Prevention and treatment of chemotherapy-induced neuropathic pain

Abstract

The present disclosure relates to meteorin and its use in the prevention and / or treatment of chemotherapy-induced neuropathic pain.Neuropathic pain resulting from chemotherapy treatment can be treated by administering meteorin to patients.Meteorin can also be used in prophylactic treatment to prevent neuropathic pain from occurring as a result of chemotherapy treatment.

Description

[Technical field]

[0001] The present invention relates to meteorin and its use in the prevention and / or treatment of chemotherapy-induced neuropathic pain. [Background technology]

[0002] Cancer is one of the leading causes of death worldwide, and despite significant efforts to implement novel chemotherapy strategies, these diseases remain a major health concern, with millions of new cases reported each year.

[0003] Chemotherapy treatments lead to improved cancer survival rates but often cause severe side effects that significantly reduce the quality of life of cancer patients.

[0004] Important anticancer drugs such as platinum-based drugs, taxanes, and vinca alkaloids are known to cause neurotoxicity in the peripheral nervous system, thereby resulting in neuropathic pain with symptoms such as allodynia, hyperalgesia, and spontaneous pain. Chemotherapy-induced neuropathic pain (CINP) is one of the most severe side effects of chemotherapy. CINP causes patients long-lasting discomfort with side effects that can last for years after the cessation of treatment, reducing the quality of life of cancer survivors. Thus, chemotherapy-induced neuropathy and pain are the most frequent non-hematological dose-limiting side effects of anticancer drugs. At too high a dose, the side effects become intolerable to those who receive it, while at low doses, effective treatment of the underlying cancer / disease is not obtained. As a result, the efficacy of many anticancer drugs is not optimal at doses where the side effects are tolerable for most patients. Thus, CINP symptoms of chemotherapy treatment may lead to a reduction in chemotherapy dose or cessation of treatment, which in turn leads to reduced survival rates.

[0005] Safe and effective therapies to prevent or treat chemotherapy-induced neuropathic pain remain an unmet clinical need for which no approved treatment exists. Medications commonly used for chronic pain conditions, such as gabapentin, tricyclic antidepressants, and opioids, are less effective and are associated with numerous side effects.

[0006] Therefore, there is a great need for preventive and therapeutic strategies for treating chemotherapy-induced neuropathic pain, preferably with no or only minor side effects and without affecting the overall health of the patient.

[0007] Meteorin is an endogenous protein previously demonstrated to be a neuronal survival factor (WO2005 / 095450). WO2012 / 041328 describes the use of meteorin for the treatment of allodynia, hyperalgesia, spontaneous pain, and phantom limb pain, based on findings in animal models of nerve injury. Summary of the Invention

[0008] The inventors of the present disclosure have surprisingly found that administering meteorin before, simultaneously with, or intermittently with chemotherapy treatment prevents chemotherapy-induced neuropathic pain (CINP). Thus, administering meteorin in conjunction with chemotherapy can prevent CINP. CINP is a severe side effect of chemotherapy, the symptoms of which, in addition to causing long-term discomfort to patients, may also lead to a reduction in the dosage of chemotherapy agents or interruption of treatment, resulting in reduced survival rates. Thus, the present invention provides a means for improving cancer treatment by allowing the use of higher doses of chemotherapy agents with reduced risk of developing neuropathic pain.

[0009] In one aspect, the present invention provides an isolated polypeptide for use in treating or preventing chemotherapy-induced neuropathic pain in a subject, comprising: i. the amino acid sequence of SEQ ID NO:3, and ii. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, which has at least 70% sequence identity to SEQ ID NO: 3; The present invention relates to an isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

[0010] In a second aspect, the present invention provides an isolated nucleic acid molecule for use in the treatment or prevention of chemotherapy-induced neuropathic pain in a subject, comprising: a. The amino acid sequence of SEQ ID NO:3; b. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, said sequence variant having at least 70% sequence identity to SEQ ID NO: 3; The present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:

[0011] In a further aspect, the present invention relates to a vector for use in the treatment or prevention of chemotherapy-induced neuropathic pain in a subject, the vector comprising a polynucleotide encoding a polypeptide according to any one of claims 1 to 6.

[0012] In a further aspect, the present invention provides a method for decreasing glutamine synthetase expression in the dorsal root ganglion in a subject in need thereof, comprising the steps of: a. The amino acid sequence of SEQ ID NO:3; b. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, said sequence variant having at least 70% sequence identity to SEQ ID NO: 3; administering a polypeptide comprising an amino acid sequence selected from the group consisting of: thereby decreasing expression of glutamine synthetase in the dorsal root ganglion.

[0013] In a further aspect, the present invention provides a method for decreasing connexin 43 expression in the dorsal root ganglion in a subject in need thereof, comprising: a. The amino acid sequence of SEQ ID NO:3; b. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, said sequence variant having at least 70% sequence identity to SEQ ID NO: 3; administering a polypeptide comprising an amino acid sequence selected from the group consisting of: thereby decreasing the expression of connexin 43 in the dorsal root ganglion. [Brief description of the drawings]

[0014] [Figure 1] Study design prophylaxis paradigm. rmMeteorin 0.5 mg / kg or 1.8 mg / kg was administered subcutaneously on days 1, 3, 5, 7, and 9 (D1, D3, D5, D7, and D9). Paclitaxel was administered intraperitoneally on days 2, 4, 6, and 8 (D2, D4, D6, and D8). Paclitaxel (PTX); intraperitoneal (ip); subcutaneous (sc); spinal cord (SC); dorsal root ganglion (DRG). [Diagram 2] Meteorin prevents paclitaxel-induced mechanical hypersensitivity in adult C57Bl6J mice. Hind paw withdrawal thresholds (PWT) were measured using von Frey filaments at baseline (BL) and then periodically throughout the experimental period up to day 57. Paclitaxel treatment was preceded by subcutaneous injections of 0.5 mg / kg (grey squares) or 1.8 mg / kg (black triangles) of rmMeteorin (MTRN) or vehicle (n=8 groups) (white circles), followed by four additional injections of each treatment as indicated by arrows. The development of mechanical hypersensitivity was substantially prevented by rmMeteorin (0.5 mg / kg and 1.8 mg / kg) compared to vehicle treatment. *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001 vs. vehicle (ANOVA mixed effects model followed by Tukey post-hoc test). Data are presented as mean±SEM. [Diagram 3]Meteorin prevents paclitaxel-induced increase in satellite glial cell density and gap junction formation. Female mice were injected with rmMeteorin (0.5 mg / kg or 1.8 mg / kg, subcutaneous) or vehicle on days 1, 3, 5, 7, and 9 alternating with paclitaxel (4 mg / kg, i.p.) administered every other day for 4 days on days 2, 4, 6, and 8 (as shown in Figure 1). Mice were sacrificed on day 24 and dorsal root ganglion (DRG) tissue was removed and subjected to immunohistochemistry with antibodies against peripherin (used to identify neuronal cell bodies - not shown), glutamine synthetase (GS), and connexin 43 (Con43). Mean grey intensity (MGI) was expressed as a function of specific staining for each antibody per μm2. *p<0.05, **p<0.01 vs. vehicle (one-way ANOVA and Tukey's multiple comparisons). Data are presented as mean±SEM. [Figure 4] Preemptive meteorin treatment prevents paclitaxel-induced loss of hindpaw intraepidermal nerve fibers. Female mice were injected with rm meteorin (0.5 mg / kg or 1.8 mg / kg, subcutaneously) or vehicle on days 1, 3, 5, 7, and 9 (D1, D3, D5, D7, and D9) alternating with paclitaxel (4 mg / kg, i.p.) administered every other day for 4 days on days 2, 4, 6, and 8 (D2, D4, D6, and D8). PGP9.5 expression was used as a specific marker to calculate intraepidermal nerve fiber (IENF) density, calculated from the number of IENFs (arrows) that were found to cross the basement membrane, normalized to the width of the epidermis (mm). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (one-way ANOVA vs. vehicle and Tukey's multiple comparisons). Data are presented as mean±SEM. [Diagram 5]Treatment paradigm of study design. Paclitaxel was administered intraperitoneally to separate cohorts of male and female mice on days 2, 4, 6, and 8 (D2, D4, D6, and D8). rmMeteorin 0.5 mg / kg or 1.8 mg / kg was administered subcutaneously on days 10, 12, 14, 16, and 18 (D10, D12, D14, D16, and D18). Paclitaxel (PTX); intraperitoneal (ip); subcutaneous (sc); spinal cord (SC); dorsal root ganglion (DRG). [Figure 6] Meteorin reverses paclitaxel-induced mechanical hypersensitivity in adult C57Bl6J mice. Following the study plan in Figure 5, separate cohorts of male and female mice were intraperitoneally injected with 4 mg / kg paclitaxel (PTX) every other day for a cumulative dose of 16 mg / kg (grey boxes). PWT was measured using von Frey filaments at baseline (BL) and then periodically throughout the experimental period up to day 54. Paclitaxel treatment was followed by five repeated subcutaneous injections of 0.5 mg / kg (grey squares) or 1.8 mg / kg (black triangles) rmMeteorin (MTRN) or vehicle (n=8 groups per family) at the time points indicated by the arrows. Data were combined for male and female treatment groups. Mechanical hypersensitivity was attenuated and resolved more rapidly with rmMeteorin treatment (0.5 mg / kg and 1.8 mg / kg) compared to vehicle treatment. *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001 vs. vehicle (ANOVA mixed effects model followed by Tukey post-hoc test). Data are presented as mean±SEM. [Figure 7]Meteorin reverses paclitaxel-induced increases in satellite glial cell density and gap junction formation. Female (top panel) or male (bottom panel) mice were administered paclitaxel (4 mg / kg, i.p.) every other day for 4 days on days 2, 4, 6, and 8. rmMeteorin (0.5 mg / kg or 1.8 mg / kg, subcutaneous) or vehicle was then administered on days 10, 12, 14, 16, and 18 (as indicated in Figure 5). Mice were sacrificed on day 24 and dorsal root ganglion (DRG) tissue was removed and subjected to immunohistochemistry with antibodies against peripherin (used to identify neuronal cell bodies - not shown), glutamine synthetase (GS), and connexin 43 (Con43). Mean grey intensity (MGI) was expressed as a function of specific staining for each antibody per μm2. *p<0.05, **p<0.01 vs. vehicle (one-way ANOVA and Tukey's multiple comparisons). Data are presented as mean±SEM. [Figure 8A] Figure 1 shows the CLUSTAL W (1.82) multiple sequence alignment of meteorin. Alignment of meteorin precursors from human (SEQ ID NO: 2), rat (SEQ ID NO: 8), and mouse (SEQ ID NO: 5). [Figure 8B] Figure 1 shows a CLUSTAL W (1.82) multiple sequence alignment of meteorin. Alignment of mature meteorin from human (SEQ ID NO: 3), rat (SEQ ID NO: 9), and mouse (SEQ ID NO: 6). [Figure 8C] Figure 1 shows a CLUSTAL W (1.82) multiple sequence alignment of meteorin. The consensus sequence of mature meteorin (SEQ ID NO: 11) generated from residues that are completely conserved in the human, mouse, and rat sequences. X represents any of the 21 naturally occurring amino acids encoded by DNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] definition As used herein, "biocompatible capsule" means that the capsule, when implanted into a host mammal, does not elicit a deleterious host response sufficient to result in rejection of the capsule or to render the capsule inoperable, e.g., by degradation.

[0016] As used herein, a "coding sequence" is a polynucleotide sequence that is transcribed and translated into a polypeptide.

[0017] As used herein, the term "expression vector" refers to vectors capable of directing the expression of genes to which they are operatively linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0018] As used herein, "immunoisolation capsule" means that the capsule, when implanted into a mammalian host, minimizes the adverse effects of the host's immune system on the cells within its core.

[0019] By "mammalian promoter" is intended a promoter that can function in a mammalian cell.

[0020] As used herein, "meteorin" refers to a polypeptide having the amino acid sequence of substantially purified meteorin from any source, whether natural, synthetic, semi-synthetic, or recombinant, obtained from any species, particularly mammals, such as chimpanzees, cows, sheep, pigs, mice, horses, and preferably humans. The term also refers to biologically active fragments of meteorin obtained from any of these species, as well as biologically active sequence variants thereof and post-translationally modified proteins.

[0021] As used herein, the term "operably linked" is intended to mean that a nucleotide sequence of interest is linked to a regulatory sequence(s) within a recombinant expression vector in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into a host cell).

[0022] As used herein, the term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals).

[0023] "Sequence identity": A high level of sequence identity indicates the likelihood that a first sequence is derived from a second sequence. Amino acid sequence identity requires identical amino acid sequences between two aligned sequences. Thus, a candidate sequence that shares 70% amino acid identity with a reference sequence requires that after alignment, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity can be determined by computer analysis, such as, but not limited to, the ClustalW computer alignment program (Higgins D., Thompson J., Gibson T., Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680) and the default parameters proposed in the literature. The ClustalW software is available from the European Bioinformatics Institute as the ClustalW WWW Service at http: / / www.ebi.ac.uk / clustalw. Use this program with default settings to align the mature (bioactive) parts of the query and reference polypeptides. Count the number of perfectly conserved residues and divide by the length of the reference polypeptide.

[0024] The ClustalW algorithm can similarly be used to align nucleotide sequences. Sequence identity can be calculated in a similar manner as shown for amino acid sequences.

[0025] The term "subject" as used herein is taken to mean any mammal to which meteorin polypeptides or polynucleotides, therapeutic cells, or biocompatible capsules may be administered. Subjects specifically intended for treatment by the methods of the present invention include humans, as well as non-human primates, sheep, horses, cows, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats, and mice, as well as organs, tumors, and cells derived from or arising from these hosts.

[0026] "Treatment" can be performed in a variety of ways, including curative treatment and / or ameliorative treatment. Curative treatment generally aims to cure an already existing clinical condition in the treated individual. Ameliorative treatment generally means treating to improve an existing clinical condition in an individual.

[0027] As used herein, the term "prevention" refers to preventing a clinical condition or reducing the risk of acquiring a condition or reducing the severity of a condition. Prevention may also be referred to herein as prophylactic or preemptive treatment.

[0028] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In this specification, "plasmid" and "vector" can be used interchangeably, since plasmid is the most commonly used form of vector. However, the present invention is intended to include other forms of such expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions.

[0029] Chemotherapy and Neuropathic Pain Chemotherapy is a type of cancer treatment that uses one or more anticancer drugs to treat cancer systemically. Chemotherapy treatment can prolong life but can also cause serious side effects. Antitumor agents in chemotherapy are designed to eliminate rapidly dividing cancer cells, but can also damage healthy structures such as the peripheral nervous system.

[0030] One of the more serious side effects of chemotherapy is chemotherapy-induced neuropathic pain, which is a category of pain that includes several forms of pain that originate from peripheral and / or central nervous system dysfunction caused by chemotherapy neurotoxicity.Symptoms of neuropathic pain include burning, tingling, electricity, pins and needles, paresthesia, dysesthesias, stiffness, numbness of the limbs, body distortion, allodynia (pain induced by normally harmless stimuli), hyperalgesia (abnormal sensitivity to pain), hyperpathy (excessive pain response that continues long after painful stimuli have ceased), and spontaneous pain.

[0031] CINP affects 30%-40% of patients undergoing chemotherapy. The prevalence of these symptoms is highest during the first month after completion of chemotherapy (68.1%), but as many as 30% of patients still report CINP symptoms 6 months after completion of chemotherapy. The severity of symptoms is generally proportional to the dose of therapeutic agent administered, and the severity of symptoms may justify a reduction in chemotherapy dose.

[0032] CINP may result from chemotherapy treatment of various cancer indications, including, but not limited to, ovarian cancer, breast cancer, gastrointestinal cancer, such as esophageal cancer, pancreatic cancer, leukemia, Hodgkin's disease, Wilms' tumor, neuroblastoma, testicular cancer, bladder cancer, lung cancer, and multiple myeloma. Thus, in one embodiment, chemotherapy-induced neuropathic pain is induced by chemotherapy treatment of ovarian cancer, breast cancer, esophageal cancer, pancreatic cancer, leukemia, Hodgkin's disease, Wilms' tumor, neuroblastoma, testicular cancer, bladder cancer, lung cancer, or multiple myeloma. In another embodiment, chemotherapy-induced neuropathic pain is induced by chemotherapy treatment of ovarian cancer, breast cancer, esophageal cancer, pancreatic cancer, leukemia, Hodgkin's disease, Wilms' tumor, neuroblastoma, testicular cancer, bladder cancer, or multiple myeloma.

[0033] CINP can be induced by various anticancer drugs, including, but not limited to, platinum-based anticancer drugs, such as carboplatin, cisplatin, and oxaliplatin; taxanes, such as paclitaxel and docetaxel; epothilones, such as ixabepilone; vinca alkaloids, such as vincristine, vinblastine, and vinorelbine; proteasome inhibitors, such as bortezomib; and immunomodulatory anticancer drugs, such as thalidomide. Thus, in one embodiment, chemotherapy-induced neuropathic pain is induced by treatment with an anticancer drug selected from the group consisting of platinum-based anticancer drugs, taxanes, epothilones, vinca alkaloids, proteasome inhibitors, and immunomodulatory anticancer drugs.

[0034] In one embodiment, chemotherapy-induced neuropathic pain is induced by treatment with a platinum-based anticancer drug, for example, by treatment with carboplatin, cisplatin, and / or oxaliplatin. In one embodiment, chemotherapy-induced neuropathic pain is induced by treatment with a taxane, for example, by treatment with paclitaxel and / or docetaxel. In one embodiment, chemotherapy-induced neuropathic pain is induced by treatment with an epothilone, for example, by treatment with ixabepilone. In one embodiment, chemotherapy-induced neuropathic pain is induced by treatment with a vinca alkaloid, for example, by treatment with vincristine, vinblastine, and / or vinorelbine. In one embodiment, chemotherapy-induced neuropathic pain is induced by treatment with a proteasome inhibitor, for example, by treatment with bortezomib. In one embodiment, chemotherapy-induced neuropathic pain is induced by treatment with an immunomodulatory anticancer drug, for example, by treatment with thalidomide.

[0035] The anti-cancer agent may be given as a monotherapy or in a combination treatment regimen in which the anti-cancer agent is administered in combination with another anti-cancer agent.

[0036] Chemotherapy-induced neuropathic pain initially manifests as an acute pain syndrome, with sensory symptoms occurring during or shortly after drug administration, and progresses to chronic neuropathy after repeated chemotherapy treatment cycles. Regarding the duration of sensory symptoms, acute neuropathy generally subsides between treatments, whereas chronic neuropathy can persist for months or years, significantly reducing the quality of life of cancer survivors.

[0037] Symptoms resulting from CINP can vary and include burning, tingling, electricity, pins and needles, paresthesia, dysesthesias, stiffness, numbness in the extremities, body distorted, allodynia, hyperalgesia, hyperpathy, and / or spontaneous pain. Thus, in one embodiment, chemotherapy-induced neuropathic pain results in burning, tingling, electricity, pins and needles, paresthesia, dysesthesias, stiffness, numbness in the extremities, body distorted, allodynia, hyperalgesia, hyperpathy, and / or spontaneous pain.

[0038] In one embodiment, chemotherapy-induced neuropathic pain results in burning, tingling, electricity, pins and needles, paresthesias, dysesthesias, stiffness, numbness in the limbs, and a feeling of the body being distorted.

[0039] In one embodiment, chemotherapy-induced neuropathic pain results in allodynia, hyperalgesia, hyperpathy, and / or spontaneous pain. In one embodiment, chemotherapy-induced neuropathic pain results in allodynia. In one embodiment, chemotherapy-induced neuropathic pain results in hyperalgesia. In one embodiment, chemotherapy-induced neuropathic pain results in hyperpathy. In one embodiment, chemotherapy-induced neuropathic pain results in spontaneous pain.

[0040] Treatment and / or prevention of chemotherapy-induced neuropathic pain Safe and effective therapies to prevent or treat chemotherapy-induced neuropathic pain remain an unmet clinical need. Medications that are usually effective against chronic pain conditions, such as gabapentin, tricyclic antidepressants, and opioids, have low efficacy and are associated with numerous side effects.

[0041] Therefore, there is a great need for preventive and therapeutic strategies for treating chemotherapy-induced neuropathic pain, preferably with minor side effects that do not affect the overall health of patients.The present invention provides for the treatment and / or prevention of CINP by administering meteorin to subjects undergoing chemotherapy treatment.Therefore, in one embodiment, the present invention relates to meteorin for use in the treatment and / or prevention of chemotherapy-induced neuropathic pain.In one embodiment, the present invention relates to meteorin for use in the treatment of chemotherapy-induced neuropathic pain.

[0042] In one embodiment, the present disclosure provides an isolated polypeptide for use in the treatment and / or prevention of chemotherapy-induced neuropathic pain in a subject, comprising: i. the amino acid sequence of SEQ ID NO:3, and ii. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, which has at least 70% sequence identity to SEQ ID NO: 3; The present invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

[0043] In one embodiment, the present invention relates to a method for the treatment and / or prevention of chemotherapy-induced neuropathic pain, comprising: i. the amino acid sequence of SEQ ID NO:3, and ii. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, which has at least 70% sequence identity to SEQ ID NO: 3; a therapeutically effective amount of an isolated polypeptide comprising an amino acid sequence selected from the group consisting of For those who need it The method relates to a method comprising administering

[0044] In one embodiment, the present disclosure provides an isolated polypeptide for the manufacture of a medicament for the treatment and / or prevention of chemotherapy-induced neuropathic pain in a subject, comprising: i. the amino acid sequence of SEQ ID NO:3, and ii. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, which has at least 70% sequence identity to SEQ ID NO: 3; The present invention provides the use of an isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

[0045] As demonstrated in Examples 1, 2, and 3 of the present disclosure, administration of meteorin prior to and / or intermittently with administration of chemotherapy agents results in reversal of chemotherapy-induced neuropathic pain. Thus, in a preferred embodiment, the present invention relates to meteorin for use in the prevention of chemotherapy-induced neuropathic pain.

[0046] In one embodiment, the present disclosure provides an isolated polypeptide for use in preventing chemotherapy-induced neuropathic pain in a subject, comprising: i. the amino acid sequence of SEQ ID NO:3, and ii. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, which has at least 70% sequence identity to SEQ ID NO: 3; The present invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

[0047] In one embodiment, the present invention provides a method for the prevention of chemotherapy-induced neuropathic pain, comprising: i. the amino acid sequence of SEQ ID NO:3, and ii. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, which has at least 70% sequence identity to SEQ ID NO: 3; a therapeutically effective amount of an isolated polypeptide comprising an amino acid sequence selected from the group consisting of For those who need it The method relates to a method comprising administering

[0048] In one embodiment, the present disclosure provides an isolated polypeptide for the manufacture of a medicament for the prevention of chemotherapy-induced neuropathic pain in a subject, comprising: i. the amino acid sequence of SEQ ID NO:3, and ii. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, which has at least 70% sequence identity to SEQ ID NO: 3; The present invention provides the use of an isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

[0049] In one embodiment, the therapeutic effect of the treatment ameliorates at least one symptom of chemotherapy-induced neuropathic pain. The at least one symptom may be selected from the group consisting of burning, tingling, electricity, pins and needles, paresthesia, dysesthesias, stiffness, numbness in the extremities, body distorted, allodynia, hyperalgesia, hyperpathy, and / or spontaneous pain. In one embodiment, the therapeutic effect of the treatment ameliorates at least one symptom selected from the group consisting of burning, tingling, electricity, pins and needles, paresthesia, dysesthesias, stiffness, numbness in the extremities, body distorted. In one embodiment, the therapeutic effect of the treatment ameliorates at least one symptom selected from the group consisting of allodynia, hyperalgesia, hyperpathy, and / or spontaneous pain.

[0050] In one embodiment, the therapeutic effect of the treatment ameliorates allodynia, hi one embodiment, the allodynia is warm allodynia, cold allodynia, thermal allodynia, and / or mechanical allodynia.

[0051] In one embodiment, the therapeutic effect of the treatment improves hyperalgesia, hi one embodiment, the hyperalgesia is mechanical hyperalgesia.

[0052] In one embodiment, the therapeutic effect of the treatment ameliorates hyperpathy.

[0053] In one embodiment, the therapeutic effect of the treatment ameliorates spontaneous pain.

[0054] Administration and Formulation Meteorin polypeptides can be administered in any medically acceptable manner, including injection by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumoral, intraperitoneal, intraventricular, epidural, intrathecal, intracerebroventricular, intracerebral, or other such as intranasal or topical. Sustained release administration by means such as depot injection or degradable implants is also specifically included in the present invention.

[0055] Administration of meteorin according to the present invention can be accomplished using any suitable delivery means, including: injection (either subcutaneous, intravenous, intraarterial, intramuscular, intrathecal, or other suitable site); pumps (e.g., Annals of Pharmacotherapy, 27:912 (1993); Cancer, 41:1270 (1993); Cancer, 41:1270 (1993); Research, 44:1698 (1984), incorporated herein by reference); microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and 5,084,350, incorporated herein by reference); slow-release polymer implants (see, e.g., Sabel, U.S. Pat. No. 4,883,666, incorporated herein by reference); encapsulated cells (see "biocompatible capsules"); non-encapsulated cell implants (see, e.g., U.S. Pat. Nos. 5,082,670 and 5,618,531, both of which are incorporated herein by reference); and inhalation.

[0056] Administration may be by periodic bolus injection of the formulation, or more continuously by intravenous or intraperitoneal administration from a reservoir either external (e.g., an intravenous bag) or internal (e.g., a biodegradable implant, a bioartificial organ, a biocompatible capsule of meteorin-producing cells, or a colony of transplanted meteorin-producing cells). See, e.g., US 4,407,957, 5,798,113, and 5,800,828, all of which are incorporated herein by reference.

[0057] Localized delivery may be by means such as delivery via a catheter into one or more arteries. In one embodiment of the present invention, localized delivery includes delivery using encapsulated cells (described in the "Biocompatible Capsules" section). A further type of localized delivery includes the localized delivery of gene therapy vectors, which are usually injected.

[0058] In a preferred embodiment of the invention, administration is by parenteral injection, preferably subcutaneous or intrathecal injection.

[0059] Although it is possible for the compounds of the present invention to be administered as raw chemicals, it is preferred to present them in the form of pharmaceutical formulations, which can be prepared by conventional methods, for example those described in Remington: The Science and Practice of Pharmacy 2005, Lippincott, Williams & Wilkins.

[0060] The term "pharmaceutical acceptable carrier" refers to one or more organic or inorganic components, natural or synthetic, that are combined with the meteorin polypeptide to facilitate its application. Suitable carriers include sterile saline, but other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutical acceptable are known to those skilled in the art.

[0061] The compound of the present invention can be formulated for parenteral administration, and can be provided in unit dose form in ampoules, prefilled syringes, small drops, or multiple dose containers, optionally with the addition of preservatives.The composition can take the form of suspension, solution, or emulsion in oily or aqueous vehicles, for example, aqueous polyethylene glycol solution.Examples of oily or non-aqueous carriers, diluents, solvents, or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g. olive oil), and injectable organic esters (e.g. ethyl oleate), and may contain agents such as preservatives, wetting agents, emulsifiers or suspending agents, stabilizers, and / or dispersants.Alternatively, the active ingredient can be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, and is composed with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0062] An "effective amount" refers to an amount that can ameliorate or slow the progression of a disease, degenerative, or damaging condition. An effective amount can be determined on an individual basis and is based, in part, on a consideration of the condition being treated and the results desired. An effective amount can be determined by one of ordinary skill in the art using such factors and using only routine experimentation.

[0063] The liposomal system may be any type of unilamellar vesicle, multilamellar vesicle, or stable plurilamellar vesicle, and may be prepared and administered according to methods well known to those skilled in the art, for example, according to the teachings of U.S. Pat. Nos. 5,169,637, 4,762,915, 5,000,958, or 5,185,154. In addition, it may be desirable to express the novel polypeptides of the present invention and other selected polypeptides as lipoproteins to enhance binding to liposomes. Recombinant meteorin protein is purified, for example, from CHO cells by immunoaffinity chromatography or any other convenient method, and then mixed with liposomes and incorporated therein with high efficiency. The liposome-encapsulated protein may be tested in vitro for any effect of stimulating cell growth.

[0064] Microencapsulation of meteorin polypeptides is contemplated when sustained release administration of meteorin polypeptides is desired in a formulation with release characteristics suitable for the treatment of any disease or disorder requiring administration of meteorin polypeptides. Microencapsulation of recombinant proteins for sustained release has been successful with human growth hormone (rhGH), interferon (rhIFN-), interleukin-2, and MN rgp120. Johnson et al. (1996); Yasuda (1993); Hora et al. (1990); Cleland, (1995), pp. 439-462; WO97 / 03692, WO96 / 40072, WO96 / 07399; and U.S. Patent No. 5,654,010.

[0065] The sustained release formulations of these proteins were developed using poly-lactic-co-glycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic acid and glycolic acid, can be rapidly excreted in the human body. Furthermore, the degradability of this polymer can be tuned from months to years depending on its molecular weight and composition. Lewis, “Controlled release of bioactive agents from lactide / glycolide polymer,” in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.

[0066] In one embodiment of the present invention, a composition comprising meteorin is contemplated.The composition may comprise the isolated polypeptide described herein, the isolated nucleic acid described herein, the expression vector encoding meteorin described herein, the cell line expressing meteorin described herein, or the biocompatible capsule secreting meteorin described herein.

[0067] Dosage Various dosing regimens for systemic administration are contemplated. In one embodiment, the method of administering a formulation comprising a meteorin polypeptide to a subject comprises administering meteorin at a dose of 1 μg to 10,000 μg per kg of subject's body weight per administration. In another embodiment, the dose is 1 μg to 7,500 μg per kg of subject's body weight per administration. In a further embodiment, the dose is 1 μg to 5,000 μg per kg of subject's body weight per administration. In a different embodiment, the dose is 1 μg to 2,000 μg per kg of subject's body weight per administration. In yet another embodiment, the dose is 1 μg to 1,000 μg per kg of subject's body weight per administration. In yet another embodiment, the dose is 1 μg to 700 μg per kg of subject's body weight per administration. In a more preferred embodiment, the dose is 5 μg to 500 μg per kg of subject's body weight per administration. In a most preferred embodiment, the dosage is 10 μg to 100 μg per kg of the subject's body weight per administration. In a preferred embodiment, the subject to be treated is a human.

[0068] Guidance regarding specific doses and methods of delivery is provided in the literature, see, for example, WO02 / 78730 and WO07 / 100898. Guidance for calculating human equivalent doses based on doses used in animal studies is provided in Reagan-Shaw et al., FASEB J, 22, 659-661 (2007).

[0069] The dose administered must be carefully adjusted to the age, weight and condition of the individual being treated, as well as the route of administration, dosage form and regimen, and the desired result, and the exact dosage should be determined by the attending physician.

[0070] In one embodiment of the invention, administration is repeated daily, hi another embodiment, administration is repeated at least 1-3 times per week, such as 2-5 times per week, for example 3-6 times per week.

[0071] In one embodiment, administration is repeated once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once every 7 days. In a preferred embodiment, administration is repeated once every 2 days.

[0072] In one embodiment, the present invention provides for the treatment of chemotherapy-induced neuropathic pain. Thus, in one embodiment, administration is initiated after the onset of symptoms of neuropathic pain.

[0073] In one embodiment, administration of the polypeptide is commenced after the initiation of chemotherapy treatment, such as 1 day, such as 2 days, such as 3 days, such as 4 days, such as 5 days, such as 8 days, such as 12 days after the initiation of chemotherapy treatment. In another embodiment, administration of the polypeptide is commenced after the initiation of chemotherapy treatment, such as 1 week, such as 2 weeks, such as 3 weeks after the initiation of chemotherapy treatment.

[0074] In one embodiment, the present invention provides for the prevention of chemotherapy-induced neuropathic pain. Thus, in one embodiment, a neurotrophic polypeptide is administered prior to, simultaneously with, or intermittently with chemotherapy treatment.

[0075] In one embodiment, administration begins prior to initiating chemotherapy treatment.

[0076] In one embodiment, administration is commenced at least 1 day prior to the initiation of chemotherapy treatment, such as at least 2 days prior to the initiation of chemotherapy treatment, such as at least 3 days prior to the initiation of chemotherapy treatment, such as at least 4 days, at least 5 days, at least 6 days, or at least 1 week prior to the initiation of chemotherapy treatment.

[0077] In one embodiment, the neurotrophic polypeptide is administered on the same day as the start of chemotherapy treatment or at least one day before the start of chemotherapy treatment, such as at least two days before the start of chemotherapy treatment, such as at least three days before the start of chemotherapy treatment, such as at least four days, at least five days, at least one week before the start of chemotherapy treatment.

[0078] Chemotherapy treatment often consists of multiple administrations of anticancer drugs at regular intervals, for example once a week, once every two weeks, for example once every three weeks, for example once a month. In one embodiment, the neurotrophic polypeptide is administered in conjunction with each administration of the anticancer drug, for example administered before, at the same time or intermittently with each administration of the anticancer drug.

[0079] In another embodiment, meteorin is administered at a relatively long interval between doses. A relatively long interval between doses is intended to include at least 2 days between doses, such as at least 3 days between doses, such as twice weekly administration. More preferably, the long interval between doses is at least 1 week, such as at least 2 weeks, more preferably at least 3 weeks, such as at least 4 weeks, or at least 1 month.

[0080] A relatively long dosing interval is intended to mean at least 2 days between doses, such as at least 3 days between doses, e.g., twice weekly dosing. More preferably, the long dosing interval is at least 1 week, such as at least 2 weeks, more preferably at least 3 weeks, such as at least 4 weeks, or at least 1 month.

[0081] In other words, the administration interval is long enough that after one dose of meteorin polypeptide, the polypeptide is no longer detectable in the serum of the treated subject when the next dose is administered. In another embodiment, the serum level is less than 10 ng / mL, such as less than 5 ng / mL, more preferably less than 1 ng / mL, such as less than 0.5 ng / mL, such as less than 0.1 ng / mL.

[0082] In some embodiments, a more frequent initial administration of meteorin is administered before a long administration range, for example, twice a day, once a day, once every 2 days, once every 3 days, or once every 4 days. This initial administration schedule may be maintained for, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 9 days, 11 days, 14 days, 21 days, or more. After completion of this administration schedule, meteorin may be administered less frequently, for example, as described above.

[0083] Accordingly, in one aspect, the present invention relates to a method of treating neuropathic pain in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of a neurotrophic polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO:3, wherein the administration is three times a week or less frequent.

[0084] Preferably, administration is weekly or less frequent. Even more preferably, administration is biweekly or less frequent.

[0085] In other words, the administration interval is long enough that after one dose of meteorin polypeptide, the polypeptide is no longer detectable in the serum of the treated subject when the next dose is administered. In another embodiment, the serum level is less than 10 ng / mL, such as less than 5 ng / mL, more preferably less than 1 ng / mL, such as less than 0.5 ng / mL, such as less than 0.1 ng / mL.

[0086] In some embodiments, the initial administration of meteorin is, for example, twice a day, once a day, once every 2 days, once every 3 days, or once every 4 days. This administration schedule may be maintained for, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 9 days, 11 days, 14 days, 21 days, or more. After completion of this administration schedule, meteorin may be administered less frequently, for example, as described above.

[0087] Meteorin The present invention relates to the use of a polypeptide identified as meteorin protein and a polynucleotide encoding said protein in the treatment of chemotherapy-induced neuropathic pain.In one embodiment, delivery is contemplated by using a capsule to deliver secreted biologically active meteorin and / or its homologues to a subject.Meteorin protein has been identified in humans (SEQ ID NO:2), mice (SEQ ID NO:5), and rats (SEQ ID NO:8), as well as various other species.

[0088] Human meteorin exists as a precursor of 293 amino acids that can be processed to generate at least one biologically active peptide. Meteorin is expressed at high levels in the nervous system and the eye, particularly in subregions of the brain. The meteorin precursors of mouse (SEQ ID NO:5) and rat (SEQ ID NO:8) consist of 291 amino acids and share 80.3 and 80.2% sequence identity with the human meteorin protein (SEQ ID NO:2), respectively (see FIG. 8).

[0089] Human meteorin contains a 23 amino acid N-terminal signal peptide sequence that is cleaved at the sequence motif ARA-GY. This signal peptide cleavage site is predicted by the SignalP method. The N-terminus of mouse meteorin has been verified by N-terminal sequencing (Jorgensen et al., 2009).

[0090] Table 1 shows the % sequence identity between full-length human meteorin and the mouse and rat sequences, see alignment in Figure 8a. [Table 1] Table 2 shows the % sequence identity between human meteorin and the mouse and rat sequences after removal of the N-terminal signal peptide, see alignment in Figure 8b. [Table 2] Based on the completely conserved residues, a consensus sequence for mature meteorin can be derived (SEQ ID NO: 11, FIG. 8c), where X is independently selected from any of the 21 naturally occurring amino acids encoded by DNA. In a preferred embodiment, the variant meteorin comprises the consensus sequence.

[0091] One of the biological functions of meteorin is its ability to induce neurite outgrowth in dissociated dorsal root ganglion (DRG) cultures, as described by Jorgensen et al. (2009) and Nishino et al. (2004).

[0092] Due to the high conservation of cysteines, these residues are predicted to play important roles in the secondary and tertiary structure of biologically active proteins. One or more of the cysteines may be involved in the formation of intra- and / or intermolecular disulfide bridges.

[0093] Meteorin Polypeptide In addition to full-length meteorin, substantially full-length meteorin, and prometeorin, the present invention provides biologically active variants of the polypeptide. A meteorin polypeptide or fragment is biologically active if it exhibits the biological activity of naturally occurring meteorin described herein, such as neurotrophic activity. It should be understood that the present invention relates to meteorin as defined herein.

[0094] The present invention relates to an isolated polypeptide molecule for use in a method for the treatment of allodynia, hyperalgesia, and / or spontaneous pain, the polypeptide comprising: a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, and 9; b) biologically active sequence variants of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, and 9, which have at least 70% sequence identity to the above SEQ ID NOs, and c) a biologically active fragment of at least 50 consecutive amino acids of either a) or b), which fragment is at least 70% identical to the above SEQ ID NOs. The amino acid sequence is selected from the group consisting of:

[0095] In one embodiment, the present invention provides a method for the preparation of a method for the treatment of atopic dermatitis comprising the steps of: i) AA of SEQ ID NO:2 30 ~AA 288 and a polypeptide having 1 to 5 extra amino acids at one or both termini compared to the native sequence, 25 ~AA 293 Up to; ii) AA of SEQ ID NO:8 28 ~AA 286 and a polypeptide having 1 to 5 extra amino acids at one or both termini compared to the native sequence, 23 ~AA 291 Up to; iii) AA of SEQ ID NO:5 31 ~AA 289 and a polypeptide having 1 to 5 extra amino acids at one or both termini compared to the native sequence, 26 ~AA 294 and iv) A variant of the above polypeptide, in which any amino acid specified in the selected sequence is changed to a different amino acid, provided that no more than 20 amino acid residues in the sequence are so changed. The present invention relates to an isolated polypeptide selected from the group consisting of:

[0096] Preferably, the biological activity is neurotrophic activity. Neurotrophically active variants may be defined by reference to one or more other in vitro and / or in vivo neurotrophic assays, in particular the DRG assay, as described above in WO2005 / 095450.

[0097] A preferred biological activity is the ability to induce substantially the same response as in the DRG assay described in Jorgensen et al. (2009). In this assay, DRG cells are grown in the presence of full-length human meteorin coding sequence (SEQ ID NO: 3). By substantially the same response in the DRG assay, it is intended that the neurite outgrowth from DRG cells is at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% of the number obtained in the DRG assay described in Jorgensen et al. (2009). The biological activity of the fragment or variant of meteorin may also be higher than the biological activity of naturally occurring meteorin (SEQ ID NO: 3).

[0098] A variant may differ from naturally occurring meteorin in amino acid sequence or in a manner unrelated to sequence, or both. A variant in amino acid sequence ("sequence variant") is generated when one or more amino acids of naturally occurring meteorin are replaced with different natural amino acids, amino acid derivatives, or non-natural amino acids. Particularly preferred variants include naturally occurring meteorin, or biologically active fragments of naturally occurring meteorin, whose sequence differs from the wild-type sequence by one or more conservative and / or semi-conservative amino acid substitutions that typically have minimal effect on the secondary and tertiary structure and hydrophobic properties of the protein or peptide. A variant may also have a sequence that differs by one or more non-conservative amino acid substitutions, deletions, or insertions that do not destroy the biological activity of meteorin. The Clustal W alignment of FIG. 8 can be used to predict which amino acid residues can be substituted without substantially affecting the biological activity of the protein. In a preferred embodiment, the variant meteorin sequence comprises the consensus sequence having SEQ ID NO: 11.

[0099] Substitutions within the following groups (Clustal W, "strong" conserved groups) should be considered as conservative substitutions within the meaning of the present invention: -S,T,A;N,E,Q,K;N,H,Q,K;N,D,E,Q;Q,H,R,K;M,I,L,V;M,I,L,F;H,Y;F,Y,W.

[0100] Substitutions within the following groups (Clustal W, "weakly" conserved groups) should be considered as semi-conservative substitutions within the meaning of the present invention: -C,S,A;A,T,V;S,A,G;S,T,N,K;S,T,P,A;S,G,N,D;S,N,D,E,Q,K;N,D,E,Q,H,K;N,E,Q,H,R,K;V,L,I,M;H,F,Y.

[0101] Other variants within the present invention are those that have modifications that improve the stability of the peptide. Such variants may, for example, include one or more non-peptide bonds (replacing peptide bonds) in the peptide sequence. Also included are variants that include residues other than naturally occurring L-amino acids, such as D-amino acids, or non-naturally occurring or synthetic amino acids, such as beta or gamma amino acids, and cyclic variants. Incorporating D-amino acids into a polypeptide instead of L-amino acids may increase resistance to proteases. See, for example, US 5,219,990. Splice variants are specifically included in the present invention.

[0102] When the outcome of a given substitution cannot be predicted with certainty, derivatives can be readily assayed for the presence or absence of neurotrophic activity according to the methods disclosed herein, preferably using the DRG assay described in Jorgensen et al., Characterization of meteorin-An evolutionary conserved neurotrophic factor, J Mol Neurosci 2009 Sep;39(1-2):104-116.

[0103] In one embodiment, the polypeptide is a naturally occurring allelic variant of a sequence selected from the group consisting of SEQ ID NOs: 3, 6, and 9. The polypeptide may comprise an amino acid sequence that is the translation of a nucleic acid sequence that differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 7.

[0104] The variant polypeptides described herein include, in one embodiment, polypeptides in which any amino acid specified in a selected sequence has been altered to provide a conservative substitution.

[0105] Variants within the scope of the present invention, in one embodiment, include proteins and peptides having an amino acid sequence that has at least 70% identity to human, mouse or rat meteorin (SEQ ID NOs: 3, 6 and 9). More preferably, the sequence identity is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

[0106] In a preferred embodiment, the sequence identity of the variant meteorin is determined with reference to the human meteorin polypeptide (SEQ ID NO:3).

[0107] In one embodiment, the variant comprises a protein comprising an amino acid sequence having at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% sequence identity to SEQ ID NO:3.

[0108] In one embodiment, preferred variants include proteins comprising an amino acid sequence having at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% sequence identity to SEQ ID NO:6.

[0109] In one embodiment, preferred variants include proteins comprising an amino acid sequence having at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% sequence identity to SEQ ID NO:9.

[0110] The neurotrophic polypeptide preferably has at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% sequence identity to the amino acid sequence of SEQ ID NO:3.

[0111] In one embodiment, the neurotrophic polypeptide comprises the consensus sequence of SEQ ID NO:11.

[0112] Preferably, the neurotrophic polypeptide has cysteine ​​residues at positions 7, 28, 59, 95, 148, 151, 161, 219, 243, and 265 relative to the amino acid sequence of SEQ ID NO:3.

[0113] In one embodiment, preferred variants of meteorin include proteins comprising 50 to 270 amino acids, more preferably 75 to 270 amino acids, more preferably 90 to 270 amino acids, more preferably 100 to 270 amino acids, more preferably 125 to 270 amino acids, more preferably 150 to 270 amino acids, more preferably 175 to 270 amino acids, more preferably 200 to 270 amino acids, more preferably 225 to 270 amino acids, more preferably 250 to 270 amino acids.

[0114] In one embodiment, the variant meteorin comprises residues marked (*) in Figure 8 as being completely conserved at the corresponding positions, more preferably the variant meteorin also comprises residues marked (*) in Figure 8 as being strongly conserved at the corresponding positions (strongly conserved groups are: S,T,A; N,E,Q,K; N,H,Q,K; N,D,E,Q; Q,H,R,K; M,I,L,V; M,I ,L,F;H,Y;F,Y,W), more preferably the variant meteorin also comprises residues marked as less conserved in FIG. 8 at the corresponding positions (the less conserved groups include: C,S,A;A,T,V;S,A,G;S,T,N,K;S,T,P,A;S,G,N,D;S,N,D,E,Q,K;N,D,E,Q,H,K;N,E,Q,H,R,K;V,L,I,M;H,F,Y). In particular, it is contemplated that the conserved cysteines must be located at the corresponding positions in the variant meteorin. Thus, in one embodiment, the variant meteorin sequence has cysteine ​​residues at positions 7, 28, 59, 95, 148, 151, 161, 219, 243 and 265 relative to the amino acid sequence of SEQ ID NO:3.

[0115] In one embodiment, the neurotrophic polypeptide comprises the consensus sequence of SEQ ID NO: 11. The consensus sequence includes amino acid residues conserved in human, mouse, and rat meteorin as shown in Figure 8. Preferably, the neurotrophic polypeptide has cysteine ​​residues at positions 7, 28, 59, 95, 148, 151, 161, 219, 243, and 265 relative to the amino acid sequence of SEQ ID NO:3.

[0116] Non-sequence modifications may include, for example, chemical derivatization in vivo or in vitro of parts of naturally occurring meteorin, as well as acetylation, methylation, phosphorylation, carboxylation, PEGylation, or glycosylation. Just as it is possible to replace protein substituents, it is also possible to replace functional groups attached to proteins with groups characterized by similar characteristics. Such modifications do not change the primary sequence. They are initially conservative, i.e. the replaced group will have approximately the same size, shape, hydrophobicity, and charge as the original group.

[0117] Many amino acids, including terminal amino acids, may be modified in a given polypeptide by natural processes such as glycosylation and other post-translational modifications, or by chemical modification techniques well known in the art. Some of the known modifications that may be present in the polypeptides of the invention are acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a polynucleotide or polynucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, protein processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginylation, and ubiquitination, to name a few.

[0118] Such modifications are well known to those skilled in the art and have been described in detail in the scientific literature. Some of the most common modifications, such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, are described in most basic textbooks, such as Creighton (1993). Numerous detailed reviews on this subject are available, such as those provided in Wold, F. (1983); Seifter et al. (1990), and Rattan et al. (1992).

[0119] In addition, the protein may contain a protein tag, allowing for subsequent purification and, optionally, removal of the tag using an endopeptidase. The tag may contain a protease cleavage site to facilitate subsequent removal of the tag. Non-limiting examples of affinity tags include poly-his tag, GST tag, HA tag, Flag tag, C-myc tag, HSV tag, V5 tag, maltose binding protein tag, cellulose binding domain tag. Preferably, for production and purification, the tag is a poly-his tag. Preferably, the tag is present at the C-terminal portion of the protein.

[0120] To increase secretion of the protein in recombinant production in other mammalian cell types, the native signal sequence of meteorin may be replaced.

[0121] Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini, Indeed, blocking of amino or carboxyl groups, or both, in a polypeptide by a covalent modification is common in naturally occurring and synthetic polypeptides, and such modifications can likewise be present in the polypeptides of the present invention.

[0122] Modifications that occur in a polypeptide often depend on the method of its production. For example, in the case of a polypeptide produced by expressing a cloned gene in a host, the nature and extent of the modification is determined in large part by the post-translational modification capabilities of the host cell and the modification signals present in the polypeptide amino acid sequence. For example, glycosylation often does not occur in bacterial hosts such as E. coli. Therefore, if glycosylation is desired, the polypeptide should be expressed in a glycosylating host, typically a eukaryotic cell. Insect cells often perform the same post-translational glycosylation as mammalian cells, and for this reason insect cell expression systems have been developed specifically to efficiently express mammalian proteins with native glycosylation patterns. Similar considerations apply to other modifications.

[0123] It will be understood that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and a given polypeptide may contain many types of modifications.

[0124] Generally, as used herein, the term polypeptide encompasses all such modifications, particularly those present in polypeptides synthesized by expressing a polynucleotide in a host cell.

[0125] Meteorin nucleotide sequence The present invention provides medical uses of genomic and cDNA encoding meteorin, for example the human cDNA nucleotide sequences (SEQ ID NO:1 and 10), the mouse cDNA sequence (SEQ ID NO:4), and the rat cDNA sequence (SEQ ID NO:7).

[0126] Variants of these sequences are also included within the scope of the present invention.

[0127] The present invention relates to an isolated nucleic acid molecule for use in a method for the treatment and / or prevention of chemotherapy-induced neuropathic pain, the nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide, the polypeptide comprising: i. the amino acid sequence of SEQ ID NO:3; ii. Biologically active sequence variants of the amino acid sequence of SEQ ID NO: 3, which have at least 70% sequence identity to SEQ ID NO: 3, and iii. A biologically active fragment of at least 50 consecutive amino acids of i) or ii), which is at least 70% identical to SEQ ID NO:3. The amino acid sequence is selected from the group consisting of:

[0128] In one embodiment, the present invention relates to an isolated nucleic acid molecule encoding a polypeptide for use in a method for the treatment and / or prevention of chemotherapy-induced neuropathic pain, the polypeptide comprising: i) AA of SEQ ID NO:2 30 ~AA 288 and a polypeptide having 1 to 5 extra amino acids at one or both termini compared to the native sequence, 25 ~AA 293 Up to; ii) AA of SEQ ID NO:8 28 ~AA 286 and a polypeptide having 1 to 5 extra amino acids at one or both termini compared to the native sequence, 23 ~AA 291 Up to; iii) AA of SEQ ID NO:5 31 ~AA 289 and a polypeptide having 1 to 5 extra amino acids at one or both termini compared to the native sequence, 26 ~AA 294 and iv) A variant of the above polypeptide, in which any amino acid specified in the selected sequence is changed to a different amino acid, provided that no more than 20 amino acid residues in the sequence are so changed. The amino acid sequence is selected from the group consisting of:

[0129] The nucleic acid molecule may comprise the nucleotide sequence of a naturally occurring allelic nucleic acid variant.

[0130] A nucleic acid molecule of the invention may encode a variant polypeptide, which has the polypeptide sequence of a naturally occurring polypeptide variant.

[0131] In one embodiment, the nucleic acid molecule differs from a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, and 10 by a single nucleotide.

[0132] Preferably, the encoded polypeptide has at least 60% sequence identity, preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 3, more preferably said polypeptide has a sequence selected from the group consisting of said SEQ ID NO: 3. Said sequence constitutes human meteorin.

[0133] In a preferred embodiment, the encoded polypeptide comprises the consensus sequence having SEQ ID NO:11.

[0134] In preferred embodiments, the encoded polypeptide has at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 95%, more preferably at least 98% sequence identity to SEQ ID NO:3, and more preferably the polypeptide has the sequence of SEQ ID NO:3.

[0135] In one aspect, the nucleic acid molecule comprises: a) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, and 10; b) a nucleotide sequence having at least 70% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, and 10; and c) a nucleic acid sequence of at least 150 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, and 10; The nucleic acid sequence comprises a nucleotide sequence selected from the group consisting of:

[0136] In one embodiment, the isolated polynucleotide of the present invention has at least 60, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% sequence identity to the polynucleotide sequence provided as SEQ ID NO:1.

[0137] In one preferred embodiment, the isolated polynucleotide of the present invention has at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% sequence identity to the polynucleotide sequence provided as SEQ ID NO:10.

[0138] In one embodiment, a preferred isolated polynucleotide variant of the present invention comprises 150 to 900 nucleic acids, more preferably 175 to 900 nucleic acids, more preferably 200 to 900 nucleic acids, more preferably 225 to 900 nucleic acids, more preferably 250 to 900 nucleic acids, more preferably 300 to 900 nucleic acids, more preferably 350 to 900 nucleic acids, more preferably 400 to 900 nucleic acids, more preferably 450 to 900 nucleic acids, more preferably 500 to 900 nucleic acids, more preferably 550 to 900 nucleic acids, more preferably 600 to 900 nucleic acids, more preferably 650 to 900 nucleic acids, more preferably 700 to 900 nucleic acids, more preferably 750 to 900 nucleic acids, more preferably 800 to 900 nucleic acids, more preferably 850 to 900 nucleic acids.

[0139] A preferred group of isolated polynucleotides includes the human meteorin cDNA sequences SEQ ID NOs: 1 and 10. Generally, cDNA sequences are much shorter than genomic sequences and are more easily inserted into an appropriate expression vector and transduced / transfected into producer cells or human cells in vivo or ex vivo.

[0140] In addition, the nucleotide sequences of the present invention include sequences that are derivatives of these sequences.The present invention also includes vectors, liposomes and other carrier vehicles that include one of these sequences or a derivative of one of these sequences.The present invention also includes proteins transcribed and translated from meteorin cDNA, preferably human meteorin cDNA, including but not limited to human meteorin and derivatives and variants.

[0141] Codon-optimized nucleic acid molecules are also contemplated for enhanced expression in a selected host cell, including, but not limited to, E. coli, yeast species, Chinese hamster, baby hamster, insect, fungi, and human.

[0142] Variant nucleic acids can be generated by modern mutagenesis methods. Methods of shuffling coding sequences from human to mouse, rat or chimpanzee are also contemplated.

[0143] Variant nucleic acids are made by replacing an amino acid present in human meteorin with an amino acid at the corresponding position present in mouse or rat meteorin, if this amino acid is different from that present in human meteorin.

[0144] Viral Vectors Broadly speaking, gene therapy aims to introduce new genetic material into a patient's cells to result in a therapeutic benefit to the patient, including the treatment or prevention of a wide range of diseases, disorders, and other conditions.

[0145] Ex vivo gene therapy approaches involve the modification of isolated cells (including, but not limited to, stem cells, neural and glial progenitor cells, and fetal stem cells) which are then injected, grafted, or otherwise transplanted into a patient. See, e.g., U.S. Patent Nos. 4,868,116, 5,399,346, and 5,460,959. In vivo gene therapy aims to directly target host patient tissue in vivo.

[0146] Viruses useful as gene transfer vectors include papovaviruses, adenoviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, and retroviruses.Suitable retroviruses include the group consisting of HIV, SIV, FIV, EIAV, and MoMLV.Another group of suitable retroviruses includes the group consisting of HIV, SIV, FIV, EAIV, and CIV.Another group of preferred viral vectors includes the group consisting of alphaviruses, adenoviruses, adeno-associated viruses, baculoviruses, HSV, coronaviruses, bovine papillomaviruses, and Mo-MLV, and is preferably adeno-associated virus.

[0147] Preferred viruses for the treatment of disorders of the nervous system are lentiviruses and adeno-associated viruses. Both types of viruses can integrate into the genome without cell division, and both types have been tested in preclinical animal studies for applications in the nervous system, particularly the central nervous system.

[0148] Methods for the preparation of AAV are described in the art, for example in US 5,677,158. Examples of AAV delivery to the central nervous system are described in US 6,309,634 and US 6,683,058.

[0149] Preferably, lentiviral vector is a replication-defective lentiviral particle.Such lentiviral particle can be produced from a lentiviral vector comprising 5' lentiviral LTR, tRNA binding site, packaging signal, promoter operably linked to polynucleotide signal encoding fusion protein, origin of second strand DNA synthesis, and 3' lentiviral LTR.The method for preparing lentivirus and administering it to neural cells in vivo is described in US20020037281 (Methods for transducing neural cells using lentiviral vectors).

[0150] Retroviral vectors are the most commonly used vectors in human clinical trials because they have 7-8 kb and the ability to infect cells and stably integrate their genetic material into host cells with high efficiency. See, for example, WO95 / 30761; WO95 / 24929. Oncovirinae require at least one round of target cell proliferation for the introduction and integration of exogenous nucleic acid sequences into the patient. Retroviral vectors randomly integrate into the patient's genome. Retroviruses can be used to target stem cells of the nervous system, because very little cell division occurs in other cells of the nervous system (especially the CNS).

[0151] Three classes of retroviral particles have been described; ecotropic, which can efficiently infect mouse cells, and amphotropic, which can infect cells of many species. The third class includes xenotropic retroviruses, which can infect cells of other species than the one that produced the virus. Their ability to integrate only into the genome of dividing cells makes retroviruses attractive for marking cell lineages in developmental studies and for delivering therapeutic or suicide genes to cancers or tumors.

[0152] For use in human patients, retroviral vectors must be replication-deficient. This prevents further production of infectious retroviral particles in the target tissue; instead, the replication-deficient vector becomes a "capture" transgene stably integrated into the target cell genome. Typically, in a replication-deficient vector, the gag, env, and pol genes have been removed (along with most of the rest of the viral genome). Heterologous DNA is inserted in place of the removed viral genes. The heterologous gene may be under the control of an endogenous heterologous promoter, another heterologous promoter that is active in the target cell, or the retroviral 5'LTR (the viral LTR is active in a variety of tissues). Typically, retroviral vectors have a transgene capacity of about 7-8 kb.

[0153] Replication-defective retroviral vectors require that the viral proteins necessary for replication and assembly are provided in trans, for example, from a genetically engineered packaging cell line. It is important that the packaging cell does not release replication-competent virus and / or helper virus. This is achieved by expressing viral proteins from RNA lacking the Ψ signal, as well as expressing the gag / pol and env genes from separate transcription units. In addition, in some second and third generation retroviruses, the 5'LTR has been replaced with a non-viral promoter that controls the expression of these genes, and the 3'promoter has been minimized to include only the proximal promoter. These designs minimize the possibility of recombination leading to the production of replication-competent vectors or helper viruses.

[0154] Expression vector The construction of vectors for recombinant expression of meteorin polypeptides for use in the present invention can be accomplished using conventional techniques that do not require detailed explanation for those skilled in the art. However, for general information, those skilled in the art can refer to Maniatis et al. (1982) if necessary. The expression vectors can be used to generate production cells for recombinant production of meteorin polypeptides for medical use, and to generate therapeutic cells that secrete meteorin polypeptides for naked or encapsulated therapy.

[0155] Briefly, recombinant expression vectors are constructed using standard ligation techniques. To analyze the constructed vector to ensure that the sequence is correct, the gene is sequenced, for example, using the method of Messing, et al. (1981), the method of Maxam, et al. (1980), or other suitable methods known to those skilled in the art.

[0156] Size separation of the cleaved fragments is carried out using conventional gel electrophoresis as described, for example, in Maniatis, et al. (pp. 133-134, 1982).

[0157] For efficient expression vector generation, the expression vector should contain the necessary regulatory sequences to express the encoded gene in the correct reading frame. Gene expression is controlled at the transcriptional, translational, or post-translational level. Transcription initiation is an early and important event in gene expression. It depends on promoter and enhancer sequences and is influenced by specific cellular factors that interact with these sequences. The transcription unit of many genes consists of a promoter and in some cases an enhancer or regulatory element (Banerji et al. (1981); Corden et al (1980); and Breathnach and Chambon (1981)). In the case of retroviruses, the control elements involved in the replication of the retroviral genome are present in the long terminal repeats (LTRs) (Weiss et al. (1982)). The LTRs of Moloney murine leukemia virus (MLV) and Rous sarcoma virus (RSV) contain promoter and enhancer sequences (Jolly et al. (1983); Capecchi et al. (1991). Other strong promoters include those derived from cytomegalovirus (CMV) and other wild-type viral promoters.

[0158] Promoter and enhancer regions of several non-viral promoters have also been described (Schmidt et al. (1985); Rossi and deCrombrugghe, (1987)). Methods for maintaining and increasing expression of transgenes in quiescent cells include the use of promoters including collagen type I (1 and 2) (Prockop and Kivirikko (1984); Smith and Niles (1980); de Wet et al. (1983)), SV40 and LTR promoters.

[0159] According to one embodiment of the present invention, the promoter is a constitutive promoter selected from the group consisting of ubiquitin promoter, CMV promoter, JeT promoter (US6,555,674), SV40 promoter, elongation factor 1 alpha promoter (EF1-alpha), RSV, CAG. Examples of inducible / repressible promoters include Tet-On, Tet-Off, rapamycin inducible promoter, Mx1, Mo-MLV-LTR, progesterone, RU486.

[0160] A group of preferred promoters includes CAG, CMV, human UbiC, JeT, SV40, RSV, Tet-regulated promoter, Mo-MLV-LTR, Mx1, Mt1, and EF-1 alpha.

[0161] In addition to using viral and non-viral promoters to promote transgene expression, enhancer sequences may be used to increase the expression level of transgenes. Enhancers can increase the transcriptional activity of not only their native genes but also some foreign genes (Armelor (1973)). For example, in the present invention, collagen enhancer sequences can be used with collagen promoter 2 (I) to increase transgene expression. In addition, enhancer elements found in SV40 virus can be used to increase transgene expression. This enhancer sequence consists of a 72 base pair repeat described in Gruss et al. (1981); Benoist and Chambon (1981), and Fromm and Berg (1982), all of which are incorporated herein by reference. This repeat sequence can increase the transcription of many different viral and cellular genes when in contiguous with various promoters (Moreau et al. (1981)).

[0162] Additional expression enhancing sequences include, but are not limited to, the woodchuck hepatitis virus post-transcriptional regulatory element, WPRE, SP163, the CMV enhancer, and the chicken beta globin insulator, or other insulators.

[0163] cell line In one aspect, the present invention relates to an isolated host cell genetically modified with a vector according to the invention.

[0164] The present invention also relates to cells suitable for biodelivery of meteorin via naked or encapsulated cells that can be genetically modified to overexpress meteorin and implanted into a patient for localized delivery of bioactive meteorin polypeptides. Such cells can be broadly referred to as therapeutic cells.

[0165] For ex vivo gene therapy, preferred groups of cells include neural cells, neural precursor cells, neural progenitor cells, neural stem cells, human glial stem cells, human progenitor cells, stem cells, and fetal cells.

[0166] For encapsulation, preferred cells include retinal pigment epithelial cells, such as ARPE-19 cells, immortalized human fibroblasts, and immortalized human astrocytes.

[0167] The ARPE-19 cell line is an excellent platform cell line for encapsulated cell-based delivery techniques, and is also useful for non-encapsulated cell-based delivery techniques. The ARPE-19 cell line is robust (i.e., the cell line is viable under harsh conditions, such as implantation in the central nervous system or intraocular environment). ARPE-19 cells can be genetically modified to secrete substances of therapeutic interest. ARPE-19 cells have a relatively long life span. ARPE-19 cells are of human origin. Furthermore, encapsulated ARPE-19 cells have good in vivo device survival rates. ARPE-19 cells can deliver effective amounts of growth factors. ARPE-19 cells induce little host immune response. Furthermore, ARPE-19 cells are non-tumorigenic. Methods for culturing and encapsulating ARPE-19 cells are described in US 6,361,771.

[0168] In another embodiment, the therapeutic cell line is selected from the group consisting of a human fibroblast cell line, a human astrocyte cell line, a human mesencephalic cell line, and a human endothelial cell line, and is preferably immortalized with TERT, SV40T, or vmyc.

[0169] Extracellular matrix The invention further includes culturing meteorin producing cells in vitro on extracellular matrix prior to transplantation into the mammalian nervous system, as pre-attaching the cells to microcarriers prior to transplantation enhances the long-term viability of the transplanted cells and provides long-term functional benefits.

[0170] Materials that may constitute the extracellular matrix include materials to which cells can adhere and grow after in vitro incubation and that may be implanted into a mammalian body without producing toxic or inflammatory reactions that may destroy the implanted cells or otherwise inhibit their biological or therapeutic activity. Such materials may be synthetic or natural chemicals, or may be of biological origin.

[0171] Matrix materials include, but are not limited to, glass and other silicon oxides, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride, polyurethane, polyalginates, polysulfones, polyvinyl alcohol, acrylonitrile polymers, polyacrylamides, polycarbonates, polypentenes, nylons, amylases, natural and modified gelatins and natural and modified collagens, natural and modified polysaccharides such as dextran and cellulose (e.g., nitrocellulose), agar, and magnetite. Either absorbable or non-absorbable materials can be used. Extracellular matrix materials well known in the art are also contemplated. Extracellular matrix materials may be commercially obtained or may be prepared by growing cells that secrete such matrix, removing the secreting cells, and allowing the transplanted cells to interact and adhere to the matrix. The matrix material on which the transplanted cells grow or with which the cells are mixed may be a resident product of the RPE cells. Thus, for example, the matrix material may be an extracellular matrix or basement membrane material produced and secreted by the transplanted RPE cells.

[0172] To improve cell adhesion, survival, and function, the solid matrix may optionally be coated on its exterior surface with factors known in the art that promote cell adhesion, growth, or survival, including cell adhesion molecules, extracellular matrices such as fibronectin, laminin, collagen, elastin, glycosaminoglycans, or proteoglycans, or growth factors.

[0173] Alternatively, if the solid matrix to which the implanted cells are attached is composed of a porous material, the growth or survival promoting factor(s) may be incorporated into the matrix material and released therefrom following in vivo implantation.

[0174] The configuration of the support is preferably spherical, such as a bead, but may also be cylindrical, elliptical, flattened sheet or strip, needle or pin shaped, etc. A preferred form of support matrix is ​​glass beads. Another preferred bead is polystyrene beads.

[0175] The size of the beads may range from about 10 μm to 1 mm in diameter, preferably from about 90 μm to about 150 μm. For descriptions of various microcarrier beads, see, for example, Fisher Biotech Source 87-88, Fisher Scientific Co., 1987, pp. 72-75; Sigma Cell Culture Catalog, Sigma Chemical Co., St, Louis, 1991, pp. 162-163; Ventrex Product Catalog, Ventrex Laboratories, 1989 (these references are incorporated herein by reference). The upper limit of the size of the beads may be set by the beads stimulating undesirable host responses that may interfere with the function of the transplanted cells or cause damage to the surrounding tissue. The upper limit of the size of the beads may also be set by the method of administration. Such limits can be readily determined by one skilled in the art. EXAMPLES

[0176] Example 1: Preemptive treatment with meteorin reverses PTX-induced neuropathic pain Materials and Methods: Female ICR / C57Bl6J mice (n=8 per group) with an average body weight of 23 g were divided into three groups; 1: Paclitaxel (PTX) and vehicle, 2: PTX and rmMeteorin (0.5 mg / kg), 3: PTX and rmMeteorin (1.8 mg / kg). As shown in Figure 1, either vehicle (Dulbecco's PBS) or rmMeteorin (0.5 mg / kg or 1.8 mg / kg) was subcutaneously injected every other day (D1, D3, D5, D7, and D9) using an insulin syringe (30G). PTX in Kholepher-ethanol (1:1) was diluted in Dulbecco's PBS and administered by intraperitoneal injection at a dose of 4 mg / kg every other day (D2, D4, D6, and D8) for a total dose of 16 mg / kg. Before the start of the experiment, the mice were acclimated to the clear acrylic behavioral chambers for 2 h. As a surrogate marker of mechanical allodynia, paw withdrawal thresholds (PWT) were tested at baseline and then every other day up to day 57 using calibrated von Frey filaments. On day 24, four animals per treatment group were euthanized and histological staining was performed (results are summarized in Example 2). Statistical analysis between groups was performed using mixed-effect ANOVA. All data are expressed as mean + / - SEM, and p<0.05 was considered significant.

[0177] result: As shown in Figure 2, on day 4, robust mechanical allodynia induced by PTX treatment occurred in all mice. With continued intermittent administration of rmMeteorin, an increase in PWT was observed at both 0.5 mg / kg (grey squares) and 1.8 mg / kg (black triangles) on days 10 and 8, respectively. Complete reversal of PWT at the 0.5 mg / kg and 1.8 mg / kg doses was then obtained on days 20 (P<0.001) and 16 (P<0.0001), respectively. This effect was substantially maintained throughout the experimental period. However, from day 32 onwards, PWT began to gradually increase towards baseline levels in vehicle-treated mice, suggesting that Meteorin-mediated reversal of PWT in PTX mice only continued significantly until day 35.

[0178] Conclusion: Preemptive treatment with repeated subcutaneous injections of rmMeteorin dose-dependently reversed paclitaxel-induced mechanical allodynia within a few days of initiating treatment, and this reversal continued even though Meteorin treatment was completed by day 8, thereby preventing the recurrence of neuropathic hypersensitivity.

[0179] Example 2: Preemptive treatment with meteorin prevents paclitaxel-induced immunohistochemical changes in hyperexcitability markers in dorsal root ganglia Materials and Methods: Mice were anesthetized with isoflurane (4%) and euthanized by decapitation. Tissues were snap frozen in OCT on dry ice, and sections (20 μm) of DRG were mounted on SuperFrost Plus slides (Thermo Fisher Scientific, Waltham, MA). They were then fixed in ice-cold 10% formalin for 15 min, followed by incubation in increasing percentages of ethanol: 50%, 70%, 100% for 5 min. Slides were then transferred to blocking solution (10% normal goat serum, 0.3% Triton-X100 in 0.1 M phosphate buffer (PB)) and gently rocked / agitated for 1 h at room temperature. Sections were incubated in primary antibodies (peripherin, glutamine synthetase, connexin 43) diluted in blocking solution for 3 h at room temperature or overnight at 4°C. Sections were washed five times with 0.1 M PB and then incubated in secondary antibodies diluted in blocking solution containing DAPI for 1 h at room temperature. Sections were washed five times with 0.1 M PB, mounted on glass slides, and cover slipped with Prolong Gold Antifade (Thermo Fisher Scientific, P36930) and sealed with nail polish. Images were taken using an Olympus FluoView 1200 confocal microscope. Analysis of immunohistochemical images obtained from three to four animals per treatment group was performed using Cellsens (Olympus).

[0180] result: Increased connectivity between satellite glial cells and neuronal cell bodies after neuropathic injury contributes to increased electrical coupling and excitability in DRG tissue, which in turn manifests as a sign of neuropathic pain. Figure 3 shows that paclitaxel (PTX)-mediated expression of glutamine synthetase enzyme (GS), a specific marker of satellite glial cells, was reduced by rmMeteorin treatment. Connexin 43 is an important gap junction protein that plays a key contribution to the hyperexcitability of DRG neurons after injury (Kim et al., 2016). Figure 3 shows that PTX-induced Connexin 43 expression encapsulating peripherin-stained neuronal cell bodies in DRG tissue was prevented by preemptive treatment with rmMeteorin.

[0181] Conclusion: Glutamine synthetase and connexin 43 are surrogate markers of PTX-induced hyperexcitability changes occurring in DRG tissues associated with behavioral neuropathic hypersensitivity. The decreased expression of both proteins after repeated subcutaneous injections of rmmeteorin suggests that decreased neuronal-glial connections are a potential pathophysiological mechanism targeted by rmmeteorin to mediate analgesia in CINP.

[0182] Example 3: Preemptive treatment with meteorin prevents loss of intraepidermal nerve fibers in the skin of paclitaxel-treated female mice.

[0183] Materials and Methods: The skin sections were post-fixed in 10% formalin solution for 24 hours, followed by 48 hours in 30% sucrose solution. 20 μm skin sections were cut using a cryostat, followed by an antigen retrieval step using 0.15 mg / ml pepsin in 0.2 M HCl. The sections were washed three times with 0.1 M PB, transferred to the primary antibody solution (PGP9.5), and subjected to immunohistochemistry processing as described in Example 2 to facilitate staining of intraepidermal nerve fibers (IENF). Analysis of images using Cellsens software was performed as described in the Materials and Methods of Example 2.

[0184] result: Treatment with paclitaxel in mice leads to the loss of IENFs from the skin of the mouse footpad in addition to the development of robust behavioral hyperalgesia (Singhmar et al., 2018). This reflects the corresponding loss of IENFs in some clinical neuropathic pain conditions. Figure 4 shows that in mice treated with both 0.5 mg / kg and 1.8 mg / kg doses of rmMeteorin, IENFs passing through the basement membrane of the epidermis stained longer and more intensely than in vehicle-treated mice.

[0185] Conclusion: Preemptive treatment with repeated subcutaneous injections of rmmeteorin prevented the loss of IENFs innervating the hindpaw skin of paclitaxel-treated female mice, reflecting a disease-modifying effect of rmmeteorin in CINP.

[0186] Example 4: Interventional treatment with meteorin reverses paclitaxel-induced neuropathic pain Materials and Methods: Adult ICR / C57Bl6J mice (n=7-8 per group) were used in two identical design experiments depicted in Figure 5. Females (body weight 27 g) were used in the first experiment, and males (body weight 34 g) were used in the second experiment. PTX in Kholepher-ethanol (1:1) was diluted in Dulbecco's PBS and administered by intraperitoneal injection at a dose of 4 mg / kg every other day (D2, D4, D6, and D8) for a total dose of 16 mg / kg, as shown in Figure 5. On day 10, mice were divided into three groups; paclitaxel (PTX) and vehicle, PTX and rmMeteorin (0.5 mg / kg), and PTX and rmMeteorin (1.8 mg / kg). Either vehicle (Dulbecco's PBS) or rmMeteorin (0.5 mg / kg or 1.8 mg / kg) was injected subcutaneously every other day (Day 10, D12, D14, D16, and D18) using an insulin syringe (30G). Mice were acclimated to the clear acrylic behavioral chambers for 2 hours before the start of the experiment. Mechanical allodynia was tested every other day (days without injections) using calibrated von Frey filaments. Cohorts of mice were tested for mechanical allodynia until all mice reached baseline. On day 24, four animals per treatment group were euthanized and histological staining was performed. Statistical analysis between groups was performed using mixed-effect ANOVA. All data are expressed as mean + / - SEM, and p<0.05 was considered significant.

[0187] result: Data from separate intervention experiments with female and male mice were combined for analysis. As shown in Figure 6, after the first PTX injection, mice already started to develop mechanical allodynia in the hind paws. After the fourth PTX injection, the reduction in PWT reached a maximum by day 9. After the first injection of rmMeteorin, both 0.5 mg / kg and 1.8 mg / kg treatment groups showed a significant reversal of PWT compared to the vehicle-treated group. This reversal continued throughout the rmMeteorin treatment period and after its cessation. Remarkably, in mice treated with rmMeteorin, the reversal of PTX-induced mechanical allodynia continued until day 48.

[0188] Conclusion: Interventional treatment with repeated subcutaneous injections of both low and high doses of rmMeteorin completely reversed paclitaxel-induced mechanical allodynia within a few days of initiating treatment, and this reversal continued even though rmMeteorin treatment was complete by day 18, thereby preventing the recurrence of neuropathic hypersensitivity.

[0189] Example 5: Interventional treatment with meteorin reverses paclitaxel-induced immunohistochemical changes in hyperexcitability markers in dorsal root ganglia Materials and Methods: On day 24, DRG tissue was harvested from female (n=9 total) and male (n=9 total) PTX mice pre-treated with either vehicle subcutaneous injection (n=3) or rm meteorin subcutaneous injection (0.5 mg / kg and 1.8 mg / kg, n=3 in each group), and immunohistochemistry for peripherin (not shown), glutamine synthetase, and connexin 43 was performed and analyzed as described in Example 2.

[0190] result: Increased connectivity between satellite glial cells and neuronal cell bodies after neuropathic injury contributes to increased electrical coupling and excitability in DRG tissue, which in turn manifests as a sign of neuropathic pain. Figure 7 shows that paclitaxel-mediated expression of glutamine synthetase enzyme (GS), a specific marker of satellite glial cells, was reduced by rmMeteorin treatment in both female and male mice. Connexin 43 is an important gap junction protein that plays a key role in the hyperexcitability of DRG neurons after injury (Kim et al., 2016). Figure 7 shows that PTX-induced Connexin 43 expression encapsulating peripherin-stained neuronal cell bodies in DRG tissue was reduced by treatment with rmMeteorin in both female and male mice.

[0191] Conclusion: Glutamine synthetase and connexin 43 are surrogate markers of PTX-induced hyperexcitability changes occurring in DRG tissues associated with behavioral neuropathic hypersensitivity. The decreased expression of both proteins after repeated subcutaneous injections of rmMeteorin intervention suggests that decreased neuronal-glial connections are a potential pathophysiological mechanism targeted by rmMeteorin to mediate analgesia in CINP in both female and male mice.

[0192] Overview of Arrays SEQ ID NO: 1: Human meteorin cDNA SEQ ID NO: 2: Full length amino acid sequence of human meteorin SEQ ID NO: 3: Human meteorin amino acid sequence without the signal peptide SEQ ID NO: 4: Mouse meteorin cDNA SEQ ID NO: 5: Mouse meteorin full length amino acid sequence SEQ ID NO: 6: Mouse meteorin amino acid sequence without the signal peptide SEQ ID NO: 7: Rat meteorin cDNA SEQ ID NO: 8: Rat meteorin full length amino acid sequence SEQ ID NO: 9: Rat meteorin amino acid sequence without the signal peptide SEQ ID NO: 10: Human codon-optimized DNA sequence SEQ ID NO: 11: Consensus sequence of mature meteorin Human meteorin cDNA (1109 base pairs; CDS=118-999) (SEQ ID NO: 1) >gi|34147349|ref|NM_024042.2|Homo sapiens protein of unknown function MGC2601 (MGC2601), mRNA [ka] Full length amino acid sequence of human meteorin (SEQ ID NO:2) >IPI00031531.1 REFSEQ_NP:NP_076947 TREMBL:Q9UJH9 ENSEMBL:ENSP00000219542 Tax_Id=9606 C380A1.2.1(novel protein) [ka] Human meteorin protein without the signal peptide (SEQ ID NO:3) [ka] Mouse meteorin cDNA, 1363 base pairs, CDS 84..959 (SEQ ID NO: 4) NM_133719. Mus musculus meteorin [gi:56550040] [ka] Mouse meteorin full length amino acid sequence (SEQ ID NO:5) ref|NP_598480.1|Meteorin [Mus musculus] [ka] Mouse meteorin protein without signal peptide (SEQ ID NO:6) [ka] Rat meteorin cDNA (1026 base pairs; CDS=1-876) (SEQ ID NO: 7) >gi|34870570|ref|XM_213261.2|1810034B16Rattus norvegicus (LOC287151), mRNA similar to Rik protein [ka] Rat meteorin full length amino acid sequence (SEQ ID NO:8) >IPI00369281.1|REFSEQ_XP:XP_213261|ENSEMBL:ENSRNOP00000026676 [ka] Rat meteorin protein without the signal peptide (SEQ ID NO: 9) [ka] Codon-optimized meteorin nucleotide sequence present in constructs pCAn.meteorin and pT2.CAn.meteorin (SEQ ID NO:10) [ka] Consensus sequence of mature meteorin (SEQ ID NO:11) [ka] X is any of the 21 amino acids that can be encoded by DNA.

[0193] References Jorgensen et al., Characterization of Meteorin-An evolutionary conserved neurotrophic factor, J mol Neurosci 2009 Sep;39(1-2):104-116. Kim YSet al.,Coupled Activation of Primary Sensory Neurons Contributes to Chronic Pain.Neuron.2016,91(5),1085-1096. Nishino et al., “Meteorin: a secreted protein that regulates glial cell differentiation and promotes axonal extension”, EMBO J., 23(9):1998-2008(2004). Johnson et al., Nat. Med., 2:795-799 (1996) Yasuda, Biomed. Ther., 27:1221-1223 (1993) Hora et al.,Bio / Technology,8:755-758(1990) Cleland,“Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems,”in Vaccine Design:The Subunit and Adjuvant Approach,Powell and Newman,eds,(Plenum Press:New York,1995),pp.439-462 Reagan-Shaw et al.,FASEB J,22,659-661(2007) Creighton,Proteins-Structure and Molecular Properties,2nd Ed.,W.H.Freeman and Company,New York,1993. Wold,F.,in Posttranslational Covalent Modification of Proteins,B.C.Johnson,Ed.,Academic Press,New York,pp1-12,1983 Seifter et al.,Meth.Enzymol.182:626-646,1990 Rattan et al.,Protein Synthesis:Posttranslational Modifications and Aging,Ann.N.Y.Acad.Sci.663:48-62,1992 Maniatis et al.,in Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,(NY 1982) Messing,et al.,(Nucleic Acids Res.,9:309-,1981), Maxam,et al.,(Methods in Enzymology,65:499,1980). Banerji et al.,Cell 27:299(1981) Corden et al.,Science 209:1406(1980) Breathnach and Chambon,Ann.Rev.Biochem.50:349(1981)). Weiss et al.,eds.,The molecular biology of tumor viruses:RNA tumor viruses,Cold Spring Harbor Laboratory,(NY 1982)). Jolly et al.,Nucleic Acids Res.11:1855(1983) Capecchi et al.,In :Enhancer and eukaryotic gene expression,Gulzman and Shenk,eds.,pp.101-102,Cold Spring Harbor Laboratories(NY 1991) Schmidt et al.,Nature 314:285(1985) Rossi and deCrombrugghe,Proc.Natl.Acad.Sci.USA 84:5590-5594(1987)). Prockop and Kivirikko,N.Eng.J.Med.311:376(1984) Smith and Niles,Biochem.19:1820(1980) de Wet et al.,J.Biol.Chem.,258:14385(1983) Gruss et al.,Proc.Natl.Acad.Sci.USA 78:943(1981) Benoist and Chambon,Nature 290:304(1981) Fromm and Berg,J.Mol.Appl.Genetics,1 :457(1982). Moreau et al.,Nucleic Acids Res.9:6047(1981) Armelor,Proc.Natl.Acad.Sci.USA 70:2702(1973) Fisher Biotech Source 87-88,Fisher Scientific Co.,1987,pp.72-75 Sigma Cell Culture Catalog,Sigma Chemical Co.,St,Louis,1991,pp.162-163 Ventrex Product Catalog,Ventrex Laboratories,1989.

Claims

1. 1. A pharmaceutical composition comprising an isolated polypeptide for use in the treatment or prevention of chemotherapy-induced neuropathic pain in a subject, the polypeptide comprising: i. the amino acid sequence of SEQ ID NO:3, and ii. a biologically active sequence variant of the amino acid sequence of SEQ ID NO:3, having at least 70% sequence identity to SEQ ID NO:3, at least 80% sequence identity to SEQ ID NO:3, at least 85% sequence identity to SEQ ID NO:3, at least 90% sequence identity to SEQ ID NO:3, at least 95% sequence identity to SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3; A pharmaceutical composition comprising an amino acid sequence selected from the group consisting of:

2. 2. The pharmaceutical composition for use according to claim 1, wherein said pharmaceutical composition is administered simultaneously or intermittently with a chemotherapy treatment.

3. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein administration of the pharmaceutical composition is initiated prior to the initiation of chemotherapy treatment, for example administration of the pharmaceutical composition is initiated at least 1 day before the initiation of chemotherapy treatment, for example at least 2 days before the initiation of chemotherapy treatment, for example at least 3 days before the initiation of chemotherapy treatment, for example at least 4 days, at least 5 days, or at least 1 week before the initiation of chemotherapy treatment.

4. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein said pharmaceutical composition is administered in conjunction with each administration of chemotherapy, for example said pharmaceutical composition is administered on the same day as the start of chemotherapy treatment or at least one day before the start of chemotherapy treatment, such as at least two days before the start of chemotherapy treatment, for example at least three days before the start of chemotherapy treatment, such as at least four days, at least five days, at least one week before the start of chemotherapy treatment.

5. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the chemotherapy treatment comprises administration of a platinum-based agent (e.g. carboplatin, cisplatin, or oxaliplatin), a taxane (e.g. paclitaxel or docetaxel), an epothilone (e.g. ixabepilone), a vinca alkaloid (e.g. vincristine or vinblastine), and a semi-synthetic analogue (e.g. vinorelbine or eribulin), a proteasome inhibitor (e.g. bortezomib), or an immunomodulatory agent (e.g. thalidomide or lenalidomide), or a combination thereof.

6. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the subject being treated is suffering from ovarian cancer, breast cancer, esophageal cancer, pancreatic cancer, leukemia, Hodgkin's disease, Wilms' tumor, neuroblastoma, testicular cancer, bladder cancer, lung cancer, or multiple myeloma.

7. the polypeptide comprises the consensus sequence of SEQ ID NO: 11, and / or the polypeptide has cysteine ​​residues at positions 7, 28, 59, 95, 148, 151, 161, 219, 243, and 265 relative to the amino acid sequence of SEQ ID NO:3; and / or the polypeptide is a variant polypeptide and any amino acid substitutions are conservative substitutions; and / or the polypeptide is capable of forming at least one intramolecular disulfide bridge, A pharmaceutical composition for use according to claim 1 or 2.

8. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the treatment results in amelioration of allodynia (e.g., thermal allodynia, e.g., cold allodynia or thermal allodynia, or mechanical allodynia), hyperalgesia (e.g., mechanical hyperalgesia), or spontaneous pain.

9. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the subject to be treated is a mammal, such as a primate, such as a human.

10. the pharmaceutical composition is administered by systemic administration, and / or The pharmaceutical composition is administered by parenteral injection, for example by subcutaneous or intrathecal injection; A pharmaceutical composition for use according to claim 1 or 2.

11. and / or the pharmaceutical composition is administered at a dose of from 1 μg / kg body weight to 10,000 μg / kg body weight, such as from 1 μg / kg body weight to 7,500 μg / kg body weight, for example from 1 μg / kg body weight to 5,000 μg / kg body weight, for example from 1 μg / kg body weight to 2,000 μg / kg body weight, such as from 1 μg / kg body weight to 1,000 μg / kg body weight, for example from 1 μg / kg body weight to 700 μg / kg body weight, for example from 5 μg / kg body weight to 500 μg / kg body weight, for example from 10 μg / kg body weight to 100 μg / kg body weight; the pharmaceutical composition is administered at least 1-3 times per week, such as 2-5 times per week, such as 3-6 times per week; A pharmaceutical composition for use according to claim 1 or 2.

12. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the pharmaceutical composition is administered every other day, or the pharmaceutical composition is administered daily.

13. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein administration of said pharmaceutical composition is initiated after the onset of symptoms of chemotherapy-induced neuropathic pain.

14. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein administration of the pharmaceutical composition is started after the start of chemotherapy treatment, such as 1 day, such as 2 days, for example 3 days, such as 4 days, for example 5 days, such as 8 days, for example 12 days, or such as 1 week, such as 2 weeks, for example 3 weeks after the start of chemotherapy treatment.

15. 1. A pharmaceutical composition comprising an isolated nucleic acid molecule for use in treating or preventing chemotherapy-induced neuropathic pain in a subject, the isolated nucleic acid molecule comprising: a. the amino acid sequence of SEQ ID NO:3; b. A biologically active sequence variant of the amino acid sequence of SEQ ID NO: 3, which has at least 70% sequence identity to SEQ ID NO: 3; A pharmaceutical composition comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:

16. 1. A pharmaceutical composition comprising a vector for use in the treatment or prevention of chemotherapy-induced neuropathic pain in a subject, the vector comprising: a. the amino acid sequence of SEQ ID NO:3, and b. A biologically active sequence variant of the amino acid sequence of SEQ ID NO:3, which has at least 70% sequence identity to SEQ ID NO:

3. A pharmaceutical composition comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:

Images