WO2000066150A1

WO2000066150A1 - New use of a substance in pns

Info

Publication number: WO2000066150A1
Application number: PCT/SE2000/000870
Authority: WO
Inventors: Thomas Lundeberg; Luigi Manni
Original assignee: Karolinska Innovations Ab; Thomas Lundeberg; Luigi Manni
Priority date: 1999-05-03
Filing date: 2000-05-03
Publication date: 2000-11-09
Also published as: SE9901578D0; AU4447400A; RU2001132588A; EP1173196A1; CN1353616A; JP2003509336A

Abstract

Peripheral neuropathies include disorders in structure and function of peripheral motor and sensory neurons, and can involve the entire neuron as well as part of it. Exogenous administration of CCK-8 and analogues thereof has been shown to induce the cellular production of neurotrophic factors, especially NGF, which can treat neuropathic states in the peripheral nervous system. Accordingly, the present invention relates to the use of a substance showing CCK-8 activity for the treatment of neuropathies in the peripheral nervous system, as well as a pharmaceutical composition comprising a substance showing CCK-8 activity.

Description

Karolinska Innovations AB

New use of a substance in PNS

The present invention relates to the use of a substance showing CCK-8 activity for the manufacture of a medicament in order to treat neuropathies in the peripheral nervous system. It also relates to a pharmaceutical composition comprising at least one substance showing CCK-8 activity.

Background of the invention

Peripheral neuropathies include disorders in structure and function of peripheral motor and sensory neurons, and can involve the entire neuron as well as part of it. Neuropathies in the peripheral nervous system can for example be induced by sur- gery, as a result of injuries, as a side effect after exposure to neurotoxic compounds or after chemical or irradiative cancer treatment. Also, diabetes mellitus patients are often distressed by peripheral neuropathies causing impairment of sensory neurons that can result in cutaneous infection and impaired wound healing.

Nerve growth factor (NGF) is the best characterized neurotrophic factor. It plays a major role in the growth and differentiation of peripheral sensory and sympathetic neurons through the modulation of neuropeptide expression. The exogenous administration of NGF has been shown to stimulate neuropeptide synthesis and, moreover, to recover neurochemical function after selective chemical impairment (Donnerer J. 1996, Neurosci Lett 221: 33-36; Donnerer J. et al., 1996, Brain Res 741: 103-108).

Thus. NGF can be used to treat peripheral neuropathy states.

However, there are some problems associated with systemic aclministration of NGF: The NGF-molecules show a short half-life when injected into blood circulation, and side effects arise when the necessarily high pharmaceutical doses are used. Also, a topical application may have a local effect, but it can normally not affect systemic disorders in the peripheral nervous system. Moreover, impurity is a problem with exogenous administration of NGF as it can result in allergic responses. A topical administration of NGF has also been shown to be toxic to the patient, and NGF has difficulties to penetrate into the peripheral nervous system. Topical aciininistration of NGF in humans has been mainly tested in clinical trial for diabetic neuropathy (Apfel SC, et al, 1998, Neurology 51: 695-702). The observed adverse events are injection site hyperalgesia, generalized myalgia and in some cases visceral pain. Topical application on mucus membrane covered tissue organs may also be painful.

To overcome these problems it would be very convenient to be able to induce the production of NGF by the administration of a substance that possesses a NGF- inducing activity. Some examples of such substances exist: (Riaz SS, et al., 1996, Prog. Neurobiol, 49: 125-143). Most of the effects of these substances as NGF in- ducers have been described using in vitro models, particularly glial cells. Although these cells are known to produce NGF, in periphery the NGF is produced mainly by tissue targets of innervation, thus the evidences in this review do not support the hypothesis that the cited substances can modulate the peripheral NGF expression. Moreover, it is not clear if the observed NGF increase in cell cultures is a specific and selective effect of the drug used or a generic, non-specific anabolic effect. The few studies describing the in vivo effects of some NGF inducers - catecholamines - are limited to a recovery of motor function in some models of peripheral neuropathy (Hanaoka Y, et al., 1992, Exp. Neurol., 115 (2):292-296; J. Neural Sci., 1994, 122(l):28-32; Saita K, et al., 1995, Neurotoxicology, 16 (3): 403-412.

EP-A2-0239716 demonstrates that the substance CCK-8 influences arterial pressure suggesting a CNS-mediated mechanism. However, this document does not show any possible involvement of growth factors, including NGF, for CCK-8.

Tirassa P. et al. (Br J Pharmacol, 1998, 123(6), 1230-1236) demonstrates that CCK- 8 induces an increase of NGF levels in brain. Part of this effect is mediated by sensory afferents. Recently, the inventors of the present invention discovered that the use of the neuropeptide CCK-8 (Asp-Tyr(S0₃H)-Met-Gly-Trp-Met-Asp-PheNH₂), (CCK-8 is the 26-33 octapeptide of the 33 amino acid long peptide cholecystokinin (CCK)) known to act as a neurotransmitter in the central nervous system, or a derivative of CCK-8 showing CCK-8 activity, can induce the production of nerve growth factor in the peripheral nervous system in a way that avoids the above mentioned problems. For example, the CCK-8 stimulation of NGF in peripheral organs and subsequent recovery of peripheral nerve injury do not require activation of sensory affer- ents (see example section). This suggests a different mechanism for CCK-8 induced NGF expression in CNS and PNS.

Summary of the invention

Accordingly, the present invention relates to the use of a substance showing CCK-8 activity for the manufacture of a medicament in order to treat neuropathies in the peripheral nervous system. In one embodiment the substance is CCK-8, in another embodiment the substance is a derivative of CCK-8 showing CCK-8 activity. The invention also relates to a pharmaceutical composition comprising the above men- tioned substances.

The inventors have shown that CCK-8 seems to effect the turn-over rate of NGF, rather than the mRNA synthesis. They have also shown that CCK-8 does not seem to give any side-effects at normal dosage. CCK-8 is very potent and need therefore only be administered in a low dose to achieve the desired effects. CCK-8 also has a calming effect on the patient.

Detailed description of the invention

As disclosed herein, by a substance showing CCK-8 activity is meant a substance with essentially CCK-8 like neurotrophin inducing activity. This means that a sub- stance showing CCK-8 activity possesses the ability to induce the cellular production of neurotrophins, especially nerve growth factor (NGF), in essentially the same manner as CCK-8, when being exogenously administered. CCK-8 activity can be determined according to the methods described in the example section of this text. Determinations of neurotrophin activity, especially NGF, can be done according to the methods described in the example section.

An object of the invention is the use of a substance showing CCK-8 activity for the manufacture of a medicament in order to treat neuropathies in the peripheral nerv- ous system. Examples of analogues to CCK-8 that can be used in this invention can be found in US-A-5631230. Other examples of substances showing CCK-8 activity are the following: gastrin, cerulein, bombesin, ceruletide, pentagastrin (and its analogues 3-leupentagastrin (3-leu-PG), 4-AspOBzl-pentagastrin (4-AspOBzl-PG)), GRP (gastrin releasing peptide), cyclic analogues of CCK-8 and CCK-4, Suc-Try- N-(Me)-Hle-Asp-Phe-NH2, BC264, A71263, U67827E, bensodiazepin, D-Tyr-Gly- [(Nle28,31)CCK-26-33], Suc-Trp-N(Me)-Nle-Asp-Phe-NH2, ARL 15849XX, ARL 16935XX, ARL 15745XX, Asp-Tyr-D-Phe-Gly-Trp-(N-Me)Nle-Asp-Phe-NH2 (SNF 9007), CCK-4 (and its analogue CCK-4(Phet)), the cyclic peptides of the H- Tyr-cyclo (D-Pen-Gly-Trp-L/D-3-transmercaptoproline)-Asp-Phe-NH2 sequence, the linear peptides of the H-Tyr-D-Val-Gly-Trp-L/D-3-trans- methylmercaptoproline-Asp-Phe- NH2 sequence, SNF 8702, the cyclic cholecysto- kinin peptide analogue JMN-320, succinyl-Tyr-(S03H)-Met-Gly-Trp-Met- phenethylamide, Boc-Trp-(Ν-Me) Nle-Asp-Phe-NH2 and Boc-Trp-(N-Me)Phe-Asp- Phe-NH2, sulphated CCK-8 (CCK-8S) and D-Tyr25-(Nle28,31)-CCK 25-33S, A- 71378, pseudogastrin [(Glu)5-Ala-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2], (des-

NH2)Tyr(S03-)-Nle-Gly-Trp-Nle-Asp-Phe-NH2, asperiicin, A71623, the phenethyl ester analogue OPE, U-67827E, Ac[X27, Nle28, Nle31]-CCK27-33 wherein X is (L,D)Phe(p-CH2C02H) or (L,D) Phe(p-CH2CONHOH), Ac[Phe(p-CH2S03H)27, Nle28, Nle31]-CCK27-33, Boc[Nle28, Nle31]-CCK27-33 (BDNL), Boc-Phe (p- CH2) S03H as a substitute for Boc-Tyr(S03H) in CCK8, acetyl-CCK-7, Ac-Phe(4- CH2C02H)-Met-Gly-Trp-Met-Asp-Phe-NH2 (28), Ac-Phe[4-(tetrazol-5-yl)]-Met- Gly-Trp-Met-Asp-Phe-NH2 (34), 3-[4-(carboxymethyl)-phenyl]propanoyl-Met-Gly- Trp-Met-Asp-Phe-NH2 (50), MK 329, L-364,718, Thr28NlE31CCK25-33(CCK9), D-Tyr25(Nle28,31)-CCK(25-33), [N-MeNle28,31]CCK26-33, Cyclic CCK analogues in which positions 28 and 31 have been replaced by lysine residues and whose side chains are bridged by a succinic moiety, Boc-[Nle28,31]-CCK-7, Boc- Trp-Leu-Asp-Phe-NH2, Tifluadom, 2-(aminomethyl)- and 3-(aminomethyl)-l,4- benzodiazepines, JMV 236 (Boc-Tyr (S03)-Nle-Gly-Tφ-Nle-Asp-Phe-NH2), CCK-JMN-180 (BOC-Tyr(S03) Ahx-Gly-Trp-Ahx-Asp2 phenylethyl ester), BOC(Νle28;Νle31)CCK27-33 (BDNL-CCK7), BC-197 (BOC-D.Asp-Tyr(S03H)- Nle-D.Lys-Trp-Nle-Asp-Phe-NH2), Boc[Nle28,Nle31JCCK27-33, Boc[D- Tyr(S03Na)27,Nle28,Nle3 l]CCK27-33, Ac[L-Phe(p- CH2S03Na)27,Nle28,Nle31JCCK27-33, Ac[D-Phe(p-

CH2S03Na)27,Nle28,Nle31]CCK27-33, Thr28 Nle31-CCK 25-33 (CCK-9), t- butyloxycarbonyl-Tyr (S03H)-Nle psi (COCH2)Gly-Trp-Nle-Asp-Phe-NH2, t- butyloxycarbonyl-[Nle28,31] CCK-8, desammo-cholecystoldnin-octapeptide (CCK 7), GE 410, N alpha-hydroxysulfonyl-[Nle28,31]CCK26-33 in which the C- terminal L-Phe33 residue has been replaced by L-Leu, D-Phe or N-methyl-L-Phe, replacement of methionine-31 in position 31 of cholecystolάnin CCK26-33 by the amino acids phenylalanine, alanine, glutamic acid, and oιτrithine and its analogue with the epsilon-amino group protected by a benzyloxycarbonyl group, D-Tyr-

Gly[(Nle28,31)CCK-26-33], pseudopeptide analogues of the C-terminal heptapep- tide of cholecystokinin Z-Tyr(S03-)-Nle-Gly-Trp-Nle-Asp psi-(CH2NH)Phe-NH2 (1), Z-Tyr(S03-)-Nle-Gly-Trp-Nle psi (CH2NH)Asp-Phe-NH2 (2), Z-Tyr(SCβ-)- Nle-Gly-Tφ psi-(CH2NH)Nle-Asp-Phe-NH2 (3), Z-Tyr(S03-)-Nle-Gly psi(CH2NH)Trp-Nle-Asp-Phe-NH2 (4), Z-Tyr(S03-)-Nle psi-(CH2NH)Gly-Trp- Nle-Asp-Phe-NH2 (5), Z-Tyr(S03-)-Met-Gly-Tφ-Nle-Asp psi(CH2NH)-Phe-NH2 (6), Z-Tyr-(S03-)-Met-Gly-Tφ-Nle psi (CH2NH)Asp-Phe-NH2 (7) and Z- Tyr(S03-)-Met-Gly-Tφ psi (CH2NH)Nle-Asp-Phe-NH2 (8), Boc-Asp-Tyr(SCβ-)- Nle-Gly-Tιp-Nle-Asp-Phe-NH2, CCK-(27-32)-NH2, analogues of acetyl-CCK- heptapeptide (Ac-Tyr(S03H)2-Met3-Gly4-Tφ5-Met6-Asp7-Phe8-NH2 ) wherein the Asp7 residue was replaced by hydroxy amino acid sulfate esters, Gly4 was sub- stituted by D-Ala. while Tφ5 and Met6 were replaced by their D enantiomer, the ceruletide (CER) analogue Nle8-CER-(4-10), carbobenzoxy-L-tyrosyl(0-sulfate)-L- methionylglycyl-L-tryptophyl-L-met onyl-L-aspartyl-beta-L-phenylalanine amine (Z-32-beta-Asp-CCK-27-33), [28-threonine,31-norleucine]- and [28-threonine,31- leucine]cholecystok-uun-pancreozymin-(25-33)-nonapeptide, Z-Arg(Z2)-

Asp(OBut)-Tyr-(S03Bal/2)-Thr(But)-Gly-Tφ-Leu-Asp(OBut)-Phe-NH2 and Z- Arg(Z2)-Asp(OBut)-Tyr(S03-Bal/2)-Thr(But)-Gly-Tφ-Nle-Asp(OBut)-Phe-NH2, Ser(S03H)-Met-Gly-Tφ-Met-Asp-Phe-NH2 (J Biol Chem 1998 May 22; 273(21): 12988-93; Pancreas 1997 Aug; 15(2): 160-7; J Clin Psychopharmacol 1996 Dec;16(6):440-5; Pharmacol Biochem Behav 1996 May;54(l):255-9; J Pharm Bio- med Anal 1996 Mar;14(5):593-600; J Med Chem 1996 Sep 27;39(20):4120-4; Psy- chopharmacology (Berl) 1996 Aug;126(4):339-44; Pharmacol Biochem Behav 1996 May;54(l):255-9; J Pharm Biomed Anal 1996 Mar; 14(5):593-600; Rocz Akad Med Bialymst 1996;41(2): 183-90; Biopolymers 1995 Oct;36(4):439-52; Pancreas 1995 Aug;l 1(2): 141-6; Peptides 1995;16(2):221-4; Peptides 1995;16(5):815-9; J Surg Oncol 1994 Sep;57(l): 11-6; Am J Physiol 1994 Jul;267(l Pt l):C220-8; J Neuro- chem 1994 Apr;62(4): 1426-31; J Med Chem 1994 Mar 4;37(5):630-5; Biopolymers 1994 Feb;34(2): 155-69; J Auton Nerv Syst 1994 Jan-Feb;46( 1-2): 65-73; Am J Physiol 1993 Nov;265(5 Pt l):G865-72; Neurosci Lett 1993 Oct 1;160(2): 193-6; J Med Chem 1992 Aug 7;35(16):2919-28; J Med Chem 1992 Jul 10;35(14):2534-42; Am J Physiol 1992 Jul;263(l Pt 2):R125-35; Biochem Biophys Res Commun 1992 Mar 16; 183(2):396-404; Am J Clin Nutr 1992 Jan;55(l Suppl):286S-290S; Int J Pept Protein Res 1992 Jan;39(l):48-57; Am J Physiol 1991 Nov;261(5 Pt 2):R1141-6; J Neurol Sci 1991 Sep;105(l): 12-3; Int J Pept Protein Res 1991 Apr;37(4):331-40; Am J Physiol 1991 Apr;260(4 Pt l):G577-85; J Med Chem 1991 Mar; 34(3): 1125- 36; J Biol Chem 1991 Feb 5;266(4):2403-8; Z Gastroenterol 1991 Feb;29(2):59-64; Neurosci Lett 1991 Jan 14;122(l):29-32; Int J Pept Protein Res 1990 Jun;35(6):566- 73; Int J Pept Protein Res 1990 May;35(5):441-51; Psy chopharmacology (Berl) 1990;101(3):384-9; J Med Chem 1990 Jan;33( l):450-5; Neuropeptides 1990 Jan; 15(l).37-41; Pain 1989 Dec;39(3):307-28; Am J Physiol 1989 Oct;257(4 Pt l):G594-600; Biochim Biophys Acta 1989 Feb 9; 1010(2): 145-50; Neuropeptides 1989 Feb-Mar; 13(2):89-94; J Med Chem 1989 Feb;32(2):445-9; Z Gastroenterol 1988 Dec;26(12):762-6; J Biol Chem 1988 Aug 5;263(22): 10641-5; Methods Find Exp Clin Pharmacol 1988 Aug; 10(8):513-20; Int J Pept Protein Res 1988 Jun;31(6):514-9; Br J Pharmacol 1988 May;94(l):246-52; J Med Chem 1988 May;31(5):966-70; Biochem Biophys Res Commun 1987 Aug 31;147(l):346-53; J Med Chem 1987 Aug;30(8): 1366-73; Proc West Pharmacol Soc 1987;30:223-6; Am J Physiol 1984 Sep;247(3 Pt l):G261-4; J Med Chem 1984 Jul;27(7):845-9; Eur J Pharmacol 1983 Oct 28;94(3-4):261-70; J Med Chem 1982 May ;25(5):589-93; Hoppe Seylers Z Physiol Chem 1981 Jul;362(7):929-42; Peptides 1981;2 Suppl 2:65-9; J Med Chem 1978 Oct;21(10): 1030-5; J Med Chem 1977 Aug;20(8):1047- 50).

Other substances showing CCK-8 activity could also be used, such as naturally occurring or artificially modified variants, analogues, and derivatives of CCK-8. Such substances could be obtained by addition, insertion, elimination or substitution of at least one amino acid in CCK-8. By substance showing a CCK-8 activity is also understood precursors, metabolites such as metabolic derivatives e.g. metabolic degradation products, agonists, or analogues of the substances mentioned herein displaying the same properties. Metabolic derivatives or metabolic degradation products may be CCK-8 like peptides e.g. with eight amino acids such as CCK-8 from which one or more amino acids has been deleted from either or both ends of the molecule. It could be ascertained that these variants are analogues of CCK-8 by immunologi- cal methods, e.g. RIA (radio-immunoassay), IRMA (radiometric methods), RIST (radioimmunosorbent test), RAST (radioallergosorbent test).

It is also a possibility to create new compounds showing CCK-8 activity by means of computer simulation. Methods for computer simulation are known by a person skilled in the ait, e.g. as described in EP 0660 210 A2.

The substances mentioned above can be made through conventional technologies, e.g. synthesized directly from the amino acid building blocks, or be obtained through direct extraction and purification from biological tissues and cell lines or by means of biotechnologies such as recombinant DNA techniques, or be bought in conventional manner from a producer or distributor of such substances.

Preferably CCK-8 is used, whereby both the D- and L-form of CCK-8 can be used in the present invention.

By neuropathies in the peripheral nervous system are meant a variety of conditions, including those associated with disorders in structure and function of peripheral motor and sensoiy neurons. Peripheral neuropathies can involve the entire neuron or part of it. Examples of states, disorders, damages and diseases that can be regarded as peripheral neuropathies and can be treated with the present invention are, for example, those selected from a group of: neuropathies associated with diabetes mellitus patients; alcohol-induced neuropathy; neuropathies associated with cancer treatment, such as cytostatica or irradiation treatment; hearing impairments, such as deafness, tinnitus; visual handicaps, such as retina damages, cornea damages; impaired wound healing; damages induced by surgery; damages as a result of injuries; damages as a side effect after exposure to neurotoxic compounds, such as antineo- plastic drugs; dystrophy; congenital and autoimmune neuropathies; or other major- diseases related syndromes. Peripheral neuropathies (PN) are a major problem in clinical practise and may be associated with pain.

The peripheral nervous system comprises the sensory (afferent) and the motor (efferent) divisions. The sensory division comprises the somatic and the visceral sen- sory neurons. The motor division comprises the autonomic nervous system (involuntary) and the somatic nervous system (voluntary). The present invention can be used to treat neuropathies in the entire peripheral nervous system.

Another object of the invention is a pharmaceutical composition in order to treat neuropathies in peripheral nervous system, comprising an effective concentration of at least one substance showing CCK-8 activity, in mixture or otherwise together with at least one pharmaceutically acceptable carrier or excipient. Examples of substances showing CCK-8 activity are CCK-8 and other substances mentioned above. Preferably, CCK-8 is used.

The pharmaceutical compositions are prepared in a manner known to a person skilled in the pharmaceutical art. The carrier or the excipient could be a solid, semi- solid or liquid material that could serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are known in the art. The pharmaceutical composition could be adapted to oral or parenteral use and could be administered to the patient as tablets, capsules, suppositories, solutions, suspensions or the like.

The pharmaceutical compositions could be administered orally, e.g. with an inert diluent or with an edible carrier. They could be enclosed in gelatin capsules or be compressed to tablets. For oral therapeutic administration the compounds according to the invention could be incoφorated with excipients and used as tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 4% by weight of the compounds according to the invention, the active ingredient, but could be varied according to the special form and could, suitably, be 4-70% by weight of the unit. The amount of the active ingredient that is contained in compositions is so high that a unit dosage form suitable for administration is obtained.

The tablets, pills, capsules, lozenges and the like could also contain at least one of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatin, excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, corn starch, and the like, lubricants such as magnesium stearate or Sterotex, glidants such as colloidal silica dioxide, and sweetening agents such as saccharose or saccharin could be added or flavourings such as peppermint, methyl salicylate or orange flavouring. When the unit dosage form is a capsule it could contain in addition of the type above a liquid carrier such as polyethylene glycol or a fatty oil. Other unit dosage forms could contain other different materials that modify the physrcal form of the unit dosage form, e g as coatings Accordmgly, tablets or pills could be coated with sugar, shellac or other enteπc coating agents A syrup could m addition to the active ingredient contam saccharose as a sweetening agent and some preservatives, dyes and flavouring agents Mateπals that are used for preparation of these different compositions should be pharmaceutically pure and non-toxic m the amounts used

For parenteral administration the compounds accordmg to the invention could be incoφoiated m a solution or suspension Parenteral administration refers to the ad- ministration not through the alimentary canal but rather by injection through some other route, as subcutaneous, intramuscular, rntraorbital, lntracapsular. intraspinal, rntrasternal, intravenous, intranasal, lntrapulmonary, through the urinary tract, through the lactiferous tract m catties, mto an organ such as bone marrow, etc Bone marrow may also be treated in vitro These preparations could contam at least 0 1% by weight of an active compound accordmg to the invention but could be varied to be approximately 0 1-50% thereof by weight The amount of the active ingredient that is contained in such compositions is so high that a suitable dosage is obtamed

The solutions or suspensions could also comprise at least one of the following adju- vants steπle diluents such as water for injection, salme, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylene diamine tetraacetic acid, buffers such as acetates, citrates or phosphates, and agents for adjustment of the tonicity such as sodium chloride or dextrose The parenteral preparation could be enclosed in ampoules, disposable syπnges or multiple dosage vessels made of glass or plastic

For topical administration the compounds accordmg to the invention could be incoφorated in a solution, suspension, or ointment These preparations could contam at least 0 1% by weight of an active compound accordmg to the mvention but could be varied to be approximately 0 1-50% thereof by weight The amount of the active ingredient that is contained in such compositions is so high that a suitable dosage is obtained. The administration could be facilitated by applying touch, pressure, massage or heat, warms, or infrared light on the skin, which leads to enhanced skin permeability. Hirvonen, J., Kalia, YN, and Guy, RH. Transdermal delivery of pep- tides by iontophoresis, Nat Bwtechnol 1996 Dec; 14(13): 1710-1713 describes how to enhance the transport of a drug via the skin using the driving force of an applied electric field. Preferably, iontophoresis is effected at a slightly basic pH.

Other administration forms are inhalation through the lungs, buccal administration via the mouth and enteral administration via the small intestine that could be effected by means known by a person skilled in the art.

For example, such a composition could be used in order to treat neuropathies in the peripheral nervous system.

Another object of the invention is a method for the treatment of a subject in need for treatment of a neuropathy in the peripheral nervous system, comprising a pharmaceutical dose of a substance showing CCK-8 activity for said subject.

By a subject is meant any mammal, including humans. A human subject is preferred.

By medicament is meant a pharmaceutical to be used in human or veterinary medicine.

CCK-8 is a gut neuropeptide widely distributed in the CNS and in the PNS. The inventors have recently demonstrated that intraperitoneal administration of this neuropeptide in doses close to the physiological circulating CCK levels (Linden A et al.. 1989). stimulated an increase of brain NGF levels and choline-acetyltransferase (ChAT) activity in forebrain cholinergic neurons of normal mice (Tirassa et al,

1998. Tirassa et al., 1999) and can counteract the cholinergic deficit in fimbria for- nix-transected mice (Tirassa et al., 1999). In this work, using models of peripheral neuropathy and sympathectomy caused by administration of neurotoxic compounds, the inventors demonstrated that CCK-8 regains the functional and biochemical impairment due to CAP and 6-OHDA treatment. These results suggest that CCK-8 might be implicated in the recovery of impaired nerve cells in PNS.

In conclusion, using animal models of sensory PN, the present study demonstrates for the first time that a peripheral neuropeptide may influence synthesis and expression of NGF and of sensory and sympathetic neuropeptide levels in peripheral tis- sues. Taken together these findings suggest that i.p. injections with low doses of

CCK-8 represents a potential useful alternative strategy to promote the recovery of normal PNS function in PN induced by surgical and chemical insults or related by other major diseases or states as mentioned above.

The invention is now described with the following example. This example is of illustrative puφose only and is not intended to limit the scope of the invention in any way.

Detailed description of the drawings

Figure 1. Hot-plate response of adult mice treated with Capsaicin (CAP) for three days. A group of CAP-treated mice and a group of control mice were treated with CCK-8 for ten days starting ten days after the last injection of Capsaicin. The latency time of response to noxious stimuli in CAP-treated mice remains higher than control for the entire observation period, while the treatment with CCK-8 induces a decrease of the response-latency time in CAP-treated mice, as revealed by ANONA on the repeated measures, reaching the baseline values after 8-10 days of treatment. The vertical lines indicate pooled SEM's derived from appropriate error mean square in the AΝONA. * p<0.05. Figure 2. Sympathetic nerve fibers in the iris of normal (A), 6-OHDA-treated mice receiving saline (B) or CCK-8 (C) for ten days. Sympathetic innervation is visualized by Glyoxilic acid-induced fluorescence (GAIF). A significant reduction of sympathetic innervation was observed after 6-OHDA treatment, while a partial re- covery was observed following CCK-8 treatment, as shown in panel D.

Figure 3. Effect of Capsaicin and CCK-8 on NGF levels in the hind paw skin. A: NGF, expressed as pg/gr of wet weight, increase immediately after CAP -treatment, reaching the higher level around 4 days from the injection of the neurotoxic com- pound. Then, the amount of the neurotrophin slowly decreases to levels lower than control, as measured 10 and 20 days after the end of CAP treatment. B: treatment for ten days with physiological amounts of CCK-8 increase NGF levels in normal mice and is able to further enhance the neurotrophin expression in CAP-lesioned mice.

Figure 4. Effects of Capsaicin and CAP+CCK treatment on NGFmRNA expression in the hind paw skin of adult mice. In situ hybridisation (A-D) shows that NGFmRNA is normally expressed in the basal epidermal layer of the skin (B). Specific NGFmRNA was confirmed by specificity test including digestion of mRNA with Rnase-A before hybridisation and hybridisation with sense NGF probe, which resulted in absence of hybridisation signal (A). The decreased expression of NGFmRNA observed after treatment with CAP (C), was completely reversed by treatment with CCK-8 (D). The histological data was confirmed by quantitative evaluation of NGFmRNA performed by densitometric analysis after RT-PCR (E-F). The vertical lines indicate pooled SEM's derived from appropriate error mean square in the ANONA. * p<0.05.

Figure 5. ΝGF-levels in the eyes of normal and 6-OHDA-treated mice receiving saline or CCK-8 for ten days. The ΝGF levels increase in both 6-OHDA and CCK-8 groups. The upregulation of ΝGF is further enhanced by CCK-8 treatment, when it is performed in 6-OHDA challenged mice. Figure 6. Effect of Capsaicin on the level of SP and CGRP in the hind paw of adult mice before and after treatment with CCK-8. The gut neuropeptide increase the level of both sensory neuropeptide only in the paw skin of CAP-treated mice. The vertical lines indicate pooled SEM's derived from appropriate error mean square in the ANONA. * p<0.05.

Figure 7. Effects of 6-OHDA and/or CCK-8 treatment on neuropeptide Y (ΝPY) concentration in peripheral tissues of adult mice. Treatment with 6-OHDA signifi- cantly reduces ΝPY concentration in eye, heart and spleen while no changes were observed in the intestine. Treatment with CCK-8 increases ΝPY contents in eyes and intestine of normal mice and recovers the 6-OHDA induced ΝPY decrease in heart and eyes.

Example 1

Animals

Adult three months old male mice of CD-I strain, purchased from C. River, Calco, Italy, were housed 4-5 per cage under a 12-12 hours light-dark cycle with water and food ad libitum. Animal care and procedures were conducted in conformity with the intramural committee and institutional guidelines in accordance with national and international laws (EEC council directive 86/609, OJ L358, 1, December 12, 1987).

Treatment with Capsaicin and CCK

To induce sensory neuropathy adult mice (n=48) were subcutaneously injected, under mild anaesthesia, with 50 mg/kg capsaicin or left untreated as controls, for two consecutive days. Given systematically to adult animals, capsaicin produces a gen- eralized desensitisation and loss of sensory nerves (Holzer P, 1991). After five days these mice were divided in four groups and treated with and without CCK-8 as fol- lowed: (i) CAP-treated, injected with CCK (n=12); (ii) CAP-treated, injected with vehicle (n=12); (iii) untreated and injected with CCK (n=12); (iv) untreated and injected with vehicle (n=12). CCK-8 (8 nmol kg^"1) or vehicle (saline) was subcutane- ously injected for ten consecutive days, starting ten days after the last CAP treat- ment, then mice were sacrified and peripheral tissue removed, immediately frozen and then used for NGF or neuropeptide determination.

Behavioural studies

To evaluate the effect of sensory denervation the hot-plate response were used. Pain reactivity was measured using a hot-plate apparatus (Socrel Hot-plate model DS37, Ugo Basile, Italy). Temperature was set at 50 ± 0,3°C, cut-off time was 60 sec. Pain reactivity was measured by scoring latency to the first episode of nociceptive heat sensitivity (jumping, forepaw or hind paw licking). Latency time was deteπriined using a digital stopwatch. All groups of mice were tested starting two days after the second injection of the neurotoxic compounds and every two days until the last CCK injection was performed.

Treatment with 6-OHDA and CCK

To induce sympathetic neuropathy, mice were injected with 100 mg/kg of 6- hydroxydopamine (6-OHDA) dissolved in physiological saline (0,85% NaCl) with 0,5 mg/ml of ascorbic acid to retard oxidation of the drug. Control mice received injections of the vehicle solution only. Animals (n=48) were treated with for two consecutive days and after five days divided in the following groups: (i) 6-OHDA- treated twice injected with CCK (=12); (ii) 6-OHDA injected with vehicle (n=12); (iii) untreated and injected with CCK (n=12); (iv) untreated and injected with vehicle (n=12). CCK-8 (8 nmol kg-1) or vehicle (saline) was subcutaneously injected for ten consecutive days, starting ten days after the last 6-OHDA treatment, then mice were sacrificed and peripheral tissue removed, immediately frozen and then used for NGF or neuropeptide determination.

Evaluation of sympathetic innervation

6-OHDA-treated and control mice were sacrificed under nembutal anaesthesia and the iris removed prepared as whole mounts and processed to evaluate the rate of noradrenergic innervation, and the number and density of neurites were deteimined moφhometrically, using the glyoxylic acid-induced fluorescence (GAIF) (Hokfelt et al, 1972). The preparations were examined under a fluorescent microscope equipped with excitation and barrier emission filters with a transmission cut-of of 470 and 500 mm, respectively. For quantitative evaluation of sympathetic innervation, the number of noradrenergic neurites in three different areas of GAIF -treated iris of each experimental group (n=8 iris per group) were counted by an unaware observer, and differences between treated and untreated rats were statistically evaluated.

NGF determination

All mice were then sacrified with an overdose of nembutal and peripheral tissues were removed and used for the evaluation of peripheral innervation, neuropeptide content and NGF levels.

The levels of NGF were measured by a highly sensitive two-site immunoenzymatic assay (Weskamp and Often, 1987) which recognizes human and murine NGF and does not cross react with brain derived neurotrophic factor (Bracci-Laudiero et al., 1992). Briefly, polystyrene 96- well immunoplates (Nunc) were coated with affinity purified polyclonal goat anti-NGF antibody which does not cross react with brain derived neurotrophic factor diluted in 0,05 M carbonate buffer (pH 9,6). Parallel wells were coated with purified goat IgG (Zymed, San Francisco, CA, USA) for evaluation of the non-specific signal. After an overnight incubation at room temperature and 2 h incubation with a blocking buffer (0,05 M carbonate buffer, pH 9,5, 1% BSA), plates were washed three times with Tris-HCl, pH 7,4, 50 mM, NaCl 200 mM, 0,5%) gelatin, 0, 1% Triton X-100. After extensive washing of the plates, the samples and the NGF standard solutions were diluted with sample buffer (0,1%, Triton X-100, 100 mM Tris-HCl, pH 7,2, 400 mM NaCl, 4 mM EDTA, 0,2 mM PMSF, 0,2 mM benzethonium chloride, 2 mM benzamidine, 40 U/ml aprotinin,

0,05%) sodium azide, 2% BSA and 0,5%> gelatin), distributed into the wells and left at room temperature overnight. The plates were then washed three times and incubated with 4 mU/well anti-β-NGF-galactosidase (Boehringer Mannheim, Germany) for 2 hours at 37°C and, after further washing, 100 μl of substrate solution (4 mg/ml of chlorophenol red, Boehringer Mannheim, Germany, substrate buffer: 100 mM

HEPES, 150 mM NaCl, 2mM MgCl₂, 0, 1% sodium azide and 1% BSA) were added to each well. After an incubation of 2 hours at 37°C, the optical density was measured at 575 nm using an ELISA reader (Dynatech), and the values of standards and samples were corrected by taking the non-specific binding into consideration. The recovery of NGF during assay procedure was estimated by adding a known amount of highly purified NGF to the samples or to the homogenization buffer, as internal control. The yield of the exogenous NGF was calculated by subtracting the amount of endogenous NGF from the value of endogenous plus exogenous values. Under these conditions the NGF recovery was over 90%. Data are represented as pg/g wet tissue and all assays were performed in triplicate.

Neuropeptide analysis

A highly specific competitive radioimmunoassay (RLA) was used (detection limit 1,5 fmol = 2 pg per incubate; detectable concentration 15 ftnol/l = 20 pg per ml). Briefly, tissue samples were cut into small pieces in the frozen state, boiled for 10 rnin in 1 mol l^"1 acetic acid and homogenized. After centriftigation at lOOOOg for 10 min, the supernatant were lyophilized and stored at -20°C before analysis. The tissue concentration of Substance P-like immunoreactivity (SP-LI) was analyzed using the C-termmally directed antiserum SP2 (Brodin et al. 1986) with ¹²⁵I-[Tyr⁸]-SP as radioligand and synthetic SP as standard. The tissue concentration of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was analyzed using antise- rum CGRP-8 raised in a rabbit against conjugated rat CGRP with 1251-histydil- rat CGRP as radioligand and rat CGRP as standard. The tissue concentration of neuropeptide Y-like immunoreactivity (NPY-LI) was analyzed using antiserum Nl wich cross-reacts 0.1% with avian pancreatic polypeptide but not with other peptide. The detection limit of the assay was 1 lpmol 1-1.

In situ hybridization for NGF mRNA.

Fourteen-micron sections from hind paw skin were cut by cryostat and mounted on poly-L-lisine-coated slides. The slices were fixed in 4% paraformaldeyde in 0.1M PBS (pH 7.4) for 10 rnin followed by repeated wash in 0.1M PBS and dehydration by 70,80,95 % ethyl alcohol. After acetylation (25% acetic anhydride in 0.1M TEA pH 8.0), the slices were incubated at 42°C for 16hrs. in a hybridization mixture containing digoxigenin-labelled NGF probes (complementary to the sequence 5 CCTGTTGAGAGTGGTGCCGGGGCATCGA3') at a final concentration of 30ng/ml hybridization buffer (50% formamide, 2XSSC,0.1%SDS, 250mg/ml denatured sheared salmon tested DNA). After washing, the slices were incubated 2h at room temperature with a 1.5U/ml sheep anti-digoxigenin POD conjugated antibody (polyclonal Fab fragment; Boheringer Mannheim). The immunoperoxidase reaction was detected using standard DAB procedure (0.6mg/ml DAB and 0.015% H20).

RT-PCR:

Total RNA was extracted by using TRIZOL kit (Gibco) following the manufacturer instruction. The tissues was omogenysed in the TRIZOL Reagent, incubated for 15 min at 4°C and then centrifuged (lOOOOg, 4°C, 15 min). 0,2 ml of chloroform for each 0,75 ml TRIZOL Reagent was added to the supernatant and, after a 15 min in- cubation at 4°C, the samples were spun for phase separation at 4°C. RNA was precipitated from the upper aqueous phase by adding 0, 1 vol. of 3M sodium acetate and an equal volume of isopropanol. After incubation for 60 min. at -20°C the precipitate was pelletted by centrifugation, washed once with 75% ethanol and redissolved in 50 ml RNase-free water. A volume (max 10 ml) of RNA solution containing 1 mg of RNA was reverse transcribed into a single stranded cDNA with the reverse tran- scription system (Promega) in a total reaction volume of 20 ml, using 250 ng

01igo(dT)15 primer, 200 units of MLV-RT (Promega) and 0,5 U RNasin ribonucle- ase inhibitor (Promega). After 60 min. incubation at 42°C, the reaction was terminated by adding 50 ml water. To compensate for the relative differences in sample size, integrity of the individual RNA samples and the variation in reverse transcrip- tion, Glyceraldehyde-3 -phosphate dehydrogenase(GAPDH) was co-amplified with murine NGF. The PCR reaction was carried out in 50 ml mixtures containing 5 ml of sample cDNA, 5 ml 10X Taq polymerase buffer (Promega), 2.5 mM MgC12, 0.2 mM of each dNTP (Pharmacia), 5 pmol each primers (NGF:5'CAGGACTCACAGGAGCAAGC3';5'GCCTTCCTGCTGAGCACACA3'. GAPDH:5'CACCACCATGGAGAAGGCC3';

5'CACCACCATGGAGAAGGCC3') and 2U Taq polymerase (Promega) on Ge- neAmp PCR System 9600 thermal cycler (Perkin Elmer) for 30 cycles (60 sec at 95°C, 60 sec at 55°C and 120 sec at 72°C). The PCR products are a 343 bases long fragment for NGF and 190 bases long fragment for GAPDH. After PCR, 10 ml of undiluted reaction product were loaded onto a 2% Agarose MP (Boehringer Mannheim) gel containing 1 mg/ml ethidium bromide. The gel was run at 1 N/cm for 15 min and then at 5 N/cm for 3 hours. The DΝA-containing bands were photographed using an ultraviolet (UN) transilluminator (Fig. 4-C). The identity of all the PCR products was confirmed by comparing to the correct size based on the known length of the DΝA sequence on agarose gel and by Southern blotting (data not showed).

Band densitometric evaluation - expressed as arbitrary units of grey level - was performed by an automatic image analyzer (Nidas System; Kontron Electronics), which determinates the optical density of the ethidium bromide stained bands using a gray scale thresholding operation. The optical density of GAPDH bands was used as normalizative factor. The data showed in fig. 4-D represent the mean ± SE of ΝGF normalized densitometric values obtained from five different RT-PCR. Statistical analysis

Data were obtained by means of analysis of variance using the SuperANONA pack- age for Macintosh (Abacus Concepts Inc., Berkeley, CA, USA), considering the treatments with saline, CAP, CCK and CAP+CCK as variables. For the hot-plate response, the effect of CAP an/or CCK were analyzed considering the repeated measures (10 tests) and the treatments (four levels: vehicle, CAP, CCK, CAP+CCK). Difference between groups was determined by Tukey-Kramer com- parison; a p<0.05 was considered statistically significant.

Results

Hot plate response

To evaluate loss of sensory innervation mice were tested for hot-plate responses. As illustrated in figure 1, mice treated with CAP display a delayed response to peripheral noxious stimuli as compared to control mice. CAP enhances the time of latency in the hot plate responses and this altered response lasts for at least one month after the treatment, suggesting deficit of sensory peripheral innervation in the paw. Subcutaneous aαlmrnistration of CCK-8 in the CAP-treated mice causes a progressive recovery of the sensory function that appears to be restored after 10 days of CCK-8 treatment (see figure 1). No differences were found in the latency time between vehicle and vehicle+CCK mice, thus no hyperalgesic effect is attributable to CCK in our experimental conditions. Moreover, CCK-8 achninistration, in our experimental conditions, does not cause loss of body weight (data not showed).

Evaluation of iris sympathetic innervation in 6-OHDA/CCK treated mice

Moφhological observation carried out on GAIF-treated irides showed that 6-OHDA causes a massive degeneration of the peripheral sympathetic nerve terminals. As shown in figure 2, the iris of control mouse (2 A) displays a dense network of sympathetic, catecholamine histofluorescent fiber, while iris of 6-OHDA-treated mice is completely devoid of these fibers (2B). Following CCK treatment, the peripheral innervation in the iris of sympathectomized mice (2C) increases significantly as compared to the iris of sympathectomized mice treated with saline. The effects of sympathectomy and CCK treatment on the iris innervation were confirmed by quantitative evaluation of the number of noradrenergic neurites in the GAIF-treated iris of each experimental group (see Fig. 2D)

Expression of NGF

To assess whether CCK treatment is able to induce NGF expression in peripheral tissue after challenge with capsaicin, the NGF levels in paw skin was measured by ELISA. The level of NGF in the paw skin increase after CAP treatment, as shown in figure 3. Likewise CCK-8 treatment increase the level of the neurotrophin as well as the CAP treatment does. The upregulation of NGF protein expression is further enhanced by CCK-8 treatment, when it is performed on CAP-challenged mice.

To identify the cells involved in the upregulation of NGF and to assess whether CCK also affect the NGFmRNAsynthesis, we analyzed NGFmRNA expression in the paw skin by means of in situ hybridization and RT-PCR. As illustrated in fig. 4B, cell localized in the basal epidermal layer express NGFmRNA. The decreased expression of NGFmRNA observed after treatment with Capsaicin (C), was completely reversed by treatment with CCK-8 (D). The quantitative evaluation, carried out by RT-PCR, demonstrates that NGFmRNA is decreased in the paw skin of

CAP-treated mice and that treatment with CCK promotes upregulation of NGF gene transcription in the CAP-treated mice (fig 4E-F).

To assess if sympathectomy and CCK-8 treatment affect the NGF expression, the levels of NGF were measured in the eyes of 6-OHDA-treated mice receiving saline or CCK-8 injections for 10 consecutive days. As shown in figure 5, the levels of NGF in the eyes increase in 6-OHDA group and in CCK-8 group. The up-regulation of NGF protein expression is further enhanced by CCK-8 treatment, when it is performed in 6-OHDA challenged mice.

Neuropeptide levels

It has been demonstrated that neuropeptide expression in peripheral tissues is affected by challenge with CAP and 6-OHDA, and that NGF is able to reverse this decrease of neuropeptide content (Donnerer J. 1996, Neurosci Lett 221: 33-36; Donnerer J. Et al, 1996, Brain Res 741: 103-108). Since our results demonstrate that CCK-8 is able to increase NGF expression in peripheral tissues, we studied whether it is also able to affect neuropeptide levels in the paw skin of CAP-treated mice and in different tissues (heart, intestine, spleen and eye) of 6-OHDA-treated mice. As reported in figure 6, our data show that the amount of sensory neuropep- tide SP and CGRP in the CAP and CCK groups are not different from the control value after 30 days, while it is increased in the CAP+CCK group, suggesting that a recovery in sensory innervation could be promoted by CCK in the CAP sensory impaired mice and that this correlates with the recovery of sensory function and to the increase in NGF gene transcription. As reported in Fig. 7, the 6-OHDA treatment reduces the NPY concentration in the eye, heart and spleen, while no changes were observed in the intestine. Ten daily injections with CCK-8 in control mice differentially affect the NPY levels in the peripheral tissues, inducing an increase in the intestine and eyes but not in heart and spleen. When the CCK-8 treatment was performed on the sympathectomised mice, it is able to recover the 6-OHDA induced deficit of NPY in heart and eyes.

Discussion

Consistent with previous findings indicating that CAP injected in adult mice causes loss of peripheral sensory innervation (Holzer P, 1991), the results of the inventors studies showed that this treatment induces an impairment of sensory response, as revealed by the hot-plate test. The inventors results showed that administration of low doses of CCK-8 for 10 days is able to promote a recovery of sensory function in CAP-treated mice. Nevertheless, administration of 6-OHDA has been showed to damage post-ganglionic terminals of noradrenergic sympathetic system (Hoeldtke R et al., 1974). The inventors data demonstrate that admimstration of CCK-8 is able to recover the 6-OHDA-damaged sympathetic innervation in the iris. Although CCK-8 is known to affect behavioral functions (Woodruff GN et al., 1991), no hyperalgesic effect and decrease of body weight were observed in mice receiving physiological doses of CCK-8. Thus the inventors data are in agreement with previous studies demonstrating that the effects of CCK-8 administration are highly dose-dependent and are subject to tolerance resulting, for example, in unchanged food intake when CCK-8 is admimstered in the long-term (Crawley JN et al., 1983). The present study demonstrates that a treatment with CCK-8 produce the induction of NGF expression in peripheral tissue and the recovery of chemical-impaired sensory and sympathetic innervation.

Claims

1. Use of a substance showing CCK-8 activity for the manufacture of a medicament in order to treat neuropathies in the peripheral nervous system.

2. Use according to claim 1, characterized in that the neuropathy to be treated is alcohol-induced neuropathy.

3. Use according to claim 1, characterized in that the neuropathy to be treated is associated with diabetes mellitus patients.

4. Use according to claim 1, characterized in that the neuropathy to be treated is associated with cancer treatment, such as cytostatica.

5. Use according to claim 1, characterized in that the neuropathy to be treated is a hearing impairment and/or a visual handicap.

6. Use according to claim 1, characterized in that the neuropathy to be treated is a damage induced by surgery.

7. Use according to claim 1, characterized in that the neuropathy to be treated is dystrophy.

8. Use according to anyone of claims 1 to 7, characterized in that the substance is CCK-8.

9. Pharmaceutical composition in order to treat neuropathies in the peripheral nervous system, characterized in that it comprises at least one substance showing CCK-8 activity, especially CCK-8, in mixture or otherwise together with at least one pharmaceutically acceptable carrier or excipient.

0. Method for the treatment of a subject in need for treatment of a neuropathy in the peripheral nervous system, comprising administrating a pharmaceutical dose of a substance showing CCK-8 activity for said subject.