EP1365785A2 - Utilisation du facteur neurotrophique ciliaire - Google Patents

Utilisation du facteur neurotrophique ciliaire

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Publication number
EP1365785A2
EP1365785A2 EP01937817A EP01937817A EP1365785A2 EP 1365785 A2 EP1365785 A2 EP 1365785A2 EP 01937817 A EP01937817 A EP 01937817A EP 01937817 A EP01937817 A EP 01937817A EP 1365785 A2 EP1365785 A2 EP 1365785A2
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EP
European Patent Office
Prior art keywords
cntf
neurotrophic factor
ciliary neurotrophic
human
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01937817A
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German (de)
English (en)
Inventor
Vivien W. Wong
Ellen M. Koehler-Stec
Neil Stahl
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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Publication date
Priority claimed from US09/577,468 external-priority patent/US6767894B1/en
Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Publication of EP1365785A2 publication Critical patent/EP1365785A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to ciliary neurotrophic factor (CNTF) and CNTF- related polypeptides useful for the treatment of neurological diseases, obesity and other diseases or disorders.
  • CNTF ciliary neurotrophic factor
  • CNTF is a protein that is required for the survival of embryonic chick ciliary ganglion neurons in vitro (Manthorpe et al., 1980, J. Neurochem. 34:69-75).
  • the ciliary ganglion is anatomically located within the orbital cavity, lying between the lateral rectus and the sheath of the optic nerve; it receives parasympathetic nerve fibers from the oculomotor nerve which innervates the ciliary muscle and sphincter pupillae.
  • CNTF is believed to induce the differentiation of bipotentia glial progenitor cells in the peri ⁇ atal rat optic nerve and brain (Hughes et al., 1988, Nature 335:70-73). Furthermore, it has been observed to promote the survival of embryonic chick dorsal root ganglion sensory neurons (Skaper and Varon, 1986, Brain Res. 389:39 46).
  • CNTF supports the survival and differentiation of motor neurons, hippocampal neurons and presympathetic spinal cord neurons (Sendtner, et al., 1990, Nature 345: 440-441; Ip, et al. 1991, J. Neurosci. 11:3124-3134; Blottner, et al. 1989, Neurosci. Lett. 105:316 320).
  • CNTF has been cloned and synthesized in bacterial expression systems, as described by Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and in International Publication No. WO 91/04316, published on April 4, 1991, both of which are incorporated by reference in their entirety herein.
  • recombinant human and rat CNTF differ in several respects.
  • the biological activity of recombinant rat CNTF in supporting survival and neurite outgrowth from embryonic chick ciliary neurons in culture is four times better than that of recombinant human CNTF (rHCNTF) (Masiakowski et al., 1991, J. Neurochem. 57:1003-1012).
  • rat CNTF has a higher affinity for the human CNTF receptor than does human CNTF (Davis et al,
  • the CNTF receptor complex contains three proteins: a specificity determining ⁇ component that directly binds to CNTF, as well as two signal transducing ⁇ components (LIFR ⁇ and gpl30) that cannot bind CNTF on their own, but are required to initiate signaling in response to CNTF.
  • the ⁇ component of the CNTFR complex is more widely distributed throughout the body than the ⁇ component.
  • the 3 components of the CNTFR complex are normally unassociated on the cell surface; CNTF induces the stepwise assembly of a complete receptor complex by first binding to CNTFR , then engaging gpl30, and finally recruiting LIFR ⁇ .
  • intracellular signaling is initiated by activating non-receptor tyrosine kinases QAK kinases) associated with the ⁇ components.
  • QAK kinases respond by phosphorylating each other and also tyrosine residues on the receptor cytoplasmic domains, creating phosphotyrosine docking sites for the Src homology 2 domains of STAT proteins. After their phosphorylation, bound STAT proteins dissociate from the receptor, dimerize, and translocate to the nucleus where they bind DNA and activate transcription (reviews: Frank, D. and Greenberg, M. (1996) Perspectives on Developmental Neurobiology 4: 3-18; Stahl, N.
  • Axokine is a mutant CNTF molecule with improved physical and chemical properties, which retains the ability to interact with and activate the CNTF receptor.
  • Leptin the product of the ob gene, is secreted by adipocy tes and functions as a peripheral signal to the brain to regulate food intake and energy metabolism (Zhang, Y., et al. (1994) Nature 372: 425-431).
  • leptin receptor a single membrane-spanning receptor
  • gpl30 Tartaglia, L., et al. (1995) Cell 83: 1263-1271.
  • Both CNTF and leptin each signals through the JAK/STAT pathway (Baumann, H., et al. (1996) Proc. Natl. Acad. Sci. USA 93: 8374-8378; Ghilardi, N., et al. (1996) Proc. Natl. Acad. Sci. USA 93: 6231- 6235).
  • Systemic administration of both CNTF and leptin results in induction of tis-11 (Gloaguen, I., et al.
  • An object of the present invention is to provide CNTF and CNTF-related proteins, collectively referred to herein as CNTF proteins, for the treatment of diseases or disorders including, but not limited to, obesity and diabetes.
  • a further object of the present invention is to provide a method for administering CNTF or CNTF-related proteins and maintaining biological activity.
  • a preferred embodiment of this invention is the administration of CNTF or a CNTF related protein to the nasal or respiratory system of a mammal to produce an increase in the level of the protein in the systemic blood circulation of the mammal.
  • a particularly preferred embodiment comprises the administration of the modified CNTF molecule, designated herein as AX-15, to the nasal passages of a patient for the treatment of obesity or diabetes.
  • FIG. 1 Alignment of CNTF protein sequences.
  • A Human, rat, rabbit mouse and chicken (Leung, et al., 1992, Neuron 8:1045-1053) sequences. Dots indicate residues found in the human sequence.
  • B Modified CNTF molecules showing human CNTF amino acid residues (dots) and rat CNTF (residues shown). The name of the purified recombinant protein corresponding to each sequence is shown on the left.
  • Figure 2 Mobility of human, rat and several modified CNTF molecules on reducing SDS-15% polyacrylamide gels. Purified recombinant proteins were loaded as indicated. Markers of the indicated MW were loaded on lane M.
  • FIG. 3 Effects of Axokine-15 (AX-15) in normal mice.
  • AX-15 Axokine-15
  • Normal C57BL/6J mice were injected subcutaneously daily for 6 days with either vehicle or AX-15 at 0.1 mg/kg, 0.3 mg/kg, or 1.0 mg/kg. Percent change in body weight in AX-15-treated versus vehicle-treated controls is shown.
  • FIG. 4 Effects of AX-15 in ob/ob mice.
  • C57BL/6J ob/ob mice were injected subcutaneously daily for 7 days with either vehicle, leptin (1.0 mg/kg) or AX-15 at 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg or 3.0 mg/kg.
  • Diet-restricted, pair-fed (PF) mice were injected with 0.3 mg/kg AX-15 to investigate the effects of food intake reduction on weight loss. Percent change in body weight in AX-15-treated and leptin-treated versus vehicle-treated controls is shown.
  • FIG. 5 Effects of AX-15 in diet-induced obesity in mice.
  • AKR/J mice were placed on a high fat diet for seven weeks prior to treatment with vehicle, leptin (1.0 mg/kg) or AX-15 at 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, or 1.0 mg/kg.
  • Diet-restricted, pair- fed AKR/J mice were injected with 0.1 mg/kg AX-15 (PF-AX-15) to investigate the effects of food intake reduction on weight loss. Percent change in body weight in AX-15 -treated and leptin-treated versus vehicle-treated controls is shown.
  • Figures 6A and 6B Effects of AX-15 and diet restriction on serum insulin and corticosterone levels in diet-induced obese AKR/J mice.
  • Figure 6A- Serum insulin levels were measured in AKR/J diet-induced obese mice following treatment with vehicle, diet restriction and AX-15 (0.1 mg/kg) (PF-AX-15) or AX-15 only (0.1 mg/kg) to determine the effects of diet and/or AX-15 treatment on obesity- associated hyperinsulinemia.
  • Figure 6B- Serum corticosterone levels were measured in AKR/J diet induced obese mice following treatment with vehicle, diet restriction and AX-15 (0.1 mg/kg) (PF-AX-15) or AX-15 only (0.1 mg/kg).
  • FIG. 7 Effect of intranasal delivery of AX-15 on body weight reduction in diet- induced obese AKR/J mice.
  • DIO mice were given daily intranasal administration of either vehicle (D) or AX-15 (94 ⁇ g/mouse in 50 ⁇ l, approximately 1.87-2.35 mg/kg) (0) for 7 days.
  • a group of mice receiving daily subcutaneous injection of AX-15 (O) at 0.1 mg/kg was also included for comparison.
  • FIG 8 Effect of intranasal delivery of AX-15 on food intake in diet-induced obese AKR/J mice.
  • DIO mice were given daily intranasal administration of either vehicle (D) or AX-15 (94 ⁇ g/mouse in 50 ⁇ l, approximately 1.87-2.35 mg/kg) (0) for 7 days.
  • a group of mice receiving daily subcutaneous injection of AX-15 (O) at 0.1 mg/kg was also included for comparison.
  • the present invention relates to a method of administering a CNTF protein to treat diseases and disorders in humans or animals.
  • CG chick ciliary ganglion
  • electrophoretic mobility data indicated that all of the modified human CNTF molecules that migrated to the same position as rat CNTF had the single amino acid substitution Gln63Arg (Q63R), in which glutamine at position 63 is replaced with arginine.
  • CNTF is characterized by its capacity to support the survival of dissociated ciliary neurons of E8 chick embryos.
  • purified recombinant rat CNTF is as active as the native protein from rat, but four times more active than recombinant human CNTF (Masiakowski, et al., 1991, J. Neurosci. 5_Z:1003-1012 and in International Publication No. WO 91/04316, published on April 4, 1991).
  • the same assay was utilized to determine the biological activity of the altered molecules prepared as described above.
  • all modified CNTF molecules that had the Q63R substitution exhibited an increased ability to support the survival of ciliary ganglion neurons as compared to the parent human CNTF protein.
  • an indication of the potential biological activity of each of the molecules may also be obtained by determining the effect of each modification on the ability of the molecules to bind to the CNTF receptor.
  • a ciliary neurotrophic factor protein As used herein, the terms "a ciliary neurotrophic factor protein”, “a CNTF protein”, “a ciliary neurotrophic factor molecule” and “a CNTF molecule” refer to human ciliary neurotrophic factor and modified ciliary neurotrophic factors.
  • modified ciliary neurotrophic factor refers to CNTF proteins and polypeptides that have certain amino acid substitutions and/or deletions in the arr ⁇ no acid residue sequence of human CNTF which result in modified CNTF proteins and polypeptides that exhibit binding to the CNTF receptor and, therefore, would be expected to have enhanced biological, immunogenic and/or purification properties.
  • CNTF proteins of the present invention may also be “pegylated” by the addition of polyethylene glycol polymers in order to enhance stability and/or bioavailability.
  • a particularly preferred "modified ciliary neurotrophic factor” according to the invention contains certain amino acid substitutions and deletions in the human CNTF protein and exhibits enhanced binding to the human CNTF receptor and therefore, would be expected to have enhanced biological properties, and specifically include AX- 13, AX-2 and AX-15, described in more detail infra.
  • the CNTF molecules useful for practicing the present invention may be prepared by cloning and expression in a prokaryotic or eukaryotic expression system as described, for example in Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and in International Publication No. WO 91/04316, published on April 4, 1991.
  • the recombinant CNTF gene may be expressed and purified utilizing any number of methods.
  • the gene encoding this protein may be subcloned into a bacterial expression vector, such as for example, but not by way of limitation, pCPHO.
  • the recombinant CNTF proteins may be purified by any technique which allows for the subsequent formation of a stable, biologically active protein.
  • they may be recovered from cells either as soluble proteins or as inclusion bodies, from which they may be extracted quantitatively by 8M guanidinium hydrochloride and dialysis. Further purification of the proteins, may be done by conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration.
  • CNTF proteins produced as described herein, or hybrids or mutants thereof may be used to promote differentiation, proliferation or survival in vitro or in vivo of cells that are responsive to CNTF, including cells that express receptors of the CNTF/IL-6/LIF receptor family, or any cells that express the appropriate signal transducing component, as described, for example, in Davis, et al., 1992, Cell 69:1121-1132. Mutants or hybrids may alternatively antagonize cell differentiation or survival.
  • the present invention may be used to treat disorders of any cell responsive to CNTF or the CNTF /CNTF receptor complex.
  • disorders of cells that express members of the CNTF/IL-6/LIF receptor family may be treated according to these methods. Examples of such disorders include, but are not limited to, obesity and diabetes.
  • the present invention provides for methods in which a patient is treated with an effective amount of the modified CNTF protein, or a hybrid or mutant thereof.
  • the modified CNTF proteins may be utilized to treat disorders or diseases as described for CNTF in International Publication No. WO91/04316 published on April 4, 1991 by Masiakowski, et al. and for the CNTF/CNTFR complex as described in International Publication No. WO91/19009 published on December 12, 1991 by Davis, et al. both of which are incorporated by reference in their entirety herein.
  • Such diseases or disorders include degenerative diseases, such as retinal degenerations, diseases or disorders involving the spinal cord, cholinergic neurons, hippocampal neurons or diseases or disorders involving motor neurons, such as amyotrophic lateral sclerosis or those of the facial nerve, such as Bell's palsy.
  • Other diseases or disorders that may be treated include obesity and diabetes
  • rHCNTF human clinical trials using recombinant human CNTF (rHCNTF) have been carried out wherein subcutaneous administration of the protein was tested for its efficacy in slowing the progression of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • rHCNTF human clinical trials using recombinant human CNTF (rHCNTF) have been carried out wherein subcutaneous administration of the protein was tested for its efficacy in slowing the progression of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • rHCNTF ⁇ C13 also designated RPN160 or RG160
  • RPN160 rHCNTF ⁇ C13
  • rHCNTF ⁇ C13 retains full biological activity and is soluble at low temperatures (5-10°C) to at least 12 mg/ml. Yet, despite this far greater solubility, rHCNTF ⁇ C13 precipitates in a PBS solution upon incubation at 37°C for several hours, even at concentrations as low as 0.1 mg/ml. It was determined that the thermal instability of rHCNTF and rHCNTF ⁇ C13 was the result of aggregation that was initiated by intermolecular disulfide bond formation and depended strongly on protein concentration and temperature.
  • modified CNTF proteins were obtained that exhibited far greater stability and maintained their biological activity after incubation for at least 7 days in PBS at 37°C.
  • This property is maintained in rHCNTF, Q63R variants which have higher potency due to the substitution of the glutamine residue at position 63 by arginine.
  • rHCNTF, C17A, Q63R, ⁇ C13 also designated RG297 shows greater biological potency than rHCNTF because of the Q63R substitution, greater solubility because of the ⁇ C13 deletion and greater stability because of the C17A substitution.
  • AX-15 Another embodiment of a modified CNTF in the present invention is AX-2, which has a C17A substitution in the amino acid residue sequence of human CNTF and a truncation of the 15 C-terminal amino acid residues of human CNTF.
  • AX-2, (rHCNTF, C17A ⁇ C15) differs from AX-15 solely in the absence of the Q63R substitution that is present in AX-15.
  • the present invention contemplates a composition comprising a ciliary neurotrophic factor protein of the invention, such as the protein described herein as AX-15, and a carrier.
  • Another object of the present invention is to provide a method of treating a disease or disorder comprising administering CNTF or a modified ciliary neurotrophic factor, such as the protein described herein as AX-15.
  • the disease or disorder treated may be a degenerative disease and/or involve the spinal cord, motor neurons, cholinergic neurons or cells of the hippocampus.
  • the method of treatment may be for treating a disease or disorder such as obesity or diabetes or treating a disease or disorder involving muscle atrophy.
  • a further object of the present invention is to provide a method of inducing weight loss in a mammal comprising administration to the mammal of a ciliary neurotrophic factor protein, particularly AX-15.
  • a specific embodiment of this invention involves inducing weight loss in a human.
  • a CNTF protein such as AX-15
  • the method of administering a CNTF protein, such as AX-15 may be used in the treatment of diet induced obesity or obesity of a genetically determined origin.
  • a modified CNTF protein, such as AX-15 described herein may also be used in a method of preventing and/or treating the occurrence of gestational or adult onset diabetes in a human.
  • any of the above-described methods involving the administration of CNTF or a modified CNTF, such as AX-15 described herein, may be practiced by administering the CNTF protein via a route of delivery selected from the group consisting of intravenous, intramuscular, intraocular, subcutaneous, intranasal, respiratory or intratracheal, such as by use of a nebulizer, and by intracolonic or vaginal suppositories.
  • a CNTF protein, such as AX-15 described herein may be administered via the implantation of cells that release the modified ciliary neurotrophic factor.
  • the present invention also provides for pharmaceutical compositions comprising CNTF or a modified CNTF protein or hybrid or mutant thereof, such as AX-15 described herein, as the sole therapeutic agent or in a complex with the CNTF receptor, in a suitable pharmacologic carrier for use in the treatment of obesity or gestational or adult onset diabetes.
  • the active ingredient which may comprise CNTF or a modified CNTF should be formulated in a suitable pharmaceutical carrier for administration in vivo by any appropriate route including, but not limited to intranasal, intratracheal, by nebulizer, intraocular and oral.
  • intranasal administration refers to delivery to the nose or nasal passageways by spray, drops, gel, inhalant or other means.
  • intratracheal administration refers to delivery to the throat or tracheal lumen by spray, propellant, atomizer, injection or other means.
  • nebulizer refers to the use of any device which reduces the formulation of the present invention to a fine spray for penetration into the lungs or nasal cavities.
  • intraocular administration refers to delivery to the eye by drop, spray, ointment or other means.
  • oral administration refers to delivery to the mouth, esophagus or stomach by pill, capsule, solution, tablet, lozenge, powder, spray or other means.
  • the active ingredient may be formulated in a liquid carrier such as saline, incorporated into liposomes, microcapsules, polymer or wax-based and controlled release preparations.
  • modified CNTF preparations are stable solutions, or formulated into tablet, pill or capsule forms.
  • the concentration of the active ingredient used in the formulation will depend upon the effective dose required and the mode of administration used. The dose used should be sufficient to achieve circulating plasma concentrations of active ingredient that are efficacious. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Effective doses are expected to be within the range of from about .001 to about 1 mg/day. EXAMPLES
  • Example 1 Electrophoretic Mobility of Modified Human CNTF Molecules Materials and Methods Preparation of Modified CNTF molecules Bacterial Strains and Plasmids E. coli K-12 RFJ26 is a strain that overproduces the lactose operon repressor.
  • the expression vectors pRPN33, which carries the human CNTF gene and pRPNHO which carries the rat CNTF gene are nearly identical (Masiakowski, et al., 1991, J. Neurosci. 57_:1003-1012 and in International Publication No. WO 91 /04316, published on April 4, 1991.)
  • Plasmid pRPN219 was constructed by first digesting pRPN33 with the restriction enzymes Nhel plus Hind3 and gel purifying the 4,081 bp fragment. The second, much smaller fragment which codes for part of the human CNTF gene was subsequently replaced with an 167 bp Nhel-Hind3 fragment that was obtained by PCR amplification from the rat gene using the primers RATTII-dniH: 5' ACGGTAAGCT TGGAGGTTCTC 3'; and RAT-Nhe-I-M: 5' TCTATCTGGC TAGCAAGGAA GATTCGTTCA GACCTGACTG CTCTTACG 3'.
  • Plasmid pRPN228 was constructed in the same manner as pRPN219, except that the 167 bp replacement fragment was amplified using the DNA primers Rat-III-dniH-L- R : 5' AAG GTA CGA TAA GCT TGG AGG TTC TCT TGG AGT CGC TCT GCC TCA GTC AGC TCA CTC CAA CGA TCA GTG 3' and Rat-Nhe-I: 5' TCT ATC TGG CTA GCA AGG AAG 3'.
  • Plasmids pRPN186, pRPN187, pRPN188, pRPN189, pRPN192, pRPN218, and . pRPN222 were generated by similar means or by direct exchange of DNA fragments using the unique restriction sites shown in Figure 1.
  • the mobilities of human, rat and several chimeric CNTF molecules on reducing SDS- polyacrylamide gels are shown in Figure 2.
  • the chimeric molecules RPN186, RPN189, RPN218 and RPN228 exhibit mobilities comparable to rat CNTF, whereas RPN187, RPN188, RPN192 and RPN222 exhibit mobilities comparable to human
  • Figure 2 also provides a measure of the purity of the different recombinant proteins. By visual inspection, purity varies from 60% for RPN189 to better than 90% for RPN228.
  • 125 TCNTF was stored at 4°C and used up to one week after preparation.
  • 125 I-CNTF approximately 10,000 cpm was mixed with a 5 mg unlabelled CNTF and analyzed by native gel electrophoresis. One major band was visible by either Coomassie staining or autoradiography.
  • 125 TCNTF also showed comparable activity to native CNTF in supporting survival of E8 chick ciliary neurons in culture.
  • SCG Superior cervical ganglia
  • NVF nerve growth factor
  • penicillin 50 U/ml
  • streptomycin 50 mg/ml
  • Cultures were maintained at 37°C in a humidified 95% air/5% C0 2 atmosphere.
  • Ganglion non-neuronal cells were eliminated by treatment with araC (10 mM) on days 1 and 3 of culture. Cultures were fed 3 times/week and were routinely used for binding assays within 2 weeks.
  • MG87/CNTFR is a fibroblast cell line transfected with the human CNTF ⁇ receptor gene (Squinto, et al.,1990, Neuron 5:757-766; Davis et al., 1991, Science 253:59-63). Binding Assays
  • Binding was performed directly on cell monolayers.
  • Cells in culture wells were washed once with assay buffer consisting of phosphate buffered saline (PBS; pH 7.4), 0.1 mM bacitracin, 1 mM PMSF, 1 mg/ml leu ⁇ Jtin, and 1 mg/ml BSA.
  • Assay buffer consisting of phosphate buffered saline (PBS; pH 7.4), 0.1 mM bacitracin, 1 mM PMSF, 1 mg/ml leu ⁇ Jtin, and 1 mg/ml BSA.
  • PBS phosphate buffered saline
  • bacitracin 1 mM PMSF
  • 1 mg/ml leu ⁇ Jtin 1 mg/ml BSA
  • BSA phosphate buffered saline
  • Non-specific binding was determined in the presence of 1,000-fold excess of unlabelled CNTF.
  • Specific binding towards MG87/CNTFR was
  • the expression plasmid pRG632 is a high copy plasmid that encodes ampicillin resistance and the gene for human CNTF-C17A,Q63R ⁇ C13 (also referred to herein as either AX-1 or AX-13) with a unique Eag I restriction enzyme recognition sequence 3' to the stop codon.
  • This plasmid was used to construct a human CNTF mutation C17A,Q63R, ⁇ C15 (designated AX-15) by PCR amplification of a 187 bp BseR I-Eagl DNA fragment that incorporates the ⁇ C15 mutation.
  • the 5' primer ⁇ C15- 5' (5'-CCAGATAGAGGAGTTAATGATACTCCT-3') ⁇ encodes the BseR I site and the 3' primer , ⁇ C15-3' ⁇ (5'-
  • GCGTCGGCCGCGGACCACGCTCATTACCCAGTCT GTGAGAAGAAATG-3' encodes the C-terminus of the AX-15 gene ending at Glyl85 followed by two stop codons and an Eag I restriction enzyme recognition sequence.
  • This DNA fragment was digested with BseR I and Eag I and ligated into the same sites in pRG632.
  • the resulting plasmid, pRG639 encodes the gene for AX- 15 (human CNTF C17A,Q63R, ⁇ C15).
  • the ⁇ C15 mutation was then transferred as a 339 bp Hind III-Eag I DNA fragment into the corresponding sites within pRG421, a high copy number expression plasmid encoding the gene for kanamycin resistance and human CNTF C17A,Q63R, ⁇ C13.
  • the resulting plasmid, pRG643, encodes the gene for AX-15 under transcriptional control of the lacUV5 promoter, and confers kanamycin resistance.
  • the AX-15 gene DNA sequence was confirmed by sequence analysis.
  • Pegylation of proteins has been shown to increase their in vivo potency by enhancing stability and bioavailability while minimizing immunogenicity. It is known that the properties of certain proteins can be modulated by attachment of polyethylene glycol (PEG) polymers, which increases the hydrodynamic volume of the protein and thereby slows its clearance by kidney filtration. (See, e.g. Clark, R., et al., " 1996, J. Biol. Chem._271: 21969-21977).
  • PEG polyethylene glycol
  • PEGylated AX-13 has better solubility and stability properties, at physiological pH, than unPEGylated AX-13. PEGylation has been shown to greatly enhance pharmacokinetic properties of AX-13 and would be expected to similarly enhance the properties of other CNTF proteins.
  • AX-13 derived from E. coli was used for these studies.
  • 20kD mPEG-SPA was obtained from Shearwater Polymers, Bicine from Sigma, and Tris-Glycine precast gels from Novex, CA.
  • a small scale reaction study was set up to determine reaction conditions.
  • 20kD mPEG SPA was reacted with purified AX-13 at a final concentration of 0.6 mg/ml, at 4°C in an amine-free buffer at a pH of 8.1. Molar ratios of PEG to protein were varied and two reaction times were used. The reaction was stopped by the addition of a primary amine in large excess. Reaction products were analyzed by reducing SDS-PAGE. The predominant modified species ran at a molecular weight of approximately 60 kD. Higher order modified bands that ran at higher molecular weights were also seen. Based on this study, an overnight reaction at a PEG-to-protein ratio of 4 was chosen.
  • AX-13 at 0.6 mg/mL was reacted with 20 kD mPEG SPA in a Bicine buffer overnight at 4°C at a pH of 8.1.
  • the reaction was stopped by the addition of a primary amine in large excess.
  • the reaction product was diluted with a low salt buffer and applied to an ion-exchange column. The column was washed with a low salt buffer and eluted with a NaCl gradient.
  • a good separation between higher order forms apparent MW >66kD on SDS-PAGE), a distinct pegylated species that ran at about 60kD and unpegylated AX-13 was obtained. Fractions corresponding to the 60kD band were tested in a bioassay.
  • a very faint band of unpegylated AX-13 was noticed in the fractions corresponding to the 60kD band.
  • the 60kD band was further purified by size exclusion chromatography (SEC) that resulted in baseline separation between unpegylated AX-13 and the 60kD band.
  • SEC size exclusion chromatography
  • E. coli strain RFJ141 containing pRG639 was grown in LB medium and expression of AX-15 protein was induced by the addition of lactose to 1% (w/v). Induced cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8.3, 5 mM EDTA, 1 mM DTT, and lysed by passage through a French pressure cell at 10,000 psi.
  • the cell lysate was centrifuged and the pellet was resuspended in 8 M guanidinium- HC1, 50 mM Tris-HCl, pH 8.3, 0.05 mM EDTA then diluted with 5 volumes of 50 mM Tris-HCl, pH 8.3, 0.05 mM EDTA (Buffer A) followed by dialysis against Buffer A.
  • the dialysate was loaded onto a Q-sepharose column equilibrated with Buffer A.
  • the AX-15 protein was eluted by a linear gradient to 1 M NaCl in 10 column volumes of buffer.
  • Fractions containing AX-15 were pooled and brought to 1 M (NH 4 ) 2 S0 4 by the slow addition of solid (NH 4 ) 2 S0 4 while maintaining the pH at 8.3 by the addition of NaOH.
  • the pool was loaded onto a phenyl-sepharose column equilibrated with 1 M (NH 4 ) 2 S0 4 in Buffer A.
  • the column was washed with 0.5 M (NH 4 ) 2 S0 4 in Buffer A, and the AX-15 protein was eluted by a linear gradient of decreasing(NH 4 ) 2 S0 4 concentration.
  • Fractions containing AX-15 protein were pooled, dialyzed against 5 mM NaP0 4 , pH 8.3, then concentrated by ultrafiltration.
  • the concentrated pool was fractionated on a Sephacryl S-100 column equilibrated with 5 mM NaP0 4 , pH 8.3.
  • a recombinant, kanamycin resistant E. coli strain RFJ141 expressing the AX-15 protein under lac promoter control (pRG643) was grown to an intermediate density of 30-35 AU 550 (Absorbance @ 550 nM) in a minimal salts, glucose medium containing 20 ⁇ g/ml kanamycin.
  • Expression of AX-15 protein was induced by addition of IPTG (isopropyl thiogalactoside) to 1.0 mM and the fermentation was continued for an additional 8 hr.
  • AX-15 protein was expressed as insoluble inclusion bodies following IPTG induction.
  • Inclusion bodies were released from the harvested cells by disruption via repeated passage of cooled (0-10°C) cell paste suspension through a continuous flow, high pressure (>8,000 psi) Niro Soavi homogenizer. The homogenate was subjected to two passages through a cooled (4-8°C) continuous flow, high speed (>17,000 x G) Sharpies centrifuge (source) to recover inclusion bodies.
  • Recovered inclusion bodies were extracted in 8.0 M guanidine HC1 with 1.0 mM DTT.
  • the AX-15 protein/ guanidine solution was diluted into 50 mM Tris-HCl, 1.0 mM DTT, 0.05 mM EDTA, pH 8.0-8.3, and diafiltered versus diluent buffer with AGT 5,000 mwco hollow fiber filters (ACG Technologies, Inc.).
  • the resulting solution, containing refolded AX-15 was filtered through a Microgon 0.22 ⁇ m hollow fiber filter (ACG Technologies, Inc.) prior to chromatographic purification. 5
  • the filtered AX-15 solution described above was loaded onto a 16.4 L DEAE Sepharose (Pharmacia) column at 10.9 mg/ml resin and washed with 50 L of 50 mM o Tris, pH 8.0-8.3, 1.0 mM DTT, and 0.05 mM EDTA buffer.
  • the AX-15 protein was eluted from the column with a 120 mM NaCl step in the same Tris buffer. Eluate exceeding a previously established 280 nM absorbance criteria of 40% maximum A 280 on the ascending portion of the peak and 20% of maximum A 2g0 on the descending portion of the peak was pooled and either stored frozen (-30°C) or used in the next 5 step of the purification procedure.
  • the AX-15 protein was eluted with a 125 mM ammonium sulfate, Tris buffer wash step. Eluate exceeding previously established 280 nM absorbance criteria of 100% maximum A 280 on the ascending portion of the peak and 20% of maximum A 2g0 on the descending portion of the peak was pooled. 5 Eluate was simultaneously diluted 1:4 into 50 mM Tris, pH 8.0 -8.3 buffer without salt to reduce its conductivity. Pooled material was stored frozen (-30°C) or used in the following step.
  • HIC hydrophobic interaction chromatography
  • the AX-15 protein was eluted with a 77.0 5 L step of 5.0 mM sodium phosphate, 130 mM NaCl, pH 7.0-7.2. The eluate was simultaneously diluted 1:5 into 10.0 mM sodium phosphate, pH 9.0-9.2 buffer without salt to reduce conductivity and increase pH. Peak material exceeding 20% maximum A 280 on the ascending portion of the peak and 20% of the maximum A 280 on the descending portion of the peak was pooled. Pooled AX-15 protein was stored frozen (-30°C) or used in the following step.
  • the pooled AX- 15 protein was filtered through Millipak 0.22 ⁇ m filters and stored at -80°C prior to dispensing or formulation.
  • the amino acid sequence of AX-15 produced follows. Alternatively, one could produce a sequence which contains a methionine residue before the initial alanine.
  • mice Normal (8 weeks) C57BL/6J mice were obtained from Taconic. The mice received daily subcutaneous injections of vehicle or AX-15. The animals were weighed daily and food intake over 24-hours was determined between days 3 and 4.
  • ob/ob mice produce a truncated, non-functional gene product (leptin). These mice are hyperphagic, hyperinsulinemic, and markedly obese.
  • mice C57BL/6J ob/ob mice were obtained from Jackson Laboratory and used for experiments at 12-14 weeks of age. The mice received daily subcutaneous injection of vehicle, AX-15, or leptin. Pair-fed group was given the average amount (g) of food consumed by animals treated with AX-15 (0.3 mg/kg). Body weights were obtained daily and food intake over 24-hours was determined between days 3 and 4. On day 8, the animals were sacrificed and carcass analysis was performed.
  • AKR/J mice have been shown to be very susceptible to diet induced obesity by increasing body fat content. Although the gene environment(diet) interaction is not completely known regarding this kind of dietary obesity, like in human obesity, the genotype is polygenic. AKR/J mice were obtained from Jackson Laboratory and put on a high fat diet (45% fat; Research Diets) at age 10-12 weeks old. All experiments commenced after 7 weeks on high fat diet. The mice received daily subcutaneous injection of vehicle, AX-15, or leptin. Pair-fed group was given the average amount (g) of food consumed by animals treated with AX-15 (0.1 mg/kg). The animals were weighed daily and food intake over 24-hours was determined between days 3 and 4. On day 8, the animals were sacrificed and sera were obtained for insulin and corticosterone measurements.
  • Recombinant human AX-15 was manufactured as set forth above and leptin was purchased from R & D Systems.
  • mice AX-15 reduced body weight in normal mice in a dose dependent manner. In 6 days, the animals lost approximately 4%, 11%, and 16% of their body weight at 0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg, respectively ( Figure 3). ob/ob mice
  • Leptin was also effective in decreasing body weight in ob/ob mice. At 1 mg/kg, leptin decreased body weight 6% in 7 days, following a course almost identical to that of AX-15 given at 0.1 mg/kg (Figure 4).
  • AX-15 reduced body weight in DIO mice dose dependently. Within one week, the animals lost approximately 14%, 26%, and 33% of their body weight when given AX- 15 at 0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg, respectively (Figure 5). Comparing the effects of the AX-15 treatment and the pair-fed control animals, there was a small but significant difference between the 2 groups, suggesting that decrease food intake was probably the primary, although not the only, cause of weight loss with AX-15 treatment. Indeed, AX-15 significantly attenuated the obesity associated hyperinsulinemia in DIO mice, whereas merely reducing food intake (pair-fed) did not (Figure 6A). In addition, AX : 15 did not cause elevation of corticosterone levels, which is a common effect of food restriction (Figure 6B).
  • DIO mice are leptin resistant; no weight loss effect was observed in these animals with daily injection of leptin (1 mg/kg; Figure 5).
  • AX-15 caused weight loss in normal mice in a dose dependent manner.
  • AX-15 induced weight loss in ob/ob mice in a dose dependent manner.
  • AX-15 (0.1 mg/kg) was as effective as leptin (1 mg/kg) in causing weight loss in ob/ob mice.
  • AX-15 caused weight loss in diet-induced obesity mice in a dose dependent manner, whereas leptin was ineffective.
  • AX-15 treatment attenuated obesity associated hyperinsulinemia in DIO mice; this effect was not observed in pair-fed control animals.
  • AX-15 was more effective in inducing weight loss in DIO mice than normal or ob/ob mice.
  • AX-15 in reducing body weight in leptin resistant mouse model suggests that AX-15 may also be effective in reducing body weight in obese humans who are resistant or unresponsive to leptin.
  • a model of human obesity is the AKR/J mice which are susceptible to diet-induced obesity (DIO) by an increase in body fat content analogous to humans, and as in human obesity the genotype is polygenic.
  • DIO diet-induced obesity
  • AX-15 administered via the intranasal route can be absorbed within the airway and/or through the mucosal lining of the nasal and respiratory tract and achieve serum levels that are in the efficacious range, based upon comparison to levels achieved following subcutaneous injection.
  • AX-15 delivered via an intranasal route was effective in reducing body weight and food intake in DIO mice. Therefore, intranasal delivery can effectively be utilized as a route of administration for AX-15 in treating appropriate disorders such as obesity and diabetes.

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Abstract

L'invention porte sur des compositions et sur des procédés d'administration par voie nasale ou respiratoire de protéines du facteur neurotrophique ciliaire, notamment dans le traitement de l'obésité et des diabètes gestationnels ou non-insulino dépendants.
EP01937817A 2000-05-24 2001-05-22 Utilisation du facteur neurotrophique ciliaire Withdrawn EP1365785A2 (fr)

Applications Claiming Priority (3)

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US09/577,468 US6767894B1 (en) 1998-02-27 2000-05-24 Use of ciliary neurotrophic factor
US577468 2000-05-24
PCT/US2001/040783 WO2001089550A2 (fr) 2000-05-24 2001-05-22 Utilisation du facteur neurotrophique ciliaire

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CN107073077A (zh) * 2014-11-07 2017-08-18 陕西麦科奥特科技有限公司 睫状神经营养因子鼻腔给药系统及其制备方法和应用

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WO2005053728A2 (fr) * 2003-12-01 2005-06-16 Xantos Biomedicine Ag Protéines associées à l'adiposité et leur utilisation à des fins thérapeutiques et diagnostiques
US7279282B2 (en) 2004-07-09 2007-10-09 Scott Mellis Methods for identifying a candidate for treatment of obesity
WO2006041922A2 (fr) * 2004-10-08 2006-04-20 Dara Biosciences, Inc. Agents, et procedes d'administration au systeme nerveux central

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IE903130A1 (en) * 1989-09-15 1991-03-27 Regeneron Pharma Ciliary neurotrophic factor
IL98304A (en) * 1990-06-01 2008-08-07 Regeneron Pharma The ciliary neurotrophic factor receptor
IT1288388B1 (it) * 1996-11-19 1998-09-22 Angeletti P Ist Richerche Bio Uso di sostanze che attivano il recettore del cntf ( fattore neurotrofico ciliare) per la preparazione di farmaci per la terapia
AU718500B2 (en) * 1997-01-23 2000-04-13 Sumitomo Pharmaceuticals Company, Limited Remedies for diabetes

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Title
See references of WO0189550A2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107073077A (zh) * 2014-11-07 2017-08-18 陕西麦科奥特科技有限公司 睫状神经营养因子鼻腔给药系统及其制备方法和应用

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