period when R5000 was administered daily for 7 days at either 0.21 or 4.2 mg / kg.

[0245] [Table 3]

[0246] 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 for up to 24 hours after the final dose. After treatment was discontinued, elevated levels of hemolysis were observed. By day 4 after the final dose was administered (264 hours in Figure 4), hemolysis was >75% of baseline. This correlates well with the measured plasma levels of the compound during and after dosing (dotted line in Figure 4). A second group of animals in the multiple dose study received a 4.2 mg / kg dose of R5000 daily. In this group, hemolysis was essentially completely inhibited (at <1%) throughout dosing and was maintained below 3% for 48 hours after the final dose (day 9; 216 hours in Figure 5). Four days after the final dose (264 hours in Figure 5), hemolysis reached approximately 10% of baseline. The results again showed inhibition of complement activity throughout the treatment period (compared to pre-treatment results), which correlated with plasma drug concentrations, and also showed excellent correlation between pharmacokinetic and pharmacodynamic values.

[0247] The complement inhibitory activity of R5000 was evaluated in a 28-day repeat-dose study in cynomolgus monkeys using an ex vivo RBC hemolysis assay. R5000 was administered daily via subcutaneous injection at either 0, 1, 2, or 4 mg / kg / day for 28 days (Day 1: Figure 6 and Day 28: Figure 7). Results showed complete inhibition of hemolysis from 2 hours after administration of the first dose through Day 28 of dosing, with percentage hemolysis being <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, with little inhibition of the complement system. The absence of complement inhibitory activity at the end of the recovery period indicated clearance of the drug from the animals.

[0248] Complement inhibition was also tested as part of a 13-week repeat-dose study in cynomolgus monkeys. Monkey samples were analyzed using an ex vivo RBC hemolysis assay. R5000 was administered daily via subcutaneous injection for 13 weeks at either 0, 0.25, 1, 2, or 10 mg / kg / day doses. Similar to the 28-day study, results from the 13-week study showed complete inhibition of hemolysis ex vivo from 2 hours after administration of the first dose through 13 weeks of dosing, with percentage hemolysis being <5% in the 0.25, 1, 2, and 10 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, with little inhibition of the complement system. The absence of complement inhibitory activity at the end of the recovery period indicated clearance of the drug from the animals.

[0249] During testing, oxidative metabolism led to the formation and detection of a hydroxylated metabolite of R5000 (referred to herein as "R5001") by ω-hydroxylation of the palmitoyl tail.

[0250] The ability of R5001 to inhibit complement-mediated lysis of red blood cells via the classical complement activation pathway was tested using antibody-sensitized sheep red blood cells in 1% normal human serum. R5001 had an IC of 4.5 nM. 50 and showed potent inhibition of complement-induced hemolysis.

[0251] Example 9. Pharmacokinetic / pharmacodynamic modeling and simulation of human pharmacokinetics A PK / PD model was constructed in silico using in vivo data obtained in cynomolgus monkeys. Model fit and accuracy were assessed by comparing simulation results with newly generated experimental data. Once validated in monkeys, the final model was used to predict human pharmacokinetics by applying non-proportional scaling to its parameters. The resulting simulations support a daily dose of 0.1 mg / kg that maintains nearly 90% target inhibition at steady state with the planned dosing interval of once daily or less frequently in humans (see Figure 8). Due to the long half-life of R5000, several doses are required to reach the final peak and trough drug concentrations. Plasma C max is expected to be approximately 3-fold higher than the initial dose after one week of daily dosing, when drug concentrations reach steady state.

[0252] Example 10. Phase 1 Single Ascending Dose Clinical Trial of R5000 A phase 1 single ascending dose clinical pharmacology study in healthy human volunteers was conducted designed to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of R5000 following subcutaneous (SC) injection. Dose volumes were determined by the dose requirement of the cohort and subject weight. Pregnant or lactating subjects and any subjects with systemic infection or colonization with Neisseria meningitides were excluded. The study was a randomized, double-blind, and placebo (PBO) controlled study with four SC single ascending dose cohorts housed within the clinical pharmacology unit for 3 days. All subjects received prophylaxis with ciprofloxacin and subjects in the highest single dose cohort (i.e., 0.4 mg / kg) were vaccinated against Neisseria meningitides for at least 14 days prior to the study. On day 1, all subjects received one dose of R5000. Four subjects (two receiving R5000 and two receiving PBO) received the lowest dose level (0.05 mg / kg), and six subjects per cohort (four receiving R5000 and two receiving PBO) received three sequentially higher dose levels (0.1, 0.2, and 0.4 mg / kg). Subject demographic information is presented in Table 4.

[0253] [Table 4]

[0254] Safety was assessed by intensive clinical monitoring, and frequent blood samples were obtained to measure R5000 concentration by liquid chromatography / high-resolution mass spectrometry and the ability to inhibit complement-mediated RBC lysis in an ex vivo antibody-sensitized sheep red blood cell hemolysis assay.

[0255] The pharmacokinetic (PK) parameters measured in this study were clearance (CL), C max (Maximum plasma drug concentration, Figure 9A), T max (the time it takes for a drug to reach its maximum plasma concentration), t 1 / 2 (half-life), AUC 0-24(area under the plasma concentration versus time curve from time 0 to 24 h; see Figure 9B for plasma concentrations over time), AUC 0-inf (area under the plasma concentration versus time curve from time 0 to infinity; see Figure 9B for plasma concentrations over time), V z (apparent volume of distribution during the terminal phase), K (excretion rate), and F (fraction). The results for each parameter are presented in Table 5.

[0256] [Table 5]

[0257] All cohorts showed C values ​​consistent with predictions from an in silico PK model developed using data from non-human primate (NHP) studies. max The plasma concentration of a single SC injection was C max The mean maximum plasma concentration (C) showed a linear relationship between C and dose level (Figure 9A), and dose-dependent exposure was observed across all dose levels (Figure 9B). max The mean area under the concentration-time curve (AUC) from 0 to 24 hours after dosing ranged from 1010 to 5873 ng / mL across doses. 0-24 ) ranges from 21,440 to 112,300 ng throughout the dose range. * These results showed that the plasma concentration (C max ) and exposure (AUC 0-24 ) is approximately proportional to the time to maximum observed plasma concentration (t max The median elimination half-life (t ) ranged from 3.0 to 4.6 hours across doses, indicating that R5000 exhibits an intermediate rate of absorption from the SC space to the central (blood) compartment. The mean apparent total body clearance (CL / F) after R5000 administration was low, ranging from 0.2481 to 0.4711 mL / hr / kg. 1 / 2 The apparent total volume of distribution (V zThe mean t / F) ranged from 61.89 to 105.1 mL / kg, indicating that R5000 was primarily localized within the circulating blood compartment with minimal extravascular distribution. 1 / 2 was determined to be 7 days.

[0258] R5000 also induced rapid dose-dependent inhibition of hemolysis [direct hemolysis (Figure 10A) and %CH 50 (FIG. 10B) and erythrocyte lysis over time in 1% plasma (FIG. 10C)] and inhibition of complement activity (as measured by WIESLAB® ELISA in all subjects after a single dose, see FIG. 11) were observed at approximately 3 hours after dosing. Results showed that at maximum plasma concentrations, maximum percent inhibition of hemolysis compared to baseline reached >90% in the 0.1, 0.2, and 0.4 mg / kg dose cohorts and 60% in the lowest dose (0.05 mg / kg) cohort. Dose-dependent inhibition of hemolysis for up to 4 days was observed in the 0.1, 0.2, and 0.4 mg / kg dose cohorts. Notably, mean hemolysis remained above baseline for up to 2 days in the 0.05 mg / kg cohort, up to 4 days in the 0.1 mg / kg cohort, and up to 7 days in the 0.2 and 0.4 mg / kg cohorts.

[0259] Similarly, analysis of complement activity showed that inhibition of complement activity remained strong for 4 days following injection of 0.4 mg / kg (see FIG. 11). Human plasma samples obtained from subjects receiving 0.4 mg / kg injections were subjected to WIESLAB® ELISA (Euro Diagnostica, Malmö, Sweden) analysis. This assay measures the alternative pathway of complement activity. Complement activity, as measured via this assay, was inhibited by 3% 3 hours post-dose and remained below 13% 96 hours after administration of R5000.

[0260] A single SC dose of R5000 was safe and well tolerated in healthy volunteers, with no clinically significant changes in vital signs, clinical laboratory parameters, physical examination, or ECG.

[0261] This study suggests that a low daily dose may achieve a steady state level suitable for >80% inhibition of hemolysis and that once weekly dosing may be sufficient. Specifically, 0.2 mg / kg may result in complete inhibition of complement activity and complete inhibition of hemolysis.

[0262] Example 11. Multiple-dose clinical trial of R5000 A phase 1 multiple-dose clinical pharmacology study in healthy human volunteers was conducted designed to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of R5000 after subcutaneous (SC) injection once daily for 7 days. The study was a single-center, randomized, double-blind, and placebo (PBO)-controlled study. Subjects received 0.2 mg / kg R5000 or matching PBO subcutaneously daily for 7 days while housed in the clinical pharmacology unit. Dose volume was determined by the dose requirement of the cohort and subject weight. Pregnant or lactating subjects and any subjects with systemic infection or colonization with Neisseria meningitides were excluded. All subjects received ciprofloxacin prophylaxis, and subjects in the multiple-dose cohort were vaccinated against Neisseria meningitides for at least 14 days prior to the study. Safety was assessed through intensive clinical monitoring, and blood samples were obtained immediately before dosing and 3 and 6 hours after dosing on each day to determine R5000 concentration by liquid chromatography / high-resolution mass spectrometry and the ability to inhibit complement-mediated RBC lysis in an in vitro antibody-sensitized sheep red blood cell hemolysis assay.

[0263] A total of six subjects were enrolled in the study (four receiving R5000 and two receiving PBO). Subject demographics are presented in Table 6.

[0264] [Table 6]

[0265] As evidenced by Table 7 and associated FIG. 12A (which shows percent hemolysis and plasma concentrations over 7 days), plasma concentrations showed a steady increase in exposure over the 7 days of dosing. From these data, the half-life of R5000 was determined to be 7 days. Plasma levels recovered to approximately 2000 ng / ml by day 15 and approximately 1000 ng / ml by day 21 (FIG. 12B).

[0266] [Table 7]

[0267] The PK parameters of R5000 after multiple dose subcutaneous administration (0.2 mg / kg / day) for 7 days are presented in Table 8. The pharmacokinetic (PK) parameters measured were clearance (CL), C max (maximum plasma drug concentration), T max (the time it takes to reach maximum plasma concentration after drug administration), t 1 / 2 (half-life), AUC tau (area under the plasma concentration-time curve from time 0 to 24 hours), AUC 0-inf (area under the plasma concentration-time curve from time 0 to infinity), V z / F (apparent volume of distribution), K el (excretion rate), and F (rate).

[0268] [Table 8]

[0269] Average C on the first day max and AUC tau were 2533ng / ml and 50,010ng, respectively. *hours / mL, which was consistent with results from the 0.2 mg / kg single dose cohort over the same post-dose period. After 7 days of daily subcutaneous dosing, C max and AUC tau Each is approximately 2.9 times (7th day C max mean = 7290 ng / ml) and 3.0-fold (day 7 AUC tau Average=151,300ng * The maximum plasma concentration on day 7 (T max The median time to T after single-dose subcutaneous administration was 3.0 hours, which is max (Day 1 T max Median = 3.0-4.6 hours), indicating a consistent rate of absorption of R5000 with repeated dosing. The mean apparent total body clearance of R5000 on day 7 (day 7 CL / F = 1.3 mL / hr / kg) was slightly increased compared to the total body clearance after a single subcutaneous dose of 0.2 mg / kg [CL / F = 0.29 mL / hr / kg at single ascending dose (SAD) of 0.2 mg / kg]. However, the elimination rate constant (K el ) were consistent after single and repeated dosing (K at 0.2 mg / kg SAD). el Average=0.0041 hours -1 0.2mg / kg MD on day 7K el Average=0.0043 hours -1 ), which indicates that the clearance of R5000 does not change significantly with repeated dosing. The apparent volume of distribution of R5000 (V z / F) showed some increase with administration of multiple doses of R5000 (V z / F average = 71.4 mL / kg; Day 7 V at MD of 0.2 mg / kg z / F average = 311.6 mL / kg). However, the V z / F was also less than the total body water, suggesting that R5000 does not distribute into the extravascular space upon repeated subcutaneous administration.

[0270] The mean percent inhibition of hemolysis was measured in subject serum samples to determine whether a low daily dose was adequate to achieve steady-state levels and complete and sustained inhibition of complement and suppression of hemolysis (see Table 9).

[0271] [Table 9]

[0272] The mean percent inhibition of hemolysis compared to baseline reached ≧95% from the first time point post-dose through 3 hours post-dose on Day 1 and continued throughout the entire 7 days of dosing. All individual subjects showed a ≧90% reduction in hemolysis at all time points. Hemolysis at Day 8 (24 hours after receiving the final dose) was found to be ≦3% in all subjects. Hemolysis returned to pre-dose levels within 2 weeks after the final dose.

[0273] The study suggests that a low daily dose achieves a steady-state level suitable for complete and sustained inhibition of complement and suppression of hemolysis. The study also suggests that weekly dosing may be sufficient to inhibit complement activity and reduce hemolysis in humans.

[0274] Complement activity in subject plasma samples was measured by WIESLAB® ELISA (Euro Diagnostica, Malmö, Sweden) assay, which measures the alternative pathway of complement activation. Inhibition of complement activity, as measured via this assay, was rapid, complete, and sustained throughout the dosing period in all subjects (see FIG. 13A and Table 10). In this table, SEM indicates standard error of the mean.

[0275] [Table 10]

[0276] Complement activity was found to be ≦5% in all subjects on day 8 (24 hours after the last dose). Complement activity returned to pre-dose levels within 2 weeks after the last dose (FIG. 13B).

[0277] R5000 was safe and well tolerated in healthy volunteers. No clinically significant changes were observed in vital signs, clinical laboratory parameters (hematology, blood chemistry, coagulation, and urinalysis), physical examination, and ECG.

[0278] R5000 and metabolite R5001 were measured in the 0.20 mg / kg dose group of the multiple dose arm of the study, and both were detected. Example 12. Pharmacokinetic / Pharmacodynamic Modeling and Simulation of Human Pharmacokinetics in Phase II Studies A PK / PD model was developed to describe the plasma concentrations of R5000 and its metabolites, as well as their activity in an in vitro erythrocyte lysis assay. The model was based on the quasi-steady-state approximation of the target-mediated pharmacokinetic model. In the multiple dose arm of the Phase I study with R5000, all subjects achieved drug concentrations consistent with the predictions from the in silico PK model generated using single dose human PK and PD data. Human PK / PD simulations of R5000 support the planned dosing schedule of a loading dose of 0.3 mg / kg followed by a daily dose of 0.1 mg / kg to achieve ≧90% target inhibition after the first dose and at steady state ( FIG. 14 ).

[0279] Example 13. Study design for Phase II clinical trials Based on the nominal predicted steady-state drug concentrations and the maximum dose (based on body weight brackets), a dose of 0.1 mg / kg / day of R5000 was selected. The results are presented in Table 11. In the table, max refers to the maximum plasma drug concentration, and AUC 0-24 indicates the area under the plasma concentration-time curve from time 0 to 24 hours, SD indicates a single dose, QD indicates a once-daily dose, and *indicates the maximum dose a patient may receive by dosing according to body weight brackets.

[0280] [Table 11]

[0281] Inhibition of hemolysis was predicted to be maintained at ≥90% at all time points when administering a daily dose of 0.1 mg / kg R5000 on the first day of dosing based on the Phase I study and a loading dose of 0.3 mg / kg R5000. This prediction was supported by overlaying the predicted R5000 plasma concentrations at steady state with the PK / PD relationships obtained from Phase I studies in healthy volunteers (Figure 15). Adequate and sustained control of hemolysis is a key component of both efficacy and safety in the treatment of paroxysmal nocturnal hemoglobinuria (PNH), in which even a moderate, transient reduction in complement blockade can result in immediate "breakthrough" intravascular hemolysis and recurrence of severe PNH symptoms. This consideration was particularly important in terms of accounting for potential dosing errors.

[0282] To efficiently achieve and maintain hemolysis control, the Phase 2 study was designed to incorporate a single loading dose of 0.3 mg / kg to adequately suppress hemolysis after the first dose. Well-tolerated single doses of up to 0.4 mg / kg subcutaneous injection in the single dose cohort of the Phase 1 study in healthy volunteers (Figure 16A) provided the rationale for the use of a 0.3 mg / kg loading dose after overlaying the expected R5000 plasma trough concentrations after the first dose with the PK / PD relationships obtained from the Phase 1 study in healthy volunteers. The loading dose was not expected to affect the expected exposure at steady state. The 0.3 mg / kg loading dose was predicted to achieve the target pharmacodynamic effect (>90% hemolysis inhibition) at trough levels and facilitate immediate control of hemolysis after the first dose (see PK / PD model presented in Figure 16B). Based on the PK / PD model, if a subject fails to dose after steady state is reached, the proposed starting dosing regimen was predicted to maintain inhibition of hemolysis by ≧90% for at least 72 hours (see model presented in FIG. 16C).

[0283] From the subsequent visit at week 2, in subjects who did not achieve an adequate response (defined as lactate dehydrogenase levels 1.5-fold below the upper limit of normal), the dose was planned to be increased to 0.3 mg / kg daily after evaluation of safety and tolerability data by the investigator and medical surveillance. If an overt breakthrough hemolytic episode (e.g., hemoglobinuria) was observed, the dose was planned to be increased to 0.3 mg / kg daily.

[0284] Example 14. Phase II Dosage, Administration and Contraindications During Phase II clinical trials, R5000 will be administered subcutaneously at a dose of 0.1 mg / kg once daily (every 24 hours) by self-administration, after an initial loading dose of 0.3 mg / kg on the first day of dosing (Study Day 1). Some subjects may receive a higher dose of 0.3 mg / kg to optimize hemolysis control. The dose (dose volume) for each injection will be determined based on the assigned dose and patient weight. Overdosage of R5000 is not expected to result in acute or specific systemic adverse events. In case of overdosage, clinically appropriate symptomatic measures will be initiated as determined by the clinical scenario and in view of medical monitoring.

[0285] Daily administration of R5000 has the potential to achieve consistent and sustained inhibition of complement, thus reducing the risk of breakthrough hemolysis. The dosing schedule of R5000 is selected to avoid the large peak-to-trough ratio in plasma concentration seen when monoclonal antibodies are administered every two weeks. With a plasma half-life of approximately 7 days, daily administration of R5000 is expected to be maintained within a narrow plasma concentration range that effectively "clamps" terminal complement activity under continuous inhibition, thus avoiding the phenomenon of "breakthrough hemolysis."

[0286] This is in contrast to the current standard of care, eculizumab. It has been reported that a proportion of patients receiving eculizumab have inadequate control of their disease resulting in the occurrence of breakthrough hemolysis near the end of the 2-week dosing interval with the approved dose of 900 mg every other week (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 treatment schedule for eculizumab is every other week, i.e., once every 12–16 days. While hemolysis is well controlled during the first day of a treatment cycle, efficacy is often reduced as eculizumab plasma levels are low during the final days before rechallenge. This lack of disease control has led to the need for eculizumab to be dose-escalated beyond the approved dose, for which no clinical trials have been conducted to establish safety or efficacy. The United Kingdom (UK) PNH National Service Reports indicate that approximately 22% of patients treated with eculizumab require nonstandard dosing due to breakthrough hemolysis at standard doses.

[0287] Subjects will be instructed to self-administer a subcutaneous dose every 24 hours at approximately the same time each day. Subjects will be instructed to inject into the abdomen, although thigh or upper arm injections may be used. The study will require subjects to record daily dosing confirmation and injection location. The currently available treatment, eculizumab, requires administration every two weeks under medical supervision as an intravenous infusion. In contrast, R5000 has been developed for subcutaneous self-administration at home, thus providing a convenient form of treatment for patients.

[0288] Subjects with hypersensitivity to R5000 or any of its excipients will not be treated with R5000. Subjects with colonization with Neisseria meningitides or unexplained or suspected infection are also prohibited from treatment with R5000.

[0289] Example 15. Safety evaluation in R5000 Phase II clinical trial The clinical trial will include safety assessments to monitor adverse events (including infusion reactions and systemic infections), clinical laboratory tests (including markers for liver and pancreatic function), ECGs, vital signs, and physical examinations. Phase II studies will incorporate frequent monitoring visits with scheduled visits weekly for the first 4 weeks and then every 2 weeks thereafter. The long-term extension study will include ongoing monitoring with monthly visits for the first 3 months and every 3 months thereafter, with the monitoring visits and visits expected to continue to collect long-term safety and tolerability data.

[0290] Example 16. Inhibition of hemolysis using R5000-related peptides in cases of red blood cells that are unresponsive to eculizumab due to C5 mutations Blood / plasma samples from two Japanese PNH patients with poor response to eculizumab were analyzed. Both patients were previously shown to have a single missense C5 heterozygous mutation c.2654G->A predicting the polymorphism p.Arg885His (for a description of this polymorphism, see Nishimura, J. et al., N Engl J Med., 2014, vol. 370, no. 7, pp. 632-9, the contents of which are incorporated herein by reference in their entirety). In this study, mR5000 was tested against patient serum samples. mR5000 is identical to R5000 except that the C-terminal lysine is unmodified. The compound mR5000 has antihemolytic activity similar to that of R5000, but has a shorter half-life when administered to subjects. Patient serum was acidified and mixed with RBCs in the presence (+) or absence (-) of mR5000. Complete inhibition of hemolysis was observed in the presence of mR5000 (Figure 17).

[0291] Example 17. R5000 treatment in patients non-responsive to eculizumab Based on the distinct binding site of R5000, R5000 is used for the treatment of PNH in patients with mutations in the C5 gene that prevent eculizumab from binding to C5. R5000 binds to a different site on C5 than eculizumab and therefore may prevent C5 activation in patients who are non-responsive to eculizumab due to mutations near its binding site on C5.

[0292] Example 18. Comparison between R5000 and antibody-based C5 inhibitors Inhibition of complement activity was assessed by hemolysis of rabbit or PNH patient red blood cells and measuring production of C5a and C5b-9 by ELISA. C5 and C5-eculizumab complex levels in PNH patient samples were measured using ELISA with a C5 binding reagent to capture C5 or the complex and detected with an anti-C5 antibody followed by an HRP-conjugated secondary antibody (total C5) or eculizumab (C5-eculizumab complex) with an HRP-conjugated secondary antibody. Inhibition of C5 binding to the alternative pathway (AP) C5 convertase was measured by SPR.

[0293] Residual hemolytic activity has been shown to be present 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 hemolytic activity and complement activity, including production of C5a and C5b-9, in several in vitro assays where residual hemolysis was evident with eculizumab (Alexion Pharmaceuticals, New Haven, CT). In serum from PNH patients treated with eculizumab, approximately 30% of the hemolytic activity of normal human serum was retained in rabbit erythrocyte lysis assays, even when excess eculizumab (up to 12.5 μM) was added. Addition of R5000 completely abolished this residual hemolytic activity. Finally, R5000 completely inhibited C5 binding to the AP C5 convertase as measured by SPR, whereas eculizumab only partially inhibited C5 binding. Overall, these results suggest that the inhibitory mechanism of R5000 promotes complete inhibition of hemolysis under conditions where eculizumab produces partial inhibition.

[0294] Example 19. Results of Cohort A in 2 Patients at Week 7 A dose-finding study was conducted to evaluate the safety, tolerability, preliminary efficacy, pharmacokinetics, and pharmacodynamics of R5000 in patients with PNH. The study was an open-label 12-week study with a long-term extension. The study program was comprehensive and designed to address three PNH populations: (Cohort A) eculizumab-naive subjects; (Cohort B) subjects switching from eculizumab to R5000; and (Cohort C) subjects with an inadequate response to eculizumab. Patients received R5000 by subcutaneous injection at 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 subsequent week 2 visit, the daily dose could be increased to 0.3 mg / kg if lactate dehydrogenase (LDH) levels were ≥ 1.5 times the upper limit of normal. The primary efficacy endpoint of the study was to obtain a change in LDH levels from baseline to the mean level from weeks 6 to 12 of the study.

[0295] At week 7, near complete inhibition of hemolytic activity was achieved in two patients (patient 001 and patient 002) in cohort A with 100% compliance with the treatment regimen (monitored remotely with a telemonitoring device for no dose interruptions). Percent hemolysis and hemoglobin levels at each time point are shown in Figure 18A and Figure 18B, respectively. Patient 001 was a 56-year-old female diagnosed with PNH in April 2007. Patient 001 had 80% granulocyte clone size and 25% RBC clone size and had not received a transfusion prior to enrollment. Patient 002 was a 65-year-old female diagnosed with PNH in April 2017. Patient 002 had 99% granulocyte clone size and 47% RBC clone size. Patient 002 was transfusion dependent prior to enrollment and received transfusions at weeks 4 and 6 of the study. A rapid decline in LDH was noted in both patients, with mean LDH at week 7 being 1.55 times the upper limit of normal (Figure 18C). R5000 levels remained relatively stable through week 6 (Figure 18D). One transient episode of breakthrough hemolysis associated with an intercurrent illness was noted. No safety or tolerability concerns were identified. No serious or relevant adverse events were noted, and no injection site reactions were observed.

[0296] Example 20. Surface Plasmon Resonance (SPR) Measurements of C5 Inhibitor Binding Binding of R5000 and eculizumab to the alternative pathway C5 convertase was examined by surface plasmon resonance (SPR) analysis. SPR measurements were performed on a ProteOn XPR36 SPR station (Bio-Rad, Hercules, CA) at 25 °C. Eculizumab was immobilized on a Bio-Rad GLH sensor chip via amine coupling (NHS / EDC coupling kit from Bio-Rad) in 10 mM sodium acetate pH 4.5 and blocked with ethanolamine (Bio-Rad) at 25 °C. Biotinylated R5000 was captured separately on a Bio-Rad NLC sensor chip. Kinetic / affinity data for binding of C5 to eculizumab and R5000 were obtained using serially diluted C5 (Complement Technologies, Inc.) and blocked with ethanolamine (Complement Technologies, Inc.). Technologies, TX) over the eculizumab or R5000 surfaces separately. The same running / assay buffer (10 mM potassium phosphate (pH 7.4 and 6.0), 150 mM NaCl, 1% DMSO, and 0.01% surfactant P-20) was used throughout. Surface regeneration was achieved with 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 1:1 Langmuir mode.

[0297] Biotinylated human complement component C3b (Complement Technologies, TX) was prepared by mixing EZ-linked maleimide-PEG2-biotin (Thermo Fisher Scientific, Waltham, MA) with C3b stock according to the supplier's protocol. Unused biotin was separated from biotinylated C3b through a desalting column. Biotinylated C3b was captured on a Bio-Rad NLC sensor chip according to standard protocols. Sequential injections of human complement components (all purchased from Complement Technologies, Tyler, TX) were performed using the simultaneous injection mode to collect alternative pathway (AP) C5 convertase on the captured C3b surface. The co-injection mode included an initial 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 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 10 mM Hepes (pH 7.4), 150 mM NaCl, 1% DMSO, and 0.01% surfactant P-20. To quantitatively analyze the binding of C5 at different inhibitor concentrations, individual SPR sensorgrams after C5 injection were normalized to the time of C5 injection and the percentage of SPR signal was calculated. The results showed reduced binding of C5 to the AP C5 convertase in the presence of R5000 and eculizumab (as evidenced by the reduced response to C5 (see FIG. 19)), with 2 μM R5000 having a more potent effect.

[0298] Example 21. Inhibition of red blood cell hemolysis in PNH patients Inhibition of hemolysis in red blood cells from PNH patients was evaluated in the presence or absence of R5000 and eculizumab. Red blood cells were collected from PNH patient blood samples by centrifugation at 1000 × g for 3 min to pellet and wash three times with Alsever's solution. The cell pellet was incubated with GVB. ++ The cells were resuspended 1:2 in buffer (Complement Technology, Tyler, TX). Donor-matched normal human serum was acidified to pH 6.4 by addition of HCl. PNH patient erythrocytes (2.5% v / v) were incubated with 50% acidified donor-matched serum and the indicated concentrations of compounds for 18 h at 37°C in a clear 96-well assay plate. Cells were then centrifuged at 1000 × g for 3 min and 100 μl of the supernatant was transferred to another clear 96-well assay plate for detection of absorbance at 412 nm.

[0299] Results from two patients shown in Figure 20 and Figure 21, respectively, show that R5000 completely blocks hemolysis of PNH patient red blood cells, while eculizumab partially inhibits hemolysis. Recombinant C5 with the known polymorphism p.Arg885His, which confers resistance to eculizumab, was used as the sole source of C5 in the alternative pathway hemolysis assay using C5-depleted serum and rabbit red blood cells. Increasing concentrations of R5000 or eculizumab were tested in the assay to evaluate their ability to inhibit hemolysis (see Figure 22). The results show that R5000 is capable of binding and inhibiting hemolysis mediated by p.Arg885His of C5, while eculizumab is ineffective.

[0300] Example 22. Measurement of C5, eculizumab, and C5 / eculizumab complex levels in plasma of PNH patients C5, eculizumab and C5 / eculizumab complex levels were measured in PNH patient plasma by one of three ELISA formats, each using a variant of R5000 as the capture agent. The variant of the R5000 assay contained an N-terminal biotinylated PEG moiety and a modified C-terminal lysine residue replaced with norvaline. 5 pmoles / well of the R5000 variant was immobilized on a neutravidin-coated 96-well microplate (Thermo Fisher Scientific, Waltham, MA), which served as the 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 h at room temperature, followed by three washes with assay buffer.

[0301] For detection of total C5, plasma was diluted 1:10,000 in assay buffer and 100 μL was added to the assay plate wells. C5 standards were prepared (3-fold serial dilutions from 100 ng / ml in assay buffer) and added to the 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 at room temperature for 1 hour to bind any C5 in the sample that was not already complexed with eculizumab. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Abcam, Cambridge, UK; 1:5000 dilution) was added as a secondary antibody to detect eculizumab bound to C5 and incubated at room temperature for 30 minutes. TMB substrate (UltraTMB ELISA, Thermo Fisher Scientific) was then added to the plate and incubated for 30 min. The reaction was stopped with 2N sulfuric acid solution and absorbance was read at 450 nm on a SPECTRAMAX® M3 plate reader (Molecular Devices, Sunnyvale, CA). The amount of total C5 was calculated by comparison to a C5 standard curve.

[0302] Detection of C5 / eculizumab complex levels was performed using the same method as above but without the addition of excess eculizumab. The assay used diluted premixed C5 / eculizumab complex (1:1 molar ratio, 3-fold serial dilutions from 100 ng / ml) as a standard.

[0303] To measure free eculizumab, 100 μl of a 1 μg / ml solution of C5 in assay buffer was added to the C5-binding peptide-coated assay plate and incubated for 1 h at room temperature. Patient plasma diluted 1:10,000 in assay buffer was added to the test wells and diluted eculizumab (3-fold serial dilutions from 100 ng / ml) was used as a standard.

[0304] The concentrations of total C5, C5 bound to eculizumab, and free eculizumab detected in samples from PNH patients treated with eculizumab or from normal human serum are presented in Figure 23. These results indicate the presence of sufficient eculizumab in PNH patient samples to saturate C5 and form C5:eculizumab complexes.

[0305] Example 23. Effect of R5000 on residual hemolysis in plasma of PNH patients treated with eculizumab In an assay designed to detect activation of the alternative pathway, the hemolytic activity of plasma from patients treated with eculizumab was tested in the presence of additional eculizumab and C5 inhibitor peptide (R5000). In 96-well clear assay plates, plasma was plated in 100 mL of 1000 mM sodium dodecyl sulfate (MS)-1000 mM trisodium fumarate (100 mM NaCl), 100 mM MgEGTA (B106; Complement Technology Inc., Tyler, TX), 12.5 μM, 4.2 μM, and 1.4 μM complement inhibitors, and assay buffer (GVB). 0 Rabbit red blood cells (Ers; #B302; Complement Technology Inc., Tyler, TX) were incubated with 25% eculizumab-treated patient plasma for 30 min at 37°C in the presence of 100% erythrocyte lysate (#Ers; #B101; Complement Technology Inc., Tyler, TX). After incubation, plates were centrifuged at 1000 × g and 100 μL of supernatant was transferred to a new 96-well clear assay plate and absorbance at 412 nm was measured. Absorbance values ​​were normalized to a plate control of 25% (final concentration) normal human serum (#NHS; Complement Technology Inc., Tyler, TX), set at 100% hemolysis.

[0306] The results shown in Figure 24 suggest that even in the presence of sufficient eculizumab to bind to any available C5 in the patient sample, sufficient activation of the alternative complement pathway still occurs to induce hemolysis of rabbit red blood cells. Treatment with additional eculizumab only resulted in moderate inhibition of hemolysis, confirming that the hemolytic activity is not due to insufficient eculizumab in the sample. However, addition of R5000 completely abolished the residual hemolytic activity, indicating that the residual activity is C5-dependent and suggesting that R5000 may further inhibit hemolysis in the presence of excess eculizumab. (Additional Note) As a preferred embodiment, the technical ideas that can be understood from the above embodiment will be described below. [Section 1] 1. A method of treating paroxysmal nocturnal hemoglobinuria (PNH) in a subject not previously treated with eculizumab, comprising: administering to said subject an initial loading dose of R5000; administering to said subject an initial therapeutic dose of R5000, wherein said initial therapeutic dose is administered two or more times at regular intervals after said initial loading dose. [Section 2] The method of claim 1, wherein lactate dehydrogenase (LDH) levels in the subject are monitored throughout treatment. [Section 3] The method of any one of the preceding claims, wherein the initial therapeutic dose is replaced with a modified therapeutic dose of R5000 after 2 or more weeks of administration of the initial therapeutic dose. [Section 4] The method of claim 3, wherein the altered therapeutic dose of R5000 comprises an increased dose of R5000. [Section 5] The method of claim 4, wherein the initial treatment dose is replaced with the altered treatment dose when the subject's LDH level is less than or equal to 1.5 times the upper limit of normal. [Section 6] The method according to any one of items 1 to 5, wherein the initial loading dose comprises about 0.1 mg / kg to about 0.6 mg / kg of R5000. [Section 7] The method according to any one of items 1 to 6, wherein the initial treatment dose comprises about 0.1 mg / kg to about 0.3 mg / kg of R5000. [Section 8] The method according to item 7, wherein the initial therapeutic dose is replaced with a modified therapeutic dose of R5000 of about 0.3 mg / kg to about 0.6 mg / kg. [Section 9] Item 9. The method according to any one of Items 1 to 8, wherein hemolysis in the subject serum is reduced. [Section 10] Item 10. The method according to any one of items 1 to 9, wherein no adverse events are observed. [Section 11] The method according to any one of paragraphs 1 to 10, wherein at least one administration of R5000 comprises self-administration. [Section 12] The method of claim 11, wherein the self-administration is monitored. [Section 13] The method of claim 12, wherein the self-administration is remotely monitored. [Section 14] 14. The method of claim 12 or 13, wherein the self-administration is monitored using a smart device. [Section 15] Item 15. The method according to any one of Items 1 to 14, wherein the regular interval is about every 12 hours to about every 168 hours. [Section 16] A method of treating PNH in a subject, comprising administering R5000 to the subject, wherein the subject is currently being treated with eculizumab or has previously been treated with eculizumab. [Section 17] 17. The method of claim 16, wherein the residual hemolytic activity in the subject is reduced or eliminated. [Section 18] 18. The method of claim 17, wherein the association between C5 and the alternative pathway C5 convertase is inhibited. [Section 19] The method according to any one of items 1 to 18, wherein R5000 is administered simultaneously with eculizumab. [Section 20] 20. The method according to any one of items 1 to 19, wherein the subject has a C5 polymorphism. [Section 21] 21. The method of claim 20, wherein the polymorphism comprises p.Arg885His. [Section 22] R5001; and A pharmaceutical composition comprising at least one pharma- ceutically acceptable excipient. [Section 23] 23. The pharmaceutical composition of claim 22, wherein the at least one pharma- ceutically acceptable excipient comprises at least one of sodium chloride and sodium phosphate. [Section 24] Item 24. The pharmaceutical composition according to item 22 or 23, comprising about 25 mM to about 100 mM sodium chloride. [Section 25] 25. The pharmaceutical composition according to any one of items 22 to 24, comprising about 10 mM to about 100 mM sodium phosphate. [Section 26] 26. The pharmaceutical composition according to any one of items 22 to 25, wherein R5001 is present at a concentration of about 1 mg / mL to about 400 mg / mL. [Section 27] A method for treating PNH in a subject, comprising administering to the subject the pharmaceutical composition according to any one of items 22 to 26. [Section 28] 28. The method of paragraph 27, wherein the subject has previously been treated with an antibody-based therapeutic. [Section 29] 29. The method of claim 28, wherein the subject is resistant or non-responsive to treatment with the antibody-based therapeutic. [Section 30] 30. The method of claim 28 or 29, wherein the antibody-based therapeutic is eculizumab. [Section 31] Item 31. The method according to any one of Items 27 to 30, wherein the pharmaceutical composition is administered at a dose of about 0.01 mg / kg to about 10 mg / kg. [Section 32] Item 32. The method according to Item 31, wherein the pharmaceutical composition is administered at a dose of about 0.1 mg / kg to about 0.3 mg / kg. [Section 33] 33. The method according to any one of items 27 to 32, wherein the pharmaceutical composition is administered daily for at least two days. [Section 34] 34. The method of claim 33, wherein the pharmaceutical composition is administered daily for about 12 weeks. [Section 35] 34. The method of claim 33, wherein the pharmaceutical composition is administered daily for at least one year. [Section 36] Item 36. The method according to any one of Items 27 to 35, wherein the pharmaceutical composition is administered subcutaneously or intravenously. [Section 37] A method for inhibiting binding of C5 to an alternative pathway C5 convertase, comprising contacting C5 with R5001. [Section 38] 38. The method of claim 37, wherein the C5 is derived from a subject having a C5 polymorphism. [Section 39] 39. The method of claim 38, wherein the C5 polymorphism comprises p.Arg885His. [Section 40] A method for inhibiting residual C5 activity in a subject with PNH, comprising administering R5000 to the subject, wherein the subject is currently being treated with eculizumab or has previously been treated with eculizumab. [Section 41] 41. The method of claim 40, wherein the association between C5 and the alternative pathway C5 convertase is inhibited. [Section 42] The method of claim 40 or 41, wherein R5000 is administered simultaneously with eculizumab. [Section 43] Item 43. The method according to any one of Items 40 to 42, wherein the subject has a C5 polymorphism. [Section 44] 44. The method of claim 43, wherein the polymorphism comprises p.Arg885His. [Section 45] 1. A method for measuring C5 levels in a sample, comprising: immobilizing a capture agent on a substrate, said capture agent binding to a site on C5 that is distinct from the eculizumab binding site; contacting the substrate with the sample, wherein the immobilized capture agent binds to C5 in the sample; contacting the substrate with a detection reagent, wherein the detection reagent binds to C5 bound to the immobilized capture agent; and measuring the level of bound detection reagent as an indication of C5 levels in the sample. [Section 46] 46. ​​The method of claim 45, wherein the detection reagent comprises a detectable label. [Section 47] 46. ​​The method of claim 45, wherein the detection reagent is detected using a secondary detection reagent. [Section 48] Item 48. The method according to any one of Items 45 to 47, wherein the detection reagent is an anti-C5 antibody. [Section 49] 49. The method according to any one of paragraphs 45 to 48, wherein both free C5 levels and C5 levels associated with eculizumab are measured. [Section 50] the substrate is contacted with a second detection reagent that binds eculizumab; 50. The method of claim 49, wherein the method comprises measuring the level of bound second detection reagent as an indication of the level of C5 associated with eculizumab. [Section 51] 51. The method of claim 50, wherein the second detection reagent comprises a detectable label. [Section 52] 51. The method of claim 50, wherein the second detection reagent is detected using a secondary detection reagent. [Section 53] Item 53. The method according to any one of Items 45 to 52, wherein the capture agent comprises a mutant of R5000. [Section 54] The R5000 mutant is an N-terminal biotinylated PEG moiety; and 54. The method of claim 53, comprising substituting the C-terminal lysine of R5000 in the R5000 mutant with norvaline. [Section 55] 1. A method for measuring free eculizumab levels in a sample, comprising: immobilizing a capture agent on a substrate, said capture agent binding to a site on C5 that is distinct from the eculizumab binding site; contacting the substrate with an excess of C5 to form a C5-capture agent complex, the C5-capture agent complex comprising immobilized C5; contacting the substrate with the sample, wherein the immobilized C5 binds to eculizumab in the sample; contacting the substrate with a detection reagent, wherein the detection reagent binds to eculizumab; and measuring the level of bound detection reagent as an indication of the level of free eculizumab in the sample. [Section 56] 56. The method of claim 55, wherein the capture agent comprises a mutant of R5000. [Section 57] The R5000 mutant is an N-terminal biotinylated PEG moiety; and 57. The method of claim 56, comprising substituting the C-terminal lysine of R5000 in the R5000 mutant with norvaline. [Section 58] Item 58. The method according to any one of Items 55 to 57, wherein the detection reagent is an antibody. [Section 59] Item 59. The method of any one of items 55 to 58, wherein the detection reagent comprises a detectable label. [Section 60] Item 59. The method of any one of items 55 to 58, wherein the detection reagent is detected using a secondary detection reagent.

[0307] [ka] [ka]

Claims

1. A pharmaceutical composition for treating paroxysmal nocturnal hemoglobinuria in a subject, comprising: A C5 inhibitor polypeptide having the core sequence of SEQ ID NO:1 with ω-hydroxylation of the palmitoyl tail; and At least one pharma- ceutically acceptable excipient Including, A pharmaceutical composition, wherein a C5 inhibitor polypeptide having a core sequence of SEQ ID NO: 1 with ω-hydroxylation of the palmitoyl tail has the ability to inhibit complement-dependent hemolysis at a half-maximal inhibitory concentration (IC 50 ) that is comparable or similar to that of a C5 inhibitor polypeptide having a core sequence of SEQ ID NO: 1 without ω-hydroxylation of the palmitoyl tail.

2. The pharmaceutical composition described in claim 1, wherein a C5 inhibitor polypeptide having a core sequence of SEQ ID NO: 1 with ω-hydroxylation of the palmitoyl tail has the ability to inhibit complement-dependent hemolysis with an IC 50 that is ±20% of the IC 50 of a C5 inhibitor polypeptide having a core sequence of SEQ ID NO: 1 without ω-hydroxylation of the palmitoyl tail.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the at least one pharma- ceutically acceptable excipient comprises at least one of sodium chloride and sodium phosphate.

4. The pharmaceutical composition of any one of claims 1 to 3, comprising about 25 mM to about 100 mM sodium chloride.

5. The pharmaceutical composition of any one of claims 1 to 4, comprising about 10 mM to about 100 mM sodium phosphate.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the C5 inhibitor polypeptide having the core sequence of SEQ ID NO:1 with ω-hydroxylation of the palmitoyl tail is present at a concentration of about 1 mg / mL to about 400 mg / mL.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the subject has previously been treated with an antibody-based therapeutic.

8. The pharmaceutical composition of claim 7 , wherein the subject is resistant or non-responsive to treatment with the antibody-based therapeutic agent.

9. 9. The pharmaceutical composition of claim 7 or 8, wherein the antibody-based therapeutic is eculizumab.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition is administered at a dose of about 0.01 mg / kg to about 10 mg / kg.

11. The method of claim 10, wherein the pharmaceutical composition is administered at a dose of about 0.1 mg / kg to about 0.3 mg / kg.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the pharmaceutical composition is administered daily for at least 2 days, for about 12 weeks, or for at least 1 year.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutical composition is administered subcutaneously or intravenously.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the subject has a C5 polymorphism.

15. 15. The pharmaceutical composition of claim 14, wherein the C5 polymorphism comprises p.Arg885His.

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