USE OF 9-OXOACRIDINE-IO-ACETIC ACID, ITS SALTS AND ESTERS FOR ENHANCING THE EFFICACY OF ANTIANDROGENIC TREATMENT

The present invention relates to a method for treatment of conditions for which decrease of androgen activity is therapeutically favourable, such as androgenic alopecia, prostate adenoma and prostate cancer as well as for prophylaxis of prostate cancer.

Androgens are male sex hormones, such as testosterone, dehydroepiandrosterone and androstenediol. Androgens are produced in testes, ovaries and adrenal cortex. Production and secretion of androgens into blood are regulated by the hypophysis. Androgens are involved in the etiopathogenesis of various conditions, including alopecia, prostate adenoma and prostate cancer. Thus, antiandrogenic treatment is used to treat symptoms arising from excessive androgen, or to prevent symptoms that are expected to be worsened by androgen, as well as in other related conditions, such as a male to female trans-sexual treatment in gender identity disorders. In particular, antiandrogenic treatment is usually used for prostate cancer or prostatic hyperplasia in men. When indication of the treatment expands, it can be applied to male baldness, female acne, hypertrichosis, gynopathy, and amenorrhea, etc.

Antiandrogenic therapy stops or blocks the effect androgen presence has on tumor cells of the prostate. Antiandrogenic agents are combined with either surgery or drug therapy that shuts down male hormone production. The common drugs used with antiandrogenic agents are known as luteinizing hormone releasing hormone (LHRH) agonists. The LHRH agonists produce side effects that the antiandrogens can keep under control; thus the combination of the two types of agents has improved survival in prostate cancer patients.Androgenic alopecia is one of well-known indications for antiandrogenic hormonal therapy. Androgenic alopecia caused by high sensitivity of cell hair follicles to androgens, particularly to dihydrotestosterone that generated from testosterone by 5-alpha reductase. For treatment of this disease most often 5-alfa- reductase blockers are used, such as finasteride, which is a synthetic 4-azasteroid compound, N-(2 methyl-2-propyl)-3-oxo-aza-5-α-androst-l-ene-17-β-carboxam ide. Benign hyperplasia (adenoma) of prostate is the most frequent benign male disease. Conversion of testosterone into dihydrotestosterone (DHT) in prostate tissue plays a significant role in pathogenesis of prostate adenoma. Therefore the use of competitive inhibitors of 5-alpha-reductase type II is effective in the treatment of benign

hyperplasia (adenoma) of prostate. For example, finasteride is used in such treatment. Finasteride decreases noticeably the level of dihydrotestosterone circulating in blood, as well as the intraprostatic dihydrotestosterone level.

Since hormonal dependence of prostate cancer was discovered more than half a century ago, hormonotherapy has become a basic method of treatment of advanced forms of this tumor.

In all cases of prostate tumor hormonotherapy, the main objective of such therapy is to reduce or completely eliminate the influence of endogenous male sexual hormones

(androgens) in the patient's organism on tumor cells. Thereto, hormones, their analogues, agonists and antagonists are used, as well as hormone synthesis stimulators or inhibitors.

Since testosterone level is regulated in the organism by a negative feedback system that involves releasing hormones from the hypothalamus and gonadotropins from the anterior pituitary, any antiandrogenic influence causes the response of the organism directed at compensation of this influence. Therefore, it would be useful to have a remedy preventing such compensatory response, as well as a remedy increasing the efficiency of antiandrogenic therapy.

The inventors of the present invention are unaware of any specially developed remedies increasing the efficiency of antiandrogenic therapy. However, it is known that some additional remedies are used to treat prostate cancer.

For example, lα,25-dihydroxycholecalciferol (calcitriol), which is a biologically active form of vitamin D ₃ (see, WO 2004/087190 (October 14, 2004), GENIX THERAPEUTICS INC.), is recommended in clinical use as a medication for prostate cancer therapy that increases the androgen therapy efficacy in treatment of prostate cancer. Calcitriol enhances the effect of luteinizing hormone-releasing hormone agonist, which has the androgen-suppressing activity being the inhibitor of gonadotropin secretion (gonadotropin secretion inhibition leads to the inhibition of the testosterone production by testicles). According to WO 2004/087190, a method of treatment of prostate cancer comprises administering an LHRH analogue and calcitriol to a patient. However, in this publication, no factual data giving evidence of efficacy of this medication and method are presented.

The object of the present invention is to provide a method and a pharmaceutical composition for increasing the efficacy of treatment of conditions for which decrease of

androgen action is therapeutically favourable, as well as to provide an enhanced method of therapy of the indicated conditions.

The inventors of the present invention have now found that 9-oxoacridine-10- acetic acid, its pharmaceutically acceptable salts and esters are particularly useful for the treatment of conditions for which decrease of androgen action is therapeutically favourable. These compounds can be also used in combination with antiandrogenic hormone therapy. In another aspect, these compounds can be used to increase the efficacy of antiandrogenic hormonotherapy.

Thus, in one aspect, 9-oxoacridine-lO-acetic acid, its pharmaceutical acceptable salts and esters are provided for use in combined therapy of conditions for which decrease of androgen action is therapeutically favourable, as well as to increase the efficacy of antiandrogenic hormone therapy aimed at decreasing androgen action.

In another aspect, use of 9-oxoacridine-10-acetic acid, its pharmaceutically acceptable salts and esters is provided in the manufacture of a medicament for the treatment of conditions for which decrease of androgen action is therapeutically favourable.

In still another aspect, use of 9-oxoacridine-10-acetic acid, its pharmaceutically acceptable salts and esters is provided in the manufacture of a pharmaceutical composition for increasing the efficacy of antiandrogenic treatment of conditions for which decrease of androgen action is therapeutically favourable, for example, in antiandrogenic hormone therapy.

In still another aspect, a pharmaceutical composition for treatment of conditions for which decrease of androgen action is therapeutically favourable, comprising (a) a compound selected from the group including 9-oxoacridine-10-acetic acid, its pharmaceutically acceptable salts and esters, and (b) at least one antiandrogenic agent, in amounts that together are efficient for the treatment or prophylaxis of said conditions, and (c) a pharmaceutically acceptable carrier or excipient.

A kit for the treatment or prophylaxis of conditions for which decrease of androgen action is therapeutically favourable, comprising a compound selected from the group including (a) 9-oxoacridine-10-acetic acid, its pharmaceutical acceptable salts and esters, in a single dosage form, and (b) at least one antiandrogenic agent, in a single dosage form and (c) instructions to administer said compounds (a) and (b) to a patient in need of such treatment, in amounts sufficient to decrease the androgen action.

In still another aspect of the present invention, a method for the treatment or prophylaxis of conditions for which decrease of androgen action is therapeutically favourable, is provided, comprising the steps of: (a) administration of an effective amount of a compound selected from the group including 9-oxoacridine - 10 - acetic acid, its pharmaceutically acceptable salts and esters to a patient in need thereof, and (b) hormonotherapy aimed to decreasing androgen action.

In still further aspect, a method of increasing the sensitivity of androgen- dependent tissues to hormone therapy aimed at decreasing androgen action is provided, comprising administration of an effective amount of a compound selected from the group including 9-oxoacridine 10-acetic acid, its pharmaceutically acceptable salts and esters.

According to the present invention, preferred salts of 9-oxoacridine- 10-acetic acid are selected from the group including sodium, meglumine, eglumine salts and the salt with 3-0-(N,N-dimethylamino-π-propyl)-l,2:5,6-di-0-isopropyliden - a,O glucofuranose.

According to the present invention, preferred esters of 9-oxoacridine- 10-acetic acid are selected from the group including ethyl, propyl, butyl, isopropyl, and amyl esters.

According to the present invention, preferred conditions, for which decrease of androgen action is therapeutically favourable, are selected from the group including alopecia, adenoma of prostate, prostate cancer and increased risk of prostate cancer relapse.

According to the present invention, an antiandrogenic agent is preferably selected from the group including non-steroidal and steroidal antiandrogens, steroid synthesis inhibitors, 5-alpha-reductase inhibitors, glucocorticoids, estrogens, luteinizing hormone - releasing hormone (LHRH) agonists (analogues) and LHRH antagonists.

Still another preferable antiandrogenic agents are selected from finasteride, which is a 5-alpha-reductase inhibitor, and a saw palmetto (Serenoa Repens) fruit extract.

In some embodiments of the invention, a method of treatment comprises a course of administration of 9-oxoacridine- 10-acetic acid, its pharmaceutically acceptable salts or esters prior to hormonotherapeutic treatment. In another embodiment of the invention, 9-oxoacridine- 10-acetic acid, its pharmaceutically acceptable salt or

ester is administered concurrently with hormonotherapy or it can be initiated prior to hormonotherapy and continued along with hormonotherapy.

9-oxoacridine-10-acetic acid is a compound having the following structural formula:

According to another nomenclature, the name of this compound is 10- (carboxymethyl)-9(10H)acridone, the CAS number is 38609-97-1, and the international nonproprietary name is cridanimod.

Certain derivatives of 9-oxoacridine-lO-acetic acid were proposed in 1971 by researchers of Hoffmann-La Roche Inc. as potent antiviral agents (US patent 3,681,360).

Nowadays, medicaments on the basis of 9-oxoacridine-10-acetic acid and its pharmaceutically acceptable salts are used for the treatment and prevention of a wide range of diseases. In particular, its immunomodulatory, interferonogenous, antibacterial, anti-promoter and radioprotective properties are well known.

Surprisingly, the inventors of the present invention have found that the use of preparations of 9-oxoacridine-10-acetic acid, its pharmaceutically acceptable salts and esters provide for the increase in efficacy of therapy aimed at decreasing the influence of endogenous androgens on the organism. Conditions, for which decrease of androgen action is desired, includes but are not limited to, alopecia, prostatic adenoma, prostate cancer and the increased risk of prostate cancer relapse.

An antiandrogenic agent can be selected from the group including but limited to, non-steroidal or steroidal antiandrogens, steroid synthesis inhibitors, in particular, androgen synthesis inhibitors, in particular, 5-alpha-reductase inhibitors, glucocorticoids, estrogens; agonists (analogues) of luteinizing hormone-releasing hormone (LHRH), and the LHRH antagonists.

The administration of any of these preparations or their combinations in a context of the present invention is considered as "hormonotherapeutic treatment aimed at decreasing androgen action".

While not wishing to be bound by any particular theory, it is believed that the mechanism of action of 9-oxoacridine-lO-acetic acid, its pharmaceutically acceptable salts and esters, which results in increase of the efficacy of antiandrogenic remedies, could be as follows. Possibly, 9-oxoacridme-10-acetic acid anion depresses the expression of additional receptors in androgen-sensitive cells that usually takes place in response to the decrease of androgen stimulation. The expression of additional receptors compensates the decrease of androgen stimulation and reduces the effect of antiandrogenic therapy. 9-oxoacridine-10-acetic acid, its pharmaceutically acceptable salts and esters prevent such expression and, hence, increase the efficacy of antiandrogenic treatment.

The use of 9-oxoacridine-10-acetic acid and its pharmaceutically acceptable salts and esters provided according to the present invention, increases the efficacy of antiandrogenic therapy and allows to reduce doses and/or duration of administration of the basic antiandrogenic medicaments conventionally used for the treatment of these conditions.

9-oxoacridine-10-acetic acid, its pharmaceutically acceptable salts and its esters can be used concurrently with antiandrogenic hormonal treatment or prior to the hormonotherapy.

When administered prior to antiandrogenic treatment, 9-oxoacridine-10-acetic acid, its pharmaceutically acceptable salts or esters, has the effect compatible to the one observed in the case when 9-oxoacridine-10-acetic acid, its pharmaceutically acceptable salts or esters are administered concurrently with antiandrogenic treatment.

When the administration of 9-oxoacridine-10-acetic acid or its pharmaceutically acceptable salts or its esters is initiated prior to antiandrogenic hormonal treatment and then it is continued along with the hormonal treatment, the effect is still more expressed.

Though is was reported that immunomodulatory effect of 9-oxoacridine-10-acetic acid, its pharmaceutically acceptable salts and its esters is observed in a dose-dependent mode within the range of 0-10 mg/kg only, surprisingly, according to the present invention, it was found that the ability to increase the effect of antiandrogenic therapy is dose dependent even at doses in excess of 10 mg/kg of body weight.

Experiments carried out by the present inventors have shown that 9-oxoacridine-

10-acetic acid, its pharmaceutically acceptable salts and esters, when used as a complementary agent to antiandrogenic therapy act through a mechanism other than immunostimulation (known as the interferon induction). This mechanism is effective in combination with antiandrogenic therapy only.

This can be proved, in particular, by the fact that antiandrogenic treatment in combination with administration of interferonhas a lesser effect than the combination of antiandrogenic treatment with administration of 9-oxoacridine-lO-acetic acid.

It shall be appreciated that, as used in the present specification, when the 9- oxoacridine-lO-acetic acid is mentioned, its pharmaceutically acceptable salts and esters are also meant.

The term "pharmaceutically acceptable salt" as used herein, means those salts, which maintain the above mentioned properties of 9-oxoacridine-10-acetic acid and which are not unacceptable biologically or unacceptable in some other way. The pharmaceutically acceptable salts derived from the salt forming bases could be obtained with inorganic or organic bases.

The salts with inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium and magnesium salts.

The salts with organic bases include, but are not limited to, salts of primary, secondary, tertiary and quaternary amines, such as alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dialkenylamines, trialkenylamines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkylamines, di(cycloalkyl)amines tri(cycloalkyl)amines, substituted cycloalkylamines, di(substituted cycloalkyl)amines, tri(substituted cycloalkyl)amines, cycloalkenylamines, di(substituted cycloalkenyl)amines, di(substituted cycloalkenyl)amines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclylamines, diheterocyclylamines, triheterocyclylamines, mixed di- and tri-amines, where at least one of the substitutes on amine differs and is selected from the group, including alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, heteroaryl, heterocyclyl, etc. Amines, in which two or three substitutes together with the nitrogen atom to which they are connected, form a heterocyclyl or a heteroaryl, also are included here.

Specific examples of appropriate amines include, in particular, isoprpylamine, trimethylamine, diethylamine, tri(isopropyl)amin, tri(rc-propyl)amme, ethanolamine, 2- dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylendiamine, glucosamine, N-alkylglucamine, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine etc.

An example of a salt of 9-oxoacridine-lO-acetic acid with an alkali metal is the sodium salt:

An example of a salt with amino compound is the salt with 1-deoxy-l- (methylamino)-D-glucitol (i.e. with meglumine, or, the same, with N- methy lglucamine) :

H ₂ -CH ₂ -(CH ₂ OH) ₄ -CH ₂ OH

Other examples of salts with various complex quaternary ammonium bases include salts with amine-substituted carbohydrates, for example, with 2-deoxy-2- amino(or 2-alkylamino)-D-glucose, where R is H or a lower alkyl:

with 1-deoxy-l-methylamino-D-glucose:

as well as salts with various esters of carbohydrates and aliphatic amino alcohols, for example

where R ₁ , R ₂ are independently alkyl, aryl, heteryl

The examples of appropriate cations are as well, in particular, cations of

3-O-(N,N-dimethylamino-«-propyl)- 1 ,2 : 5 ,6-di-O-isopropyliden-α,D- glucofuranose,

1-deoxy -l-(ethylamino)-D-glucitol (i.e. eglumine),

1- deoxy -l-(propylamino)-D- glucitol,

1- deoxy -l-(butylamino)-D- glucitol,

1- deoxy -l-(methylamino)-L- glucitol,

1- deoxy -l-(ethylamino)-L- glucitol,

1- deoxy -l-(propylamino)-L- glucitol, and

1- deoxy -l-(butylamino)-L- glucitol.

According to the present specification, esters of 9-oxoacridine-lO-acetic acid include compounds obtained by hydrogen atom substitution in acid OH-group with an organic group R.

Examples of suitable esters include but are not limited to, esters of 9-oxoacridine- 10-acetic acid with lower alkyls (namely with (C ₁ -C ₁₂ )alkyls, in particular ethyl, propyl, isopropyl, butyl and amyl esters), as well as with choline and other lypophilic alcohols.

After rapid penetration through biological membranes in vivo, these compounds are easily hydrolyzed to free 9-oxoacridine-lO-acetic acid.

Commonly, the term an «antiandrogen» or «antiandrogenic agent» as used in its most general sense, refers to any substance which is able to inhibit biosynthesis, secretion and transport of androgens or depress their action. In view of the above, in the context of the present specification, the term «antiandrogenic therapy» is used along with the term «the therapy aimed at decrease the androgen action on the organism)).

In the present specification, the terms «antiandrogenic hormonotherapy)), «hormonotherapy» and «antiandrogenic therapy» are interchangeable.

Antiandrogens include steroid and non-steroid compounds that are able to inhibit the endogenous androgens physiological activity. The action of such compounds is related to androgen receptors blocking in the target tissues; they do not interfere with biosynthesis and secretion of androgens.

An example of steroidal antiandrogens is Cyproterone.

Examples of non-steroidal antiandrogens include flutamide, bicalutamide, nilutamide. Antiandrogenic activity could be inherent to a certain degree in a number of endogenous steroid compounds, including progestins and estrogens. Their synthetic derivatives possess antiandrogenic activity, as well as some androgen derivatives.

Steroid synthesis inhibitors include compounds, which inhibit production of steroids, including androgens production, by altering the activity of steroidogenic enzymes; for example, aminoglutetimide and ketoconazole are steroid synthesis inhibitors.

The compounds that inhibit 5α-reductase and therefore testosterone transformation into dihydrotestosterone possess a specific type of antiandrogenic activity. They do not bind to the androgen receptors, but they are also considered by pharmacologists as a antiandrogenic agent.

5-alpha-reductase inhibitors include, in particular, finasteride (Proscar) or saw palmetto (βerenoa Repens) fruit extract (Permixon).

Glucocorticosteroid preparations (glucocorticoids) could, in particular, be selected from a group including hydrocortisone, prednisolone, dexamethasone.

Ethynilestradiol, diethylstilbestrol and hexestrol in particular belong to estrogens (estrogenic agents). Hypothalamic factors ("releasing-factors"), releasing pituitary hormones

(gonadorelin analogues), namely, luteinizing hormone-releasing hormone agonists, which inhibit androgen synthesis are represented, in particular, by triptorelin, buserelin, leuprolid (leuprorelin) and goserelin. As a luteinizing hormone-releasing hormone antagonist, abarelix could be used. Hormone therapy used for the treatment of benign prostatic hyperplasiacan include, for example, the use of 5-alpha-reductase inhibitors, in particular, finasteride, its analogues and derivatives, or, for example, the extract from plant Serenoa Repens, for example Permixon, as well as various combinations thereof. In case of benign prostatic hyperplasia for relaxation of prostate smooth muscle tone, alpha - adrenoreceptor antagonists (i.e. blockers of alpha-adrenoreceptors) are often administered, such as terazosin, doxazosin, tamsulozin or their analogues, along with 5- alpha-reductase inhibitors.

In the treatment of benign prostatic hyperplasia, 9-oxoacridine-lO-acetic acid or its pharmaceutical acceptable salts and esters can be easily administered topically, for example in the form of suppositories.

Pharmaceutically acceptable salts of the 9-oxoacridine-10-acetic acid can be used in a single dose from 0.5 to 100 mg/kg body weight (calculated based on 9-oxoacridine- 10-acetic acid), preferably from 4 to 20 mg/kg body weight. The daily dose can vary from 2 to 1000 mg/kg, preferably from 2 to 200 mg/kg. In each particular case, the preparation dosage can be calculated by a specialist on the basis of the specification and the examples.

It shall be appreciated that the used amounts of the 9-oxoacridine-10-acetic acid or its pharmaceutically acceptable salt or ester, and of an antiandrogenic agent are synergistically effective. By the term "effective amount" of an agent as provided herein is meant a nontoxic but sufficient amount of the agent to provide the desired effect, such as antiandrogenic effect. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity

of the condition being treated, and the particular compound or agent and its mode of administration, and the like. Thus, it is not possible to specify an exact "effective amount." However, an appropriate effective amount may be determined by one of ordinary skill in the art using only routine experimentation. By "pharmaceutically acceptable" is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected antiandrogenic agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. It shall be also appreciated, that each of inventive compositions and medicaments may alternatively include, compise, or be substantially composed of any suitable components disclosed in the present specification, and such compositions and medicaments, including pharmaceutical compositions, and a kit according to the invention, may additionally or alternatively be prepared in such a way that a component, a material, an ingredient or an object could be excluded therefrom, which was used in a corresponding medicament or composition known in the prior art, or which is not necessary to achieve the technical effect of the present invention.

The same refers to method of treatment according to the invention, which alternatively may include, comprise, or be substantially composed of any matching stages disclosed in the present specification, and such inventive methods may additionally or alternatively exclude some steps or objects, which is used in a method, known in the prior art, or which is not necessary to achieve the technical effect of the present invention.

Further, the invention is illustrated by particular examples of implementation.

To designate the 9-oxoacridine-lO-acetic acid further the abbreviation CMA will be used.

Commercially available preparations of CMA salts, for example sodium SMA salt (preparation Neovir, Pharmsynthez, Russia), meglumine CMA salt (preparation

Cycloferon, NTFF Polysan, Russia), as well as commercially available CMA (Sigma,

USA, cat. # 17927, catalogue of year 2005), among others, were used in the experiments and clinical studies carried out by the present inventors.

Some other CMA salts as well as esters were synthesized by known, relatively simple methods (see for example: Inglot A.D. et al., Archivum Imrnunologiae et Therapiae Experimentalis, 1985, vol. 33, pp. 275-285; RU 2135474; RU 2036198; RU 2033413). In some cases, suppositories containing CMA or its pharmaceutically acceptable salt or ester, were prepared on the basis of widely used for this purpose suppository masses, such as Witepsol (Witepsol W 35, E 75), in a manner illustrated by the presented examples for rectal administration in clinic.

EXAMPLE 1. Preparation of a pharmaceutical composition for topical (rectal) use, comprising an unionized CMA as an active compound

250 g of finely crystalline CMA were mixed during heating up to 60 degrees Celsius with a suppository mass, consisting from a widely used basis for suppositories Witepsol W 35 and twin 80 emulsifier, the mixture was homogenized in a mixer and suppository mass with total weight of 1.5 kg was obtained; 970 suppositories were molded, each weighing 1.5 g and having the following composition: 9-oxoacridine-10- acetic acid - 0.250 g; twin 80 - 0.05 g; Witepsol W 35 - 1.2 g. The yield was 97%.

EXAMPLE 2. Preparation of a a pharmaceutical composition for topical (rectal) use, comprising CMA ester as an active compound.

250 g of ethyl ester of CMS, a basis for suppositories, consisting from a widely used suppository mass Witepsol W 35 and Tween 80 emulsifier, were mixed during heating to 60 degrees Celsius; the mixture was homogenized in a mixer to obtain a suppository mass with total weight 1.5 kg; 970 suppositories were molded, each weighing 1.5 g and having the following composition: CMA - 0.280 g; Tween 80 - 0,05 g; Witepsol W 35 - 1,17 g. The yield was 97%.

EXAMPLE 3. Preparation of a pharmaceutical composition comprising CMA and finasteride

500 g of crystalline CMA (CAS 38609-97, Fluka, 17927) were mixed with 5 g of finasteride that is N-(2-methyl-2-propyl)-3-oxo-aza-5-α-androst-l-ene-17-β-

carboxamide CAS Ne 98319-26-7 (Sigma, F1293). After thorough mixing in a drum shredder the mixture was encapsulated in the solid gelatinous capsules, and 980 capsules were obtained. Each capsule contained 500 mg of CMA and 5 mg of finasteride. The yield was 97%.

EXAMPLE 4. The study of the influence of CMA and its pharmaceutical acceptable salts and esters on the increase of inhibitory effect of finasteride as the 5- alpha-reductase inhibitor on testosterone stimulated prostate growth

The classic model of testosterone growth stimulation prostate of previously castrated animals was used. This study used 12 groups of animals with 10 rats per group. Male Sprague-Dawley rats weighting 220 to 230 g were used. AU animals were castrated under ke-. tamine anesthesia. Three days after castration the animals (of group

NaNo 1-12) were started to receive testosterone enantat injections (Testoviron Depot,

Chering, Germany) at a dose 30 mg/kg per week for 2 weeks. The animals from the first group served as a negative control and received per os the vehicle only i.e. starch gel.

For 5 days of the first week after castration and then for 4 days of the second week others groups of animals were injected daily with corresponding preparations in the following way:

The rats in the groups 2, 3, 4, 5, 6 and 7 were administered finasteride (Proscar, Merck Sharp & Dohme B. V.) at a dose 10 mg/kg per os in starch gel.

Additionally to finasteride administration, rat in the groups 3-7 were injected with the following preparations:

- The rats in the group 3 were injected subcutaneously with interferon beta- Ia (Avonex, Biogen B.V., Netherlands) at a dose 1 x 10 ⁵ IU/kg; -The rats in the group 3 received subcutaneously CMA sodium salt in physiological solution at a dose 5 mg/kg that was equimolar with 4.6 mg/kg of CMA;

- The rats in the group 5 received subcutaneously CMA meglumine salt in physiological solution at a dose 8.2 mg/kg that was equimolar with 4.6 mg/kg of CMA;

- The rats in the group 6 received subcutaneously fine-dyspersated suspension of CMA in physiological solution at a dose 4.6 mg/kg.

- The rats in the group 7 received subcutaneously fine-dyspersated suspension of ethyl ester of CMA in physiological solution at a dose 5.1 mg/kg, that was equimolar with 4.6 mg/kg of CMA;

The rats of groups 8, 9, 10, 11 and 12 did not receive finasteride but received, correspondingly, interferon beta- Ia ₅ sodium salt of CMA, meglumine salt of CMA, nonsolubilized CMA and ethyl ester of CMA at the doses, regimens and in manners of administration which were the same as those for rats of groups 3, 4, 5, 6 and 7. The 11 day from the beginning of treatments (that is in a day after last injection) the animals were killed by ether anesthesia, ventral prostates were dissected out and weighed; the average of ventral prostate weight was calculated in each group. Then relative weight (in percentage terms) of ventral prostate in each group was calculated

(with respect to average ventral prostate weight for group 2, which was the negative control group).

The results of this series of experiments are presented in Table 1.

Table 1.

CMA and its pharmaceutical acceptable salts and its ester increases inhibitory effect of 5-alpha-reductase inhibitor finasteride on testosterone stimulated prostate growth.

According to findings presented in Table 1, 9-oxoacridine-lO-acetic acid (CMA) itself, its sodium and meglumine salt and its ethyl ester, as well interferon beta - Ia did not influence practically on the investigated parameter reflecting level of inhibition of androgenic stimulation. At the same time combination of finasteride with CMA as well as with its sodium and meglumine salts, increase this effect obviously, and more strongly than the combination finasteride with interferon beta - Ia.

In other series of experiments designed in a similar manner, other compounds of CMA were investigated. In particular, other CMA esters, including butyl ester as well as other salts of CMA, were studied. For example, these compounds were: salts formed by CMA and cyclic amino sugars and their esters, in particular, the salt formed by CMA and 3-O-(N,N,-dimethylamino-«-propyl)- 1 ,2:5,6-di-O-isopropyliden- α,D-

glucofuranose. This salt is described in patent RU 2197248 (and named as Anandine preparation).

AU pharmaceutical acceptable salts and esters of CMA showed similar ability to increase effect of antiandrogenic treatment on prostate weight. The CMA esters at the same mass doses had more strong effect than interferon but, as a rule, they had smaller effect (up to 10-15%) than the one observed for CMA salts. As in experiments which illustrated in Table 1 as well in other experiments realized by the inventors the ability of CMA, its pharmaceutically acceptable salts and its esters to influence independently on growth of normal, hyperplasic and malignant androgen-dependent tissues out of hormonal exposure, was investigated specially. However CMA, its pharmaceutically acceptable salts and esters do not have such influence by themselves. Thus, CMA itself, its pharmaceutical acceptable salts and its esters have no proper hormonal and/or antitumor activity as for androgen-dependent tissues (including hyperplasic tissues) as for tumors derived from these tissues.

EXAMPLE 5. The ability of sodium salt of CMA to increase effect of antiandrogenic therapy on the model of hormone-sensitive variant of a rat prostatic adenocarcinoma.

Male Copenhagen rats weighing 200-230 g were used. To model tumor process, under ketamine anesthesia, the 5 mm ³ volume pieces of androgen-sensitive variant (Invariant) of Dunning R3327 rat prostatic adenocarcinoma were transplanted subcutaneously into the right thigh. Hormone therapy was started 2.5 months after transplantation when the tumor volume reached 0.5- 1.5 CM ³ . CMA (as sodium salt) (NaCMA) treatment or interferon beta- Ia treatment were realized as preliminary sensitization regimen (i.e. "pre-sensitization") or as therapy regimen (synchronously with hormone therapy). In the first variant, treatment (regimen I) was started 2-2V-. weeks before hormone therapy commencement and was finished at the day before hormone therapy commencement. In the second variant, treatment (regimen II) was started synchronously with hormone therapy commencement and was continued to the end of the experiment. Also, in one of experiment series prior to hormone therapy commencement the animals were started to receive NaCMA or interferon beta Ia, correspondingly, and then the animals were continued to be treated

with above preparations during the hormone therapy course (regimen III). Under all treatment regimen (I, II or III) CMA (as sodium salt) was given every day at dose 10 mg per kg of animal weight, and interferon beta - Ia was given every day at dose 1 x 10 ⁵ UI per kg of animal weight. Immediately before hormone therapy and then once per week, size of the tumor was measured and tumor volume was calculated. Treatment efficacy was evaluated with Tumor Growth Inhibition index (TGI). TGI was determined as the ratio of the difference between average tumor volume from treated animals and average tumor volume from negative control animals, divided by average tumor volume from negative control animals. TGI was evaluated in percentage points.

Avg.tumor vol.in the neg. cntrl. group-Avg.tumor vol.in the i QO ₀ / GI (%)= group

Average tumor vol. in the neg. cntrl. group

In the experiments an antiandrogen, flutamide (Flucinom, Schering Plough, USA) was used and it was given perorally in starch gel at dose 15 mg/kg per day each day.

The LHRH analogue, leuprolide acetate (leuprorelin) (Lucrin Depot, Abbott Laboratories Ltd., USA) was used also and it was administered subcutaneously at dose 0.1 mg/kg once a week for 4 weeks.

To estimate possible effects of CMA on tumor sensitivity to chemohorrnonetherapy an agent having binary, - estrogenic and direct cytotoxic -action was used. For this purpose estramustine (Estracyt, Pharmacia & Upjohn, USA) was used. Estramustine was given intraperitoneally at a dose 100 mg/kg 3 times a week for

4 weeks.

The testectomy was one of variants of hormone therapy.

The variants of hormone therapy and treatment regimens as well as the results of the experiment are illustrated in Table 2.

Table 2.

The ability of sodium salt of CMA to increase effect of antiandrogenic therapy on the model of hormone-sensitive variant of a rat prostatic adenocarcinoma

* See explanation in the text of the specification.

The more TGI index, the more effective inhibition of tumor growth takes place and therefore, the combination and therapy regimen to be used are more effective.

From presented data it is evidence that NaCMA very effective increase hormone sensitivity of hormone-sensitive variant of prostate cancer. Its effectiveness is more than the one of interferon beta- Ia since in all experiments for the combinations comprising NaCMA, the index is higher than those in experiments without using CMA or in experiments for combinations comprising interferon beta- Ia.

Moreover, NaCMA in contrast to interferon beta- Ia, appeared to be able to "presensitize" the tumor to following hormonal or chemohormonal treatment.

The most powerful effect was observed in those variant of regimen when NaCMA was administered before hormone treatment and then its administration was continued under ongoing hormone therapy or chemohormone therapy (regimen III).

EXAMPLE 6. The ability of meglumin salt of CMA to increase effect of antiandrogenic therapy on the model of hormone-sensitive variant of a rat prostatic adenocarcinoma.

The experiments were conducted out according to design described in Example 5, but instead of flutamide, an antiandrogen nilutamide was used and it was administered orally at dose 7 mg/kg/day (Anandron, Rousel Uclaf) in starch gel; buserelin was used instead of leuprolide at dose 0.1 mg/kg once every four weeks; meglumin salt of CMA was used as pharmaceutical acceptable salt of CMA.

The results of experiments are illustrated in Table 3.

Table 3.

The ability of meglumin salt of CMA to increase effect of antiandrogenic therapy on the model of hormone-sensitive variant of a rat prostatic adenocarcinoma.

* See explanation in the text.

From the data presented in the table it is evident that effects of meglumine salt and sodium salt of CMA are similar (see. Example 5).

EXAMPLE 7. Benign prostatic hyperplasia (adenoma) treatment.

Therapeutic effect of the claimed method was studied in patients with benign prostatic hyperplasia (adenoma). Patients were between 50 and 62 years old, benign prostatic hyperplasia diagnosis was based on anamnesis, prostate digital investigation, Doppler-graphic examination, estimation of residual urine volume, determination of serum level of prostate specific antigen (PSA). The patients were randomly assigned to four groups.

Two groups received treatment with variants of claimed method as followed: The first group was treated with inhibitor 5-alpha reductase finasteride (5 mg per day before doing to bed) and with CMA as tablets of its meglumine salt at dose 600 mg (calculated based on CMA) (prepeparation Cycloferon, NTFF Polysan, Russia) orally twice a week.

The second group was treated with preparation of extract of Serenoa Repens possessing to inhibit 5-alpha reductase (Permixon, Pierre Fabre) at dose 160 mg twice daily. Addditioanlly second group were treated additionally per rectum as well 2 times per week with 1 suppository containing of 250 mg of CMA and made as it is described in the Example 1. At that the suppositories was started to be used two weeks before plant inhibitor 5-alpha reductase (Permoxon) therapy commencement.

Third and fourth groups underwent the same therapy but they were administered with subcutaneous injection of 3 million IU of recombinant interferon beta -Ia (Avonex, Biogen B. V., Netherlands) 2 times per week in place of CMA preparations.

Treatment effect was determined after 12 weeks of the therapy. The changes of the following parameters (as compared with basal indexes before start of hormone therapy) were estimated: prostate size, IPSS score, maximum urinary flow rate, residual urine volume. The side effects rate was estimated also. All four groups of patients were similar in age, intensity of benign prostatic hyperplasia (adenoma) clinical presentations.

The result is presented in the Table 4.

Table 4.

Benign prostatic hyperplasia (adenoma) treatment

From the data presented in the table it is evident that use of CMA and its pharmaceutically acceptable salts and its esters decrease significantly the clinic signs of benign prostatic hyperplasia (adenoma) under antiandrogenic influence. Moreover, their therapeutic effect is more potent and less number of side effects was registered than in case of interferon beta - 1 a use.

The following side effects of the therapies were:

- in patient treated with finasteride: ejaculation disorder, libido decrease, impotence;

- in patient treated with the preparation of saw palmetto (Serenoa Repens ) fruit extract: sickness, heartburn, gastric pains;

- in patient treated with interferon beta -Ia: sickness, vomiting, fever. There were no specific adverse reactions caused by CMA.

EXAMPLE 8. Benign prostatic hyperplasia (adenoma) treatment

Two groups of patients were treated with the preparation of saw palmetto (Serenoa Repens ) fruit extract that has ability to inhibit 5 -alpha reductase (Permixon, Pierre Fabre) at doses of 160 mg twice daily.

Patients of the first group were administered additionally with subcutaneous injection of 3 million IU of recombinant interferon beta -Ia (Avonex, Biogen B. V., Netherlands) 2 times per week.

Patients of the second group were administered as well additionally per rectum 2 times per week with 1 suppository containing of 280 mg of ethyl ester of CMA and made as it is described in the Example 2. At that the suppositories were started to be administered at the same time as a plant 5 -alpha reductase inhibitor was started to be administered.

The results are presented in the Table 5.

Table 5.

Benign prostatic hyperplasia (adenoma) treatment

From the data presented in the table it is evident that use of CMA and its pharmaceutically acceptable salts and its esters decrease significantly the clinic signs of benign prostatic hyperplasia (adenoma) under antiandrogenic influence.

EXAMPLE 9. Prevention of cancer prostate relapse.

5 years ago a patient, male of 69 old was diagnosed with prostate cancer (on the basis of prostate digital investigation, biopsy and tomography of small pelvis organs). Before surgery Gilson score was 8, PSA level was 45 ng/ml, distant and regional metastases were no observed. The patient underwent radical prostatectomy with dissection of lymph nodes of small pelvis. The histology examination showed the lesions of the lymph nodes. Postoperative level of PSA was 0.1 ng/ml.

To decrease of prostate cancer relapse risk after surgery the patients was administered with antiandrogen flutamide (750 mg per day orally, parted to three dose) and CMA sodium salt (2 times per week 4 ml of 12.5% sterile solution for injection).

At a later time (5 years) serum PSA remained on undetectable level, the signs of the tumor relapse were no observed. Hence, inventive method is high effective for prevention of prostate cancer relapse.