WO2005018620A2 - Use of nitric oxide synthase (nos) cofactor for the treatment of sexual dysfunction - Google Patents

Abstract

Compositions and methods are provided for the treatment of sexual dysfunction in a mammal, including male sexual dysfunction, such as erectile dysfunction, and female sexual dysfunction. In particular, the use of a NOS cofactor in the preparation of a medicament for the treatment and/or prevention of sexual dysfunction is described.

Description

Use of nitric oxide synthase (NOS) cofactor for the treatment of sexual dysfunction

Field of the invention

The present invention relates to the technical field of treatment of sexual dysfunction. In particular, the present invention relates to the use of a nitric oxide synthase (NOS) cofactor for the preparation of a pharmaceutical composition for the treatment of sexual dysfunction of a subject, including both male erectile dysfunction and female sexual dysfunction, including methods and formulations for the use and administration of the same.

Background of the invention

Both males and females can suffer from sexual dysfunction. Sexual dysfunctions are relatively common in the general population; see O'Donohue et al., Clin. Psychol. Rev. 17 (1997), 537-566. The disorder may relate to seeking sexual behavior (proceptivity) and/or to acceptance of sexual behavior, accompanied by sexual arousal (receptivity). The prevalence of sexual problems is higher in populations receiving medicaments, in particular antidepressants and anti- hypertensives. A need for pharmacotherapy for sexual dysfunction is increasing, but there has been very little research effort directed at finding drugs to treat sexual dysfunction. Sexuai dysfunctions include erectile dysfunctions (ED) of organic and psychogenic origin (Benet and Melman, Urol. Clin. N. Amer. 22 (1995), 699-709) as well as hypoactive sexual desire disorders, sexual arousal disorders, anorgasmy and sexual pain disorders (Berman et al., Urology 54 (1999), 385-391).

In males, impotence can be defined as an inability to achieve penile erection or ejaculation. Its prevalence is claimed to be between 2 % and 7 % of the human male population, increasing with age up to 50 years, and between 18 % and 80 % between 55 and 80 years of age. In the USA alone, for example, it has been estimated that there are up to 10 million impotent males, with the majority suffering from problems of organic rather than of psychogenic origin. Although many different drugs have been shown to induce penile erection, they were only effective after direct injection into the penis, e.g., intraurethrally or intracavernosally (i.e.) and were not approved for erectile dysfunction. US-A-5, 576,290 discloses peptides which are stated to induce erection, but they have to be given subcutaneously, e.g., by injection, and if an excessive dose is given they produce an exaggerated erectile response and stomach discomfort. Impotence treatment was revolutionized by the unexpected discovery that cGMP PDE5 inhibitors, e.g. pyrazolo[4,3-d]pyrimidin-7-ones were useful in the treatment of erectile dysfunction and could be administered orally, therefore obviating the disadvantages associated with l c. administration.

In addition to oral medication with PDE5 inhibitors, intracavernous prostaglandin injection therapy (SKAT) is also a well-established component of the therapy cascade. All treatment options entail specific side effects or contraindications; see Table 1. Table 1: Treatment options, side effects and problems, contraindications

In recent years, therapy for erectile dysfunction has focused mainly on the PDE5 inhibitor sildenafil (Viagra©). The success rate for sildenafϊl varies in the group of ED patients with an organically caused disease from 43 % in patients who have undergone radical prostatectomy to 85 % in patients with a neurologically related disorder, and amounts to an average of 82 % (range 43-85 %, 100 mg) (Gresser and Gleiter, Eur. J. Med. Res. 7 (2002), 435-446). A certain proportion of the patient population, such as diabetics, does not gain significant benefit from PDE5 inhibitors, possibly due to a lack of endogenous nitric oxide. Therefore, new treatment modalities based on the absence of endogenous nitric oxide have been developed. Among them are Rho-kinase inhibitors, soluble guanylate cyclase activators and nitric oxide- releasing PDE5 inhibitors (Cellek et al., Expert Opin. Investig. Drugs 11 (2002), 1563-1573. All of these are not directly linked to the reason behind erectile dysfunction. Thus, there is a need foi novel medications in the treatment of male sexual dysfunction.

Thirty to 50 % of American women complain of sexual dysfunction. Ageing, menopause, and decline in circulating estrogen levels significantly increase the incidence of sexual complaints. In a recent publication, Berman et al., (Int. J. Impot. Res. 11 (1999), S31-38), describe methodology for evaluating physiologic and subjective components of the female sexual response in the clinical setting and determine the effects of age and estrogen status on them. Low or absent sexual drive/desire constitutes the commonest problem in the female population (Laumann et al., JAMA 281 (1999), 537-544), but no therapy is available other than psychotherapy or empirical approaches. In a further publication (Bonney et al., Scrip's Complete Guide to Women's Healthcare, PJB Publications Ltd., London, 2000) the causes and management of female sexual dysfunction are discussed, including the use of tibolone (Livial; Organon) which is a synthetic steroid that mimics the effects of estrogen and has been reported to have mild androgenic properties, and the use of testosterone.

Most current female sexual dysfunction (FSD) research focuses on vasoactive agents for treatment of female sexual arousal disorder. The underlying assumption is that genital blood flow is responsible for female arousal, and thus vasoactive agents that increase flow could improve arousal deficiency (Davis, Curr. Psychiatry Rep. 2 (2000), 21 1-214). Preliminary results from small pilot studies suggest vasoactive agents may be efficacious. Laboratory evidence indicates that phosphodiesterase type 5 modulates human clitoral smooth muscle tone (Park, Biochem. Biophys. Res. Commun. 249 (1998), 612-617). Thus, phosphodiesterase inhibitors such as sildenafil might have impact on sexual response. The success of the different drugs for female sexual dysfunction, however, was low (Perelman, J. Sex. Marital Ther. 28 (2002), 195-204; Modelska, Am. J. Obstet. Gynecol 188 (2003), 286-293). It seems therefore necessary to find new therapeutic strategies in the treatment of female sexual dysfunction.

The solution to the above described problems is achieved by providing the embodiments characterized in the claims, and described further below. Summary of the invention

The present invention is based on the realization that substances that act as a cofactor of nitri< oxide synthase (NOS) have utility in the treatment of sexual dysfunction, including th< behavioral component thereof, in both male and female subjects. In other words, they cai provide a treatment for erectile dysfunctions of organic and psychogenic origin as well a: hypoactive sexual desire disorders, sexual arousal disorders, anorgasmy and sexual pair disorders.

The invention therefore provides a method of treating sexual dysfunction which comprise: administering to a subject suffering therefrom and in need of treatment an effective amount of ∑ cofactor of nitric oxide synthase (NOS); said cofactor preferably being tetrahydrobiopterir (BH4) or a precursor or analogue thereof. The invention further provides the use of a cofactor ol nitric oxide synthase (NOS) in the manufacture of a medicament for preventing or treating ma sexual dysfunction or female sexual dysfunction.

The use of the NOS cofactor in accordance with the present invention may be accompanied b) the use of further therapeutic agents such as a phosphodiesterase (PDE) inhibitor, a prostaglandir (PG), apomorphine, papaverin or phentolamin. Therefore, the present invention also concerns z pharmaceutical composition comprising a NOS cofactor such as BH4 and a second therapeutic such as sildenafil or vardenafil.

Hence, in one aspect of the present invention substances and compositions are provided, which can improve female sexual function and maintenance of functional and structural integrity ol vaginal and clitoral tissue. Those substances and compositions are based on nitric oxide synthase enzyme cofactors, in particular tetrahydrobiopterin (BH ), its precursors and its analogues. Those substances and compositions can advantageously be administered by systemic application. The, up to now, used therapeutics for treatment of female sexual dysfunction do not prevent reasons of female sexual dysfunction, i.e. the possible reduced or diminished NO release by endothelial NOS (eNOS) that in accordance with the present invention has surprisingly been found to be a main cause for sexual dysfunction. Furthermore, previous treatments lack effect in a distinct percentage of patients and no long-time protection of vaginal and clitoral tissue has been obtained. BH4 is an important factor for eNOS-dependent NO release. A reduction or lack of BH4 leads tc a dysfunction of eNOS, which causes a switch from NO release to formation of oxygen free radicals. In accordance with the present invention, it was found that the increased formation ol free radical reaction products, without increase of NO/oxygen free radical reaction producl nitrotyrosine in corpus cavernosum tissue indicates that an imbalance of NO release and oxygen free radical formation by eNOS is the final mechanism leading to erectile dysfunction and injury of erectile tissue in male and can lead to functional and structural alteration of vaginal and clitoral tissue. Accordingly, a causal therapy with BH4, its precursors or analogues can improve female sexual function and can prevent injury of vaginal and clitoral tissue. Therefore BH4 should have direct effects for improvement of female sexual function alone or in combination with othei downstream working therapeutics like PDE-5 inhibitors and a long-time effect by prevention oi oxygen free radical damage of vaginal and clitoral tissue. Two further advantages of BH4 treatment should be also considered, the low side effects and the positive effects on disease which are often associated with female sexual dysfunction.

In accordance with the above, a further aspect of the present invention relates to the use of nitric oxide synthase cofactors such as tetrahydrobiopterin (BH4), and its precursors and analogues, to manufacture compositions for improving erectile function and maintenance of functional and structural integrity of corpus cavernosum tissue. Those compositions can be used in systemic administration as well. The therapeutic options in use until now for the treatment of erectile dysfunction (ED) do not prevent the condition underlying erectile dysfunction in accordance with the present invention: namely, the reduced or diminished NO release by endothelial NOS (eNOS). In addition, present-day therapies are unable to ameliorate ED in a distinct percentage of patients. Furthermore, no long-term protection of cavernosal tissue is obtained.

A causal therapy using BH4, or its precursors or analogues, can improve erectile function and prevent injury of erectile tissue. BH4 should hence have beneficial effects for the improvement of erectile function, either alone or in combination with other downstream therapeutics such as PDE5 inhibitors, in addition to the long-term effect of preventing oxygen free radical damage of erectile tissue. Two further advantages of BH4 treatment should also be considered: the low side effects and the positive effects on diseases that are often associated with erectile dysfunction.

It is an object of the present invention to provide a method for improving female sexual function, desire and/or maintenance of functional and structural integrity of clitoral and vaginal tissue, for the treatment of benign prostatic hyperplasia, or for improving erectile function and/or maintenance of functional and structural integrity of corpus cavernosum tissue, comprising the step of administering to a subject having or suspected to get a sexual dysfunction a therapeutic amount of a NOS cofactor such as BH4, optionally in combination with a second therapeutic agent such as those mentioned above.

It is another object of the present invention to provide a use of a NOS cofactor as single or co- medication for the treatment of subvesical obstruction caused by prostatic hyperplasia or bladder neck sclerosis, wherein said co-medication is, for example, a prostate-selective medication such as a prostate-selective alpha reductase inhibitor or alpha receptor inhibitor.

Another object of the invention is to provide the use of a NOS cofactor as single or co- medication for the treatment of interstitial cystitis, low bladder capacity disorders or of micturition disorders as urge incontinence.

These and other objects of the invention are achieved by providing the embodiments as described further below.

Detailed description of the invention The present invention relates to the use of a nitric oxide synthase (NOS) cofactor for the preparation of a pharmaceutical composition for the treatment of sexual dysfunction of a subject. Preferably, said cofactor is tetrahydrobiopterin (BH4) or a precursor or analogue thereof

The present invention is based on the observation that the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin and its precursors can be used to produce a substance that can improve erectile function and maintenance of functional and structural integrity of corpus cavernosum tissue; see Example 2. In particular, patients with erectile dysfunction have been treated for 3 to 6 months with BH4 or sepiapterin, and the alteration in erectile function was monitored. In this study the sole effect of BH4 or sepiapterin, as well as the effect of BH4 or sepiapterin in combination with a PDE5 inhibitor, has been investigated in erectile dysfunction and it could surprisingly be shown that BH4 considerably improves penile response.

Without intending to be bound by theory it is believed that BH4, its precursors and its analogues could improve erectile function by enhancing NO-dependent relaxation of corpus cavernosum smooth muscle and protect corpus cavernosum tissue from degeneration, which entails a reduction of erectile tissue when smooth muscle and endothelium are replaced by non-erectile fibrous tissue. The experiments performed in accordance with the present invention led to the conclusion that BH4 and other NOS cofactors could ameliorate the NOS-dependent imbalance of NO release and oxygen radical formation in the human corpus cavernosum. The substances can be used alone or in combination with other substances that work by means of increased activation or support of the NO/cGMP pathway. The increased formation of oxygen free radicals was observed when an increased amount of the free radical reaction product 8-isoprostane was detected in corpus cavernosum tissue of 10 patients with erectile dysfunction. All of the patients showed an increased amount of 8-isoprostane in smooth muscle and sinus endothelium, the main localizations of endothelial NOS in corpus cavemosum tissue (Bloch et al., Urol. Res. 26 (1998), 129-135), compared to patients with normal erectile function. In contrast to the increased 8- isoprostane, no alteration in nitrotyrosine formation could be found, although an increase in oxygen radicals should lead, with an unchanged NO level, to increased peroxynitrite production and subsequently to increased nitrotyrosine formation. Since there was no significant alteration in endothelial NOS expression in corpus cavernosum tissue of patients with erectile dysfunction (Bloch et al. 1998), this gives strong evidence for an imbalance in NO release and oxygen radical formation by endothelial NOS in the corpus cavernosum tissue of patients with erectile dysfunction. In accordance with the present invention, the main reason for the imbalance in NO release and oxygen free radical production is expected to be due to a lack of BH4. The current literature concerning the biochemistry and the role of nitric oxide synthase in NO release and oxygen free radical production is discussed with respect to the theory thought to be behind the present invention in the example section below.

The therapeutic use of BH4, its precursors or its analogues, could ameliorate the NOS-dependent imbalance of NO release and oxygen radical formation in the human corpus cavernosum. This could improve erectile function by enhancing NO-dependent relaxation of corpus cavernosum smooth muscle and protect corpus cavernosum tissue from degeneration, which entails a reduction of erectile tissue when smooth muscle and endothelium are replaced by non-erectile fibrous tissue. Although there is no direct information about the role of free radicals in female sexual dysfunction, the structural and physiological intercourses makes it very likely that oxygen free radicals play a role in the aetiology of female sexual dysfunction as well. Thus, the present invention relates to methods for treating sexual dysfunction in a patient comprising administering to the patient an effective amount of BH4 or of derivatives of BH4.

The term "sexual dysfunction" is used herein in its broadest sense as it applies to both males and females. Generally, the term denotes the inhibition of one or more of the phases of sexual response including appetite, excitement, orgasm or resolution. In males, sexual dysfunction encompasses, for example, decreased sexual desire, the inability to sustain an erection, the inability to ejaculate and/or the inability to experience orgasm. In females, sexual dysfunction refers, for example, to pain or discomfort during sexual intercourse, diminished vaginal lubrication, delayed vaginal engorgement, increased time for arousal, diminished ability to reach orgasm, and/or diminished clitoral sensation. It will be appreciated that treatment of sexual dysfunction as used herein in reference to both males and females encompasses the enhancement of sexual pleasure and stimulation. For example, an orgasmic woman seeking a more pronounced sexual response can be treated according to the present methods. A more pronounced sexual response or enhancement of sexual pleasure includes, but is not limited to, decrease in the amount of foreplay, decrease in the period between orgasms, decrease in the intercourse time required for orgasm, and achievement of multiple orgasms.

The term "patient" as used herein refers generally to both male and female members of the animal kingdom. Because the present methods include inducement of sexual stimulation, they have application in, for example, animal husbandry. The present methods are therefore not limited to applications for humans.

In a particularly preferred embodiment of the present invention said cofactor is a compound of formula (I):

wherein R and R each represent a hydrogen atom or, taken together with each other, represent a single bond, while R³ represents -CH(OH)CH(OH)CH₃, -CH(OCOCH₃)CH(OCOCH₃)CH₃, -CH , -CH₂OH, or a phenyl group when R¹ and R² each represent a hydrogen atom, or -COCH(OH)CH₃ when R and R together represent a single bond, or a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable composition containing either entity. Preferred are compounds wherein R³ is L-erythro-CH(OH)CH(OH)CH₃.

In a particularly preferred embodiment of the present invention, said compound is selected from the group consisting of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), (6R,S)-5,6,7,8- tetrahydrobiopterin, ,2'-diacetyl-5,6,7,8-tetrahydrobiopterin, sepiapterin, 6-methyl-5,6,7,8- tetrahydropterin, 6-hydroxymethyl-5,6,7,8-tetrahydropterin, 6-phenyl-5,6,7,8-tetrahydropterin, or a precursor of any one thereof. Most preferably, said compound is a 5,6,7,8- tetrahydrobiopterin or a salt thereof; e.g., BH4 or a salt thereof.

The above described compounds of the formula (I) used as effective ingredients in the present invention are mostly known compounds; see, for example, European patent application EP 0 908 182-A and Japanese patent publication disclosures (KOKAI) Nos. 25323/84, 76086/84, 277618/86 and 267781/88. These compounds may be used as appropriate salts with pharmacologically non-toxic acids, including mineral acids such as hydrochloric acid, phosphoric acid, sulfuric acid, boric acid; and organic acids such as acetic acid, formic acid, maleic acid, fumaric acid, mesylic acid.

In European patent application EP 0 908 182-A compounds of formula (I) are described in context with preventing or ameliorating diseases caused by dysfunction of NOS, in particular for the treatment of hypertension and renal diseases. However, the specific utility of those compounds for the treatment of sexual dysfunction such as erectile dysfunction or dysfunction of sexual arousal has neither been considered nor suggested.

A pharmaceutical composition of the present invention is prepared by formulating a NOS cofactor such as a compound of formula (I) with generally used carriers by conventional procedures into a dosage form suitable for oral, intracavernous, rectal or parenteral administration (including administration into vein and cerebrospinal fluid). The carriers used for these pharmaceutical formulations generally include excipients, binders, disintegrators, etc. depending on the dosage form chosen. Typical examples of excipients include starch, lactose, sucrose, glucose, mannitol and cellulose, and examples of binders include polyvinylpyrrolidone, starch, sucrose, hydroxypropyl cellulose, Arabic gum. Examples of disintegrators include starch, agar, gelatin powder, cellulose, CMC, but any other conventional excipients, binders and disintegrators may also be used. In addition to such carriers, the pharmaceutical composition of the present invention may also contain antioxidants for stabilizing effective ingredients. Antioxidants can be appropriately selected from those conventionally used for pharmaceutical preparations, such as ascorbic acid, N-acetylcysteine, L-cysteine, dl-alpha-tocopherol, natural tocopherol etc. They are used in an amount that stabilizes the active ingredient(s) (one or more) and generally, the ratio of an antioxidant averages between 0.2 and 2.0 parts by weight to 1 part by weight of the active ingredient(s).

Formulations of the present invention suitable for oral administration may be provided in the form of tablets, sublingual tablets, capsules, powders, granules or fine granules, or suspensions in a non-aqueous liquid such as syrups, emulsions or draft (pro re nata preparation) that contain the prescribed amount of the active ingredient (one or more).

For example, granules are prepared by homogeneously mixing the active ingredient(s) (one or more) with one or more auxiliary ingredients such as carriers and antioxidants as mentioned above, followed by granulation and sieving to uniform grain size. Tablets can be prepared by compressing or molding the active ingredient(s) (one or more) optionally together with one or more auxiliary ingredients. Capsules are prepared by filling powder or granules of active ingredient (one or more) optionally mixed homogeneously with an auxiliary ingredient (one or more) into appropriate capsules using a capsule filling machine or the like. Formulations for rectal administration can be provided as suppositories using conventional carriers such as cocoa butter. Parenteral formulations can be provided as dry solids of the active ingredient(s) (one or more) sealed in a nitrogen-filled sterilized containers. Such dry solid preparations can be administered to patients after dispersing or dissolving them into a determined amount of sterilized water just prior to administration.

In manufacturing these formulations, antioxidants as mentioned above may preferably be added to a mixture of an effective ingredient and conventional carriers, and optionally one or more auxiliary ingredients selected from buffers, flavors, surfactants, viscosants, lubricants, etc. can also be added, if needed. The dosage of active ingredients, i.e. NOS cofactor may vary with the administration route, the symptom to be treated and the patient condition, and the final determination of the dosage should be made by an attendant physician. For example, an appropriate dosage for treating erectile dysfunctions ranges from 0.1 to 1000 mg/kg (b.w.)/day, and representative optimal dosage is 10 to 100 mg/kg (b.w.)/day. A desired dosage of said active ingredients may be administered once a day or in divided doses of two to four times a day at appropriate intervals. Active ingredients may be administered alone or in combination with pharmaceutical formulations containing other active ingredients suitable for the disease under treatment to facilitate control of the dosage, for example.

In addition to NOS cofactors such as compounds of formula (I) as effective ingredients, formulations of the present invention may contain at least one auxiliary effective ingredient selected from the substrates or coenzyme or cofactor for NOS such as L-arginine, flavins, for example, FAD, FMN, etc., or calcium. More excellent therapeutic effects can be expected when NOS cofactors are mixed with these effective ingredients than when used alone. The proportion of each of said auxiliary effective ingredients in formulations of the present invention is not specifically limited. For example, the weight ratio of at least one formulation selected from L- arginine, flavins and calcium to 1 part of the NOS cofactor such as compounds of formula (I) may be within the range from 0.1 to 10, preferably 0.5 to 2. For example, an appropriate dosage of such mixed formulations for treating sexual dysfunctions ranges from 0.1 to 1000 mg/kg (b.w.)/day, preferably 10 to 100 mg/kg (b.w.)/day, in terms of the total amount of active ingredients.

A physician may appropriately choose formulations containing compounds of formula (I) alone or in combination with other active ingredients, depending on the age, condition or other factors of the patient.

The most preferable active ingredients used in the present invention are (6R)-L-erythro-5,6,7,8- tetrahydrobiopterin (BH4) and salts thereof, but (6R,S)-5,6,7,8-tetrahydrobiopterin, l',2'- diacetyl-5,6,7,8-tetrahydrobiopterin, sepiapterin, 6-methyl-5,6,7,8-tetrahydropterin, 6- hydroxymethyl-5,6,7,8-tetrahydropterin or 6-phenyl-5,6,7,8-tetrahydropterin and salts thereof may also be used. Needless to say, BH4, naturally occurring component is most preferable. BH4 dihydrochloride has little toxicity to rats judging from the fact that an acute toxicity was more than 2 g/kg (b.w.). An optically inactive analogue, (6R,S)-5,6,7,8-tetrahydrobiopterin is also only slightly toxic as reported in Japanese Patent Public Disclosure No. 25323/84 for the treatment of Parkinson's disease, so that it can also be used for the therapy according to the present invention. Other compounds of formula (I) also show little or no acute toxicity.

Hence, the appropriate concentration of the therapeutic agent might be dependent on the particular agent. The therapeutically effective dose has to be compared with the toxic concentrations; the clearance rate as well as the metabolic products play a role as do the solubility and the formulation. Therapeutic efficacy and toxicity of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50 % of the population) and LD50 (the dose lethal to 50 % of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio LD50/ED50.

The pharmaceutical composition may be administered by any means known in the art, including administration by intravenous and intracavernous injection, for administration through mucous membranes, buccal or sublingual administration, oral administration, dermal administration, inhalation administration and nasal administration. In a preferred embodiment, administration is effected by systemic administration of a metered amount of a formulation including an aqueous buffer, which buffer may be a saline or citrate buffer. Most preferably, the composition is formulated as a tablet and comprises ascorbic acid and/or N-acetyl-L-cysteine as a diluent.

For preparing pharmaceutical compositions from the compounds of this invention, inert pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, sachets, and suppositories. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain 5 % to about 70 % of the active component. Suitable carriers are magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and the like. Liquid form preparations include solutions, suspensions, and emulsions. Sterile water or water- propylene glycol solutions of the active compounds may be mentioned as an example of liquid preparations suitable for parenteral administration. Liquid preparations can also be formulated in solution in aqueous polyethylene glycol solution. Aqueous solutions for oral administration can be prepared by dissolving the active component in water and adding suitable colorants, flavoring agents, stabilizers, and thickening agents as desired. Aqueous suspensions for oral use can be made by dispersing the finely divided active component in water together with a viscous material such as natural synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other suspending agents known to the pharmaceutical formulation art.

Preferably the pharmaceutical preparation is in unit dosage form. In such form, the preparation is divided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, sachet, or tablet itself, or it can be the appropriate number of any of these packaged forms.

For preparing suppository preparations, a low-melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into conveniently sized moulds and allowed to cool and solidify.

Furthermore, the active ingredient(s) can be designed to be released in an active form over time. General methods for the preparation of retard compositions, i.e. compositions for controlled release of drugs are known in the art; see, e.g., Gupta et al., Drug Discov. Today 7 (2002), 569-579. Controlled release can be achieved for example by chemical modification of the agent or application as a prodrug, whereby the prodrug is inactive and rendered active by a metabolic reaction at the site of application. Similarly various formulations of agents are possible tailoring the release according to needs. In the compositions of the present invention the therapeutic agent can be cross-linked with the resorbable substance such that it is released when the substance is degraded. Compositions that are adapted for oral administration to humans are preferred, especially such compositions in unit dosage form.

As mentioned before, the pharmaceutical compositions of the present invention can be used for the treatment of sexual dysfunction of both, male and female. Female sexual dysfunctions are highly prevalent but not well defined and understood. Existing definitions and classifications of female sexual dysfunctions are reevaluated and revised at present. Contemporary classification was expanded to include psychogenic and organic causes of desire, arousal, orgasm and sexual pain disorders. Female sexual dysfunction (FSD) was recently recognized as arising from multiple organic etiologies; it is not primarily a psychological symptom as believed previously. A symptom-related complex resulting in physiologic changes, FSD can respond to either treatment of the underlying condition or supportive measures (Lightner, Mayo. Clin. Proc. 77 (2002), 698-702). Limited studies are available concerning physiological and biochemical mechanisms involved in female sexual function and dysfunction (Traish et al., Arch. Sex. Behav. 31 (2002), 393-400). A comprehensive and integral understanding of female sexual function requires detailed investigation of the vascular, neurological (central and peripheral), and structural components of this complex physiological process. The female sexual response cycle is initiated by neuro transmitters which increase pelvic blood flow, vaginal lubrication, clitoral and vaginal reactivity. These changes are regulated by the tone of vascular and non-vascular smooth muscle in the vagina and clitoris. Recent studies have suggested that several physiological modulators such as vasoactive intestinal polypeptide (VIP), nitric oxide (NO), and prostaglandin E (PGE) regulate vaginal smooth muscle contractility. Data from experimental models have provided a preliminary understanding of the mechanisms of the female sexual arousal response (for review see Min et al., Int. J. Impot. Res. 13 (2001), 151-156).

Under consideration of some morphological and structural intercourses between female and male outer sexual organs, it seems appropriate to extend knowledge from regulation of male outer sexual organs to female. Especially the nearly unknown regulation of female clitoral and vaginal tissue, which contains comparable to the male also cavernosal tissue, makes it necessary to give some evidences from regulation of male outer sexual organs. This is further supported by the observation that electrical field stimulation elicited clitoral relaxation by a NO-cGMP-dependent pathway (Vemulapalli and Kurowski, Life Sci. 26 (2000), 23-29), which also imply that alteration of NO-cGMP pathway may be useful to treat female sexual dysfunction. Furthermore, an increased prevalence of female sexual arousal disorder has been found in 6 studies of 6 comparing diabetic to non diabetic females. Its main symptom was a deficient vaginal lubrication, making sexual intercourse unpleasant. This disorder is the female equivalent to erectile dysfunction. It probably results from similar mechanisms, involving damages in the vascular and autonomic nervous systems, as well as alteration in the nitric oxide production and efficacy (Buvat and Lemaire, Diabetes Metab. 27 (2001) 67-75).

Based on numerous clinical observations of humans, the course of an erection can be divided into various stages. Initially, sexual arousal through visual, imagined or tactile stimulation activates the parasympathetically dominated cerebral sexual centers in the limbic system and the hypothalamus. This causes various stimulating neuro transmitters to be released, such as dopamine, oxytocin, ACH and others, setting in motion an "erection cascade". Penile erection is a hemodynamic process, involving increased arterial inflow and restricted venous outflow, in coordination with corpus cavernosum smooth muscle relaxation (Anderson et al., Physiol. Rev. (1997), 191-236). Although this process is generally accepted to be under neuroregulatory control (Pickert et al., Br. J. Urol. 75 (1995), 516-522), biochemical mediators released locally from the endothelium and/or smooth muscle also participate in initiating and maintaining erection (Burnett et al., Science 257 (1992), 401-403). Nitric oxide (NO), which is produced both in cavernosal nerves, smooth muscle and the endothelium, has been recognized as playing a key role in the physiology of penile erection (Pickert et al., Br. J. Urol. 75 (1995), 516-522). Additionally, it has been shown that endothelial NO synthase (eNOS) is one of the main sources of nitric oxide in the cavernosal tissue, which is then available in endothelial cells and cavernosal smooth muscle cells (Bloch et al, Urol. Res. 26 (1998), 129-135). Also in female rats eNOS was localized to vascular endothelium and perivascular smooth muscle fibers of vaginal tissue and N-NOS immunoreactivity was localized to nerve fibers supplying vaginal smooth muscle, perivascular nerve plexuses, and lamina propria. The expression of the NOS isoform in the rat vaginal tissue is estrogen-dependent and it was suggested that NOS is involved in estrogen deficiency-dependent apoptosis and vaginal atrophy. This cellular distribution of NOS in the rat vagina suggests that NO may modulate both vaginal blood supply and vaginal smooth musculature. Estrogen appears to play a critical role in concomitantly regulating vaginal NOS expression and apoptosis in nerves, smooth muscle, vascular endothelium, and epithelium of the rat vagina (Berman et al., Urology 51 (1998), 650-656). A further study by Giraldi et al., Int. J. Impot. Res. 14 (2002), 271-282, suggests that in the rat the L-arginine/NO system not only plays an important role in the regulation of vaginal smooth muscle tone, but also affects blood flow and may have sensory functions. These findings may have significant clinical implications for the pathophysiology of postmenopausal female sexual dysfunction (Berman et al., Urology 51 (1998), 650-656). During an erection NO is released to the parasympathetic nerve terminals in the muscles of the corpus cavernosum, activating the cGMP/cAMP mechanism. The end result is intracellular calcium depletion in the corpus cavernosum leading to relaxation of the muscles; this in turn decreases the peripheral resistance in both the muscles and the cavernous and helicine arteries. Thus, blood flow in the penis increases and erection is initiated.

Diminishing NO synthesis in the corpora cavernosa as an age-specific change is part of the reason for the high prevalence of ED with advancing age (Porst, Manual der Impotenz, 1. Ed. - Bremen: UNI-MED, 2000, ISBN 3-89599-455-3). This causes a reduced and delayed reaction on the part of the corpus cavernosum to central and peripheral erection-inducing nerve impulses, with a drop in the maximum achievable intracavemosal pressure. The described regulation of male cavernosal tissue and the expression of NOS isoforms in rat vaginal endothelial and smooth muscle cells could possibly explain alteration of blood flow and maintenance of tissue integrity in female outer sexual organs, although the signal pathways in the human female outer sexual organs are nearly unknown at physiological, biochemical and morphological level.

Under consideration of some morphological and structural intercourses between female and male outer sexual organs, it seems appropriate to extend knowledge from regulation of male outer sexual organs, too. The alteration of NO-cGMP pathway may be useful to treat female sexual dysfunction. In accordance with the present invention, medication or co-medication with NOS cofactors such as BH4 and analogues thereof offers a promising solution here.

Accordingly, in one aspect of the present invention, the pharmaceutical compositions described herein are useful in the treatment of female sexual dysfunction, and this includes female sexual dysfunction associated with hypoactive sexual desire disorders, sexual arousal disorders, orgasmic disorders or anorgasmy, or sexual pain disorders.

This aspect of the present invention is based on the observation that the therapeutic use of BH4, its precursors or its analogues could ameliorate the NOS-dependent imbalance of NO release and oxygen radical formation in the human female outer sexual organs. This could ameliorate female sexual function by improved NO-dependent blood flow, smooth muscle relaxation and sensory function as well as degeneration, which causes a reduction of functional vaginal tissue by replacement of nerve fibers, smooth muscle and endothelium by non-functional fibrous tissue. The NOS cofactors can be used alone or in combination with other substances, which work by increased activation or support of the NO/cGMP pathway. Although no information exists about the female vaginal tissue, the present findings from male outer sexual organs suggest under consideration of the above described functional and structural intercourses between female and male a relation of pathomechanisms in male and female. As described before, the increased formation of oxygen free radicals was observed by detection of an increased amount of free radical reaction product 8-isoprostane in corpus cavemosum tissue of 10 patients with erectile dysfunction. In accordance with the present invention, the main reason for endothelial NOS-dependent imbalance of NO release and oxygen free radical production is a lack of BH4. In view of the experiments performed in accordance with the present invention, it is reasonable to assume that also in females BH4 plays a central role in the functional regulation and maintenance of vaginal tissue. Therefore female patients with sexual dysfunction are treated for 3 to 6 months with BH4 or sepiapterin and the alteration of sexual function is monitored. In this study the sole effect of BH4 or sepiapterin as well as the effect of BH4 or sepiapterin in combination with PDE5 inhibitor is investigated in female sexual dysfunction and preliminary results indicate similar beneficial effects as for the corresponding treatment of males.

Advantages of the present invention comprise, inter alia, that the treatment with NOS cofactors such as BH4, its precursors or its analogues, have only limited side effects. Furthermore, treatment with BH4 is a causal therapy, directly linked to the impaired endothelial NOS activity leading to female sexual dysfunction, and in a BH4-saturated condition no further effect of BH4 treatment for NO release by endothelial NOS occurs. As mentioned before, it has been observed that the increased oxygen radical production in the corpus cavemosum from patients with erectile dysfunction leads to an increase of oxygen free radical reaction products such as 8-isoprostane and a strong effect of oxygen radicals for cell and tissue vitality. Without intending to be bound by theory, it is believed that this observation should also be considered for therapy of female sexual dysfunction. In general, the mostly used therapies of female sexual dysfunction are not focused to prevention of tissue damage. The results assessed in accordance with the present invention give evidence for an imbalance between oxygen radical formation and NO release in eNOS-containing cells in males and suggest a comparable alteration in females. This imbalance is hypothesized to elicit from functional lack of BH4. Therefore, the advantages discussed with respect to the application of NOS cofacors for the treatment of sexual dysfunction of males such as long-term treatment and combination therapy also apply to the treatment of sexual dysfunction of females. This is all the more true since hitherto sexual dysfunction including sexual arousal disorders has mainly been associated to physiological disorders that are not amenable to therapeutic treatment that is aiming at ameliorating organic dysfunctions.

Sexual arousal disorders can be characterized as persistent or recurrent inability to achieve or maintain adequate sexual excitement, causing personal distress. Common problems include lack of or diminished vaginal lubrication, decreased clitoral and labial sensation, decreased clitoral and labial engorgement and lack of vaginal smooth muscle relaxation. Orgasmic disorders can be characterized as persistent or recurrent difficulty or delay in attaining orgasm after adequate sexual stimulation and arousal, causing personal distress. Sexual pain disorders can be characterized by dyspareunia, (characterized by recurrent or persistent genital pain associated with sexual intercourse), vaginismus (characterized by recurrent or persistent involuntary spasm of the muscles of the outer third of the vagina which interferes with vaginal penetration, causing personal distress) and other pain disorders (characterized by recurrent or persistent genital pain induced by non-coital sexual stimulation). In one embodiment, the said dysfunction is related to the maintenance of functional and structural integrity of clitoral and vaginal tissue.

The pharmaceutical composition may be administered in accordance with the dosage regimen described above. For example, the administration of said composition to the female can be in a dosage which is in the range of from 0.1 to 500 mg/kg/day and wherein said administration is at intervals of 1 to 3 times a day during a period of at least 12 weeks; said treatment regimen may be combined and/or repeated at one or several intervals. Preferably, the dosage is in the range of 10 to 100 mg/kg/day.

In a further embodiment, the present invention comprises the use of a second agent capable of ameliorating sexual dysfunction for the preparation of a pharmaceutical composition, see also supra, wherein if said NOS cofactor and said second agent are comprised in a first and second pharmaceutical composition, said first and second pharmaceutical composition are applicable simultaneously or sequentially.

Experiments performed in accordance with the present invention demonstrated that a significant dosage reduction of drugs commonly used in the treatment of sexual dysfunction can be achieved when simultaneously the NOS cofactor BH4 is given to the patient. In particular, the dosage of the PDE5 inhibitor Levitra© (vardenafil) could be reduced by 50 %, i.e. from 20 to 10 mg in case 100 mg BH4 was applied at the same time. Since NOS cofactors such as BH4 are not toxic and have less side effects, if any, compared to drugs such as PDE5 inhibitors and PGEs used so far, the co-medication of a NOS cofactor in the treatment of sexual dysfunction provides an important advantage since contraindications of other drugs can be avoided while the overall beneficial effect remains substantially the same or may even be improved. Thus, without wishing to be bound by any particular theory or teaching, it is believed that NOS cofactors can generally be used as part of a medicament in combination with one or more other therapeutic agents such as a vasodilator, hormone therapy or neurotransmitter modulator used or tested in the treatment of sexual dysfunction.

Vasodilators for the treatment of sexual dysfunctions of organic (rather than psychogenic) origin, act at the penis, clitoris or vagina level on local blood flow or lubricant secretions. Vasodilators useful for the treatment of sexual dysfunction include alprostadil or phentolamine, NO (nitric oxide) enhancers such as L-arginine, and PDE5 inhibitors such as sildenafil or a pharmaceutically acceptable salt thereof (see EP 0 702 555 and references cited therein; Scrip's Complete Guide to Women's Healthcare (2000), 194-205; Sachs, Neuroscience and Biobehavioral Review 24 (2000), 541-560, Benet and Melman, Urol. Clin. N. Amer. 22 (1995), 699-709), VIP (Vaso-Intestinal Peptide) enhancers (Scrip's Complete Guide to Women's Healthcare (2000), 194-205) or angiotensin-2 receptor antagonists such as losartan (Ferrario and Levy, J. Clin. Hypertens. 4 (2002), 424-432).

Hormone therapies useful in the treatment of sexual dysfunction of organic and psychogenic nature include modulators of steroid hormones, steroid hormones or hormone products (including synthetic hormones) including estrogen (Scrip's Complete Guide to Women's Healthcare (2000), 194-205), or androgens such as testosterone (Scrip's Complete Guide to Women's Healthcare (2000), 194-205; Sachs, Neuroscience and Biobehavioral Review 24 (2000), 541-560), which act in areas of the CNS associated with sexual desire and sexual arousal (Wilson, Pharmacological targets for the control of male and female sexual behaviour. In: Sexual Pharmacology, Riley et al. (Eds.), Clarendon Press, Oxford (1993), 1-58). Furthermore, melanocortin receptor-specific compounds have been explored for use in treatment of sexual dysfunction as described and summarized in US patent US-A-6, 579,968.

Neurotransmitter modulators useful in the treatment of both psychogenic and organic sexual dysfunction include neurotransmitter agonists and antagonists such as catecholamine agonists such as the D2 agonist quinelorane, selective dopamine D3 receptor agonists (see WO03/051370), 5HT2 antagonists such as ritanserin, monoamine synthesis modifiers such as treatments that reduce endogenous 5HT activity, including inhibition of 5HT synthesis using parachlorophenylalanine, monoamine metabolism or uptake modifiers that inhibit catecholamine metabolism or uptake, such as tricyclic antidepressants, e.g. imipramine; see Wilson (1993), supra.

Furthermore, substances that act as bombesin receptor antagonists are described in WO02/40022 and references cited therein to have utility in the treatment of sexual dysfunction.

All of the above mentioned compounds and therapies can be combined and used with a NOS cofactor for the treatment of a sexual dysfunction in accordance with the present invention. The use of this combination therapy includes the preparation of therapies that would allow administration of both components of the medicament, i.e. a NOS cofactor and a vasodilator, hormone therapy medicament or neurotransmitter modulator medicament in a single dose. A preferred formulation would allow oral administration. However, administration by suppository, cream, transdermal patch or injection is also part of this invention.

Alternatively, the invention envisages formulations which allow administration of the NOS cofactor via a separate route to that of the vasodilator, hormone therapy medicament or neurotransmitter modulator medicament. Such routes could include, for example, oral administration of the NOS cofactor and transdermal patch application of the vasodilator. For example, a transdermal delivery system designed to deliver therapeutic levels of drugs which address sexual dysfunction caused by impotency in males and vaginal dryness in females to specific sites below the dermal level of the skin is described in US patent application US2003/138494. Thus, there may be provided a kit in which unit doses of NOS cofactor occur in association with unit doses of the vasodilator, hormone therapy medicament or neurotransmitter modulator medicament. For example, in the case of a kit where a NOS cofactor is formulated as a tablet, capsule or other unit dosage form for oral administration and the vasodilator is provided as a transdermal patch, the two dosage forms could be provided in the form of a two-row tear-off strip in which compartments containing the tablets, etc. occur above compartments containing the transdermal patches. Other forms of packaging in which the two dosage forms are spatially associated so as to make it easy for patients to take them together and to be reminded when they have done so will readily occur to those skilled in the art. The kit will also contain instructions as to when and how the individual components of the kit should be administered.

In such co-medication said second agent is preferably a phosphodiesterase (PDE) inhibitor, a prostaglandin (PG), papaverin or phentolamin, wherein said PDE inhibitor is preferably a PDE5 or PDE6 inhibitor and said PG is PGE1 or PGE2. Most preferably, said second agent is vardenafil, tadalafil, or sildenafil. In a particularly preferred embodiment said co-medication comprises the use of BH4 or a salt thereof, and as said second agent sildenafil or vardenafil.

As discussed before, the present invention is inter alia based on experiments performed on male patients suffering from sexual dysfunction; see also Example 2.

The psychogenic component of male sexual dysfunction has been classified by the nomenclature committee of the International Society for Impotence Research (and is illustrated in Sachs, Neuroscience and Biobehavioral Review 24 (2000), 541-560) as generalized type, characterized by a general unresponsiveness or primary lack of sexual arousal, and ageing-related decline in sexual arousability, characterized by generalized inhibition or chronic disorders of sexual intimacy. The inventors believe that there are common mechanisms underlying the pathologies of male and female psychogenic sexual dysfunctions.

Hence, the pharmaceutical compositions of the present invention described herein are useful in the treatment of male sexual dysfunction, especially erectile dysfunction and psychogenic male sexual dysfunction associated with generalized unresponsiveness and ageing-related decline in sexual arousability, in particular wherein said dysfunction is related to benign prostatic hyperplasia, erectile function and/or maintenance of functional and structural integrity of corpus cavemosum tissue. Advantages of the present invention comprise inter alia that the treatment with NOS cofactors such as BH4, its precursors or its analogues, have only limited side effects. Furthermore, treatment with BH4 is a causal therapy, directly linked to the impaired endothelial NOS activity leading to erectile dysfunction; in a BH4-saturated condition BH4 treatment has no further effects on NO release by endothelial NOS.

As discussed before, the increased oxygen radical production in the corpus cavemosum of patients with erectile dysfunction, leading to an increase in oxygen free radical reaction products such as 8-isoprostane, and the strong effect of oxygen radicals on cell and tissue vitality (for review, see Drδge, Physiol. Rev. 82 (2002), 47-95) should also be considered for therapy of erectile dysfunction. In general the most commonly used therapies for erectile dysfunction are not focused on the prevention of tissue damage. The results obtained in accordance with the present invention give evidence for an imbalance between oxygen radical formation and NO release in eNOS-containing cells. This imbalance could be elicited by a functional lack of BH4. Therefore, the long-term effect of BH4 should also be considered, as previously shown for the prevention of tissue injury and facilitation of recovery in endothelial dysfunction and/or other chronic diseases (Pieper, J. Cardiovasc. Pharmacol. 29 (1997), 8-15; Meininger et al., Biochem. 349 (2000), 353-356; Sun et al, Am. J. Physiol. Heart Circ. Physiol. 281 (2002), 863-869). This gives evidence for a further advantage of BH4 therapy: the positive side effects that are often found in patients with associated chronic disease. Furthermore, the mode with which BH4 works also makes a combination therapy with other downstream therapeutics, e.g. PDE5 inhibitors, effective.

As described for the treatment of sexual disorders of the male, the pharmaceutical composition may be administered in accordance with the dosage regimen described above. For example, the administration of said composition to the female can be in a dosage which is in the range of from 0.1 to 500 mg/kg/day and wherein said administration is at intervals of 1 to 3 times a day during a period of at least 12 weeks; said treatment regimen may be combined and/or repeated at one or several intervals. Preferably, the dosage is in the range of 10 to 100 mg/kg/day.

The response rate to PDE5 inhibitors as a treatment for erectile dysfunction of purely organic origin is approx. 40-60 %. There are marked individual differences here, depending on the degree of severity of the damage in neuronal signaling and/or smooth muscle relaxation. Due to the systemic side effects (headache, flushing, nasal congestion, etc.) and contraindications (manifest cardiac insufficiency, nitrate co-medication) associated with PDE5 inhibitors, an increase in dosage is not always feasible, and the medication cannot be used at all for some patients. This results in the need to find an alternative medication for such patients, i.e. one that has few side effects but is nonetheless effective, or, if possible, to find a specific co-medication that allows a reduction in the PDE5 inhibitor dosage. As demonstrated in the examples and described above, medication or co-medication with NOS cofactors such as preferably BH4 offers a promising solution here.

Thus, in a further embodiment, the present invention comprises the use of a second agent capable of ameliorating sexual dysfunction for the preparation of a pharmaceutical composition, see also supra, wherein if said NOS cofactor and said second agent are comprised in a first and second pharmaceutical composition, said first and second pharmaceutical composition being applicable simultaneously or sequentially.

As mentioned before, experiments performed in accordance with the present invention demonstrated that a significant dosage reduction of drugs commonly used in the treatment of sexual dysfunction can be achieved when simultaneously the NOS cofactor BH4 is given to the patient. In particular, the dosage of the PDE5 inhibitor Levitra© (vardenafil) could be reduced by 50 %, i.e. from 20 to 10 mg in case 100 mg BH4 was applied at the same time. Since NOS cofactors such as BH4 are not toxic and have less side effects, if any, compared to drugs such as PDE5 inhibitors and PGEs used so far, the co-medication of a NOS cofactor in the treatment of sexual dysfunction provides an important advantage since contraindications of other drugs can be avoided while the overall beneficial effect remains substantially the same or may even be improved as well.

Thus, without wishing to be bound by any particular theory or teaching, it is believed that NOS cofactors can generally be used as part of a medicament in combination with one or more other therapeutic agents such as a vasodilator, hormone therapy or neurotransmitter modulator used or tested in the treatment of sexual dysfunction; see supra.

In such co-medication said second agent is preferably a phosphodiesterase (PDE) inhibitor, a prostaglandin (PG), papaverin, apomorphine or phentolamin, wherein said PDE inhibitor is preferably a PDE5 or PDE6 inhibitor and said PG is PGEl or PGE2. Most preferably, said second agent is vardenafil, tadalafil, or sildenafil. In a particularly preferred embodiment said co- medication comprises the use of BH4 or a salt thereof, and as said second agent sildenafil, tadalafil, or vardenafil. In view of the particularly advantageous co-medication as demonstrated in corresponding experiments, see supra, the present invention also relates to pharmaceutical compositions comprising a NOS cofactor as defined above and a second agent capable of ameliorating sexual dysfunction such as those described hereinbefore. Thus, the present invention generally relates to the use of a NOS cofactor as defined herein, optionally in combination with a second agent capable of ameliorating sexual dysfunction such as those described hereinbefore, for the preparation of a pharmaceutical composition for improving female sexual function, desire and/or maintenance of functional and structural integrity of clitoral and vaginal tissue, for the treatment of benign prostatic hyperplasia, or for improving male erectile function and/or maintenance of functional and structural integrity of corpus cavemosum tissue.

Likewise, the present invention relates to a method for improving female sexual function, desire and/or maintenance of functional and structural integrity of clitoral and vaginal tissue, for the treatment of benign prostatic hypeφlasia, or for improving erectile function and/or maintenance of functional and structural integrity of coφus cavemosum tissue, comprising the step of administering to a subject having or suspected to get a sexual dysfunction a therapeutic amount a NOS cofactor as defined herein, optionally in combination with a second agent capable of ameliorating sexual dysfunction such as those described hereinbefore.

A further cause for sexual dysfunction in men can be benign prostatic hypeφlasia. Benign prostatic hypeφlasia (BPH), a nonmalignant enlargement of the prostate, is the most common benign tumor in men. Approximately 50 % of all men older than 65 years have some degree of BPH and a third of these men have clinical symptoms consistent with bladder outlet obstruction (Hieble and Caine, Fed. Proc. 45 (1986), 2601). In the U.S., benign and malignant diseases of the prostate are responsible for more surgery than diseases of any other organ in men over the age of fifty.

There are two components of BPH, a static and a dynamic component. The static component is due to enlargement of the prostate gland, which may result in compression of the urethra and obstruction to the flow of urine from the bladder. The dynamic component is due to increased smooth muscle tone of the bladder neck and the prostate itself (which interferes with emptying of the bladder) and is regulated by alpha 1 adrenergic receptors ([ ]l-ARs). The medical treatments available for BPH address these components to varying degrees, and the therapeutic choices are expanding. Surgical treatment options address the static component of BPH and include transurethral resection of the prostate (TURP), transurethral incision of the prostate (TUIP), open prostatectomy, balloon dilatation, hyperthermia, stents and laser ablation. TURP is the preferred treatment for patients with BPH and approximately 320,000 TURPs were performed in the U.S. in 1990 at an estimated cost of $ 2.2 billion (Weis et al, Prostate 22 (1993), 325-334). Although an effective treatment for most men with symptomatic BPH, approximately 20-25 % of patients do not have a satisfactory long-term outcome (Lepor and Rigaud, J. Urol. 143 (1990), 533-537). Complications include retrograde ejaculation (70-75 % of patients), impotence (5-10 %), postoperative urinary tract infection (5-10 %), and some degree of urinary incontinence (2-4 %) (Mebust et al., J. Urol. 141 (1989), 243-247). Furthermore, the rate of re-operation is approximately 15-20 % in men evaluated for 10 years or longer (Wennberg et al., JAMA 257 (1987), 933-936).

Apart from surgical approaches, there are some drug therapies which address the static component of this condition. Finasteride (Proscar^(R), Merck), is one such therapy which is indicated for the treatment of symptomatic BPH. This drug is a competitive inhibitor of the enzyme 5a-reductase which is responsible for the conversion of testosterone to dihydrotestosterone in the prostate gland (Gormley et al., N. Engl. J. Med. 327 (1992), 1185-1191). Dihydrotestosterone appears to be the major mitogen for prostate growth, and agents which inhibit 5a-reductase reduce the size of the prostate and improve urine flow through the prostatic urethra. Although finasteride is a potent 5a-reductase inhibitor and causes a marked decrease in serum and tissue concentrations of dihydrotestosterone, it is only moderately effective in treating symptomatic BPH (Oesterling, N. Engl. J. Med. 332 (1995), 99-109). The effects of finasteride take 6-12 months to become evident and for many men the clinical improvement is minimal (Barry et al., J. Urol. 158 (1997), 488-492).

The dynamic component of BPH has been addressed by the use of adrenergic receptor blocking agents ([α] l-AR blockers) which act by decreasing the smooth muscle tone within the prostate gland itself. A variety of [α] l-AR blockers (terazosin, prazosin, and doxazosin) have been investigated for the treatment of symptomatic bladder outlet obstruction due to BPH, with terazosin HC1 (Hytrin^(C)) and doxazosin mesylate (Cardura^(C)) being the most extensively studied. Although the [α]l-AR blockers are well-tolerated, approximately 10-15 % of patients develop a clinically adverse event (Lepor, J. Clin. Endocrinol. Metab. 80 (1995), 750-753). The undesirable effects of all members of this class are similar, with postural hypotension being the most commonly experienced side effect (Lepor et al., J. Urol. 148 (1992), 1467-1474). In comparison to the 5a-reductase inhibitors, the [α] l-AR blocking agents have a more rapid onset of action (Steers, Dis. Mon. 41 (1995), 437-497). However, their therapeutic effect, as measured by improvement in the symptom score and the peak urinary flow rate, is moderate. (Oesterling, N. Engl. J. Med. 332 (1995), 99-109).

Furthermore, administration of saw palmetto extracts has been investigated; see for review, e.g., Gerber, J. Urol. 163 (2000), 1408-1412. Various compositions comprising saw palmetto extract as well as a controlled delivery system is described in US patent application US2003/096024. Furthermore, the treatment of benign prostatic hypeφlasia with oral administration of 13 cis- retinoic acid has been described in WO03/024394.

As mentioned before, the dynamic component is due to increased smooth muscle tone of the bladder neck and the prostate itself. Without intending to be bound by theory it is believed that in view of the above described observations in patients suffering from sexual dysfunction nitric oxide (NO) plays an important role in maintaining the balance of the smooth muscle tone, too. Therefore, it is expected that administration of a NOS cofactor such as BH4 ameliorates the conditions associated with BPH as well, such as subvesical obstruction, but also any other symptom of BPH described above.

Accordingly, in a further aspect the present invention relates to the use of a NOS cofactor as defined hereinabove as single or co-medication for the treatment of subvesical obstruction caused by prostatic hypeφlasia or bladder neck sclerosis. The formulation of the pharmaceutical compositions for the medication can be done as described for the preceding embodiments as well as concurrent or sequential administration of other therapeutic compounds; see supra. Hence, administration of a NOS cofactor such as BH4 can be combined with any one of the afore described drugs for the treatment of BPH. Preferably, said co-medication is a prostate-selective medication such as described above, e.g., a prostate-selective alpha reductase inhibitor or alpha receptor inhibitor. From the above it is also evident that the present invention relates to the use of a NOS cofactor as defined herein as single or co-medication for the treatment of interstitial cystitis, low bladder capacity disorders or of micturition disorders as urge incontinence. It is understood that the entire description concerning the preparation of pharmaceutical compositions with one or more active ingredients described in the preceding sections also applies for the single and co-medication of those kinds of sexual dysfunction. In this respect, the uses, methods and compositions disclosed herein are described and preferably applied to humans. However, the treatment of other mammals is envisaged and can be beneficial as well, in particular the treatment of domestic animals such as horses, cows, sheep, dogs, etc., wherein the treatment according to the present invention may help in animal breeding programs, but maybe also useful for helping keeping animal population, e.g., in the zoo, for example in case animals are hesitant to copulate because of hospitalization.

These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the materials, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using, for example, electronic devices. For example, the public database "Medline" may be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352- 364.

The above disclosure generally describes the present invention. Several documents are cited throughout the text of this specification. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc.) are hereby expressly incoφorated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for puφoses of illustration only and are not intended to limit the scope of the invention. EXAMPLES

The examples which follow further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed descriptions of conventional methods, such as those employed herein can be found in the cited literature; see also "The Merck Manual of Diagnosis and Therapy" Seventeenth Ed. by Beers and Berkow (Merck & Co., Inc. 2003).

Example 1: Pharmaceutical compositions of the present invention for the treatment of sexual dysfunction Pharmaceutical compositions for use in the present invention can be prepared according to known methods such as those described in the prior art referred to in the general section above. Preferred formulations are, e.g., those described in Examples 1 to 9 of European patent application EP 0 908 182 A, the disclosure content of which is incoφorated herein by reference. Particularly preferred compositions of the present invention are 10 mg and 50 mg (6R)-BH4 tablets. Each 10 mg tablet contains (6R)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride (6R- BH₄-2HC1) , ascorbic acid, 5 mg N-acetyl-L-cysteine and other excipients (no lactose is added and no materials are used that are derived from animal or cell culture techniques). Each 50 mg tablet contains 50 mg (6R)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride (6R-BH -2HC1) , ascorbic acid, 25 mg N-acetyl-L-cysteine and other excipients (no lactose is added and no materials are used that are derived from animal or cell culture techniques).

Those tablets are particularly suited for oral administration of 1 to 20 mg tetrahydrobiopterin/kg/day. However, greater dosages can be used as well as described in Example 2. For example, the tablets can be dissolved in a little water, orange juice or other liquid, and mixed gently. Administration of the suspension is preferably done within 30 minutes. On the other hand, the tablets may be swallowed undissolved.

Furthermore, co-medication of a NOS cofactor and another therapeutic agent, i.e. for use in the treatment of sexual dysfunction, can be done, for example, by swallowing a 5 to 20 mg Levitra© (vardenafil) tablet with some water together or sequentially with a 10 to 50 mg BH4 tablet, see supra, preferably 20 to 60 minutes before the sexual activity. Similar comedication can be made with, for example, a 25, 50 or 100 mg Viagra© (sildenafil) tablet. However, other agents such as those described in the general section may be used instead as well. Thus, a BH4 and, e.g., silenafil or vardenafil composition may be present in the same composition or contained, e.g., as tablets in a kit. Example 2: NOS cofactor tetrahydrobiopterin BH4 increases penile rigidity and tumescence in erectile dysfunction patients

In early clinical studies, single oral doses of 5-2000 mg tetrahydrobiopterin were tolerated in healthy men. To confirm that tetrahydrobiopterin would be effective in humans during sexual stimulation, this study was carried out to objectively measure, using RigiScan technology, the penile responses to visual sexual stimulation while patients were treated with a placebo or 10 mg, 20 mg, 50 mg, lOOmg, 200 mg or 500mg of tetrahydrobiopterin. The subjects suffered from mild to moderate ED according to the international index of erectile function (IIEF) (Rosen et al., Urology 49 (1997), 822-830).

Study design

The influence of single oral doses of a placebo or 10 mg, 20 mg, 50 mg, 100 mg, 200 mg and 500 mg of tetrahydrobiopterin on penile rigidity and tumescence during visual sexual stimulation was evaluated through objective measurements using the RigiScan device. The study was a single-center, randomized, placebo-controlled, double-blind, 7-way cross-over design with each patient receiving each dose with at least 5 days in-between. After baseline measurements, the subject waited 20 min after taking the drug before being shown a sexually graphic video for 20 min. The degree of erection was measured while the patient watched and throughout a subequent rest period of 20 min and three further 20-min viewing sessions.

Subjects

Volunteers were male, Caucasian, 18-60 years of age, of normal body weight (Broca index: within 20 % of normal), and diagnosed with ED as evaluated on the basis of a standardized questionnaire (IIEF) (Rosen et al., Urology 49 (1997), 822-830). In the 6 months prior to start of the study the patients needed to have had a penile erection in response to visual sexual stimulation. Subjects with no response or only weak tumescence were excluded.

Subjects with any of the following findings were excluded: ED known to be due to neurological or endocrine causes, or an anatomical deformity such as severe penile fibrosis, or a spinal cord injury or radical prostatectomy. Patients were excluded if they had diabetes mellitus, or any further relevant comorbidity or major psychiatric illness. Additionally, subjects were excluded if blood pressure and heart rate were too abnormal or other laboratory test results indicated a second or third degree AV-block, QRS duration > 120ms or QT interval >500 ms. Subjects were not allowed to be taking any other medication likely to interact with the compound under investigation.

Treatments The treatments under investigation were administered as oral drinking solutions containing either 10 mg, 20 mg, 50 mg, 100 mg, 200 mg or 500 mg of tetrahydrobiopterin, or a placebo solution. The medication was followed by 100 ml tap water. The investigated treatments were administered in the morning after a light breakfast.

RigiScan measurements under visual sexual stimulation

The RigiScan (RigiScan Ambulatory Rigidity and Tumescence Monitor, Dacomed Coφ., Minneapolis, USA) was used to monitor penile rigidity and tumescence. The subjects were connected to the RigiScan device from 0,5 h before until 3 h after dosing, using a conventional set-up with a cuff around the base and another around the tip of the penis. Radial rigidity, expressed as a percentage of complete rigidity, was determined automatically every 15 s by the device after internal calibration. Tumescence was also automatically calculated by the device. The visual sexual stimulation consisted of sexually graphic videos (a set of different tapes was supplied to the subject from which he could select). The subjects were instructed to try to prevent ejaculation and were asked not to stimulate themselves manually during the whole session. The primary endpoint was the total duration of erections with greater than 60 % rigidity, as calculated using the RigiScan software. Secondary endpoints were duration of >80 % rigidity, Rigidity Activity Units (RAU) and Tumescence Activity Units (TAU) (Benet et al., J. Urol. 156 (1996), 1947-1950). The average erection rigidity, duration of event, circumference and average event tumescence were evaluated as ancillary criteria. All values were calculated separately for the base and the tip of the penis.

Safety surveillance and criteria of safety and tolerability

The volunteers spontaneously reported any signs or symptoms, but they were also evaluated by non-leading questions while under close medical surveillance. Adverse events (AEs) were defined as illnesses, subjective or objective signs or symptoms (including clinically significant changes of laboratory results) that appeared or worsened during the course of a study, independently of a relationship to study medication. Data analysis

Based on experiences in published studies with sildenafil (Boolell et al., Int. J. Impot. Res. 8 (1996), 47-52) and vardenafil (World J. Urol. 19 (2001), 32-39; Stark et al., Eur. Urol. 40 (2001), 181-188), the chosen sample size was considered to be adequate and sufficient for exploratory puφoses. Relevant pharmacodynamic data were fitted to a general linear model with effects for sequence, subject within sequence, period and treatment by analysis of variance (ANOVA). On the basis of the resulting variance estimates, point and 95 % confidence interval (CI) estimates of the pair-wise mean treatment differences were calculated for exploratory puφoses (without adjustment for multiplicity).

Subject disposition

10 subjects were available for evaluation of the pharmacodynamic study criteria.

Demography 10 men, 22-54 years of age (mean 36,6 +/- 9,1 years), with a body weight of 64-101 kg (mean 78,8 +/- 7,9 kg) and a Broca-index of 0,88-1,14 (mean 1,00 +/- 0,08) were enrolled and randomized.

Pharmacodynamic effects The maximal time of measurement was 3 h, although visual stimulation was only supplied for 80 min. Out of this time period it was noted that under placebo treatment the average time of erection with rigidity greater than 60 % was 39,2 min at the base and 26,3 min at the tip. Under 10 mg tetrahydrobiopterin treatment, the average time of erection with rigidity greater than 60 % was 46,9 min at the base and 30,9 min at the tip. With 20 mg tetrahydrobiopterin, the duration was statistically prolonged up to 48 min and 32,9 min at the base and the tip of the penis. With 50 mg tetrahydrobiopterin, the duration was statistically significantly prolonged up to 59,2 min and 53,1 min at the base and the tip of the penis, respectively (P < 0,01). Similary, the 100-mg dose showed marked prolongation of the time that the penis exceeded 60 % rigidity compared with that of the placebo, reaching 69,5 min and 64,9 min at the base and tip, respectively (P < 0,001). With 200 mg tetrahydrobiopterin, the duration was statistically significantly prolonged up to 78,6 min and 67,4 min at the base and the tip of the penis, respectively (P < 0,01). Similarly, the 500-mg dose showed marked prolongation of the time that the penis exceeded 60 % rigidity compared with that of the placebo, reaching 81,1 min and 69,1 min at the base and tip, respectively (P < 0,001). Example 3: Role and biochemistry of nitric oxide synthase (NOS)

Nitric oxide synthase catalyses the production of NO and L-citrulline from L-arginine, O₂, and NADPH-derived electrons. NOS utilizes 6 different cofactors and prosthetic groups: thiolate- bound heme, FAD and FMN, calmodulin Ca²⁺, (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), and Zn²⁺. There are three distinct isoforms of the enzyme; the two Ca²⁺-dependent enzymes, i.e. the neuronal NOS (nNOS) and the endothelial NOS (eNOS), as well as the Ca²⁺-independent NOS the inducible (iNOS).

NOS catalyses the formation of NO in two consecutive steps. First L-arginine is oxidized to N- hydroxy-L-arginine (NHA), in a reaction that consumes one molecule of O₂ and two NADPH- derived electrons. Subsequently, NHA is converted into L-citrulline and NO with consumption of one more NADPH-derived electrons and another molecule of O₂ (Stuehr et al., J. Biol. Chem. 266 (1991), 6259-6263).

The gene for NOS was found to contain a binding site for (6R)-L-erythro-5, 6,7,8- tetrahydrobiopterin (also referred to as "BH4" and "BH " interchangeably herein) included in compounds of the formula (I) below as active ingredients of the present invention, in addition to those for coenzymes such as calmodulin (CaM), flavin, NADPH. Both the conversion of L- arginine into NHA, and the transformation of NHA into L-citrulline and NO, require the presence of BH4. NOS will oxidize NADPH in the absence of substrate or pterin, but in that case the reaction products are O₂ ^* and H₂O₂. The NOS-catalyzed reduction by NADPH of O₂ into O₂ ^" /H₂O₂ is referred to as uncoupled catalysis, since in this reaction NADPH oxidation and O₂ reduction are uncoupled from arginine hydroxylation and NO formation (Mayer et al., FEBS Lett. 288 (1991), 187-191 ; Pou et al., J. Biol. Chem. 267 (1992), 24173-24176; Klatt et al., J. Biol. Chem. 268 (1993), 14781-14787; Gorren and Mayer, Curr. Drug Metabol. 2 (2002), 133- 157). Altogether the uncoupling of the L-arginine-nitric oxide pathway by insufficiency or impaired metabolism of BH4, results in increased formation of superoxide anion and reduced nitric oxide production (Vasquez-Vivar et al, Proc. Natl. Acad. Sci. 95 (1998), 9220-9225; Xia et al., J. Biol. Chem. 273 (1998), 25804-25808; Cosentino and Luscher, Cardiovasc. Res. 43 (1999), 274-278; Fleming and Busse, Cardiovasc. Res. 43 (1999), 532-541). It is indicated that NOS itself can be a potential source for endothelial production of oxygen free radicals and that decreased availability of BH4 might cause a shift in balance between ability of protective NO and toxic oxygen free radicals (Vasquez-Vivar et al., Proc. Natl. Acad. Sci. 95 (1998), 9220- 9225). A further reduction of avaible NO occurs by the reaction of oxygen free radicals as O₂- and H₂O₂ with NO. One of the reaction products is peroxynitrite, which leads in comparison to NO to a opposite contractile effect on vascular smooth muscle (for review Drδge, Physiol. Rev. 82 (2002), 47-95). The imbalance of the release of oxygen radical and NO could result in endothelial dysfunction and oxidative vascular injury, as described in a number of vascular diseases (Lϋscher and Noll, Atherosclerosis 1 18 (1995), 81-90; Li and Fόrstermann, J. Pathol. 190 (2000), 244-254). In this regard recent investigations proposed that endothelial NOS- dependent superoxide formation plays an important role in pathologic conditions, such as diabetes, hypertension, and atheroscleorsis (Shen et al., Am. J. Physiol. 281 (2001), H838-H846; Wever et al., Circulation 97 (1998), 108-112;), which would be related to reduced levels of BH4 (Cosentino and Katusic; Circulation 91 (1995), 139-144). In different human and animal studies it could be shown that BH4 or its precursor sepiapterin ameliorates impaired NOS-dependent vasoreactivity of different large arterial and venous vessels (Higman et al., Arterioscler. Thromb. Vase. Biol. 16 (1996), 546-552; Tiefenbacher et al., Circulation 102 (1996), 2172-2179; Pieper, J. Cardiovasc. Pharmacol. 29 (1997), 8-15; Shinozaki et al., Diabetes 48 (1999), 2437-2445; Heitzer et al., Diabetologia 43 (2000), 1430-1438; Meininger et al., Biochem. J. 349 (2000), 353- 356; Sun et al., Am. J. Physiol. Heart Circ. Physiol. 281 (2001), H1863-H1869). Beside the reduced smooth muscle relaxation leading to impaired vasodilation under lack of BH4, it is suggested that dysfunctional NOS caused by insufficiency of BH4 participates in oxidative injury, especially under pathologic conditions, including ischemia and reperfusion. In fact, administration of exogenous BH4 has been shown to reduce post-ischemic endothelial dysfunction, post-transplantation lung edema, ischemia reperfusion injury and OFR injury in grafts, and ischemic renal injury (Tiefenbacher et al., Circulation 102 (1996), 2172-2179; Schmid et al., J. Thorac. Cardiovasc. Surg. 118 (1999), 726-732; Kakoki et al., J. Am. Soc. Nephrol. 1 1 (2000), 301-309; Yamashiro et al., J. Thorac. Cardiovasc. Surg. 124 (2002), 775- 784). An imbalance of NO and free oxygen radical production leads to tissue injury and pronounced aging by increase of apoptotic cell death, alteration of cell function, such as inhibition of mitochondrial activity, as well as by alteration of cell signalling and transcription (for review Drδge, Physiol. Rev. 82 (2002), 47-95).

An increased production of oxygen radicals and peroxynitrite can be investigated in human tissue by detection of radical reaction products such as 8-isoprostane for oxygen radicals and nitrotyrosine for peroxynitrite (Viera et al., Methods Enzymol. 301 (1999), 373-381 ; Morrow, Drug Metab. Rev. 32 (2000), 377-385). Recently, an immunohistochemical increase of 8- isoprostane and nitrotyrosine formation during cardiac surgery in human heart could be detected (Mehlhom et al, J. Thorac. Cardiovasc. Surg. 125 (2002), 178-183). Evidence from basic scientific studies indicates that oxidative stress mediated through the superoxide radical (superoxide) and other reactive oxygen species (ROS) may be central to impaired cavernosal function in erectile dysfunction (ED). Increased inactivation of NO by superoxide results in impaired penile NO transmission and smooth muscle relaxation. Furthermore, propagation of endothelial dysfunction by ROS may result in chronic impairment of penile vascular function, a process analogous to early atherogenesis. Indeed, ED and atherosclerosis are closely linked through shared risk factors. Given the current understanding of ED pathophysiology, antioxidants may be of benefit in both the short- and long-term treatment. This gives evidence that antioxidant therapy can be used for prevention or treatment of ED (Jones et al., Expert Opin. Pharmacother. 3 (2002), 889-897).

Conclusion

Hitherto the sources of oxygen radical in coφus cavemosum tissue had not been recognized, especially the involvement of eNOS was not known, although a recent study could show that the oxygen radical scavenger superoxide dismutase prevents dysfunction of cavernosal smooth muscle of diabetic rabbits (Khan et al., BJU Int. 88 (2001), 303-304). Furthermore, there was no direct information about the role of free radicals in female sexual dysfunction. According to the present invention, however, it was found that by coping with the problem of imbalance of NO and free oxygen radical production through the use of NOS cofactor BH4 erectile function could be considerably improved and that the stmctural and physiological intercourses provide a reasonable expectation that oxygen radical plays a role in the aetiology of female sexual dysfunction as well. Therefore, the present invention provides novel uses and compositions that effectively prevent and/or ameliorate sexual dysfunction. In addition, the active ingredients of the pharmaceutical compositions of the present invention based on NOS cofactor, in particular BH4 and derivatives thereof, have no adverse effects or the like over long- term use because they are substances that inherently exist in vivo.

Claims

Claims

Use of a nitric oxide synthase (NOS) cofactor for the preparation of a pharmaceutical composition for the treatment of sexual dysfunction of a subject.

The use of claim 1 , wherein said cofactor is tetrahydrobiopterin (BH4) or a precursor or analogue thereof.

The use of claim 1 or 2, wherein said cofactor is a compound of the formula (I):

wherein R¹ and R² each represent a hydrogen atom or, taken together with each other, represent a single bond, while R³ represents -CH(OH)CH(OH)CH₃, -CH(OCOCH₃)CH(OCOCH₃)CH₃, -CH₃, -CH₂OH, or a phenyl group when R¹ and R² each represent a hydrogen atom, or -COCH(OH)CH₃ when R¹ and R² together represent a single bond, or a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable composition containing either entity.

The use of claim 3, wherein R³ is L-erythro-CH(OH)CH(OH)CH₃.

The use of claim 3, wherein said compound is selected from the group consisting of (6R)- L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), (6R,S)-5,6,7,8-tetrahydrobiopterin, l',2'- diacetyl-5,6,7,8-tetrahydrobiopterin, sepiapterin, 6-methyl-5,6,7,8-tetrahydropterin, 6- hydroxymethyl-5,6,7,8-tetrahydropterin, 6-phenyl-5,6,7,8-tetrahydropterin, or a precursor of any one thereof.

6. The use of claim 3, wherein said compound is a 5,6,7,8-tetrahydrobiopterin or a salt thereof.

7. The use of claim 6, wherein said compound is BH4 or a salt thereof.

8. The use of any one of claims 1 to 7, wherein said composition is designed for systemic administration.

9. The use of any one of claims 1 to 8, wherein said composition is designed to be administered orally, intravenously, intracavemously, sublingually, or buccally.

10. The use of any one of claims 1 to 9, wherein said composition comprises ascorbic acid and/or N-acetyl-L-cysteine as a diluent.

11. The use of any one of claims 1 to 10, wherein said composition is designed for direct treatment as a single dose in the amount of 10 to 1000 mg/kg.

12. The use of any one of claims 1 to 11, wherein said subject is a female.

13. The use of claim 12, wherein said dysfunction is related to the maintenance of functional and structural integrity of clitoral and vaginal tissue.

14. The use of claim 12 or 13, wherein the administration of said composition to the female is in a dosage which is in the range of from 0.1 to 500 mg/kg/day and wherein said administration is at intervals of 1 to 3 times a day during a period of at least 12 weeks; said treatment regimen may be combined and/or repeated at one or several intervals.

15. The use of claim 14, wherein the dosage is in the range of 10 to 100 mg/kg/day.

16. The use of any one of claims 12 to 15, comprising the use of a second agent capable of ameliorating sexual dysfunction for the preparation of a pharmaceutical composition, wherein if said NOS cofactor and said second agent are comprised in a first and second pharmaceutical composition, said first and second pharmaceutical composition are applicable simultaneously or sequentially.

17. The use of claim 16, wherein said second agent is a phosphodiesterase (PDE) inhibitor, a prostaglandin (PG), papaverin or phentolamin.

18. The use of claim 17, wherein said PDE inhibitor is a PDE5 or PDE6 inhibitor or said PG is PGEl or PGE2.

19. The use of claim 18, wherein said second agent is vardenafil, tadalafil, or sildenafil.

20. The use of any one of claims 16 to 20, wherein said NOS cofactor is BH4 or a salt thereof, and said second agent is sildenafil or vardenafil.

21. The use of any one of claims 1 to 11 , wherein said subject is a male.

22. The use of claim 21, wherein said dysfunction is related to benign prostatic hypeφlasia, erectile function and/or maintenance of functional and structural integrity of coφus cavemosum tissue.

23. The use of claim 21 or 22, wherein the administration of said composition to the male is in a dosage which is in the range of from 0.1 to 500 mg/kg/day and wherein said administration is at intervals of 1 to 3 times a day during a period of at least 12 weeks; said treatment regimen may be combined and or repeated at one or several intervals.

24. The use of claim 23, wherein the dosage is in the range of 10 to 100 mg/kg/day.

25. The use of any one of claims 21 to 24, comprising the use of a second agent capable of ameliorating sexual dysfunction for the preparation of a pharmaceutical composition, wherein if said NOS cofactor and said second agent are comprised in a first and second pharmaceutical composition, said first and second pharmaceutical composition are applicable simultaneously or sequentially.

26. The use of claim 25, wherein said second agent is a phosphodiesterase (PDE) inhibitor, a prostaglandin (PG), apomoφhine, papaverin or phentolamin.

27. The use of claim 26, wherein said PDE inhibitor is a PDE5 or PDE6 inhibitor or said PG is PGEl or PGE2.

28. The use of claim 27, wherein said second agent is vardenafil, tadalafil, or sildenafil.

29. The use of any one of claims 25 to 28, wherein the administration of said composition to the male is in a dosage which is in the range of from 10 to 100 mg/kg/day of NOS cofactor and 1 to 100 mg/kg/day of said second agent.

30. The use of any one of claims 25 to 29, wherein said NOS cofactor is BH4 or a salt thereof, and said second agent is sildenafil or vardenafil.

31. A pharmaceutical composition comprising a NOS cofactor as defined in any one of claims 1 to 7 and a second agent as defined in any one of claims 16 to 19 or 26 to 28.

32. Use of a NOS cofactor as defined in any one of claims 1 to 7, optionally in combination with a second agent as defined in any one of claims 16 to 19 or 26 to 28, for the preparation of a pharmaceutical composition for improving female sexual function, desire and/or maintenance of functional and stmctural integrity of clitoral and vaginal tissue, for the treatment of benign prostatic hypeφlasia, or for improving male erectile function and/or maintenance of functional and stmctural integrity of coφus cavemosum tissue.

33. A method for improving female sexual function, desire and/or maintenance of functional and stmctural integrity of clitoral and vaginal tissue, for the treatment of benign prostatic hypeφlasia, or for improving erectile function and/or maintenance of functional and stmctural integrity of coφus cavemosum tissue, comprising the step of administering to a subject having or suspected to get a sexual dysfunction a therapeutic amount of a NOS cofactor as defined in any one of claims 1 to 7, optionally in combination with a second agent as defined in any one of claims 16 to 19 or 26 to 28.

34. Use of a NOS cofactor as defined in any one of claims 1 to 7 as single or co-medication for the treatment of subvesical obstruction caused by prostatic hypeφlasia or bladder neck sclerosis.

35. The use of claim 34, wherein said co-medication is a prostate-selective medication.

36. The use of claim 35, wherein the therapeutic agent in said prostate-selective medication is a prostate-selective alpha reductase inhibitor or alpha receptor inhibitor.

37. Use of a NOS cofactor as defined in anyone of claims 1 to 7 as single or co-medication for treatment of interstitial cystitis, low bladder capacity disorders or of micturition disorders as urge incontinence.