EP1137460A2 - Treatment of erectile dysfunction in diabetes patients - Google Patents

Treatment of erectile dysfunction in diabetes patients

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Publication number
EP1137460A2
EP1137460A2 EP99959520A EP99959520A EP1137460A2 EP 1137460 A2 EP1137460 A2 EP 1137460A2 EP 99959520 A EP99959520 A EP 99959520A EP 99959520 A EP99959520 A EP 99959520A EP 1137460 A2 EP1137460 A2 EP 1137460A2
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EP
European Patent Office
Prior art keywords
diabetic
nitrergic
synthase
inhibitor
rats
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99959520A
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German (de)
French (fr)
Inventor
Selim The Wolfson Inst. for Bio. Res. CELLEK
Salvador The Wolfson Inst. for Bio. Res. MONCADA
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University College London
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University College London
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Publication of EP1137460A2 publication Critical patent/EP1137460A2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/223—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of alpha-aminoacids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to the prevention or reduction of nitrergic nerve degeneration in diabetic mammals and in particular to the prevention of erectile dysfunction in male diabetic mammals.
  • Erectile dysfunction is a common problem among diabetic men, particularly those suffering from diabetes mellitus. The likelihood of erectile dysfunction developing and the severity of the dysfunction both increase with the duration of diabetes.
  • penile prostheses are available to assist such patients in achievement of an erection and the short term success rate is good. However, such treatment often leads to problems with infection and ischaemia and alternative treatments are therefore required.
  • nitric oxide NO
  • Andersson et al Phyisol. Rev. 75, 191-235 (1995).
  • NOS nitric oxide synthase
  • both NO itself and activators of NOS may be suitable candidates for therapeutic treatment of erectile dysfunction.
  • erectile dysfunction in diabetics is due to selective degeneration in the nitrergic nerves innervating the penis.
  • NO itself is responsible for the degeneration of the relevant nitrergic nerves in diabetic men.
  • the present invention provides the use of a pharmaceutically acceptable inhibitor of NO synthase in the manufacture of a medicament for use in preventing or reducing degeneration of nitrergic nerves in a preventing or reducing degeneration of nitrergic nerves in a diabetic mammal.
  • Also provided is a method of preventing or reducing degeneration of nitrergic nerves in a diabetic mammal which method comprises the administration to the said mammal of an effective amount of a pharmaceutically acceptable inhibitor of NO synthase. Typically, a safe and effective amount is administered.
  • an agent for the prevention or reduction of degeneration of nitrergic nerves in a diabetic mammal comprising a pharmaceutically acceptable inhibitor of NO synthase.
  • the mammal is suffering from diabetes mellitus.
  • the present invention is particularly suitable for preventing or reducing degeneration of nitrergic nerves containing neuronal NO synthase.
  • the cytotoxic effect of NO on nitrergic nerves in diabetics may be due to overproduction of NO, leading to overstimulation and thus degeneration of nitrergic nerves. It may also be due to an alteration in endogenous anti-oxidant mechanisms which could render nitrergic nerves prone to the cytotoxic effects of NO.
  • the said diabetic mammal is preferably a human.
  • the said medicament or said agent is for use, or the said method is effective, in preventing erectile dysfunction in a male mammal.
  • Erectile dysfunction may be defined as an inability to obtain or sustain an erection adequate for intercourse.
  • a reduction of nitrergic nerve degeneration is achieved by application of an NO synthase inhibitor if subsequent degeneration occurs to a lesser extent than if the NO synthase inhibitor had not been applied.
  • substantially no degeneration takes place during treatment with the NO synthase inhibitor.
  • degeneration of nitrergic nerves includes any morphological or functional impairment.
  • nitrergic nerves is intended to include any nerves whose transmitter function depends on the release of NO.
  • the said medicament or said agent is for use, or the said method is effective, in preventing or reducing degeneration of the nitrergic nerves innervating the genitals of a male or female mammal.
  • Any pharmaceutically acceptable inhibitor of NO synthase can be used in the present invention.
  • Competitive, non-competitive, reversible and irreversible inhibitors are suitable.
  • the inhibitor may inhibit iNOS, eNOS and/or nNOS. Preferably, it inhibits eNOS and/or nNOS.
  • Suitable inhibitors include L-arginine analogues, thiocitrullines, indazole derivatives, imidazole derivatives, hydrazine derivatives, thioureas, thiazoles, biotin derivatives and phenyl-substituted thiopene amidines.
  • L-arginine analogues examples include methyl-L-arginine, N G - nitro-L-arginine methyl esther (L-NAME), N G -monomethyl-L-arginine (L-NMMA), N G -amino-L-arginine (L-NAA), N w ,N w -dimethyl-L-arginine ( ADMA), N W ,N* 2 - dimethyl-L-arginine (SDMA), N ⁇ efhyl-L-arginine (L-NEA), N w -methyl-L- homoarginine (L-NMHA), N -nitro-L-arginine (L-NOARG), N ⁇ -iminoethyl-L- ornithine (L-NIO), N ⁇ -iminoethyl-L-lysine (L-homo-NIO) and L-canavanine (L- CAN).
  • suitable thiocit examples
  • SMTC L-thiocitrulline
  • Et-TC L-S-ethyl-thiocitrulline
  • suitable indazole derivatives include indazole and 7-substituted indazoles such as 7-nitroindazole and 3-bromo-7-nitroindazole.
  • hydrazine derivatives examples include aminoguanidine.
  • suitable imidazole derivatives include phenyl substituted imidazoles such as 1-phenyl-imidazole.
  • thioureas examples include S-methylisothiourea sulphate, ⁇ -(S- methylisothioureido)-L-norvaline (L-MLN), S-ethylisothiourea (SETU) and S- isopropylisothiourea (SIPT).
  • suitable thiazoles include 2-amino-thiazole and 2-amino-4,5- dimethyl thiazole.
  • biotin derivatives examples include 2-iminobiotin.
  • Preferred NOS inhibitors are selective inhibitors of neuronal NOS.
  • Such selective inhibitors include N -nitro-L-arginine (L-NOARG), N w -nitro-L-arginine methyl ester (L-NAME), N ⁇ -iminoethyl-L-ornithine (L-NIO), L-thiocitrulline (L-
  • TC S-methyl-L-thiocitrulline
  • Et-TC L-S-ethyl-thiocitrulline
  • 2-amino- thiazole 2-amino-4,5-dimethyl thiazole, 7-nitroindazole (7-NI) and phenyl- substituted thiopene amidines.
  • the above NOS inhibitors are commercially available, or may be made by analogy with known methods.
  • the inhibitor may be a pharmaceutically acceptable salt of one the above compounds.
  • Suitable salts include salts with pharmaceutically acceptable acids, both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succininc, tartaric, benzoic, acetic, methanesulphonic, efhanesulphonic, benzenesulphonic or p-toluenesulphonic acid.
  • Salts may also be formed with pharmaceutically acceptable bases such as alkali metal (eg sodium or potassium) and alkali earth metal (eg calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines or heterocyclic amines.
  • Inhibitors of NOS can be identified by: (a) contacting a candidate compound with NOS and a substrate and co- factor therefor, under conditions under which NOS activity, in the absence of an inhibitor, would be expected to occur; and
  • a suitable such assay for identifying inhibitors of NOS is a microtiter plate assay in which NOS activity is measured by determining the change in absorbance as
  • This assay comprises:
  • the buffer is a HEPES buffer capable of maintaining a pH of about 7, preferably about 7.4.
  • the cofactors comprise oxyhemoglobin, NADPH and BH 4 . They may also comprise CaCl 2 , MgCl 2 , FMN, FAD and/or CaM.
  • the NOS may be a naturally occurring form of eNOS, iNOS, or nNOS or may be a variant which retains NOS activity, for example variants produced by mutagenesis techniques.
  • NOS used in the assay is preferably of mammalian origin, for example rodent (including rat and mouse) or primate (such as human).
  • the NOS is of human origin.
  • the NOS may be obtained from mammal cellular extracts or produced recombinantly from, for example, bacteria, yeast or higher eukaryotic cells including mammalian cell lines and insect cell lines.
  • NOS used in the assay is recombinant. More preferably, it is obtained by expression in S/21 cells according to the methodology in Charles et al, Methods in Molecular Biology (edited by M.A.
  • Step (c) of the assay may be carried out by reading the difference in absorbance between 420 and 405 nm. Typically, this is done by a spectrometer.
  • NOS inhibitors used in the present invention typically achieve at least 50% NOS inhibition, more preferably at least 80% NOS inhibition. Ideally, they achieve substantially complete NOS inhibition.
  • Selective inhibitors of neuronal NOS can be identified by conducting the above microtiter assay using different purified NOS isoforms.
  • Compounds which achieve a higher inhibition of nNOS than of eNOS or iNOS are selective nNOS inhibitors.
  • Preferred selective nNOS inhibitors are those which inhibit nNOS at least twice as strongly, preferably at least five times as strongly, more preferably at least ten times as strongly, as eNOS and/or iNOS.
  • a selective nNOS inhibitor substantially does not inhibit eNOS or iNOS.
  • the inhibitors of NO synthase may be administered in a variety of dosage forms.
  • a NO synthase inhibitor is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g.
  • binding agents e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone
  • disaggregating agents e.g. starch, alginic acid, alginates or sodium starch glycolate
  • dyestuffs effervescing mixtures
  • dyestuffs e
  • Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginte, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • a therapeutically effective amount of a compound of the invention is administered to a patient.
  • a typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration.
  • daily dosage levels are from 5 mg to 2 g.
  • administration of an NOS inhibitor is applied to a patient on an on-going basis, to provide continuous protection against degeneration of nitrergic nerves.
  • an erection may be achieved by reducing the amount of NOS inhibitor, or ceasing administration of NOS inhibitor, in good time before an erection is required.
  • the dosage is reduced or the treatment suspended no more than 3 days before an erection is required. In some cases, dosage reduction or suspension of treatment up to 1 day before an erection is required will suffice.
  • a further compound capable of inducing erection may be administered.
  • Suitable such compounds include L-arginine, inhibitors of cyclic guanosine 3',5'-monophosphate phosphodiesterases (described in EP-B-702555) and activators of soluble guanylate cyclase (sGC).
  • sGC soluble guanylate cyclase
  • An example of an sGC activator is 3-(5'-hydroxymethyl-2'-furyl)-l- benzylindazole (YC-1) (Hobbs, A.J., TiPS, December 1997, Vol 18, p.484).
  • the compound capable of inducing erection is L-arginine.
  • L- arginine is the substrate for NOS and will therefore be capable of reversing the inhibition in most cases. This is confirmed in Czech et al, Pharmacol. Biochem. Behav. 1998 (60) p 91-96 and Luiking et al Am. J. Physiol. 1998 (274) G984-G991.
  • the further compound capable of inducing erection can be formulated as set out above and administered at the dosages set out above.
  • the further compound is L-arginine, it is preferably formulated in a form suitable for oral administration and delivered at a dosage of about 50 mg per kg of body weight.
  • the following Examples illustrate the invention. Reference Example 1
  • the first and second groups were injected with streptozotocin (STZ) (75 mg kg '1 , i.p.).
  • the third and fourth groups were injected with sterile saline (1 ml kg "1 , i.p.).
  • the second and fourth groups were given N G -nitro-L-arginine methyl ester (L- NAME) (0.05 mg ml "1 and 0.1 mg ml "1 respectively) in drinking water beginning 72 hours after the injection of STZ or saline. The 72 hour period was sufficient for diabetes to be established.
  • Diabetic rats were observed to drink approximately 100 ml day 1 whereas non-diabetic rats drank approximately 50 ml day 1 ; thus each animal in the second and fourth groups received 5 mg L-NAME each day. This dose has been shown to inhibit NO synthase activity but not to alter the renal function and catecholamine and insulin concentrations in the rat (Navarro et al, Am J. Physiol. 267, R1516 - R1521 (1994).
  • FIG 1 shows sections stained using the polyclonal antibody against nNOS.
  • (a) shows a stained section from a Group III (saline) control rat
  • (b) shows a stained section from a 4 week diabetic rat (note the varicosity formation and breakage in the nerve fibres)
  • (c) shows a stained section from an 8 week diabetic rat (note that the nitrergic nerves became very sparse)
  • (d) shows a stained section from an 8 week diabetic rat treated with L-NAME (note that the structure and number of the nitrergic nerve fibres are preserved during diabetes).
  • the parameters of the electrical field stimulation were 50 V, 0.3 ms pulse duration, 1-25 Hz, for 5 s, every 2 minutes.
  • EFS electrical field stimulation
  • EFS were recorded after treatment of the tissues with scopolamine (5 ⁇ M) and guanethidine (5 ⁇ M) followed by elevation of the tone with phenylephrine (EC 80 ) as described in Kasakov et al, Br. J. Pharmacol., 115, 149-1154 (1995). These responses were affirmed by total inhibition by 300 ⁇ M N G -nitro-L-arginine (L- NOARG) at the end of the experiment.
  • concentration-response curves to exogenous sodium nitroprusside (SNP) in control and diabetic animals were prepared and were found to be indistinguishable.
  • Concentration-response curves to exogenous noradrenaline were also prepared for control and diabetic animals and found to be indistinguishable. This excludes the possibility that the enhanced noradrenergic contraction might be due to hyperresponsiveness of the smooth muscle to noradrenaline.
  • Rats prepared in Reference Example 1 were sacrificed at 1 week, 2 weeks, 3 weeks, 4 weeks and 8 weeks for the purpose of an NO synthase activity assay.
  • the whole penis and anococcygeus muscles of the rats were frozen in liquid nitrogen and pulverised in a stainless steel pestle and mortar.
  • the homogenisation was performed using HESD buffer (20 mM HEPES pH:7.2, 1 mM ' EDTA, 0.2 M sucrose, 5 mM dithiothreitol, 0.1 mM PMSF, 20 ⁇ g/ml leupeptin and soya bean trypsin inhibitor, 5 ⁇ g ml "1 pepstatin A, E-64, bestatin, aprotinin and 3,4- dichloroisocoumarin) and centrifuging at 13,000 g for 30 mins at 4°C.
  • the protective effect of L-NAME on the erectile function of the rats prepared in Reference Example 1 was investigated by studying the effects of in vivo electrical stimulation of the cavernous nerve. Anaesthesia was induced in the rats with sodium thiopentone (120 mg kg "1 , i.p.) and was maintained by a subsequent injection of sodium pent ⁇ barbital (5 mg kg' 1 , i.p.) as required. The right external jugular vein and left carotid artery were cannulated for saline infusion (50 ⁇ l min '1 ) and for blood pressure monitoring, respectively. The penile shaft and cavernous nerve were exposed as described in Rehman et al, Am. J. Physiol. 272, H1960-H1971 (1997).
  • ICP Intracavernous pressure
  • a 25-gauge cannula connected to a pressure transducer.
  • the cavernous nerve was stimulated by bipolar platinum hook electrodes (0.5 mm in diameter). Square pulses of 10 Hz, 0.3 ms pulse duration, for 1-3 minutes, 0.05-0.5 mA were applied via a Grass stimulator coupled to a Grass constant current unit.
  • FIG. 4 The results are shown in Figure 4, in which a shows typical traces from a Group III (saline) control rat (upper trace), a 12 week diabetic rat (middle trace) and a 12 week diabetic rat treated with L-NAME (lower trace). There was no significant difference between Group III and Group IV animals. Note that the increase in intracavernous pressure (ICP) in the diabetic rat could not be maintained during the stimulation period.
  • the vertical scale corresponds to 100 cmH 2 O ICP.
  • the horizontal scale corresponds to 2 minutes.
  • Nitrergic relaxation responses to EFS were recorded after treatment of the tissues with scopolamine (5 ⁇ M) and guanethidine (5 ⁇ M) followed by elevation of the tone with phenylephrine (EC 80 ) as described in Kasakov et al, Br. J. Pharmacol, 115, 149-1154 (1995). These responses were affirmed by total inhibition by 300 ⁇ M N G -nitro-L-arginine (L- NOARG) at the end of the experiment. The results are shown in Figure 5, in which a shows typical traces of the corpus cavernosum in a control rabbit (1), a 24 week (2) and a 30 week (3) diabetic rabbit.
  • nitrergic relaxation responses in the anococcygeus muscle from 24 and 30 week diabetic rabbits were significantly lower than in the anococcygeus muscle from the control animals (EFS at 5 Hz elicited relaxations of 89.1+9.2%, 54.6+6.8%) and 36.7+8.2% of the maximum tone in control, 24 week diabetic and 30 week diabetic rabbits respectively; E ⁇ 0.05 vs control; n-4-6).
  • concentration response curve to exogenous SNP or noradrenaline in t either anococcygeus muscle or corpus cavernosum of the diabetic animals were found to be no change in the concentration response curve to exogenous SNP or noradrenaline in t either anococcygeus muscle or corpus cavernosum of the diabetic animals.
  • rabbits prepared in Reference Example 2 were sacrificed after 30 weeks.
  • the corpus cavernosum of the rabbits were frozen in liquid nitrogen and pulverised in a stainless steel pestle and mortar.
  • the homogenisation was performed using HESD buffer (20 mM HEPES pH:7.2, 1 mM EDTA, 0.2 M sucrose, 5 mM dithiothreitol, 0.1 mM PMSF, 20 ⁇ g/ml leupeptin and soya bean trypsin inhibitor, 5 ⁇ g ml "1 pepstatin A, E-64, bestatin, aprotinin and 3,4-dichloroisocoumarin) and centrifuging at 13,000 g for 30 min at 4°C. Endogenous L-arginine in the supernatant was removed using activated Dowex-50W resin.
  • NO synthase was assayed in the supernatant by the formation of [U- 14 C]-citrulline from L-[U- 14 C]- arginine as described in Knowles et al, In Methods in Molecular Biology, (ed. Titheradge, MA) (Humana Press, Totowa, 1997). The results showed a significant decrease in the activity of the enzyme at the

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Abstract

Prevention or reduction of nitrergic nerve degeneration in diabetic men can be effected by use of an inhibitor of nitric oxide synthase. Such prevention or reduction of nitrergic nerve degeneration can prevent erectile dysfunction in diabetic man, as such dysfunction is caused by a selective defect in the nitrergic nerves innervating the penis.

Description

TREATMENT OF ERECTILE DYSFUNCTION IN DIABETES PATIENTS
The present invention relates to the prevention or reduction of nitrergic nerve degeneration in diabetic mammals and in particular to the prevention of erectile dysfunction in male diabetic mammals. Erectile dysfunction is a common problem among diabetic men, particularly those suffering from diabetes mellitus. The likelihood of erectile dysfunction developing and the severity of the dysfunction both increase with the duration of diabetes. A variety of penile prostheses are available to assist such patients in achievement of an erection and the short term success rate is good. However, such treatment often leads to problems with infection and ischaemia and alternative treatments are therefore required.
Mammalian erection is brought about by the nervi erigentes, which relax the muscle coat of the arterioles of the penis and of the spongy tissue of the corpora cavernosa. Such relaxation is known to be effected in some way by release of nitric oxide (NO);- see for example Andersson et al, Phyisol. Rev. 75, 191-235 (1995).
It is also known that inhibitors of the enzyme nitric oxide synthase (NOS) block relaxation of the corpora cavernosa and hence block erection;- see for example Burnett et al, Science 257, 401-403 (1992) and Cellek et al, Proc. Natl. Acad. Sci. USA 94, 8226-8231 (1997). Further, Benelli et al Eur J Pharmacol (1995) Dec 29;294(2-3):505-10 report that intraperitoneal administration of L-arginine (the natural substrate for NOS) both increased the percentage of copulating in sexually naive rats and improved the indexes of sexual performance in sexually experienced rats. Thus the art indicates that both NO itself and activators of NOS may be suitable candidates for therapeutic treatment of erectile dysfunction. However, it has now been found that erectile dysfunction in diabetics is due to selective degeneration in the nitrergic nerves innervating the penis. Further, it has also been found that, surprisingly, NO itself is responsible for the degeneration of the relevant nitrergic nerves in diabetic men. These findings indicate that NO production during diabetes is implicated in nitrergic neurodegeneration in diabetics. Accordingly, the present invention provides the use of a pharmaceutically acceptable inhibitor of NO synthase in the manufacture of a medicament for use in preventing or reducing degeneration of nitrergic nerves in a preventing or reducing degeneration of nitrergic nerves in a diabetic mammal.
Also provided is a method of preventing or reducing degeneration of nitrergic nerves in a diabetic mammal, which method comprises the administration to the said mammal of an effective amount of a pharmaceutically acceptable inhibitor of NO synthase. Typically, a safe and effective amount is administered.
Also provided is an agent for the prevention or reduction of degeneration of nitrergic nerves in a diabetic mammal, comprising a pharmaceutically acceptable inhibitor of NO synthase.
Typically, the mammal is suffering from diabetes mellitus. The present invention is particularly suitable for preventing or reducing degeneration of nitrergic nerves containing neuronal NO synthase. The cytotoxic effect of NO on nitrergic nerves in diabetics may be due to overproduction of NO, leading to overstimulation and thus degeneration of nitrergic nerves. It may also be due to an alteration in endogenous anti-oxidant mechanisms which could render nitrergic nerves prone to the cytotoxic effects of NO.
The said diabetic mammal is preferably a human. Preferably, the said medicament or said agent is for use, or the said method is effective, in preventing erectile dysfunction in a male mammal. Erectile dysfunction may be defined as an inability to obtain or sustain an erection adequate for intercourse. In the context of the present application, a reduction of nitrergic nerve degeneration is achieved by application of an NO synthase inhibitor if subsequent degeneration occurs to a lesser extent than if the NO synthase inhibitor had not been applied. Ideally, substantially no degeneration takes place during treatment with the NO synthase inhibitor. In the context of the present application, degeneration of nitrergic nerves includes any morphological or functional impairment.
Reference to nitrergic nerves is intended to include any nerves whose transmitter function depends on the release of NO. Preferably, the said medicament or said agent is for use, or the said method is effective, in preventing or reducing degeneration of the nitrergic nerves innervating the genitals of a male or female mammal. Any pharmaceutically acceptable inhibitor of NO synthase can be used in the present invention. Competitive, non-competitive, reversible and irreversible inhibitors are suitable. The inhibitor may inhibit iNOS, eNOS and/or nNOS. Preferably, it inhibits eNOS and/or nNOS. Suitable inhibitors include L-arginine analogues, thiocitrullines, indazole derivatives, imidazole derivatives, hydrazine derivatives, thioureas, thiazoles, biotin derivatives and phenyl-substituted thiopene amidines.
Examples of suitable L-arginine analogues include methyl-L-arginine, NG- nitro-L-arginine methyl esther (L-NAME), NG-monomethyl-L-arginine (L-NMMA), NG-amino-L-arginine (L-NAA), Nw,Nw-dimethyl-L-arginine ( ADMA), NW,N*2- dimethyl-L-arginine (SDMA), N^efhyl-L-arginine (L-NEA), Nw-methyl-L- homoarginine (L-NMHA), N -nitro-L-arginine (L-NOARG), Nδ-iminoethyl-L- ornithine (L-NIO), Nδ-iminoethyl-L-lysine (L-homo-NIO) and L-canavanine (L- CAN). Examples of suitable thiocitrullines include S-methyl-L-thiocitrulline
(SMTC), L-thiocitrulline (L-TC) and L-S-ethyl-thiocitrulline (Et-TC).
Examples of suitable indazole derivatives include indazole and 7-substituted indazoles such as 7-nitroindazole and 3-bromo-7-nitroindazole.
Examples of suitable hydrazine derivatives include aminoguanidine. Examples of suitable imidazole derivatives include phenyl substituted imidazoles such as 1-phenyl-imidazole.
Examples of suitable thioureas include S-methylisothiourea sulphate, δ-(S- methylisothioureido)-L-norvaline (L-MLN), S-ethylisothiourea (SETU) and S- isopropylisothiourea (SIPT). Examples of suitable thiazoles include 2-amino-thiazole and 2-amino-4,5- dimethyl thiazole.
Examples of suitable biotin derivatives include 2-iminobiotin.
Preferred NOS inhibitors are selective inhibitors of neuronal NOS. Such selective inhibitors include N -nitro-L-arginine (L-NOARG), Nw-nitro-L-arginine methyl ester (L-NAME), Nδ-iminoethyl-L-ornithine (L-NIO), L-thiocitrulline (L-
TC), S-methyl-L-thiocitrulline (SMTC), L-S-ethyl-thiocitrulline (Et-TC), 2-amino- thiazole, 2-amino-4,5-dimethyl thiazole, 7-nitroindazole (7-NI) and phenyl- substituted thiopene amidines.
The above NOS inhibitors are commercially available, or may be made by analogy with known methods. The inhibitor may be a pharmaceutically acceptable salt of one the above compounds. Suitable salts include salts with pharmaceutically acceptable acids, both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succininc, tartaric, benzoic, acetic, methanesulphonic, efhanesulphonic, benzenesulphonic or p-toluenesulphonic acid. Salts may also be formed with pharmaceutically acceptable bases such as alkali metal (eg sodium or potassium) and alkali earth metal (eg calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines or heterocyclic amines. Inhibitors of NOS can be identified by: (a) contacting a candidate compound with NOS and a substrate and co- factor therefor, under conditions under which NOS activity, in the absence of an inhibitor, would be expected to occur; and
(b) determining whether, or to what extent, NOS activity takes place. A suitable such assay for identifying inhibitors of NOS is a microtiter plate assay in which NOS activity is measured by determining the change in absorbance as
NADPH is converted to NADP*. This assay comprises:
(a) adding a candidate compound, a known NOS inhibitor (for example L-NMMA) and a buffer solution to separate microtiter wells;
(b) adding to each well NOS enzyme, cofactors therefor, L-arginine and buffer; and
(c) determining the change in absorbance in each well.
Typically, the buffer is a HEPES buffer capable of maintaining a pH of about 7, preferably about 7.4. The cofactors comprise oxyhemoglobin, NADPH and BH4. They may also comprise CaCl2, MgCl2, FMN, FAD and/or CaM. The NOS may be a naturally occurring form of eNOS, iNOS, or nNOS or may be a variant which retains NOS activity, for example variants produced by mutagenesis techniques. NOS used in the assay is preferably of mammalian origin, for example rodent (including rat and mouse) or primate (such as human).
Preferably, the NOS is of human origin.
The NOS may be obtained from mammal cellular extracts or produced recombinantly from, for example, bacteria, yeast or higher eukaryotic cells including mammalian cell lines and insect cell lines. Preferably, NOS used in the assay is recombinant. More preferably, it is obtained by expression in S/21 cells according to the methodology in Charles et al, Methods in Molecular Biology (edited by M.A.
Titheradge, Humana Press, Totowa), vol 100, pgs 51-60. Step (c) of the assay may be carried out by reading the difference in absorbance between 420 and 405 nm. Typically, this is done by a spectrometer.
Comparison of the well containing the candidate compound with the control wells containing a known NOS inhibitor (100% inhibition) and no inhibitor (0% inhibition) allows % inhibition achieved by the candidate compound to be calculated. A microtiter assay as set out above is described in detail in Dawson &
Knowles, Methods in Molecular Biology (edited by M.A. Titheradge, Humana Press,
Totowa), vol 100, Chapt. 22, pgs 237-242.
Any compound which is identified as an NOS inhibitor using an assay as described above can be used in the present invention. The NOS inhibitors used in the present invention typically achieve at least 50% NOS inhibition, more preferably at least 80% NOS inhibition. Ideally, they achieve substantially complete NOS inhibition.
Selective inhibitors of neuronal NOS can be identified by conducting the above microtiter assay using different purified NOS isoforms. Compounds which achieve a higher inhibition of nNOS than of eNOS or iNOS are selective nNOS inhibitors. Preferred selective nNOS inhibitors are those which inhibit nNOS at least twice as strongly, preferably at least five times as strongly, more preferably at least ten times as strongly, as eNOS and/or iNOS. Ideally, a selective nNOS inhibitor substantially does not inhibit eNOS or iNOS. The inhibitors of NO synthase may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The inhibitors may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The inhibitors may also be administered as suppositories. A NO synthase inhibitor is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tab letting, sugar-coating, or film coating processes.
Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginte, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. A therapeutically effective amount of a compound of the invention is administered to a patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g. Typically, administration of an NOS inhibitor is applied to a patient on an on-going basis, to provide continuous protection against degeneration of nitrergic nerves.
In some cases, administration of an NOS inhibitor in accordance with the invention may make it difficult for a patient to achieve an erection. In this event, an erection may be achieved by reducing the amount of NOS inhibitor, or ceasing administration of NOS inhibitor, in good time before an erection is required.
Typically, the dosage is reduced or the treatment suspended no more than 3 days before an erection is required. In some cases, dosage reduction or suspension of treatment up to 1 day before an erection is required will suffice.
If it is not possible to predict when an erection will be required in sufficient time to reduce the dosage or suspend treatment as described above, a further compound capable of inducing erection may be administered. Suitable such compounds include L-arginine, inhibitors of cyclic guanosine 3',5'-monophosphate phosphodiesterases (described in EP-B-702555) and activators of soluble guanylate cyclase (sGC). An example of an sGC activator is 3-(5'-hydroxymethyl-2'-furyl)-l- benzylindazole (YC-1) (Hobbs, A.J., TiPS, December 1997, Vol 18, p.484).
Preferably, the compound capable of inducing erection is L-arginine. L- arginine is the substrate for NOS and will therefore be capable of reversing the inhibition in most cases. This is confirmed in Czech et al, Pharmacol. Biochem. Behav. 1998 (60) p 91-96 and Luiking et al Am. J. Physiol. 1998 (274) G984-G991. The further compound capable of inducing erection can be formulated as set out above and administered at the dosages set out above. When the further compound is L-arginine, it is preferably formulated in a form suitable for oral administration and delivered at a dosage of about 50 mg per kg of body weight. The following Examples illustrate the invention. Reference Example 1
Induction of diabetes in rats
Male Wistar rats (200-250 g, n=320) were divided into four groups (Table I).
The first and second groups were injected with streptozotocin (STZ) (75 mg kg'1, i.p.). The third and fourth groups were injected with sterile saline (1 ml kg"1, i.p.). The second and fourth groups were given NG-nitro-L-arginine methyl ester (L- NAME) (0.05 mg ml"1 and 0.1 mg ml"1 respectively) in drinking water beginning 72 hours after the injection of STZ or saline. The 72 hour period was sufficient for diabetes to be established.
Diabetic rats were observed to drink approximately 100 ml day 1 whereas non-diabetic rats drank approximately 50 ml day 1; thus each animal in the second and fourth groups received 5 mg L-NAME each day. This dose has been shown to inhibit NO synthase activity but not to alter the renal function and catecholamine and insulin concentrations in the rat (Navarro et al, Am J. Physiol. 267, R1516 - R1521 (1994).
The blood glucose and body weights of the rats were monitored weekly and the results are shown in Table 1. Any animal in the first and second group with a blood glucose concentration lower than 20 mM at the end of the first week was excluded from the study. Another group of rats (weight-matched rats; 180-200 g: n=8) was also evaluated in pharmacological experiments; no significant difference was found between them and Group III (age-matched control rats). L-NAME was withdrawn 72 hours prior to the experiments detailed in the following Examples. This time period is sufficient to reduce the L-NAME concentrations in the blood by
80-90%. The experiments detailed in the following Examples were carried out in the absence of L-NAME. TABLE1
Example 1
After 4, 8, 12 and 16 weeks, some of the animals prepared in Reference Example 1 were sacrificed and perfused with formaldehyde for immunohistological studies. Rats were perfused through the left ventricle with 4% paraformaldehyde in
0.1 M phosphate buffer. Serial cryosections from the penises and anococcygeus muscles at 40 μm intervals were stained with a polyclonal antibody against nNOS (1:3000 dilution; prepared as described in Rodrigo et al, Phil. Trans. R. Soc. Lond. B, 345, 175-221 (1994)) or with a polyclonal tyrosine hydroxylase antibody (1:1000 dilution; Pel-Freez Biologicals, USA). The stained sections were visualised with biotinylated secondary antibodies and with avidin-biotin peroxidase complex and nickel-enhanced diaminobenzidine procedures. The results are shown in Figure 1. In Figure 1, (a) to (d) show sections stained using the polyclonal antibody against nNOS. (a) shows a stained section from a Group III (saline) control rat, (b) shows a stained section from a 4 week diabetic rat (note the varicosity formation and breakage in the nerve fibres), (c) shows a stained section from an 8 week diabetic rat (note that the nitrergic nerves became very sparse) and (d) shows a stained section from an 8 week diabetic rat treated with L-NAME (note that the structure and number of the nitrergic nerve fibres are preserved during diabetes).
(e) to (g) show sections stained with the polyclonal tyrosine hydroxylase antibody, (e) shows a stained section from a Group III (saline) control rat, (f) shows a stained section from an 8 week diabetic rat and (g) shows a stained section from an 8 week diabetic rat treated with L-NAME. Scale is 0.33 μm for panel c, 0.43 μm for others. The results are typical of those observed in 6 to 12 animals in each group. Sections from Group IV (saline + L-NAME) rats were not found to be significantly different from the sections from Group III rats.
The immunohistochemical study of the penis and anococcygeus muscles from 4 week diabetic rats showed varicosity formation and breakage in the nitrergic nerve fibres (Fig. lb). This degenerative process was more pronounced in the tissues from 8 week diabetic animals, thus leading to a decreased number of nitrergic nerve fibres (Fig. lc). Noradrenergic nerve fibres were intact, showing no degeneration even at 8 weeks (Fig. le-g). In tissues obtained from diabetic rats which had received L- NAME, however, the degenerative process was not observed in the nitrergic nerves (Fig. Id).
Example 2
The above morphological findings were supported with functional studies performed on the anococcygeus muscle of the rats prepared in Reference Example 1. Animals were sacrificed at 4 weeks and at 8 weeks for this study. Bilateral anococcygeus muscles from the rats were placed in a horizontal supervision chamber and superfused with modified Krebs' solution containing dexamethasone (5 μM) and indomethacin (5 μM) as described in Kasakov et al, Br. J. Pharmacol, 115, 149-1154 (1995).
The parameters of the electrical field stimulation (EFS) were 50 V, 0.3 ms pulse duration, 1-25 Hz, for 5 s, every 2 minutes. Nitrergic relaxation responses to
EFS were recorded after treatment of the tissues with scopolamine (5 μM) and guanethidine (5 μM) followed by elevation of the tone with phenylephrine (EC80) as described in Kasakov et al, Br. J. Pharmacol., 115, 149-1154 (1995). These responses were affirmed by total inhibition by 300 μM NG-nitro-L-arginine (L- NOARG) at the end of the experiment.
The results are shown in Figure 2.
In Figure 2, (a) shows that noradrenergic contractions elicited by EFS (5 Hz) were enhanced in the anococcygeus muscle of diabetic rats but not of the diabetic rats treated with L-NAME. (*significantly different from control; EO.001; n=6-12). (b) shows EFS - induced nitrergic relaxations in the anococcygeus muscle of Group III
(saline) control rats (closed squares), of 8 week diabetic rats (closed triangles) and of 8 week diabetic rats treated with L-NAME (open triangles) (*significantly different from control; O.05; n=6-12). There was no significant difference between Group III and Group IV animals. This functional study demonstrates that noradrenergic contractions were enhanced in the tissues from diabetic animals and, moreover, nitrergic relaxant responses were significantly decreased in diabetic animals. In tissues from diabetic animals treated with L-NAME, however, the noradrenergic contractile and nitrergic relaxant responses were not significantly different from control animals. To establish that defective nitrergic relaxation was not due to changes in the responsiveness of the smooth muscle to NO, concentration-response curves to exogenous sodium nitroprusside (SNP) in control and diabetic animals were prepared and were found to be indistinguishable. Concentration-response curves to exogenous noradrenaline were also prepared for control and diabetic animals and found to be indistinguishable. This excludes the possibility that the enhanced noradrenergic contraction might be due to hyperresponsiveness of the smooth muscle to noradrenaline. These results suggest that nitrergic neurofransmission becomes defective due to morphological changes in the nitrergic nerves which is prevented when the animals are treated with L-NAME.
Example 3
Rats prepared in Reference Example 1 were sacrificed at 1 week, 2 weeks, 3 weeks, 4 weeks and 8 weeks for the purpose of an NO synthase activity assay.
The whole penis and anococcygeus muscles of the rats were frozen in liquid nitrogen and pulverised in a stainless steel pestle and mortar. The homogenisation was performed using HESD buffer (20 mM HEPES pH:7.2, 1 mM' EDTA, 0.2 M sucrose, 5 mM dithiothreitol, 0.1 mM PMSF, 20 μg/ml leupeptin and soya bean trypsin inhibitor, 5 μg ml"1 pepstatin A, E-64, bestatin, aprotinin and 3,4- dichloroisocoumarin) and centrifuging at 13,000 g for 30 mins at 4°C. Endogenous L-arginine in the supernatant was removed using activated Dowex-50W resin. NO synthase was assayed in the supernatant by the formation of [U-1 C]-citrulline from L-[U-14C]-arginine as described in Knowles et al. In methods in Molecular Biology (ed. Titheradge, M.A.) (Humana Press, Totowa, 1997).
The results are shown in Figure 3 a, which demonstrates that constitutive (Ca2+-dependent) NO synthase activity decreased gradually during the course of diabetes in the anococcygeus muscle of rats (black columns) but did not change in the L-NAME-treated diabetic animals (white columns). The columns of the control group represent the results from saline-injected animals and saline-injected animals which were treated with L-NAME for 8 weeks respectively. (* significantly different from control: PO.05; n=6-20).
Inducible NO synthase activity was not detectable in any of the groups.
Example 4
Animals prepared in Reference Example 1 were sacrificed at 12 weeks, and homogenates were prepared in the same way as in Example 3. Equal amounts (39 μg lane 1) of the homogenates were run on 7.5% polyacrylamide SDS gels, then transferred to nitrocellulose filters. The blots were incubated for 1 hour with mouse anti-human nNOS (1 :2000 dilution; Transduction Labs, UK) and aήti-actin (1 :2000 dilution; Boehringer-Mannheim, UK) monoclonal antibodies. Final incubation was performed for 1 hour with horse radish peroxidase-conjugated goat anti-mouse IgG (1 :3000 dilution; Vectors Labs, UK). The reactive bands were detected with a luminol-based kit (ECL, Amersham, UK). The optimal X-ray exposure was selected, scanned and the density of each band was calculated by densitometry using a computer analysis program.
The results are shown in Figure 3b for Group III (saline) control rats, 12 week diabetic (STZ) rats and 12 week diabetic rats treated with L-NAME (STZ+L- NAME). There was no significant difference between Group III and Group IV animals. The graph represents the relative densitometric values as calculated for the intensities of the main 160 K bands on the blots divided by the corresponding intensity of the actin bands (*significantly different from control; PO.05; n=3). The analysis of the main band with an Mr of 160 K revealed a significant 48% reduction of the band intensity in the diabetic rats. This effect was not observed in the diabetic animals treated with L-NAME.
Example 5
The protective effect of L-NAME on the erectile function of the rats prepared in Reference Example 1 was investigated by studying the effects of in vivo electrical stimulation of the cavernous nerve. Anaesthesia was induced in the rats with sodium thiopentone (120 mg kg"1, i.p.) and was maintained by a subsequent injection of sodium pentόbarbital (5 mg kg'1, i.p.) as required. The right external jugular vein and left carotid artery were cannulated for saline infusion (50 μl min'1) and for blood pressure monitoring, respectively. The penile shaft and cavernous nerve were exposed as described in Rehman et al, Am. J. Physiol. 272, H1960-H1971 (1997).
Intracavernous pressure (ICP) in the right crus of the penis was monitored using a 25-gauge cannula connected to a pressure transducer. The cavernous nerve was stimulated by bipolar platinum hook electrodes (0.5 mm in diameter). Square pulses of 10 Hz, 0.3 ms pulse duration, for 1-3 minutes, 0.05-0.5 mA were applied via a Grass stimulator coupled to a Grass constant current unit.
The results are shown in Figure 4, in which a shows typical traces from a Group III (saline) control rat (upper trace), a 12 week diabetic rat (middle trace) and a 12 week diabetic rat treated with L-NAME (lower trace). There was no significant difference between Group III and Group IV animals. Note that the increase in intracavernous pressure (ICP) in the diabetic rat could not be maintained during the stimulation period. The vertical scale corresponds to 100 cmH2O ICP. The horizontal scale corresponds to 2 minutes.
In Figure 4b, the increase in ICP (measured as the area under the curve and expressed as arbitrary units) is plotted against the amplitude of the electrical stimulation of the cavernous nerve in the Group III (saline) control rats (squares), 12 week diabetic rats (triangles) and 12 week diabetic rats treated with L-NAME (circles) (*significantly different from control; PO.05; n=6-10). There was no significant difference between Group III and Group IV animals.
These results show that L-NAME can provide protection of the erectile function in vivo. Reference Example 2
Male New Zealand white rabbits (2.5-3.0 kg; n=64) were divided into two groups. After sedation with a mixture of fentanyl citrate (0.315 mg ml"1) and fluanison (10 mg ml"1) (0.3 ml kg"1; i.m.), the first group was injected with alloxan, as described in Howell et al, J. Endocrinol. 37, 421-427 (1967) (95 mg kg 1; i.v.). The second group was injected with sterile saline (95 ml kg"1; i.v.). Any animal with a blood glucose concentration lower than 20 mM at the end of the first week was excluded from the study.
At the end of 30 weeks, alloxan- injected rabbits failed to gain weight compared to saline-injected rabbits (2.96+0.70 kg (n=30) and 4.86±0.15 kg (n=12) respectively; p<0.05) and had higher plasma glucose levels (43.1+1.07 mM (n=30) and 6.1+0.5 mM (n=12) respectively; p<0.05). No significant difference was found in the pharmacological experiments between control rabbits (age-matched, as described above) and weight-matched rabbits (2.5-2.8 kg, n=6).
Example 6
To investigate changes in the nitrergic i nervation of alloxan-induced diabetic rabbits, animals prepared in Reference Example 2 were sacrificed 24 and 30 weeks after injection of alloxan. Bilateral anococcygeus muscles and penile corpus cavernosum from the rabbits were placed in a horizontal superfusion chamber and superfused with modified Krebs' solution containing dexamethasone (5 μM) and indomethacin (5 μM) as described in Kasakov et al, Br. J. Pharmacol, 115, 149-1154 (1995). The parameters of the electrical field stimulation (EFS) were 50 V, 0.3 ms pulse duration, 1-25 Hz, for 5 s, every 2 minutes. Nitrergic relaxation responses to EFS were recorded after treatment of the tissues with scopolamine (5 μM) and guanethidine (5 μM) followed by elevation of the tone with phenylephrine (EC80) as described in Kasakov et al, Br. J. Pharmacol, 115, 149-1154 (1995). These responses were affirmed by total inhibition by 300 μM NG-nitro-L-arginine (L- NOARG) at the end of the experiment. The results are shown in Figure 5, in which a shows typical traces of the corpus cavernosum in a control rabbit (1), a 24 week (2) and a 30 week (3) diabetic rabbit. Note that the lag-period between the end of EFS and the beginning of the contraction became negative in diabetic animals; i.e. the noradrenergic contraction started during EFS. Moreover, the noradrenergic contractions became larger in magnitude as diabetes progressed. The vertical scale corresponds to 1 g of force.
Figure 5b shows the lag-period between the end of EFS and the beginning of the contraction of the anococcygeus muscle (black columns) and corpus cavernosum (white columns) in age-matched control (1), weight-matched control (1W), 24 week (2) and 30 week (3) diabetic rabbits (*significantly different from control; PO.001; n=6-8).
The absence of a lag-period following the nerve stimulation in diabetic rabbits demonstrated a predominance of noradrenergic versus nitrergic innervation (Cellek et al, Proc. Natl. Acad. Sci. USA, 94, 8226-8231 (1997)). As shown in Figure 5a, this phenomenon could be observed at 24 weeks and became maximum at 30 weeks.
Furthermore, nitrergic relaxation responses in the anococcygeus muscle from 24 and 30 week diabetic rabbits were significantly lower than in the anococcygeus muscle from the control animals (EFS at 5 Hz elicited relaxations of 89.1+9.2%, 54.6+6.8%) and 36.7+8.2% of the maximum tone in control, 24 week diabetic and 30 week diabetic rabbits respectively; E<0.05 vs control; n-4-6). There was found to be no change in the concentration response curve to exogenous SNP or noradrenaline in t either anococcygeus muscle or corpus cavernosum of the diabetic animals. These results suggest that nitrergic transmission became defective during the course of diabetes in alloxan-administered rabbits which caused a nitrergic-noradrenergic imbalance, confirming the results in STZ-induced diabetic rats.
Example 7
In order to investigate biochemical analysis of the constitutive NO synthase in the corpus cavernosum of diabetic rabbits, rabbits prepared in Reference Example 2 were sacrificed after 30 weeks. The corpus cavernosum of the rabbits were frozen in liquid nitrogen and pulverised in a stainless steel pestle and mortar. The homogenisation was performed using HESD buffer (20 mM HEPES pH:7.2, 1 mM EDTA, 0.2 M sucrose, 5 mM dithiothreitol, 0.1 mM PMSF, 20 μg/ml leupeptin and soya bean trypsin inhibitor, 5 μg ml"1 pepstatin A, E-64, bestatin, aprotinin and 3,4-dichloroisocoumarin) and centrifuging at 13,000 g for 30 min at 4°C. Endogenous L-arginine in the supernatant was removed using activated Dowex-50W resin. NO synthase was assayed in the supernatant by the formation of [U-14C]-citrulline from L-[U-14C]- arginine as described in Knowles et al, In Methods in Molecular Biology, (ed. Titheradge, MA) (Humana Press, Totowa, 1997). The results showed a significant decrease in the activity of the enzyme at the
30th week of diabetes (20.07+2.78 pmol/mg protein/min in control rabbits vs 11.34+2.56 pmol/mg protein/min in 30 week diabetic rabbits; PO.05; n=6). These results suggest that nitrergic transmission became defective during the course of diabetes in alloxan- administered rabbits.

Claims

1. Use of a pharmaceutically acceptable inhibitor of NO synthase in the manufacture of a medicament for use in preventing or reducing degeneration of nitrergic nerves in a diabetic mammal.
2. Use according to claim 1, wherein the medicament is for use in preventing or reducing degeneration of nitrergic nerves containing neuronal NO synthase.
3. Use according to claim 1 or 2, wherein the medicament is for use in preventing erectile dysfunction in a male mammal.
4. Use according to any one of the preceding claims, wherein the mammal is suffering from diabetes mellitus.
5. Use according to any one of the preceding claims, wherein the inhibitor of NO synthase is a selective inhibitor of neuronal NO synthase.
6. Use according to any one of the preceding claims, wherein the inhibitor of NO synthase is an L-arginine analogue, a thiocitrulline, an indazole derivative, an aminoguanidine derivative or a thiourea.
7. Use according to any one of the preceding claims, wherein the diabetic mammal is a human.
8. A method of preventing or reducing degeneration of nitrergic nerves in a diabetic mammal, which method comprises the administration to the said mammal of an effective amount of a pharmaceutically acceptable inhibitor of NO synthase.
9. An agent for the prevention or reduction of degeneration of nitrergic nerves in a diabetic mammal, comprising a pharmaceutically acceptable inhibitor of NO synthase.
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