WO2000075176A1 - Petits analogues cycliques du facteur neurotrophique derive du cerveau (bdnf) - Google Patents

Petits analogues cycliques du facteur neurotrophique derive du cerveau (bdnf) Download PDF

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WO2000075176A1
WO2000075176A1 PCT/AU2000/000641 AU0000641W WO0075176A1 WO 2000075176 A1 WO2000075176 A1 WO 2000075176A1 AU 0000641 W AU0000641 W AU 0000641W WO 0075176 A1 WO0075176 A1 WO 0075176A1
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loop
compound according
compound
monomeric
bdnf
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PCT/AU2000/000641
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Richard Anthony Hughes
Paul O'leary
Richard Zwar
Alison Hunt-Sturman
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The University Of Melbourne
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Priority to EP00930886A priority Critical patent/EP1212353A4/fr
Priority to AU49016/00A priority patent/AU780408B2/en
Priority to JP2001502457A priority patent/JP2003502295A/ja
Priority to CA002376729A priority patent/CA2376729A1/fr
Publication of WO2000075176A1 publication Critical patent/WO2000075176A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular 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
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to methods and compositions for promoting nerve cell growth and in particular to agonists of brain-derived neurotrophic factor.
  • the invention relates more particularly to agonists which are derivatives of peptides based on the structures of the solvent-exposed loops 2 and 4 of brain- derived neurotrophic factor.
  • Brain-derived neurotrophic factor is a member of the neurotrophin family of neurotrophic factors, which includes nerve growth factor (NGF) , neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and neurotrophin-6 (Thoenen, 1991; G ⁇ tz et al, 1994) .
  • BDNF has been shown to promote neuronal survival during embryonic development, and to prevent neuronal degeneration resulting from disease or injury. Furthermore, several BDNF-responsive neuronal populations have been implicated in human neurodegenerative disease.
  • BDNF acts as a potent neurotrophic factor for cranial and spinal motor neurons which degenerate in amyotrophic lateral sclerosis (Thoenen et al, 1993), as well as for dopaminergic neurons of the substantia nigra which are lost in Parkinson's disease (Spina et al, 1992) .
  • BDNF has neurotrophic actions on small fibre sensory neurons involved in several types of sensory neuropathy (Lindsay, 1994) .
  • BDNF and the other neurotrophins are mediated by binding to two classes of cellular receptor: members of the trk family of receptor tyrosine kinases, and the low affinity neurotrophin receptor, p75.
  • Specific neurotrophins bind with high affinity (Ka approximately 10 "11 M) to particular trk members expressed by responsive neurons: thus NGF and NT-3 bind to trkA; BDNF and NT-4/5 bind to trkB; NT-3 binds to trkC.
  • Binding of a neurotrophin to its specific-trk receptor causes receptor homodimerisation, triggering the intrinsic kinase domains of the receptors to autophosphorylate intracellular tyrosine residues, and thus initiating signal transduction cascades leading to neuronal survival (Barbacid, 1994) .
  • p75 acts as a common low affinity receptor for the neurotrophins, and binds each with comparable affinity (Ka approximately 10 ⁇ 9 M) ; p75 is expressed widely on central and peripheral neurons as well as on other cell types, such as Schwann cells (for review see Chao and Hempstead, 1995) .
  • p75 While the role of the trk members in signalling the neurotrophic effects of the neurotrophins is well established, the function of p75 remains controversial. Although there is compelling evidence that p75 either modulates responses mediated by trk members or itself plays a part in survival signalling, the final effect of p75 appears to depend on the relative levels of expression of p75 and trk (Kaplan and Miller, 1997). Of particular interest are the observations that p75 may, under certain circumstances, cause apoptosis either in the absence (Rabizadeh et al, 1993; Barrett and Bartlett, 1994) or presence (Frade et al, 1996) of bound neurotrophin. This "death signal" of p75 may be mediated by an intracellular region homologous to the death-signalling domains of tumour necrosis factor (TNF) receptor-1 and Fas (Chapman, 1995).
  • TNF tumour necrosis factor
  • the neurotrophins are homodimers which consist of two identical protomers of approximately 120 amino acids, held together by hydrophobic interactions.
  • the overall amino acid homology between the different neurotrophins is approximately 50%, and sequence alignment between the members reveals a common pattern of sequence homology and variability (Iba ⁇ ez et al, 1993).
  • X-ray crystal structures have been determined for the NGF homodimer (McDonald et al, 1991) and for a BDNF/NT-3 heterodimer (Robinson et al, 1995), revealing a common fold for the neurotrophins. This structure is depicted in Figure 1.
  • the regions of high sequence homology exist as seven ⁇ -strands, which contribute to three longitudinal anti-parallel twisted ⁇ -sheets. This structure is locked by a "cystine knot" of three disulphide bridges. The six cysteine residues which participate in the cystine knot structure are fully conserved in all the neurotrophins.
  • the three pairs of ⁇ -strands are linked by three ⁇ -hairpin loops (loop 1, loop 2 and loop 4) and a longer loop (loop 3), which correspond predominantly to the regions of sequence variability.
  • ⁇ -hairpin loop regions of the neurotrophins are responsible for the specificity of different trk receptors, and thus are important regions in receptor binding and activation.
  • structure-activity data obtained from the neurotrophins support this hypothesis.
  • Site-directed mutagenesis studies have identified amino acid residues of in loop 2 of BDNF, which are important for binding to trkB and for biological activity. Insertion of this region of BDNF into NGF gave a chimeric protein which, unlike native
  • NGF bound to trkB and displayed BDNF-like biological activity (Iba ⁇ ez et al, 1993) .
  • Additional residues in loop 3 (Gin 84 ) and loop 4 (Lys 96 and Arg 97 ) have been shown to be important in activation of trkB, but are thought not to be involved in receptor binding.
  • these residues When mapped on to the three- dimensional structure of BDNF, these residues are solvent- accessible, and together form a binding surface that almost exclusively spans the top half of the molecule.
  • neurotrophic factors such as BDNF
  • BDNF neurotrophic factor
  • neurodegenerative diseases such as motor neuron disease and peripheral neuropathies
  • neurodegenerative diseases such as motor neuron disease and peripheral neuropathies
  • neurotrophins and other neurotrophic factors offer exciting prospects for the treatment of neurodegenerative diseases, such as motor neuron disease and peripheral neuropathies (for review see Hefti, 1994) .
  • neurotrophic factors are orally inactive, are unable to cross the blood-brain barrier, and typically have a short half-life in plasma (Dittrich et al, 1994) .
  • the recombinant human neurotrophic factors themselves are unlikely to be optimal agents for the long-term treatment of neurodegenerative disease.
  • the peptides consist of two monocyclic peptides connected by a linking moiety. It is surprising that the linking moiety in some of the dimers found to be active is incorporated at sites other than those predicted to be optimal on the basis of the structure-activity relationships. In addition, the linker distance found to give optimal activity was shorter than that predicted from our design template.
  • the invention provides a cyclic compound of one or more cyclic moieties, which has a biological activity of brain-derived neurotrophic factor (BDNF) .
  • BDNF brain-derived neurotrophic factor
  • the compound is a bicyclic dimeric compound (that is, a compound composed of two monocyclic compounds connected by a chemical linker) based on loop 2 of BDNF, of general formula (I) :
  • monomeric loop 2 sequence means a sequence of amino acid residues or functional equivalents thereof, which is substantially homologous to the loop 2 region of BDNF, and which comprises all or part of the following sequence:
  • constraint means any chemically and biologically compatible grouping of atoms serving to limit the flexibility of the monomeric loop 2 sequence; and linker means any chemically and biologically compatible grouping of atoms serving to link two monomeric loop 2 sequences and their associated constraints to give a bicyclic, dimeric compound.
  • the preferred linking groups have 0 to 20 carbon atoms, and 0 to 10 heteroatoms (N, O, S, P etc.), and may be straight chain or branched, may contain saturated, unsaturated and/or aromatic rings, may contain single and/or double bonds, and may contain chemical groups such as amide, ester, disulphide, thioether, ether, phosphate, amine and the like.
  • the "constraint” can be obtained by several methods, including but not limited to:
  • residues contributing the side chains may be derived from the monomeric loop 2 sequence itself, or may be incorporated into or added on to the monomeric loop 2 sequence for this purpose; and,
  • the compound is a tricyclic dimeric compound (that is, a compound composed of two monocyclic compounds connected by two chemical linkers) based on loop 2 of BDNF of general formula (II) : monomeric-1inker1-monomeric loop 2 sequence-linker2-loop 2 sequence
  • monomeric loop 2 sequence means a sequence of amino acid residues or functional equivalents thereof, which is substantially homologous to the loop 2 region of BDNF, and which comprises all or part of the following sequence : Glu 40 -Lys 41 -Val 42 -Pro 43 -Val 44 -Ser 45 -Lys 46 -Gly 7 -Gln 48 -
  • linker means any chemically and biologically compatible grouping of atoms serving to link two monomeric loop 2 sequences and their associated constraints to give a tricyclic, dimeric compound.
  • linkers may be the same or different .
  • the preferred linking groups have 0 to
  • the "constraint” can be obtained by several methods, including but not limited to: (i) cyclising the N-terminal amine with the C-terminal carboxyl acid function either directly via an amide bond between the N-terminal nitrogen and C-terminal carbonyl, or indirectly via a spacer group, for example by condensation with an ⁇ -amino carboxylic acid;
  • residues contributing the side chains may be derived from the "monomeric loop 2 sequence" itself, or may be incorporated into or added onto the monomeric loop 2 sequence for this purpose; and,
  • the compound is a monomeric, monocyclic compound based on the p75-binding region of loop 4 of BDNF and incorporating a molecular spacer of the general formula (III) : monomeric loop 4 sequence
  • monomeric loop 4 sequence means a sequence of amino acid residues or functional equivalents thereof, which is substantially homologous to the p75-binding region of loop 4 of BDNF, and comprises all or part of the following sequence:
  • Lys 95 -Lys 96 -Arg 97 means any chemically and biologically compatible grouping of atoms serving to limit the flexibility of the monomeric loop 4 sequence.
  • constraint' means any chemically and biologically compatible grouping of atoms serving to limit the flexibility of the monomeric loop 4 sequence. For example by covalently linking all or part of the "monomeric loop 4 sequence" to form a cyclic structure (ring) .
  • the "constraint *" can be derived by several methods, including but not limited to:
  • residues contributing the side chains may be derived from the "monomeric loop 4 sequence" itself, or may be incorporated into or added on to the "monomeric loop 4 sequence” for this purpose;
  • the compounds of the invention include peptide analogues, including but not limited to the following: 1. Compounds in which one or more amino acids is replaced by its corresponding D-amino acid. The skilled person will be aware that retro-inverso amino acid sequences can be synthesised by standard methods. See for example Chorev and Goodman, 1993; 2. Peptidomimetic compounds, in which the peptide bond is replaced by a structure more resistant to metabolic degradation. See for example Olson et al, 1993. 3. Compounds in which individual amino acids are replaced by analogous structures, for example gem- diciminoalkyl groups or alkylmalonyl groups, with or without modified termini or alkyl, acyl or amine substitutions to modify their charge.
  • the substitution is conservative, i.e., an amino acid is replaced by one of similar size and with similar charge properties.
  • bicyclic dimers are of formula (IV) to (VI) : (L2-8P2C) 2
  • dimeric bicyclic peptides (L2-8P2C) 2 and (L2-8S4C) 2 consist of monomeric loop 2 sequences constrained by disulphide bonds formed between cysteine residues added to the loop 2 sequence and joined by a linker consisting of a disulphide bond formed between cysteine residues substituted into the loop 2 sequence, or
  • dimeric bicyclic peptide (L2-8&E+K) 2 consists of monomeric loop 2 sequences constrained by disulphide bonds formed between cysteine residues added to the loop 2 sequence and joined by a linker consisting of an amide bond formed between a glutamate and a lysine residue added to the loop 2 sequence.
  • the tricyclic dimer is of formula (VII) : (L2- 8S4C&E+K) 2
  • 8S4C&E+K 2 consists of monomeric loop 2 sequences constrained by disulphide bonds formed between cysteine residues added to the loop 2 sequence, and joined by one linker consisting of a disulphide bond formed between cysteine residues substituted into the loop 2 sequence and a second substituted consisting of an amide bond formed between a glutamate and a lysine residue added to the loop 2 sequence.
  • the monomeric, monocyclic compound is of formula (VIII) :
  • the invention further provides a pharmaceutical composition, comprising a compound according to the invention together with a pharmaceutically acceptable carrier.
  • composition may be formulated so as to be suitable for a variety of routes of administration, for example intravenous, subcutaneous, intramuscular or intrathecal or intraventricular injection, oral or for topical administration.
  • routes of administration for example intravenous, subcutaneous, intramuscular or intrathecal or intraventricular injection, oral or for topical administration.
  • compositions will depend on the individual route of administration.
  • Methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 17 th Edition, Mack Publishing Company, Easton, Pennsylvania, USA.
  • Pharmaceutically acceptable carriers include conventional carriers which are suitable for use with peptide-based drugs, including diluents, excipients, and preservatives and the like.
  • carriers such as dextrose, mannitol, sucrose, or lactose, buffer systems such as acetate, citrate and phosphate, and bulking agents such as serum albumin, preferably human serum albumin, may be used.
  • the invention also provides a culture medium additive for promotion of growth of neuronal cells in vitro, comprising a compound according to the invention together with a carrier or diluent which does not adversely effect the growth of cells in culture.
  • a carrier or diluent which does not adversely effect the growth of cells in culture.
  • Suitable carriers and diluents will be well known to the person skilled in art, and include physiologically acceptable fluids such as water, saline solution, or buffer solutions.
  • the optimal concentration of compound will vary according to the cell type and the culture conditions, but will generally be in the range l-500 ⁇ M, preferably l-100 ⁇ M.
  • the invention further provides a method of treatment of a condition characterised by neuronal deficit or neuronal death, comprising the step of administering an effective amount of a compound of the invention to a subject in need of such treatment.
  • the method of the invention is suitable for treatment of conditions including but not limited to neurodegenerative diseases such as motor neurone disease (amyotrophic lateral sclerosis), progressive spinal muscular atrophy, infantile muscular atrophy, Charcot-Marie-Tooth disease, Parkinson's Disease, Parkinson-Plus syndrome, Guamanian Parkinsonian dementia complex, progressive bulbar atrophy, Alzheimer's disease and the like, other neurodegenerative conditions such as those arising from ischaemia, hypoxia, neural injury, surgery, exposure to neurotoxins such as N-methyl-4-phenyl- 1, 2, 3, 6-tetrahydropyridine) , and peripheral sensory neuropathies, including those resulting from exposure to drugs (such as cis-platin) and toxins and resulting from diabetes, for example mononeuropathy multiplex.
  • neurodegenerative diseases such as motor neurone disease (amyotrophic lateral sclerosis), progressive spinal muscular atrophy, infantile muscular atrophy, Charcot-Marie-Tooth disease, Parkinson's Disease, Parkinson-Plus syndrome, Guamanian Parkinsonian dementia complex
  • the dose required will depend on the nature and severity of the condition to be treated, and the route of administration, and will be at the discretion of the attending physician or surgeon.
  • a suitable route, frequency of administration, and dosage can readily be established using conventional clinical trial methodology.
  • amino acid sequence variants of said sequence are encompassed.
  • one or more of the amino acids Glu 40 -Lys 41 -Val 42 -Pro 43 -Val 44 - Ser 45 -Lys 46 -Gly 47 -Gln 48 -Leu 49 -Lys 50 -Gln 51 may be deleted, and optionally substituted by one or more amino acid residues; or wherein an amino acid residue has been covalently modified so that the resulting product is a non-naturally occurring amino acid.
  • Amino acid sequence variants may be made synthetically, for example, by peptide synthesis, or may exist naturally.
  • an amino acid sequence variant of the monomeric loop 2 or loop 4 sequence of BDNF is included within the scope of the invention provided that it is functionally active.
  • “functionally active” and “functional activity” in reference to the monomeric loop 2 or loop 4 sequence of BDNF means that the compound generated therefrom is able to promote or enhance the growth, survival, function, and/or differentiation of neurons and glia, especially axon fasciculation and process outgrowth, whether the neurons be central, peripheral, motorneurons, or sensory neurons, e.g. photoreceptors, vestibular ganglia, spinal ganglia and auditory hair cells.
  • monomeric loop 2 or loop 4 amino acid sequence variants generally will share at least about 75% (preferably greater than 80% and more preferably greater than 90%) sequence identity with the amino acid sequence Glu 0 -Lys 1 -Val 42 -Pro 43 -Val 4 -Ser 45 -Lys 46 -Gly 47 -Gln 48 -Leu 49 -Lys 50 - Gln 51 , after aligning the sequences to provide for maximum homology, as determined, for example, by the Fitch, et al . , Proc. Nat . Acad. Sci . USA 80:1382-1386 (1983), version of the algorithm described by Needleman, et al . , J. Mol . Biol . 48:443-453 (1970).
  • Amino acid sequence variants of the monomeric loop 2 or loop 4 sequence of BDNF are prepared by introducing appropriate amino changes into amino acid sequence, or by in vitro synthesis.
  • Such variants include, for example, deletions from, or insertions or substitutions of, amino acid residues within Glu 40 -Lys 41 -Val 42 -Pro 3 -Val 44 - Ser 45 -Lys 46 -Gly 7 -Gln 8 -Leu 49 -Lys 50 -Gln 51 . Any combination of deletion, insertion, and substitution may be made to arrive at an amino acid sequence variant of the monomeric loop 2 or loop 4 sequence of BDNF, provided that such variant possesses the desired characteristics described herein.
  • functionally active amino acid sequence variants of the monomeric loop 2 or loop 4 sequence of BDNF may be selected, for example, by substituting one or more amino acid residues in the amino acid sequence Glu 0 -Lys 41 -Val 42 -Pro 43 -Val 4 -Ser 45 -Lys 46 -Gly 47 - Gln 48 -Leu 9 -Lys 50 -Gln 51 with other amino acid residues of a similar or different polarity or charge.
  • an amino acid residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and, by means of recombinant DNA technology, replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • a neutral or negatively charged amino acid most preferably alanine or polyalanine
  • Amino acid sequence deletions generally range from about 1 to 6 residues, more preferably about 1 to 3 residues, and typically are contiguous. Generally, the number of consecutive deletions will be selected so as to preserve the tertiary structure of the monomeric loop 2 or loop 4 sequence of BDNF.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions, or intrasequence insertions (i.e., insertions made within the amino acid sequence Glu 40 -Lys 41 -Val 4 -Pro 43 -Val 4 -Ser 45 -Lys 46 -Gly 7 -Gln 48 - Leu 9 -Lys 50, -Gln 51 ) may range generally from about 1 to 10 residues, more preferably 1 to 5, most preferably 1 to 3.
  • the third group of variants are those in which at least one amino acid residue in the amino acid sequence Glu 40 -Lys 41 -Val 42 -Pro 43 -Val 44 -Ser 5 -Lys 4s -Gly 7 -Gln 8 -Leu 49 -Lys 50 - Gln 51 , preferably one to four, more preferably one to three, even more preferably one to two, and most preferably only one, has been removed and a different residue inserted in its place.
  • the sites of greatest interest for making such substitutions are those sites most likely to be important to the functional activity of the monomeric loop 2 or loop 4 sequence of BDNF.
  • Val . [V) ile; leu; met; phe; ala; norleucine leu changes, denominated exemplary substitutions in Table A, or as further described below in reference to amino acid classes, may be introduced and the resulting variant the monomeric loop 2 or loop 4 sequence of BDNF analyzed for functional activity.
  • Insertional, deletional, and substitutional changes in the amino acid sequence Glu 40 -Lys 41 -Val 42 -Pro 43 - Val 4 -Ser 5 -Lys 46 -Gly 7 -Gln 48 -Leu 49 -Lys 50 -Gln 51 may be made to improve the stability of the monomeric loop 2 or loop 4 sequence of BDNF.
  • Covalent modifications of the monomeric loop 2 or loop 4 sequence of BDNF are also included within the scope of this invention.
  • covalent modifications are introduced into the monomeric loop 2 or loop 4 sequence of BDNF by reacting targeted amino acid residues of the monomeric loop 2 or loop 4 sequence of BDNF with an organic derivatizing agent that is capable of reacting with selected amino acid side chains or the N- or C-terminal residues .
  • Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ - bromo- ⁇ - (5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2- chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa- 1, 3-diazole.
  • Histidyl residues are derivatized by reaction with diethyl-pyro-carbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Suitable reagents for derivatizing ⁇ -amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4- pentanedione; and transaminase-catalyzed reaction with glyoxylate .
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2, 3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatisation of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • aromatic diazonium compounds or tetranitromethane Most commonly, N-acetylimidizole and tetranitromethane are used to form O- acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • R and R' are different alkyl groups, such as l-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or l-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide.
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions .
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • substantially homologous means that an amino acid sequence is quite similar to that of the monomeric loop 2 or loop 4 sequence of BDNF, and have at least about 85% (preferably at least about 90%, and most preferably at least about 95%) of the amino acids matching with at least 7 of the amino acids found in the sequence Glu 40 -Lys 1 -Val 42 -Pro 3 -Val 44 -Ser 45 -Lys 46 -Gly 47 -Gln 8 -Leu 49 -Lys 50 - Gin 51 .
  • more resistant to metabolic degradation means that the compound of the invention has been modified such that the resulting compound is more stable under acidic conditions than the "native" sequence of the monocyclic loop 2 or loop 4 sequence of BDNF.
  • amino acid substitutions as discussed previously may be undertaken which produce compounds more resistant to metabolic degradation. It is well known in the art that D- amino acids, and amino acids analogues are more resistant to acidic environmen . Conjugates of small peptides and cholic acid have reduced metabolic degradation problems.
  • biological activity with reference to BDNF means a biological activity which is normally promoted, either directly or indirectly, by the presence of BDNF, and includes, but is not limited to, BDNF binding to the p75 receptor or the trkB receptor, neuron survival, neuron differentiation, including neuron process formation and neurite outgrowth, and biochemical effects such as induction of enzymes which are stimulated by BDNF.
  • biological activities can be measured by conventional in vitro and in vivo assays, such as the chick dorsal root ganglion assay described herein by Barde et al (1980) and the neurite outgrowth, and in vivo kindling assays described in W097/15593 and by Riopelle et al (1982).
  • the compounds of the invention may be synthesised by any suitable method.
  • Solid phase methods such as those developed for synthesis of peptides and peptidomimetic compounds are preferred, including but not limited to the Fmoc solid phase peptide synthesis method described herein, the Boc solid phase peptide synthesis method, and PIN synthesis methods (for review, see Maeji et al . , 1995).
  • Boc solid phase peptide synthesis method for review, see Maeji et al . , 1995.
  • PIN synthesis methods for review, see Maeji et al . , 1995.
  • Figure 1 shows the backbone trace of the three- dimensional structure of BDNF dimer (one monomer in black, the other grey), showing the positions of the loop 2 (trkB binding) and loop 4 (predominantly p75 binding) regions. Side chains of the p75 binding tripeptide in loop 4 (Lys- Lys-Arg) are shown.
  • Figure 2 illustrates the molecular modelling of monomeric cyclic loop 2 analogues.
  • An ⁇ -carbon to ⁇ - carbon trace of the native loop 2 of BDNF is shown, superimposed with low-energy conformations of loop 2 analogues L2-12, L2-10, L2-8 and L2-6, each of which is constrained by a disulphide bridge (indicated by arrows) .
  • Figure 3 shows the concentration-response curves of monomeric cyclic loop 2 analogues in competition with BDNF.
  • the monomeric cyclic loop 2 analogues L2-12 (closed triangles), L2-12a (open triangles), L2-10 (open squares), L2-8 (closed diamonds) and L2-6 (open diamonds) and the monomeric linear peptide L2-12b (closed squares) were assayed in competition with BDNF (4 x 10 ⁇ xx M) in cultures of E8 to E10 chick sensory neurons.
  • Figure 4 shows the concentration-response curves of monomeric cyclic peptide L2-12, alone and in competition with NGF.
  • Surviving neurons were counted after 48 hrs in culture, and these counts expressed as a percentage of originally-plated viable neurons and normalised such that survival in cultures containing NGF alone (P; positive control; closed circle) was set to 100% and survival in cultures with neither NGF nor L2-12 (N; negative control; open circle) to 0%.
  • P positive control
  • N negative control
  • L2-12 closed triangles
  • L2-12 closed triangles
  • L2-12 closed triangles
  • Data are expressed as the mean ⁇ SEM.
  • Figure 5 shows the effect of a monomeric cyclic loop 2 analogue (L2-12a) on the concentration-response curve of BDNF.
  • L2-12a (1 x 10 "7 M) was assayed in competition with BDNF (1.8 x 10 "13 to 1.8 x 10 "10 M, 0.51 og increments) in cultures of E8-E10 sensory neurons.
  • Figure 6 illustrate the maximal inhibition of BDNF-mediated survival of cultured sensory neurons by monomeric cyclic loop 2 analogues systematically substituted with alanine.
  • Loop 2 analogues were assayed in competition with BDNF (4 x 10 "11 M) in cultures of E8-E10 sensory neurons.
  • Surviving neurons were counted after 48 hrs in culture, and these counts were expressed as a percentage of originally-plated viable neurons then normalised such that survival of cultures containing BDNF was set as 100%, while that for cultures with neither BDNF nor loop 2 analogue was set to 0%.
  • Maximal inhibition of BDNF-mediated survival was calculated by subtracting the lowest value for BDNF-mediated survival from that of BDNF alone (100%) .
  • Figure 7A shows a schematic view of the two loop two regions in the model of the three-dimensional structure of the BDNF dimer, showing the interatomic distances (A) between ⁇ -carbon atoms of selected residues.
  • Figure 7B shows a schematic view of the disulphide bridge of the cysteine residue, showing the average interatomic distance and 90% confidence interval (90% Cl) of ⁇ -carbon atoms, determined by conformational analysis.
  • Figure 8 shows a graph of the survival of sensory neurons in the presence of the bicyclic dimeric peptides (L2-8P2C) 2 , (L2-8V3C) 2 and (L2-8S4C)2.
  • Neurons were prepared from chick dorsal root ganglia from embryonic chicks (E8-E10), and surviving neurons counted after 48 hrs in culture. Data are presented as a percentage of the number of cells supported by BDNF (lng/ml; 100%) after the same period in culture. Survival in negative control cultures was set to 0%. Highly significant differences in neuronal survival in the presence of (L2-8P2C) 2 , and (L2- 8S4C) 2 were observed compared to survival in negative controls (ANOVA, *** p ⁇ 0.001, Bonferroni multiple comparisons test) .
  • Figure 9 shows a graph of the survival of sensory neurons in the presence of the monomeric cyclic peptides
  • L2-8P2C(Acm) and L2-8S4C (Acm) Neurons were prepared from dorsal root ganglia from embryonic chicks (E8-E10), and surviving neurons counted after 48 hours in culture. Data are presented as a percentage of the number of cells supported by BDNF (lng/ml; 100%) after the same period in culture. Survival in negative control cultures was set to 0%. Data were obtained from at least two independent experiments.
  • Figure 10 shows a graph of the survival of sensory neurons in the presence of the amide-linked dimeric bicyclic peptide (L2-8&E+K) 2 .
  • Neurons were prepared from chick dorsal root ganglia obtained from embryonic chicks (E8-E10), and surviving neurons counted after 48 hours in culture. Data are expressed as a percentage of the number of cells supported by BDNF (lng/ml; 100%) after the same period in culture. Survival in negative control cultures was set to 0%. A highly significant difference in neuronal survival in the presence of (L2-8&E+K) 2 was observed compared to survival in negative controls (ANOVA, p ⁇ 0.001, Bonferroni multiple comparisons test).
  • Figure 11 shows a graph of the survival of sensory neurons in the presence of the dimeric tricyclic loop 2 analogue (L2-8S4C&E+K) 2 .
  • Neurons were prepared from chick dorsal root ganglia obtained from embryonic chicks (E8-E10), and surviving neurons counted after 48 hrs in culture. Data are expressed as a percentage of the number of cells supported by BDNF (lng/ml; 100%) after the same period in culture. Survival in negative control cultures was set to 0%. A highly significant difference in neuronal survival in the presence of (L2-8S4C&E+K) 2 was observed compared to survival in negative controls (ANOVA, p ⁇ 0.001, Bonferroni multiple comparisons test) .
  • Figure 12 shows a graph of the survival of sensory neurons in the presence of the monomeric cyclic loop 4-derived L4-3pA(II) (closed circles) .
  • Neurons were prepared from dorsal root ganglia from embryonic chicks (E8-E10) and surviving neurons counted after 48 hrs in culture.
  • B positive control (BDNF 1 ng/ml) ;
  • N negative control (no peptide) .
  • Figure 14 shows a graph of the effect of the monomeric cyclic loop 4-derived peptides L4-3pA(I) (open diamonds), L4-3pA(II) (open squares) and L4-3Hx (open triangles), and their linear homologues L4-3pAa (crosses) and L4-3Hxa (asterisks) on the neuronal survival effect mediated by BDNF (1 ng/ml) .
  • BDNF (1 ng/ml
  • Figure 15 shows a graph of the effect of the monomeric cyclic loop 4-derived peptides L4-3pA(I) (open diamonds), L4-3pA(II) (open squares) and L4-3Hx (open triangles), and their linear homologues L4-3pAa (crosses) and L4-3Hxa (asterisks) on the neuronal survival effect mediated by NGF (1 ng/ml) .
  • NGF (1 ng/ml
  • Figure 16 shows a graph of the survival of sensory neurons in the presence of the monomeric cyclic loop 4 peptides, L4-3Ap(I), L4-3Ap(II), L4-3AP(I) and L4- 3AP(II). All peptides were added at a concentration of 10 " 6 M. BDNF was added at a concentration of 1 ng/ml. Neg shows the survival of control cultures containing neither BDNF nor peptide. None of the peptides exhibited an effect on neuronal survival that was significantly different to that seen in negative control cultures.
  • Figure 17 shows a graph of the survival of sensory neurons in the presence of the monomeric cyclic loop 4 peptides L4-3K3ApA and L4-3K4ApA.
  • the peptides were added at a concentration of 10 "6 M.
  • BDNF was added at a concentration of 1 ng/ml.
  • Neg shows the survival of control cultures containing neither BDNF nor peptide. Neither of the peptides exhibited an effect on neuronal survival that was significantly different to that seen in negative control cultures.
  • Figure 18 shows the solution structure of peptide L4-3pA(II) derived by nuclear magnetic resonance (NMR) spectroscopy. Depicted is an overlay of the twenty conformations of peptide L4-3pA(II) with the lowest target function in the software package DYANA, following 10,000 steps of simulated annealing followed by 2,000 steps on minimisation using the NMR-derived distance and dihedral angle constraints. Residues are labelled and numbered.
  • Figure 19 shows the effects of peptide L4-3pA(II) of the invention on neuronal loss following peripheral nerve lesion. This was accomplished by comparing the number of sensory neurones in the C8 dorsal root ganglia and motor neurons in the central region of the spinal cord in the lesioned side versus that in the intact contralateral side five days following nerve lesion, and administration of the peptide to the distal nerve stump.
  • Mouse recombinant BDNF was a kind gift from Dr R Kolbeck and Professor Y-A Barde (Max-Planck-Institute for Psychiatry, Martinsried, Federal Republic of Germany) .
  • NGF, purified from male mouse submaxillary gland was purchased from Boehringer-Mannheim (Mannheim, Federal Republic of Germany) .
  • Fertilised chicken eggs were obtained from Research Poultry Farms (Research, Victoria, Australia) , trypsin from Worthington (Freehold, NJ, U.S.A.), L-15 from GIBCO BRL (Grand Island, NY, U.S.A.), horse serum from CSL (Parkville, Victoria, Australia), Nunclon 10 cm tissue culture dishes from Nalge Nunc International (Roskilde, Denmark), Falcon Multiwell 48-well tissue culture plates from Becton Dickinson (Franklin Lakes, NJ, U.S.A.) and mouse laminin, isolated from Englebreth-Holm-Swarm tumour cells, from Collaborative Biomedical Products (Bedford, MA, U.S.A.).
  • Fmoc-amino acids and Wang resin were purchased from Auspep (Parkville, Victoria, Australia) , PR-500 resin from Calbiochem- NovaBiochem (Alexandria, New South Wales, Australia) and Econosil irregular packed HPLC columns from Alltech
  • a model of the three-dimensional structure of murine BDNF was obtained by protein homology modelling techniques from murine NGF. This was performed by the Swiss-Model automated protein homology server running at the Glaxo Institute for Molecular Biology in Geneva, Switzerland, accessed via the Internet (http://expasy.hcuge.ch/swissmod/SWISS-MODEL.html, Peitsch, 1995) . Briefly, a three-dimensional model of the target sequence is produced in the following manner: Swiss-Model searches the Brookhaven Protein Data Bank for the sequences of homologous proteins of known three-dimensional structure. Once a template sequence is found, Swiss-Model produces a structural framework for the target sequence, using a combination of sequence alignment tools and three- dimensional superimposition.
  • Homologous regions of the template protein form the structural backbone of the target sequence, while non-conserved regions are built using the three-dimensional structures of related sequences in the Brookhaven Protein Data Bank.
  • Side chains not present on the template protein are inserted, and all side chains are corrected using a library of allowed rotamers.
  • the model is then optimised by energy minimisation using the CHARM force-field.
  • the co-ordinates of the BDNF monomer were downloaded and the BDNF dimer constructed by superimposing two monomers over the co-ordinates of selected conserved ⁇ - carbon atoms in the NGF dimer, using the PC-based molecular modelling software Hyperchem version 4.0 (Hypercube, Ontario, Canada) .
  • Cyclic Loop 2 Analogues The molecular modelling of peptide analogues was carried out using Hyperchem, as follows: After visual inspection of the model obtained by homology modelling, loop 2 was defined and excised from the three-dimensional structure of BDNF, and various means of constraining peptides to the native loop 2 conformation were investigated. Each constraint was built between the terminal residues of the peptide, and these residues geometrically optimised using the Polak-Ribiere algorithm and MM+ force-field to a local low-energy conformation. These modelled peptides were assessed for their ability to mimic the native conformation by measuring the root mean square deviation of the peptide backbone to that of the native loop following least squares superimposition.
  • the second ⁇ -hairpin loop (loop 2) was defined as Glu 40 -Lys 41 -Val 4 -Pro 43 -Val 4 -Ser 45 - Lys 46 -Gly 47 -Gln 48 -Leu 49 -Lys 50 -Gln 51 , where the amino acid numbering is the same as in mature BDNF.
  • Peptide analogues of this loop were modelled to investigate
  • the peptide analogues were synthesised with free amino and carboxyl termini, except L2-12a, which had acetylated amino and amidated carboxyl termini .
  • the linear peptide L2-12b was synthesised without terminal Cys residues, to ensure that it remained in a linear form during biological assays.
  • the peptides L2-12E1 ⁇ A to L2- 12Q12 ⁇ A contain alanine substitution at the indicated positions in the L2-12 sequence.
  • the peptide analogues synthesised are listed in Table 1.
  • Amino acids are represented by their one letter code, reading left to right from amino to carboxyl termini.
  • the analogue code for example L2-12K9 ⁇ A, refers to 12 residues from the native loop 2 sequence of BDNF with lysine at position 9 substituted with alanine. Cysteine residues not found in the native BDNF sequence were incorporated to form disulphide bridges, which are represented by lines between side chains. "peptide L2-12P4 ⁇ A not synthesised. Example 4 Inhibition of BDNF-Mediated Sensory Neuron
  • dorsal root ganglia were dissected from four embryonic day 8-10 chicks (E8-E10), treated with 0.1% trypsin for 20 min at 37°C, washed twice with 2 ml medium (L-15 (C0 2 ), 5% horse serum, 60 ⁇ g/ml streptomycin and 100 ⁇ g/ml penicillin) and gently triturated.
  • Non-neuronal cells were removed by pre-plating the neuronal suspension on a 10 cm tissue culture dish for 3 h at 37°C, 5% C0 2 .
  • phase-bright healthy neurons with neurites at least twice the length of the cell soma were counted in 20-30 fields at 200x magnification, and counts expressed as a percentage of the original number of viable neurons plated (% neuronal survival) .
  • Percentage neuronal survival data was normalised, such that neuronal survival in the presence of neurotrophin (4 x 10 "11 M; positive control) was set to 100%, while survival in the absence of both neurotrophin and monomeric cyclic loop 2 peptide analogue (negative control) was set to 0%. Values were expressed as mean ⁇ SEM. Data from different experiments were analysed for lack of significant variation using a parametric one-way analysis of variance (ANOVA) before being grouped. Statistics were performed using Instat version 2.04a (GraphPad, San Diego, CA, U.S.A.). Prism software (GraphPad, San Diego, CA, U.S.A.) was used to fit sigmoidal curves to the data.
  • Maximal inhibition refers to the greatest % reduction in normalised % BDNF-mediated neuronal survival.
  • pIC 50 values were calculated from logistic sigmoidal curves fitted to the data given in Figure 3.
  • the monomeric linear peptide L2- 12b did not show significant inhibition of BDNF-mediated survival over the concentration range tested (1 x 10 "11 to 1 x 10 "4 M; Figure 3) .
  • the peptide also caused a small rightward shift of the BDNF concentration-response curve (pECso of BDNF alone: 11.2 ⁇ 0.2; BDNF + L2-12a: 10.9 ⁇ 0.3), although this was not statistically significant.
  • Example 8 Identification of Amino Acids Important for the Inhibitory Effect of Monomeric Cyclic Loop 2 Peptides The contribution of individual residues within the monomeric cyclic L2-12 sequence towards BDNF-inhibitory activity was examined by conducting an alanine scan (Ala scan) , and testing the resulting monomeric cyclic peptides from 1 x 10 '11 to 1 x 10 "4 M in log increments for their ability to modulate BDNF-mediated survival at 4 x 10 "11 M. The sequences of the alanine substituted peptides are shown in Table 1 above, and the results are shown in Figure 6.
  • Dimeric Bicyclic Loop 2 Analogues The two loop 2 regions of BDNF are juxtaposed in the three-dimensional model of the dimer ( Figure 1) , which allows design of small dimeric peptides that mimic this spatial arrangement.
  • Figure 1 On the basis of observations made in the highly analogous NGF-trkA receptor system, we predicted that small dimeric loop 2 analogues could act as agonists if they could facilitate dimerization of trkB. It has been shown that divalent antibodies to trkA can cause the homodimerisation of this receptor, leading to signal transduction and NGF-like biological activity in vitro (Clary et al, 1994) .
  • a small peptide mimetic of erythropoietin produced by a recombinant library technique, possesses full erythropoietin-like biological activity as a result of self-association to form a dimer which dimerises the erythropoietin receptor (Wrighton et al, 1996) .
  • Examination of the X-ray crystal structure of the peptide-erythropoietin receptor complex (Livnah et al, 1996) reveals that the structure of the bound dimeric peptide bears a striking resemblance to the loop 2 regions of BDNF in our three-dimensional model.
  • peptide L2-8 The most effective of the monomeric, cyclic, disulphide-linked loop 2 peptides which were shown in Examples 4, 5, 7 and 8 were able to inhibit BDNF neuronal survival activity, peptide L2-8, was chosen as the basis for the design of dimeric peptides.
  • This peptide consists of 8 amino acid residues of BDNF plus the two terminal cysteine residues oxidised to cyclic disulphide, i.e. a total of 10 residues.
  • Examination of the model of the three-dimensional structure of BDNF revealed three amino acid positions in which the two loop 2 regions in BDNF are in close proximity, thus presenting an opportunity to create dimeric analogues of peptide L2-8.
  • Disulphide-Linked Dimeric Bicyclic Loop 2 Analogues The disulphide-linked dimeric bicyclic loop 2 peptides were analysed for their ability to promote the survival of sensory neurons in cultures prepared from dorsal root ganglia obtained from embryonic day 8-10 chicks, as described in Example 4.
  • Peptides (L2-8P2C) 2 and (L2-8S4C) 2 each displayed concentration-dependent neuronal survival activity, maximally promoting the survival of 28% and 30% of the neurons that would be supported by BDNF itself . These results are shown in Figure 8.
  • peptide (L2-8V3C) 2 was inactive, as shown in Figure 8, despite the likelihood that it could best accommodate the cysteine residue, at least in terms of interatomic distance.
  • the amide-linked dimeric bicyclic loop 2 peptide (L2-8&E+K) 2 was assayed in cultures of sensory neurons prepared from dorsal root ganglia obtained from embryonic chicks, as described in Example 4. Peptide displayed concentration dependent neuronal survival activity, supporting the survival of 28% of those neurons supported by BDNF (lng/ml) with an EC 50 in the order of 10 "8 M. The results are shown in Figure 10.
  • the dimeric tricyclic peptide (L2-8S4C&E+K) 2 was prepared as shown in Scheme 3 from two cyclic N-acetylated, C-amidated, partially-protected monomers synthesised by standard solid phase techniques on Rink amide MBHA resin as described in Example 14.
  • Example 18 Intrinsic Neuronal Survival Activity of a Dimeric Tricyclic Loop 2 Analogue
  • the dimeric tricyclic loop 2 peptide (L2- 8S4C&E+K) 2 was assayed in cultures of sensory neurons prepared from dorsal root ganglia obtained from embryonic chicks, as described in Example 4.
  • Peptide displayed concentration dependent neuronal survival activity, supporting the survival of 35% of those neurons supported by BDNF (lng/ml) with an EC 50 in the order of 10 "10 M. The results are shown in Figure 11.
  • the maximal neuronal survival promoting effect of the dimeric tricyclic loop 2 peptide (L2-8S4C&E+K) 2 is similar to that of the dimeric bicyclic loop 2 analogues.
  • peptide (L2-8S4C&E+K) 2 is approximately two orders of magnitude more potent than the dimeric bicyclic analogues. This activity is consistent with the hypothesis that the presence of two dimerising constraints (Cys-to-Cys disulphide and Lys-to-Glu amide) would create a molecule which much better mimics the spatial arrangement of the two loop 2 moieties than any of the dimeric bicyclic compounds, which contain only a single dimerising constraint.
  • Loop 4 of BDNF The three positively-charged residues thought to be important for the binding of BDNF to the low affinity neurotrophin receptor p75 are contiguous (Lys 95 -Lys 96 -Arg 97 ) and are located on loop 4, as shown in Figure 1. This gave us the opportunity to propose small monomeric cyclic peptides that might mimic the conformation of this tripeptide sequence, using the computer-aided molecular design approach described in Example 2.
  • L4-3pA a pentapeptide incorporating a relatively conformationally restricted DPro residue
  • L4-3Hx a tetrapeptide incorporating a conformationally flexible 6- aminohexanoyl residue. Both peptides were cyclised by condensing their amino-terminus with their carboxy-terminus (head-to-tail cyclisation) .
  • Example 20 Synthesis of Monomeric Cyclic Analogues of the p75 Binding Region of Loop 4 of BDNF
  • the monomeric cyclic loop 4 peptides were synthesised from 9-fluorenylmethoxycarbonyl (Fmoc) amino acids, using standard solid phase synthesis protocols as described in Example 3.
  • linear side chain-protected peptides L4-3pAa and L4-3Hxa suitable for head-to-tail cyclisation to give the monomeric cyclic peptides L4-3pA and L4-3Hx, respectively, were obtained by treating peptides synthesised on acid-labile 2-chlorotrityl derivatised resin (NovaBiochem, Australia) with acetic acid/trifluoroethanol/dichloromethane (1:1:8) for 30 minutes (Barlos et al . , 1991).
  • the cyclic peptides were obtained by stirring the appropriate linear side chain- protected peptide (0.1 to 0.5 mg/ml) in dichloromethane in the presence of the standard peptide bond-formation reagents 2- (lH-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyl uronium hexafluorphosphate (HBTU) , 1-hydroxybenzotriazole (HOBt) and diisopropylamine (DIEA) (HBTU:HOBt :DIEA 1:1:1.5 equivalents relative to peptide) .
  • Treatment of the product of this reaction with TFA/scavengers yielded the desired fully-deprotected product.
  • the corresponding linear homologues were prepared by treating the appropriate side chain-protected linear peptide with TFA/scavengers, without prior cyclisation.
  • Example 21 Intrinsic Neuronal Survival Activity of a Monomeric Cyclic Analogue of the p75 Binding Region of Loop 4 of BDNF
  • the monomeric cyclic loop 4 peptide, L4-3pA(II) displayed concentration-dependent neuronal survival activity.
  • the monomeric cyclic peptides L4-3K3ApA and L4- 3K4ApA were assayed in cultures of sensory neurons prepared from embryonic chicks as described in Example 4. Compared to L4-3pA(II), peptides L4-3K3ApA and L4-3K4ApA displayed only marginal neuronal survival activity. These data, shown in Figure 17, suggest that the two Lys residues of the cyclic monomeric peptide L4-3pA are required for neuronal survival activity.
  • Analogue of the p75 Binding Region of Loop 4 of BDNF The neuronal survival activity of the monomeric cyclic analogues of the p75 binding region of loop 4 of
  • BDNF is confined almost exclusively to peptide L4-3pA(II) .
  • a HPLC pure sample of peptide L4-3pA(II) was lyophilised then taken up in 550 ml of 10% 2 H 2 0/90%H 2 0 and the pH adjusted to 5.3. The solution was then transferred into a 5 m NMR tube.
  • NMR spectra were acquired at 400 MHz on a Varian Inova 400 MHz NMR spectrometer.
  • One-dimensional X H spectra were acquired with a sweep-width of 4000 Hz over 8K points.
  • Solvent suppression was achieved with selective low-power presaturation. Spectra were acquired at a series of sample temperatures (30°C, 15°C and 5°C) to check for temperature dependence of the peptide spectrum. The peptide did not show significant temperature dependence.
  • the conformation of the backbone of peptide L4-3pA(II) is uniquely defined in solution.
  • side chain of Lys 4 adopts a single conformation up to its gamma-carbon atom, while the conformation of the side chain of Arg 5 is uniquely defined to the delta-nitrogen.
  • the presence of a single backbone conformation and well-defined side chains for peptide L4- 3pA is consistent with the biological data showing that compounds of closely related sequence to L4-3pA show either markedly reduced, or no neuronal survival activity in cell culture experiments. This exceptionally well-defined conformation of L4-3pA will be used as a template for the design of non-peptidic molecules with neuronal survival promoting activity.
  • peptide L4-3pA(II) The ability of peptide L4-3pA(II) to prevent or slow neurodegeneration in vivo was tested in a model of peripheral nerve lesion. To do this, newborn (24-48 hrs) Wistar rat pups (4 per treatment group) were rendered unconscious by ice-induced hypothermia. The median and ulnar nerve in the right forelimb was exposed, transected and wrapped with a piece of gel foam containing 10 ⁇ l a solution in PBS of L4-3pA(II) at one of two doses (10 ⁇ g/ ⁇ l or 1 ⁇ g/ ⁇ l) or PBS alone.
  • Pups were re-united with their mothers and after 5 days were killed with a lethal injection of sodium pentobarbital (150 mg/kg) and perfused with a buffered 4% solution of parafor aldehyde.
  • Spinal cords and DRGs were dissected out and embedded in paraffin, and serial transverse sections were cut, mounted on glass slides and stained with 0.5% cresyl violet.
  • Neurons displaying prominent nucleoli were counted in every fifth section to include the entire rostrocaudal length of the DRG. Effects of peptides on neuronal loss were determined by comparing the number of neurons in the experimental side versus that in the intact contralateral side. Statistical comparisons between treatments was determined by one way ANOVA followed by post hoc Tukey's test.
  • both doses of peptide L4-3pA(II) significantly reduce the loss of of sensory (panel A: lOO ⁇ g, 31 ⁇ 5% loss; lO ⁇ g, 23 ⁇ 3% loss) and motor (panel B: lOO ⁇ g, 16 ⁇ 2% loss; lO ⁇ g, 11 ⁇ 6% loss) neurons that would otherwise die (panel A: sensory neurons 45 ⁇ 2% loss; panel B: motor neurons 35 ⁇ 2% loss) as a result of the lesion.
  • the degree of rescue is similar to that seen with other neurotrophic factors, such as LIF

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Abstract

La présente invention concerne des procédés et des compositions permettant de favoriser la croissance de cellules nerveuses. Cette invention concerne notamment des agonistes du facteur neurotrophique dérivé du cerveau. De façon plus spécifique, cette invention concerne des composés cycliques qui comprennent un ou plusieurs groupes fonctionnels cycliques et présentent une activité biologique de facteur neurotrophique dérivé du cerveau (BDNF).
PCT/AU2000/000641 1999-06-08 2000-06-07 Petits analogues cycliques du facteur neurotrophique derive du cerveau (bdnf) WO2000075176A1 (fr)

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EP00930886A EP1212353A4 (fr) 1999-06-08 2000-06-07 Petits analogues cycliques du facteur neurotrophique derive du cerveau (bdnf)
AU49016/00A AU780408B2 (en) 1999-06-08 2000-06-07 Small cyclic mimics of brain-derived neurotrophic factor (BDNF)
JP2001502457A JP2003502295A (ja) 1999-06-08 2000-06-07 脳由来神経栄養因子の小さな環状模擬体
CA002376729A CA2376729A1 (fr) 1999-06-08 2000-06-07 Petits analogues cycliques du facteur neurotrophique derive du cerveau (bdnf)

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EP1248642A1 (fr) * 2000-01-18 2002-10-16 Ludwig Institute For Cancer Research Inhibiteur peptidomimetique de vegf-d/vegf-c/vegf
GB2387386A (en) * 2002-04-03 2003-10-15 Purzer Pharmaceutical Co Ltd Cyclic Peptides and their Preparation
WO2010093284A1 (fr) 2009-02-16 2010-08-19 Учреждение Российской Академии Медицинских Наук Научно-Исследовательский Институт Фармакологии Имени В.В.Закусова Рамн Mimétiques dipeptidiques de neurotrophines ngf et bdnf
RU2477144C2 (ru) * 2010-03-22 2013-03-10 Федеральное государственное бюджетное учреждение "Научно-исследовательский институт фармакологии имени В.В. Закусова" Российской академии медицинских наук Лекарственное средство с антиаритмическим и антифибрилляторным действием
US8557960B2 (en) 2009-11-06 2013-10-15 Sungkyunkwan University Peptide for augmenting and expression of BDNF and pharmaceutical composition for prevention and treatment of neurodegenerative diseases including Alzheimer's disease or Parkinson's disease, comprising the same
RU2613314C2 (ru) * 2013-06-28 2017-03-15 Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт фармакологии имени В.В. Закусова" Малые молекулы с NGF-подобной активностью, обладающие антидиабетическими свойствами
WO2020150275A1 (fr) * 2019-01-14 2020-07-23 New York University Dimères peptidiques cycliques
RU2800369C1 (ru) * 2022-04-05 2023-07-20 Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт фармакологии имени В.В. Закусова" Димерные дипептидные миметики нейротрофина-3

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US20230312651A1 (en) * 2020-07-10 2023-10-05 The University Of Tokyo Cyclic Peptide, Peptide Complex, and Drug Composition Containing Said Cyclic Peptide and/or Said Peptide Complex

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EP1248642A4 (fr) * 2000-01-18 2005-05-18 Ludwig Inst Cancer Res Inhibiteur peptidomimetique de vegf-d/vegf-c/vegf
US7045133B2 (en) 2000-01-18 2006-05-16 Ludwig Institute For Cancer Research VEGF-D/VEGF-C/VEGF peptidomimetic inhibitor
GB2387386A (en) * 2002-04-03 2003-10-15 Purzer Pharmaceutical Co Ltd Cyclic Peptides and their Preparation
FR2838443A1 (fr) * 2002-04-03 2003-10-17 Purzer Pharmaceutical Co Ltd Pentapeptides cycliques et leur preparation
GB2387386B (en) * 2002-04-03 2005-11-09 Purzer Pharmaceutical Co Ltd Cyclic pentapeptides useful as anti-thrombotic and vasodilator agents
US7053047B2 (en) * 2002-04-03 2006-05-30 Purzer Pharmaceutical Co., Ltd. Cyclic pentapeptides and their preparation
DE10315030B4 (de) * 2002-04-03 2009-12-17 Purzer Pharmaceutical Co., Ltd. Zyklische Peptide, Verfahren zu deren Herstellung und deren medizinische Verwendung
WO2010093284A1 (fr) 2009-02-16 2010-08-19 Учреждение Российской Академии Медицинских Наук Научно-Исследовательский Институт Фармакологии Имени В.В.Закусова Рамн Mimétiques dipeptidiques de neurotrophines ngf et bdnf
US9683014B2 (en) 2009-02-16 2017-06-20 Uchrezhdenie Rossiiskoi Akademii Meditsynskikh Nauk Nauchno-Issledovatelsky Institut Farmakologii Imeni V.V.Zakusova Ramn Dipeptide mimetics of NGF and BDNF neurotrophins
US8557960B2 (en) 2009-11-06 2013-10-15 Sungkyunkwan University Peptide for augmenting and expression of BDNF and pharmaceutical composition for prevention and treatment of neurodegenerative diseases including Alzheimer's disease or Parkinson's disease, comprising the same
RU2477144C2 (ru) * 2010-03-22 2013-03-10 Федеральное государственное бюджетное учреждение "Научно-исследовательский институт фармакологии имени В.В. Закусова" Российской академии медицинских наук Лекарственное средство с антиаритмическим и антифибрилляторным действием
RU2613314C2 (ru) * 2013-06-28 2017-03-15 Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт фармакологии имени В.В. Закусова" Малые молекулы с NGF-подобной активностью, обладающие антидиабетическими свойствами
WO2020150275A1 (fr) * 2019-01-14 2020-07-23 New York University Dimères peptidiques cycliques
US20210395311A1 (en) * 2019-01-14 2021-12-23 New York University Cyclic peptide dimers
RU2800369C1 (ru) * 2022-04-05 2023-07-20 Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт фармакологии имени В.В. Закусова" Димерные дипептидные миметики нейротрофина-3
RU2800369C9 (ru) * 2022-04-05 2023-10-04 Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт фармакологии имени В.В. Закусова" Димерные дипептидные миметики нейротрофина-3

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AUPQ084899A0 (en) 1999-07-01
EP1212353A1 (fr) 2002-06-12
CA2376729A1 (fr) 2000-12-14
EP1212353A4 (fr) 2004-10-20
JP2003502295A (ja) 2003-01-21

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