COMPSTATIN ANALOGS, COMPOSITION THAT UNDERSTANDS THEM AND USE OF THE SAME

专利摘要:
compstatin analogs, composition that comprises them and their use. in some aspects, the present invention provides cell reaction compstatin analogs and compositions comprising cell reaction compstatin analogs. in some aspects, the invention further provides methods for using cell reaction compstatin analogs, for example, for inhibiting complement-mediated damage to a cell, tissue or organ. in some respects, the invention provides long-acting compstatin analogs and compositions comprising long-acting compstatin analogs. in some aspects, the invention further provides methods for using long-acting compstatin analogs, for example, for inhibiting complement-mediated damage to a cell, tissue or organ. in some aspects, the invention provides targeted compstatin analogs and compositions comprising targeted compstatin analogs. in some respects, the invention further provides methods for the use of compstatin analogs directed, for example, to inhibit complement-mediated damage to a cell, tissue or organ.
公开号:BR112013028816B1
申请号:R112013028816-7
申请日:2012-05-11
公开日:2020-12-15
发明作者:Cedric François；Pascal Deschatelets
申请人:Apellis Pharmaceuticals, Inc；
IPC主号:

专利说明:

Cross Reference to Related Orders
[001] The present invention claims priority to United States provisional patent application No. 61 / 484,836, filed on May 11, 2011, all of which is incorporated by reference herein. Background of the Invention
[002] The complement is a system composed of more than 30 proteins bound per cell and plasma that plays an important role in innate and adaptive immunity. Complement system proteins act in a series of enzymatic cascades, through a variety of protein cleavage events and interactions. Complement activation occurs through three main pathways: classical antibody-dependent pathway, the alternative pathway and the mannose-binding lectin (MBL) pathway. Inadequate or excessive activation of complement is an underlying cause or contributing factor to several serious conditions and diseases, and considerable effort has been devoted over the past few decades to explore various complement inhibitors as therapeutic agents. However, there is still a need for innovative approaches to inhibit complement activation for a variety of therapeutic purposes. Summary of the Invention
[003] In some aspects, the invention provides compostatin analogs of cellular reaction. For example, the invention provides cell reaction compstatin analogs, compositions comprising cell reaction compstatin analogs and methods of producing, identifying, characterizing and / or using cell reaction compstatin analogs. In some aspects, the invention provides a physiologically acceptable composition comprising a cell reaction compstatin analog. In some aspects, the invention provides a pharmaceutical grade composition comprising a cell reaction compostatin analogue.
[004] In some aspects, the invention provides long-acting compostatin analogs. For example, the invention provides long-acting compstatin analogs, compositions comprising long-acting compstatin analogs and methods of producing, identifying, characterizing and / or using long-acting compstatin analogs. In some aspects, the invention provides a physiologically acceptable composition comprising a long-acting compstatin analogue. In some aspects, the invention provides a pharmaceutical grade composition comprising a long-acting compstatin analogue.
[005] In some aspects, the invention provides targeted compostatin analogs. For example, the invention provides targeted compstatin analogs, compositions comprising targeted compstatin analogs and methods of producing, identifying, characterizing and / or using targeted compstatin analogs. In some aspects, the invention provides a physiologically acceptable composition comprising a targeted compstatin analog. In some aspects, the invention provides a pharmaceutical grade composition comprising a targeted compstatin analogue.
[006] The invention also provides methods of protecting a cell against complement-mediated data. In some embodiments, the methods comprise contacting the cell with a cell reaction compstatin analog. The Cell can be any type of cell in various modalities. For example, in some embodiments, the cell is a blood cell. In some embodiments, the blood cell is a red blood cell (RBC), also called an erythrocyte. In some embodiments, the cell has an abnormally low expression, surface density and / or activity of one or more complement regulatory proteins. For example, the cell may have a mutation in a gene encoding that protein, where the mutation results in reduced or absent expression and / or reduced activity of the encoded protein. The cell can be of any type or species of animal in various modalities. For example, the cell can be mammalian, for example, primates (human or non-human primate), rodents (for example, mouse, rat, rabbit), ungulates (for example, pig, sheep, cow), canids or felines. In several modalities, the protection is a complement of primates, for example, a complement of humans. In some modalities, contact with the cell is made ex vivo (outside a subject's body). In some modalities, contact with the cell is made in vivo (in a subject, for example, a human). In some embodiments, the cell must be transplanted into a subject or has been transplanted into or subject. In some aspects, the invention provides an isolated cell with a compstatin analog covalently attached to it. In some aspects, the invention provides an isolated tissue or organ with a compstatin analogue bound to at least some of its cells.
[007] The invention provides methods of treating a subject in need of treatment for a complement-mediated disease. In modalities, the method comprises the administration of a compstatin analogue of cellular reaction to the subject. In modalities, the method comprises the administration of a long-acting compstatin analogue to the subject. In modalities, a long-acting compstatin analog is a cell reaction compstatin analog. In some modalities, complement-mediated disease is paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic-uremic syndrome (HUS), or another disease associated with complement-mediated hemolysis. In some modalities, the disease is an ischemia / reperfusion (I / R) injury (for example, due to myocardial infarction, thromboembolic stroke or surgery). In some modalities, the disease is trauma. In some modalities, the disease is transplant rejection.
[008] All articles, books, patent applications, patents, other publications, websites and databases mentioned in this application are incorporated by reference into this document. In case of conflict between the specification and any of the incorporated references, the specification (including any changes to it) will prevail. Unless otherwise indicated, meanings of terms and abbreviations accepted in the art are used in this document. Brief Description of Drawings
[009] Figure 1 is a graph showing the percentage of complement inhibiting activity of compostatin analog CA28 (SEQ ID NO: 28) and three long acting compstatin analogs (CA28-1, CA28 -2, CA28-3), as a function of peptide concentration (μM). Complement activation inhibition was tested in vitro using a classic complement inhibition assay. The graph shows values obtained by averaging the result of two sets of measurements. CA28 (circles; red), CA28-1 (crosses (x); blue); CA28-2 (triangles, green), CA28-3 (squares, purple).
[0010] Figure 2 is a graph showing the percentage of CA28 complement activation inhibiting activity and the long-acting compstatin analogs CA28-2 and CA28-3, as a function of peptide concentration (μM). CA28 (squares, light gray), CA28-2 (diamonds, black), CA28-3 (circles, dark gray). CA28-3 is a compound that contains several portions of peptide. Although the activity per peptide portion is less than the activity of an individual CA28 molecule, the total activity of CA28-3 exceeds the activity of CA28 on a molar basis.
[0011] Figure 3 is a graph showing the plasma versus time concentrations of CA28 and long-acting compstatin CA28-2 and CA28-3 in Cynomolgus monkeys after a single intravenous injection. CA28 was administered at 200 mg / kg. CA28-2 and 3-CA28 were administered at 50 mg / kg. In calculating the doses for these experiments, it was assumed that the substances and 3-CA28 administered consisted of 80% w / w of the active compound based on dry weight. However, during sample analysis, the standard curve assumed 100% w / w of the active compound based on dry weight, by an estimate of 30%. Thus, Cmax values overestimate the actual Cmax. CA28 (squares, light gray), CA28-2 (triangles, black), CA28-3 (circles, dark gray).
[0012] Figure 4 is a graph showing the percentage of CA28 complement activation inhibitory activity and CA28-4 long-acting compstatin analogs, as a function of peptide concentration (μM). Complement activation inhibition was tested in vitro using a classic complement inhibition assay. The graph shows values obtained by averaging the result of four sets of measurements for CA28-4.CA28 (squares, light gray), CA28-4 (crosses, black).
[0013] Figure 5 is a graph showing the plasma versus time concentrations of CA28 and long-acting compstatin CA28-2, CA28-3 and CA28-4 in Cynomolgus monkeys after a single intravenous injection. CA28 was administered at 200 mg / kg. CA28-2, 3- CA28 and CA28-4 were administered at 50 mg / kg. In calculating the doses for these experiments, it was assumed that the substances and 3-CA28 administered consisted of 80% w / w of the active compound based on dry weight. However, during sample analysis, the standard curve assumed 100% w / w of the active compound based on dry weight. Thus, the Cmax values overestimate the Cmax that would be achieved if these compounds were administered at the doses indicated on a dry mass basis, by an estimate of 30%. CA28 (squares, light gray), CA28-2 (triangles, black), CA28-3 (circles, dark gray), CA28-4 (inverted triangles, black). Detailed Description of Certain Modalities of the Invention I. Definitions
[0014] The terms "approximately" or "about" in reference to a number generally include numbers that fall within ± 10%, in some ± 5% modalities, in some ± 1% modalities, in some ± 0 modalities, 5% of the number, unless otherwise indicated or evident to the contrary through the context (except when such number inadmissibly exceeds 100% of a possible value).
[0015] A "complement component" or "complement protein" is a protein that is involved in the activation of the complement system or participates in one or more complement-mediated activities. Components of the classic complement pathway include, for example, C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9 and the C5b-9 complex, also referred to as the membrane attack complex (MAC ) and active fragments or enzymatic cleavage products from any of the above (for example, C3a, C3b, C4a, C4b, C5a, etc.). Components of the alternative pathway include, for example, factors B, D and properdin. Components of the lectin pathway include, for example, MBL2, MASP-1 and MASP-2. Complement components also include cell-bound receptors for soluble complement components, where that receptor mediates one or more biological activities of such a soluble complement component following the binding of the soluble complement component. Such receivers include, for example, C5a receptor (C5aR), C3a receptor (C3aR), Complement Receptor 1 (CR1), Complement Receptor 2 (CR2), Complement Receptor 3 (CR3, also known as CD45), etc. It will be appreciated that the term "complement component" is not intended to include those molecules and molecular structures that serve as a "trigger" for complement activation, for example, antigen-antibody complexes, foreign structures found on microbial or artificial surfaces, etc. .
[0016] A "complement-mediated disease" is a disease in which complement activation is known or suspected to be a contributing and / or at least partially causative factor in at least some subjects suffering from the disease, for example, disease where complement activation results in tissue damage. Non-restrictive examples of complement-mediated diseases include, but are not limited to, (i) various diseases characterized by hemolysis or hemolytic anemia, such as atypical hemolytic-uremic syndrome, cold agglutinin disease, paroxysmal nocturnal hemoglobinuria, transfusion reactions; (ii) transplant rejection (eg, hyperacute or acute transplant rejection) or post-transplant dysfunction; (iii) diseases involving ischemia / reperfusion injury, such as trauma, surgery (for example, aneurysm repair), myocardial infarction, ischemic stroke; (iv) diseases of the respiratory system, such as asthma and chronic obstructive pulmonary disease (COPD); (v) arthritis, for example, rheumatoid arthritis; (vi) eye diseases such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma and uveitis. "Disease" is used interchangeably here with similar words "disorder" and "condition" to refer to any impairment of the health or abnormal state of functioning of an organism, for example, any state in which medical and / or surgical management is indicated or for which a subject appropriately seeks medical and / or surgical treatment. It should also be understood that listing a specific disease in a given category is for convenience and is not intended to limit the invention. It will be understood that certain diseases could be properly listed in several categories.
[0017] A "complement regulatory protein" is a protein involved in the regulation of complement activity. A complement regulatory protein may fail to regulate complement activity through, for example, inhibiting complement activation or deactivating or accelerating the decay of one or more activated complement proteins. Examples of complement regulatory proteins include C1 inhibitor, C4 binding proteins, clu- terine, vitronectin, CFH, factor I and CD46, CD55, CD59, CR1, CR2 and CR3 cell-bound proteins.
[0018] "Linked", as used here with respect to two or more moieties, means that the moieties are physically associated or linked to each other to form a molecular structure that is sufficiently stable for the moieties to remain associated in the conditions in which the bond is formed and, preferably, in the conditions in which the new molecular structure is used, for example, physiological conditions. In certain preferred embodiments of the invention, the bond is a covalent bond. In other embodiments, the bond is non-covalent. Portions can be linked directly or indirectly. When two portions are directly connected, they are also covalently linked to each other or are close enough so that intermolecular forces between the two portions maintain their association. When two parts are indirectly linked, they are linked to the other also covalently or non-covalently to a third portion, which maintains the association between the two portions. In general, when two parts are referred to as being connected by a "bonding part" or "bonding part", the bond between the two bonded parts is indirect, and normally each of the bonded parts is covalently bonded to the bonding part. Two portions can be linked using a "linker". A binder can be any appropriate portion that reacts with the entities to be bound within a reasonable period of time, under conditions consistent with the stability of the entities (portions that can be protected as appropriate, depending on conditions) and in sufficient quantity, to produce a reasonable income. Normally, the linker will contain at least two functional groups, one of which reacts with a first entity and the other reacts with a second entity. It will be appreciated that after the linker has reacted with the entities to be linked, the term "linker" can refer to the part of the resulting structure that originated from the linker, or at least the portion that does not include the functional groups of the reaction. A linking portion may include a portion that does not participate in a connection with the entities being linked, and whose main purpose is for entities to spatially separate entities from one another. Such a part can be called a "spacer".
[0019] "Polypeptide", as used herein, refers to a polymer of amino acids, optionally including one or more amino acid analogs. A protein is a molecule composed of one or more polypeptides. A peptide is a relatively short polypeptide, typically between 2 and 60 amino acids in length, for example, between 8 and 40 amino acids in length. The terms "protein", "polypeptide" and "peptide" can be used interchangeably. Polypeptides used in this document may contain amino acids such as those that are naturally found in proteins, amino acids that are not naturally found in proteins and / or analogues of amino acids other than amino acids. As used here, an "analog" of an amino acid can be a different amino acid that structurally resembles amino acids, or a compound that is not an amino acid that structurally resembles the amino acid. A large number of analogs recognized in the 20-amino acid technique commonly found in proteins are known (the "standard" amino acids). One or more of the amino acids in a polypeptide can be modified, for example, by the addition of a chemical entity such as a group of carbohydrates, a phosphate group, a farnesyl group, an isopharnesyl group, a group of fatty acids, a linker for conjugation, functionalization, or other modification, etc. Certain suitable non-restrictive analogs and modifications are described in WO2004026328 and / or below. The polypeptide can be acetylated, for example, the N-terminal and / or amidated, for example, at the C-terminal.
[0020] "Reactive functional groups" as used in this document refers to groups, including, but not limited to, olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates , thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, acetals, anhydrides, sulfates, sulfene acids nonic, isonitriles, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids, thiohydroxamic acids, alenes, orthoesters, sulfites, enamines, inamines, ureas, isoureas, semicarbazides, carbodiimides, carbamates, imines, azides, azides compounds, azo compounds and nitrous compounds, N-Hydroxysuccinimide esters, maleimides, sulfhydryls and the like. Methods for preparing each of these functional groups are well known in the art and their application or modification for a specific purpose is within the ability of an individual skilled in the art (see, for example, Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989, and Hermanson, G., Bioconjugate Tech-niques, 2nded., Academic Press, San Diego, 2008).
[0021] "Specific binding" generally refers to a physical association between a target polypeptide (or, more generally, a target molecule) and a binding molecule such as an antibody or ligand. The association is generally dependent on the presence of a specific structural feature of the target such as an antigenic determinant, epitope, pocket or binding cavity, recognized by the binding molecule. For example, if an antibody is specific for epitope A, the presence of a polypeptide containing epitope A or the presence of unlabeled free A in a reaction containing both unlabeled free A and the binding molecule that binds to it will reduce the amount of labeled A that binds to the binding molecule. It should be understood that specificity does not have to be absolute, but it generally refers to the context in which the connection occurs. For example, it is known in the art that numerous antibodies cross-react with other epitopes in addition to those present in the target molecule. Such cross-reactivity may be acceptable depending on the application for which the antibody is to be used. An individual skilled in the art will be able to select antibodies or ligands with a sufficient degree of specificity to perform properly in a given application (for example, for the detection of a target molecule, for therapeutic purposes, etc.). It should also be understood that specificity can be assessed in the context of additional factors, such as the affinity of the binding molecule with the target versus the affinity of the binding molecule for other targets, for example, competitors. If a binding molecule has a high affinity for a target molecule to be detected and a low affinity for non-target molecules, the antibody will likely become an acceptable reagent. Once the specificity of a binding molecule is established in one or more contexts, it can be used in other, preferably similar contexts, without necessarily reevaluating its specificity. In some embodiments, the affinity (measured by the equilibrium dissociation constant, Kd) of two molecules that have specific binding is 10-3M or less, for example, 10-4M or less, for example, 10-5M or less, for example, 10-6M or less, 10-7M or less, 10-8M or less, or 10-9M or less in the conditions tested, for example, under physiological conditions.
[0022] A "subject" treated according to the instantaneous invention is usually a human, a non-human primate or an inferior animal (for example, a mouse or rat), which expresses or contains at least some primate C3 component ( for example, human) of the complement and, optionally, one or more additional primate component (s) of the complement. In some modalities, the subject is male. In some modalities, the subject is female. In some modalities, the subject is an adult, for example, a human being at least 18 years old, for example, between 18 and 100 years old.
[0023] "Treat", as used here when it comes to treating a subject, refers to the provision of treatment, that is, provision of any type of medical or surgical management of a subject. Treatment can be provided to reverse, alleviate, inhibit progression, prevent or reduce the likelihood of a disease, or to reverse, alleviate, inhibit or prevent progression, prevent or reduce the likelihood of one or more symptoms or manifestations of a disease. disease. "Avoid" refers to preventing an illness or symptom or manifestation of an illness from occurring for at least a period of time in at least some individuals. Treating may include administration of a compound or composition to the subject after the development of one or more symptoms or indicative manifestations of a disease, for example, in order to reverse, alleviate, reduce the severity of, and / or inhibit or prevent progression disease and / or to reverse, alleviate, reduce the severity of, and / or inhibit or one or more symptoms or manifestations of the disease. A compound or composition can be administered to a subject who has developed a disease, or is at an increased risk of developing the disease in relation to a member of the general population. A compound or composition can be administered to a subject who has developed a disease, or is at an increased risk of developing one or more symptoms or specific manifestations of the disease or an exacerbation of the disease in relation to other individuals diagnosed with the disease, or in relation to the typical or average risk of the subject for such symptom or manifestation or exacerbation. For example, the subject may have been exposed to a "trigger" that puts the subject at increased risk (for example, temporarily increased risk) of suffering an exacerbation. A compound or composition can be administered prophylactically, that is, before the development of any symptoms or manifestation of the disease. Usually in this case the subject will be at risk of developing the disease, for example, in relation to a member of the general population, optionally matched in terms of age, sex and / or other demographic variable (s).
[0024] As used here, the term "aliphatic" denotes a group of hydrocarbons that can be a linear (i.e., unbranched), branched or cyclic (including fused polycyclic, bridge and spiro fusion) and can be completely saturated or it may contain one or more units of unsaturation, but it is not aromatic. Unless otherwise specified, aliphatic groups contain 1-30 carbon atoms. In some embodiments, aliphatic groups contain 1-10 carbon atoms. In other embodiments, aliphatic groups contain 1-8 carbon atoms. In still other embodiments, aliphatic groups contain 1-6 carbon atoms, and in still other embodiments, aliphatic groups contain 1-4 carbon atoms. Suitable aliphatic groups include, but are not limited to, alkyl, alkenyl, alkynyl, linear or branched groups, and hybrids thereof, such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl or (cycloalkyl) alkenyl.
[0025] As used herein, "alkyl" refers to a linear, branched or cyclic saturated hydrocarbon having from about 1 to about 22 carbon atoms (and all combinations and sub-combinations of specific ranges and numbers of carbon atoms in it), with about 1 to about 12, or about 1 to about 7 carbon atoms being preferred in certain embodiments of the invention. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl, cycle -octyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl.
[0026] As used here, "halo" refers to F, Cl, Br or I.
[0027] As used here, "alkanoyl" refers to an optionally substituted, linear or branched acyclic aliphatic residue having about 1 to 10 carbon atoms (and all combinations and subcombination of specific ranges and numbers of carbon atoms) in it, for example, from about 1 to 7 carbon atoms which, as will be appreciated, are attached to a C = O terminal group with a single bond (and can also be referred to as an "acyl group") . Alkanoyl groups include, but are not limited to, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, isopentanoyl, 2-methyl-butyryl, 2,2-dimethoxypropionyl, hexanoyl, heptanoyl, octanoyl and the like, and for the purposes of the present invention, a group formyl is considered an alkanoyl group. "Lower alkanoyl" refers to an optionally substituted, linear or branched acyclic aliphatic residue having about 1 to about 5 carbon atoms (and all combinations and subcombination of specific ranges and numbers of carbon atoms) . Such groups include, but are not limited to, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, isopentanoyl, etc.
[0028] As used here, "aryl" refers to an optionally substituted mono- or bicyclic aromatic ring system, having from about 5 to about 14 carbon atoms (and all combinations and subcombination of specific ranges and numbers of carbon atoms in it), with preferably about 6 to 10 carbons. Non-restrictive examples include, for example, phenyl and naphthyl.
[0029] As used in this document, "aralkyl" refers to alkyl radicals with aryl substituent and having from about 6 to about 22 carbon atoms (and all combinations and subcombination of specific ranges and numbers of carbon atoms in it) ), with about 6 to about 12 carbon atoms being preferable in certain embodiments. Aralkyl groups can be optionally substituted. Non-restrictive examples include, for example, benzyl, naphthylmethyl, diphenylmethyl, triphenylmethyl, phenylethyl and diphenylethyl.
[0030] In this document, the terms "alkoxy" and "alkoxy" refer to an optionally substituted alkyl-O- group where alkyl is as previously defined. Exemplary alkoxy and alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy and heptoxy.
[0031] As used in this document, "carboxy" refers to a -C (= O) OH group.
[0032] As used in this document, "alkoxycarbonyl" refers to a group -C (= O) O-alkyl, where alkyl is defined as above.
[0033] As used in this document, "aroila" refers to a group at -C (= O) -aryl, where aryl is defined as previously. Exemplary aroyl groups include benzoyl and naphthyl.
[0034] The term "cyclic ring system" refers to an aromatic or non-aromatic ring system, partially unsaturated or fully saturated, with 3 to 10 members, which includes single rings with 3 to 8 atoms and ring and bi systems - and tricyclics which may include 5 or 6-membered aromatic aryl or aromatic heterocyclic groups fused to a non-aromatic ring. These heterocyclic rings include those that have 1 to 3 heteroatoms selected independently from the group consisting of oxygen, nitrogen and sulfur. In certain embodiments, the term heterocyclic refers to a 5, 6, or 7-membered non-aromatic ring or polycyclic group in which at least one atom of the ring is a heteroatom selected from the group consisting of O, S and N, including , among others, a bi or tricyclic group, comprising rings of six members fused with between one and three hetero atoms independently selected from the group consisting of oxygen, sulfur and nitrogen. In some embodiments, "cyclic ring system" refers to a cycloalkyl group which, in this document, refers to groups having 3 to 10, for example, 4 to 7 carbon atoms. Cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which is optionally substituted. In some embodiments, "cyclic ring system" refers to a portion of cycloalkenyl or cycloalkynyl, which is optionally substituted.
[0035] Typically, substituted chemical moieties include one or more substituents that replace hydrogen. Exemplary substituents include, for example, halo, alkyl, cycloalkyl, aralkyl, aryl, sulfhydryl, hydroxyl (-OH), peroxyl, cyano (-CN), carboxyl (-COOH), -C (= O) O-alkyl, aminocarbonyl (-C (= O) NH2), N-substituted aminocarbonyl (-C (= O) NHR "), CF3, CF2CF3, and the like. Regarding the aforementioned substituents, each R" of the portion can be independently, any H, alkyl, cycloalkyl, aryl or aralkyl, for example.
[0036] As used in this document, "L-amino acid" refers to any of the naturally occurring levorotatory alpha-amino acids normally present in proteins or the alkyl esters of such alpha-amino acids. The term "D-amino acid" refers to dextrorotatory alpha-amino acids. Unless otherwise specified, all amino acids referred to in this document are L-amino acids.
[0037] As used in this document, an "aromatic amino acid" is an amino acid comprising at least one aromatic ring, for example, comprising an aryl group.
[0038] As used in this document, an "aromatic amino acid analog" is an amino acid analog that comprises at least one aromatic ring, for example, comprises an aryl group. II. Overview
[0039] The present invention provides compstatin analogs of cellular reaction and methods relating thereto, for example, methods of using them. Compstatin analogues of cell reaction are compounds that make up a portion of compstatin analog and a cell reaction functional group that is capable of reacting with a functional group exposed on the surface of a cell, for example, under physiological conditions, to form a bond covalent. The cell reaction compstatin analogue is thus covalently bound to the cell. Without intending to be limited by any specific theory, a compstatin analog bound to the cell protects the cell from complement-mediated damage, for example, by binding to C3 (which may be in the form of C3 (H2O)) on the cell surface and / or in the vicinity of the cell and inhibits C3 cleavage and activation, and / or binding to C3b and inhibiting its deposition in the cell or participation in the complement activation cascade. In some aspects of the invention, isolated cells are brought into contact with an ex vivo (outside the body) cell reaction compstatin analog. In some aspects of the invention, cells are present in an isolated tissue or organ, for example, a tissue or organ to be transplanted into a subject. In some aspects of the invention, cells are brought into contact with a cell reaction compstatin analog in vivo, by administering the cell reaction compstatin analog to a subject. The cell reaction compstatin analogue is thus covalently bound to the cell in vivo. In some ways, the inventive approach protects cells, tissues and / or organs from the deleterious effects of complement activation for at least two weeks, without the need for further treatment during that time.
[0040] In some aspects, the invention provides compstatin analogs, comprising a targeting portion that binds non-covalently to a target molecule present on the surface of cells or tissues or an extracellular substance not bound to cells or tissues . Such compstatin analogs are referred to herein as "target compstatin analogs". Often the target molecule is a protein or carbohydrate attached to the cell membrane and exposed on the cell surface. The targeting portion directs the compstatin analog to a cell, tissue or location susceptible to complement activation. In some aspects of the invention, isolated cells are brought into contact with a compstatin analog directed ex vivo (outside the body). In some aspects of the invention, cells are present in an isolated tissue or organ, for example, a tissue or organ to be transplanted into a subject. In some aspects of the invention, a targeted compostatin analogue is administered to a subject and becomes non-covalently bound to a cell, tissue or extracellular substance in vivo. In some respects, the inventive approach protects cells, tissues and / or organs against the deleterious effects of complement activation for at least two weeks, without the need for further treatment during that time. In some embodiments, a targeted compstatin analog comprises a targeting portion and a cellular reaction portion. The targeting portion directs the compstatin analog, for example, to a specific cell type, by non-covalent binding to a molecule in such cells. The cell reaction portion then covalently attaches itself to the cell or extracellular substance. In other embodiments, a targeted compstatin analog does not comprise a cell reaction portion.
[0041] In some respects, the invention provides long-acting compostatin analogs, wherein the long-acting compstatin analogs make up a portion such as polyethylene glycol (PEG) that prolongs the life of the compound in the body (for example, example, reducing your blood clearance). In other embodiments, a long-acting compstatin analog does not comprise a targeting portion or a cellular reaction portion. In some embodiments, a long-acting compstatin analogue comprises a targeting portion and / or a cellular reaction portion. III. Complement System
[0042] To facilitate the understanding of the invention and without the intention to limit the invention in any way, this section provides an overview of the complement and its activation pathways. More details are found, for example, in Kuby Immunology, 6thed., 2006; Paul, W.E., Fundamental Immunology, Lippincott Williams &Wilkins; 6thed., 2008; and Walport MJ., Complement. First of two parts. N Engl J Med., 344 (14): 1058-66, 2001.
[0043] Complement is an arm of the innate immune system that plays an important role in defending the body against infectious agents. The complement system comprises more than 30 whey and cellular proteins that are involved in three main pathways, known as the classic, alternative and lectin pathways. The classical pathway is usually triggered by binding an antigen complex and the IgM or IgG antibody to C1 (although certain other activators may also initiate the pathway). Activated C1 cleaves C4 and C2 to produce C4a and C4b, in addition to C2a and C2b. C4b and C2a combine to form C3 convertase, which cleaves C3 to form C3a and C3b. The binding of C3b to C3 convertase produces C5 convertase, which cleaves C5 to C5a and C5b. C3a, C4a and C5a are anaphylotoxins and mediate various reactions in the acute inflammatory response. C3a and C5a are also chemotactic factors that attract immune cells such as neutrophils.
[0044] The alternative pathway is initiated by and amplified on, for example, microbial surfaces and various complex polysaccharides. In this way, hydrolysis from C3 to C3 (H2O), which occurs spontaneously at a lower level, leads to the binding of factor B, which is cleaved by factor D, generating a fluid phase C3 convertase that activates the complement by cleavage of C3 in C3a and C3b. C3b binds to target just like cell surfaces and forms a complex with factor B, which is subsequently cleaved by factor D, resulting in a C3 counteract. Surface-bound C3 convertases cleave and activate additional C3 molecules, resulting in rapid deposition of C3b in close proximity to the activation site and leading to the formation of additional C3 convertase, which in turn, generates additional C3b. This process results in a C3 cleavage cycle and C3 convertase formation that significantly amplifies the response. Cleavage of C3 and binding of another C3b molecule to C3 convertase gives rise to a C5 convertase. C3 and C5 convertases in this pathway are regulated by host cell molecules CR1, DAF, MCP, CD59 and fH. The mode of action of these proteins involves any decay accelerating activity (ie, the ability to dissociate convertases), the ability to serve as cofactors in the degradation of C3b or C4b by factor I, or both. Normally, the presence of complement regulatory proteins on host cell surfaces prevents significant complement activation from occurring on it.
[0045] The C5 convertases produced in both pathways cleave C5 to produce C5a and C5b. C5b then binds to C6, C7 and C8 to form C5b-8, which catalyzes the polymerization of C9 to form the membrane attack complex (MAC) C5b-9. MAC is inserted into the membranes of target cells and causes cell lysis. Small amounts of MAC in the cell membrane can have a variety of consequences other than cell death.
[0046] The lectin complement pathway is initiated by the binding of mannose-binding lectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates. The MB1-1 gene (known as LMAN-1 in humans) encodes an integral type I membrane protein, located in the intermediate region between the Golgi complex and the endoplasmic reticulum. The MBL-2 gene encodes the soluble mannose-binding protein found in serum. In the human lectin pathway, MASP-1 and MASP-2 are involved in the proteolysis of C4 and C2, leading to a C3 convertase described above.
[0047] Complement activity is regulated by several mammalian proteins, known as complement control proteins (CCP) or complement activation activation proteins (RCA) (US Patent No. 6,897,290). These proteins differ with respect to the specificity of the ligand and complement inhibition mechanism (s). They can accelerate normal conversion deterioration and / or function as cofactors for factor I, to enzymatically cleave C3b and / or C4b into smaller fragments. CCPs are characterized by the presence of multiple (typically 4-56) homologous motifs known as short consensus repeats (SCR), complement control protein modules (CCP), or SUSHI domains, approximately 50-70 amino acids long that contain a conserved motif, including four disulfide-linked cysteines (two disulfide bonds), proline, tryptophan and many hydrophobic residues. The CCP family includes the type 1 complement receptor (CR1; C3b: C4b receptor), complements the type 2 receptor (CR2), membrane cofactor protein (MCP; CD46), decay accelerating factor (DAF), complement factor H (fH) and C4b binding protein (C4bp). CD59 is a membrane-bound complement regulatory protein not structurally related to CCPs. Complement regulatory proteins usually serve to limit the activation of complement that might otherwise occur in mammalian cells and tissues, for example, the human host. Thus, "auto" cells are normally protected against the deleterious effects that would otherwise occur if complement activation continued in those cells. Deficiencies or defects in the complement regulatory protein (s) are involved in the pathogenesis of a variety of complement-mediated diseases, for example, as discussed in this document. IV. Compstatin analogues
[0048] Compstatin is a cyclic peptide that binds to C3 and inhibits complement activation. US Patent No. 6,319,897 describes a peptide having the sequence Ile- [Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys] -Thr (SEQ ID NO: 1), with the disulfide bond between the two cysteines denoted by square brackets. It will be understood that the name "compsatin" was not used in US Patent No. 6,319,897, but was later adopted in the scientific and patent literature (see, for example, Morikis, et al., Protein Sci., 7 ( 3): 619-27, 1998) to refer to a peptide with the same sequence as SEQ ID NO: 2 disclosed in US Patent No. 6,319,897, but amidated at the C-terminus as shown in Table 1 (SEQ ID NO: 8). The term "compstatin" is used in this document consistently with such use (ie, to refer to SEQ ID NO: 8). Compstatin analogs that have a superior complement inhibiting compstatin activity have been developed. See, for example, WO2004 / 026328 (PCT / US2003 / 029653), Morikis, D., et al., Biochem Soc Trans. 32 (Pt 1): 28-32, 2004, Mallik, B., et al., J. Med. Chem., 274-286, 2005; Katragadda, M., et al. J. Med. Chem., 49: 4616-4622, 2006; WO2007062249 (PCT / US2006 / 045539); WO2007044668 (PCT / US2006 / 039397), WO / 2009/046198 (PCT / US2008 / 078593); WO / 2010/127336 (PCT / US2010 / 033345) and discussion below.
[0049] Compstatin analogs can be acetylated or starched, for example, at the N-terminal and / or C-terminal. For example, compstatin analogs can be acetylated at the N-terminal and amidated at the C-terminal. Consistent with the use in the technique, "compstatin" as used in this document and the activities of compstatin analogs described here in relation to those of compstatin, refer to amidated compstatin in the C-terminal (Mallik, 2005, supra).
[0050] Concatamers or multimers of compstatin or a corresponding complement inhibitor analog are also useful in the present invention.
[0051] As used herein, the term "compstatin analog" includes compstatin and any complement inhibitor analog thereof. The term "compstatin analogue" encompasses compstatin and other compounds designed or identified on the basis of compstatin and whose complement inhibitory activity is at least 50% as great as that of compstatin as measured, for example, using any accepted complement activation assay in the technique or substantially similar or equivalent tests. Certain suitable assays are described in US Patent No. 6,319,897, WO2004 / 026328, Morikis, supra, Mallik, supra, Katragadda 2006, supra, WO2007062249 (PCT / US2006 / 045539); WO2007044668 (PCT / US2006 / 039397), WO / 2009/046198 (PCT / US2008 / 078593); and / or WO / 2010/127336 (PCT / US2010 / 033345). The assay can, for example, measure alternative or classical mediated erythrocyte lysis or be an ELISA test. In some embodiments, an assay described in WO / 2010/135717 (PCT / US2010 / 035871) is used.
[0052] The activity of a compstatin analogue can be expressed through its IC50 (the concentration of the compound that inhibits the activation of complement by 50%), with a lower IC50 indicating greater activity, as recognized in the art. The activity of a preferred compstatin analogue for use in the present invention is at least as great as that of compstatin. It is noted that certain modifications known to reduce or eliminate complement the inhibiting activity and can be explicitly excluded from any modality of the invention. The IC50 of compstatin was measured as 12 μM using an alternative mediated erythrocyte lysis assay (WO2004 / 026328). It will be appreciated that the exact IC50 value measured for a given compstatin analogue will vary with experimental conditions (for example, the serum concentration used in the assay). Comparative values, for example, obtained from experiments in which IC 50 is determined by several different compounds under substantially identical conditions, are useful. In one embodiment, the IC50 of the compstatin analog is no more than the IC50 of compstatin. In certain embodiments of the invention, the activity of the compstatin analogue is between 2 and 99 times that of compstatin (that is, the analogue has a lower IC50 than the IC50 of compstatin by a factor between 2 and 99). For example, the activity can be between 10 and 50 times as great as that of compstatin, or between 50 and 99 times as great as that of compstatin. In certain embodiments of the invention, the activity of the compstatin analogue is between 99 and 264 times that of compstatin. For example, the activity can be 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, or 264 times as large as that of compstatin . In certain modalities, the activity is between 250 and 300, 300 and 350, 350 and 400, or 400 and 500 times as great as that of compstatin. The invention also contemplates compstatin analogs with activities between 500 and 1000 times that of compstatin, or more. In certain embodiments, IC50 of the compstatin analogue is between about 0.2 μM and about 0.5 μM. In certain embodiments, IC50 of the compstatin analogue is between about 0.1 μM and about 0.2 μM. In certain embodiments, IC50 of the compstatin analogue is between about 0.5 μM and about 0.1 μM. In certain embodiments, the IC50 of the compstatin analogue is between about 0.001 μM and about 0.05 μM.
[0053] Kd of compstatin binding to C3 can be measured using isothermal titration calorimetry (Katragadda, et al., J. Biol. Chem., 279 (53), 54987-54995, 2004). The binding affinity of a variety of compstatin analogs for C3 was correlated with its activity, with a lower Kd, indicating a higher binding affinity, as recognized in the art. A linear correlation between binding affinity and activity was shown for certain tested analogs (Katragadda, 2004, supra; Katragadda 2006, supra). In certain embodiments of the invention, the compstatin analog binds to C3 with a Kd between 0.1 μM and 1.0 μM, between 0.05 μM and 0.1 μM, between 0.025 μM and 0.05 μM, between 0.015 μM and 0.025 μM, between 0.01 μM and 0.015 μM or between 0.001 μM and 0.01 μM.
[0054] Compounds "designed or identified on the basis of compostatin" include, among others, compounds that make up a chain of amino acids, the sequence of which is obtained by (i) modifying the compstatin sequence (for example, replacing one or more amino acids of the compstatin sequence by a different amino acid or amino acid analog, inserting one or more amino acids or amino acid analogs in the compstatin sequence or excluding one or more amino acids from the compstatin sequence); (ii) selection of a phage display peptide library in which one or more compstatin amino acids are randomized and, optionally, additionally modified according to method (i); or (iii) identification by screening for compounds that compete with compstatin or any analog thereof obtained by methods (i) or (ii) for binding with C3 or a fragment thereof. Many useful compstatin analogs make up a hydrophobic cluster, a β-turn and a disulfide bridge.
[0055] In certain embodiments of the invention, the compstatin analog sequence comprises or essentially consists of a sequence that is obtained by making 1, 2, 3 or 4 substitutions in the compstatin sequence, that is, 1, 2, 3 or 4 amino acids following compstatin they are replaced by a different standard amino acid or a non-standard amino acid. In certain embodiments of the invention, the amino acid at position 4 is changed. In certain embodiments of the invention, the amino acid at position 9 is changed. In certain embodiments of the invention, the amino acids at positions 4 and 9 are changed. In certain embodiments of the invention, only the amino acids at positions 4 and 9 are changed. In certain embodiments of the invention, the amino acid at position 4 or 9 is altered, or in certain embodiments, both amino acids 4 and 9 are altered, and furthermore, up to 2 amino acids located at selected positions of 1, 7, 10, 11 and 13 are changed. In certain embodiments of the invention, the amino acids at positions 4, 7 and 9 are changed. In certain embodiments of the invention, the amino acids at position 2, 12, or both are changed, provided that the change preserves the ability of the compound to be cyclized. Such change (s) in positions 2 and / or 12 can be in addition to the change (s) in position 1, 4, 7, 9, 10, 11 and / or 13. Optionally, the sequence of any of the compstatin analogs whose sequence is obtained by replacing one or more amino acids in the compstatin sequence further includes up to 1, 2 or 3 additional amino acids at the C-terminus. In one embodiment, the additional amino acid is Gly. Optionally, the sequence of any of the compostatin analogs whose sequence is obtained by substituting one or more amino acids from the compstatin sequence further includes up to 5, or up to 10 additional amino acids at the C-terminus. It should be understood that compstatin analogs may have one or more of the characteristics or features of the various modalities described herein, and characteristics or features of any modality may additionally characterize any other modality described in this document, unless otherwise indicated or evidenced by the context. In certain embodiments of the invention, the sequence of the compstatin analog comprises or essentially consists of a sequence identical to that of compstatin, except in the positions corresponding to positions 4 and 9 in the sequence of compstatin.
[0056] Compstatin and certain compstatin analogs with slightly higher activity than compstatin contain only standard amino acids ("standard amino acids" are glycine, leucine, isoleucine, valine, alanine, phenylalanine, tyrosine, tryptophan, aspartic acid, asparagagin, glutamic acid, glutamine, cysteine, methionine, arginine, lysine, proline, serine, threonine and histidine). Certain compstatin analogs with enhanced activity incorporate one or more non-standard amino acids. Useful non-standard amino acids include halogenated amino acids once or multiple times (for example, fluorinated gases), D-amino acids, homo-amino acids, N-alkyl amino acids, dehydro amino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan ), ortho-, meta- or para-aminobenzoic acid, phosphoamino acids, methoxylated and α amino acids, α-disubstituted amino acids. In certain embodiments of the invention, a compstatin analog is made by replacing one or more L-amino acids in a compstatin analog described elsewhere with the corresponding D-amino acid. Such compounds and methods of using them are an aspect of the invention. Exemplary non-standard amino acids in use include 2-naphthylalanine (2-NaI), 1-naphthylalanine (1-NaI), 2-indanylglycine (2Ig1) carboxylic acid, dihydrotryptophan (Dht), 4-benzoyl-L-phenylalanine (Bpa) , 2-α-aminobutyric acid (Abu-2), 3-α-aminobutyric acid (Abu-3), 4-α-aminobutyric acid (4-Abu), cyclohexylalanine (Cha), homocyclohexylalanine (hCha), 4 -fluoro-L-tryptophan (4fW), 5-fluoro-L-tryptophan (5fW), 6-fluoro-L-tryptophan (6fW), 4-hydroxy-L-tryptophan (4OH-W), 5-hydroxy-L -tryptophan (5OH-W), 6-hydroxy-L-tryptophan (6OH-W), 1-methyl-L-tryptophan (1MeW), 4-methyl-L-tryptophan (4MeW), 5-methyl-L-tryptophan (5MeW), 7-aza-L-tryptophan (7aW), α-methyl-L-tryptophan (αMeW), β-methyl-L-tryptophan (βMeW), N-methyl-L-tryptophan (NMeW), ornithine ( orn), citrulline, norleucine, Y-glutamic acid, etc.
[0057] In certain embodiments of the invention, the compstatin analogue comprises one or more Trp analogs (for example, at position 4 and / or 7 with respect to the compstatin sequence). Exemplary Trp analogs are mentioned above. See also Beene, et. Al. Biochemistry41: 10262-10269, 2002 (describing, among others, halogenated Trp analogs once or multiple times); Babitzke & Yanofsky, J. Biol. Chem.270: 12452-12456, 1995 (describing, among others, methylated and halogenated Trp and other analogs of Trp and indole); and US patents 6,214,790, 6,169,057, 5,776,970, 4,870,097, 4,576,750 and 4,299,838. Other analogs of Trp include variants that are replaced (for example, by a methyl group) on α or β carbon and, optionally, also in one or more positions of the indole ring. Amino acids comprising two or more aromatic rings, including unsubstituted, unsubstituted or alternatively substituted variants of them, are of interest as Trp analogs. In certain embodiments of the invention, the Trp analogue, for example, at position 4, is 5-methoxy, 5-methyl-, 1-methyl- or 1-formyl-tryptophan. In certain embodiments of the invention, a Trp analogue (for example, at position 4) comprising a 1-alkyl substituent, for example, a lower alkyl substituent (for example, C1-C5) is used. In certain embodiments, N (α) methyl tryptophan or 5-methyltryptophan is used. In some embodiments, an analog comprising a 1-alkanoyl substituent, for example, a lower alkanoyl (for example, C1-C5) is used. Examples include 1-acetyl-L-tryptophan and L-β-tryptophan.
[0058] In certain modalities, the Trp analogue increased the hydrophobic character in relation to Trp. For example, the indole ring can be replaced by one or more alkyl groups (for example, methyl). In certain modalities, the Trp analogue participates in a hydrophobic interaction with C3. Such a Trp analogue can be located, for example, at position 4 with respect to the compstatin sequence. In certain embodiments, the Trp analogue comprises a substituted or unsubstituted bicyclic aromatic ring component or two or more substituted or unsubstituted monocyclic aromatic ring components.
[0059] In certain modalities, the Trp analogue increased propensity for the formation of hydrogen bonds with C3 in relation to Trp, but it does not have a greater hydrophobic character in relation to Trp. The Trp analogue may have a greater polarity in relation to Trp and / or more ability to participate in an electrostatic interaction with a hydrogen bonding donor in C3. Certain exemplary Trp analogs with a higher hydrogen bond formation feature comprise an electronegative substituent on the indole ring. Such a Trp analogue can be located, for example, at position 7, relative to the compstatin sequence.
[0060] In certain embodiments of the invention, the compstatin analogue comprises one or more Ala analogs (for example, at position 9 relative to the compstatin sequence), for example, Ala analogs that are identical to Ala, except that they include one or more side chain CH2 groups. In certain embodiments, the Ala analogue is a single methyl branched amino acid, such as 2-Abu. In certain embodiments of the invention, the comppatin analogue comprises one or more Trp analogs (for example, at position 4 and / or 7 with respect to the compstatin sequence) and an Ala analogue (for example, at position 9 in the compostatin sequence).
[0061] In certain embodiments of the invention, the compstatin analogue is a compound comprising a peptide that has a sequence of (X'aa) n- Gln - Asp - Xaa - Gly- (X "aa) m, (SEQ ID N °: 2) where each X'aa and each X "aa is an independently selected amino acid or amino acid analog, where Xaa is Trp or a Trp analogue, and where n> 1 in> 1 and n + m is between 5 and 21. The peptide has a core sequence of Gln - Asp-Xaa - Gly, where Xaa is Trp or a Trp analogue, for example, a Trp analogue, having increased the propensity to form hydrogen bonds with an H bond donor in relation to Trp, but, in certain modalities, not having a greater hydrophobic character in relation to Trp. For example, the analogue can be one in which the Trp indole ring is replaced with an electronegative portion, for example, a halogen like fluorine. In one embodiment, Xaa is 5-fluorotryptophan. In the absence of evidence to the contrary, a person skilled in the art would recognize that any unnatural peptide, whose sequence comprises this nucleus sequence and which inhibits complement activation and / or binds to C3, will have been constructed based on the compstatin sequence. In an alternative embodiment, Xaa is an amino acid or an amino acid analog other than a Trp analogue that allows the Gln - Asp - Xaa - Gly peptide to form a β-turn.
[0062] In certain embodiments of the invention, the peptide has a core sequence of X'aa-Gln - Asp - Xaa - Gly (SEQ ID NO: 3), where X'aa and Xaa are selected from Trp and analogues of Trp. In certain embodiments of the invention, the peptide has a core sequence of X'aa-Gln - Asp - Xaa - Gly (SEQ ID NO: 3), where X'aa and Xaa are selected from Trp, analogues of Trp and other amino acids or amino acid analogs comprising at least one aromatic ring. In certain embodiments of the invention, the nucleus sequence forms a β-turn in the context of the peptide. The β-turn can be flexible, allowing the peptide to assume two or more conformations as assessed, for example, using nuclear magnetic resonance (NMR). In certain embodiments, X'aa is a Trp analogue comprising a substituted or unsubstituted bicyclic aromatic ring component or two or more substituted or unsubstituted monocyclic aromatic ring components. In certain embodiments of the invention, X'aa is selected from the group consisting of 2-naptylalanine, 1-naptylalanine, 2-indanylglycine carboxylic acid, dihydrotryptophan and benzoylphenylalanine. In certain embodiments of the invention, X'aa is a Trp analogue that has a greater hydrophobic character than Trp. For example, X'aa can be 1-methyltryptophan. In certain embodiments of the invention, Xaa is a Trp analogue that is more likely to form hydrogen bonds in relation to Trp, but, in certain embodiments, not having a greater hydrophobic character in relation to Trp. In certain embodiments of the invention, the Trp analogue that is more likely to form hydrogen bonds than Trp comprises a modification of the Trp indole ring, for example, at position 5, as a substitution of a halogen atom for a H atom at position 5. For example, Xaa can be 5-fluorotryptophan.
[0063] In certain embodiments of the invention, the peptide has a core sequence of X'aa-Gln - Asp - Xaa - Gly-X "aa (SEQ ID NO: 4), where X'aa and Xaa are independently selected from Trp and analogs of Trp and X "aa is selected from His, Ala, analogs of Ala, Phe and Trp. In certain embodiments of the invention, X'aa is a Trp analogue that has increased hydrophobic character over Trp, such as 1-methyltryptophan or another Trp analogue with an alkyl substituent on the indole ring (for example, at position 1, 4 , 5, or 6). In certain embodiments, X'aa is a Trp analog comprising a substituted or unsubstituted bicyclic aromatic ring component or two or more substituted or unsubstituted monocyclic aromatic ring components. In certain embodiments of the invention, X'aa is selected from the group consisting of 2-naptilalanine, 1-naptilalanine, 2-indanylglycine carboxylic acid, dihydrotryptophan and benzoylphenylalanine. In certain embodiments of the invention, Xaa is a Trp analogue that is more likely to form hydrogen bonds with C3 compared to Trp, but, in certain embodiments, not having a greater hydrophobic character compared to Trp. In certain embodiments of the invention, the Trp analogue that is more likely to form hydrogen bonds than Trp comprises a modification of the Trp indole ring, for example, at position 5, as a substitution for a halogen atom for an H atom at position 5. For example, Xaa can be 5-fluorotryptophan. In certain embodiments, X "aa is Ala or an analogue of Ala, such as Abu or another single methyl branched amino acid. In certain embodiments of the invention, the peptide has a core sequence of X'aa-Gln - Asp - Xaa - Gly-X "aa (SEQ ID NO: 4), where X'aa and Xaa are independently selected from Trp, Trp analogs and amino acids or amino acid analogs comprising at least one aromatic side chain, and X" aa is selected from His, Ala, Ala, Phe and Trp analogs. In certain embodiments, X "aa is selected from Trp analogs, aromatic amino acids and aromatic amino acid analogs.
[0064] In certain preferred embodiments of the invention. The peptide is cyclic. The peptide can be cyclized through a link between any two amino acids, one of which is (X'aa) n and the other which is located within (X "aa) m. In certain embodiments, the cyclic part of the peptide is between 9 and 15 amino acids in length, for example, 10-12 amino acids in length In certain embodiments, the cyclic part of the peptide is 11 amino acids in length, with a bond (for example, a disulfide bond) between amino acids at positions 2 and 12 For example, the peptide can be 13 amino acids long, with a link between amino acids at positions 2 and 12, resulting in a cyclic portion 11 amino acids long.
[0065] In certain embodiments, the peptide comprises or consists of the sequence X'aa1 - X'aa2 - X'aa3 - X'aa4 -Gln-Asp-Xaa-Gly- X "aa1- X" aa2- X "aa3- X "aa4- X" aa5 (SEQ ID N °: 5). In certain modalities, X 'aa4 and Xaa are selected from Trp and analogues of Trp, and X'aa1, X'aa2, X'aa3, X "aa1, X" aa2, X "aa3, X" aa4, and X "aa5 are independently selected from amino acids and amino acid analogues. In certain embodiments, X'aa4 and Xaa are selected from aromatic amino acids and aromatic amino acid analogues. Any one or more of one of X'aa1, X'aa2, X'aa3, X "aa1, X" aa2, X "aa3, X" aa4, and X "aa5 can be identical to the amino acid in the corresponding position on compstatin. In one embodiment, X "aa1 is Ala or a single methyl branched amino acid. The peptide can be cyclized through a covalent bond between (i) X’aa1, X’aa2, or X’aa3; and (ii) X "aa2, X" aa3, X "aa4 or X" aa5. In one embodiment, the peptide is cyclized through a covalent bond between X'aa2 and X "aa4. In one embodiment, the covalently linked amino acids are each Cys and the covalent bond is a disulfide bond (SS). modalities, the covalent bond is a CC, CO, CS or CN bond In certain embodiments, one of the covalently bound residues is an amino acid or amino acid analog with a side chain comprising a primary or secondary amine, the other covalently bound residue is an amino acid or amino acid analog with a side chain comprising a carboxylic acid group, and the covalent bond is an amide bond Amino acids or amino acid analogs with a side chain comprising a primary or secondary amine include lysine and diaminocarboxylic acids of structure general NH2 (CH2) nCH (NH2) COOH such as 2,3-diaminopropionic acid (dapa), 2,4-diaminobutyric acid (daba) and ornithine (orn), where n = 1 (dapa), 2 (daba) , and 3 (orn), respectively. Examples of amino acids with a side chain comprising a carboxylic acid group include dicarboxylic amino acids such as glutamic acid and aspartic acid. Analogs such as beta-hydroxy-L-glutamic acid can also be used. In some embodiments, a peptide is cyclized with a thioether bond, for example, as described in PCT / US2011 / 052442 (WO / 2012/040259). For example, in some embodiments, a disulfide bond in either peptide is replaced with a thioether bond. In some embodiments, a cystathionine is formed. In some embodiments, cystothionine is a delta-cystathionine or a gamma-cystathionine. In some embodiments, a modification involves replacing a Cys-Cys disulfide bond between cysteines in X'aa2 and X "aa4 in SEQ ID NO: 5 (or corresponding positions in other sequences) with the addition of a CH2 to form a homocysteine in X'aa2 or X "aa4 and the introduction of a thioether bond to form a cystathione. In one embodiment, cystathionine is a gamma-cystathionine. In another embodiment, cystathionine is a delta-cystathionine. Another modification in accordance with the present invention comprises replacing the disulfide bond with a thioether bond without the addition of a CH2, thus forming a lanthionine. In some embodiments, a compstatin analog with a thioether in place of a disulfide bond has greater stability, at least under some conditions, compared to the compstatin analog with a disulfide bond.
[0066] In certain embodiments, the compstatin analogue is a compound comprising a peptide having a sequence: Xaa1 - Cys - Val - Xaa2 - Gln - Asp - Xaa2 * - Gly - Xaa3 - His - Arg - Cys - Xaa4 (SEQ ID NO: 6); on what:
[0067] Xaa1 is Ile, Val, Leu, B1- Ile, B1-Val, B1-Leu or a dipeptide comprising Gly-Ile or B1- Gly-Ile and B1 represents a first blocking vapor;
[0068] Xaa2 and Xaa2 * are selected independently from Trp and Trp analogs;
[0069] Xaa3 is His, Ala or an analogue of Ala, Phe, Trp or an analogue of Trp;
[0070] Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from Thr-Ala and Thr-Asn or a tripeptide comprising Thr-Ala-Asn, where a -OH carboxyl terminus any of the L-Thr, D-Thr, Ile, Val, Gly, Ala or Asn is optionally substituted by a second blocking portion B2; and
[0071] the two Cys residues are joined by a disulfide bond. In some embodiments, Xaa4 is Leu, Nle, His, or Phe or a dippeptide selected from Xaa5-Ala and Xaa5-Asn or a Xaa5-Ala-Asn tripeptide, where Xaa5 is selected from Leu, Nle , His or Phe and in which a carboxy-terminal -OH of any of L-Thr, D-Thr, Ile, Val, Gly, Leu, Nle, His, Phe, Ala, or Asn is optionally substituted by a second portion of B2 block; and the two Cys residues are joined by a disulfide bond.
[0072] In other embodiments, Xaa1 is absent or is any amino acid or amino acid analog, and Xaa2, Xaa2 *, Xaa3, and Xaa4 are as defined above. If Xaa1 is absent, the Cys doo N-terminal residue may have a blocking portion B1 attached to it.
[0073] In another embodiment, Xaa4 is any amino acid or amino acid analog and Xaa1, Xaa2, Xaa2 * and Xaa3 are as defined above. In another embodiment, Xaa4 is a dipeptide selected from the group consisting of: Thr-Ala and Thr-Asn, where the carboxy-terminal -OH or Ala or Asn is optionally substituted by a second B2 blocking portion.
[0074] In any of the modalities of the compostatin analog of SEQ ID NO: 6, Xaa2 can be Trp.
[0075] In any of the modalities of the compostatin analog of SEQ ID NO: 6, Xaa2 can be a Trp analog comprising a substituted or unsubstituted bicyclic aromatic ring component or two or more monocyclic aromatic ring components click substituted or unsubstituted. For example, the Trp analogue can be selected from 2-naphthylalanine (2-NaI), 1-naphthylalanine (1-NaI), 2-indanylglycine (Ig1) carboxylic acid, dihydrotropithopan (Dht) and 4-benzoyl-L -phenylalanine.
[0076] In any of the modalities of the compostatin analog of SEQ ID NO: 6, Xaa2 can be a Trp analog with increased hydrophobic character in relation to Trp. For example, the Trp analogue can be selected from 1-methyltryptophan, 4-methyltryptophan, 5-methyltryptophan and 6-methyltryptophan. In one embodiment, the Trp analogue is 1-methyltryptophan. In one embodiment, Xaa2 is 1-methyltryptophan, Xaa2 * is Trp, Xaa3 is Ala and the other amino acids are identical to those of compstatin.
[0077] In any of the modalities of the comps-tatin analogue of SEQ ID NO: 6, Xaa2 * can be a Trp analogue such as a Trp analogue with a propensity for increased hydrogen bond formation with C3 over to Trp, which, in certain modalities, did not increase the hydrophobic character in relation to Trp. In certain embodiments, the Trp analogue comprises an electronegative substituent on the indole ring. For example, the Trp analogue can be selected from 5-fluorotryptophan and 6-fluorotryptophan.
[0078] In certain embodiments of the invention, Xaa2 is Trp and Xaa2 * is an analog of Trp that is more likely to form hydrogen bonds with C3 than Trp, which, in certain modalities, is not more hydrophobic in character. relation to Trp. In certain embodiments of the compstatin analogue of SEQ ID NO: 6, Xaa2 is a Trp analogue with increased hydrophobic character over Trp, such as a Trp analogue selected from 1-methyltryptophan, 4-methyltryptophan, 5-methyltryptophan and 6-methyltryptophan, and Xaa2 * is a Trp analogue with a propensity for formation of increased hydrogen bonding with C3 in relation to Trp, which, in certain modalities, did not increase the hydrophobic character in relation to Trp. For example, in one embodiment, Xaa2 is methyltryptophan and Xaa2 * is 5-fluorotryptophan.
[0079] In some of the modalities mentioned above, Xaa3 is Ala. In some of the above mentioned modalities, Xaa3 is a single methyl branched amino acid, for example, Abu.
The invention further provides compstatin analogs of SEQ ID NO: 6, as described above, wherein Xaa2 and Xaa2 * are independently selected from Trp, Trp analogs and other amino acids or amino acid analogs comprising at least one aromatic ring, and Xaa3 is His, Ala or an analogue of Ala, Phe, Trp, an analog of Trp or another aromatic amino acid or aromatic amino acid analog.
[0081] In certain embodiments of the invention, the N- or C-terminal blocking portion of any of the compstatin analogs described herein is any group that stabilizes a peptide against degradation that would otherwise occur in the blood or mammalian interstitial fluid (for example, human or non-human primate). For example, the blocking portion B1 could be any portion that alters the N-terminal structure of a peptide in order to inhibit the cleavage of a peptide bond between the N-terminal amino acid of the peptide and the adjacent amino acid. The blocking portion B2 could be any portion that alters the C-terminal structure of a peptide in order to inhibit cleavage of a peptide bond between the C-terminal amino acid of the peptide and the adjacent amino acid. Any suitable blocking portions known in the art could be used. In certain embodiments of the invention, the blocking portion B1 comprises an acyl group (i.e., the portion of a carboxylic acid that remains after removal of the - OH group). The acyl group generally comprises between 1 and 12 carbons, for example, between 1 and 6 carbons. For example, in certain embodiments of the invention, the blocking portion B1 is selected from the group consisting of: formyl, acetyl, proprionyl, butyryl, isobutyryl, valeryl, isovaleryl, etc. In one embodiment, the blocking portion B1 is an acetyl group, that is, Xaa1 is Ac-Ile, Ac-Val, Ac-Leu, or Ac-Gly-Ile.
[0082] In certain embodiments of the invention, the blocking portion B2 is a primary or secondary amine (-NH2 or -NHR1, where R is an organic portion, such as an alkyl group).
[0083] In certain embodiments of the invention, the blocking portion B1 is any group that neutralizes or reduces the negative charge which, otherwise, may be present at the N-terminus at physiological pH. In certain embodiments of the invention, the blocking portion B2 is any group that neutralizes or reduces the negative charge that might otherwise be present at the C-terminus at physiological pH.
[0084] In certain embodiments of the invention, the compstatin analog is acetylated or amidated from the N-terminus and / or C-terminus, respectively. A compstatin analog can be acetylated at the N-terminal, amidated at the C-terminal, and / or either acetylated at the N-terminal or amidated at the C-terminal. In certain embodiments of the invention, a compstatin analog comprises an alkyl or aryl group at the N-terminus, instead of an acetyl group.
[0085] In certain embodiments, the compstatin analogue is a compound that comprises a peptide having a sequence: Xaa1 - Cys - Val - Xaa2 - Gln - Asp - Xaa2 * - Gly - Xaa3 - His - Arg - Cys - Xaa4 (SEQ ID NO: 7); on what:
[0086] Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide comprising Gly-Ile or Ac-Gly-Ile;
[0087] Xaa2 and Xaa2 * are selected independently from Trp and Trp analogs;
[0088] Xaa3 is His, Ala or an analogue of Ala, Phe, Trp or an analogue of Trp;
[0089] Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from Thr-Ala and Thr-Asn or a tripeptide comprising Thr-Ala-Asn, in which a -OH carboxyl terminus any of the L-Thr, D-Thr, Ile, Val, Gly, Ala or Asn is optionally substituted by -NH2; and the two Cys residues are joined by a disulfide bond. In some embodiments, Xaa4 is Leu, Nle, His, or Phe or a dippeptide selected from Xaa5-Ala and Xaa5-Asn or a Xaa5-Ala-Asn tripeptide, where Xaa5 is selected from Leu, Nle , His or Phe and in which a carboxy-terminal -OH of any of L-Thr, D-Thr, Ile, Val, Gly, Leu, Nle, His, Phe, Ala, or Asn is optionally substituted by a second portion of B2 block; and the two Cys residues are joined by a disulfide bond.
[0090] In some embodiments, Xaa1, Xaa2, Xaa2 *, Xaa3 and Xaa4 are as described above for the various embodiments of SEQ ID NO: 6. For example, in certain embodiments, Xaa2 * is Trp. In certain embodiments, Xaa2 is a Trp analogue with increased hydrophobic character over Trp, for example, 1-methyltryptophan. In certain embodiments, Xaa3 is Ala. In certain embodiments, Xaa3 is a single methyl branched amino acid.
[0091] In certain embodiments of the invention, Xaa1 is Ile and Xaa4 is L-Thr.
[0092] In certain embodiments of the invention, Xaa1 is Ile, Xaa2 * is Trp and Xaa4 is L-Thr.
[0093] The invention further provides compstatin analogs of SEQ ID NO: 7, as described above, wherein Xaa2 and Xaa2 * are independently selected from Trp, Trp analogs and other amino acids or amino acid analogs, and Xaa3 is His, Ala or an analogue of Ala, Phe, Trp, an analogue of Trp or another aromatic amino acid or aromatic amino acid analog.
[0094] In certain embodiments of any of the compstatin analogs described in this document, a Phe analogue is used instead of Phe.
[0095] Table 1 provides a list of non-restrictive compstatin analogs useful in the present invention. The analogues are referred to in an abbreviated form in the left column, indicating specific changes in the designated positions (1-13), in comparison with the parental peptide, compstatin. Consistent with the use in the art, "compstatin" as used in this document and the activities of compstatin analogs described here in relation to those of compstatin, refer to the amidated compstatin peptide at the C-terminus. Unless otherwise indicated, peptides in Table 1 are amidated at the C-terminus. Bold text is used to indicate certain changes. The activity in relation to compstatin is based on published data and trials described there (WO2004 / 026328, WO2007044668, Mallik, 2005; Katra-gadda, 2006). Where several publications reporting an activity have been consulted, the most recently published value is used, and it will be recognized that values can be adjusted in case of differences between the tests. It will also be appreciated that, in certain embodiments of the invention, the peptides listed in Table 1 are cyclized through a disulfide bond between the two Cys residues when used in the therapeutic compositions and methods of the invention. Alternative means for cyclizing the peptides are also within the scope of the invention. As noted above, in various embodiments of the invention, one or more amino acids (s) of a compostatin analogue (for example, any of the compstatin analogs disclosed herein) may be an N-alkyl amino acid (for example, a amino acid N-methyl). For example, and without limitation, at least one amino acid within the cyclic part of the peptide, at least one amino acid from the N-terminal to the cyclic part, and / or at least one amino acid at the C-terminal to the cyclic part can be an amino acid N-alkyl, for example, an N-methyl amino acid. In some embodiments of the invention, for example, a compstatin analogue comprises an N-methyl glycine, for example, in the position corresponding to position 8 of compstatin and / or in the position corresponding to position 13 of compstatin. In some embodiments, one or more of the compstatin analogs in Table 1 contain at least one N-methyl glycine, for example, in the position corresponding to the 8 position of compstatin and / or in the position corresponding to the 13 position of compstatin. Table 1


ND = not available
[0096] In certain embodiments of the compositions and methods of the invention, the compstatin analog has a sequence selected from sequences 9-36. In certain embodiments of the compositions and methods of the invention, the compstatin analogue has a sequence selected from SEQ ID NO: 14, 21,28, 29, 32, 33, 34 and 36. In certain embodiments of the compositions and / or methods of the invention, the compstatin analog has a sequence selected from SEQ ID NO: 30 and 31. In one embodiment of the compositions and methods of the invention, the compstatin analog has a sequence of SEQ ID NO: 28. In one embodiment of the compositions and methods of the invention, the compstatin analogue has a sequence of SEQ ID NO: 32. In one embodiment of the compositions and methods of the invention, the compstatin analog has a sequence of SEQ ID NO: 34. In one embodiment of the compositions and methods of the invention, the compstatin analogue has a sequence of SEQ ID NO: 36.
[0097] In certain embodiments of the compositions and methods of the invention, the compstatin analog has a sequence as set out in Table 1, but where the group Ac- is replaced by an alternative blocking portion B1, as described above. In some embodiments, the -NH2 group is replaced by an alternative blocking portion B2, as described above.
[0098] In one embodiment, the compstatin analog binds substantially to the same region of the human C3 β chain as does compstatin. In one embodiment, the compstatin analog is a compound that binds to a fragment of the C-terminal part of the human C3 β chain having a molecular weight of about 40 kDa, to which compstatin binds (Soulika, AM, et al., Mol. Immunol., 35: 160, 1998; Soulika, AM, et al., Mol. Immunol. 43 (12): 2023-9, 2006). In certain embodiments, the compstatin analogue is a compound that binds to the compstatin binding site as determined in a compstatin-C3 structure, for example, a crystalline structure or 3D structure derived from NMR. In certain embodiments, the compstatin analogue is a compound that can replace compstatin in a compstatin-C3 structure and would form substantially the same intermolecular contacts with C3 as compstatin. In certain embodiments, the compstatin analogue is a compound that binds to a peptide binding site having a sequence set out in Table 1, for example, SEQ ID NO: 14, 21,28, 29, 32, 33, 34 or 36 in a C3 peptide structure, for example, a crystalline structure. In certain embodiments, the compostatin analog is a compound that binds to a peptide binding site having SEQ ID NO: 30 or 31 in a C3 peptide structure, for example, a crystalline structure. In certain embodiments, the compstatin analog is a compound that can replace the peptide of SEQ ID NO: 9-36, for example, a compound that can replace the peptide of SEQ ID NO: 14, 21, 28, 29, 32, 33, 34 or 36 in a C3-peptide structure and would form substantially the same intermolecular contacts with C3 as the peptide. In certain embodiments, the compstatin analog is a compound that can replace the peptide of SEQ ID NO: 30 or 31 in a compstatin-C3 structure and would form substantially the same intermolecular contacts with C3 as the peptide.
[0099] A person skilled in the art will readily be able to determine whether a compstatin analog binds to a fragment of the C-terminal part of the β chain of C3 using routine experimental methods. For example, a person skilled in the art could synthesize a photo-crosslinkable version of the compstatin analog, including a photo-crosslinking amino acid, such as p-benzoyl-L-phenylalanine (Bpa) in the compound, for example, at the C-terminal of sequence (Soulika, AM, et al, supra). Optionally additional amino acids, for example, an epitope tag such as a FLAG tag or an HA tag could be included to facilitate detection of the compound, for example, by Western blotting. The compstatin analog is incubated with the fragment and crosslinking is started. The colocalization of the compstatin analog and the C3 fragment indicates binding. Surface plasmon resonance can also be used to determine whether a compstatin analogue binds to the C3 binding site or a fragment thereof. An individual skilled in the art would be able to use molecular modeling software to predict whether a compound would form substantially the same intermolecular contacts with C3 that compstatin would form or a peptide with the sequence of any of the peptides in Table 1, for example, SEQ ID N °: 14, 21, 28, 29, 32, 33, 34 or 36 or, in some embodiments, SEQ ID NO: 30 or 31.
[00100] Compstatin analogs can be prepared by various synthetic methods of peptide synthesis known in the art through the condensation of amino acid residues, for example, in accordance with conventional peptide synthesis methods, they can be prepared by in vitro expression or into living cells of suitable nucleic acid sequences, encoding them using methods known in the art. For example, peptides can be synthesized using standard solid phase methodologies, as described in Malik, supra, Katragadda, supra, WO2004026328, and / or WO2007062249. Potentially reactive moieties, such as amine and carboxyl groups, reactive functional groups, etc., can be protected and subsequently unprotected using various protective groups and methodologies known in the art. See, for example, "Protective Groups in Organic Synthesis", 3rded. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York: 1999. Peptides can be purified using standard approaches, such as reverse phase HPLC. The separation of diastereomeric peptides, if desired, can be carried out using known methods, such as reverse phase HPLC. The preparations can be lyophilized, if desired, and then dissolved in a suitable solvent, for example, water. The pH of the resulting solution can be adjusted, for example, to physiological pH, using a base such as NaOH. Peptide preparations can be characterized by mass spectrometry, if desired, for example, to confirm mass and / or disulfide bond formation. See, for example, Mallik, 2005, and Katragadda, 2006.
[00101] The compstatin analogue, optionally linked to a cell reaction portion or targeting portion, can be modified by adding a molecule such as polyethylene glycol (PEG) or similar molecules to stabilize the compound, reduce its immunogenicity, increase its lifespan in the body, increase or decrease its solubility, or increase its resistance to degradation. PEGylation methods are well known in the art (Veronese, F.M. & Harris, Adv. Drug Deliv. Rev. 54, 453-456, 2002; Davis, F.F., Adv. Drug Deliv. Rev. 54, 457-458, 2002); Hinds, K.D. & Kim, S.W. Adv. Drug Deliv. Rev. 54, 505-530 (2002; Roberts, M.J., Bentley, M.D. & Harris, J.M. Adv. Drug Deliv. Rev. 54, 459-476; 2002); Wang, Y.S. et al. Adv. Drug Deliv. Rev. 54, 547-570, 2002). A wide variety of polymers such as modified PEGs and PEGs, including derivatized PEGs to which the polypeptides can conveniently be attached, are described in Nektar Advanced Pegylation 2005-2006 ProductCatalog, Nektar Therapeutics, San Carlos, CA, which also provides details of suitable procedures conjugation. In another embodiment, a compstatin analog is fused to the Fc domain of an immunoglobulin or a part of it. In some other embodiments, a compstatin analog is conjugated to a portion of albumin or an albumin-binding peptide. Thus, in some embodiments, a compstatin analog is modified with one or more polypeptide or non-polypeptide components, for example, the compstatin analog is PEGylated or conjugated to another portion. In some embodiments, the component is not the Fc domain of an immunoglobulin or a part of it. A compstatin analogue can be supplied as a multimer or as part of a supramolecular complex, which can include a single molecular species or multiple different species (for example, several different analogs).
[00102] In some embodiments, a compstatin analog is a multivalent compound comprising a plurality of portions of compstatin analog covalently or non-covalently attached to a polymeric backbone or support. The analogous portions of compstatin can be identical or different. In certain embodiments of the invention, the multivalent compound comprises multiple instances, or copies, of a single analogous portion of compstatin. In other embodiments of the invention the multivalent compound comprises one or more instances of each of two of more non-identical compstatin analog portions, for example, 3, 4, 5, or more different compstatin analog portions. In certain embodiments of the invention, the number of portions of compstatin analog ("n") is between 2 and 6. In other embodiments of the invention, n is between 7 and 20. In other embodiments of the invention, n is between 20 and 100 In other modalities, n is between 100 and 1,000. In other embodiments of the invention, n is between 1,000 and 10,000. In other embodiments, n is between 10,000 and 50,000. In other embodiments, n is between 50,000 and 100,000. In other modalities, n is between 100,000 and 1,000,000.
[00103] The compstatin analog portions can be attached directly to the polymeric support or can be connected via a linker portion that connects the compstatin analog portion to the polymeric support. The binding portion can be attached to a single analogous portion of compstatin and to the polymeric support. Alternatively, a linker portion may have multiple portions of compostatin analog joined to it so that the linker portion links multiple portions of compstatin analog to the polymeric support.
[00104] In some embodiments, the compstatin analogue comprises an amino acid having a side chain comprising a primary or secondary amine, for example, a Lys residue. For example, a Lys residue, or a sequence comprising a Lys residue, is added at the N-terminus and / or C-terminus of the compstatin analog. In some embodiments, the Lys residue is separated from the cyclic portion of the compstatin analog by a flexible or rigid spacer. The spacer may, for example, comprise a saturated or unsaturated, substituted or unsubstituted alkyl chain, oligo (ethylene glycol) chain, and / or other moieties, for example, as described in Section VI with respect to binders. The chain length can be, for example, between 2 and 20 carbon atoms. In other embodiments, the spacer is a peptide. The peptide spacer may be, for example, between 1 and 20 amino acids in length, for example, between 4 and 20 amino acids in length. Suitable spacers may comprise or consist of multiple Gly residues, Ser residues, or both, for example. Optionally, the amino acid that has a side chain comprising a primary or secondary amine and / or at least one amino acid in a spacer is a D-amino acid. Any of a variety of polymeric main structures or supports could be used. For example, the polymeric backbone or support may be a polyamide, polysaccharide, polyanhydride, polyacrylamide, polymethacrylate, polypeptide, polyethylene oxide, or dendrimer. Suitable polymeric main structures and methods are described, for example, in WO98 / 46270 (PCT / US98 / 07171) or WO98 / 47002 (PCT / US98 / 06963). In one embodiment, the polymeric backbone or support comprises multiple reactive functional groups, such as groups of carboxylic acids, anhydride, or succinimide. The polymeric backbone or support is reacted with compstatin analogs. In one embodiment, the compstatin analogue comprises any one of a number of different reactive functional groups, such as groups of carboxylic acids, anhydride or succinimide, which are reacted with appropriate groups in the polymeric backbone. Alternatively, monomeric units that could be joined to one another to form a polymeric backbone or support are first reacted with the compstatin analogs and the resulting monomers are polymerized. In another embodiment, short chains are prepolymerized, functionalized, and then a mixture of short chains of different composition is assembled into longer polymers. V. Compstatin Mimetics
[00105] The structure of compstatin is known in the art, and NMR structures for a number of compstatin analogs having higher activity than compstatin are also known (Malik, supra). Structural information can be used to design compstatin mimetics.
[00106] In one embodiment, the compstatin mimetic is any compound that competes with compstatin or any compstatin analogue (for example, a compstatin analogue whose sequence is provided in Table 1) for binding to C3 or a fragment thereof (for example, a 40 kD fragment of the □ chain to which compstatin binds). In some modalities, the compostatin mimetic has an activity equal to or greater than that of compstatin. In some embodiments, the compstatin mimetic is more stable, available orally, or has better bioavailability than compstatin. The compstatin mimetic can be a peptide, nucleic acid, or small molecule. In certain embodiments, the compstatin mimetic is a compound that binds to the compstatin binding site as determined in a compstatin-C3 structure, for example, a crystal structure or a 3-D structure derived from experiments on NMR. In certain embodiments, the compstatin mimetic is a compound that could replace compostatin in a compstatin-C3 structure and would form substantially the same intermolecular contacts with C3 as compstatin. In certain embodiments, the compstatin mimetic is a compound that binds to a peptide binding site having a sequence set out in Table 1, for example, SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36 or in certain embodiments SEQ ID NO: 30 or 31, in a C3 peptide structure. In certain embodiments, the compstatin mimetic is a compound that could replace a peptide having a sequence set out in Table 1, for example, SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36 or in certain embodiments SEQ ID NO: 30 or 31, in a C3-peptide structure and would form substantially the same inter-molecular contacts with C3 as the peptide. In certain embodiments, the compstatin mimetic has a non-peptide backbone, but has side chains arranged in a sequence designed based on the compstatin sequence.
[00107] A person skilled in the art will appreciate that once a specific desired conformation of a short peptide has been verified, methods for designing a peptide or peptidomimetic to fit that conformation are well known. See, for example, G.R. Marshall (1993), Tetrahedron, 49: 3547-3558; Hruby and Nikiforovich (1991), in Molecular Conformation and Biological Interactions, P. Balaram & S. Ramasehan, eds., Indian Acad. of Sci., Bangalore, PP. 429455), Eguchi M, Kahn M., Mini Rev Med Chem., 2 (5): 447-62, 2002. Of specific relevance to the present invention, the design of peptide analogs can be further refined, considering the contribution of several side chains of amino acid residues, for example, for the purpose of functional groups or for aesthetic considerations as described in the technique for compstatin and analogues thereof, among others.
[00108] It will be appreciated by those skilled in the art that a peptide mimic can serve equally well as a peptide in order to provide the conformation of the specific main structure and side chain functionalities necessary to bind C3 and inhibit complement activation. In that sense, it is contemplated as being within the scope of the present invention to produce and use C3 binding, complement inhibitory compounds through the use of naturally occurring amino acids, amino acid derivatives, analogs or non-amino acid molecules capable of being joined to form the conformation of the appropriate main structure. A non-peptide analogue, or an analogue comprising peptide and non-peptide components, is sometimes referred to in this document as “peptidomimetic” or “isosteric mimetic”, to denote substitutions or derivations of a peptide that has many of the same conformational characteristics. main structure and / or other functionalities, in order to be sufficiently similar to the exemplified peptides to inhibit complement activation. More generally, a compostatin mimetic is any compound that would position pharmacophores similar to its positioning on compstatin, even if the main structure is different.
[00109] The use of peptidomimetics for the development of high-affinity peptide is well known in the art. Assuming similar rotational restrictions of amino acid residues, within a peptide, analogues comprising portions of non-amino acid can be analyzed, and their conformational motifs verified, using the Ramachandran graph (Hruby & Nikiforovich 1991), among other known techniques.
[00110] A person skilled in the art will readily be able to establish adequate screening assays to identify additional compstatin mimetics and select those having desired inhibitory activities. For example, compstatin or an analog of it could be labeled (for example, with a radioactive or fluorescent label) and placed in contact with C3 in the presence of different concentrations of a test compound. The ability of the test compound to decrease binding of the compstatin analogue to C3 is assessed. A test compound that significantly decreases the binding of the compstatin analogue to C3 is a candidate compstatin mimetic. For example, a test compound that decreases the steady-state concentration of a compstatin-C3 analog complex, or that decreases the rate of formation of a compstatin-C3 analog complex by at least 25%, or by at least 50%, is a candidate compstatin mimetic. One skilled in the art will recognize that a number of variations of this screening assay can be employed. The compounds to be screened include natural products, aptamer libraries, phage display libraries, compound libraries synthesized using combining chemistry, etc. The invention encompasses synthesizing a combinatorial library of compounds based on the core sequence described above and screening the library to identify compstatin mimetics. Any of these methods can also be used to identify new compstatin analogs, having a higher inhibitory activity than the compstatin analogs tested so far. It will be appreciated that compstatin mimetics could be used in the cell reactive compounds of the invention, and the invention provides such cell reactive compstatin mimetics. SAW. Compstatin Analogs of Extended Action or Cell Reactives
[00111] The invention provides a variety of cellular reactive compostatin analogs. In some respects, a cell reactive compostatin analogue comprises a compound of the formula ALM, where A is a moiety comprising a cell reactive functional group J, L is an optionally present linker moiety, and M comprises a moiety analogous moiety compstatin. The compstatin analogue portion can comprise any compstatin analogue, for example, any compstatin analogue described above, in various embodiments. The formula ALM encompasses modalities in which AL is present at the N-terminus of the compstatin analog portion, modalities in which AL is present at the C-terminus of the compstatin analog portion, modalities in which AL is attached to a side chain of a amino acid of the compstatin analogue portion, and modalities where the same or different A-Ls are present at both ends of M. It will be appreciated that when determined compstatin analogue (s) is (are) present as a portion of the compstatin analog in a compound of formula ALM, a functional group of the compstatin analog will have reacted with a functional group of L to form a covelente bond to A or L. For example, a reactive cellular compstatin analog in which the portion of compstatin analogue comprises a compstatin analogue that contains an amino acid with a side chain containing a primary amine group (NH2) (whose compstatin analogue can be represented by the formula R1— (NH2)), it can have a formula R1 — NH— L- A in which a new covalent bond to L (for example, N — C) has been formed and hydrogen has been lost. Thus, the term "compstatin analog portion" includes molecular structures in which at least one atom of a compstatin analog participates in a covalent bond with a second portion, which can, for example, make a modification of a side chain. Similar considerations apply to portions of the compstatin analog present in the multivalent compound described above In some embodiments, an N-terminal or C-terminal blocking portion of a compostatin analog, for example, a described compstatin analog in Section IV above, it is replaced by AL in the structure of a cell reactive compostatin analog In some embodiments, A or L comprises a blocking portion In some embodiments, a cell reactive compstatin analog has a molar hair activity minus 10%, 20% or 30%, for example, between 30% and 40%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30 % and 90% or more of the activity of a compstatin analog corresponding have the same amino acid sequence (and, if applicable, one or more blocking moieties, but not comprising a reactive cellular moiety). In some embodiments in which a cell reactive compstatin analog comprises multiple portions of compstatin analog, the molar activity of the cell reactive compstatin analog is at least about 10%, 20%, or 30%, for example, between 30% and 40%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, or more, of the sum of the activities of said portions of compstatin analog.
[00112] Cell reactive portion A may comprise any of a variety of different cell reactive functional groups J, in various embodiments. In general, a reactive cell functional group can be selected based at least in part on factors such as (a) the specific functional group to be targeted; (b) the ability of the reactive functional group to react with the targeting functional group under physiologically acceptable ex vivo conditions (for example, physiologically acceptable pH and osmolarity) and / or in vivo conditions (for example, in the blood); (c) the specificity of the reaction between the reactive functional group and the targeting functional group under physiologically acceptable ex vivo and / or in vivo conditions; (d) the stability (for example, under in vivo conditions) of the covalent bond that would result from the reaction of the reactive functional group with its targeting functional group; and the facility to synthesize a cell reactive compstatin analogue comprising the reactive functional group, etc. In some embodiments, a reactive functional group that reacts with its chemical targeting group without releasing an exit group is selected. In some embodiments, a reactive functional group that results in the release of an exit group in reaction with a target is selected. Compounds containing such groups can be useful, for example, to monitor the extent and / or progress of a reaction. In some embodiments, a leaving group is physiologically acceptable for cells, tissues, or organs in the amount generated (for example, based on the concentration and / or absolute amount generated) and / or is clinically acceptable for an individual in the amount generated in vivo (for example, based on the concentration in a relevant body fluid such as blood and / or based on the absolute amount generated). In some embodiments, an ex vivo generated leaving group is at least partly removed, for example, by washing the cells or by washing or perfusing a tissue or organ, for example, with saline.
[00113] In many embodiments, a cellular reactive functional group used in the invention reacts with a side chain of an amino acid residue and / or with an N-terminal amino group or C-terminal carboxyl group of a protein. In some modalities, the cell reactive functional group is reactive with sulfhydryl groups (-SH), which are found in the side chains of cysteine residues. In some embodiments, a maleimide group is used. Maleimide groups react with sulfhydryl groups of protein cysteine residues at physiological pH and form a stable thioether bond. In some embodiments, a haloacetyl group, such as an iodoacetyl group or a bromoacetyl group, is used. Haloacetyls react with sulfhydryl groups at physiological pH. The reaction of the iodoacetyl group proceeds by nucleophilic substitution of iodine with a sulfur atom of a sulfhydryl group resulting in a stable thioether bond. In other embodiments, a group of iodoacetamide is used. In some modalities, the cell reactive functional group reacts with amino groups (- NH2), which are present in the N-terminal of proteins and in the side chain of lysine residues (α-amino group). In some embodiments, an activated ester, for example, a succinimidyl ester (i.e., NHS ester) is used. For example, N-Hydroxysuccinimide (NHS) or its water-soluble analog (sulfo-NHS) can be used in the synthesis, whereby the resulting cellular reactive compstatin analog comprises an NHS ester. In some modalities, the cell reactive functional group reacts with carboxyl groups (-COOH), which are present in the C-terminal of proteins and in the side chains of various amino acid residues. In some embodiments, the cellular reactive compstatin analog is reactive with hydroxyl groups (-OH), which are present in the side chains of various amino acids and in carbohydrate portions of glycosylated proteins.
[00114] In general, the linking portion L may comprise any one or more aliphatic and / or aromatic moieties consistent with the formation of a stable compound joining the linked moieties. The term "stable", as used herein, preferably refers to compounds that have sufficient stability to permit manufacture and that maintain the integrity of the compound for a sufficient period of time, for example, to be useful for one or more of the purposes described in this document. In some embodiments, L comprises a saturated or unsaturated, substituted or unsubstituted, branched or unbranched aliphatic chain having a length between 1 and 30, between 1 and 20, between 1 and 10, between 1 and 6, or 5 or less carbon atoms, where length refers to the number of C atoms in the main (longest) chain. In some modalities, the aliphatic chain comprises one or more heteroatoms (O, N, S), which can be independently selected. In some embodiments, at least 50% of the atoms in the L main chain are carbon atoms. In some embodiments, L comprises a portion of saturated alkyl (CH2) n, where n is between 1 and 30.
[00115] In some modalities, L comprises one or more heteroatoms and has a length between 1 and 1000, between 1 and 800, between 1 and 600, between 1 and 400, between 1 and 300, between 1 and 200, between 1 and 100, between 1 and 50, between 1 and 30 or between 1 and 10 of total carbon atoms in a chain. In some embodiments, L comprises a portion of oligo (ethylene glycol) (- (O-CH2-CH2-) n), where n is between 1 and 500, between 1 and 400, between 1 and 300, between 1 and 200 , between 1 and 100, between 10 and 200, between 200 and 300, between 100 and 200, between 40 and 500, between 30 and 500, between 20 and 500, between 10 and 500, between 1 and 40, between 1 and 30 , between 1 and 20 or between 1 and 10.
[00116] In some embodiments, L comprises an unsaturated portion such as -CHCH- or -CH2-CHCH-; a portion comprising a non-aromatic cyclic ring system (for example, a cyclohexyl portion), an aromatic portion (for example, an aromatic cyclic ring system such as a phenyl portion); an ether portion (-C-O-C-); a portion of amide (-C (O) -N-); an ester portion (- CO-O-); a carbonyl portion (-C (O) -); an imine portion (-CN-); a thioether portion (-C-S-C-); an amino acid residue; and / or any portion that may be formed by the reaction of two compatible reactive functional groups. In certain embodiments, one or more portions of a cell binding or reactive portion is / are replaced by independent substitution of one or more hydrogen atoms (or others) with one or more portions including, but not limited to, aliphatic; aromatic, aryl; alkyl, aralkyl, alkanoyl, aroyl, alkoxy; uncle; F; C1; Br; I; -NO2; -CN; -CF3; -CH2CF3; -CHC12; - CH2OH; -CH2CH2OH; -CH2NH2; -CH2SO2CH3; - or -GRG1 where G is -O-, -S-, -NRG2-, -C (= O) -, -S (= O) -, -SO2-, -C (= O) O-, - C (= O) NRG2-, -OC (= O) -, -NRG2C (= O) -, -OC (= O) O-, -OC (= O) NRG2-, - NRG2C (= O) O- , -NRG2C (= O) NRG2-, -C (= S) -, -C (= S) S-, -SC (= S) -, - SC (= S) S-, -C (= NRG2) -, -C (= NRG2) O-, -C (= NRG2) NRG3-, - OC (= NRG2) -, -NRG2C (= NRG3) -, -NRG2SO2-, -NRG2SO2NRG3-, or -SO2NRG2-, in that each occurrence of RG1, RG2 and RG3 independently includes, but is not limited to, a portion of hydrogen, halogen, or an optionally substituted aliphatic, aromatic, or aryl. It will be appreciated that cyclic ring systems when present as substituents can optionally be linked via a linear portion. Combinations of substituents and variables provided for by this invention are preferably those that result in the formation of stable compounds useful in any one or more of the methods described in this document, for example, useful for the treatment of one or more disorders and / or for contacting with a cell, tissue, or organ, as described in this document, and / or useful as intermediates in the manufacture of one or more compounds.
[00117] L can comprise one or more of any of the portions described in the previous paragraph, in various modalities. In some embodiments, L comprises two or more different portions bonded together to form a structure normally having a length of between 1 to about 60 atoms, between 1 to about 50 atoms, for example, between 1 and 40, between 1 and 30, between 1 and 20, between 1 and 10 or between 1 and 6 atoms, where length refers to the number of atoms in the main (longest) chain. In some embodiments, L comprises two or more different portions, linked together to form a structure normally having between 1 to about 40, for example, between 1 and 30, for example, between 1 and 20, between 1 and 10 or between 1 and 6 carbon atoms in the main (longest) chain. In general, the structure of such a cell reactive compstatin analog can be represented by the formula A- (LPj) j-M, where j is normally between 1 and 10, and each LPj is independently selected from the portions described in the previous paragraph. In many embodiments, L comprises one or more carbon-containing chains such as- (CH2) n- and / or - (O-CH2-CH2-) n, which are covalently joined to each other and / or to a cellular reactive functional group or compstatin analog, for example, by portions (for example, portions of amide, ester, or ether) that result from the reaction of two compatible reactive functional groups. In some embodiments, L comprises a portion of oligo (ethylene glycol) and / or a saturated alkyl chain. In some modalities, L comprises - (CH2) mC (= O) -NH- (CH2CH2O) n (CH2) pC (= O) - or - (CH2) mC (= O) -NH- (CH2) p (OCH2CH2) nC (= O) -. In some modalities, m, n, and p are selected so that the number of carbons in the chain is between 1 and 500, for example, between 2 and 400, between 2 and 300, between 2 and 200, between 2 and 100, between 2 and 50, between 4 and 40, between 6 and 30, or between 8 and 20. In some modalities, m is between 2 and 10, n is between 1 and 500, and / or p is between 2 and 10. In some modalities modalities, m is between 2 and 10, n is between 1 and 400, and / or p is between 2 and 10. In some modalities, m is between 2 and 10, n is between 1 and 300, and / or p is between 2 and 10. In some modalities, m is between 2 and 10, n is between 1 and 200, and / or p is between 2 and 10. In some modalities, m is between 2 and 10, n is between 1 and 100, and / or p is between 2 and 10. In some modalities, m is between 2 and 10, n is between 1 and 50, and / or p is between 2 and 10. In some modalities, m is between 2 and 10, n is between 1 and 25, and / or p is between 2 and 10. In some modalities, m is between 2 and 10, n is between 1 and 8, and / or p is between 2 and 10. Optionally and, at least one -CH2- is replaced by CH-R, where R can be any substituent. Optionally, at least one -CH2- is replaced by a portion of heteroatom, cyclic ring system, amide, ester, or ether. In some embodiments, L does not comprise an alkyl group having more than 3 carbon atoms in the longest chain. In some embodiments, L does not comprise an alkyl group having more than 4, 5, 6, 7, 8, 9, 10, or 11 carbon atoms in the longest chain.
[00118] In some embodiments of the invention, A comprises a cell reactive functional group J and an L1 linker comprising an LP1 linker portion and a reactive functional group which reacts with the compstatin analog to generate A-M. In some embodiments, a bifunctional L2 ligand comprising two or more reactive functional groups and an LP2 ligand portion is used. The reactive functional groups of L react with appropriate reactive functional groups of A and M to produce an A-L-M cell reactive compstatin analogue. In some embodiments, the compstatin analog comprises an L3 linker comprising an LP3 linker portion. For example, as discussed below, a linker comprising a reactive functional group can be present at the N- or C-terminal or a portion comprising a reactive functional group can be linked to the N- or C-terminal via a linker. Thus, L may contain multiple binding portions LP contributed, for example, by A, the ligand (s) used to join A and M, and / or the compstatin analogue. It will be understood that, when present in the ALM structure, certain reactive functional group (s) present (s) present before the reaction in L1, L2, L3, etc., will have to be subjected to the reaction, so that only a portion of said functional group (s) is present in the final ALM structure, and the compound will contain portions formed by reaction of said (s) ) functional group (s). In general, if a compound contains two or more binding moieties, the binding moieties can be the same or different, and can be independently selected in several embodiments. Multiple LP binding moieties can be linked together to form a larger binding moiety L, and at least some of these linking moieties may have one or more analogous compstatin and / or reactive functional group (s) ( s) cell (s) connected to them. In molecules comprising multiple compstatin analogs, compstatin analogs can be the same or different and, if different, can be independently selected. The same applies to binding portions and reactive functional groups. The invention encompasses the use of multivalent compstatin analogs comprising one or more cell reactive functional group (s) and the use of compstatin analogue concatamers comprising one or more functional group (s) reactive cell (s). In some embodiments, at least one bond is a stable non-covalent bond such as an avidin biotin (strept) bond or other non-covalent bond of approximately equivalent strength.
[00119] In some embodiments, a cell reactive compstatin analog comprises a compstatin analog in which any of SEQ ID NOs: 3-36 is extended by one or more amino acids at the N-terminal, C-terminal, or both, wherein at least one of the amino acids has a side chain comprising a reactive functional group such as a primary or secondary amine, a sulfhydryl group, a carboxyl group (which may be present as a carboxylate group), a guanidino group, a phenol group, an indole ring, a thioether, or an imidazole ring. In some embodiments, the amino acid (s) is / are L-amino acids. In some embodiments, any one or more of the amino acid (s) is a D-amino acid. If multiple amino acids are added, the amino acids can be selected independently. In some embodiments, the reactive functional group (for example, a primary or secondary amine) is used as a target for the addition of a moiety comprising a cellular reactive functional group. Amino acids, having a side chain comprising a primary or secondary amine include lysine (Lys) and diaminocarboxylic acids of the general structure NH2 (CH2) nCH (NH2) COOH such as 2,3-diaminopropionic acid (dapa), 2,4- diaminobutyric (daba), and ornithine (orn), where n = 1 (dapa), 2 (daba), and 3 (orn), respectively. In some embodiments, at least one amino acid is cysteine, aspartic acid, glutamic acid, arginine, tyrosine, tryptophan, methionine, or histidine. Cysteine has a side chain comprising a sulfhydryl group. Aspartic acid and glutamic acid have a side chain comprising a carboxyl group (ionizable to a carboxylate group). Arginine has a side chain comprising a guanidino group. Tyrosine has a side chain comprising a phenol group (ionizable to a phenolate group). Tryptophan has a side chain comprising an indole ring that includes, for example, tryptophan. Methionine has a side chain comprising a thioether group that includes, for example, methionine. Histidine has a side chain comprising an imidazole ring. A wide variety of non-standard amino acids having side chains comprising one or more of these reactive functional groups is available, including naturally occurring amino acids and amino acids not found in nature. See, for example, Hughes, B. (ed.), Amino Acids, Peptides and Proteins in Organic Chemistry, Volumes 1-4, Wiley-VCH (2009-2011); Blaskovich, M., Handbook on Syntheses of Amino Acids General Routes to Amino Acids, Oxford University Press, 2010. The invention encompasses modalities in which one or more non-standard amino acid (s) and / are used to provide a target for addition of a portion comprising a cell reactive functional group. Any one or more of the amino acid (s) can be protected as appropriate during the synthesis of the compound. For example, one or more amino acid (s) can be protected during reaction (s) involving the targeting amino acid side chain. In some embodiments, where a sulfhydryl-containing amino acid is used as a target for the addition of a moiety comprising a reactive cell functional group, the sulfhydryl is protected while the compound is being cyclized by the formation of an intramolecular disulfide bond, among others. amino acids such as cisteins.
[00120] In the discussion of this paragraph, an amino acid having a side chain containing an amine group is used as an example. The invention encompasses analogous modalities in which an amino acid having a side chain containing a different reactive functional group is used. In some embodiments, an amino acid having a side chain comprising a primary or secondary amine is linked directly to the N-terminal or C-terminal of any of SEQ ID NOs: 3-36 via a peptide bond. In some embodiments, an amino acid having a side chain comprising a primary or secondary amine is attached to the N- or C-terminal of any of SEQ ID NOs: 3-36 via a linker portion, which can contain either one or more of the binding portions described above. In some embodiments, at least two amino acids are attached to either or both ends. The two or more attached amino acids can be joined to each other by the peptide bonds or at least some of the attached amino acids can be joined to each other by a linker portion, which can contain any of the one or more linker portions described in this document. Thus, in some embodiments, a cell reactive compstatin analog comprises a portion of compstatin M analog of formula B1-R1-M1-R2-B2, where M1 represents any of SEQ ID NOs: 3-36, any R1 or R2 may be absent, at least one of R1 and R2 comprises an amino acid having a side chain containing a primary or secondary amine, and B1 and B2 are optionally present blocking portions. R1 and / or R2 can be joined to M1 by a peptide bond or a non-peptide bond. R1 and / or R2 can comprise a linking portion LP3. For example, R1 can have the formula M2-LP3 and / or R2 can have the formula LP3-M2 where LP3 is a linker portion, and M2 comprises at least an amino acid having a side chain comprising a primary or secondary amine. For example, M2 can be Lys or an amino acid chain comprising Lys. In some embodiments, LP3 comprises or consists of one or more amino acids. For example, LP3 can be between 1 and about 20 amino acids in length, for example, between 4 and 20 amino acids in length. In some embodiments, LP3 comprises or consists of multiple residues of Gly, Ser, and / or Ala. In some embodiments, LP3 does not comprise an amino acid that comprises a reactive SH group, such as Cys. In some embodiments, LP3 comprises a portion of oligo (ethylene glycol) and / or a saturated alkyl chain. In some embodiments, LP3 is linked to the N-terminal amino acid of M1 via an amide bond. In some embodiments, LP3 is linked to the C-terminal amino acid of M1 via an amide bond. The compound can be further extended at either or both ends by the addition of additional linker (s) and / or amino acid (s). Amino acids can be the same or different and, if different, can be independently selected. In some embodiments, two or more amino acids having side chains comprising reactive functional groups are used, wherein the reactive functional groups can be the same or different. The two or more reactive functional groups can be used as targets for adding two or more portions. In some embodiments, two or more reactive cell moieties are added. In some modalities, a reactive cell portion and a targeting portion are added. In some embodiments, a ligand and / or cell reactive moiety is linked to an amino acid side chain after incorporating the amino acid into a peptide chain. In some embodiments, a ligand and / or cell reactive moiety is already linked to the amino acid side chain before the use of the amino acid in the synthesis of a cell reactive compstatin analog. For example, a Lys derivative having a linker attached to its side chain can be used. The linker can comprise a reactive cell functional group or can subsequently be modified to comprise a reactive cell functional group.
[00121] Certain cellular reactive compstatin analogs are described in more detail below. In the following discussion, a peptide having the amino acid sequence Ile-Cys * -Val- (1Me) Trp-Gln- Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr (SEQ ID NO: 37) (corresponding to the compstatin analog of SEQ ID NO: 28, where the asterisks in SEQ ID NO: 37 represent cysteines joined by a disulfide bond in the active compound, and (1Me) Trp represents 1-methyl-tryptophan)), is used as a portion of exemplary compstatin analog; maleimide (abbreviated Mal) is used as an example of a reactive cell functional group; (CH2) n and (O-CH2-CH2) n are used as examples of linking moieties; lysine is used as an example of an amino acid comprising a reactive functional group (in some compounds), and acetylation and amidation of the N- and C-terminals, respectively, are used as exemplary blocking moieties optionally present in some compounds and are represented in italic, that is, as Ac and NH2 respectively. It will be appreciated that the compounds can be prepared using a variety of synthetic approaches and using a variety of precursors. The discussion of the various synthetic approaches and precursors below is not intended to limit the invention. In general, any of the characteristics of any of the compounds described below can be freely combined with the characteristic (s) of other compounds described below or elsewhere in this document, and the invention encompasses such modalities.
[00122] In some embodiments, the cell reactive moiety is provided by a cell reactive compound comprising a macheimide group (such as a cell reactive functional group) and an alkanoic acid (RCOOH), where R is an alkyl group. For example, 6-maleimidocaproic acid (Mal- (CH2) 5-COOH), shown below, can be used.

[00123] In some embodiments, the reactive cell portion is provided by an alkanoic acid derivative in which the carboxylic acid portion has been activated, for example, the OH portion has been converted into a better leaving group. For example, the carboxyl group of compound I can be reacted with EDC, followed by reaction with NHS (which can optionally be supplied as water-soluble sulfo-NHS), resulting in a 6-maleimidocaproic acid N-hydroxysuccinimide ester derivative , that is, N-Hydroxysuccinimide (NHS) ester of 6-maleimidohexanoic acid (shown below).

[00124] The compound of SEQ ID NO: 37 can be modified at the N- and / or C-terminal to generate a cell reactive compstatin analog. For example, compound II can be used to generate the following cell reactive compstatin analogue by reaction with the N-terminal amino group of Ile.
[00125] Maleimide- (CH2) 5-C (= O) -Ile-Cys * -Val- (1Me) Trp-Gln-Asp- Trp-Gly-Ala-His-Arg-Cys * -Thr-NH2 (SEQ ID NO: 38). It will be appreciated that in SEQ ID NO: 38 the -C (= O) moiety is linked to the C-terminal amino acid (Ile) immediately, via a C-N bond, where N is part of the amino acid and is not shown.
[00126] In other embodiments, a maleimide group is attached to Thr at the C-terminal, resulting in the following cell reactive compstatin analog:
[00127] Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr- (C = O) - (CH2) 5-maleimide (SEQ ID NO: 39).
[00128] In some embodiments, a cell reactive compstatin analogue can be synthesized using a bifunctional ligand (for example, a heterobifunctional ligand). An exemplary heterobifunctional linker comprising portions of (CH2-CH2-O) n and (CH2) m (where m = 2) is shown below:

[00129] Compound III comprises a maleimide group as a cell reactive functional group and an NHS ester moiety that reacts readily with an amino group (e.g., an N-terminal amino group or an amino group on an amino acid side chain ).
[00130] An embodiment of compound III, where n = 2 can be used to generate the following cell reactive compstatin analog using the compstatin analog of SEQ ID NO: 37:
[00131] Maleimide- (CH2) 2-C (= O) -NH- CH2CH2OCH2CH2OCH2CH2C (= O) -Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp- Gly-Ala-His-Arg- Cys * -Thr-NH2 (SEQ ID NO: 40)
[00132] It will be appreciated that in the compound of SEQ ID NO: 40 a portion of -C (= O) is linked to the N-terminal amino acid (Ile residue through a CN bond, where N is part of the amino acid and not In some embodiments, a linker has the formula of Compound III where n> 1. Exemplary values for n in a portion of (CH2-CH2-O) n are provided in this document.
[00133] In some embodiments, the alkyl chain that connects the maleimide portion to the rest of the molecule contains more or less methylene units, the oligo (ethylene glycol) portion contains more or less ethylene glycol units, and / or there is more or less methylene units that flank one or both sides of the oligo (ethylene glycol) portion, compared to the compound of SEQ ID NO: 39 or SEQ ID NO: 40. Exemplary cell reactive compstatin analogs illustrative of some of these variations are shown below (SEQ ID NOs: 41-46):
[00134] Maleimide- (CH2) 2-C (= O) -NH-CH2CH2OCH2CH2C (= O) -Ile- Cys * -Val- (1Me) Trp -Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-NH2 (SEQ ID NO: 41)
[00135] Maleimide- (CH2) 3-C (= O) -NH-CH2CH2OCH2CH2OCH2C (= O) - Ile-Cys * -Val- (1Me) Trp -Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-NH2 (SEQ ID NO: 42)
[00136] Maleimide- (CH2) 5-C (= O) -NH-CH2CH2OCH2CH2OCH2C (= O) - Ile-Cys * -Val- (1Me) Trp -Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-NH2 (SEQ ID NO: 43)
[00137] Maleimide- (CH2) 4-C (= O) -NH- CH2CH2OCH2CH2OCH2CH2C (= O) -Ile-Cys * -Val- (1Me) Trp -Gln-Asp-Trp- Gly-Ala-His-Arg- Cys * -Thr-NH2 (SEQ ID NO: 44)
[00138] Maleimide- (CH2) 2-C (= O) -NH- CH2CH2OCH2CH2OCH2CH2C (= O) -Ile-Cys * -Val- (1Me) Trp -Gln-Asp-Trp- Gly-Ala-His-Arg- Cys * -Thr-NH2 (SEQ ID NO: 45)
[00139] Maleimide- (CH2) 5-C (= O) -NH-CH2CH2OCH2CH2OCH2C (= O) - Ile-Cys * -Val- (1Me) Trp -Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-NH2 (SEQ ID NO: 46)
[00140] In some embodiments, SEQ ID NO: 37 is extended to comprise a Lys residue at the N- or C-terminal of the peptide, for example, as exemplified below for a C-terminal bond:
[00141] Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-Lys-NH2 (SEQ ID NO: 47).
[00142] In some embodiments, a Lys residue is linked to the N- or C-terminal of SEQ ID NO: 37 via a peptide linker, for example, as exemplified below by a C-terminal link:
[00143] Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr- (Gly) 5-Lys-NH2 (SEQ ID NO: 48 ).
[00144] In some embodiments, a linker comprising a primary or secondary amine is added to the N- or C-terminal of a compstatin analog. In some embodiments, the linker comprises an alkyl chain and / or a portion of oligo (ethylene glycol). For example, NH2 (CH2CH2O) nCH2C (= O) OH (for example, 8-amino-3,6-dioxaoctanoic acid (AEEAc) or 11-amino-3,6,9-trioxaundecanoic acid) or an NHS ester of even (for example, an NHS ester of 8-amino-3,6-dioxaoctanoic acid or 11-amino-3,6,9-trioxaundecanoic acid), can be used. In some embodiments, the resulting compound is as follows (where the portion contributed by the linker is shown in bold):
[00145] NH2 (CH2) 5C (= O) -Ile-Cys-Val- (1Me) Trp-Gln-Asp-Trp-Gly- Ala-His-Arg-Cys-Thr-NH2 (SEQ ID NO: 49)
[00146] NH2 (CH2CH2O) 2CH2C (= O) -Ile-Cys-Val- (1Me) Trp-Gln-Asp- Trp-Gly-Ala-His-Arg-Cys-Thr-NH2 (SEQ ID NO: 50)
[00147] In some embodiments, a Lys residue is attached to the N- or C-terminal of SEQ ID NO: 37 via a linker comprising a non-peptide portion. For example, the linker may comprise an alkyl chain, oligo (ethylene glycol) chain, and / or cyclic ring system. In some embodiments, 8-AEEAc or an NHS ester thereof is used, resulting (in the case of Lys binding at the C-terminal) in the following compound (where the portion contributed by 8-AEEAc is shown in bold):
[00148] Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C (= O) -Lys-NH2 ( SEQ ID NO: 51)
[00149] It will be appreciated that in SEQ ID NOs: 49 and 50, a portion of -C (= O) is attached to the adjacent Ile residue via a CN bond, where N is part of the amino acid and is not shown . Similarly, in SEQ ID NO: 51, a portion of -C (= O) is attached to the adjacent Lys residue via a C-N bond, where N is part of the amino acid and is not shown. It will be appreciated that in SEQ ID NO: 51 the NH portion is linked to the N-terminal amino acid (Thr) immediately, via a C-N bond, where C is the carbonyl carbon of the amino acid and is not shown.
[00150] The compounds of SEQ ID NOs: 47-51 can be readily modified in the primary amine group to produce a cell reactive compstatin analogue. For example, compounds of SEQ ID NOs: 47-51 (or other compounds comprising a primary or secondary amine and a portion of compstatin analog) can be reacted with 6-maleimidocaproic acid N-succinimidyl ester to produce the following analogs of reactive cell compstatin:
[00151] Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-Lys- (C (= O) - (CH2) 5- Mal) -NH2 (SEQ ID NO: 52).
[00152] Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr- (Gly) 5-Lys - (C (= O) - (CH2) 5-Mal) -NH2 (SEQ ID NO: 53).
[00153] Mal- (CH2) 5- (C (= O) -NH (CH2) 5C (= O) -Ile-Cys-Val- (1Me) Trp- Gln-Asp-Trp-Gly-Ala-His- Arg-Cys-Thr-NH2 (SEQ ID NO: 54)
[00154] Mal- (CH2) 5- (C (= O) NH (CH2CH2O) 2CH2C (= O) -Ile-Cys-Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg -Cys-Thr-NH2 (SEQ ID NO: 55)
[00155] Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C (= O) -Lys- (C (= O) - (CH2) 5-Mal) - NH2 (SEQ ID NO: 56)
[00156] In another embodiment, a cell reactive compstatin analogue is represented as: Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp- Gly-Ala-His-Arg-Cys * -Thr- Lys-C (= O) -CH2 (OCH2CH2) 2NH (C (= O) - (CH2) 5-Mal) -NH2 (SEQ ID NO: 57).
[00157] The invention provides variants of SEQ ID NOs: 38-57 where -Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr- is replaced by an amino acid sequence comprising the amino acid sequence of any other compstatin analogue, for example, any of SEQ ID NOs 3-27 or 29-36, with the proviso that the blocking portions present in N - and / or C-terminal of a compstatin analogue may be absent, replaced by a linker (which may comprise a blocking portion), or linked to a different N- or C-terminal amino acid present in the variant (s) ( corresponding).
[00158] Other bifunctional crosslinkers, comprising a maltimide as a cell reactive moiety and an NHS ester as a cell reactive moiety for use in various embodiments of the invention, include, for example, 4- (p-maleimidophenyl) butyrate (SMPB ); Succinimidyl 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC); N-Y-maleimidobutyryl-oxisuccinimide ester (GMBS). The addition of a sulfonate to the NHS ring results in water-soluble analogues such as sulfo-succinimidyl (4-iodoacetyl) -aminobenzoate (sulfo-SIAB), sulfo 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate -succinimidyl (sulfo-SMCC), sulfo-succinimidyl 4- (p-maleimidophenyl) butyrate (sul-fo-SMPB), sulfo-N — Y — maleimidobutyryl-oxisuccinimide ester (sulfo-GMBS) etc., which can prevent the need for an organic solvent. In some embodiments, a long-chain version of any of the above, comprising a spacer arm between the NHS ester portion and the rest of the molecule, is used. The spacer may comprise, for example, an alkyl chain. An example is succinimidyl-4- [N-Maleimidomethyl] cyclohexane-1-carboxy- [6-amidocaproate].
[00159] In some embodiments, a bifunctional linker comprising an NHS ester (as a reactive amine moiety) and an iodoacetyl group (reactive with sulfhydryl groups) is used. Such binders include, for example, N-succinimidyl (4-iodoacetyl) -aminobenzoate (SIAB); 6 - succinimidyl [(iodoacetyl) -amino] hexanoate (SIAX); Succinimidyl 6- [6- (((iodoacetyl) amino) -hexanoyl) amino] hexanoate (SI-AXX); 4 - ((iodoacetyl) amino) methyl) -cyclohexane-1-succinimidyl (SIAC); 6 - succinimidyl hexanoate (SIACX) (((((4- (iodoacetyl) amino) methyl-cyclohexane-1-carbonyl) amino) hexanoate;
[00160] In some embodiments, a bifunctional linker comprising an NHS ester (as a reactive amine moiety) and a pyridyl disulfide group (as a reactive cellular reactive moiety with sulfhydryl groups) is used. Examples include N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP); succinimidyloxycarbonyl-α-methyl-α- (2-pyridyldithio) toluene (SMPT) and versions comprising a sulfonate in the NHS ring and / or a spacer comprising an alkyl chain between the NHS ester portion and the rest of the molecule (for example, succinimidyl 6- (3- [2-pyridyldithio] -propionamido) hexanoate) (LC-SPDP). Variations of such binders that include additional or different portions could be used. For example, a longer or shorter alkyl chain could be used in a spacer, or a portion of oligo (ethylene glycol) instead of an alkyl chain.
[00161] In general, a cell reactive compstatin analog can be synthesized using a variety of approaches. Cell reactive compounds comprising a cell reactive functional group and a linker can often be purchased as preformed building blocks. For example, 6-maleimidocaproic acid and N-hydroxysuccinimide ester of 6-malemeidocaproic acid can be purchased from various suppliers. Alternatively, such compounds can be synthesized using methods known in the art. See, for example, Keller O, Rudinger J. Helv Chim Acta. 58 (2): 531-41, 1975 and Hashida S, et al., J Appl Biochem., 6 (1-2): 56-63, 1984. See also, Hermanson, G. supra, and references therein, for discussion of methods and reagents for use in the synthesis of conjugates. In general, the invention encompasses any method of producing a compound comprising a component of compostatin analogue and a cell reactive functional group, and the resulting compounds.
[00162] In some embodiments, an amino acid having a linker attached to a side chain is used in the synthesis of a linear peptide. The linear peptide can be synthesized using standard methods for peptide synthesis known in the art, for example, standard solid phase peptide synthesis. The linear peptide is then cyclized (for example, by oxidation of the Cys residues to form an intramolecular disulfide). The cyclic compound can then be reacted with a linker comprising a reactive cell functional group. In other embodiments, a portion comprising a cell reactive functional group is reacted with a linear compound prior to cyclization thereof. In general, reactive functional groups can be adequately protected to prevent unwanted reactions from each other during the synthesis of a cellular reactive compstatin analog. The reactive cell functional group, any of the amino acid side chains, and / or any or both or both ends of the peptide can be protected during the reaction and subsequently unprotected. For example, SH groups of Cys residues and / or reactive portions of SH such as maleimides can be protected until after cyclization to prevent reaction between them. Reaction conditions are selected based, at least in part, on the requirements of the specific functional group (s) to achieve reasonable yield within a reasonable period of time. The temperature, pH, and concentration of the reagents can be adjusted to achieve the desired point or rate of reaction. See, for example, Hermanson, supra. The desired product can be purified, for example, to remove the unreacted compound comprising the cell reactive functional group, unreacted compstatin analogue, linker (s), products other than the desired cell reactive compstatin analogue that may have been generated in the reaction, other substances present in the reaction mixture, etc. Compositions and methods for making cell reactive compstatin analogs, and intermediates in synthesis, are aspects of the invention.
[00163] In some aspects of the invention, the binder (s) described above is / are used in the production of compstatin analogs comprising a portion such as polyethylene glycol (PEG) chain or other ( s) polymer (s) that, for example, stabilize the compound, increase its life in the body, increase its solubility, decrease its immunogenicity, and / or increase its resistance to degradation. Without limiting the invention in any way, such a portion may be referred to herein as a "release reducing portion" (CRM), and a compstatin analog comprising such portion may be referred to as a "long-acting compstatin analog". In some embodiments, a long-acting compstatin analogue has an average plasma half-life of at least 1 day, for example, 1 to 3 days, 3 to 7, 7 to 14 days, or 14 to 28 days, when administered IV at a dose of 10 mg / kg to humans or non-human primates. In some embodiments, the mean plasma half-life of a long-acting compstatin analogue after IV administration at a dose of 10 mg / kg to humans or non-human primates is increased by at least a factor of 2, for example example, by a factor of 2 to 5, 5 to 10, 10 to 50 or 50 to 100 times compared to a corresponding compstatin analog having the same amino acid sequence (and, if applicable, one or more portions block (s), but not understanding CRM. As noted above, in some embodiments, a compstatin analog of any SEQ ID NOs: 3-36 is extended by one or more amino acids at the N-terminus, C-terminus, or both, where at least one of the amino acids has a side chain comprising a reactive functional group such as a primary or secondary amine, a sulfhydryl group, a carboxyl group (which may be present as a carboxylate group), a guanidino group, a phenol group, a ring indole, a thioether, or an imidazole ring, which facilitate conjugation with a reactive functional group to link a CRM to the compstatin analogue. It will be understood that a corresponding compstatin analog not comprising CRM may also lack one or more of those amino acids that are present in the long-acting compstatin analog to which it corresponds. Thus, a corresponding compstatin analogue comprising any of SEQ ID NOs: 3-36 and not having a CRM will be understood as "having the same amino acid sequence" as SEQ ID NOs: 3-36, respectively. For example, a corresponding compstatin analogue comprising the amino acid sequence of SEQ ID NO: 14, 21, 28, 29, 32, 33, 34 or 36 and not having a CRM will be understood as "having the same amino acid sequence "as SEQ ID NO: 14, 21, 28, 29, 32, 33, 34 or 36, respectively. In some embodiments, a plasma half-life is a terminal half-life after the administration of a single IV dose. In some embodiments, a plasma half-life is a terminal half-life after the stationary state has been achieved after multiple IV doses. In some embodiments, a long-acting compstatin analogue achieves a Cmax in plasma at least 5 times greater than that of a corresponding compstatin analogue not comprising CABG, for example, between 5- and 50 times greater, after administration of a single IV dose to a primate, or after multiple IV doses. In some embodiments, a long-acting compstatin analogue achieves a Cmax in the plasma between 10 and 20 times greater than that of a cor-respondent compstatin analogue not comprising CABG after the administration of a single IV dose to a primate, or after administration of multiple doses IV. In some modalities a primate is a human being. In some embodiments, a primate is a non-human primate, for example, a monkey, such as a Cynomolgus monkey or a Rhesus monkey. In some embodiments, the renal release of a long-acting compstatin analog during the first 24 hours after IV administration at a dose of 10 mg / kg to humans or non-human primates is reduced by at least a factor of 2 , for example, by a factor of 2 to 5, 5 to 10, 10 to 50 or 50 to 100 times compared to the renal release of a corresponding compostatin analog. The concentration of the compstatin analogue can be measured in blood and / or urine samples using, for example, UV, HPLC, mass spectrometry (MS) or antibody to the CRM, or combinations of such methods, such as LCMS or LCMSMS. Pharmacokinetic parameters, such as half-life and release, can be determined using methods known to those skilled in the art. Pharmacokinetic analysis can be performed, for example, with WinNonlin software v 5.2 (Pharsight Corporation, St. Louis, MO).
[00164] In some embodiments, a long-acting compstatin analogue has a molar activity of at least 10%, 20%, 30%, for example, between 30% and 40%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, or more, of the activity of a corresponding compstatin analog having the amino acid sequence (and, if applicable, one or more blocking portions, but not comprising a CRM). In some embodiments, where a long-acting compstatin analogue comprises multiple portions of compstatin analogue, the molar activity of the long-acting compstatin analogue is at least about 10%, 20%, or 30%, for example, between 30% and 40%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, or more, of the sum of the referred activities portions of compstatin analog. In some embodiments, a polyethylene glycol (PEG) comprises a portion of (CH2CH2O) n having a molecular weight of at least 500 daltons. In some embodiments, a linker described above comprises a portion of (CH2CH2O) having an average molecular weight of between about 500; 1,000; 1,500; 2,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; and 100,000 daltons. “Average molecular weight" refers to the number of average molecular weight. In some embodiments, the polydispersity D of a portion of (CH2CH2O) n is between 1,0005 and 1.50, for example, between 1.005 and 1.10 , 1.15, 1.20, 1.25, 1.30, 1.40, or 1.50, or any value between 1,0005 and 1.50.
[00165] In some embodiments, a portion of (CH2CH2O) n is monodispersed and the polydispersity of a portion of (CH2CH2O) n is 1.0. Such mono-dispersed (CH2CH2O) n portions are known in the art and are commercially available from Quanta BioDesign (Powell, OH), and include, by way of non-limiting example, mono-dispersed portions where n is 2, 4, 6, 8, 12, 16, 20 or 24.
[00166] In some embodiments, a compound comprises multiple portions of (CH2CH2O) n in which the total molecular weight of said portions of (CH2CH2O) n is between about 1,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In some embodiments, the compound comprises multiple portions of (CH2CH2O) n having defined lengths, for example, n = 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 or more. In some embodiments, the compound comprises a sufficient number of (CH2CH2O) n portions having defined lengths to result in a molecular weight of said (CH2CH2O) n portions of between about 1,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In some embodiments n is between about 30 and about 3000. In some embodiments a portion of compstatin analog is attached to each end of a linear PEG. A bifunctional PEG having a reactive functional group at each end of the chain can be used, for example, as described above. In some modalities reactive functional groups are identical while in some modalities different reactive functional groups are present at each end. In some embodiments, multiple portions of (CH2CH2O) n are provided as a branched structure. The branches can be attached to a main structure of the linear polymer (for example, as a comb-like structure) or they can emanate from one or more central core groups, for example, as a star-like structure. In some embodiments, a branched molecule has 3 to 10 (CH2CH2O) n chains. In some embodiments, a branched molecule has 4 to 8 (CH2CH2O) n chains. In some embodiments, a branched molecule has 10, 9, 8, 7, 6, 5, 4, or 3 (CH2CH2O) n chains. In some embodiments, a star-shaped molecule has 10 to 100, 10 to 50, 10 to 30, or 10 to 20 (CH2CH2O) n chains that emanate from a central nucleus group. In some embodiments, a long-acting compstatin analogue can thus comprise, for example, 3 to 10 portions of compstatin analogue, for example, 4 to 8 portions of compstatin analogue, each linked to a (CH2CH2O) chain ) n via a functional group at the end of the chain. In some embodiments, a long-acting compstatin analogue may comprise, for example, 10 to 100 portions of compstatin analogue, each linked to a (CH2CH2O) n chain through a functional group at the end of the chain. In some embodiments, the branches (sometimes referred to as "arms") of a branched or star-shaped PEG contain about the same number of portions of (CH2CH2O). In some embodiments, at least some of the branching components may be different. It will be understood that in some embodiments one or more (CH2CH2O) n chains do not have a compstatin analog portion attached to them. In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the chains have a portion of compstatin analog attached to them.
[00167] In genera and compounds represented here, a portion of polyethylene glycol is removed with the oxygen atom on the right side of the repeat unit or on the left side of the repeat unit. In cases where only one orientation is withdrawn, the present invention encompasses both orientations (i.e., (CH22CHO) ne (OCH2CH2) n) of portions of polyethylene glycol for a given compound or genus, or in cases where a compound or genus contains multiple portions of polyethylene glycol, all combinations of guidelines are encompassed by the present disclosure.
[00168] Formulas for some exemplary monofunctional PEGs comprising a reactive functional group are illustrated below. For illustrative purposes, formulas in which the reactive functional group (s) comprise an NHS ester are represented, but other reactive functional groups could be used, for example, as described above. In some embodiments, (CH22CHO) n is represented as a left-ended termination with a methoxy group (OCH3), but it will be understood that the chains represented below and elsewhere in this document may end with a different OR portion (for example, an aliphatic group, an alkyl group, a lower alkyl group or any other suitable PEG terminal group) or an OH group. In addition, it will be appreciated that portions other than those described can connect the portions of (CH2CH2O) n with the NHS group in various modalities.
[00169] In some modalities, a monofunctional PEG is of formula A:

[00170] where the "reactive functional group" and n are as defined above and described in classes and subclasses in this document;
[00171] R1is hydrogen group, aliphatic, or any suitable terminal group; and
[00172] T is a covalent bond or a straight or branched C1-12 hydrocarbon chain in which one or more carbon units of T are optionally and independently replaced by -O-, -S-, - N (Rx ) -, -C (O) -, -C (O) O-, -OC (O) -, -N (Rx) C (O) -, -C (O) N (Rx), - S (O ) -, -S (O) 2-, -N (Rx) SO2-, or -SO2N (Rx) -; and
[00173] each Rx is independently hydrogen or C1-6 aliphatic
[00174] Exemplary monofunctional PEGs of formula A include:

[00175] In Formula I, a portion comprising the reactive functional group has the general structure -CO- (CH2) m-COO-NHS, where m = 2. In some embodiments, monofunctional PEGs have the structure of Formula I, where m is between 1 and 10, for example, between 1 and 5. For example, in some modalities m is 3, as shown below:


[00176] In Formula II, a portion comprising the functional-recreational group has the general structure - (CH2) m-COO-NHS, where m = 1. In some embodiments, a monofunctional PEG has the formula II structure, where m is between 1 and 10 (for example, where m is 5, as shown in Formula III below), or where m is 0 (as shown below in Formula IIIa).

[00177] In some embodiments a linear bifunctional PEG comprises a portion comprising a reactive functional group at each of its ends. The reactive functional groups can be the same (homobifunctional) or different (heterobifunctional). In some embodiments, the structure of a bifunctional PEG can be symmetrical, in which the same portion is used to connect the reactive functional group to oxygen atoms at each end of the - (CH2CH2O) n chain. In some embodiments, different portions are used to connect the two reactive functional groups to the PEG portion of the molecule. The structures of the exemplary bifunctional PEGs are shown below. For illustrative purposes, formulas in which the reactive functional group (s) comprise an NHS ester are represented, but other reactive functional groups could be used.
[00178] In some modalities, a bifunctional linear PEG is of formula B:

[00179] where each T and "reactive functional group" are independently, as defined above and, described in classes and subclasses in this document, and n is as defined above and described in classes and subclasses in this document.
[00180] Exemplary bifunctional PEGs of formula B include:

[00181] In Formula IV, the portion comprising the reactive functional group has the general structure - (CH2) m-COO-NHS, where m = 1. In some modalities, bifunctional PEGs have the structure of Formula IV, where m is between 1 and 10, for example, between 1 and 5.

[00182] In Formula V, the portion comprising the reactive functional group has the general structure -CO- (CH2) m-COO-NHS, where m = 2. In some modalities, bifunctional PEGs have the structure of Formula V, where m is between 1 and 10, for example, between 1 and 5.
[00183] In certain embodiments, the present invention provides compstatin analog conjugates of compounds and genera containing PEG represented in this document. In some embodiments, a functional group (for example, an amine, hydroxyl or thiol group) in a compstatin analog is reacted with a compound containing PEG having a "reactive functional group", as described herein, to generate such conjugates . For example, Formulas III and IV, respectively, can form compstatin analog conjugates having the structure:

[00184] where,
represents the point of attachment of an amine group to a compstatin analog. In certain embodiments, an amine group is a lysine side chain group. It will be appreciated that corresponding conjugates can be formed with any of the compounds and genera containing PEG described in this document, depending on the choice of the reactive functional group and / or the functional group of compstatin.
[00185] In some embodiments, a combed, comb-type, or star-shaped PEG comprises a portion comprising a reactive functional group at the end of each of the multiple (- CH-2CH2O) n chains. The reactive functional groups can be the same or they can be at least two different groups. In some embodiments, a branched, comb-type, or star-shaped PEG is of the following formulas:

[00186] Where each R2 is independently a "reactive functional group" or R1, and each T, n, and "reactive functional group" is independently as defined above and described in classes and subclasses in this document. The structure of exemplary branched PEGs (having 8 arms, or branches) comprising portions of NHS as reactive functional groups is described below:

[00187] The structure of exemplary branched PEGs (having 4 arms, or branches) comprising portions of NHS as reactive functional groups is described below:


[00188] The number of branches emanating from the main structure can be varied. For example, the number 4 in formulas VI and VII above can be changed to any other integer between 0 and 10 in various ways. In certain embodiments, one or more branches do not contain a reactive functional group and the branch ends with a -CH2CH2OH or -CH2CH2OR group, as described above.
[00189] In some embodiments a branched PEG has the structure of Formula VII, VIII, or IX (or variants thereof having different numbers of branches), with the proviso that x is

[00190] In some embodiments a branched PEG has the structure of Formula VII, VIII, or IX (or variants thereof having different numbers of branches) with the proviso that x is

[00191] Evidently the methylene group (CH2) in the x-portion above may, instead, comprise a longer alkyl chain (CH2) m, where m is up to 2, 3, 4, 5, 6, 8, 10, 20 or 30, or may comprise one or more other portions described herein.
[00192] In some embodiments, exemplary branched PEGs having NHS or maleimide reactive groups are described below:

[00193] In some embodiments, a variant of Formula X or XI is used, in which 3 or each of the four branches comprise a reactive functional group.
[00194] Still other examples of PEGs can be represented as follows:

[00195] As noted above, it will be appreciated that, as described in this document, in various embodiments, any of a variety of portions can be incorporated between the peptide component and the (CH2CH2O) nR portion of a compstatin analog. long-acting, such as linear alkyl, ester, amide, aromatic ring (e.g., substituted or unsubstituted phenyl), substituted or unsubstituted cycloalkyl structure, or combinations thereof. In some embodiments, such portions can make the compound more susceptible to hydrolysis, which can release the peptide portion of the CRM compound. In some embodiments, such release may increase tissue penetration in vivo and / or compound activity. In some embodiments, hydrolysis is general hydrolysis (for example, acid-base). In some embodiments, hydrolysis is catalyzed by enzyme, for example, catalyzed by esterase. Naturally, both types of hydrolysis can occur. Examples of PEGs comprising one or more of these moieties and an NHS ester as a reactive functional group are as follows:

[00196] In some embodiments a branched PEG (multiple arms) or star-shaped PEG comprises a pentaerythritol nucleus, hexaglycerin nucleus, or tripentaerythritol nucleus. It will be understood that not all branches can emanate from a single point in certain modalities.
[00197] Monofunctional, bifunctional, branched, and others PEGs comprising one or more reactive functional groups can, in some embodiments, be obtained from, for example, NOF America Corp. White Plains, NY or BOC Sciences 45-16 Ramsey Road Shirley, NY 11967, USA, among others, or can be prepared using methods known in the art.
[00198] In some embodiments of the invention, a compstatin analogue comprises a reactive cell functional group and a CRM. In some aspects, the invention provides variants of the molecules of any of the aforementioned cell reactive compstatin analogs in which a cell reactive moiety or group is replaced by a moiety of (CH2CH2O) n having a molecular weight of at least 500 daltons, for example. example, at least 1,500 daltons to about 100,000 daltons (for example, an average molecular weight of about 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; or 100,000 daltons).
[00199] Exemplary long-acting compstatin analogs are set out below, where n is sufficient to provide an average molecular weight of about 500; 1,000; 1,500; 2,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; and 100,000 daltons. (CH2CH2O) nC (= O) -Ile-Cys-Val- (1Me) Trp-Gln-Asp-Trp-Gly- Ala-His-Arg-Cys-Thr-NH2) (SEQ ID NO: 58) Ac-Ile -Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C (= O) -Lys-C (= O) - (CH2CH2O ) n- NH2 (SEQ ID NO: 59) Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-Lys-C (= O ) - (CH2CH2O) n -NH2 (SEQ ID NO: 60). Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr- (Gly) 5-Lys-C (= O) - (CH2CH2O) n -NH2 (SEQ ID NO: 61) Ac- (CH2CH2O) nC (= O) Lys- (Gly) 5-Ile- Cys * -Val- (1Me) Trp- Gln-Asp-Trp-Gly-Ala-His- Arg-Cys * -Thr - NH2) (SEQ ID NO: 62) Ac- (CH2CH2O) nC (= O) Lys-Ile- Cys * -Val- (1Me) Trp-Gln-Asp- Trp-Gly-Ala- His-Arg-Cys * -Thr - NH2) (SEQ ID NO: 63)
[00200] In SEQ ID NO: 58, (CH2CH2O) n is coupled via an amide bond to the N-terminal amino acid. In SEQ ID NOs: 59-63, the (CH2CH2O) portion n is coupled via an amide bond to a Lys side chain; thus, it will be understood that the NH2 at the C-terminal in SEQ ID NOs: 59, 60, and 61, represents amidation of the C-terminal of the peptide, and it will be understood that in SEQ ID NOs: 62 and 63, the Ab in the N- terminal represents acetylation of the N-terminal of the peptide, as described above. It will be appreciated by those skilled in the art that a free end of a (CH2CH2O) n portion normally ends with an (OR) where the underlined O represents the O atom in the terminal group (CH2CH2O). (OR) is often a moiety such as a hydroxyl (OH) or methoxy (-OCH3) group, however, other groups (for example, other alkoxy groups) could be used. Thus, SEQ ID NO: 59, for example, can be represented as Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His- Arg-Cys * -Thr-NH -CH2CH2OCH2CH2OCH2-C (= O) -Lys- (C (= O) - (CH2CH2O) nR) -NH2 (SEQ ID NO: 64) where R is, for example, either H or CH3 in the case of a PEG linear. In the case of a bifunctional, branched or star-shaped PEG, R represents the remainder of the molecule. In addition, it will be understood that the portion comprising the reactive functional group may vary, as described in this document (for example, according to any of the formulas described in this document). For example, long-acting compstatin analogs comprising the same peptide sequence as SEQ ID NO: 64, wherein the portion comprising the reactive functional group comprises an ester and / or alkyl chain can be represented as follows Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C (= O) -Lys- (C (= O) - (CH2) m - (CH2CH2O) nR) -NH2 (SEQ ID NO: 65); Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C (= O) -Lys- (C (= O ) - (CH2) m- C (= O) - (CH2CH2O) nR) -NH2 (SEQ ID NO: 66) Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala -His-Arg- Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C (= O) -Lys- (C (= O) - (CH2) m- C (= O) - (CH2) j (CH2CH2O) nR) - NH2 (SEQ ID NO: 67)
[00201] In SEQ ID NOs: 65-67 m can vary from 1 to about 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or 30 in various modalities. SEQ ID NO: 67 may vary from 1 to about 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or 30 in various embodiments.
[00202] Furthermore, it will be appreciated that, as described in this document, in various embodiments other portions can be incorporated between Lys- (C (= O) - and (CH2CH2O) nR, such as an amide, aromatic ring (for example, a substituted or unsubstituted phenyl), or a substituted or unsubstituted cycloalkyl structure.
[00203] The invention provides variants of SEQ ID NOs: 58-67, where -Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr- is replaced by an amino acid sequence comprising the amino acid sequence of any other compstatin analogue, for example, any of SEQ ID NOs 3-27 or 29-36, with the proviso that the blocking portions present in the N- and / or C-terminal of a compstatin analog may be absent, replaced by a linker (which may comprise a blocking portion), or linked to a different N- or C-terminal amino acid present in (s) ) corresponding variant (s).
[00204] Any compstatin analogue, for example, any compound comprising any of SEQ ID NOs: 3-37 can, in various embodiments, be linked through or near its N-terminal or C-terminal end (for example , through a side chain of an amino acid at or near its N-terminal or C-terminal amino acid) directly or indirectly, to any portion comprising a reactive functional group, for example, any compound of Formulas I-XVI or Formulas AH .
[00205] In some embodiments, the CRM comprises a polypeptide that occurs in human serum, or a fragment thereof or a substantially similar variant of the polypeptide or fragment thereof. In some embodiments, the polypeptide, fragment, or variant has a molecular weight of between 5 kD and 150 kD, for example, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 kD, or more, for example, between 100 and 120, or 120 and 150 kD. In some embodiments, producing a long-acting compstatin analog comprises reacting a compstatin analog comprising a reactive functional group with one or more amino acid side chains of the polypeptide, wherein the side chain comprises a compatible functional group. In some embodiments, producing a long-acting compstatin analogue comprises reacting a compstatin analogue comprising a functional group reactive with the N-terminal amine and / or the C-terminal carboxyl group of the polypeptide. In some embodiments, producing a long-acting compstatin analogue comprises reacting a compstatin analogue comprising an amine reactive functional group with amino acids having a side chain comprising a primary amine (e.g., lysine) and / or with the N- terminal of the polypeptide. In some embodiments, producing a long-acting compstatin analogue comprises reacting a compstatin analogue comprising a reactive carboxyl group with the C-terminal carboxyl group of the polypeptide. In some embodiments, a portion of compstatin analog is attached at each end of the polypeptide and, optionally, to the side chain of one or more internal amino acids. In some embodiments, producing a long-acting compstatin analog comprises reacting a compstatin analog comprising a reactive sulfhydryl group with one or more sulfhydryl groups of the polypeptide.
[00206] In some embodiments, at least one reactive functional group is introduced into the polypeptide. For example, in some embodiments at least one side chain of the polypeptide is modified to convert a first reactive functional group to a different reactive functional group, prior to reaction with the compstatin analog. In some embodiments, a thiol is introduced. Several methods are available for introducing thiols into biomolecules, including the reduction of intrinsic disulfides, as well as the conversion of amine, aldehyde or carboxylic acid groups to thiol groups. Cross-linking of cysteine disulfide in proteins can be reduced to cysteine residues by dithiothreitol (DTT), tris- (2-carboxyethyl) phosphine (TCEP), or -tris- (2-cyanoethyl) phosphine. The amines can be indirectly thiolated by the reaction with 3- (2-pyridyldithium) succinimidyl (SPDP) followed by reduction of the 3- (2-pyridyldithium) propionyl conjugate with DTT or TCEP. The amines can be indirectly thiolated by the reaction with succinimidyl acetylthioacetate followed by removal of the acetyl group with 50 mM hydroxylamine or hydrazine at a pH close to neutral. The amines can be directly thiolated by reaction with 2-iminothiolane, which preserves the total charge of the molecule and introduces a free thiol. Tryptophan residues in proteins without thiol can be oxidized to mercaptotryptophan residues, which can be modified by iodoacetamides or maleimides. A polypeptide comprising one or more thiols can be reacted with a compostatin analog comprising a maleimide group, such as Ac-Ile-Cys * -Val-Trp (1-Me) -Gln-Asp-Trp-Gly-Ala- His-Arg-Cys * -Thr-AEEAc-Lys- (C (= O) - (CH2) 5-Mal) -NH2 (SEQ ID NO: 68) to generate a long-acting compostatin analogue.
[00207] In some embodiments, the polypeptide is produced recombinantly. In some embodiments, the polypeptide is at least partly produced recombinantly (for example, in bacteria or in eukaryotic host cells such as fungi, insects, plants, or vertebrate animals) and / or at least in part, produced using chemical synthesis. In some embodiments, the polypeptide is puf. In some embodiments, the polypeptide is glycosylated. In some embodiments, the polypeptide is non-glycosylated. In some embodiments, the polypeptide is human serum albumin (HSA). In some embodiments, a substantially similar variant of the polypeptide is sufficiently similar to the polypeptide that it is a variant so as not to be recognized as foreign by an individual's normal immune system, for example, a human individual. In some embodiments changes in the sequence of the substantially similar variant when compared to the polypeptide of which it is a variant are selected in order to avoid MHC Class I epitopes. Various methods known in the art can be used to predict whether a sequence comprises an epitope MHC Class I.
[00208] The invention additionally provides multimers, for example, concatamers, comprising two or more (for example, between 2 and 10) compstatin analogs comprising a CRM, wherein the average molecular weight of the resulting molecule (or components of the CRM) is between 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In some embodiments, compstatin analogs comprising a CRM can be linked using any of the linker portions described above. VII. Targeted Compstatin Analogs
[00209] The invention provides targeted compstatin analogs that comprise a targeting portion and a compstatin analogue portion, wherein the targeting portion non-covalently binds to a target molecule. In some aspects, the invention provides targeted compstatin analogs analogous to the cell reactive compstatin analogs described in Section VI, wherein the compounds comprise a targeting portion in addition to or instead of a cellular reactive portion. The targeting portion may comprise, for example, an antibody, polypeptide, peptide, nucleic acid (e.g., an aptamer), carbohydrate, small molecule, or supramolecular complex, which specifically binds to the target molecule. In some embodiments, the affinity (as measured by the equilibrium dissociation constant, Kd) of the targeting portion for the target molecule (as measured by the equilibrium dissociation constant, Kd) is 10-3M or less, for example, 10- 4M or less, for example, 10-5M or less, for example, 10-6M or less, 10-7M or less, 10-8M or less, or 10-9M or less under the conditions tested, for example, under conditions physiological.
[00210] In the embodiments of the invention in which the targeting moiety is an antibody, the antibody can be any immunoglobulin or a derivative thereof, which maintains the binding capacity, or any protein having a binding domain that is homologous or homologous in large part to an immunoglobulin binding domain. These proteins can be derived from natural sources, or partially or totally synthetically produced (for example, using recombinant DNA techniques, chemical synthesis, etc.). The antibody can be of any species, for example, human, rodent, rabbit, goat, chicken, etc. The antibody can be a member of any class of immunoglobulin, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In various embodiments of the invention, the antibody can be a fragment of an antibody such as Fab ', F (ab'). Sub.2, scFv (single chain variable) or another fragment that maintains an antigen or binding site a recombinantly produced scFv fragment, including recombinantly produced fragment. See, for example, Allen, T., Nature Reviews Cancer, Vol. 2, 750-765, 2002, and references therein. Monovalent, bivalent or multivalent antibodies can be used. The antibody can be a chimeric antibody in which, for example, a variable domain of rodent origin is fused to a constant domain of human origin, thereby maintaining the specificity of the rodent antibody. In some embodiments, a human antibody or part of it is generated, for example, in rodents whose genome incorporates human immunoglobulin genes, using a display technology such as phage display, etc. In some embodiments, a humanized antibody is generated by grafting one or more regions to determine the complementarity of a non-human species (eg mouse) in a human antibody sequence. The antibody can be partially or completely humanized. See, for example, Almagro JC, Fransson J. Humanization of antibodies. Front Biosci. 13: 1619-33 (2008) for reviewing different methods of obtaining humanized antibodies that can be used to obtain a portion of targeting use in the invention. An antibody can be polyclonal or monoclonal, although for the purposes of the present invention monoclonal antibodies are generally preferred. In certain embodiments of the invention an F (ab ') 2 or F (ab') fragment is used while in other embodiments antibodies comprising an Fc domain are used. Methods for producing antibodies that specifically bind to virtually any molecule of interest are known in the art. For example, monoclonal or polyclonal antibodies can be purified from natural sources, for example, from blood or ascites fluid from an animal that produces the antibody (for example, after immunization with the molecule or an antigenic fragment from it) or can be produced recombinantly, in cell culture. Methods of generating antibody fragments, for example, by digestion, disulfide reduction, or synthesis are known in the art.
[00211] In various embodiments of the invention a targeting moiety can be any molecule that specifically binds to a target molecule through a mechanism other than an antigen-antibody interaction. This targeting portion is referred to as a "binder". For example, in various embodiments of the invention a linker can be a polypeptide, peptides, nucleic acid (for example, DNA or RNA), carbohydrate, lipid or phospholipid or small molecule. In some embodiments, a small molecule is an organic compound, whether naturally or artificially created, that has relatively low molecular weight and is not a protein, polypeptide, nucleic acid, or lipid, typically with a molecular weight of less than about 1500 g / mol and usually having multiple carbon-carbon bonds. In general, an aptamer is an oligonucleotide (for example, RNA or DNA, optionally comprising one or more modified nucleosides (for example, bases or sugars other than standard 5 bases (A, G, C, T, U) or sugars ( ribose and deoxyribose), more commonly found in RNA and DNA), or modified internucleoside bonds (for example, non-phosphodiester bonds) that, for example, stabilize the molecule, for example, by making it more resistant nuclease degradation) that binds to a specific protein. In some embodiments, an oligonucleotide is up to about 100 nucleosides in length, for example, between 12 and 100 nucleosides in length. Aptamers can be derived using an in vitro evolution process called SE-LEX, and methods for obtaining specific aptamers for a protein of interest are known in the art. See, for example, Brody E N, Gold L. J Biotechnol. 2000 March; 74 (1): 5-13. In some embodiments, a blocked peptide nucleic acid or nucleic acid is used.
[00212] In certain embodiments of the invention a targeting portion comprises a peptide. In some embodiments, a peptide that binds to a target molecule of interest is identified using display technology such as phage display, ribosome display, yeast display, etc.
[00213] Small molecules can be used as binders. Methods for identifying such ligands are known in the art. For example, in vitro screening of small molecule libraries, including combinatorial libraries, computer-based screening and, for example, identifying small organic compounds that bind to concave surfaces (packages) of proteins, can identify small molecule binders for numerous proteins of interest (Huang, Z., Pharm. Ther. 86: 201-215, 2000).
[00214] In certain embodiments of the invention, targeting portions are not proteins or molecules that are normally used as carriers and conjugated to antigens for the purpose of creating antibodies. Examples are proteins or carrier molecules such as bovine serum albumin, caliphaphine keyhole limpet hemocyanin, and diphtheria toxin. In certain embodiments of the invention the targeting portion is not an Fc portion of an immunoglobulin molecule. In some embodiments, a targeting portion is part of a complex comprising one or more additional portions to which it is covalently or non-covalently attached.
[00215] In various embodiments of the invention, a target molecule can be any molecule produced by a cell (including any forms expressed on the cell surface or modified forms thereof resulting at least in part from extracellular modification). In some embodiments, a target molecule is an extracellular substance present within or on a tissue. In some embodiments, a target molecule is characteristic of a particular sick or physiological state or characteristic of one or more type (s) of cell or type (s) of tissue. A target molecule is often a molecule, at least partially present on the cell surface (for example, a transmembrane or a protein attached to the membrane in another way), so that at least part of the molecule is accessible for binding by an agent extracellular binding as an antibody. A target molecule can, but need not be, a specific type of cell. For example, a target molecule of a specific cell type is often a protein, peptide, mRNA, lipid or carbohydrate that is present at a higher level in or in a specific cell type or cell types than in many other types of cells. In some cases, a target molecule of a specific type of cell is present at detectable levels only on or within a specific type of cell of interest. However, it will be possible to note that a target molecule of a specific type of useful cell does not have to be absolutely specific for the type of cell of interest to be considered the specific type of cell. In some embodiments, a target molecule of a specific cell type for a specific cell type is expressed at levels of at least 3 folds greater in that cell type than in a reference population of cells that may consist of, for example, a mixture containing cells from a plurality (e.g., 5-10 or more) of different tissues or organs in approximately equal amounts. In some embodiments, the target molecule of a specific cell type is present at levels of at least 4-5 folds, between 5-10 folds, or more than 10 folds greater than its average expression in a reference population. In some embodiments, detection or measurement of a target molecule of a specific cell type allows one skilled in the art to distinguish one type or types of cell of interest from cells of many, most or all other types. In general, the presence and / or abundance of most target molecules can be determined using one or more standard techniques such as Northern blotting, in situ hybridization, RT-PCR, sequencing, immunological methods such as immunoblotting, immunodetection (for example, by immuno- histochemistry) or fluorescence detection after staining with fluorescent labeled antibodies (for example, using FACS), the oligonucleotide or cDNA microarray or matrix membrane, protein microarray analysis, mass spectrometry etc.
[00216] In some embodiments, a target molecule is a channel, transporter, receptor or other molecule, at least in part, exposed on the cell surface. In some embodiments, a target molecule is a water channel or anion carrier (for example, an aquaporin protein).
[00217] In some embodiments, the target molecule is a protein, at least in part, exposed on the surface of red blood cells, such as a glucophorin (for example, glucophorin A, B, C or D) or band 3.
[00218] In some embodiments, the target molecule is a protein, at least in part, exposed on the surface of endothelial cells. In some embodiments, the target molecule is present on the surface of the normal healthy vasculature. In some embodiments, the target molecule is present on the surface of activated endothelial cells. In some embodiments, the target molecule is present on the surface of activated endothelial cells, but not on the surface of unactivated endothelial cells. In some embodiments, a target molecule is a molecule whose expression or exposure is induced by a stimulus such as injury or inflammation. In some embodiments, a target molecule would be recognized as "non-self" by a recipient who receives a transplant containing cells that express the target molecule. In some embodiments, the target molecule is a carbohydrate xenoantigen to which antibodies are commonly found in humans. In some embodiments, the carbohydrate comprises a blood group antigen. In some embodiments, the carbohydrate is composed of a xenoantigen, for example, an alpha-gal epitope (Galalpha1-3Galbeta1- (3) 4GlcNAc-R) (See, for example, Macher BA and Galili U. The Galalpha1, 3Galbeta1,4GlcNAc- R (alpha-Gal) epitope: a carbohydrate of unique evolution and clinical relevance.Bio-chim Biophys Acta. 1780 (2): 75-88 (2008).
[00219] In some embodiments of the invention, a compstatin analogue comprises a targeting fraction and a CRM.
[00220] In some modalities, a directed compstatin analogue comprises several targeting fractions, which can be the same or different. Different targeting fractions can bind to the same target molecule or different target molecules. The invention provides a targeted compstatin analogue that is versatile with respect to the targeting fraction, the compstatin analogue, or both.
[00221] In general, the invention encompasses any method of producing a compound consisting of a fraction of a compstatin analogue and a fraction of targeting and the resulting compounds. In some embodiments, a targeted compstatin analog can be produced using methods generally similar to those described in section VI, where a targeting fraction is used instead of, or in addition to, a reactive cell fraction. In some embodiments, a targeted compstatin analog composed of a peptide as a targeting fraction is synthesized as a polypeptide chain, comprising a fraction of a compostatin analog and a targeting peptide fraction. Optionally, the polypeptide chain comprises one or more spacer peptides between the fraction of compstatin analog and the targeting fraction.
[00222] In some embodiments, a targeted compstatin analogue has a molar activity of at least about 10%, 20% or 30%, for example, between 30% and 40%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90% or more, of the activity of a corresponding compstatin analog with the same amino acid sequence (and, if applicable, a or more blocking fractions), but not comprising a targeting fraction. In some embodiments in which a targeted compstatin analogue comprises several fractions of compstatin analogue, the molar activity of the targeted compstatin analogue is at least about 10%, 20% or 30%, for example, between 30% and 40 %, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90% or more, of the sum of the activities of the fractions of the mentioned compstatin analogue. VIII. Uses
[00223] Cell reactive, long-acting or targeted compstatin analogs have a wide variety of uses. Without restricting the invention in any way, certain uses of targeted cell reactive, long-acting or compstatin analogs and aspects of the invention are described herein. In some modalities, a reactive cell analog, long-acting or directed compostatin is administered to a subject who is suffering from or at risk of damage mediated by complement to an organ, tissue or cells. In some embodiments, a cell reactive compstatin analogue is contacted with an organ, tissue or cells ex vivo and becomes covalently bound to it. The organ, tissue or cells are introduced into a subject and are protected against the damage that would be caused by the recipient's complement system.
[00224] Compstatin analogs that do not bind covalently to cells can be used for the purposes described herein. For example, a modified compstatin analogue with a group that increases the life of the compound in the body and / or a compstatin analogue composed of a group that targets the compstatin analogue to a susceptible cell type or location to complement the activation can be used, and the invention covers such uses. In some embodiments, a long-acting compstatin analogue is used. In some embodiments, a compstatin analog composed of a targeting fraction is used. In some embodiments, a compstatin analog comprising a fraction that extends the life of the compound in the body and a targeting fraction is used. When the discussion below relates to a cell reactive compstatin analogue, the invention provides compositions and analogous methods relating to targeted compstatin analogs and (at least in aspects relating to the administration of a compstatin analogue to a subject) modalities in which a compstatin analogue does not comprise a targeting fraction or a reactive cell fraction, optionally a long-acting compstatin analogue, is used instead of, or in addition to, a cellular reactive compstatin analogue.
[00225] Certain uses of interest include: (1) protecting red blood cells (red blood cells) from complement-mediated damage in individuals with disorders such as nocturnal paroxysmal hemoglobinuria or atypical hemolytic-uremic syndrome or other disorders characterized by red cell lysis complement-mediated; (2) protect transplanted cells, tissues and organs from complement-mediated damage; (3) reducing ischemia / reperfusion (I / R) injury (for example, in individuals suffering from trauma, vascular obstruction, myocardial infarction or other situations in which I / R may occur); and (4) protecting various body structures (for example, the retina) or membranes (for example, the synovial membrane) that can be exposed to complement the components of complement-mediated damage in any of a variety of different complement-mediated disorders . The beneficial effects of inhibiting complement activation on the cell surface or other body structures are not limited to those resulting directly from protecting the cells or structures themselves against damage mediated by direct complement (for example, preventing cell lysis). For example, inhibiting complement activation using a cell reactive compstatin analogue can reduce anaphylatoxin generation and the resulting influx / activation of neutrophils and other pro-inflammatory events and / or reduce potentially harmful release of intracellular content, thus potentially with beneficial effects on remote organ systems or throughout the body. A. Protection of blood cells
[00226] In some embodiments of the invention, a cell reactive compstatin analogue, targeted cell compstatin analogue and / or an undirected compstatin analogue (e.g., an undirected long-acting compstatin analogue) is used to protect the blood cells against complement-mediated damage. Blood cells can be any cellular component of blood, for example, red blood cells (erythrocytes), leukocytes (white blood cells) and / or platelets. In some embodiments, a targeted cell compstatin analog is targeted at a target molecule exposed on the cell surface of red blood cells such as glucophorin or band 3. A variety of disorders are associated with complement-mediated damage to blood cells. Such disorders may result, for example, from deficiencies or defects in one or more soluble or cellular CRPs of an individual, for example, due to (a) mutations in the genes encoding those proteins; (b) mutations in the genes necessary for the production or proper function of one or more CRPs, and / or (c) the presence of autoantibodies to one or more CRPs. Complement-mediated red cell lysis may result from the presence of autoantibodies against red cell antigens that may arise due to a diverse set of causes (often being idiopathic). Individuals who have such mutations in genes encoding CRPs and / or with antibodies against CRPs or against their own red blood cells are at increased risk for disorders involving complement-mediated red cell damage. Individuals who have had one or more episodes characteristic of a disorder are at increased risk of recurrence.
[00227] Paroxysmal nocturnal hemoglobinuria (PNH) is a relatively rare disease comprising acquired hemolytic anemia, characterized by complement-mediated intravascular hemolysis, hemoglobinuria, bone marrow failure and thrombophilia (propensity to develop blood clots). It affects an estimated 16 individuals per million worldwide, occurs in both sexes and can occur at any age, often affecting young adults (Bessler, M. & Hiken, J., Hematology Am Soc Hematol EducProgram, 104- 110 (2008); Hillmen, P. Hematology Am Soc Hematol Educ Program, 116-123 (2008)). PNH is a chronic and debilitating disease, punctuated by acute hemolytic episodes and results in significant morbidities and reduced life expectancy. In addition to anemia, many patients have abdominal pain, dysphagia, erectile dysfunction and pulmonary hypertension and are at increased risk for thromboembolic events and renal failure.
[00228] HPN was first described as a distinct entity in the 1800s, but it was only in the 1950s, with the discovery of the alternative pathway of complement activation, that the cause of HPN hemolysis was firmly established (Parker CJ. Paroxysmal nocturnal hemoglobinuria : an historical overview. Paroxysmal nocturnal hemo-globinuria: an historical overview. 93-103 (2008)). CD55 and CD59 are normally attached to the cell membrane using glycosyl phosphatidylinositol (GPI) anchors (glycolipid structures that anchor certain proteins to the plasma membrane). PNH arises as a result of the benign clonal expansion of hematopoietic stem cells that have acquired a somatic mutation in the PIGA gene, which encodes a protein involved in the synthesis of GPI anchors (Takeda J, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria, Cell 73: 703-711 (1993)). The progeny of such stem cells are deficient in anchored GPI proteins, including CD55 and CD59. This defect renders these cells susceptible to complement-mediated red cell lysis. Flow cytometry analysis using antibodies to anchored GPI proteins is often used for diagnosis. It detects the deficiency of GP proteins anchored on the cell surface and allows the determination of the degree of deficiency and the proportion of affected cells (Brodsky RA. Advances in the diagnosis and therapy of nocturnal paroxysmal hemoglobinuria. Blood Rev. 22 (2): 65-74 (2008) HPN type III red blood cells are completely deficient in GPI-bound proteins and are highly sensitive to complement whereas HPN type II red blood cells are partially deficient and are less sensitive. FLAER is an inactive variant marked fluorescence of proaerolysin (a bacterial toxin that binds GPI anchors) and is increasingly used in conjunction with flow cytometry for PNH diagnosis. Lack of FLAER binding to granulocytes is sufficient for the diagnosis of PNH. In some embodiments, a cell reactive compstatin analog protects HPN red blood cells from C3b deposition.
[00229] In some modalities, a reactive cell analog, long-acting or directed compstatin is administered to a subject suffering from atypical hemolytic syndrome (aHUS). aHUS is a chronic disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure and is caused by inadequate complement activation, often due to mutations in genes that encode complement regulatory proteins (Warwicker, P., et al. Kidney Int 53, 836-844 (1998); Kavanagh, D. & Goodship, T. Pediatr Nephrol 25, 2431-2442 (2010). Mutations in the complement factor H (CFH) gene are the most common genetic anomaly in patients with aHUS and 60-70% of these patients die or reach the final stage of renal failure within one year after the onset of the disease (Kavanagh & Goodship, supra.) Mutations in factor I, factor B, C3, H-related protein factor 1-5 and thrombomodulin have also been described. Other causes of aHUS include autoantibodies against complement regulatory proteins such as CFH. In some modalities, a reactive cell, long-acting analogue or targeted compstatin is administered to a subject that has been identified as having a mutation in factor I, factor B, C3, H 1-5 related protein factor or thrombomodulin or that has been identified as having antibodies against a complement regulatory protein, for example, CFH.
[00230] Complement-mediated hemolysis occurs in a diverse group of other conditions, including autoimmune hemolytic anemias involving antibodies that bind to red blood cells and lead to complement-mediated hemolysis. For example, such hemolysis can occur in primary chronic cold agglutinin disease and certain reactions to drugs and other foreign substances (Berentsen, S., et al., Hematology 12, 361-370 (2007); Rosse, WF, Hillmen, P . & Schreiber, AD Hematology Am Soc Hematol Educ Program, 48-62 (2004)). In some embodiments of the invention, a cell reactive compstatin analogue is administered to a subject suffering from or at risk for chronic cold agglutinin disease. In another embodiment, a cell reactive compstatin analogue is used to treat a subject suffering from or at risk for HELLP syndrome, which is defined by the presence of hemolysis, elevated liver enzymes and low platelet count and is associated with regulatory somatic mutations of complement in at least some individuals (Fakhouri, F., et al., 112: 4542-4545 (2008)).
[00231] In other embodiments, cellular reactive compstatin analogs are used to protect red blood cells or other cellular components from the blood to be transfused into a subject. Certain examples of such uses are discussed below. As noted above, long-acting and / or targeted compstatin analogs can be used in the above methods to inhibit complement-mediated hemolysis and / or red cell damage. In some embodiments, a long-acting compstatin analogue composed of a fraction (CH2CH2O) is used to treat PNH or aHUS. B. Transplantation
[00232] Transplantation is a therapeutic approach of increasing importance, providing a means to replace organs and tissues that have been damaged by trauma, illness or other conditions. Kidneys, liver, lungs, pancreas and heart are among the organs that can be successfully transplanted. Tissues that are often transplanted include bones, cartilage, tendons, cornea, skin, heart valves and blood vessels. Pancreatic islets or islet cell transplantation is a promising approach for the treatment of diabetes, for example, type I diabetes. For the purposes of the invention, an organ, tissue, or cell (or population of cells) that is transplanted , is being transplanted or has been transplanted can be referred to as a "graft". For these purposes, a blood transfusion is considered a "graft".
[00233] Transplantation submits the graft to a variety of harmful events and stimuli that can contribute to graft dysfunction and, potentially, failure. For example, ischemia-reperfusion injury (I / R) is a common and significant cause of morbidity and mortality in the case of many grafts (particularly solid organs) and can be an important determinant of the likelihood of graft survival. Transplant rejection is one of the greatest risks associated with transplantation between genetically different individuals and can lead to failure and the need to remove the graft from the graft recipient.
[00234] In some embodiments of the invention, a cell reactive compstatin analogue, targeted cell compstatin analogue and / or an undirected compstatin analogue is used to protect a graft against complement-mediated damage. A cell reactive compstatin analogue reacts with cells from the graft, becomes covalently bound to it and inhibits complement activation. A targeted cell compstatin analogue binds to a target molecule in the graft (for example, expressed by endothelial cells or other cells in the graft) and inhibits complement activation. A target molecule can be, for example, a molecule whose expression is induced or stimulated by a stimulus such as injury or inflammation, a molecule that would be recognized as "not self" by the receptor, a xenoantigen of carbohydrates to which antibodies are commonly found in humans as a blood group antigen or a xenoantigen, for example, a molecule composed of an alpha-gal epitope. In some embodiments, a reduction in complement activation can be demonstrated by a reduction in the average deposition of C4d in the blood vessels of the grafts that were contacted with a compstatin analogue, for example, a cell reactive compstatin analogue, compared to average level of C4d deposition in grafts that have not been contacted with a compstatin analogue (for example, in individuals who are compared to grafts and other therapy they receive).
[00235] A graft can be contacted with a cell reactive, long-acting analogue or targeted compstatin before, during, and / or after being transplanted, in various modalities of the invention. For example, before transplantation of a graft taken from a donor, it can be contacted with a liquid composed of a reactive cell analogue, long-acting or directed compstatin. For example, the graft can be bathed in or painted with the solution. In another embodiment, a reactive cell analog, long-acting or directed compstatin is administered to a donor prior to removal of the graft. In some embodiments, a reactive cellular, long-acting or directed compstatin analogue is administered to a recipient during and / or after the introduction of the graft. Some modalities, a reactive cell analog, long-acting or directed compstatin is delivered locally to the transplanted graft. In some embodiments, a cell reactive compstatin analogue is administered systemically, for example, intravenously.
[00236] The invention provides a composition comprising: (a) an isolated graft; and (b) reactive cell analog, long-acting or targeted compstatin. In some embodiments, the composition further comprises a liquid solution suitable for contacting (for example, suitable for rinsing, washing, bathing, spraying, maintaining or storing) a graft (for example, an organ) as an isolated graft that has been removed from a donor and is waiting for the transplant to a recipient. In some embodiments, the invention provides a composition comprising: (a) a liquid solution suitable for contacting a graft (for example, an organ); and (b) a reactive cellular, long-acting or targeted compstatin analogue. The liquid solution can be any physiologically acceptable liquid solution for the graft (for example, suitable non-cytotoxic osmotic composition) and medically acceptable taking into account the subsequent introduction of the prosthesis to the recipient (for example, preferably sterile or at least reasonably free of microorganisms or other contaminants) and compatible with the cellular reactive compstatin analogue (ie, it will not destroy the reactivity of the compstatin analog) or compatible with the long-acting or targeted compstatin analogue. In some embodiments, a solution is any solution of its own in the art for such purposes. In some embodiments, a liquid solution is Marshall or hyperosmolar citrate (Soltran®, Baxter Healthcare), University of Wisconsin (UW) solution (ViaSpan ™, Bristol Myers Squibb), histidine tryptophan ketoglutarate (HTK) solution (Custodial®, Kohler Medical Limited), EuroCollins (Fresenius) and Celsior ® (Sangstat Medical), Polisol, IGL-1 or AQIX ®RS-1. Of course, other solutions, for example, containing equivalent or similar ingredients, in the same or different concentrations can be used within the scope of physiologically acceptable compositions. In some embodiments, a solution does not contain ingredients with which the cellular reactive compstatin analogue would react significantly and any solution can be modified or designed in the absence of these ingredients. In some embodiments, the cellular reactive compstatin analog is present in the graft-compatible solution at a concentration of, for example, between 0.01 mg / ml and 100 mg / ml or can be added to the solution to achieve such a concentration.
[00237] In some embodiments, the invention provides a kit comprising: (a) a reactive cell, long-acting or directed compstatin analogue; and (b) a solution compatible with a graft (for example, powder) or solid components thereof. The reactive cell, long-acting or targeted compstatin analogue can be supplied in solid form (for example, powder) or at least in part dissolved in a solution. In some embodiments, the reactive cell analog, long-acting or directed compstatin and / or the graft compatible solution supplied in predetermined quantities, so that, when combined, a solution of adequate concentration to come into contact with a graft with the reactive cell analog, long-acting or directed compstatin is produced. In many embodiments, the cell reactive, long-acting or targeted compstatin analogue and the graft-compatible solution or its solid components (for example, powder) are in separate containers within the kit. In some modalities, the reactive cell analog, long-acting or directed compstatin and the graft compatible solution are supplied in solid form (for example, powder) or in separate or mixed containers. In some embodiments, the kit consists of instructions for use, for example, instructions for adding a reactive cell, long-acting or targeted compstatin analog and the graft compatible solution and / or instructions for placing a graft in contact with a cell reactive compstatin analog. Optionally, the kit contains a label approved by a government agency responsible for regulating products used in transplantation, cell therapy and / or blood transfusion.
[00238] The invention further provides a method of covalently attaching a compstatin analogue to an isolated composite graft bringing the isolated graft into contact with a reactive cellular compstatin analogue. The invention further provides an isolated graft with a compstatin analog covalently linked thereto. Typically, the isolated graft has many compostatin analog molecules attached to it. In some embodiments, a graft is or comprises a solid organ such as a kidney, liver, lung, pancreas or heart. In some modalities, a graft is or comprises bone, cartilage, fascia, tendon, ligament, cornea, sclera, pericardium, skin, heart valve, blood vessels, amniotic membrane or dura mater. In some embodiments, a graft comprises several organs such as a heart-lung or pancreas-kidney graft. In some embodiments, a graft comprises less than a complete organ or tissue. For example, a graft may contain a part of an organ or tissue, for example, a liver lobe, vasosanguineous section, piece of skin or heart valve. In some embodiments, a graft consists of a preparation made up of isolated cells or tissue fragments that have been isolated from their original tissue, but maintain at least some tissue architecture, for example, pancreatic islets. In some embodiments, a preparation comprises isolated cells that are not connected to each other via connective tissue, for example, hematopoietic stem cells or progenitor cells derived from peripheral and / or blood cable, or whole blood or any blood product. - blood cells like red blood cells (erythrocytes) or platelets. In some modalities, a graft is obtained from a deceased donor (for example, a "donation after brain death" (DBD) or "donation after cardiac death" donor). In some modalities, depending on the type of graft, a graft is obtained from a living donor. For example, sections of kidneys, liver, blood cells, are among the types of grafts that can often be obtained from a living donor without undue risk to the donor and consistent with good medical practice.
[00239] In some modalities, a graft is a xenotransplant (that is, the donor and the recipient are of different species). In some modalities, a graft is an autograft (that is, a graft from one part of the body to another part of the body in the same individual). In some embodiments, a graft is an isograft (that is, the donor and recipient are genetically identical). In most modalities, the graft is an allograft (that is, the donor and recipient are genetically non-identical members of the same species). In the case of an allograft, the donor and recipient may or may not be genetically related (for example, family members). Normally, the donor and recipient have compatible blood groups (at least ABO compatibility and, optionally, Rh, Kell and / or other blood cell antigen compatibility). The recipient's blood may have been designed for alloantibodies to the graft and / or the recipient and the donor provided that the presence of such antibodies can lead to hyperacute rejection (ie, rejection begins almost immediately, for example, within a few minutes after the graft comes into contact with the recipient's blood). A complement-dependent cytotoxicity (CDC) assay can be used to display a subject's serum for detection of anti-HLA antibodies. The serum is incubated with a panel of lymphocytes of the known HLA phenotype. If the serum contains antibodies against HLA molecules in the target cells, cell death occurs due to complement-mediated lysis. Using a selected panel of target cells allows specificity to be assigned to the detected antibody. Other techniques useful for determining the presence or absence of anti-HLA antibodies and, optionally, determining their HLA specificity, include ELISA assays, flow cytometry assays, Micro-account matrix technology (eg Luminex technology). The methodology for carrying out these tests is well known and a variety of kits for carrying them out are commercially available.
[00240] In some modalities, a reactive cell analog, long-acting or directed compstatin inhibits complement-mediated rejection. For example, in some modalities, a reactive cell analog, long-acting or directed compstatin inhibits hyperacute rejection. Hyperacute rejection is caused, at least in part, by antibody-mediated activation of the receptor's complement system via the classical pathway and the resulting MAC deposition in the graft. It usually results from the presence in the receptor of pre-existing antibodies that react with the graft. While it is desirable to try to avoid hyperacute rejection by combining appropriately before transplantation, it may not always be possible to do so, for example, due to time and / or resource constraints. In addition, some recipients (for example, multiplying transfused individuals, individuals who have previously received transplants, women who have had multiple pregnancies) may already have so many preformed antibodies, potentially including antibodies against antigens that are not normally tested, that can be difficult or perhaps almost impossible to confidently obtain a compatible graft in a timely manner. Such individuals have a higher risk of hyperacute rejection.
[00241] In some modalities, a reactive cell analog, long-acting or directed compstatin inhibits acute rejection or graft failure. As used here, "acute rejection" refers to rejection occurring for at least 24 hours, usually at least several days to a week, after a transplant, up to 6 months after the transplant. Acute antibody-mediated rejection (AMR) often involves an acute increase in specific donor allo-antibody (DSA) in the first few weeks after transplantation. Without wishing to be bound by any theory, it is possible that pre-existing plasma cells and / or the conversion of memory B cells to new plasma cells play a role in increased DSA production. Such antibodies can result in damage mediated by complement to the graft, which can be inhibited by putting the graft in contact with a cell reactive compstatin analog. Without wanting to be bound by any theory, inhibiting the activation of the complement to the graft can reduce leukocyte infiltration (eg, neutrophils), another collaborator of acute graft failure.
[00242] In some modalities, a reactive cell analogue, of prolonged action or of directed compstatin inhibits I / R injury mediated by an excerpt. As discussed below, I / R injury can occur after reperfusion of tissue whose blood supply has been temporarily interrupted, as in transplanted organs. Reducing I / R injury would reduce the likelihood of acute graft dysfunction or reduce its severity and reduce the likelihood of acute graft failure.
[00243] In some modalities, a reactive cell analog, long-acting or directed compstatin inhibits chronic acute rejection or chronic graft failure. As used here, "chronic rejection or graft failure" refers to rejection or failure occurring at least 6 months post-transplant, for example, between 6 months and 1, 2, 3, 4, 5 years or more post-transplant , often after months to years of good graft function. It is caused by a chronic inflammatory and immune response against the graft. For the purposes of this, chronic rejection may include chronic allograft vasculopathy, a term used to refer to fibrosis of the internal blood vessels in the transplanted tissue. As immunosuppressive regimes have reduced the incidence of acute rejection, chronic rejection is becoming more prominent as a cause of graft dysfunction and failure. There is growing evidence that the production of allo-antibody B cells is an important element in the genesis of chronic rejection and graft failure (Kwun J. and Knechtle SJ, Transplantation, 8: 955-61 (2009). Damage prior to graft can be a contributing factor, leading to chronic processes, such as fibrosis that can ultimately lead to chronic rejection, so inhibiting such previous damage using a cell reactive compstatin analogue can delay or reduce the likelihood or severity of chronic graft rejection.
[00244] In some embodiments, a long-acting compstatin analogue is administered to a graft recipient to inhibit graft rejection and / or graft failure. C. Ischemia / Reperfusion injury
[00245] Ischemia-Reperfusion Injury (I / R) is a major cause of tissue damage after trauma and other conditions associated with temporary interruption of blood flow, such as myocardial infarction, stroke, serious infection, illness vascular, aneurysm repair, cardiopulmonary bypass and transplantation.
[00246] In the scenario of trauma, hypotension, systemic hypoxemia and local interruption of blood supply resulting from compartment syndrome, bruises and vascular lesions cause ischemia that damages metabolically active tissues. Restoration of the blood source triggers an intense systemic inflammatory reaction that is often more harmful than the ischemia itself. Once the ischemic region is perfused, factors that are produced and released locally enter the circulatory system and reach remote locations, sometimes causing significant damage to organs not affected by the original ischemic insult, such as the lungs and intestine, leading to to single and multiple organ dysfunction. Complement activation occurs shortly after reperfusion and is a key mediator of post-ischemic damage, both directly and through its stimulatory and chemo-attracting effects on neutrophils. All three main complement pathways are activated and, acting cooperatively or independently, are involved in I / R related to adverse events that affect many organ systems. In some embodiments of the invention, a reactive cellular, long-acting or directed compstatin analogue is administered to a subject who has recently (for example, within the previous 2, 4, 8, 12, 24 or 48 hours) experienced trauma, for example example, a trauma that puts the subject at risk of I / R injury, for example, due to systemic hypoxemia, hypotension and / or local interruption of blood supply. In some modalities, the cellular reactive compstatin analogue can be administered intravascularly, optionally in a blood vessel that supplies an injured body part or directly to the body part. In some modalities, the subject suffers from spinal cord injury, traumatic brain injury, burns and / or hemorrhagic shock.
[00247] In some modalities, a reactive cell analog, long-acting or directed compstatin is administered to a subject before, during or after a surgical procedure, for example, a surgical procedure that is expected to temporarily interrupt blood flow for a tissue, organ or body part. Cardiopulmonary bypass, angioplasty, repair / replacement of heart valves, aneurysm repair or other vascular surgeries are examples of such procedures. The cell reactive compstatin analogue can be administered before, after, or over a period of overlap with the surgical procedure.
[00248] In some modalities, a reactive cell analog, long-acting or directed compstatin is administered to a subject who has suffered an MI, thromboembolic effusion, deep venous thrombosis or pulmonary embolism. The cell reactive compstatin analog can be administered in combination with a thrombolytic agent such as tissue plasminogen activator (tPA) (eg, alteplase (Activase), reteplase (Retavase), tenecteplase (TNKase)), anistreplase (Eminase), streptokinase ( Kabikinase, Streptase) or urokinase (Abbokinase). The reactive cell, long-acting or targeted compstatin analogue can be administered before, after or during a period of overlap with the thrombolytic agent.
[00249] In some modalities, a reactive cell analog, long-acting or directed compstatin is administered to a subject to treat I / R injury. D. Other Complement-Mediated Disorders
[00250] In some incarnations, a reactive cell analog, long-acting or directed compstatin is introduced into the eye for the treatment of an eye disease, such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma or uveitis. For example, a cell reactive compstatin analogue can be introduced into the vitreous cavity (for example, by intravitreal injection), to treat a subject not suffering or at risk for AMD. In some embodiments, a reactive cell analog, long-acting or directed compstatin is introduced into the anterior chamber, for example, to treat anterior uveitis.
[00251] In some embodiments, a cell reactive, long-acting or targeted compstatin analogue is used to treat a subject who is suffering from or at risk for an autoimmune disease, for example, an autoimmune mediated disease, at least in part, by antibodies against one or more of its own antigens.
[00252] Cell reactive, long-acting or targeted compstatin analogs can be introduced into the synovial cavity, for example, in a subject suffering from arthritis (for example, rheumatoid arthritis). Of course, they can be administered systemically in addition or
[00253] In some embodiments, a reactive cell analog, long-acting or targeted compstatin is used to treat a subject suffering from or at risk of an intracerebral hemorrhage.
[00254] In some modalities, a reactive cell analog, long-acting or directed compstatin is used to treat a subject suffering from or at risk of myasthenia gravis.
[00255] In some modalities, a reactive cell analog, long-acting or directed compstatin is used to treat a subject suffering from or at risk of neuromyelitis optica (NMO),
[00256] In some embodiments, a reactive cellular, long-acting or targeted compstatin analogue is used to treat a subject suffering from or at risk for membrane glomerulitis (MPGN), for example, MPGN type I, type MPGN II or MPGH type III.
[00257] In some modalities, a reactive cell analog, long-acting or directed compstatin is used to treat a subject suffering from or at risk of a neurodegenerative disease. In some modalities, a cell reactive, long-acting or targeted compstatin analogue is used to treat a subject suffering from neuropathic pain or at risk of developing neuropathic pain. In some modalities, a reactive cell analog, long-acting or directed compstatin is used to treat a subject suffering from or at risk of rhinosinusitis or nasal polyposis. In some embodiments, a cell reactive, long-acting, or targeted compstatin analogue is used to treat a subject suffering from or at risk for cancer. In some modalities, a reactive cell analog, long-acting or targeted compstatin is used to treat a subject suffering from or at risk of sepsis. In some modalities, a reactive cell analog, long-acting or targeted compstatin is used to treat a subject suffering from or at risk for adult respiratory distress syndrome.
[00258] In some modalities, a reactive cell analog, long-acting or directed compstatin is used to treat a subject suffering from or at risk of anaphylaxis or infusion reaction. For example, in some modalities, a subject may be pre-treated before, during or after receiving a drug or vehicle that may cause an anaphylaxis or infusion reaction. In some modalities, a subject at risk or suffering from anaphylaxis of a food (for example, peanuts, crustaceans or other food allergens), insect bites (for example, bee, wasp), is treated with a reactive cell analog, of action prolonged or directed compstatin.
[00259] The reactive cell analog, long-acting or directed compostatin can be administered locally or systemically, in various modalities of the invention.
[00260] In some modalities, a reactive cell analog, long-acting or directed compstatin is used to treat a respiratory disease, for example, asthma or obstructive pulmonary disease (COPD). The reactive cell analog, long-acting or directed compstatin can be administered to the respiratory tract by inhalation, for example, as a dry powder or via nebulization, or it can be administered by injection, for example, intravenously, in various modalities . In some modalities, a reactive cell analog, long-acting or targeted compstatin is used to treat severe asthma, for example, asthma that is not sufficiently controlled by bronchodilators and / or corticosteroid inhalation. IX. Compositions and Administration
[00261] The invention provides a variety of compositions comprising a cell reactive, long-acting or directed compstatin analogue. In various embodiments, a composition can have any feature or combination of features discussed here as long as they are not mutually exclusive. The invention provides modalities of such compositions and methods of using them, wherein the compstatin analogue is any compstatin analogue.
[00262] In some embodiments, a composition comprises a reactive cell analog, long-acting or purified directed compstatin. Purification can be achieved using a variety of approaches that can be selected by a person skilled in the art based on achieving a desired level of purity with respect to the various components present in the composition prior to purification. For example, filtration, high performance liquid chromatography, affinity chromatography and / or other approaches and their combinations can be used. In some embodiments, the composition comprises at least 80%, 85%, 90%, 95%, 98%, 99% or more of reactive cell, long-acting or directed compstatin analog in percentage of the total compstatin analog by weight. In some embodiments, the composition comprises at least 80%, 85%, 90%, 95%, 98%, 99% or more of reactive cell, long-acting or directed compstatin analogue as a percentage of the total compstatin analogue on a basis molar. In some embodiments, a composition consists or consists essentially of a reactive cell analog, long-acting or targeted compstatin.
[00263] In some embodiments, a composition comprising a cell reactive compstatin analog and a compound comprising a cell reactive functional group is characterized in that the ratio of the cell reactive compstatin analog to the compound that make up the cell reactive functional group on a base molar is at least 10: 1, 20: 1, 50: 1, 100: 1, 500: 1, 1,000: 1 or more. In some embodiments, the composition comprises at least 80%, 85%, 90%, 95%, 98%, 99% or more of cell reactive compstatin analogs as a percentage of the total weight compstatin analog In some embodiments, the composition comprises at least 80%, 85%, 90%, 95%, 98%, 99% or more of cellular reactive compstatin analogue as a percentage of the total compstatin analogue on a molar basis. In some embodiments, a composition comprises at least 80%, 85%, 90%, 95%, 98%, 99% or more of cellular reactive compostatin analog by weight. In some embodiments, a composition comprises at least 80%, 85%, 90%, 95%, 98%, 99% or more of long-acting compstatin analog by weight. In some embodiments a composition comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or more of weight-directed compstatin analog. In some embodiments, a composition comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or more of weight-directed compstatin analog. In some modalities, weight is dry weight.
[00264] In some aspects, the invention provides a pharmaceutical grade composition, consisting of a reactive cell analog, long-acting or directed compstatin. The pharmaceutical grade composition can have any of the above-mentioned characteristics in terms of purity in various embodiments. The pharmaceutical grade composition is sufficiently free of endotoxins, heavy metals and unidentified and / or uncharacterized substances to be acceptable, without further purification, as it is a pharmaceutical composition suitable for administration to a human subject or for the manufacture of a pharmaceutical composition to be administered to a human subject. In some embodiments, the pharmaceutical grade composition is sterile.
[00265] Appropriate preparations, for example, substantially pure preparations of a reactive cell analog, long-acting or targeted compstatin or other active agent, can be combined with pharmaceutically acceptable carriers or vehicles etc. to produce a suitable pharmaceutical composition. The term "pharmaceutically acceptable carrier or vehicle" refers to a non-toxic carrier or vehicle that does not destroy the compound pharmacological activity with which it is formulated. One skilled in the art will understand that a carrier or vehicle is "non-toxic" if it is compatible with administration to a subject in an adequate amount to deliver the compound without causing undue toxicity. Pharmaceutically acceptable carriers or vehicles that can be used in the compositions of this invention include, but are not limited to, water, saline, Ringer's solution, sodium acetate or potassium acetate solution, 5% dextrose and the like. The composition can include other components as appropriate for the desired formulation, for example, as discussed in this document. Complementary active compounds, for example, compounds independently useful for treating a subject who is suffering from a complement-mediated disorder can also be incorporated into the compositions. The invention provides such pharmaceutical compositions comprising a cell reactive, long-acting or targeted compstatin analogue and, optionally, a second active agent useful for treating a subject who is suffering from a complement-mediated disorder.
[00266] In some embodiments, the invention provides a pharmaceutically acceptable composition suitable for administration to humans, packaged together with a label approved by a government agency responsible for regulating pharmacists, for example, the U.S. Food & Drug Administration. In some embodiments, the invention provides a pharmaceutical package or kit comprising: (a) a reactive cell, long-acting, or pharmaceutically acceptable analogue in solid state analog; (b) a pharmaceutically acceptable carrier or vehicle. Optionally, the kit or packaging contains instructions for the cell reactive, long-acting analogue or compstatin directed on the carrier. In some embodiments, a pharmaceutical package or kit is provided. The package or kit comprises a sufficient amount of pharmaceutical composition for at least 1 dose, for example, between 1 and 200 doses or any number of intervening or sub-intervals. In some embodiments, a pharmaceutical package or kit comprises one or more needles and, optionally, one or more syringes. In some embodiments, at least one pre-filled syringe is provided. In some embodiments, one or more unit dosage forms or pre-measured rates are provided. In some modalities, instructions for administration are provided, which, in some modalities, include instructions for self-administration.
[00267] The pharmaceutical composition can be administered to a subject by any appropriate route of administration, including, but not limited to, intravenous, intramuscular, subcutaneous, inhalation, nasal, intrathecal, intracranial, intraarterial, via oral, rectal, transdermal, intradermally, under the skin etc. In some embodiments, a composition comprising a reactive cellular, long-acting or directed compstatin analogue is administered intravenously. In some embodiments, a composition comprising a reactive cell, long-acting or directed compstatin analogue is administered intraarterially. The composition can be administered locally, within the vascular system, supplying an organ or tissue, or extravascularly in the vicinity of an organ or tissue. It will be understood that "administration" encompasses directly administering a compound or composition to a subject, instructing a third party to administer a compound or composition to a subject, prescription or suggesting a compound or composition to a subject (for example, for self-administration), self-administration and, if necessary, other means of making a compound or making a compound available to a subject.
[00268] Pharmaceutical compositions suitable for injectable use (for example, intravenous administration) or by pump or catheter usually include sterile aqueous solution (where water is soluble) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions and or dispersion. Sterile solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent, optionally with one or a combination of ingredients such as buffers such as acetates, citrates, lactates or phosphates; agents for the regulation of tonicity, such as sodium chloride or dextrose; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid, glutathione or sodium bisulfite; chelating agents such as ethylenediamine-tetra-acetic acid; and other suitable ingredients, etc., as desired, followed by filter-based sterilization. One skilled in the art will be aware of the numerous physiologically acceptable compounds that can be included in a pharmaceutical composition. Other useful compounds include, for example, carbohydrates, such as glucose, sucrose, lactose; dextrans; amino acids, such as glycine; polioli, such as mannitol. These compounds can, for example, serve as bulking agents and / or stabilizers, for example, in a powder and / or when part of the manufacturing or storage process involved in filming. Surfactant (s) like Tween-80, Pluronic-F108 / F68, deoxycholic acid, phosphatidylcholine etc. they can be included in a composition, for example, to increase solubility or to provide micro-emulsion to deliver hydrophobic drugs. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, if desired. The parenteral preparation can be placed in ampoules, disposable syringes or infusion bags or multi-dose vials made of glass or plastic. Preferably, solutions for injection are sterile and acceptably free of endotoxins.
[00269] Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and other appropriate ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods may include vacuum drying and lyophilization which produces a powder of the active ingredient plus any additional desired ingredient, for example, from a previously filtered sterile solution thereof.
[00270] Oral administration can be used in certain modalities. Oral compositions generally include an inert diluent or an edible carrier. For the purpose of administering oral therapy, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules, for example, gelatin capsules. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant like magnesium stearate or Sterotes; a fluidizer, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as mint, methyl salicylate or orange flavoring. A liquid composition can also be administered orally. Formulations for oral delivery may incorporate agents to improve stability within the gastrointestinal tract and / or to improve absorption.
[00271] For administration by inhalation, a compostatin analogue can be delivered in the form of an aerosol spray from a pressed container or dispenser that contains an appropriate propellant, for example, a gas such as carbon dioxide. A metered dose inhaler or nebulizer can be used. The aerosol can comprise liquid particles or dry aerosol (for example, dry powders, large porous particles, etc.).
[00272] For topical application, a compstatin analogue can be formulated in an appropriate ointment that contains the active component suspended or dissolved in one or more carriers. Topical administration carriers include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, compound polyoxypropylene, wax emulsion and water. Alternatively, pharmaceutically acceptable compositions can be formulated as an appropriate lotion or cream that contains a compstatin analogue suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
[00273] Systemic administration can also be through trans-mucous or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art and include, for example, fusidic acid derivatives, bile salts, detergents and transmucosal administration. Transmucosal administration can be performed, for example, with the use of nasal sprays or suppositories. For transdermal administration, the active compounds are usually formulated in ointments, gels or creams as generally known in the art.
[00274] The compounds can also be prepared in the form of suppositories (for example, with conventional suppository bases such as cocoa butter and other glycerins) or retention enemas for rectal delivery.
[00275] In certain embodiments of the invention, a compstatin analogue or other active compound is prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including microencapsulated implants and delivery systems. For example, a compstatin analogue can be incorporated or encapsulated in a microparticle or nanoparticle formulation. Biodegradable, biocompatible polymers can be used, such as vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poly-polyesters, polyethers, polylactic acid, PLGA etc. Liposomes or other lipid-based particles can be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 and / or other references listed in this document. Deposit formulations containing a compstatin analogue can be used. The compstatin analogue is released from the deposit over time, for example, in order to provide a therapeutic concentration for longer than if the compound was administered intravenously. Those skilled in the art will observe that the materials and methods selected for the preparation of a controlled release formulation, implant etc. must preserve the activity of the compost.
[00276] It will be appreciated that analogs of compstatin and / or additional active agents can be provided as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable organic and inorganic bases and acids. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulphate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, Digluconate, dodecylsulfate, ethanesulfonate, formiate, fumarate, glycate, gluten glycolic, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, iodhydrate, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, phenylate, persulfate, 3 , picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Also, pharmaceutically acceptable salts can be prepared as alkali metal or alkaline earth salts, such as sodium, potassium or calcium salts, if appropriate depending on the identity of the active agent.
[00277] It will be understood that the pharmaceutically acceptable carriers, compounds and methods of preparation mentioned in this document are exemplary and not restrictive. See, for example, Re-mington: The Science and Practice of Pharmacy. 21st Edition. Phila-delphia, PA. Lippincott Williams & Wilkins, 2005, for further discussion of pharmaceutically acceptable compounds and methods of preparing pharmaceutical compositions of various types.
[00278] A pharmaceutical composition can be administered in an effective amount to achieve a desired beneficial effect. In some embodiments, an effective amount is sufficient to provide one or more of the following benefits:
[00279] In certain embodiments of the invention, a pharmaceutical composition consisting of a reactive cell analog, of prolonged action or of directed compstatin is administered by parenteral route. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered by intravenous injection. In some embodiments, the composition is administered as an IV bolus or an intravenous infusion. In some embodiments, the composition is administered as an IV drip. In some embodiments, the composition is administered as an IV bolus followed by an intravenous infusion or IV drip. In some embodiments, an IV infusion is administered for about 1, 2, 3, 4, 5, 15, 20, 30, 60 or 120 minutes. In some embodiments, an IV drip is administered over more than about 60 minutes, for example, over about 1, 2, 3 or more hours. In some embodiments, a total amount of between about 0.1 mg / kg / day and about 2,000 mg / kg / day of compstatin analogue is administered, for example, between about 1 mg / kg / day and about 1,000 mg / kg / day, for example, between about 5 mg / kg / day and about 500 mg / kg / day. In some embodiments, a total amount of between about 10 mg / kg / day and about 100 mg / kg / day of compstatin analogue is administered, for example, between about 10 mg / kg / day and about 50 mg / kg / day, for example, between about 10 mg / kg / day and about 20 mg / kg / day. It will be appreciated that a variety of different dosage regimens could be used to deliver a desired total daily amount. For example, a desired amount of compstatin analogue could be administered in a single administration or in multiple administrations, for example, over a period of 24 hours. For example, a subject can receive two or more doses within a 24-hour period, which can be administered over the same period of time or over different periods of time. In some modalities, a reactive cell analog, long-acting or directed compstatin is administered at intervals greater than 24 hours. For example, doses can be administered on average every two days, every 3-4 days, weekly, every two weeks etc., in various modalities. In some modalities, covalently linked, cellular reactive, long-acting or targeted compstatin analogues protect cells, tissues, organs, for a period of weeks or months without the need for retreatment. For example, subjects can be retreated at intervals of 1-2 weeks, 2-4 weeks, 4-6 weeks, 6-8 weeks or even longer. In some embodiments, subcutaneous administration is used to deliver at least a few doses. For example, the administration of approximately 0.1-5 mg / kg / day, for example, about 0.5-2 mg / kg / day is contemplated in some modalities, for example, in a volume of about 0.25 ml - 2 ml, for example, a volume of about 1 ml. In some embodiments, the concentration is from about 50 mg / ml to about 300 mg / ml, for example, about 50 mg / ml - about 100 mg / ml or about 100 mg / ml - about 200 mg / ml. In some modalities it is daily administration. In some embodiments, intramuscular administration is used to deliver similar amounts of compound. It will be understood that there may be an initial treatment phase during which treatment is more frequent and / or at which higher doses are administered. For example, a subject with paroxysmal nocturnal hemoglobinuria or aHUS, may require several doses to achieve protection for a substantial fraction of the subject's red blood cells. Thereafter, lower doses and / or less frequent dosing could be used, for example, to protect the newly formed red blood cells and / or to replace the protection of existing red blood cells. Naturally, similar approaches can be followed to treat any disease, if applicable. In some modalities, treatment is started using IV administration and then changed to subcutaneous, intramuscular or intradermal for maintenance therapy. Depending on the disease, treatment may continue at intervals of, for example, months, years or indefinitely. Appropriate doses and administration regimen depend on the potency and half-life of the compstatin analogue (or other active agent) at least in part and, optionally, can be adapted to the specific recipient, for example, by administering increasing doses up to that a pre-selected desired response is achieved, such as a desired level of complement inhibition and / or cell protection. If desired, the specific dose level for any specific subject can be selected based at least in part on a variety of factors, including the activity of the specific compound employed, the particular condition being treated, age, body weight, general health, administration, the rate of excretion, any combination of drugs and / or the degree of expression of complement proteins or activity measured in one or more samples obtained from the subject.
[00280] The invention encompasses the administration of a compstatin analog in combination with additional therapy. Such additional therapy may include the administration of any agent used in the art or potentially useful for the treatment of a subject suffering from the disease.
[00281] When two or more therapies (for example, compounds or compositions) are used or administered "in combination" with each other, they can be given at the same time, within overlapping periods of time or in sequence (for example, separated up to 2 weeks in time), in various modalities of the invention. They can be administered via the same route or different routes. In some embodiments, the compounds or compositions are administered within 48 hours of each. In some embodiments, a compstatin analogue can be given before or after administration of the additional compounds, for example, in sufficiently close time so that the compstatin analogue and the additional compounds are present at useful levels within the body at least once. In some embodiments, the compounds or compositions are administered close enough to each other in time so that no more than 90% of the composition previously administered has been metabolized to inactive metabolites or eliminated, for example, excreted, from the body, at the time when the second compound or composition is administered.
[00282] In some embodiments, a composition that includes a cell reactive compstatin analogue and additional compounds is administered. Example 1: Development of PEG-bonded Compstatin Analogs that Retain Substantial Complement Inhibiting Activity
[00283] A compstatin analogue with the amino acid sequence of the compstatin analogue of SEQ ID No.: 28, but incorporating an AEEAc-Lys fraction located at the C-terminal to the Thr residue of SEQ ID No.: 28 for purposes of further conjugation of an NHS activated PEG ester to the amino group of the lysine side chain. The compound was synthesized using standard methods. Briefly, amino acids (including AEEAc) were obtained as Fmoc-protected amino acids, where the amino group of each amino acid was protected with Fmoc. Groups of side-chain functionalities have also been blocked with several suitable protection groups. The synthesis was carried out following the solid phase methodology, described by Merrifield (J. Amer. Chem. Soc. 85, 2149 (1963)). The assembly of the chain was carried out in the solid phase, with the conclusion that the N-terminal was acetylated; the peptide was then cleaved from the solid phase and simultaneously deprotected via acylolysis using TFA and conversion to amide. The linear peptide was then oxidized and purified. The resulting compstatin analog is represented by Ac-Ile-Cys * -Val- (1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys * -Thr-AEEAc-de Lys - NH2 (SEQ ID No. 51), abbreviated as CA28-AEEAc-Lys. Note that for the sake of brevity, the N-terminal acetyl group and C-terminal amino acid groups are omitted in this abbreviation. Activated PEGs of monofunctional linear NHS-ester with molecular weights of 30 kD and 40 kD (NOF America Corp. White Plains, NY, Cat. No. SUNBRIGHT ® ME-400GS), respectively, were coupled to the CA28- lysine side chain. AEEAc-Lys, resulting in long-acting compstatin analogs represented as follows: CA28-AEEAc-Lys- (PEG30k) and CA28-AEEAc-Lys- (PEG40k) and purified. Note that the number after the term "PEG" and preceding the letter "k" represents the molecular weight of the PEG fraction in kilodaltons and "k" is an abbreviation for kD). CA28-AEEAc-Lys- (PEG30k) is also called CA28-1. CA28-AEEAc-Lys- (PEG40k) is also called CA28-2.
[00284] The inhibitory activity of the synthesized compounds was assessed by measuring the effect of the compounds on complement activation through the classical route using a standard complement inhibition assay. The protocol measures C3b deposition in an ELISA format. C3b deposition monitored using this method is generated through the complement activated by the classical route. Soon, 96-well plates are coated with BSA. Human plasma, chicken ovalbumin (OVA), polyclonal anti-OVA antibodies and compound to be tested (called "drug") are added and incubated, followed by the addition of conjugated anti-human C3 HRP antibody. After further incubation, the substrate is added and the signal is detected. The protocol details are as follows: Protocol for Classical Complement Inhibition Test Materials: • Ninety-six well plates (polystyrene plate, Thermo Scientific, 9205) • Chicken OVA (Sigma A5503-5G) • Rabbit OVA (Abcam ab1221) • Blocking buffer (Startingblock buffer, Thermo Scientific 37538) • Veronal buffer (5 X concentration, Lonza 12-624E) • Human plasma (collected with Lepirudin in a final concentration of 50 ug / ml) • Antibody conjugated to goat anti-human C3 horseradish peroxidase (MP Biomedicals, 55237) • Tween-20 Wash Buffer (0.05% Tween 20-PBS buffer) • TMB (Peroxidase substrate, BD 555214) - 1: 1 mixture of BD 51-2607KC and 51-2606KC. • 1M H2SO4 Protocol: 1. Add 100 µl / well of 1% chicken OVA (in PBS) 2. Incubate overnight @ 4 ° C or at room temperature for 1-2 hours. 3. Remove by shaking and tapping the plate. 4. Block adding 200ul of the blocking buffer 5. Incubate for 1 h at room temperature 6. Remove by shaking and tapping the plate 7. Add 100 µl dilution of 1: 1000 polyclonal antifungal OVA in blocking buffer 8. Incubate by 1 h at room temperature 9. Wash twice with wash buffer 10. Add 50 ul VB + + to wells # 2 to 12 11. Add 100ul of dilution of the starting drug (2 X in VB + +) to well 1. 12. Serially dilute the drug from wells 1 to 10 as follows at. Take 50ul of the original well solution b. Add this to the next well c. Mix by pipetting several times d. Repeat until well # 10 Observe: from well # 10 remove 50ul and discard. 13. Add 50ul of 2 X plasma dilution (1: 37.5 dilution of the original plasma) to wells 1 to 11 14. Incubate for 1h 15. Wash with wash buffer 16. Add 100ul of 1/1000 dilution of strong root anti-C3-Peroxidase antibody in blocking buffer 17. Incubate for 1h 18. Wash with wash buffer 19. Add 100ul of TMB to all wells 20. Incubate for 5-10 min in the dark 21. Add 50 ul 1M H2SO4 22. Read plate at 450nm • B + + Formula: Barbital 5 mM NaCl 72.5 mM MgCl2 0.5 mM CaCl2 0.15 mM PH 7.4 Standard solutions: Veronal Buffer (5 X)
Mg-Cl2 (200X)
CaCl2 (500x)
To prepare 50 ml of working buffer: • Weigh 210 mg of NaCl • Add 10 ml of 5 X GB • Add 100ul of CaCl2 (500 X) • Add 250ul of MgCl (200 X) • Adjust volume to 50 ml with H2O • Adjust the pH to 7.4
[00285] The data were analyzed using the GraphPad Prism5 software. Data sets from each experiment were normalized for percentage activation compared to the 100% activation control corresponding to the well to which no compound is added. Drug concentration values (X values) were transformed into their logarithms and the percent activation (Pa) (Y values) was transformed to percent inhibition (Pi) using the following formula Pi = 100-Pa (Yi = 100-Ya) . Percent inhibition was plotted against drug concentration and the resulting data set was suitable for a sigmoidal dose response function [Y = + Low (Up-Down) / (1 + 10 ((Log EC-X) ))]. IC50 values were obtained from the adjustment parameters.
[00286] The results are shown in Figure 1, and the IC50 values are shown in Table 2 (in Example 2). As indicated, CA28-1 and 2-CA28 exhibited about 30% of CA28 activity on a molar basis. Example 2: Development of Long-acting Compstatin Analogs That Demonstrate Increased Molar Activity
[00287] PEG with eight arms activated with NHS ester with a molecular weight of 40 kD (NOF America Corp. White Plains, NY, Cat. No. SUNBRIGHT ® HGEO-400GS; chemical formula: octave hexaglycerol (succinimidyloxyglutaryl) polyoxyethylene) coupled to the lysine side chain of CA28-AEEAc-Lys, resulting in long-acting compstatin analogs represented as follows: (CA28-AEEAc) 8-PEG40k, is also called CA28-3.
[00288] CA28-3 complement inhibition activity was tested using the assay described in Example 1. The results are plotted in Figure 1 and the IC50 value is listed in Table 2, both as a function of the CA28 concentration. The concentration of CA28 was calculated using the extinction coefficient of CA28 at 283 nm (10208.14 L ^ mol-1 ^ cm-1). Based on other analyzes (UV absorption vs mass of material and analysis of elementary CHN%), it was concluded that there are 7.5 fractions of CA28 per molecule of CA28-3. Thus, the activity of CA28-3 on a molar basis is 7.5 times greater than that shown in Figure 1 and Table 2. Thus, the IC50 value in Table 2 is 7.5 times greater than the real IC50 of CA28-3 on molar basis. The IC50 of CA28-3 on a molar basis is calculated to be about 0.26 (less than that of the parent compound CA28). Figure 2 shows the percentage activation of the complement inhibiting the activity of CA28 and CA28-2 and 3-CA28 of long-acting compstatin analogs, as a function of the concentration of CA28-3 (μM), that is, the activity of CA28 -3 was corrected to take into account the fact that the compound contains 7.5 fractions of CA28. On a molar basis, CA28-3 complement inhibition activity exceeds that of CA28. Table 2

[00289] The solubility of CA28-1, CD28-2 and 3-CA28 in water with or without a variety of buffer substances and / or excipients exceeds that of the parent compound CA28 was observed. Example 3: Compstatin Analogs of Extended Action that Demonstrate Plasma Half-Life and Dramatically Increased Cmax
[00290] This example describes the determination of pharmacokinetic parameters of CA28-2 and CA28-3 of long-acting compstatin analogs after administration to Cynomolgus monkeys. Dosage and Sample Collection
[00291] CA28-2 and 3-CA28 were administered at time 0 through intravenous injection in female Cynomolgus monkeys (three per group, 2-5 years old, 2.9-3.5 kg). Compounds were administered at 50 mg / kg, in 5% dextrose in water at a concentration of 25 mg / ml. Blood specimens (~ 1 ML each) were collected from the femoral vein for the following moments: Pre-dose, 5 min, 15 min, 30 min, 1 hour (h), 4 h, 8 h, 24 h, 48 h, 96 h (4 days) and 192 h (8 days) post dose. Specimens were collected by direct venipuncture and placed in a red top serum tube containing no anticoagulant and kept at room temperature for at least 30 minutes. Blood samples were centrifuged at a temperature of 4 ° C to 3000 x g for 5 minutes. The samples were kept refrigerated throughout the processing. Serum samples were collected after centrifugation and placed in sample tubes. The samples were stored in a freezer to maintain -60 ° C-80 ° C. All animals showed normal activity throughout the study. No anomalies related to the compound were observed in the animals throughout the study.
[00292] Analysis of the Sample. The plasma samples obtained as described above were analyzed by LC / MS / MS using the following methods to determine the concentration of the compound: 50 μL of sample was mixed with internal standard (CA28-AEEAc-Arg) and 100 μl of 1 M NH4OAc, pH 3.5 with HOAc was added and mixed. Then, 250 μl of acetonitrile was added and mixed. The sample was centrifuged and supernatant poured into another tube and dried. The sample was reconstituted and injected into the LC / MS / MS system. Mobile Phase A was 5mm NH4OAc with 0.1% FA and Mobile Phase B was 90:10 (ACN: 50 mM NH4OAc) with 0.1% FA. The LC column was the WP-RP Intra 2 x 150 mm, 3 μ. The quantification was based on a triple quadripolar mass spectrometer of applied API-4000 biosystem operated in positive ion mode. collision-induced dissociation at the origin (ICD) was used to fragment the compound in the mass spectrometer source and the 144 m / z ion was selected by mass in Q1, fragmented and the 77 m / z ion selected by mass in Q3 and detected. Data were processed using the Analyst 1.4.2 software.
[00293] Results. The serum concentrations in micrograms / ml of CA28-2 and 3-CA28 at each time point are shown in Table 3 below. Data for each of the 3 monkeys that received the indicated compound are shown. The average values and standard deviations are calculated promptly. There was remarkable consistency between animals. CA28 are historical data obtained in a previous study in which CA28 was administered intravenously to Cynomo-lgus monkeys. In that study, CA28 was detected in samples using HPLC. Table 3

[00294] The results for each compound were averaged and are plotted in Figure 3. There was a noticeable increase in half-life and Cmax for CA28-2 and CA28-3 compared to CA28. The terminal half-lives of CA28-2 and CA28-3 were around 4 - 4.5 days. Based on these data, it is expected that intravenous administration at dosage intervals of approximately 1-2 weeks will provide sustained levels of compound and will effectively inhibit complement activation in humans, although shorter or longer dosing intervals may be used. Example 4: Long-acting Compstatin Analog Understanding HSA as a Clearing Reducing Portion
[00295] Human serum albumin (HSA) side chain lysines were converted to thiols using 2-iminothiolane and reacted with a compstatin analogue comprising a maleimide as a reactive functional group: Ac-Ile-Cys * -Val-Trp ( 1-Me) -Gln-Asp- Trp-Gly-Ala-His-Arg-Cys * -Thr-AEEAc-Lys- (C (= O) - (CH2) 5-Mal) -NH2 (SEQ ID NO: 68 ). The resulting long-acting compstatin analog (CA28-4) was tested in vitro for complement inhibitory activity (Figure 4), as described in Example 1 and in vivo for pharmacokinetic properties as described in Example 3. CA28-4 pharmacokinetic parameters after administration to Cynomolgus monkeys they were determined as described in the previous example. The results are shown in Figure 5 (together with the results for CA28, CA28-1, CA28-2 and CA28-3). PK data for CA28-4 are shown in Table 4. Table 4 Serum concentration in ug / mL
Example 5: Compstatin Analogs of Extended Action in Patients with PNH
[00296] A cohort of individuals diagnosed with PNH is divided into 4 groups. Subjects in Groups 1 and 2 are treated with intravenous administration of CA28-2 or CA28-3, respectively, at a dose of between 5 mg / kg and 20 mg / kg, at intervals of between 1 and 2 weeks. Optionally, treatment is started at more frequent intervals and then reduced in frequency for maintenance therapy. Subjects in Group 3 are treated with eculizumab, according to the recommended dosage regimen. Intravascular hemolysis (based on LDH measurement and / or labeling (51) Cr of red blood cells), reticulocytosis (an indicator of anemia), hematocrit, hemoglobin concentration in the blood, opsonization of red blood cells (deposition of products activation of C3, such as C3b, in red blood cells, which can be detected using flow cytometry), PNH symptoms, transfusion needs, thromboembolic events, quality of life and survival are monitored over time. The results are compared between the groups and with the historical data of control PNH patients obtained in eculizumab clinical trials. An improvement in persistent anemia (for example, as evidenced by reduced reticulocyte, reduced evidence of hemolysis, increased hematocrit, increased hemoglobin), higher quality of life, reduced symptoms of nocturnal paroxysmal hemoglobinuria, reduced transfusion requirements, reduced thromboembolic events, increased quality of life and / or increased survival, in individuals receiving CA28-2 (Group 1) or CA28-3 (Group 2), compared to individuals in Group 4 is indicative of effectiveness. Example 6: Compstatin Analogs of Extended Action in Patients with PNH
[00297] Example 4 is repeated with the modification that subjects are individuals with PNH who remain transfusion dependent and / or continue to have a hemoglobin below a threshold (such as 9.0 g / dL) despite treatment with eculizumab. The results are compared between groups. Example 7: Compstatin Analogs of Extended Action in Patients with aHUS
[00298] A cohort of individuals diagnosed with aHUS is divided into 4 groups. Subjects in groups 1 and 2 are treated with intravenous administration of CA28-2 or CA28-3, respectively, at a dose of between 5 mg / kg and 20 mg / kg, at intervals of between 1 and 2 weeks. Optionally, treatment is started at more frequent intervals and then reduced in frequency for maintenance therapy. Subjects in Group 3 are treated with eculizumab, according to the recommended dosage regimen. Intravascular hemolysis (based on LDH measurement), opsonization of red blood cells (deposition of C3 activation products, such as C3b, in red blood cells), symptoms of aHUS, kidney function, need for plasma exchange or dialysis, quality of life and survival are monitored over time. The results are compared between groups and with the historical data of control aHUS patients obtained in eculizumab clinical trials. Reduced evidence of hemolysis, improved quality of life, reduced aHUS symptoms, less need for plasma or dialysis replacement, higher quality of life or increased survival in individuals receiving CA28-2 or CA28-3 compared to individuals in the group 4 are indicative of effectiveness. Example 8: Examples 5-7 are repeated using additional long-acting compstatin analogs. Example 9: Examples 5-7 are repeated using cell reactive compstatin analogs.
[00299] Those skilled in the art will recognize or be able to verify using no more than routine experimentation, many equivalents for the specific modalities of the invention described here. The scope of the present invention is not intended to be limited to the above description, but is preferably as set out in the added claims. It will be noted that the invention does not depend in any way on specific results achieved in any specific example or with any specific modality. Articles such as "the" and "one" may mean one or more of one, unless otherwise indicated or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one or all members of the group are present in, employed in or otherwise relevant to a particular product or process, unless otherwise indicated or otherwise evident from the context. The invention includes modalities in which exactly one member of the group is present in, used in or otherwise relevant to a particular product or process. For example, and without restriction, it is understood that when the claims or description indicate that a residue in a specific position can be selected from a certain group of amino acids or amino acid analogs, the invention includes individual embodiments in which the residue in which the position is any of the listed amino acids or amino acid analogues. The invention also includes modes in which more than one or all members of the group are present in, used in or otherwise relevant to a particular product or process. In addition, it should be understood that the invention encompasses all variations, combinations and permutations in which one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the claims listed or from the description above, another claim is introduced. For example, any claim that is dependent on another claim can be modified to include one or more elements, limitations, clauses or descriptive terms found in any other claim that is dependent on the same underlying claim. In addition, when the claims cite a composition, it is to be understood that methods for administering the composition according to any of the methods disclosed in this document and methods for using the composition for any of the purposes disclosed in this document are included in the scope of the invention, and methods of making the composition according to any of the manufacturing methods disclosed in this document are included within the scope of the invention, unless otherwise indicated or unless it is evident to one skilled in the art that a contradiction or inconsistency would arise. Methods of treating a subject may include a step of caring for a subject who needs such treatment (for example, a subject who has, or is at increased risk of, having a disease), a step of diagnosing a subject as having a disease and / or a step of selecting a subject for treatment with a cell reactive compound analog.
[00300] When the elements are presented as lists, it should be understood that each subgroup of the elements is also disclosed, and any element can be removed from the group. For the sake of brevity, only a portion of these modalities has been specifically cited here, but the invention includes all of these modalities. It should also be understood that, in general, where the invention, or aspects of the invention, is / are said to comprise specific elements, features etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, resources etc. The discussion of various diseases, disorders and conditions under various headings here is for convenience and is not intended to limit the invention.
[00301] When intervals are given, terms are included. In addition, it should be understood that unless stated otherwise, or otherwise evident from the context and understanding of the person skilled in the art, values that are expressed as intervals can assume any specific value or subscale within the stated scales in different embodiments of the invention, to the tenth of the unit of the lower limit of the scale, unless the context clearly dictates otherwise. Any modality, aspect, element, resource, etc. specific to the present invention can be explicitly excluded from the claims even if that exclusion is not explicitly stated in this document. For example, any compstatin analogue, functional group, ligand parcel, disease or indication can be explicitly excluded.

权利要求:
Claims (21)
[0001]
1. Compstatin analogue, characterized by the fact that it comprises: one or more portions reducing the clearance linked to one or more portions of compstatin analogue, wherein each portion of compstatin analogue comprises a cyclic peptide having an amino acid sequence defined in any of SEQ ID NOs: 3-36, extended by one or more terminal amino acids at the N-terminal, C-terminal or both; wherein the one or more amino acids has a side chain comprising a primary or secondary amine and is separated from the cyclic peptide by a rigid or flexible spacer comprising an oligo (ethylene glycol) portion which is (- (O-CH2CH2-) n), where n is between 1 and 500, so that the portion analogous to compstatin comprises the cyclic peptide, linked through its N-terminal or C-terminal to the spacer, which is linked to one or more amino acids; and each scrubbing reducing portion comprises a polyethylene glycol (PEG) and is covalently linked via a side chain binding portion comprising a primary or a secondary amine, wherein the linking portion comprises an unsaturated alkyl portion, a moiety comprising a non-aromatic cyclic ring system, an aromatic portion, an ether portion, an amide portion, an ester portion, a carbonyl portion, an imine portion, a thioether portion, or an amino acid residue, wherein the clearance reducing portion is and the spacer are separated by one or more amino acids comprising a side chain having a primary or secondary amine, so that the compstatin analog comprises the cyclic peptide linked through its N-terminal or C-terminal to the spacer, which is linked to one or more amino acids, which in turn is linked to the binding portion and the clearance reducing portion.
[0002]
2. Compstatin analogue, characterized by the fact that it comprises one or more portions reducing the clearance linked to one or more portions of compstatin analogue, in which: each analogue portion of compstatin comprises a cyclic peptide having an amino acid sequence defined in SEQ ID NO: 28, 32 or 34, extended by one or more terminal amino acids at the N-terminal, the C-terminal or both; wherein the one or more amino acids has a side chain comprising a primary or secondary amine and is separated from the cyclic peptide by a rigid or flexible spacer comprising an oligo (ethylene glycol) moiety which is (- (O-CH2CH2-) n ), where n is between 1 and 500, so that the compstatin-like portion comprises the cyclic peptide, linked through its N-terminal or C-terminal to the spacer, which is linked to one or more amino acids; and each clearance-reducing portion is or comprises a polyethylene glycol (PEG), the Fc domain of an immunoglobulin, an albumin portion, or an albumin-binding peptide and is covalently linked via a side-chain binding portion comprising a primary or secondary amine, wherein the linking portion comprises an unsaturated alkyl portion, a portion comprising a non-aromatic cyclic ring system, an aromatic portion, an ether portion, an amide portion, an ester portion, a carbonyl portion, a portion imine, a thioether moiety or an amino acid residue, wherein the clearance reducing moiety and the spacer are separated by one or more amino acids comprising a side chain having a primary or secondary amine, so that the compstatin analog comprises the peptide cyclic peptide linked via its N-terminal or C-terminal to the spacer, which is linked to one or more amino acids, which in turn is linked to the binding portion and the reducing portion of the clearance.
[0003]
Compstatin analogue according to claim 2, characterized in that the one or more terminal amino acids is or comprises a Lys.
[0004]
Compostatin analogue according to claim 2, characterized in that the oligo (ethylene glycol) portion is (- (O-CH2-CH2-) n) where n is between 1 and 10.
[0005]
Compostatin analog according to claim 2, characterized by the fact that the spacer comprises - (CH2) m- and - (O-CH2-CH2-) n covalently bonded, where m is between 1 and 10 and n is between 1 and 10.
[0006]
Compostatin analog according to claim 2, characterized in that the spacer comprises NH2 (CH2CH2O) nCH2C (= O) OH, where n is between 1 and 500, or an NHS ester thereof.
[0007]
Compostatin analogue according to claim 6, characterized in that the spacer comprises 8-amino-3,6-dioxaoctanoic acid (AEEAc), 11-amino-3,6,9-trioxaundecanoic acid, or an ester NHS of both.
[0008]
Compostatin analogue according to claim 2, characterized in that the clearance reducing portion is or comprises a PEG.
[0009]
Compostatin analog according to claim 8, characterized by the fact that PEG is a linear PEG.
[0010]
10. Compostatin analog according to claim 8, characterized by the fact that PEG is a branched PEG.
[0011]
Compostatin analogue according to claim 8, characterized in that the compstatin analogue comprises multiple modified PEG or PEG portions.
[0012]
Compostatin analogue according to claim 8, characterized in that the compstatin analogue comprises one or more portions of compstatin analogue linked to a bifunctional PEG portion.
[0013]
13. Compostatin analog according to claim 2, characterized in that the compstatin analog comprises multiple portions analogous to compstatin.
[0014]
Compostatin analogue according to claim 13, characterized in that it is a multivalent compound comprising a plurality of compostatin analog portions covalently linked to a polymeric backbone or support.
[0015]
Compstatin analog according to claim 9, characterized in that an analogous portion of compstatin is attached to each end of a linear PEG.
[0016]
Compstatin analog according to claim 1, characterized in that the cyclic peptide has an amino acid sequence as defined in SEQ ID NO: 28, 32 or 34.
[0017]
17. Compostatin analog according to claim 1, characterized by the fact that the cyclic peptide has an N-methylGly in a position corresponding to position 8 of SEQ ID NO: 8.
[0018]
18. A compostatin analogue according to claim 2, characterized in that the cyclic peptide has an amino acid sequence as defined in SEQ ID NO: 28.
[0019]
19. Compostatin analogue according to claim 2, characterized by the fact that it has a plasma half-life of at least 2 days when injected intravenously into a primate.
[0020]
20. Compostatin analogue according to claim 19, characterized by the fact that it has a plasma half-life of at least 3 days when injected intravenously into a primate.
[0021]
21. Compostatin analog according to claim 20, characterized by the fact that it has a plasma half-life of at least 4 days when injected intravenously into a primate.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-03-19| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-18| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-09-15| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-15| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 11/05/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161484836P| true| 2011-05-11|2011-05-11|

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US61/484,836|2011-05-11|

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PCT/US2012/037648|WO2012155107A1|2011-05-11|2012-05-11|Cell-reactive, long-acting, or targeted compstatin analogs and uses thereof|

---