WO2015042580A1 - Compositions and methods for treatment of neuropathic pain - Google Patents

Abstract

Disclosed are methods of treating neuropathic pain in a human subject by administering a neublastin polypeptide at a dosage of 100-1,600 μg/kg body weight of the human subject.

Description

COMPOSITIONS AND METHODS FOR TREATMENT OF NEUROPATHIC PAIN

Technical Field

The invention relates to protein chemistry, molecular biology, and vascular biology.

Background

Neublastin, also known as artemin and enovin, is a 24 kDa homodimeric, secreted protein that promotes the outgrowth and survival of neurons of the peripheral and central nervous system (Baudet et al, 2000, Development, 127:4335; Masure et al., 1999, Eur. J. Biochem., 266:892; Rosenblad et al, 2000, Mol. Cell Neurosci., 15(2): 199). Neublastin mRNA is expressed predominantly in embryonic kidney and lung, and in adults, is expressed highest in pituitary gland, trachea, and placenta (Baudet et al, 2000, Development,

127:4335).

Neublastin is a member of the glial cell line-derived neurotrophic factor (GDNF) ligand family. GDNF ligands activate both Ras and phosphatidylinositol-3 -kinase signal transduction pathways by engaging the membrane-bound c-RET receptor tyrosine kinase. This c-RET-mediated signaling requires an additional co-receptor, a glycosylphosphatidyl inositol (GPI)-anchored GDNF family receptor alpha (GFRa) protein, which confers ligand specificity to c-RET. Four GFRa co-receptor proteins have been identified (GFRa 1-4). Neublastin shows highest affinity for GFRa3 in vitro, however in studies using human fibroblasts, neublastin can stimulate c-RET-dependent signaling through either GFRa3 or GFRal (Baudet et al, 2000, Development, 127:4335; Masure et al, 1999, Eur. J. Biochem. 266:892; Rosenblad et al, 2000, Mol. Cell Neurosci., 15(2): 199).

Neublastin and the other GDNF family members are members of the transforming growth factor beta (TGF beta) superfamily and thus, are characterized by the presence of seven conserved cysteine residues with similar spacing which form the structure of a cysteine knot (Saarma, 1999, Microsc. Res. Tech., 45:292). Each monomer contains two disulfide bonds that form a closed loop structure encircling the third disulfide to form a tight knot structure. The seventh cysteine contained within each monomer forms an intermolecular disulfide bond, covalently linking the monomers to form the final dimer product (Rattenholl et al 2000, J. Mol. Biol, 305:523). Summary

The present invention is based, at least in part, on the discovery that administration of neublastin within a dosage range of 100-1,600 μg/kg body weight elicits a reduction in pain in human subjects with painful lumbar radiculopathy.

The invention features a method of treating neuropathic pain in a human subject in need thereof by administering to the human subject a polypeptide comprising an amino acid sequence that is at least 80% identical to amino acids 15-113 of SEQ ID NO: l, wherein the polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET, and wherein the polypeptide is administered at a dosage of 100-1,600 μg/kg body weight of the human subject. Also disclosed is a polypeptide comprising an amino acid sequence that is at least 80% identical to amino acids 15-113 of SEQ ID NO: l, wherein the polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET for treating neuropathic pain in a human subject when administered at a dosage of 100-1,600 μg/kg body weight of the human subject.

The amino acid sequence contained in the polypeptide can optionally be at least 90% identical (e.g., at least 95% or 98% identical) to amino acids 15-113 of SEQ ID O: l . In some embodiments, the polypeptide contains or consists of amino acids 10-1 13 of SEQ ID NO: l, amino acids 15-1 13 of SEQ ID NO: l, amino acids 15-113 of SEQ ID NO:2, amino acids 15-113 of SEQ ID O:3, amino acids 15-1 13 of SEQ ID O:4, amino acids 15-113 of SEQ ID NO:5, amino acids 15-113 of SEQ ID NO:8, or amino acids 15-1 13 of SEQ ID NO:9. For example, the polypeptide can contain or consist of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO:2, the amino acid sequence of SEQ ID NO:3, the amino acid sequence of SEQ ID NO:4, the amino acid sequence of SEQ ID NO:5, the amino acid sequence of SEQ ID NO:8, or the amino acid sequence of SEQ ID NO:9.

In some embodiments, the polypeptide is administered at a dosage of 200-1,200 μg/kg body weight of the human subject.

In some embodiments, the polypeptide is administered at a dosage of 200-800 μg/kg body weight of the human subject.

In some embodiments, the polypeptide is administered at a dosage of 400-800 μg/kg body weight of the human subject.

In some embodiments, the polypeptide is administered at a dosage of 300-500 μg/kg body weight of the human subject. In some embodiments, the polypeptide is administered at a dosage of 150, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1, 100, 1,200, 1,300, 1,400, 1,500, or 1,600 μg/kg body weight of the human subject.

The polypeptide can be administered to the human subject, for example, via systemic administration (e.g., subcutaneous or intravenous administration).

A variety of dosing regimens can be applied according to the methods described herein. In some instances, the method comprises multiple administrations of the polypeptide. For example, the multiple administrations can include at least three administrations of the polypeptide within one week (e.g., every other day). In this example, the second

administration can optionally be 44-52 hours (e.g., about 48 hours) after the first

administration and the third administration can optionally be 44-52 hours (e.g., about 48 hours) after the second administration. In another example, the multiple administrations can include at least three administrations of the polypeptide within a period of three weeks. In this example, the second administration can optionally be 6-8 days (e.g., 7 days) after the first administration and the third administration can optionally be 6-8 days (e.g., 7 days) after the second administration. In another example, the multiple administrations can include at least two administrations of the polypeptide within one week. The same dosage of the polypeptide can optionally be provided to the human subject at each of the multiple administrations described above.

After an initial dosing or an initial dosing period with the polypeptide as described above (e.g., at least three administrations of the polypeptide within one week), the polypeptide may subsequently be administered at the same or at a different dose and/or dosing frequency as a means to maintain a therapeutic effect. In some instances, the dose level of the polypeptide is increased (as compared to the initial dosing) but the dosing frequency is decreased (as compared to the initial dosing). In one example, after three administrations of the polypeptide within one week (e.g., by intravenous administration), the polypeptide is subsequently administered at an interval of once per month (e.g., by intravenous administration), optionally for a period of at least 2, 3, 4, 5, or 6 months. In another example, after three administrations of the polypeptide within one week (e.g., by intravenous administration), the polypeptide is subsequently administered at an interval of once per week (e.g., by subcutaneous administration), optionally for a period of at least 2, 3, 4, 5, or 6 months. In another example, after three administrations of the polypeptide within one week (e.g., by intravenous administration), the polypeptide is subsequently administered at an interval of once every two weeks (e.g., by subcutaneous administration), optionally for a period of at least 2, 3, 4, 5, or 6 months. In another example, after three administrations of the polypeptide within one week (e.g., by intravenous administration), the polypeptide is subsequently administered three times per week (e.g., by subcutaneous administration), optionally for a period of at least 2, 3, 4, 5, or 6 months. In another example, after three administrations of the polypeptide within one week (e.g., by intravenous administration), the polypeptide is subsequently administered three times per week every other week (e.g., by subcutaneous administration), optionally for a period of at least 2, 3, 4, 5, or 6 months.

In some embodiments of the methods described herein, the neuropathic pain is associated with painful lumbar radiculopathy, painful diabetic neuropathy, post-herpetic neuralgia, post-traumatic neuralgia, post-surgical neuralgia, or a complex regional pain syndrome, or is induced by a therapeutic drug.

In some embodiments of the methods described herein, the neuropathic pain is associated with leprosy, Lyme disease, infection by a virus, or a cancer.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Brief Description of the Drawings

Fig. 1 is an alignment of wild type human (SEQ ID NO: 10), mouse (SEQ ID NO: 1 1), and rat (SEQ ID NO: 12) pre pro neublastin polypeptides. The left and right vertical lines indicate, respectively, the start of the mature 1 13 amino and 104 amino acid forms. The RRXR heparin binding motif is boxed.

Figs. 2A-2B are graphs depicting Likert general sciatica pain assessment scores by week for subjects in Part I of the study (Fig. 2 A) and Part II of the study (Fig. 2B).

Figs. 3A-3B are graphs depicting Likert back pain assessment scores by week for subjects in Part I of the study (Fig. 3A) and Part II of the study (Fig. 3B). Figs. 4A-4B are graphs depicting Likert leg pain assessment scores by week for subjects in Part I of the study (Fig. 4A) and Part II of the study (Fig. 4B).

Figs. 5A-5D are graphs depicting Likert general sciatica pain assessment scores by time point for subjects in Part I of the study from day 0 to day 17 (Fig. 5A), Part I of the study from day 0 to day 71 (Fig. 5B), Part II of the study from day 0 to day 7 (Fig. 5C), and Part II of the study from day 0 to day 61 (Fig. 5D).

Figs. 6A-6D are graphs depicting Likert back pain assessment scores by time point for subjects in Part I of the study from day 0 to day 17 (Fig. 6A), Part I of the study from day 0 to day 71 (Fig. 6B), Part II of the study from day 0 to day 7 (Fig. 6C), and Part II of the study from day 0 to day 61 (Fig. 6D).

Figs. 7A-7D are graphs depicting Likert leg pain assessment scores by time point for subjects in Part I of the study from day 0 to day 17 (Fig. 7A), Part I of the study from day 0 to day 71 (Fig. 7B), Part II of the study from day 0 to day 7 (Fig. 7C), and Part II of the study from day 0 to day 61 (Fig. 7D).

Figs. 8A-8B are graphs depicting Short-Form McGill Pain Questionnaire sciatica pain assessment scores by week for subjects in Part I of the study (Fig. 8A) and Part II of the study (Fig. 8B).

Detailed Description

The present invention provides compositions and methods for treating neuropathic pain in a human subject. As disclosed in the accompanying examples, administration of neublastin was found to elicit pain reduction in human subjects with painful lumbar radiculopathy. Neublastin Polypeptides

A mature form of wild-type human neublastin is 113 amino acids in length and has the

following amino acid sequence: AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDE LVRFRFCSGSCRRARSPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDV NSTWRTVDRLSATACGCLG (SEQ ID NO: 1). Polypeptides having the amino acid sequence of SEQ ID NO: 1 or biologically active variants of thereof can be used in the methods described herein. A variant neublastin polypeptide can contain one or more additions, substitutions, and/or deletions, as detailed in the following sections. Wild-type neublastin polypeptides and biologically active variants thereof are collectively referred to herein as "neublastin polypeptides."

A variant neublastin polypeptide can vary in length from the corresponding wild-type polypeptide. Although the mature human neublastin polypeptide of SEQ ID NO: 1 consists of the carboxy terminal 1 13 amino acids of pre pro neublastin (SEQ ID NO: 10), not all of the 113 amino acids are required to achieve useful neublastin biological activity. Amino terminal truncation is permissible. Thus, a variant neublastin polypeptide can contain, for example, the carboxy terminal 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 11 1, 112, or 113 amino acids of SEQ ID NO: 1 (i.e., its length can be 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 1 1, 112, or 113 amino acids).

A variant neublastin polypeptide can also vary in sequence from the corresponding wild-type polypeptide. In particular, certain amino acid substitutions can be introduced into the neublastin sequence without appreciable loss of a neublastin biological activity. In exemplary embodiments, a variant neublastin polypeptide (i) contains one or more amino acid substitutions, and (ii) is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 1 (or 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to amino acids 15-113 of SEQ ID NO: 1). A variant neublastin polypeptide differing in sequence from SEQ ID NO: l (or differing in sequence from amino acids 15-113 of SEQ ID NO: l) may include one or more amino acid substitutions (conservative or non-conservative), one or more deletions, and/or one or more insertions.

Fig. 1 is an alignment of the wild type human, mouse, and rat pre pro neublastin polypeptides. The vertical lines in Fig. l indicate the start of the mature 1 13 amino acid form (left vertical line) and 104 amino acid form (right vertical line) of neublastin. The RRXR heparin binding motif is boxed. This alignment of bioactive forms of neublastin indicates specific exemplary residues (i.e., those that are not conserved among the human, mouse, and rat forms) that can be substituted without eliminating bioactivity.

Percent identity between amino acid sequences can be determined using the BLAST 2.0 program. Sequence comparison can be performed using an ungapped alignment and using the default parameters (Blossom 62 matrix, gap existence cost of 1 1, per residue gap cost of 1, and a lambda ratio of 0.85). The mathematical algorithm used in BLAST programs is described in Altschul et al, 1997, Nucleic Acids Research 25:3389-3402.

A conservative substitution is the substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The non-polar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution.

Non-conservative substitutions include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, He, Phe or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

A biologically active variant neublastin polypeptide, when dimerized, binds to a ternary complex containing GFRa3 and RET. Any method for detecting binding to this complex can be used to evaluate the biological activity a variant neublastin polypeptide. Exemplary assays for detecting the ternary complex-binding ability of a variant neublastin polypeptide are described in WO00/01815 (the content of which is incorporated herein by reference).

A variant neublastin polypeptide can also be assessed to evaluate its ability to trigger the neublastin signaling cascade. For example, the Kinase Receptor Activation (KIRA) assay can be used to assess the ability of a variant neublastin polypeptide to induce RET autophosphorylation (See also, Sadick et al, 1996, Anal. Biochem., 235(2):207).

Substitutions at one or more of the following amino acid residues are expected to result in a variant neublastin polypeptide having reduced or absent heparin binding ability as compared to wild type neublastin: Arg 48, Arg 49, Arg 51, Ser 46, Ser 73, Gly 72, Arg 39, Gin 21, Ser 20, Arg 68, Arg 33, His 32, Val 94, Arg 7, Arg 9, or Arg 14. Reference to a neublastin amino acid reside by position number refers to the numbering of residues relative to SEQ ID NO: l . A neublastin amino acid residue designated for substitution (e.g., an arginine residue at position 48, 49, and/or 51) can be substituted with a non-conservative amino acid residue (e.g., glutamic acid) or a conservative or amino acid residue. Exemplary amino acids that can be substituted at a residue identified herein (e.g., position 48, 49, and/or 51) include glutamic acid, aspartic acid, and alanine.

Examples of variant neublastin polypeptides that exhibit reduced or absent heparin binding are disclosed in Table 1 and in WO 2006/023781 (the content of which is incorporated herein by reference). Amino acid residues of the variant neublastin

polypeptides that are mutated as compared to the corresponding wild type position are bolded and underlined in Table 1. In addition, the neublastin polypeptide (e.g., 113, 99, or 104 amino acids in length) used as the background for the substitution is depicted in Table 1.

Table 1 : Variant Neublastin Polypeptides

SEQ Position Length of Amino Acid Sequence

ID NO Substituted Polypeptide

2 Arg 48 113 AGGPGSRARAAGARGCRLRSQLVPVRA

LGLGHRSDELVRFRFCSGSCERARSPHD

LSLASLLGAGALRPPPGSRPVSQPCCRPT

RYEAVSFMDVNSTWRTVDRLSATACGC

LG

3 Arg 49 113 AGGPGSRARAAGARGCRLRSQLVPVRA

LGLGHRSDELVRFRFCSGSCREARSPHD

LSLASLLGAGALRPPPGSRPVSQPCCRPT

RYEAVSFMDVNSTWRTVDRLSATACGC

LG

4 Arg 51 113 AGGPGSRARAAGARGCRLRSQLVPVRA

LGLGHRSDELVRFRFCSGSCRRAESPHD

LSLASLLGAGALRPPPGSRPVSQPCCRPT

RYEAVSFMDVNSTWRTVDRLSATACGC

LG

5 Arg 48 and 113 AGGPGSRARAAGARGCRLRSQLVPVRA

Arg 49 LGLGHRSDELVRFRFCSGSCEEARSPHD

LSLASLLGAGALRPPPGSRPVSQPCCRPT

RYEAVSFMDVNSTWRTVDRLSATACGC

LG

6 Arg 48 and 99 GCRLRSQLVPVRALGLGHRSDELVRFRF

Arg 49 CSGSCEEARSPHDLSLASLLGAGALRPPP

GSRPVSQPCCRPTRYEAVSFMDVNSTW RTVDRLSATACGCLG

7 Arg 48 and 104 AAGARGCRLRSQLVPVRALGLGHRSDE

Arg 49 LVRFRFCSGSCEEARSPHDLSLASLLGAG

ALRPPPGSRPVSQPCCRPTRYEAVSFMD VNSTWRTVDRLSATACGCLG

8 Arg 49 and 113 AGGPGSRARAAGARGCRLRSQLVPVRA

Arg 51 LGLGHRSDELVRFRFCSGSCREAESPHD

LSLASLLGAGALRPPPGSRPVSQPCCRPT RYEAVSFMDVNSTWRTVDRLSATACGC SEQ Position Length of Amino Acid Sequence

ID NO Substituted Polypeptide

LG

9 Arg 48 and 1 13 AGGPGSRARAAGARGCRLRSQLVPVRA

Arg 51 LGLGHRSDELVRFRFCSGSCERAESPHD

LSLASLLGAGALRPPPGSRPVSQPCCRPT

RYEAVSFMDVNSTWRTVDRLSATACGC

LG

A polypeptide can optionally contain heterologous amino acid sequences in addition to a neublastin polypeptide. "Heterologous," as used when referring to an amino acid sequence, refers to a sequence that originates from a source foreign to the particular host cell, or, if from the same host cell, is modified from its original form. Exemplary heterologous sequences include a heterologous signal sequence (e.g., native rat albumin signal sequence, a modified rat signal sequence, or a human growth hormone signal sequence) or a sequence used for purification of a neublastin polypeptide (e.g., a histidine tag).

Neublastin polypeptides can be isolated using methods known in the art. Naturally occurring or recombinantly produced neublastin polypeptides can be isolated from cells or tissue sources using standard protein purification techniques. Alternatively, mutated neublastin polypeptides can be synthesized chemically using standard peptide synthesis techniques. The synthesis of short amino acid sequences is well established in the peptide art. See, e.g., Stewart, et ah, Solid Phase Peptide Synthesis (2d ed., 1984).

In some embodiments, neublastin polypeptides are produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding a neublastin polypeptide can be inserted into a vector, e.g., an expression vector, and the nucleic acid can be introduced into a cell. Suitable cells include, e.g., mammalian cells (such as human cells or CHO cells), fungal cells, yeast cells, insect cells, and bacterial cells (e.g., E. coli). When expressed in a recombinant cell, the cell is preferably cultured under conditions allowing for expression of a neublastin polypeptide. The neublastin polypeptide can be recovered from a cell suspension if desired. As used herein, "recovered" means that the mutated polypeptide is removed from those components of a cell or culture medium in which it is present prior to the recovery process. The recovery process may include one or more refolding or purification steps. Buffers and methods for inducing folding of a denatured neublastin polypeptide are described in, e.g., WO 2006/023782.

Variant neublastin polypeptides can be constructed using any of several methods known in the art. One such method is site-directed mutagenesis, in which a specific nucleotide (or, if desired a small number of specific nucleotides) is changed in order to change a single amino acid (or, if desired, a small number of predetermined amino acid residues) in the encoded variant neublastin polypeptide. Many site-directed mutagenesis kits are commercially available. One such kit is the "Transformer Site Directed Mutagenesis Kit" sold by Clontech Laboratories (Palo Alto, CA).

Pharmaceutical Compositions

A neublastin polypeptide can be incorporated into a pharmaceutical composition containing a therapeutically effective amount of the polypeptide and one or more adjuvants, excipients, carriers, and/or diluents. Acceptable diluents, carriers and excipients typically do not adversely affect a recipient's homeostasis (e.g., electrolyte balance). Acceptable carriers include biocompatible, inert or bioabsorbable salts, buffering agents, oligo- or

polysaccharides, polymers, viscosity-improving agents, preservatives and the like. One exemplary carrier is physiologic saline (0.15 M NaCl, pH 7.0 to 7.4). Another exemplary carrier is 50 mM sodium phosphate, 100 mM sodium chloride. Further details on techniques for formulation and administration of pharmaceutical compositions can be found in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.).

Administration of a pharmaceutical composition containing a neublastin polypeptide can be systemic or local. Pharmaceutical compositions can be formulated such that they are suitable for parenteral and/or non-parenteral administration. Specific administration modalities include subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, intrathecal, oral, rectal, buccal, topical, nasal, ophthalmic, intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterine administration.

Administration may be by periodic injections of a bolus of the pharmaceutical composition or may be made more continuous by intravenous or intraperitoneal

administration from a reservoir which is external (e.g., an IV bag) or internal (e.g., a bioerodable implant, a bioartificial organ, or a colony of implanted neublastin production cells). See, e.g., U.S. Pat. Nos. 4,407,957, 5,798, 1 13, and 5,800,828, each incorporated herein by reference. Administration of a pharmaceutical composition may be achieved using suitable delivery means such as: a pump (see, e.g., Annals of Pharmacotherapy, 27:912 (1993); Cancer, 41 : 1270 (1993); Cancer 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, herein incorporated by reference); continuous release polymer implants (see, e.g., Sabel, U.S. Pat. No. 4,883,666, incorporated herein by reference); macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881, 4,976,859 and 4,968,733 and published PCT patent applications W092/19195, WO 95/05452, each incorporated herein by reference); injection, either subcutaneously, intravenously, intra-arterially, intramuscularly, or to other suitable site; or oral administration, in capsule, liquid, tablet, pill, or prolonged release formulation.

Examples of parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, pump delivery, encapsulated cell delivery, liposomal delivery, needle-delivered injection, needle-less injection, nebulizer, aeorosolizer, electroporation, and transdermal patch.

Formulations suitable for parenteral administration conveniently contain a sterile aqueous preparation of the neublastin polypeptide, which preferably is isotonic with the blood of the recipient (e.g., physiological saline solution). Formulations may be presented in unit-dose or multi-dose form.

An exemplary formulation contains a neublastin polypeptide described herein and the following buffer components: sodium succinate (e.g., 10 mM); NaCl (e.g., 75 mM); and L- arginine (e.g., 100 mM).

Methods of Treatment

A neublastin polypeptide can be used for treating or preventing neuropathic pain. A neublastin polypeptide (or a pharmaceutical composition comprising same) can be used in the treatment of a number of peripheral neuropathies associated with neuropathic pain, including: (a) trauma-induced neuropathies, such as those caused by physical injury or disease state, physical damage to the brain, physical damage to the spinal cord, stroke associated with brain damage, and neurological disorders related to neurodegeneration, (b) chemotherapy-induced neuropathies, (c) toxin-induced neuropathies (including but not limited to neuropathies induced by alcoholism, vitamin B6 intoxication, hexacarbon intoxication, amiodarone, chloramphenicol, disulfiram, isoniazide, gold, lithium,

metronidazole, misonidazole, or nitrofurantoin), (d) drug-induced neuropathies, including therapeutic drug-induced neuropathic pain (such as caused by anti-cancer agents, particularly anti-cancer agents selected from the group consisting of taxol, taxotere, cisplatin, nocodazole, vincristine, vindesine and vinblastine; and such as caused by anti-viral agents, particularly anti-viral agents selected from the group consisting of ddl, DDC, d4T, foscarnet, dapsone, metronidazole, and isoniazid), (e) vitamin-deficiency-induced neuropathies (including but not limited to vitamin B 12 deficiency, vitamin B6 deficiency, and vitamin E deficiency), (f) idiopathic neuropathies, (g) diabetic neuropathies, (h) pathogen-induced nerve damage, (i) inflammation-induced nerve damage, (j) neurodegeneration, (k) hereditary neuropathy (including but not limited to Friedreich ataxia, familial amyloid polyneuropathy, Tangier disease, and Fabry disease), (1) metabolic disorders (including but not limited to renal insufficiency and hypothyroidism), (m) infectious and viral neuropathies (including but not limited to neuropathic pain associated with leprosy or Lyme disease or neuropathic pain associated with infection by a virus, particularly a virus selected from the group consisting of a herpes virus (e.g. herpes zoster which may lead to post-herpetic neuralgia), a human immunodeficiency virus (HIV), and a papilloma virus), (n) auto-immune neuropathies (including but not limited to Guillain-Barre syndrome, chronic inflammatory de-myelinating polyneuropathy, monoclonal gammopathy of undetermined significance and

polyneuropathy), (o) trigeminal neuralgia and entrapment syndromes (including but not limited to Carpel tunnel), and (p) other neuropathic pain syndromes including post-traumatic neuralgia, phantom limb pain, multiple sclerosis pain, complex regional pain syndromes (including but not limited to reflex sympathetic dystrophy and causalgia), neoplasia- associated pain, vasculitic/angiopathic neuropathy, and painful lumbar radiculopathy

(commonly referred to as sciatica).

Exemplary neuropathic pain conditions that can be treated or prevented by administration of a neublastin polypeptide are reviewed in the following sections.

Painful Lumbar Radiculopathy

Radicular pain, or radiculopathy, is defined as spinal nerve root dysfunction causing dermatomal pain and paresthesias, myotomal weakness, and/or impaired deep tendon reflexes. Lumbar radiculopathy refers to a pathologic process involving the lumbar nerve roots causing radicular symptoms into a lower extremity. The nerve root pathology arises primarily from direct neural compression irrespective of whether the etiology is an acute herniated or displaced disc, bony spurs, foraminal stenosis, central stenosis, or hypermobility of a vertebral segment.

Painful lumbar radiculopathy is diagnosed mainly by history and clinical examination, where the patient describes pain radiating along a dermatomal distribution in the leg. The patient may also describe associated sensory or motor symptoms. The neurological examination will find ipsilateral straight leg raising induced pain in case of fifth lumbar root (L5) or first sacral root (SI) compression, and usually detects neurological deficits localizing to a single root distribution. The most useful test for confirming the presence of a radiculopathy is needle electromyogram (EMG); however, the needle EMG examination can identify only the root or roots that are physiologically involved, not the precise anatomic site of pathology in the lumbar spinal canal. The most accurate imaging study to assess neural structures within the lumbar spine is magnetic resonance imaging (MRI) scanning.

Painful Diabetic Neuropathy

Individuals with diabetes can develop nerve damage throughout the body, which in some cases results in neuropathic pain. The pain can appear in the extremities, e.g., the toes, feet, legs, hands, arms, and/or fingers. Treatment and prevention of painful diabetic neuropathy is contemplated. A neublastin polypeptide can be administered to reduce the severity of neuropathic pain that has already appeared. Prophylactic treatment would commence after determination of the initial diagnosis of diabetes or diabetes-associated symptoms and before the onset of neuropathic pain. Prophylactic treatment may also commence upon determining that a subject is at risk for developing diabetes or diabetes- associated symptoms. During prophylactic treatment, a neublastin polypeptide is administered to prevent the appearance of neuropathic pain or reduce the severity of the pain if it occurs.

Infection-Associated Neuropathic Pain

Treatment and prevention of infectious infection-associated neuropathic pain is contemplated. A neublastin polypeptide is administered to treat or prevent appearance of neuropathic pain including but not limited to neuropathic pain associated with leprosy or Lyme disease or neuropathic pain associated with infection by a virus, particularly a virus selected from the group consisting of a herpes virus (and more particularly by a herpes zoster virus, which may lead to post-herpetic neuralgia), a human immunodeficiency virus (HIV), and a papilloma virus). Prophylactic treatment is indicated after determination of infection and before onset of neuropathic pain.

Symptoms of acute viral infection often include the appearance of a rash. Other symptoms include, for example, the development of persistent pain in the affected area of the body, which is a common complication of a herpes zoster infection (shingles). Post-herpetic neuralgia can last for a month or more, and may appear several months after any rash-like symptoms have disappeared. Dosage and Frequency of Administration

A neublastin polypeptide (or a pharmaceutical composition comprising same) can be administered to a human subject experiencing neuropathic pain (e.g., painful lumbar radiculopathy) at a dosage of 100-1,600 μg/kg body weight of the human subject, per dose. Administration can be, for example, by intravenous or subcutaneous administration.

Exemplary dosage ranges include: 200-1,200 μg/kg body weight of the human subject, per dose; 200-800 μg/kg body weight of the human subject, per dose; 400-800 μg/kg body weight of the human subject, per dose; and 300-500 μg/kg body weight of the human subject, per dose. For example, a neublastin polypeptide can be administered at a dosage of 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1, 100, 1, 150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,550, or 1,600 μg/kg body weight of the human subject.

The above neublastin polypeptide doses can be administered to the human subject in a single administration or repeatedly over the course of multiple administrations of the polypeptide. Examples of dosing regimens comprising multiple administrations include, e.g., two administrations of the polypeptide within one week, three administrations of the polypeptide within one week, and three administrations of the polypeptide within a period of three weeks. The same or different dosages of the neublastin polypeptide can be

administered to the human subject at each of the multiple administrations.

After an initial dosing (single administration) or an initial dosing period (multiple administrations) with the polypeptide, the polypeptide may subsequently be administered to the human subject to maintain a therapeutic effect by administration at the same or at a different dose, at the same or at a different dosing frequency, and by the same or different route of administration. Examples of subsequent dosing regimens include subsequent administration at an interval of once per month, subsequent administration at an interval of once per week, subsequent administration at an interval of once every two weeks, subsequent administration three times per week, and subsequent administration three times per week every other week. The subsequent administrations can continue for any period of time, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer.

The efficacy of neublastin in treating neuropathic pain can be evaluated by questioning human subjects on the status of their neuropathic pain symptoms pre- and post- treatment. The Examples below describe means of assessing the effectiveness of neublastin treatment by use of the Likert Numerical Pain Rating Scale and the Short-Form McGill Pain Questionnaire.

The following are examples of the practice of the invention. They are not to be construed as limiting the scope of the invention in any way.

Examples

A Multi-Centered, Randomized, Blinded, Placebo-Controlled, Serial-Cohort, Multiple Ascending Dose Study of BGOOOIO (Neublastin) in Subjects with Sciatica

Study Design

This was a Phase 1, randomized, double-blinded, placebo-controlled, serial-cohort multiple ascending dose study of the safety, tolerability, pharmacokinetics, and efficacy of BGOOOIO in subjects with sciatica (alternatively referred to as painful lumbar radiculopathy). The study was conducted in two parts:

Part I assessed administration of BGOOOIO or placebo once weekly for three weeks (Cohorts A to D); and

Part II assessed administration of BGOOOIO or placebo three times weekly for one week (Cohorts E to G). In Part II, subjects were dosed every 48 hours.

Up to 28 subjects were planned, and in total, 28 subjects were randomized, treated, and had evaluable data. Subjects were enrolled sequentially cohort by cohort. For each cohort, all subjects were randomized to receive three intravenous (IV) injections of study treatment (three subjects receiving BGOOOIO and one subject receiving placebo). No subject in the study received more than three IV injections of study treatment. The starting dose in Part II per the protocol was required to be at least one dose level below the maximum tolerated dose in the once weekly schedule, and no more than 400 μg/kg.

Description of BGOOOIO

BGOOOIO is a soluble recombinant protein produced by Chinese hamster ovary

(CHO) cells stably transfected with a plasmid encoding the carboxyl-terminal, 104 amino acids of human neublastin (hNBN) linked to a signal peptide. hNBN is expressed as a

103/104 amino acid homodimer whose tertiary structure resembles that of other transforming growth factor beta superfamily members. Each BGOOOIO monomer contains one asparagine- linked glycosylation site, therefore, as expressed in CHO cells, the BG00010 dimer has a potential for two, one, or no glycosylations. The species with two glycosylations per dimer is the predominant form produced with a lesser amount of monoglycosylated product and little nonglycosylated material.

Treatments

BG00010 was supplied as a liquid drug product in vials containing 1.6 mg/mL of BG00010 formulated with 10 mM sodium succinate, 75 mM sodium chloride, and 100 mM L-arginine hydrochloride, at pH 5.5. BG00010 was stored at 2 to 8°C (36 to 46°F), in a monitored, locked refrigerator, and was protected from light, protected from freezing, and was not to be shaken. Saline provided by the study site was used as placebo.

Selection and Timing of Dose for Each Subject

On the morning of dosing, subjects were served a standard breakfast. The IV injections were given between 45 and 90 minutes following that meal. Subjects then fasted for approximately three hours after which a standard lunch was served. A standard dinner was served approximately six hours after lunch. A standard breakfast was given the following morning. All subjects were served identical meals during their stay in the unit.

Beverages were permitted, in moderation, upon request. Subjects were allowed two caffeine-containing beverages per day while confined to the unit. Vigorous exercise (i.e., aerobic exercise for greater than 30 minutes) was prohibited from 48 hours prior to administration of study treatment until 24 hours after administration of study treatment. Alcohol use was prohibited from 24 hours prior to administration of study treatment until one week after administration of study treatment (Day 7), and for 24 hours prior to other visits.

Efficacy Evaluation

Clinical efficacy was assessed using numerical pain rating assessments as measured by an 11 -point Likert scale assessing general, leg, and back pain, and by measuring pain on the Visual Analog Scale (VAS) of the Short-Form McGill Pain Questionnaire (SF-MPQ). Subjects were asked to evaluate their pain in three areas (general pain, back pain, and leg pain) in order to explore the potential of BG00010 to reduce pain following multiple dose administration. Likert Numerical Pain Rating Scale

The change in Likert Pain Rating Assessment scores (general pain, back pain, and leg pain) from baseline were summarized by week and at each time point. The average score before first dose was calculated and treated as the baseline score when computing the change from baseline. Graphical presentations of change from baseline by week are displayed for general sciatica pain (Fig. 2A is subjects in Part I; Fig. 2B is subjects in Part II), back pain (Fig. 3A is subjects in Part I; Fig. 3B is subjects in Part II), and leg pain (Fig. 4A is subjects in Part I; Fig. 4B is subjects in Part II). Graphical presentations of change from baseline by time point are displayed for general sciatica pain (Fig. 5A is subjects in Part I, from day 0 to day 17; Fig. 5B is subjects in Part I, from day 0 to day 71; Fig. 5C is subjects in Part II, from day 0 to day 7; Fig. 5D is subjects in Part II, from day 0 to day 61), back pain (Fig. 6A is subjects in Part I, from day 0 to day 17; Fig. 6B is subjects in Part I, from day 0 to day 71; Fig. 6C is subjects in Part II, from day 0 to day 7; Fig. 6D is subjects in Part II, from day 0 to day 61), and leg pain (Fig. 7A is subjects in Part I, from day 0 to day 17; Fig. 7B is subjects in Part I, from day 0 to day 71 ; Fig. 7C is subjects in Part II, from day 0 to day 7; Fig. 7D is subjects in Part II, from day 0 to day 61).

In Part I of the study (one dose of study treatment per week for three weeks), during the first week of study treatment (after one dose), placebo-treated subjects reported a mean change in general sciatica pain from baseline of -1.37 on the Likert scale. These subjects continued to report a decrease in general sciatica pain during the dosing period with the highest level of mean pain reduction being reported following their third dose of study medication (-2.16 points mean decrease in pain from baseline at Week 3). A change from baseline of -4.16 points was seen for one placebo-treated subject at Week 10.

Subjects who received BG00010 during Part I of the study showed a similar response to their placebo counterparts after receiving a single dose of BG00010. However, after receiving a second dose of BG00010, subjects in the dosing groups 400 μg/kg and above began to report greater levels of general sciatica pain reduction than the placebo-treated subjects. The greatest level of pain reduction was observed at Weeks 7 and 11 in the 800 μg/kg dose group, with a change from baseline in the mean pain score of approximately -3.80 points. However, it should be noted that data for only one subject were available at these time points.

The placebo-treated subjects in Part II of the study (three doses of study treatment per week for one week) showed a change from baseline in the mean general sciatica pain score during Weeks 1 and 2 of -0.51 and -0.84 points. These were the greatest decreases from baseline seen in Part II of the study for placebo-treated subjects.

The BGOOOlO-treated subjects in Part II of the study showed a decrease in general sciatica pain that was observed at Week 1 and was maintained as a greater change from baseline than placebo-treated subjects for the duration of the study. The greatest level of mean pain reduction was observed at Week 2 in the subjects that received 400 μg/kg, with a change from baseline in the mean pain score of -4.24 points.

The results for leg pain and back pain as measured by the Likert Pain Rating

Assessment were similar to those seen for general sciatica pain.

Short-Form McGill Pain Questionnaire

A graphical presentation of mean VAS scores over time at each dose level is presented in Figs. 8A-8B (Fig. 8A is subjects in Part I; Fig. 8B is subjects in Part II).

In Part I of the study, the 800 μg/kg BG00010 dose group showed the greatest mean decrease from baseline in the VAS score, with the greatest mean decrease seen at Day 43 (change of -34 points). The greatest mean decrease from baseline for placebo-treated subjects was seen at Day 71 (change of -20.67 points). For the other dose groups in Part I of the study, no notable benefit in pain reduction versus placebo-treated subjects was seen.

In Part II of the study, the greatest mean decrease from baseline was seen 56 days post third dose in the 600 μg/kg dose group (change of -33.33 points). The greatest mean decrease from baseline for placebo-treated subjects was seen 56 days post third dose (change of -3.67 points). Notable reduction from baseline in pain was seen in the 600 and 1200 μg/kg BG00010 dose groups versus placebo. Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:

1. A method of treating neuropathic pain in a human subject in need thereof, comprising administering to the human subject a polypeptide comprising an amino acid sequence that is at least 80% identical to amino acids 15-113 of SEQ ID NO: 1, wherein the polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET, and wherein the polypeptide is administered at a dosage of 100-1,600 μg/kg body weight of the human subject.

2. The method of claim 1, wherein the method comprises multiple administrations of the polypeptide.

3. The method of claim 2, wherein the multiple administrations comprise at least three administrations of the polypeptide within one week.

4. The method of claim 3, wherein the three administrations of the polypeptide within one week occur every other day.

5. The method of claim 3 or 4, wherein after the at least three administrations of the polypeptide within one week, the polypeptide is subsequently administered at an interval of once per month.

6. The method of claim 3 or 4, wherein after the at least three administrations of the polypeptide within one week, the polypeptide is subsequently administered at an interval of once per week.

7. The method of claim 3 or 4, wherein after the at least three administrations of the polypeptide within one week, the polypeptide is subsequently administered at an interval of once every two weeks.

8. The method of claim 3 or 4, wherein after the at least three administrations of the polypeptide within one week, the polypeptide is subsequently administered three times per week.

9. The method of claim 3 or 4, wherein after the at least three administrations of the polypeptide within one week, the polypeptide is subsequently administered three times per week every other week.

10. The method of claim 2, wherein the multiple administrations comprise at least three administrations of the polypeptide within a period of three weeks.

11. The method of claim 2, wherein the multiple administrations comprise at least two administrations of the polypeptide within one week.

12. The method of any one of claims 1 to 11, wherein the polypeptide is administered at a dosage of 200-1,200 μg/kg body weight of the human subject

13. The method of any one of claims 1 to 11, wherein the polypeptide is administered at a dosage of 200-800 μg/kg body weight of the human subject.

14. The method of any one of claims 1 to 11, wherein the polypeptide is administered at a dosage of 400-800 μg/kg body weight of the human subject.

15. The method of any one of claims 1 to 11, wherein the polypeptide is administered at a dosage of 300-500 μg/kg body weight of the human subject.

16. The method of any one of claims 1 to 11, wherein the polypeptide is administered at a dosage of 150, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1, 100, or 1,200 μg/kg body weight of the human subject.

17. The method of any one of claims 1 to 11, wherein the polypeptide is administered at a dosage of 400 μg/kg body weight of the human subject.

18. The method of any one of claims 1 to 17, wherein the neuropathic pain is associated with painful lumbar radiculopathy, painful diabetic neuropathy, post-herpetic neuralgia, post-traumatic neuralgia, post-surgical neuralgia, or a complex regional pain syndrome, or is induced by a therapeutic drug.

19. The method of any one of claims 1 to 17, wherein the neuropathic pain is associated with leprosy, Lyme disease, infection by a virus, or a cancer.

20. The method of any one of claims 1 to 19, wherein the polypeptide is administered to the human subject via intravenous administration.

21. The method of any one of claims 1 to 19, wherein the polypeptide is administered to the human subject via subcutaneous administration.

22. The method of any one of claims 1 to 21, wherein the amino acid sequence is at least 90% identical to amino acids 15-1 13 of SEQ ID NO: 1.

23. The method of any one of claims 1 to 21, wherein the amino acid sequence is at least 95% identical to amino acids 15-1 13 of SEQ ID NO: 1.

24. The method of any one of claims 1 to 21, wherein the polypeptide comprises amino acids 15-113 of SEQ ID NO: 1, amino acids 15-1 13 of SEQ ID NO:2, amino acids 15-113 of SEQ ID NO:3, amino acids 15-113 of SEQ ID NO:4, amino acids 15-113 of SEQ ID NO:5, amino acids 15-1 13 of SEQ ID NO:8, or amino acids 15-113 of SEQ ID NO:9.

25. The method of any one of claims 1 to 21, wherein the polypeptide comprises amino acids 10-113 of SEQ ID NO: l .

26. The method of any one of claims 1 to 21, wherein the polypeptide consists of amino acids 10-113 of SEQ ID NO: l .