WO1997015593A1 - Antagonistes de la neurotrophine - Google Patents

Antagonistes de la neurotrophine Download PDF

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Publication number
WO1997015593A1
WO1997015593A1 PCT/CA1995/000603 CA9500603W WO9715593A1 WO 1997015593 A1 WO1997015593 A1 WO 1997015593A1 CA 9500603 W CA9500603 W CA 9500603W WO 9715593 A1 WO9715593 A1 WO 9715593A1
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Prior art keywords
peptide
amino acid
neurotrophin
cysteine
bicyclic
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PCT/CA1995/000603
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English (en)
Inventor
Richard John Riopelle
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Queen's University At Kingston
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Application filed by Queen's University At Kingston filed Critical Queen's University At Kingston
Priority to AU36952/95A priority Critical patent/AU3695295A/en
Priority to PCT/CA1995/000603 priority patent/WO1997015593A1/fr
Publication of WO1997015593A1 publication Critical patent/WO1997015593A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to neurotrophin antagonists.
  • the present invention relates to neurotrophin-derived peptides which inhibit or reduce undesirable neurotrophin activity.
  • neurotrophins A family of structurally and functionally related neurotrophic factors exist which are collectively known as neurotrophins.
  • the family of neurotrophins include the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) neurotrophin-5 (NT-5) and neurotrophin-6 (NT-6).
  • the neurotrophins exhibit similar structural conformations, including three surface ⁇ -hairpin loops, a ⁇ -strand, an internal reverse turn region, and N- and C- termini. With respect to sequence similarities, the neurotrophins share approximately 50% amino acid identity.
  • the neurotrophins are also functionally similar in that they each exhibit low affinity binding to a receptor known as the "p75 nerve growth factor receptor" or p75 N ⁇ FR .
  • Each neurotrophin also exhibits binding to a receptor of the tyrosine kinase (trk) family which is of higher affinity than the binding to the p75 receptor. This interaction is believed to be related to neuron survival, but is also involved with neuron differentiation including process formation.
  • trk tyrosine kinase
  • Trk receptor- neurotrophin interaction has been found to be more selective than neurotrophin interaction with the p75 N ⁇ FR receptor.
  • NGF binds only a trk receptor known as the TrkA receptor, while BDNF, NT-4 and NT-5 exhibit exclusive binding to a TrkB receptor.
  • TrkA receptor a trk receptor known as the TrkA receptor
  • BDNF, NT-4 and NT-5 exhibit exclusive binding to a TrkB receptor.
  • NT-3 is less selective and, although it binds primarily with a TrkC receptor, it also exhibits some binding to the TrkA and TrkB receptors (Ibanez et al., EMBO J. 1993, 12:2281).
  • the neurotrophins function primarily to promote survival of certain classes of peripheral and central neurons both during development and following neuronal
  • NGF in particular, is involved with the development of neurons in the peripheral nervous system and supports neuronal survival, as well as enhancing and maintaining the differentiated state of neurons.
  • the neurotrophins may also support inappropriate neurite outgrowth thereby facilitating the progression of a disease condition.
  • neurotrophins promote the undesirable sprouting of hippocampal "mossy fibres". Such inappropriate sprouting of mossy fibres is a common accompaniment of epilepsy in humans.
  • pathological states such as Alzheimer's disease, aberrant process growth, known as dystrophic neurite formation, is a strong correlate of disease severity.
  • neurotrophins are essential for the normal development and growth of neurons, they may be detrimental under certain circumstances.
  • ligands capable of inhibiting or reducing selected neurotrophin-mediated activities would be desirable therapeutically to treat neurodegenerative disease and repair of nervous system injury.
  • the present invention provides bicyclic neurotrophin-derived peptides, or functional equivalents thereof, which inhibit a neurotrophin-mediated activity.
  • Another aspect of the present invention provides a peptide comprising amino acids from positions 58-68 and 108-110 of a neurotrophin, or a functional equivalent thereof, wherein the amino acid from position 58 is covalently bound to the amino acid from position 108 and the amino acid from position 68 is covalently bound to the amino acid at position 110 to form a bicyclic structure.
  • SUBSTITUTE SHEET includes a carrier and a peptide comprising amino acids from positions 58-68 and 108-110 of a neurotrophin, or a functional equivalent thereof, wherein the amino acid from position 58 is covalently bound to the amino acid from position 108 and the amino acid from position 68 is covalently bound to the amino acid at position 110 to form a bicyclic structure.
  • a method for inhibiting a neurotrophin-mediated activity comprising the step of exposing neurons to a composition as described above including a bicyclic peptide in combination with a suitable carrier.
  • a further aspect of the present invention provides a method for inhibiting neurotrphin-mediated activity in a mammal comprising the step of administering to said mammal a therapeutically effective amount of a composition which includes a bicyclic neurotrophin-derived peptide in combination with a pharmaceutical carrier.
  • Figure 1 illustrates generally the structure of a neurotrophin
  • Figure 2 illustrates linear, cyclic and bicyclic peptides prepared from the 68- 58/108-110 region of NGF
  • Figure 3 graphically illustrates the effect of the bicyclic NGF peptide of Fig. 2 on neurite outgrowth
  • Figure 4 illustrates the effects of the peptides of Fig. 2 on kindling-induced seizures.
  • the present invention relates to bicyclic neurotrophin-derived peptides, or functional equivalents thereof, which inhibit a neurotrophin-mediated activity.
  • neurotrophin refers to neurotrophic factors that are structurally homologous to NGF, i.e. include three surface ⁇ -hairpin loops, a ⁇ - strand, an internal reverse turn region, and N- and C- termini as illustrated in Fig. 1, and which promote at least one of neuron survival and neuron differentiation, as determined using assays of conventional design such as the in vitro assay exemplified herein and described by Riopelle et al. in the Can. J. of Phys. and Pharm. , 1982, 60:707.
  • Mammalian nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) and neurotrophin-5 (NT-5) are examples of neurotrophins.
  • neurotrophin-derived refers to peptides comprising an amino acid sequence native to a given mammalian neurotrophin.
  • “Functional equivalents" of neurotrophin-derived peptides in accordance with the present invention are peptides which differ from a neurotrophin-derived peptide, by deletion, replacement or modification of one or more of its amino acids, but which retains the activity of the neurotrophin-derived peptide, i.e. is capable of inhibiting a neurotrophin-mediated activity.
  • Functional equivalents of a neurotrophin-derived peptide in accordance with the present invention may comprise, for example, conservative amino acid replacements of native amino acids, e.g. an amino acid of the neurotrophin-derived peptide may be replaced with an amino acid having a similar charge such as replacement of an arginine residue with a lysine residue.
  • the neurotrophin-derived peptide may include derivatized internal or terminal amino acids, as discussed in more detail herein, to yield a peptide which retains the activity of the neurotrophin-derived peptide.
  • Neurotrophin-mediated activity is a biological activity that is normally promoted, either directly or indirectly, in the presence of a neurotrophin.
  • Neurotrophin-mediated activities include, for example, neurotrophin binding to the p75 NOFR receptor or neurotrophin binding to one of the trk receptors, neuron survival, neuron differentiation including neuron process formation and neurite outgrowth, and biochemical changes such as enzyme induction.
  • a biological activity that is mediated include, for example, neurotrophin binding to the p75 NOFR receptor or neurotrophin binding to one of the trk receptors, neuron survival, neuron differentiation including neuron process formation and neurite outgrowth, and biochemical changes such as enzyme induction.
  • SUBSTITUTE SHEET by a particular neurotrophin, e.g. NGF is referred to herein by reference to that neurotrophin, e.g. NGF-mediated activity.
  • a bicyclic peptide, or a functional equivalent thereof, to inhibit a neurotrophin-mediated activity conventional in vitro and ]n vivo assays can be used.
  • a receptor affinity cross-linking assay such as the assay described in herein in Example 2 can be used to assess the extent to which a bicyclic peptide inhibits neurotroph in/receptor binding.
  • Peptide inhibition of neurite survival and outgrowth can be determined using the m vitro assay described by Riopelle et al. in the Can. J. of Phys. and Pharm., 1982, 60:707, exemplified herein in Example 3, or using the in vivo kindling experiment described in Example 4.
  • bicyclic is used herein to refer to a peptide in which there exists two ring closures.
  • the ring closures are formed by covalent linkages between amino acids in the peptide.
  • a covalent linkage between two non-adjacent amino acids constitutes a ring closure, as does a second covalent linkage between a pair of adjacent amino acids which are already linked by a covalent peptide linkage.
  • the covalent linkages forming the ring closures may be amide linkages, i.e. the linkage formed between a free amino on one amino acid and a free carboxyl of a second amino acid, or linkages formed between the side chains or "R" groups of amino acids in the peptides.
  • bicyclic peptides in accordance with the present invention may be "true" bicyclic peptides, i.e. peptides cyclized by the formation of a peptide bond between the N-terminus and the C-terminus of the peptide, or they may be "depsi- bicyclic" peptides, i.e. peptides in which the terminal amino acids are covalently linked through their side chain moities.
  • the bicyclic peptide is consistent with the internal reverse turn region of the selected neurotrophin.
  • the reverse turn region of a neurotrophin extends from the amino acid at position 58 to the amino acid at position 68, and includes also the region extending from the amino acid at position 108 to the amino acid at position 110, as illustrated in Figure 1.
  • the "reverse turn” results from the dual linkage occurring in this region.
  • the dual linkage includes a first covalent linkage between the amino acid at position 58 and the amino acid at position 108, and
  • Depsi-bicyclic peptides in accordance with the present invention result from the formation of covalent linkages between the side chains of the amino acids from positions 58, 68, 108 and 110.
  • the amino acid residues from these positions have side chains that will readily react to form such covalent linkages.
  • cysteine residues are particularly suitable amino acids for this purpose since the free thiol R groups of cysteine residues readily oxidize to form covalent disulfide bridges.
  • the R groups of the amino acids in these positions can be derivatized to yield groups, such as free thiol groups, which will readily react to form the desired covalent linkages.
  • amino acids from positions 58 and 108, and positions 68 and 110 can be selected to have R groups, or derivatized to yield R groups, which will form amide linkages.
  • an amide linkage can be formed between the amino acids from positions 58 and 108 if the amino acid at one of these positions yields a free amino group, while the amino acid at the other position yields a free carboxyl group.
  • amino acids which yield a free amino group suitable for the formation of an amide bond are lysine, asparagine and giutamine.
  • amino acids which yield a free carboxyl group suitable for the formation of an amide bond are glutamic acid and aspartic acid.
  • N- and C- termini remain as free amino and free carboxyl residues, respectively, since it is the side chains of the terminal amino acids which are involved in the covalent cyclizing linkage.
  • the free terminal amino and carboxyl groups may also be derivatized or altered without affecting the activity of the peptide as an inhibitor of a neurotrophin- mediated activity.
  • the termini may be derivatized to include a non- peptidic blocking group that will prevent potential degradation at the N- and C- terminal ends from occurring.
  • non-peptidic groups include protecting groups such as those conventionally used in the art of peptide synthesis which will not adversely affect the in vitro and m vivo uses of the bicyclic peptide.
  • suitable non-peptidic N-terminal blocking groups can be introduced by alkylation or
  • N-terminal blocking groups include C r C 5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof. Amino acid analogues lacking the amino functionality are also useful to block the N-terminus.
  • Suitable non-peptidic C- terminal blocking groups in which the carboxyl group of the C-terminus may be either inco ⁇ orated or not, include esters, ketones or amides.
  • Ester or ketone-for ing alkyl groups particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH 2 ), and mono- and di ⁇ alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups.
  • Amino acid analogues lacking the carboxyl functionality are also useful C-terminal blocking groups such as agmatine. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the bicyclic peptide to yield desamino and descarboxylated forms thereof without affect on peptide activity.
  • True bicyclic peptides are also peptides in accordance with the present invention. Such peptides result from the formation of a peptide linkage between the N-terminal amino group of the amino acid from position 68 and the C-terminal carboxyl group of the amino acid from position 110.
  • Bicyclic peptides in accordance with the present invention may be derived from any mammalian neurotrophin due to the highly homologous nature of neurotrophins among different species with regard to both conformation and amino acid sequence.
  • the amino acid residues of neurotrophins at positions 58, 68 and 108-110 are conserved across species and have been found to play an important role in peptide conformation.
  • the amino acid residues in positions 59-67 do not appear to be important for peptide conformation, and further have not been found to participate in NGF interactions (Drinkwater et al., J. Biol. Chem. 1993, 268(31):23202).
  • bicyclic peptides of the present invention may be derived from the 58-68/108-110 amino acid region of, for example, human, mouse or rat NGF. Likewise, the bicyclic peptide may be derived from the 58-68/108-110 region
  • AA 68 is selected from cysteine, the desamino form thereof, and an N- terminally blocked cysteine;
  • AA 110 is selected from cysteine, descarboxylate cysteine, and a C- terminally blocked cysteine;
  • XX represents a peptide comprising from 1-10 amino acid residues.
  • the bicyclic peptide has an amino acid sequence represented by formula (II):
  • AA 68 and AA 110 are as defined above;
  • X is an acidic amino acid
  • X 2 is selected from the group consisting of a non-polar amino acid and an uncharged polar amino acid
  • X 3 is selected from the group consisting of an acidic amino acid and a non-polar amino acid.
  • acidic amino acid refers to an amino acid which is negatively charged at pH 6.0
  • a non-polar amino acid refers to an amino acid having a non-polar side-chain such as alanine, leucine, methionine and proline
  • an uncharged polar amino acid refers to an amino acid having an uncharged polar side-chain such as glycine, serine, cysteine and asparagine.
  • bicyclic peptide has an amino acid sequence represented generally by formula (II):
  • AA 68 and AA 110 are as defined above;
  • X is selected from glutamic acid and aspartic acid
  • X 2 is selected from proline and serine
  • X 3 is selected from aspartic acid and alanine.
  • Specific peptide sequences in accordance with the present invention derived from mouse and human neurotrophins include:
  • NGF m C 68 -G-S-E-V-P-N-S-A-R-C 58 -C 108 -V-C"° (SEQ ID NO: 3)
  • NGF hum C 68 -G-S-D-V-P-N-P-D-R-C 58 -C 108 -V-C 110 (SEQ ID NO:4)
  • a bicyclic peptide derived from the 58-68/108-110 region of mouse NGF, as illustrated in Figure 2 was prepared and found to inhibit rat NGF-mediated activity.
  • the bicyclic peptide inhibited cross-linking of NGF to both the p75 NOFR receptor and the trkA receptor, and inhibited NGF- mediated neurite outgrowth as determined both in vitro and vivo.
  • the bicyclic peptides of the present invention may be readily prepared by standard, well-established solid-phase peptide synthesis (SPPS) as described by Stewart et al. in Solid Phase Peptide Synthesis. 2nd Edition, 1984, Pierce Chemical Company, Rockfor, Illinois; and as described by Bodanszky and Bodanszky in The Practice of Peptide Synthesis. 1984, Springer- Verlag, New York.
  • SPPS solid-phase peptide synthesis
  • SUBSTITUTE SHEET derivatized, insoluble polymeric support such as cross-linked polystyrene or polyamide resin.
  • "Suitably protected” refers to the presence of protecting groups on both the ⁇ -amino group of the amino acid, and on any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions which will not affect the final peptide product. Stepwise synthesis of the oligopeptide is carried out by the removal of the N-protecting group from the initial amino acid, and coupling thereto of the carboxyl end of the next amino acid in the sequence of the desired peptide. This amino acid is also suitably protected.
  • the carboxyl of the incoming amino acid can be activated to react with the N-terminus of the support- bound amino acid by formation into a reactive group such as formation into a carbodiimide, a symmetric acid anhydride or an "active ester” group such as hydroxybenzotriazole or pentafluorophenyl esters.
  • a reactive group such as formation into a carbodiimide, a symmetric acid anhydride or an "active ester” group such as hydroxybenzotriazole or pentafluorophenyl esters.
  • solid phase peptide synthesis methods include the BOC method which utilizes tert-butyloxycarbonvl as the ⁇ -amino protecting group, and the FMOC method which utilizes 9-fluorenylmethyloxycarbonyl to protect the ⁇ -amino of the amino acid residues, both methods of which are well-known by those of skill in the art.
  • N- and/or C- protecting groups can also be achieved using protocols conventional to solid phase peptide synthesis methods.
  • synthesis of the desired peptide is typically performed using, as solid phase, a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C- terminal protecting group.
  • a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C- terminal protecting group.
  • synthesis is performed using a p- methylbenzhydry lamine (MBH A) resin so that, when peptide synthesis is completed, treatment with hydrofluoric acid releases the desired C-terminally amidated peptide.
  • MSH A p- methylbenzhydry lamine
  • SUBSTITUTE SHEET terminus by esterification can also be achieved using conventional procedures. This entails use of resin/blocking group combination that permits release of side-chain protected peptide from the resin, to allow for subsequent reaction with the desired alcohol, to form the ester function.
  • FMOC protecting groups in combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this pu ⁇ ose, with cleavage from the support being effected by TFA in dicholoromethane. Esterification of the suitably activated carboxyl function e.g. with DCC, can then proceed by addition of the desired alcohol, followed by deprotection and isolation of the esterified peptide product.
  • Inco ⁇ oration of N-terminal protecting groups can be achieved while the synthesized peptide is still attached to the resin, for instance by treatment with suitable anhydride and nitrile.
  • the resin-coupled peptide can be treated with 20% acetic anhydride in acetonitrile.
  • the N-protected peptide product can then be cleaved from the resin, deprotected and subsequently isolated.
  • Recombinant techniques can also be used to prepare peptides in accordance with the present invention.
  • DNA encoding the desired peptide is prepared and inserted into an appropriate expression vector.
  • the vector is used to transfect a suitable host organism for production of the peptide. Conventional techniques are then used to culture the host and to isolate the peptide product from the cell culture media.
  • amino acid composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide.
  • amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequenators, which sequentially degrade the peptide and identify the amino acids in
  • SUBSTITUTE SHEET order may also be used to determine definitely the sequence of the peptide.
  • a bicyclic peptide in accordance with the present invention, it is then cyclized to form an active bicyclic peptide in accordance with the present invention.
  • Many techniques are available for appropriately cyclizing the peptide, and the protocol used will depend on the type of linkages used to form the bicyclic product.
  • there are numerous covalent linkages which are suitable to cyclize the peptide including, for example, side chain linkages such as disulfide linkages and peptide or amide linkages.
  • disulfide linkages were used to form a bicyclic peptide as illustrated in Fig. 2.
  • the internal cysteine residues of the linear peptide were first protected in order to conduct the cyclization reactions in a stepwise fashion.
  • the protected peptide was then air oxidized to allow a disulfide linkage to form between the terminal cysteine residues.
  • the protecting groups were removed from the internal cysteine residues, and the peptide was again subjected to oxidizing conditions to allow a disulfide linkage to form between the internal cysteine residues thereby resulting in the bicyclic peptide.
  • the bicyclic peptide Prior to its use to inhibit neurotrophin-mediated activity, the bicyclic peptide is purified to remove contaminants which may adversely affect its activity.
  • strict standards of purity such as those required for pharmaceutical compounds, may not be required for use of the present compounds in vitro.
  • a compound according to the present invention if it is to be used in a pharmaceutical sense, it must be purified so as to meet the standards set out by the appropriate regulatory agencies. Any one of a number of conventional purification procedures may be used to attain the required level of purity including, for example, reversed-phase high-pressure liquid chromatography (HPLC) using an alkylated silica column such as Q-, C 8 - or C lg - silica.
  • HPLC reversed-phase high-pressure liquid chromatography
  • a gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid.
  • Ion-exchange chromatography can also be used to separate peptides based on their charge.
  • a composition comprising an effective amount of a neurotrophin- derived bicyclic peptide and a suitable carrier.
  • suitable carrier is meant a carrier which admixes with the selected bicyclic peptide to yield a composition suitable for the application for which it is to be used.
  • effective amount is meant an amount of bicyclic peptide sufficient to inhibit an undesired neurotrophin-mediated activity by about 50% as determined using assays of conventional design such as those described herein in the specific examples.
  • the present bicyclic peptides have use as media supplements to prevent undesirable neurotrophin-mediated activity of neuron cells in vitro.
  • primary sensory neurons require NGF for survival in cell culture; however, NGF also influences neuron differentiation, notably process formation and outgrowth, which are undesirable for the use of primary sensory neurons in cell culture.
  • NGF is added to the cell culture media along with a bicyclic peptide.
  • the bicyclic peptide is first combined with a carrier which will not adversely affect the growth of the cells in culture.
  • Such carriers will include, for example, physiologically acceptable fluids such as water or any other fluid suitable for addition to the cell culture.
  • the peptide can be combined with media suitable for culturing neuronal cells prior to being added to the cell culture.
  • concentration of the peptide in the cell culture will be in the range of from about 1-500 ⁇ M, and preferably from about 1-100 ⁇ M.
  • concentration of bicyclic peptide for use in preventing neuron differentiation in cell culture will, of course, vary in each independent case, and will depend on the extent of inhibition desired as well as the type of neuronal cells involved.
  • compositions for in vivo administration e.g. for treating neurological conditions such as epilepsy or Alzheimer's disease, are also contemplated.
  • Such compositions comprise a therapeutically effective amount of a bicyclic peptide
  • a pharmaceutically acceptable carrier means acceptable for use in the pharmaceutical and veterinary arts, i.e. non-toxic and not adversely affecting the activity of the bicyclic peptide.
  • therapeutically effective amount means an amount of the compound sufficient to reduce undesirable neurotrophin-mediated activity, as determined using assays of conventional design such as the assays described herein in the specific examples, in an inflicted individual without causing adverse effects.
  • compositions for in vivo administration include conventional carriers generally selected for combination with peptide-based drugs such as diluents, excipients and the like. Reference may be made to "Remington's Pharmaceutical Sciences", 17th Ed. , Mack Publishing Company, Easton, Penn., 1985, for guidance on drug formulations generally. As will be appreciated, the pharmaceutical carriers used to prepare compositions in accordance with the present invention will depend on the administrable form to be used to treat the inflicted individual.
  • the compounds are formulated for administration by injection intraventricularly, and are accordingly provided as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic.
  • the compounds may be administered in distilled water or, more desirably, in saline or 5% dextrose solution.
  • Water solubility of these and other compounds of the invention may be enhanced, if desired, by inco ⁇ orating into the composition a solubility enhancer, such as cetyltrimethylammonium bromide or chloride.
  • Lyoprotectants such as mannitol, sucrose or lactose and buffer systems, such as acetate, citrate and phosphate may also be included in the formulation, as may bulking agents such as serum albumin.
  • the compounds of the present invention may be formulated for administration by routes other than injection.
  • oral dosage forms such as tablets, capsules and the like, formulated in accordance with standard
  • a pharmaceutical composition appropriate for treatment can readily be established in appropriately controlled trials, and will correspond to an amount of bicyclic peptide that reduces undesirable neurotrophin-mediated activity without causing intolerable side effects to the individual being treated. It is anticipated that an effective treatment regimen for patients will involve the intraventricular administration of dosages which achieve a level of peptide in the spinal fluid of the individual being treated of about 1-500 ⁇ M. It will be appreciated, of course, that the dosage sizes required to attain this in vivo concentration will vary according to the route of administration, the frequency of administration, on the individual being treated and on the neurological condition being treated.
  • the bicyclic peptide illustrated in Fig. 2 was prepared by first synthesizing the linear form thereof using the solid phase synthesis method.
  • An automated synthesizer e.g. Applied Biosystems 430A, was used with a Wang resin (available from NovaBiochem). All amino acid side chains were protected with Mtr (4- methoxy-2,3,6-trimethyl-benzene-sulfonyl) groups, with the exception of the cysteines from positions 58 and 108 which were protected with ACM (acetamidomethyl) groups.
  • a TFA-cleavage (1-2 hrs) from the resin yielded the linear peptide retaining only the ACM protecting groups.
  • the peptide was dissolved in 0.1 M ammonium bicarbonate buffer, pH 8.3, at a concentration of 0.1 mg/mL.
  • the reaction mixture was stirred at room temperature, and the progression of the reaction was monitored by HPLC.
  • the HPLC solvent comprising solvent A of water with 0.1 % TFA and solvent B of
  • the mono-cyclized product (0.1 mmol) was then cyclized at the ACM protected sites.
  • the product was dissolved in 1.5 mL of methanol. This solution was added dropwise to 2.5 mL of methanol containing 63.5 mg of iodine over 30 minutes with stirring at room temperature. The stirring was continued (approx. 3 hrs) while the progression of the reaction was determined using HPLC.
  • the reaction was quenched by the addition of solid zinc powder (1-2 mg).
  • the mixture was diluted with 10 mL of water, filtered and lyophilized.
  • the bicyclic product was purified using HPLC, and its structure was confirmed by standard methods.
  • the ability of the peptides derived from the 68-58/108-110 region of NGF to antagonize NGF interaction with the p75 and trkA receptors was determined.
  • the peptides tested were bicyclic (BC) 68-58/108-110, cyclic (C) 68-58/108-110, and linear (L) 68-58/108-110, each of which are illustrated in Figure 2.
  • PC12 rat pheochromocytoma cells (ATCC CRL 1721) were independently incubated in RPMI (GIBCO)/ 10% fetal calf serum (GIBCO) with 20 ⁇ M and 200 ⁇ M solutions of NGF peptide, prepared as described in detail in Example 1, in the presence of 125 I-NGF (isolated from mouse submaxillary gland as described in Mobley et al., 1976, Biochemistry, 15:1543) for 2 hours at 4°C.
  • Control PC12 cells were incubated in RPMI/ 10% fetal calf serum in the presence of 125 I-NGF only (no NGF peptide).
  • the cells were solubilized in 1 ml of lysis buffer containing 10 mM tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1 % NP-40, 0.01 mg/ml aprotinin, 0.5 mM orthovanadate, 0.5 ⁇ l/ml leupeptin, and 2 mM phenyl-methyl-sulfonylfluoride at 4 °C for 15 min. Cellular debris was removed by centrifugation. TrkA antibody (supplied by W. Mobley, University of California, San Francisco) or p75 antibody (supplied by E. Shooter, Stanford U.
  • the immunoprecipitated proteins were then washed with lysis buffer and eluted from the affinity gels using reducing SDS sample buffer, and solubilized in the reducing buffer for 10 min at 95 °C.
  • Samples were separated on a modified Laemmli discontinuous acrylamide gel system (Laemmli, Nature, 1970, 227:680) using 4% SDS PAGE staking gel and a gradient urea polyacrylamide separating gel ranging from 4.5% acrylamide/18% urea to 7.5% acrylamide/48 % urea.
  • the gels were fixed and processed for autoradiography using -70°C exposure with Kodak XAR film and manual processing.
  • the ganglia were then triturated using a 5 mL narrow-tip pipette to a single cell suspension.
  • the cell suspension was washed through a 500- ⁇ l FCS undercut (700 x g for 5 min at 4° C) and resuspended in 4 mL of Ham's F12 medium plus 5% FCS.
  • FCS undercut 700 x g for 5 min at 4° C
  • FCS F12 medium Ham's F12 medium plus 5% FCS.
  • the cell suspension was then preplated on a 100-mm Flacon culture dish and incubated for 45-60 min at 37°C in a 5 % CO 2 humidified atmosphere. Cells enriched in neurons were decanted for the bioassay, since non-neuronal cells of DRG preferentially stick to the culture substrate.
  • the inside wells of 96-well Falcon microculture plates were coated with polylysine (0.1 mg/mL) (Sigma) for 4 h at 37°C (the outside wells were filled with distilled water to provide humidity) and, following a rinse with tissue culture media, 100 ⁇ L of neuron-rich cell suspension was added to each well at 10 5 cells/mL.
  • the bioassays were read using a Leitz Diavert microscope with phase optics. To afford adequate optics, the meniscus effect of each well was removed by filling the well with a balanced salt solution until a flat, air-filled interface was achieved at the top of the well. At least 100 neurons per well were counted, and the assay was scored as the ratio of cells bearing neurites greater than one cell diameter to total viable (phase-bright) cells.
  • IC* i.e. the concentration of bicyclic peptide required to inhibit neurite growth on 50% of the cells
  • Cells enriched for sensory neurons were prepared from ED8 chick DRG as described above. The cells were plated at a density of 800-1000 cells per well in Terasaki plates treated with poly-D-lysine and laminin in tissue culture medium containing 1 ng/ml NGF and 68-58/108-110 depsibicyclic peptide in the amounts shown below. Following a 20-22 hr. incubation at 37°C, 5% CO 2 , the cells were fixed in 4% formaldehyde in PBS and cells on the tissue culture surface were counted as a percentage of total cells.
  • Kindling is a phenomenon in which repeated low-intensity (subconvulsive) electrical stimulation of forebrain areas leads to a progressive and permanent amplification of seizure activity, and is thus, widely accepted as a model for human temporal lobe epilepsy.
  • the effect of the present neurotrophin-derived peptides on kindling was determined as follows.
  • mice Male Long-Evans hooded rats (300-400 g) were used. The animals were housed individually, maintained on an ad lib feeding schedule and kept on a 12 h on/ 12 h off light cycle.
  • the rats were anesthetised with 0.1 ml per 100 g body weight of 100 mg/ml ketamine hydrochloride (Rogar/STB Inc., London, Canada) and 0.05 ml per 100 g body weight of 20 mg/ml xylocaine 2% hydrochloride (Astra, Mississauga, Canada), and then placed in a stereotaxic holder.
  • the rats were implanted unilaterally with a bipolar twisted, teflon-coated, stainless steel electrode with an exposed tip (wire diameter 190 ⁇ m) in the right amygdala at stereotaxic coordinates of 3.3 mm caudal and 8.0 mm lateral to bregma and 8.5 mm ventral to the brain surface (selected from Paxinos and Watson, 1982, "The rat brain in stereotaxic coordinates", Academic
  • a cannula (Alzet brain infusion kit, Alza Co ⁇ .) was implanted in the lateral ventricle, 5 mm below the skull surface, at 0.6 mm caudal to bregma and 1.3 mm lateral to the midline. It was firmly attached to the skull with dental cement and anchored with three stainless steel screws.
  • An osmotic pump (Alzet model 2002, flow speed 0.5 ⁇ l/h, effective maximally for 14 days) was connected to the cannula via polyethylene tubing and placed subcutaneously in the neck area. Histological examination of lateral ventricle sections was done to confirm that the cannula was correctly placed.
  • ⁇ M of peptide Forty-five (45) ⁇ M of peptide, in a physiologically acceptable buffer, was delivered throughout the duration of the experiment to each test animal. There were five groups of test animals, 5 animals per peptide test group, 10 animals in the negative control group and 12 animals in the positive control group. Each test group was administered one peptide selected from the linear, cyclic and bicyclic 68-58/108-110 peptides. The negative control group was infused with control serum IgGs, and the positive control group was infused with 100 ⁇ g/day of anti-NGF antibody.
  • the anti-NGF antibody was obtained from sheep immunized with 0.5 mg of 2.5S NGF (prepared from male mouse salivary glands according to the method of Mobley et al.
  • the kindling stimulations were started.
  • the animals received a one-second train of one-millisecond pulses at a frequency of 60 Hz and a pulse intensity of 200-400 ⁇ A. These pulses were sufficient to trigger an epileptiform afterdischarge (AD) following each stimulation.
  • AD epileptiform afterdischarge
  • Each animal was stimulated in this fashion twice a day over a period of 11 days. Progression of kindling was monitored behaviorally and electrophysiologically by recording the
  • the number of stimulations to reach stage-5 seizures for control rats and rats receiving the linear, cyclic and bicyclic peptides is illustrated graphically in Fig. 4.
  • the results illustrate that the bicyclic peptide has a potency which is approximately equal to that of the anti-NGF IgG in delaying the onset of kindling in comparison to the control serum IgG, linear peptide and cyclic peptide.

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Abstract

L'invention a trait à un nouvel antagoniste de la neurotrophine. Cet antagoniste est un peptide bicyclique, dérivé, de préférence, d'une région interne à spire inversé d'un membre de la famille des neurotrophines, similaire d'un point de vue structurel, au nombre desquels on compte NGF, BDNF, NT-3, NT-4, NT-5 et NT-6. Ces antagonistes comprennent, en règle générale, des acides aminés se trouvant placés entre les positions 58-68 et 108-110 d'une neurotrophine, l'acide aminé se trouvant en position 58 possédant une liaison covalente avec l'acide aminé de la position 108 tandis que l'acide aminé occupant la position 68 est lié de manière covalente avec celui qui est situé à la position 110, de manière à constituer une structure bicyclique. Les antagonistes dérivés de la neurotrophine s'avèrent des plus utiles pour inhiber une activité indésirable à médiation neurotrophinique comme une excroissances des axones et des dendrites survenant lors de certains états pathologiques neurodégénératifs.
PCT/CA1995/000603 1995-10-25 1995-10-25 Antagonistes de la neurotrophine WO1997015593A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055868A2 (fr) * 1998-04-24 1999-11-04 Genentech, Inc. Proteines fizz
US6291247B1 (en) 1994-05-11 2001-09-18 Queen's University At Kingston Methods of screening for factors that disrupt neurotrophin conformation and reduce neurotrophin biological activity
FR2807660A1 (fr) * 2000-04-13 2001-10-19 Warner Lambert Co Utilisation d'antagonistes du ngf pour la prevention ou le traitement de douleurs viscerales chroniques
WO2004032870A2 (fr) 2002-10-08 2004-04-22 Rinat Neuroscience Corp. Procedes de traitement d'une douleur post-chirurgicale par administration d'un antagoniste du facteur de croissance neuronal, et compositions renfermant celui-ci
JP2007137811A (ja) * 2005-11-17 2007-06-07 Lion Corp 神経栄養因子−４（Ｎｅｕｒｏｔｒｏｐｈｉｎ−４）アンタゴニスト
EP2100902A1 (fr) 2002-10-08 2009-09-16 Rinat Neuroscience Corp. Méthode pour le traitement de la douleur par administration d'un anticorps antagoniste du facteur de croissance neuronal et d'un analgésique opioide, et compositions les contenant
EP2191846A1 (fr) 2003-02-19 2010-06-02 Rinat Neuroscience Corp. Methode de traitement de la douleur consistant a administrer un antagoniste du facteur de croissance neuronale ainsi que des medicaments anti-inflammatoires non steroidiens (nsaid) et composition les contenant
EP2206728A1 (fr) 2004-04-07 2010-07-14 Rinat Neuroscience Corp. Méthodes de traitement de la douleur associée au cancer des os par administration d'un anticorps antagoniste des facteurs de croissance neuronale
EP2305711A2 (fr) 2005-04-11 2011-04-06 Rinat Neuroscience Corp. Procédés de traitement de la douleur de l'arthrose en administrant un antagoniste de facteur de croissance nerveuse et compositions le contenant
US9505829B2 (en) 2010-08-19 2016-11-29 Zoetis Belgium S.A. Anti-NGF antibodies and their use
US9617334B2 (en) 2012-06-06 2017-04-11 Zoetis Services Llc Caninized anti-NGF antibodies and methods thereof
US10323086B2 (en) 2002-12-24 2019-06-18 Rinat Neuroscience Corp. Methods for treating osteoarthritis pain by administering a nerve growth factor antagonist and compositions containing the same
US10982002B2 (en) 2018-03-12 2021-04-20 Zoetis Services Llc Anti-NGF antibodies and methods thereof
US11008386B2 (en) 2002-12-24 2021-05-18 Rinat Neuroscience Corp. Anti-NGF antibodies and methods using same
WO2023212596A1 (fr) 2022-04-27 2023-11-02 Regeneron Pharmaceuticals, Inc. Traitement de l'arthropathie basé sur la stratification du score de risque polygénique de l'arthrose

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K RASHID ET AL.: "Peptide mimics of an NGF domain inhibit kindling and neuronal sprouting in rats", SOCIETY FOR NEUROSCIENCE ABSTRACTS, 24TH ANNUAL MEETING OF THE SOCIETY FOR NEUROSCIENCE, MIAMI BEACH, FLORIDA, USA, NOVEMBER 13-18, 1994, vol. 20, no. 1-2, 1994, pages 678, XP002007155 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6291247B1 (en) 1994-05-11 2001-09-18 Queen's University At Kingston Methods of screening for factors that disrupt neurotrophin conformation and reduce neurotrophin biological activity
WO1999055868A3 (fr) * 1998-04-24 2000-11-09 Genentech Inc Proteines fizz
WO1999055868A2 (fr) * 1998-04-24 1999-11-04 Genentech, Inc. Proteines fizz
FR2807660A1 (fr) * 2000-04-13 2001-10-19 Warner Lambert Co Utilisation d'antagonistes du ngf pour la prevention ou le traitement de douleurs viscerales chroniques
WO2001078698A2 (fr) * 2000-04-13 2001-10-25 Warner-Lambert Company Utilisation des antagonistes du ngf dans la prevention et le traitement de la douleur viscerale chronique
WO2001078698A3 (fr) * 2000-04-13 2002-04-25 Warner Lambert Co Utilisation des antagonistes du ngf dans la prevention et le traitement de la douleur viscerale chronique
WO2004032870A2 (fr) 2002-10-08 2004-04-22 Rinat Neuroscience Corp. Procedes de traitement d'une douleur post-chirurgicale par administration d'un antagoniste du facteur de croissance neuronal, et compositions renfermant celui-ci
EP2100902A1 (fr) 2002-10-08 2009-09-16 Rinat Neuroscience Corp. Méthode pour le traitement de la douleur par administration d'un anticorps antagoniste du facteur de croissance neuronal et d'un analgésique opioide, et compositions les contenant
US10323086B2 (en) 2002-12-24 2019-06-18 Rinat Neuroscience Corp. Methods for treating osteoarthritis pain by administering a nerve growth factor antagonist and compositions containing the same
US11008386B2 (en) 2002-12-24 2021-05-18 Rinat Neuroscience Corp. Anti-NGF antibodies and methods using same
EP2191846A1 (fr) 2003-02-19 2010-06-02 Rinat Neuroscience Corp. Methode de traitement de la douleur consistant a administrer un antagoniste du facteur de croissance neuronale ainsi que des medicaments anti-inflammatoires non steroidiens (nsaid) et composition les contenant
EP2206728A1 (fr) 2004-04-07 2010-07-14 Rinat Neuroscience Corp. Méthodes de traitement de la douleur associée au cancer des os par administration d'un anticorps antagoniste des facteurs de croissance neuronale
EP3372614A1 (fr) 2004-04-07 2018-09-12 Rinat Neuroscience Corp. Méthodes de traitement de la douleur associée au cancer des os par administration d'un antagoniste des facteurs de croissance neuronale
EP3272358A1 (fr) 2005-04-11 2018-01-24 Rinat Neuroscience Corporation Procédés de traitement de la douleur de l'arthrose en administrant un antagoniste de facteur de croissance nerveuse et compositions le contenant
EP2305711A2 (fr) 2005-04-11 2011-04-06 Rinat Neuroscience Corp. Procédés de traitement de la douleur de l'arthrose en administrant un antagoniste de facteur de croissance nerveuse et compositions le contenant
JP2007137811A (ja) * 2005-11-17 2007-06-07 Lion Corp 神経栄養因子−４（Ｎｅｕｒｏｔｒｏｐｈｉｎ−４）アンタゴニスト
US9505829B2 (en) 2010-08-19 2016-11-29 Zoetis Belgium S.A. Anti-NGF antibodies and their use
US10093725B2 (en) 2010-08-19 2018-10-09 Zoetis Belgium S.A. Anti-NGF antibodies and their use
US10125192B2 (en) 2010-08-19 2018-11-13 Zoetis Belgium S.A. Caninized anti-NGF antibodies and their use
US9617334B2 (en) 2012-06-06 2017-04-11 Zoetis Services Llc Caninized anti-NGF antibodies and methods thereof
US9951128B2 (en) 2012-06-06 2018-04-24 Zoetis Services Llc Caninized anti-NGF antibodies and methods thereof
US10982002B2 (en) 2018-03-12 2021-04-20 Zoetis Services Llc Anti-NGF antibodies and methods thereof
WO2023212596A1 (fr) 2022-04-27 2023-11-02 Regeneron Pharmaceuticals, Inc. Traitement de l'arthropathie basé sur la stratification du score de risque polygénique de l'arthrose

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