WO2014184564A1 - Nouveaux conjugués polymères - Google Patents

Nouveaux conjugués polymères Download PDF

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WO2014184564A1
WO2014184564A1 PCT/GB2014/051493 GB2014051493W WO2014184564A1 WO 2014184564 A1 WO2014184564 A1 WO 2014184564A1 GB 2014051493 W GB2014051493 W GB 2014051493W WO 2014184564 A1 WO2014184564 A1 WO 2014184564A1
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trkad5
conjugate
polymer
group
ngf
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Keith Powell
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Abzena UK Ltd
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Polytherics Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Definitions

  • Novel Polymer Conjugates This invention relates to novel polymer conjugates, and a process for their preparation.
  • the neurotrophin Nerve Growth Factor has been recognised as a key modulator of inflammatory and nociceptive responses (Watson JJ et al., BioDrugs. 2008;22(6):349-59).
  • NGF neurotrophin Nerve Growth Factor
  • an association between increased NGF levels and a variety of inflammatory conditions has been observed in human patients as well as in several animal models. These include systemic lupus erythematosus (Bracci-Laudiero et al., Neuroreport 4:563-565 (1993)), multiple sclerosis (Bracci-Laudiero et al., Neurosci. Lett. 147:9-12 (1992)), psoriasis (Raychaudhuri et al., Acta Derm.
  • Chronic pain arising from inflammatory, neuropathic, or cancer pain presents a huge economic and social burden and consumes a large amount of healthcare resources around the world.
  • an estimated 70 million people are thought to live with chronic pain, at a cost of more than US$100 billion in lost productivity and direct medical costs annually (A Call to Revolutionize Chronic Pain Care in America: An Opportunity in Health Care Reform. The Mayday Fund Report. 2009).
  • At least three pharmacologic strategies are under development that target NGF-TrkA signalling and that have shown promise in preclinical models. These are: sequestration of NGF or inhibiting its binding to TrkA, antagonising TrkA so as to block NGF from binding to TrkA, and blocking TrkA kinase activity (Mantyh, PW et al., Anesthesiology. 2011 Jul;115(l): 189-204).
  • WO2008/013782 describes how NGF, through its cognate receptor TrkA, induces Sp35 expression, resulting in inhibition of axonal myelination and oligodendrocyte differentiation via Sp35 function. Based on these discoveries, WO2008/013782 discloses methods of promoting myelination and increased neuronal survival and treating demyelination by administration of a TrkA antagonist.
  • the TrkA antagonist may be any molecule which interferes with the ability of TrkA to negatively regulate CNS myelination and/or neuronal survival and/or inhibiting or decreasing Sp35 expression.
  • TrkA-IgG fusion proteins were administered have shown that sequestration of endogenous NGF is able to block the hyperalgesia associated with inflammation (McMahon SB et al., Nat Med. 1995 Aug;l(8):774-80).
  • Tanezumab mainly reduces the chronic pain associated with a condition, but the underlying cause of the disease persists in many cases. Accordingly, overuse of the joints after improvements in pain due to tanezumab therapy is one possible explanation. Tanezumab shows tight binding to NGF and the complex has a half-life of more than 100 hours (Abdich, YN et al., Protein Sci. 2008 Aug; 17(8): 1326-35). Other possible explanations include a direct anti-NGF effect on bone, or negative effects of NGF blockage on blood vessels leading to avascular necrosis (Garber, K., Nat Biotechnol. 2011 Mar;29(3): 173-4). In March 2012, the FDA recommended the reversal of the clinical hold, with additional safeguards.
  • TrkA domain 5 A small, 13.5 kDa domain of the TrkA receptor (TrkA domain 5, or TrkAd5), capable of sequestering NGF in an in vivo model of cystitis and an in vitro model of allergic asthma and of reversing symptoms associated with these conditions has also been described (Watson, JJ et al., J Pharmacol Exp Ther. 2006 Mar;316(3): 1122-9). TrkAd5 has also been shown to be effective against pain in experimentally induced osteoarthritis in rats (McNamee, KE et al., Pain. 2010 May;149(2):386-92).
  • TrkAd5 (referred to as TrkAIg2)
  • TrkAIg2 TrkAd5
  • TrkAIg2 TrkAd5
  • TrkAIg2 TrkAd5
  • TrkAIg2 TrkAd5
  • TrkAIg2 TrkAd5
  • TrkAIg2 TrkAd5
  • TrkAIg2 TrkAd5
  • TrkAIg2 TrkAd5
  • TrkAIg2 TrkAd5 protein has a short half-life in vivo. Accordingly, while TrkAd5 has previously been shown to sequester NGF and alleviate the associated hyperalgesia in preclinical animal models, its short half-life meant that TrkAd5 was not suitable as a therapeutic for alleviating pain, especially chronic pain, where a prolonged analgesic effect is required.
  • the ability of TrkAd5 to sequester NGF has been used to encourage growth and/or repair of neuronal tissue.
  • WO2004/071545 discloses that, under normal conditions, levels of circulating growth factors in the body are too low to be effective for nerve regeneration. To address this, biotinylated TrkAd5 is used to immobilise NGF to the surface of a
  • biocompatible scaffold providing a structure upon, which or through which nerve cells may be supported for growth, regeneration or repair. Also disclosed is use of the implantable polymeric scaffold as a reservoir for the controlled release of TrkAd5, for treating a condition associated with elevated neurotrophin levels such as inflammation or pain.
  • therapeutically active molecules for example proteins and peptides
  • proteins and peptides do not possess the properties required to achieve efficacy in medical use.
  • some molecules which might find utility as active therapeutic agents in medicines are systemically toxic or lack optimal bioavailability and pharmacokinetics.
  • proteins clear from the blood circulation quickly they typically have to be administered to the patient frequently. Frequent administration further increases the risk of toxicity, especially immunologically derived toxicities.
  • PEG polyethylene glycol
  • PEGylation and many PEGylating reagents are known, for example from WO 99/45964, WO 2005/007197, and WO 2009/047500.
  • PEGylating reagents have been shown to alter the pharmacokinetics of therapeutic proteins favourably by prolonging circulation time and decreasing clearance rates by increasing the effective size of the protein, decreasing systemic toxicity, and in several cases, displaying increased clinical efficacy.
  • PEGylation impairs the pharmacological activity of the protein, resulting from loss in binding affinity due to steric interference with the drug-target binding interaction (Fishburn CS. J Pharm Sci. 2008 Oct;97(10):4167-83). It is also commonly found that PEGylation can reduce the intrinsic in vivo activity of some proteins. This loss of potency is offset by the prolonged circulation time, and thus the combination of decreased activity at the target receptor or enzyme with increased plasma half-life can translate to increased in vivo efficacy. The level of in vivo efficacy shown for a PEGylated molecule depends on the balance between the pharmacodynamic (PD) and pharmacokinetic (PK) properties of the conjugate.
  • PD pharmacodynamic
  • PK pharmacokinetic
  • PEGylated-TrkAd5 retains NGF sequestration activity at levels at least as efficacious as those shown by the unconjugated protein.
  • the present invention provides a conjugate of a TrkAd5 polypeptide with a water-soluble polymer, wherein the TrkAd5 comprises the amino acid sequence of residues 1 to 97 of SEQ ID NO: 1 or an analogue thereof.
  • conjugates according to the invention are expected to display favourable
  • TrkAd5 polypeptide should be understood to include any polypeptide comprising or consisting of the amino acid sequence of TrkA domain 5, also known as the Ig-like sub-domain 2 or TrkAIg2 of the extracellular region of the human TrkA receptor, and any analogue thereof, which has the ability to sequester NGF.
  • the putative extracellular region of human TrkA is 375 amino acids long (Holden, PH. et al (1997) Biotechnology 15: 668-672; Robertson AG, et al., Biochem Biophys Res Commun. 2001 Mar 23;282(1): 131-41).
  • a schematic representation of the TrkA extracellular region is shown in Figure 1A of Holden, PH.
  • TrkAd5 is shown as residues 253- 349 of the extracellular region, while the amino acid sequence of TrkAd5 corresponds to amino acids 120-216 as shown in Figure IB of Holden, PH et al.
  • the full amino acid canonical sequence of the human TrkA receptor is provided in UniProt database
  • TrkAd5 corresponds to amino acids 285-381 of that sequence.
  • the TrkAd5 polypeptide used in the present invention may additionally comprise the amino acid sequence of one or more parts of the TrkA receptor, such as the proline rich region of the extracellular region of TrkA (C-terminal to TrkAd5).
  • this proline rich region corresponds to residues 350-375 of the extracellular region shown in Figure 1A of Holden, PH. et al (1997), and has the amino acid sequence of amino acids 217-242 shown in Figure IB of Holden, PH et al.
  • This sequence corresponds to amino acids 382-407 of the TrkA-I Isoform of the human TrkA receptor provided in UniProt database under Identifier: P04629-2).
  • TrkA (UniProt Identifier: P04629-1, TrkA-II Isoform)
  • TrkA-II Isoform An alternatively spliced form of TrkA (UniProt Identifier: P04629-1, TrkA-II Isoform) is known, containing a six-amino acid insert VSFSPV in the proline rich region (Robertson AG, et al., Biochem Biophys Res Commun. 2001 Mar 23;282(1): 131-41).
  • the proline rich region of this splice form corresponds to amino acids 382-413 of the sequence of UniProt Identifier: P04629-1.
  • TrkAd5 polypeptides are referred to herein as "TrkAd5".
  • the TrkAd5 used in the present invention comprises or consists of the amino acid sequence of residues 1 to 97 of SEQ ID NO: 1 or an analogue thereof.
  • the TrkAd5 comprises or consists of the amino acid sequence of residues 1 to 123 of SEQ ID NO: 1 or residues 1 to 129 of SEQ ID NO: 2 or analogues thereof.
  • SEQ ID NO: 1 shows the amino acid sequence of human TrkAd5 and the proline rich region.
  • SEQ ID NO: 2 shows the amino acid sequence of a splice variant of TrkAd5 including a six amino acid insert in the proline rich region.
  • additional terminal amino acid(s) may be present, and such sequences should be understood to be included in the definition of the TrkAd5 polypeptide used herein.
  • the TrkAd5 used in the present invention may also optionally be modified by the addition of one or more amino acids at the N or C terminus of the chain.
  • the TrkAd5 polypeptide may additionally comprise one or more of the amino acid residues flanking the core sequence of amino acids 285-381 of UniProt Identifier: P04629.
  • the N- terminal end of the TrkAd5 may comprise amino acids 284, 283-284, 282-284 or 280-284 of the TrkA sequence of UniProt Identifier: P04629.
  • the C- terminal end of the TrkAd5 may comprise amino acids 382, 382-383, 382-384 or 382-386 of the TrkA sequence of UniProt Identifier: P04629.
  • the TrkAd5 used in the present invention may optionally also include a polyhistidine tag, in which a number of histidine residues, for example 2 or more, for example up to 12, preferably up to 9, more preferably up to 6, histidine residues may be introduced.
  • a polyhistidine tag in which a number of histidine residues, for example 2 or more, for example up to 12, preferably up to 9, more preferably up to 6, histidine residues may be introduced.
  • analogue used in relation to TrkAd5 refers to functional portions and derivatives of the natural TrkAd5 sequence, and any polypeptide substantially homologous thereto, that differs by the substitution, insertion or deletion of one or more amino acids, but that has substantially the same activity or function as the unmodified sequence or partial sequence, i.e., the ability to sequester NGF. Methods for testing the activity or function of analogues of TrkAd5 are described in the Examples below.
  • Derivatives of TrkAd5 also include sequences from other biological sources such as mammals, birds (for example chicken), insects, reptiles or amphibian.
  • substantially homologous denotes a characteristic of an amino acid sequence, wherein a selected amino acid sequence has at least about 70 or about 75 percent sequence identity as compared to a selected reference amino acid sequence.
  • the amino acid sequence has a sequence identity of at least 80%, more preferably at least 90% and most preferably at least 95% compared to a selected reference amino acid sequence. More preferably still, highly homologous sequences often share greater than at least about 96, 97, 98, or 99 percent sequence identity between the selected sequence and the reference sequence to which it was compared.
  • the percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions which total less than about 25 percent or so of the chosen reference sequence, for example using sequence comparison algorithms well-known to those of skill in the art, such as, the FASTA program analysis described by Pearson and Lipman (1988) and the gapped BLAST algorithm (e.g., Altschul et al. Nucleic Acid Res. (1997)25: 3389) which weights sequence gaps and sequence mismatches according to the default weightings provided by the National Institutes of Health/ NCBI database (Bethesda, MD; see www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast).
  • amino acid changes in the polypeptide sequence are conservative.
  • “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, and valine; glycine, and alanine; asparagine and glutamine; and serine, threonine,
  • phenylalanine, and tyrosine Other groups of amino acids that may represent conservative changes include (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • Amino acids may be classified according to the nature of their side groups.
  • Amino acids with nonpolar alkyl side groups include glycine, alanine, valine, leucine, and isoleucine.
  • Serine and threonine have hydroxyl groups on their side chains, and because hydroxyl groups are polar and capable of hydrogen bonding, these amino acids are hydrophilic.
  • Sulfur groups may be found in methionine and cysteine.
  • Carboxylic acid groups are part of the side chain of aspartic acid and glutamic acid, which because of the acidity of the carboxylic acid group, the amino acids are not only polar but can become negatively charged in solution.
  • Glutamine and asparagine are similar to glutamic acid and aspartic acid, except the side chains contain amide groups. Lysine, arginine, and histidine have one or more amino groups in their side chains which can accept protons, and thus these amino acids act as bases. Aromatic groups may be found on the side chains of phenylalanine, tyrosine, and tryptophan. Tyrosine is polar because of its hydroxyl group, but tryptophan and phenylalanine are non-polar. A
  • polypeptide may also, or alternatively, contain non-conservative changes.
  • the polypeptide chain of TrkAd5 is unmodified from the full human sequence or partial sequence, or is unmodified other than by the addition of a polyhistidine tag, for example at the N or C terminus, or elsewhere along the main chain.
  • TrkAd5 may exist in monomeric, dimeric, or aggregate form. In the conjugates of the invention, TrkAd5 is preferably in monomeric form.
  • TrkAIg2 Methods for the preparation and purification of TrkAd5 are described in WO 99/53055 and an improved purification method is described in WO 03/025016 (in each of which TrkAd5 is referred to as TrkAIg2), the contents of which are herein incorporated by reference.
  • the polymer may be bonded to TrkAd5 at any suitable point.
  • the polymer chain may be bonded via amine groups in the TrkAd5 (e.g. via lysine or histidine residues), via the N-terminal group, via thiol groups (i.e.
  • TrkAd5 via cysteine residues
  • non-naturally encoded amino acids incorporated into TrkAd5 such as those disclosed in paragraphs [21] and [22] of WO 2006/009901.
  • the polymer may be bonded to residues present in the unmodified TrkAd5 sequence, or residues that have been artificially introduced. Where the bonding is via amine groups, these are preferably artificially introduced as histidine residues in the form of a polyhistidine tag.
  • the attachment can be on the N-terminal group of the unmodified TrkAd5; such attachment may be achieved for example by reductive amination.
  • the bonding is via thiol groups, these groups are preferably provided by reduction of a disulfide bridge or via free cysteine residues present in the native or modified sequence.
  • the conjugates of the invention may contain one or more separate polymer chains, and a polymer chain may be linear or branched. If two or more chains are present, these may be the same or different.
  • a polymer chain may be bonded to a single amino acid residue, or to two amino acid residues. Advantages may be obtained when a polymer chain is bonded to two amino acid residues.
  • the conjugate comprises one polymer chain bound to a polyhistidine tag.
  • the polymer is preferably bound to a 6 x His tag, present at the N or C terminus, or elsewhere along the main chain.
  • the conjugate comprises one polymer chain bound via a disulfide bridge.
  • a polymer may for example be a polyalkylene glycol, a polyvinylpyrrolidone, a polyacrylate, for example polyacryloyl morpholine, a polymethacrylate, a polyoxazoline, a
  • polyvinylalcohol a polyacrylamide or polymethacrylamide, for example
  • polycarboxymethacrylamide or a HPMA copolymer.
  • the polymer may be a polymer that is susceptible to enzymatic or hydrolytic degradation.
  • Such polymers include polyesters, polyacetals, poly(ortho esters), polycarbonates, poly(imino carbonates), and polyamides, such as poly(amino acids).
  • a polymer may be a homopolymer, random copolymer or a structurally defined copolymer such as a block copolymer, for example it may be a block copolymer derived from two or more alkylene oxides, or from poly(alkylene oxide) and either a polyester, polyacetal, poly(ortho ester), or a poly(amino acid).
  • Polyfunctional polymers that may be used include copolymers of divinylether-maleic anhydride and styrene-maleic anhydride.
  • Naturally occurring polymers may also be used, for example polysaccharides such as chitin, dextran, dextrin, chitosan, starch, cellulose, glycogen, poly(sialylic acid), hyaluronic acid and derivatives thereof.
  • a protein may be used as the polymer.
  • Polymers such as polyglutamic acid may also be used, as may hybrid polymers derived from natural monomers such as saccharides or amino acids and synthetic monomers such as ethylene oxide or methacrylic acid.
  • the polymer is a polyalkylene glycol, this is preferably one containing C 2 and/or C 3 units, and is especially a polyethylene glycol.
  • a polymer, particularly a polyalkylene glycol may contain a single linear chain, or it may have branched morphology composed of many chains either small or large.
  • Pluronics are an important class of PEG block copolymers. These are derived from ethylene oxide and propylene oxide blocks. Substituted, or capped, polyalkylene glycols, for example methoxypolyethylene glycol, may be used.
  • the polymer may, for example, be a comb polymer produced by the method described in WO 2004/113394, the contents of which are incorporated herein by reference.
  • the polymer may be a comb polymer having a general formula:
  • A may or may not be present and is a moiety capable of binding to a protein or a polypeptide
  • D where present, is obtainable by additional polymerisation of one or more olefinically unsaturated monomers which are not as defined in E;
  • E is obtainable by additional polymerisation of a plurality of monomers which are linear, branched, or star-shaped substituted or non-substituted, and have an olefinically unsaturated moiety;
  • F where present, is obtainable by additional polymerisation of one or more olefinically-unsaturated monomers which are not as defined in E;
  • d and f are an integer between 0 and 500;
  • e is an integer of 0 to 1000;
  • Water soluble polymers are widely used to conjugate therapeutically active molecules such as proteins.
  • Water soluble polymers are generally those that are soluble in water or an aqueous medium under ambient conditions of room temperature and atmospheric pressure at a pH of between about 6 and 8, i.e., at about neutral or physiological pH.
  • the polymer has a solubility in water, measured at 25°C, of at least about 0.1 gram/litre (g/L), preferably more than 0.3 gram/litre, most preferably more than 0.5 gram/litre.
  • the polymers have solubility in water, measured at 25°C, of from about 0.1 gram/litre (g/L) to about 500 grams/litre (g/L).
  • the polymer may optionally be derivatised or functionalised in any desired way.
  • the polymer carries a diagnostic agent, an additional therapeutic agent, or a labelling agent, or a binding agent capable of binding a diagnostic agent, additional therapeutic agent, or labelling agent.
  • Reactive groups may be linked at the polymer terminus or end group, or along the polymer chain through pendent linkers; in such case, the polymer is for example a polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate, or a maleic anhydride copolymer.
  • Multimeric conjugates that contain more than one biological molecule can result in synergistic and additive benefits.
  • the conjugate may comprise two or more TrkAd5 molecules.
  • the polymer may be coupled to a solid support using conventional methods.
  • the optimum molecular weight of the polymer will of course depend upon the intended application and duration of analgesic effect that is required.
  • Long-chain polymers may be used, for example the number average molecular weight may be in the range of from
  • each chain may have the same or different molecular weight as any other.
  • the one or more polymer chains may have a molecular weight of at least 5, 10, 15, 20, 30, or 40 kDa.
  • Very small oligomers consisting for example of as few as 2 repeat units, for example from 2 to 20 repeat units, are useful for some applications.
  • the conjugate is intended to leave the circulation and penetrate tissue, for example for use in the treatment or alleviation of inflammatory pain caused by malignancy, infection or autoimmune disease, or by trauma, it may be advantageous to use a lower molecular weight polymer in the range up to
  • the present invention also provides a process for the preparation of a conjugate according to the invention, which comprises reacting TrkAd5 with a polymer conjugating reagent. Any suitable conjugation reagent may be used.
  • PEG-benzotriazolyl carbonate PEG carboxylates and PEG esters (e.g. PEG-succinimidyl ester and PEG-p-nitrophenyl ester and their derivatives); PEG aldehydes; PEG-tresyl or -tosyl; PEG-dichlortriazine or -chlorotriazine; PEG vinyl sulfone; PEG maleimide; and PEG- iodoacetamide; and corresponding reagents containing polymers other than PEG.
  • PEG-benzotriazolyl carbonate PEG carboxylates and PEG esters (e.g. PEG-succinimidyl ester and PEG-p-nitrophenyl ester and their derivatives); PEG aldehydes; PEG-tresyl or -tosyl; PEG-dichlortriazine or -chlorotriazine; PEG vinyl sulfone
  • each L is a leaving group, for example one of those mentioned below.
  • Preferred leaving groups include halogen atoms, for example chlorine, bromine or iodine atoms, -S.CH 2 CH 2 OH groups, and -S-phenyl groups.
  • a further group of reagents include polymers functionalised with a linker having an N- succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with the TrkAd5, as well as a maleimido- or haloacetyl- based moiety for reaction with the polymer.
  • Crosslinking reagents comprising a maleimido-based moiety include, but is not limited to, N- succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N- maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate), which is a "long chain" analog of SMCC (LC-SMCC), ⁇ -maleimidoundecanoic acid N-succinimidyl ester (KMUA), ⁇ - maleimidobutyric acid N-succinimidyl ester (GMBS), ⁇ -maleimidocaproic acid N- hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(a-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl
  • Cross-linking reagents comprising a haloacetyl-based moiety include N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N- succinimidyl 3-(bromoacetamido propionate (SBAP).
  • SIAB N-succinimidyl-4-(iodoacetyl)-aminobenzoate
  • SIA N-succinimidyl iodoacetate
  • SBA N-succinimidyl bromoacetate
  • SBAP N- succinimidyl 3-(bromoacetamido propionate
  • the reagent may be one which conjugates to thiol groups or amine groups, for example lysine or histidine groups.
  • the conjugation reagent is one which conjugates to histidine groups present in a polyhistidine tag, or to thiol groups, especially to two thiol groups derived from a disulfide bridge.
  • Thiol conjugation reagents include for example compounds of the formula (I), (II) and (III) defined below, maleimides, including those of formula VI above, the "PermaLink" (Trade Mark) reagents, and compounds of the formula IV and V above.
  • Polymer conjugation reagents in which the polymer chain becomes bound to two amino acid residues, for example to two histidine residues in a polyhistidine tag, or to two cysteine residues, are particularly preferred.
  • Such reagents include those of formula I, II, III and VI.
  • the reagent may be one of the reagents described in WO 99/45964, WO 2005/007197, or WO 2010/100430, the contents of which are incorporated herein by reference.
  • a preferred polymer conjugating reagent has the formula I, II or III below:
  • W represents an electron- withdrawing group, for example a keto group, an ester group
  • X-Q-W together may represent an electron withdrawing group
  • A represents a Ci_5 alkylene or alkenylene chain
  • B represents a bond or a Ci_ 4 alkylene or alkenylene chain
  • each L independently represents a leaving group
  • R 1 represents a hydrogen atom or a Ci_ 4 alkyl group, R 1 represents a hydrogen atom, and each of L and L' independently represents a leaving group; or
  • R 1 represents a hydrogen atom or a Ci_ 4 alkyl group, L represents a leaving group, and R 1 and L' together represent a bond; or
  • R 1 and L together represent a bond and R 1 and L' together represent a bond
  • R represents a hydrogen atom or a Ci_ 4 alkyl group.
  • TrkAd5 is linked to the rest of the molecule either via a polyhistidine tag, or via two thiol groups derived from the disulfide bond present in TrkAd5.
  • a linking group Q may for example be a direct bond, an alkylene group (preferably a Ci_io alkylene group), or an optionally-substituted aryl or heteroaryl group, any of which may be terminated or interrupted by one or more oxygen atoms, sulfur atoms, -NR groups (in which R represents a hydrogen atom or an alkyl (preferably Ci_ 6 alkyl), aryl (preferably phenyl), or alkyl-aryl (preferably Ci_ 6 alkyl -phenyl) group), keto groups, -O-CO- groups, -CO-O- groups, -0-CO-O, -0-CO-NR-, -NR-CO-0-, -CO-NR- and/or -NR.CO- groups.
  • Suitable aryl groups include phenyl and naphthyl groups, while suitable heteroaryl groups include pyridine, pyrrole, furan, pyran, imidazole, pyrazole, oxazole, pyridazine, primidine and purine.
  • linking groups Q are heteroaryl or, especially, aryl groups, especially phenyl groups, terminated adjacent the polymer X by a -CO.NR- or, especially, an -NR.CO- group.
  • the linkage to the polymer may be by way of a hydrolytically labile bond, or by a non-labile bond.
  • W may for example represent a keto group CO, an ester group -O-CO- or a sulfone group -SO 2 -; or, if X-Q-W- together represent an electron withdrawing group, this group may for example be a cyano group.
  • W represents a keto group.
  • X represent a polymer
  • X'-Q- represents a hydrogen atom
  • Substituents which may be present on an optionally substituted aryl or heteroaryl group include for example one or more of the same or different substituents selected from alkyl (preferably Ci_ 4 alkyl, especially methyl, optionally substituted by OH or C0 2 H), -CN, -N0 2 , -C0 2 R, -COH, -CH 2 OH, -COR, -OR, -OCOR, -OC0 2 R, -SR, -SOR, -S0 2 R, -NHCOR,
  • the presence of electron withdrawing substituents is especially preferred.
  • Preferred substituents include for example CN, N0 2 , -OR, -OCOR, -SR, -NHCOR, -NR.COR, -NHOH and -NR.COR.
  • An especially preferred polymer conjugation reagent is one of the general formula I which includes the functional group:
  • a specific, highly preferred conjugation reagent has the formula:
  • the PEG may optionally carry a diagnostic agent, a therapeutic agent, or a labelling agent, or a binding agent capable of binding a diagnostic agent, a therapeutic agent, or a labelling agent.
  • the immediate product of the conjugation process using one of the reagents described above is a conjugate which contains an electron- withdrawing group W.
  • the process of the invention is reversible under suitable conditions. This may be desirable for some applications, for example where rapid release of the protein is required, but for other applications, rapid release of the protein may be undesirable. It may therefore be desirable to stabilise the conjugates by reduction of the electron-withdrawing moiety W to give a moiety which prevents release of the protein. Accordingly, the process may comprise an additional optional step of reducing the electron withdrawing group W in the conjugate.
  • borohydride for example sodium borohydride, sodium cyanoborohydride, potassium borohydride or sodium triacetoxyborohydride
  • reducing agent include for example tin(II) chloride, alkoxides such as aluminium alkoxide, and lithium aluminium hydride.
  • a moiety W containing a keto group may be reduced to a moiety containing a CH(OH) group; an ether group CH.OR may be obtained by the reaction of a hydroxy group with an etherifying agent; an ester group CH.O.C(0)R may be obtained by the reaction of a hydroxy group with an acylating agent; an amine group CH.NH 2 , CH.NHR or CH.NR 2 may be prepared from a ketone by reductive amination; or an amide CH.NHC(0)R or CH.N(C(0)R) 2 may be formed by acylation of an amine.
  • a sulfone may be reduced to a sulfoxide, sulfide or thiol ether.
  • a cyano group may be reduced to an amine group.
  • a key feature of using the conjugation reagents of formulae I, II or III described above is that an a-methylene leaving group and a double bond are cross-conjugated with an electron withdrawing function that serves as a Michael activating moiety. If the leaving group is prone to elimination in the cross-functional reagent rather than to direct displacement and the electron- withdrawing group is a suitable activating moiety for the Michael reaction then sequential intramolecular bis-alkylation can occur by consecutive Michael and retro Michael reactions. The leaving moiety serves to mask a latent conjugated double bond that is not exposed until after the first alkylation has occurred and bis-alkylation results from sequential and interactive Michael and retro-Michael reactions.
  • the electron withdrawing group and the leaving group are optimally selected so bis-alkylation can occur by sequential Michael and retro-Michael reactions. It is also possible to prepare cross -functional alkylating agents with additional multiple bonds conjugated to the double bond or between the leaving group and the electron withdrawing group.
  • the reaction is carried out in an aqueous reaction medium.
  • the solvent may consist of water, or co- solvents may additionally be present.
  • the polymer conjugation reagents of formulae I, II or III may be used to conjugate either to a polyhistidine tag, or to two thiol groups derived from a disulfide bridge.
  • reaction involves reducing the disulfide bond and subsequently reacting the reduced product with the conjugating reagent.
  • Suitable reaction conditions are given in the references mentioned above.
  • the process may for example be carried out in a solvent or solvent mixture in which all reactants are soluble, suitably, in an aqueous reaction medium. This reaction medium may also be buffered, depending on the pH requirements of the nucleophile.
  • the optimum pH for the reaction will generally be at least 4.5, typically between about 5.0 and about 8.5, preferably about 5.0 to 7.5.
  • the optimal reaction conditions will of course depend upon the specific reactants employed. Reaction temperatures between 3-37°C are generally suitable. Reactions conducted in organic media (for example THF, ethyl acetate, acetone) are typically conducted at temperatures up to ambient.
  • the TrkAd5 can be effectively conjugated with the desired reagent using a stoichiometric equivalent or an excess of reagent. Excess reagent and the product can be easily separated during routine purification, for example by standard chromatography methods, e.g. ion exchange chromatography or size exclusion chromatography, diafiltration, or, when a polyhistidine tag is present, by separation using metal affinity chromatography, e.g. based on nickel or zinc.
  • standard chromatography methods e.g. ion exchange chromatography or size exclusion chromatography, diafiltration, or, when a polyhistidine tag is present, by separation using metal affinity chromatography, e.g. based on nickel or zinc.
  • Conjugates of a TrkAd5 polypeptide with a polymer, wherein the TrkAd5 comprises the amino acid sequence of residues 1 to 97 of SEQ ID NO: 1 or an analogue thereof, which are circulatable in the blood, are expected to display favourable pharmacokinetics due to prolonged circulation time and decreased clearance rate.
  • the remarkable finding that such conjugates retain NGF sequestration activity at levels at least as efficacious as those shown by the unconjugated protein makes the conjugates particularly suitable for use in therapy.
  • the conjugates of the invention may be used for treating or alleviating NGF- associated disorders, especially inflammatory conditions and acute and chronic pain, including lupus erythematosus, contact dermatitis, eczema, shingles, postherpetic neuralgia, hyperalgesia, irritable bowel disease, Crohn's disease, colitis, bladder cystitis, pancreatitis, multiple sclerosis, asthma, psoriasis, and arthritis, including chronic arthritis, osteo arthritis and rheumatoid arthritis, and pain of any etiology, including acute and chronic pain, in particular pain arising from inflammatory and neuropathic disorders, or cancer.
  • NGF-associated disorders especially inflammatory conditions and acute and chronic pain, including lupus erythematosus, contact dermatitis, eczema, shingles, postherpetic neuralgia, hyperalgesia, irritable bowel disease, Crohn's disease, colitis, bladder cystitis, pancre
  • the present invention therefore also provides a conjugate according to the invention for use in therapy, especially for treating or alleviating an inflammatory condition and/or pain; the use of a conjugate according to the invention for the manufacture of a medicament for the treatment or alleviation of an inflammatory conditions and/or pain; and a method of treating or alleviating an inflammatory condition and/or pain which comprises the administration to a patient, especially a human patient, of a conjugate according to the invention.
  • the conjugate can be administered to the patient prior to, simultaneously with, or after administration of another active agent, such as low dose NSAIDs or opioids.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a conjugate according to the invention together with a pharmaceutically acceptable carrier, optionally together with an additional active agent, for example in a form suitable for injection or infusion.
  • a pharmaceutically acceptable carrier optionally together with an additional active agent, for example in a form suitable for injection or infusion.
  • Particular pain conditions for which the present invention finds utility include conditions linked to acute, chronic and inflammatory pain which are associated with an increase in NGF levels, such as interstitial cystitis/painful bladder syndrome; pancreatitis; chronic
  • Figure 1 is a gel showing the PEGylation reaction mixture of a 6His-TrkAd5 protein and a linear 20kDa bis-sulfone PEG reagent
  • Figure 2 is a gel showing purified fractions of the PEGylated-TrkAd5 product
  • Figure 3 shows a representation of the plate layout of an NGF-TrkAd5 ELISA assay
  • Figure 4 shows graphs depicting NGF binding curves of (A) an NGF standard calibration curve (top), and (B) the results of an NGF-TrkAd5 ELISA assay (bottom);
  • Figure 5 shows further graphs depicting NGF binding curves of (A) an NGF standard calibration curve (top), (B) the results of a NGF-TrkAd5 ELISA assay (bottom);
  • Figure 6 shows the results of a cell imaging analysis demonstrating the ability of 20 kDa monoPEGylated TrkAd5 to sequester NGF and to inhibit NGF-induced ERK phosphorylation in PC 12 cells expressing full-length TrkA receptors;
  • Figure 7 shows the results of an in vitro assay demonstrating the ability of 20 kDa monoPEGylated TrkAd5 to sequester NGF and to inhibit NGF-dependent neurite outgrowth in PC 12 cells.
  • Example 1 PEGylation of 6His-TrkAd5 protein.
  • Recombinant 6His-TrkAd5 protein was produced in E. coli BL21 (DE3) cells using the method described in W0 99/53055 in the section headed "Expression of TrkAIgl2, TrkAIgl and TrkAIg2" on page 18 of the specification, and incorporating a 6-histidine tag to the N- terminus of the polypeptide.
  • 6His-TrkAd5 protein in 20 mM Tris, 100 mM NaCl pH 8.2 (3.15 mg/mL, 0.5 mL) was buffer-exchanged for 50 mM sodium phosphate, 150 mM NaCl, pH 6.1 using Nap-5 column (0.5 mL protein was loaded and the flow discarded, protein was then eluted with 1 mL of the buffer), diluted to 1.3 mL with 50 mM sodium phosphate, 150 mM NaCl, pH 6.1 and quantitated by UV ( ⁇ 2 8 ⁇ 0.895, 1.053 mg/mL).
  • lane M indicates Novex Protein Standards
  • lane 1 indicates 6His-TrkAd5
  • lane 2 indicates PEGylation reaction mixture.
  • the unconjugated TrkAd5 migrates as a band at about 15kDa, while the conjugated protein migrates as a band at about 54kDa.
  • the column was then washed with buffer A (50 mM Tris buffer, pH 8.0) in order to remove the unbound PEG reagent and the conjugate eluted from the anion exchange column using 60 min gradient 0-100 % of buffer B (50 mM Tris buffer, pH 8.0, 1 M NaCl) at lmL/min. Separation was confirmed by SDS-PAGE.
  • buffer A 50 mM Tris buffer, pH 8.0, 1 M NaCl
  • the fractions containing 20 kDa mono-PEGylated product were pooled and purified by size exclusion chromatography using Superdex 200 pep grade column and 50 mM Phosphate buffer, 150 mM NaCl, pH 7.4 as a mobile phase at lmL/min flow rate.
  • Lane M indicates Novex Protein Standards
  • lanes 2-8 indicate multi-PEGylated 6His-TrkAd5 species
  • lanes 9-15 indicate 20 kDa mono-PEGylated product.
  • the fractions that contained pure product (lanes 10-15) were pooled and used for ELISA analysis.
  • Example 2 ELISA Assay.
  • the 20 kDa monoPEGylated TrkAd5 was analysed using modified version of NGF Emax® ImmunoAssay System supplied by Promega.
  • Nunc Maxisorp ELISA plate was coated with 100 ⁇ ⁇ per well of capture Anti-NGF pAb at 1: 1000 dilution from stock into carbonate coating buffer. The wells were sealed with a plate sealer and incubated overnight at 4°C. The next day, the Block & Sample IX Buffer was prepared by diluting Block & Sample 5X Buffer in ultrapure water. The coated plate was removed from the refrigerator and the contents of each well were ejected by turning upside down over a sink.
  • the inverted plate was then patted down on a paper towel three times to empty each well completely. All wells were washed with TBST (Tris-Buffered Saline & Tween 20) wash buffer (100 ⁇ ⁇ per well). The contents of the plate were again ejected as described above. 200 ⁇ ⁇ of Block & Sample IX Buffer was added to each well using a multichannel pipettor. The plate was incubated at room temperature for one hour without shaking.
  • TBST Tris-Buffered Saline & Tween 20
  • Rows A-C were designated for NGF + TrkAd5 and rows D-F for NGF + 20 kDa
  • This step was carried out during NGF + TrkAd5/ 20 kDa monoPEGylated TrkAd5 sample incubation.
  • the supplied NGF Standard was diluted (stock 1 ⁇ g/mL) 1 :2000 in Block & Sample IX Buffer to achieve a concentration of 500 pg/mL.
  • Serial 1 :2 dilutions were then carried out using Block & Sample IX Buffer.
  • the contents of the plate were ejected by turning upside down over a sink.
  • the inverted plate was then patted down on a paper towel three times to remove residual liquid and washed once with TBST wash buffer. Two rows (row G and H, Figure 3) were designated for the standard curve.
  • the wells were sealed with a plate sealer and incubated for 2.5 hours at room temperature with shaking (500 + 100 rpm). During this incubation, the TMB One Solution was equilibrated to room temperature. After incubation, all wells were washed five times with TBST wash buffer. 100 ⁇ ⁇ of the room temperature TMB One Solution was added to each well using a multichannel pipette. The plate was incubated at room temperature with shaking for 5-10 minutes. The reaction was stopped by adding 100 ⁇ ⁇ of 1 M HC1 to the wells in the same order in which TMB One Solution was added. The absorbance at 450 nm was recorded on a plate reader within 15 minutes of stopping the reaction.
  • Figure 4A shows the NGF standard calibration curve
  • Figure 4B shows NGF binding curves for the conjugated protein (20 kDa monoPEG-TrkAd5) and the unconjugated protein (TrkAd5).
  • the conjugated TrkAd5 retains NGF- sequestration activity at levels at least as efficacious as those shown by the unconjugated protein.
  • Example 3 ELISA Assay
  • Examples 1 and 2 were repeated following the protocols described therein, with the exception that double the amount of the 6His-TrkAd5 and 20 kDa monoPEGylated TrkAd5 test samples were used in the ELISA assay.
  • the test samples were prepared in serial dilutions (in triplicate) starting from 400 ng/mL - 20.4 pg/mL, 200 ⁇ ⁇ for each dilution, in Block & Sample IX Buffer.
  • NGF was prepared at 400 pg/mL, 5.5 mL in Block & Sample IX Buffer. NGF was then mixed with each prepared TrkAd5/ 20 kDa monoPEGylated TrkAd5 samples (1:1, 200 ⁇ : 200 ⁇ ). This gave final concentrations for test samples ranging from 200 ng/mL - 10.2 pg/mL (each had 200 pg/mL of NGF to be sequestered).
  • Figure 5A shows the NGF standard calibration curve
  • Figure 5B shows NGF binding curves for the conjugated protein (20 kDa monoPEG-TrkAd5) and the unconjugated protein (TrkAd5).
  • the conjugated TrkAd5 showed higher activity in sequestering NGF (83%) compared to that of the unconjugated protein (42%).
  • the unconjugated mPEG had no effect on NGF sequestration activity (data not shown).
  • TrkAd5 A 450 % of % of NGF monoPEG- % of % of NGF (200 (OD avge) NGF sequestered TrkAd5 NGF sequestered pg/mL) binding by TrkAd5 (OD avge) binding by
  • Example 4 IN Cell analysis demonstrating the ability of 20 kDa monoPEGylated TrkAd5 to sequester NGF and to inhibit NGF-induced ERK phosphorylation in PC12 cells expressing full-length TrkA receptors.
  • PC 12 cells were plated at 2xl0 4 cells in 200 pL per well in complete media [10% horse serum, 5% foetal calf serum, DMEM, penicillin, streptomycin and 2 mM glutamine] on collagen coated black walled 96-well plates and incubated at 37°C overnight in 5% C0 2 . Media was changed to 100 ⁇ 1 ⁇ 0 ⁇ % FCS media per well [0.1% foetal calf serum, DMEM, penicillin, streptomycin and 2 mM glutamine] and incubated at 37°C overnight in 5% C0 2 . All test articles were assayed in three separate experiments, each carried out in triplicate.
  • the 20 kDa monoPEGylated TrkAd5 protein was tested in the concentration range 0.1-4000 nM. This was in a final volume of 200 ⁇ , incubated at 37°C in 4% C0 2 for 5 minutes. Activity was compared to non-pegylated 6His- TrkAd5 and any non-specific effect due to the unconjugated mPEG and dilution buffer was monitored.
  • the assay mix was removed, cells were placed on ice and washed in ice cold 1 x PBS.
  • Non-PEGylated 6His-TrkAd5 had an IC50 of 7.30 nM + 1.06 (mean + SEM). ⁇ 20 kDa monoPEGylated TrkAd5 sequestered NGF slightly better than unconjugated 6His- TrkAd5 (Two-way ANOVA p ⁇ 0.05). Bonferroni post-test analysis indicated significant differences at 10 nM NGF (p ⁇ 0.001) and 4 nM NGF (p ⁇ 0.05).
  • Figure 6 shows collated data from three separate experiments, each data point measured in triplicate (+SEM). Mean calculated EC50s are given. Significant differences marked as asterisks are only seen between 20 kDa monoPEGylated TrkAd5 (black, left) and 6His- TrkAd5 (grey, right)) at 10 nM (***p ⁇ 0.001) and 4 nM (*p ⁇ 0.05). Values with >4 nM mPEG (grey, top)) were highly significantly different from other Test Articles. All data points from all experiments are included. Concentrations of Test Article were originally used in the range of 0.4 nM - 4 ⁇ for experiment 1. Due to high sequestration effect subsequent experiments were adjusted to the range of 100 pM - 1 ⁇ . Control (zero Test Article) values are plotted on the left of the x-axis.
  • Example 5 In vitro assay demonstrating the ability of 20 kDa monoPEGylated TrkAd5 to sequester NGF and to inhibit NGF-dependent neurite outgrowth in PC12 cells.
  • PC 12 cells extend neurites in response to NGF. During this assay growth occurs over 48 hours when NGF is added at a concentration of 1 ng/niL (38.5 pM). Previous studies have shown non-PEGylated (i.e., unconjugated) 6His-TrkAd5 is able to achieve complete sequestration of this dose of NGF at concentrations above 70 nM. The 20 kDa
  • DMEM + 10% horse serum, 5% foetal calf serum, penicillin, streptomycin and 2 mM glutamine DMEM + 10% horse serum, 5% foetal calf serum, penicillin, streptomycin and 2 mM glutamine
  • the cell number was adjusted to 4 x 10 4 /mL in complete medium and cells were seeded at 0.5ml / well in 24 well plates (collagen coated).
  • 1 ng human recombinant NGF (Sigma) was added to 0.5 mL of test article as appropriate and incubated for 10 minutes at room temperature. This was then added to cells and incubated at 37°C in 5% C0 2 for 48 hours.
  • Neurite outgrowth was assessed after 48 hours using a semi-quantitative measurement.
  • the scale of 0-4 was employed using the following criteria:

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Abstract

La présente invention concerne un conjugué d'un polypeptide Trk Ad5 avec un polymère hydrosoluble, le Trk Ad5 comprenant la séquence d'acides aminés des résidus 1 à 97 de SEQ ID NO: 1 ou un analogue de celle-ci.
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US10835616B2 (en) 2014-10-14 2020-11-17 Polytherics Limited Process for the conjugation of a peptide or protein with a reagent comprising a leaving group including a portion of PEG
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10835616B2 (en) 2014-10-14 2020-11-17 Polytherics Limited Process for the conjugation of a peptide or protein with a reagent comprising a leaving group including a portion of PEG
KR20230158134A (ko) 2014-10-14 2023-11-17 폴리테릭스 리미티드 Peg의 일부를 포함하는 이탈기를 포함하는 시약을 사용한, 펩티드 또는 단백질의 접합방법
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CN113840831A (zh) * 2019-03-20 2021-12-24 金德雷德生物科学股份有限公司 医用ngf拮抗剂
JP2022525754A (ja) * 2019-03-20 2022-05-19 キンドレッド バイオサイエンシズ インコーポレイテッド 医療用ｎｇｆアンタゴニスト
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RU2829812C2 (ru) * 2019-03-20 2024-11-06 Эланко Юс Инк. Антагонисты ngf для медицинского использования

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