WO2013190443A1

WO2013190443A1 - Anti-mullerian hormone

Info

Publication number: WO2013190443A1
Application number: PCT/IB2013/054938
Authority: WO
Inventors: Pietro Giulio SIGNORILE; Alfonso Baldi
Original assignee: Signorile Pietro Giulio; Alfonso Baldi
Priority date: 2012-06-18
Filing date: 2013-06-17
Publication date: 2013-12-27
Also published as: ITRM20120285A1

Abstract

The present description relates to antimullerian hormone or biologically active form thereof for use in the treatment and/or slowing of endometriosis, as well as to pharmaceutical compositions comprising said hormone and/or biologically active form thereof for use in the treatment and/or slowing of endometriosis.

Description

ANTI-MULLERIAN HORMONE

DESCRIPTION

STATE OF THE PRIOR ART

Endometriosis is defined as a recurrent and benign gynecological disorder, characterized by the presence of endometrial tissue (glands and stroma) present outside of the uterine cavity. Endometriotic tissue is most commonly, found on the peritoneal surface inside the female pelvis, on ovaries, in the rectovaginal septum, and more rarely in the pericardium, in the pleura and even in the brain (Giudice LC , and Kao LC: Endometriosis. The Lancet, 364: 1789-1799, 2004).

Currently, a definite diagnosis for endometriosis can be formulated only through surgical practices and this makes an exact assessment of its prevalence in female population difficult. Recent estimates indicate a 6-10% prevalence; however, in patients with pain and/or infertility such prevalence rises to 35-60% (Houston DE: Evidence for the risk of pelvic endometriosis by age, race, and socioeconomic status. Epidemiol Rev, 6: 167-191 , 1984). Actually, endometriosis is generally associated to infertility and pelvic pain, like e.g. dysmenorrhea, abdominal and pelvic intermenstrual pain, back pain, dysuria and dyspareunia (Giudice LC, and Kao LC: Endometriosis. The Lancet, 364: 1789-1799, 2004). The disorder has been singled out as a nosological entity per se since 1860, by Austrian pathologist Von Rokitansky (Von Rokitansky C: Ueber uterusdrusen-neubildung in uterus and ovarilsarcomen. Z Ges Aertze Wein, 37: 577-593, 1860). In spite of its being very common in the female gender and known since about 150 years ago, it is oft-times underestimated, its pathogenetic mechanisms are less than clear and diagnostic and therapeutic strategies are still inadequate.

Antimullerian hormone (AMH) is a dimeric glycoprotein belonging to the Transforming Growth Factor-beta (TGF-beta) superfamily. AMH is produced by Sertoli cells in male fetus and is responsible for regression of Muller's ducts (La Marca A et al.: Anti-Mullerian hormone (AMH): what do we still need to know? Hum Reproduct, 24: 2264-2275, 2009). In the female fetus, AMH begins to be expressed from the 32^nd week of gestation in follicular granulosa cells. AMH expression in ovarian follicles is maintained over a woman's entire fertile life, to then decay in the menopause, and AMH levels are considered indicators of the active ovarian reserve (Lee MM et al.: Mullerian inhibiting substance in humans: normal levels from infancy to adulthood. J Clin Endocrinol Metab, 81 : 571-576, 1996). It is a substance present in human female body, and is increased in the serum of women suffering from ovarian polycystosis, its increase causing no negative organic or biological effect on female organs. Moreover, an anti-cancer action was proposed for AMH in epithelial tumours of the ovary, and various experimental evidences seem to support its cytotoxic effect on tumour cells (La Marca A., Volpe A: The anti-Mullerian Hormone and ovarian cancer. Hum Reproduct., 13: 265-273, 2007).

Considering the very low toxicity of said substance in the human body, the possibility of use as anti-cancer drug is of great clinical interest.

Recent studies demonstrated that AMH, as well as a receptor (MISRII) thereof, are expressed in the adult female also at endometrial level, where they probably carry on a paracrine-type function.

Endometriosis to date is still a disorder for which the sole effective therapeutic strategy is the surgical removal of endometriotic lesions: there is no pharmacological therapy able to eliminate or reduce endometriotic lesions, and the sole pharmacological treatments used by the medico-scientific community are merely able to act on symptoms, relieving them.

Therefore, the need to single out a novel therapeutic strategy allowing to radically treat endometriosis by directly acting on all endometriotic lesions at cell level, and differently from surgery, which not only is an invasive method but enables to intervene merely on visible lesions and not on microscopic ones, is direly felt in the state of the known art.

SUMMARY OF THE INVENTION

The present description relates to the use of antimullerian hormone (AMH) and/or biologically active form thereof in the treatment and/or slowing of endometriosis in a subject in need thereof.

In the present invention, by "slowing of endometriosis" it is meant the decrease or the non-variation over time of the number of endometriotic lesions observed and identified by the clinician in a subject suffering from endometriosis. By "treatment of endometriosis" it is meant instead the elimination, over time, of all endometriotic lesions present in a subject suffering from endometriosis, therefore, in other terms, in the present description it is meant the eradication, over time, of endometriosis itself in the subject undergoing therapy with AMH.

The invention subject of the present description is based on the observation, performed by the same Inventors, that the treatment of endometriotic cells (obtained from the glandular or stromal component of the endometriotic lesion) with the antimullerian hormone causes, on the one hand, treated cells blockage in the G1/G2 phase of the cell cycle, with entailed decrease of the S phase of the cycle itself, and, on the other hand, increase of apoptosis-induced cell death. In particular, therefore, the Inventors of the present Invention have demonstrated for the first time that it is possible to slow the growth or induce apoptosis of endometriotic cells by treatment with the antimullerian hormone. Accordingly, the antimullerian hormone can advantageously be employed to slow the development of endometriotic lesions already present in the patient and/or to treat in a radical manner said pathology with no need to resort to endometriotic lesion removal by surgery.

Therefore, the main advantage associated to the invention subject of the present invention is to have identified for the first time in the antimullerian hormone a pharmacological instrument extremely effective in the noninvasive treatment of endometriosis, treatment that, as already mentioned in the preceding section related to the state of the known art, had only been possible by surgery.

Therefore, objects of the present application are:

-antimullerian hormone or biologically active form thereof for use in the treatment and/or slowing of endometriosis in a subject in need thereof, wherein said hormone or said biologically active form thereof is administered by oral, transdermal or transvaginal route;

- a pharmaceutical composition comprising antimullerian hormone and/or biologically active form thereof and at least one pharmaceutically acceptable excipient for use in the treatment and/or slowing of endometriosis in a subject in need thereof, wherein said composition is administered by oral, transdermal or transvaginal route;

- a kit comprising aliquots for sequential or concomitant use of the components of the composition as previously defined for use in the treatment and/or slowing of endometriosis.

Further advantages, as well as the features and the modes of employ of the present invention will be made apparent in the following detailed description of some preferred embodiments thereof, given merely by way of illustration and not for limitative purposes. DETAILED DESCRIPTION OF THE FIGURES

Figure 1 shows the effect of antimullerian hormone on the different phases of the cell cycle in treated cells.

Figure 2 shows, over time, the degree of apoptosis induced in endometriotic cells after treatment with antimullerian hormone.

Figure 3 shows how MISRII anti-mullerian hormone receptor is actually expressed at RNA level in endometriotic cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antimullerian hormone (AMH) or biologically active form thereof for use in the treatment and/or slowing of endometriosis in a subject.

Antimullerian hormone is a homodimeric disulfide-linked glycoprotein with a molecular weight of 140 kDa belonging to the Transforming Growth Factor-beta (TGF- beta) family. The nucleotide sequence and the amino acid sequence coding for AMH, both of human and bovine origin, are described in the state of the known art, and reported, e.g., in U.S. Pat. No. 5047336.

To the ends of the present invention, AMH may be obtained by recombinant DNA techniques, or by cloning of the nucleotide sequence of interest in a cloning vector, transformation of a host cell with said vector, expression of the protein of interest in said host cell, purification of the recombinant protein obtained. The techniques for obtaining recombinant proteins are widely known and described in various laboratory manuals, like, e.g., in Maniatis et al. (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY), therefore in the present description it is not necessary to delve into the techniques by which AMH can be obtained in the form of recombinant protein. Moreover, U.S. Pat. No. 5047336 incorporated herein as reference describes in detail a method for producing recombinant AMH from nucleotide sequences, both of human and bovine origin. Therefore, the technician in the field could synthesize AMH according to any one conventional technique, or even, if desired, by placing an order for synthesis at companies providing, among their services, synthesis of proteins of interest.

Alternatively, AMH hormone may be isolated and purified from a suitable biological sample of an animal in which the antimullerian protein is expressed. Merely by way of a non-limiting example, the biological sample may be blood or plasma and said animal may be a mammal, such as a human being, or bovines, pigs, equines, dogs, cats, rabbits. Therefore, in general, the antimullerian hormone useful for the treatment and/or slowing of endometriosis may be of any origin, as also indicated above, and obtained by genetic engineering techniques or purification from suitable biological samples. Preferably, said hormone is of human or bovine origin.

By "biologically active form" it is meant, in the present description, a modified form of the protein of interest characterized by the ability to induce the same biological effects of the wild-type protein, since its biological action will be induced via the same molecular mechanism stimulated by the wild-type form. Therefore, by way of a non- limiting example, biologically active forms of the antimullerian hormone are proteins which, compared to the wild-type form, have amino acid deletions, insertions, point mutations or substitutions with an amino acid of the same type/group (e.g., aspartate with glutamate, arginine with lysine, etc.), or additional sequences for detection/purification/stability.

In a preferred embodiment of the invention, said biologically active form comprises or consists in the C-terminal (carboxy-terminal) portion of AMH. The carboxy-terminal portion of AMH corresponds to a 12.5 kDa fragment (about 25 kDa under non-reducing conditions, therefore under conditions favourable to existence of the dimeric form) obtained by proteolytic cleavage at residue 427 of the amino acid sequence of 535 aa of monomeric human AMH (monomeric human AMH amino acid sequence: Accession number P03971 in Bank UniProtKB/Swiss-Prot Bank, version 133, last modified on May 16, 2012; http://www.uniprot.org/). Instead, the residue at which proteolytic cleavage occurs is 443 of the bovine protein of 551 aa (bovine AMH amino acid sequence: Accession number P03972 in UniProtKB/Swiss-Prot Bank, version 91 , last modified on May 16, 2012; http://www.uniprot.org/).

Analogously to the above, also the C-terminal portion may be obtained by genetic engineering techniques, or isolated and purified from a biological sample of an animal. Alternatively, it may be simply obtained by proteolytic cleavage, e.g. by utilizing plasmin enzyme, from the corresponding wild-type form of the amino acid sequence of AMH. Moreover, to the ends of the present invention, the carboxy-terminal portion may in turn be utilized in a biologically active modified form thereof, as previously described above for wild-type AMH.

The present invention refers to the use of said antimullerian hormone or said biologically active form thereof for the treatment and/or slowing of endometriosis in a subject in need thereof. In other terms, AMH can advantageously be used to decrease, eliminate, or keep unvaried over time the number of endometriotic lesions in a subject suffering from endometriosis. By "subject suffering from endometriosis" it is meant any animal subject in which it is possible to diagnose and/or highlight endometriotic lesions, like e.g. a human being or a horse.

Antimullerian hormone for use according to what is described herein is preferably used at an amount effective to the ends of the treatment and/or slowing of endometriosis. The effective dosage of AMH or biologically active form thereof to be administered may be preset in daily unitary doses, or at intervals of plural days or of weeks, or may be assessed by the medical staff according to the patient's disorder state, weight, gender and age.

By "effective dose" or "therapeutically effective dose" it is meant a dose allowing the researcher or physician to observe the therapeutic effect in the treated patient. In the specific instance, a therapeutically effective dose will be a dose (administered in one or more unitary dosages over time) leading to a reduction, elimination or non-variation of the number of endometriotic lesions observed with respect to time t=0, i.e. prior to start of the therapy with AMH or biologically active form thereof.

At the molecular level, the biological effect observed in the treated patient, i.e. the antimullerian hormone ability to act directly on the endometriotic lesion, is due to the presence of receptors for AMHMISRII hormone in endometriotic cells. In fact, experimental data reported below also demonstrate that endometriotic cells express the MISRII receptor specific for antimullerian hormone, and, therefore, that such cells are an ideal target for said hormone. Moreover, it is reported in the literature that MISRII receptor saturation occurs at concentrations of 75-100 nmol/L and that the dissociation constant varies in a range of 6-12 nmol/L (Masiakos PT, MacLaughlin DT, Maheswaran S, Teixeira J, Fuller AF Jr, Shah PC, et al. Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) type II receptor, bind, and are responsive to MIS. Clin Cancer Res 1999; 5: 3488-99).

However, the effective dose will essentially depend on how the substance will be pharmacologically defined, considering also that AMH probably exerts a paracrine action on target tissues, and that it will probably be necessary to modify the molecule so as to obtain high concentrations just at the target sites.

In particular, experimental data reported below demonstrate how in AMH- treated endometriotic cells a cell cycle blockage in the G1/G2 phase is observed, with consequent decrease of the S phase of the cycle itself, and an increase of cell death by apoptosis. Such data support the ability of AMH or biologically active form thereof in slowing or treating endometriosis. The present invention also relates to a pharmaceutical composition comprising antimullerian hormone and/or biologically active form thereof and at least one pharmaceutically acceptable excipient for use in the treatment and/or slowing of endometriosis in a subject in need thereof.

In a preferred embodiment of the compositions described herein, the biologically active form is a form that comprises the C-terminal portion of said hormone, as defined above.

The hormone or its modified form, in accordance with the aforedescribed, together with an excipient, adjuvant, carrier, diluent conventionally used in preparations/compositions for pharmaceutical use, may be put in the composition itself in any kind of dosage deemed suitable to the ends of the present invention, therefore, e.g., in unitary-type doses.

By "unitary dose" it is meant the dose suitable to reach the therapeutically effective amount as defined above, and is usually set also on the basis of age, gender and health conditions of the patient to be treated.

The unitary dose could vary depending on the administration regimen selected by the physician. Evidently, the administration regimen should be suitable for achieving the desired effect, be it therapeutic and/or slowing of endometriosis, which will typically be determined by a physician, in light of the relevant circumstances, including the site of disorder onset, the administration route selected, the individual patient's age, weight and response, the severity of the patient's symptoms, and the like.

As described above, the pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable excipient, adjuvant and/or vehicle that, as used herein, includes any and all solvents, diluents or other liquid carriers, aiding agents for dispersion or suspension, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suitable for the particular form of dosage desired. Remington's Pharmaceutical Sciences, 16^th Ed. , E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) describes various carriers used in the formulation of pharmaceutically acceptable compositions and the known techniques for their preparation.

Some exemplary materials that may serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, aluminium, aluminium stearate, lecithin, serum proteins, such as human serum albumine, buffer substances (phosphate, sorbic acid or potassium sorbate), partial mixtures of glycerids of vegetable saturated fatty acids, water, salt or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, fatty tissue, sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and derivatives thereof, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax, oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, corn oil and soybean oil; glycols, such as propylene glycol or polyethyl glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminium hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution; ethyl alcohol and phosphate-buffered salines, as well as other compatible non-toxic lubricants, such as sodium lauryl sulphate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweeteners, flavors and perfuming agents, preservatives and antioxidants, may also be present in the composition according to the judgment of who prepares the formulation.

The pharmaceutical compositions described herein may also comprise at least one active ingredient different from AMH and known to be useful in the treatment of endometriosis. Merely by way of example, hereinafter some active ingredients (principles) are indicated, some of which are already used in the treatment of endometriosis, which may be used in combination with AMH.

Preferably, said at least one active ingredient is selected from the group comprising the following classes of compounds:

a) non-steroidal antiphlogistics, like: aryl acidic anti-inflammatory agents, non- acidic anti-inflammatory agents, anthelmintics, sulfide compounds, protease inhibitors, synthetic antimalarics, gold salts, griseofulvin, various substances having a bland antiphlogistic action, such as glucosamine, organ lysates, SAMe, heparin, heparinoids, azulene derivatives, flavonoids, coumarins, aescin, calcitonin, D-glycofuranoside, proteolytic enzymes, antifibrinolytics, bametan, bufenine, salbutamol, glycyrrhizinic acid, immunosuppressants;

b) steroidal antiphlogistics (natural and synthetic glucocorticoids);

c) folliculoid or estrogens hormones and (natural and synthetic) inhibitors thereof; d) luteoid or progestin hormones and (natural and synthetic) inhibitors thereof; e) (natural and synthetic) associations of estro-progestins;

f) testicular hormone (testosterone) and (natural and synthetic) inhibitors thereof; g) agonists and antagonists of GHRH (gonadotropin-releasing hormone), GH-IH (somatostatin), CRH (ACTH-releasing hormone), PIH and PRH (respectively, prolactin- inhibiting and releasing hormones), MIH and MRH (respectively, MSH-inhibiting and releasing hormones), LH-RH and FSH-RH (respectively, LH- and FSH-release stimulating hormones), all of them natural and synthetic;

h) (natural and synthetic) agonists and antagonists of STH (somatotropic hormone); (natural and synthetic) agonists and antagonists of ACTH (corticotropic hormone); (natural and synthetic) agonists and antagonists of TRH (thyrotrophic hormone); (natural and synthetic) agonists and antagonists of FSH and LH (gonadotropic hormones); (natural and synthetic) agonists and antagonists of MSH (melanophore-stimulating hormones); (natural and synthetic) agonists and antagonists of vasopressin and of oxytocin;

I) (natural and synthetic) aromatase inhibitors;

m) antineoplastic drugs, like, e.g., alkylating agents, antimetabolites, antibiotics, plant-derived mitostatics, enzymatic agents, hormones, immunogens, and various ones such as nocodazole, razoxane, azelaic acid;

n) biological antineoplastic drugs, like e.g. drugs belonging to the two families of -MAB and -INIB;

o) antibiotics, like e.g. pre-sulfonamides, sulfonamides, post-sulfonamides, penicillins, cephalosporins, macrolides, lincosamides, novobiocin, phosphomycin, rifampicin, ristocetin, vancomycin, fusidic acid, streptogramin, chloramphenicol, tiamphenicol, tetracycline, aminoglycoside, cyclic polypeptides, albomycin and monomycin, antimycobacterials, antimycotics, antiluetics, antiamebics, antimalarics, antitrypanosoma agents, antibilharziosics, local parasiticides;

p) coagulants and anticoagulants;

q) antianemic and anemic agents; platelet genetic agents and platelet lowering agents;

r) vasoconstrictors, anti-hypertensive-hypotensive;

s) cardiotrophics, heart stimulants, like e.g. digitalis drugs, camphor drugs, methylated xanthines, adrenalinics, antiarrhytmics.

t) diuretics and antidiuretics;

u) organic and inorganic substances used as tracers in the diagnosis and the therapy with physical means, such as, by way of example, X-rays, magnetic waves, ultrasounds, PET, etc.

v) immunostimulators, immunosuppressants and immunomodulants.

The concentration of the further one or more active ingredients in the composition could easily be established by persons skilled in the art on the basis of the concentrations normally used. The pharmaceutically acceptable compositions may be administered by oral, parenteral, intravenous, aerosol, rectal, transdermal, subcutaneous, intracisternal, intramuscular, vaginal, intraperitoneal, topical, perilingual and intranasal route, and according to all administration routes known to a technician in the field and suitable to the ends of the present invention.

In an embodiment of the invention, the administration route is selected among oral, transdermal or transvaginal administration route.

By way of a non-limiting example, hereinafter some dosages by oral, transdermal or transvaginal route of the AMH hormone or the pharmaceutical compositions comprising it are reported:

oral administration route, e.g. in the form of tablets, capsules, film-coated tablets, etc., from 0.3 mg to 6 mg per day;

transdermal administration route, e.g. in the form of patch with continuous release, from 0.25 mg to 4 mg in the 24 hours (patch duration, from 1 to 7 days);

transdermal administration route, e.g. in the form of removable subcutaneous implant, with release of 0.25 mg to 4 mg per day;

transdermal administration route: e.g. in the form of single-dose gel, from 0.25 mg to 4 mg per day;

transvaginal administration route: e.g. in the form of tablets, ovules, single-dose gel, from 0.5 mg to 6 mg, one per day;

In case of use of the carboxy-terminal active portion, the above dosages can also be reduced to those reported herebelow:

oral administration route, e.g. in the form of tablets, capsules, film-coated tablets, etc., from 0.03 mg to 6 mg per day;

transdermal administration route, e.g. in the form of patch with continuous release, from 0.025 mg to 4 mg in the 24 hours (patch duration, from 1 to 7 days); transdermal administration route, e.g. in the form of removable subcutaneous implant, with release of from 0.025 mg to 4 mg per day;

transdermal administration route: e.g. in the form of single-dose gel, from 0.025 mg to 4 mg per day;

transvaginal administration route: e.g. in the form of tablets, ovules, single-dose gels, from 0,05 mg to 6 mg, one per day; Moreover, in connection with the seriousness of the endometriosis to be treated and to the extension and localization of the lesions in the female body, such doses may be increased up to ten times the illustrated ones.

The compositions for oral administration may take the form of liquid solutions or suspensions, or powders. The compositions could be presented in the form of unitary dose, so as to facilitate dosage.

The term "form of unitary dose" refers to a discrete physical unit suitable for unitary doses for human subjects, each unitary dose containing a predetermined amount of active material calculated for producing the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical forms of unitary doses include vials or syringes of the liquid composition, or pills, tablets, capsules or the like in case of solid compositions and transdermal patches.

Liquid forms suitable for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, gels, microemulsions, solutions, suspensions, syrups and elixirs and could contain diluents commonly used in the state of the art. The oral liquid forms can therefore include a suitable aqueous or non-aqueous vehicle with buffers, aiding agents for suspension and dispersion, emulsifying agents, solvents, colorants, flavors and the like.

E.g., they could include water or other solvents, solubilizing and emulsifying agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, (wheat) germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl acid, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions could also include adjuvants such as humectants, emulsifying and suspending agents, sweetening, flavoring and parfuming agents.

The liquid forms could also be injectable preparations, e.g., sterile injectable aqueous and oleaginous suspensions, and could be formulated according to the known art by using suitable dispersing agents, humectants and suspending agents. The sterile injectable preparations could also be an injectable sterile solution, suspension or emulsion in a parenterally acceptable diluent or solvent, like, e.g., a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be used there are water, Ringer's solution, USP and isotonic sodium chloride solution. Furthermore, sterile fixed oils are conventionally used as solvent or suspending means. Moreover, fatty acids like oleic acid are used in injectable preparations. Preservatives and other additives such as antimicrobials, antioxidants, chelating agents and inert gases could also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16^th Edition).

The injectable compositions could be sterilized, e.g., by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that may be dissolved or dispersed in sterile water or other injectable sterile means before use.

Injectable slow-release forms can be made also by forming microincapsulated matrices of the active principle(s) in biodegradable polymers such as polylactide- polyglycolide. Depending on the active principle(s)/polymer ratio and the nature of the specific polymer used, release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable slow-release formulations can also be prepared by trapping the active principle(s) in liposomes or microemulsions compatible with body tissues.

Semiliquid (waxy) formulations. The compositions for rectal or vaginal administration are preferably suppositories that can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which is solid at room temperature but liquid at body temperature and therefore dissolves in the rectum or in the vaginal cavity, releasing the active compound.

Solid-dose forms for oral administration include capsules, tablets, pills, powders and granules. In such solid forms the active principle(s) is/are mixed with at least one pharmaceutically acceptable inert excipient or carrier, like, e.g., sodium citrate or calcium phosphate citrate and/or fillers or extenders (such as starches, lactose, sucrose, glucose, mannitol, and silicic acid) binders; (such as, e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose); humectants (e.g., glycerol); disintegrating agents (such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates and sodium carbonate); retarding agents (e.g., paraffin) absorption accelerators (such as quaternary ammonium compounds); wetting agents (such as, e.g., cetyl alcohol and glycerol monostearate); absorbents (such as kaolin and bentonite clay); lubricants (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate) and mixtures thereof.

In case of capsule, tablets and pills, the form of dose could also comprise buffering agents. The solid compositions as indicated above can also be employed to fill capsules of rigid or soft gelatin using excipients like lactose or milk sugar, as well as high-molecular weight polyethylene glycols and the like. The solid dose forms of tablets, film-coated tablets, capsules, pills, and granules, can be prepared with coatings such as enteric coatings and other coating agents known in the art of pharmaceutical formulations. Optionally, they could be formulated so as to release the active ingredient(s) solely or preferentially in certain parts of the intestinal tract, optionally in a delayed manner.

The compositions of the present invention could also be formulated for topical administration in the form of ointments, pastes, lotions, gels, powders, solutions, sprays, inhalants, ophthalmic or otic drops, or plasters. The active component (or components) is mixed under sterile conditions with a pharmaceutically acceptable carrier and, needwise, with any required preservative or buffer.

Transdermal patches could be used to provide a controlled release. Absorption enhancers could also be used, to increase the flow of the compound through the skin. The release rate could be controlled by providing a rate-controlled membrane or dispersing the compound into a polymer matrix or a gel. Therefore, as indicated above, the composition comprising antimullerian hormone and/or biologically active form thereof may moreover contain one or more further active principles, could also be administered in prolonged-release forms or with systems for administration of prolonged-release drugs. A description of materials for such embodiment can also be found in the materials incorporated in Remington's Pharmaceutical Sciences.

The above-described components for oral administration or injectable compositions are merely representative. Further materials as well as processing techniques and the like are shown in Part 8 of Remington's Pharmaceutical Sciences, 17^th Edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein as reference.

The components of the composition, like AMH and/or its biologically active form and/or other active principles present can be lyophilized for storage and reconstituted in a suitable carrier before use. For this embodiment, lyophilizing and reconstituting techniques known in the state of the art can be used.

For what observed and demonstrated by the Inventors of the present invention in connection with antimullerian hormone effectiveness in eliminating and/or reducing the endometriotic lesions, AMH can also be used in a method for the treatment and/or slowing of endometriosis comprising the step of:

e) administering effective amounts of antimullerian hormone or a composition comprising it, as defined in the present description, to a patient in need thereof.

The present invention also comprises a pharmaceutical kit for concomitant or sequential administration of the various components comprised in the pharmaceutical composition, as defined above, and present in the kit in the form of aliquots. Therefore, by way of example, when the composition comprises as active principles AMH and biologically active form thereof, the kit may comprise one or more separate aliquots for said AMH and of said form. Each aliquot of the kit described herein could further comprise one or more of pharmaceutically acceptable carriers, adjuvants or vehicles that, as used herein, include any and all solvents, diluents or other liquid vehicles, aiding agents for dispersion or suspension, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suitable for the specific dose form desired.

The kit could be formulated so as to be able to administrate said aliquots for oral, parenteral, intravenous, aerosol, rectal, transdermal, subcutaneous, intracisternal, intramuscular, vaginal, intraperitoneal, topical, perilingual and intranasal use.

The pharmaceutical kit as described could be used in the treatment and/or slowing of endometriosis in a subject.

Aim of the next section is to illustrate data obtained on antimullerian hormone and the activities observed on the latter, and therefore to clarify the invention without, of course, being limitative of the same.

EXAMPLES

EXAMPLE 1 : AMH effectiveness in blocking endometriotic cell growth. Primary cultures of immortalized endometriotic cells, both of the glandular component and the stromal one, previously defined and characterized as described in Boccellino et al (Boccellino et al: In vitro model of stromal and epithelial immortalized endometriotic cells. Journal of Cellular Biochemistry, doi: 10.1002/jcb.24000. [Epub ahead of print]) were used as in vitro cell model to test pharmacological effectiveness of antimullerian hormone.

Immortalized epithelial and stromal cell (ESC) lines were resuspended in DMEM/F12 culture medium enriched with 10% FBS (FetalSerum Bovine), 100 III/ mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin and 200 mM L- Glutamine. They were plated in Corning T-25 flasks in the presence of puromycin (final concentration: 500 ng/mL) and incubated at 37°C under a 5% C0₂ atmosphere. Then, cell lines were trypsinized and transferred into 6-well multiwell at a density of about 100.000 cells/well and incubated at 37°C under a 5% C0₂ atmosphere. After 24 hours medium was sucked off, cells were washed with sterile PBS 1X and only three wells were induced with Recombinant Human MIS/AMH by R&D system, Catalog # 1737-MS (reconstituted to 10 ug/ ml_ in sterile PBS containing 0.1 % BSA) for 72 hours at three different final concentrations (10-100-1000 ng). After 24 hours, other three wells were induced with Recombinant Human MIS/AMH at the three different concentrations. After other 24 hours, further three wells were induced with the same indications. At the end of the 72 hours, medium was sucked off, cell were washed with sterile PBS 1X, trypsinized, transferred into specific tubes for cytofluorimeter FACS reading and centrifuged at 1000 rpm for 5 min at 4°C; obtained pellet was subjected to analysis of cell cycle and apoptosis. Each cell pellet obtained after treatment with Recombinant Human MIS/AMH for 72, 48 and 24 hours at different concentrations was resuspended and lysed in about 500 μΙ_ of hypotonic Buffer (containing 0.1 % sodium citrate, 0.1 % NP-40, 50 μg/mL Propidium iodide (PI) ). Cells were incubated in the dark, at room temperature for about 30 minutes. Then, the Inventors proceeded with sample acquisition at the FACS-Calibur cytofluorimeter, using Cell Quest software (Becton Dickinson). Samples were analyzed with standard procedures using Cell Quest and ModFit version 3 (Verity) software.

As evident from Figure 1 , regardless of AMH concentrations used, at +48 hours the treatment causes a significant cell cycle blockage in G1 , with entailed decrease of phase S; such blockage then significantly becomes in G2 at +48 hours, with an entailed decrease of phase S.

Moreover, analysis of apoptosis extent in said cells demonstrated, as evident in Figure 2, that already from +24 hours the number of apoptotic cells increases with respect to control, reaching the highest values at +72 hours for the treatment with 1000 ng/mL.

Example 2: Expression of Ml SRI I gene, receptor for AMH, in endometriotic cells.

From epithelial ESC and Stromal ESC line, total RNA was extracted by using Trizol reagent according to Invitrogen protocol. Obtained DNA was retro-transcribed in complementary DNA (cDNA) by using the Superscript Vilo kit (Invitrogen) according to the manufacturer's instructions. The resulting cDNA, along with synthesized primers MISRII (1) and (2) was analyzed through PCR.

PCR reaction occurred according to the EuroClone protocol (EuroTaq; 100 mMdATP- dTTP- dCTP- dGTP; 10X Reaction Buffer (Magnesium Free); 50 mM MgCI2; 10 μΜ Oligont Reverse eForward) under the conditions: 1) 94°C for 10min; 94°C for 30 sec; 59°C for 30 sec; 72°C for 30 sec; repeated for 35 cycles.

2) 94°C for 10min; 94°C for 30 sec; 62°C for 30 sec; 72°C for 30 sec; repeated for 40 cycles.

PCR products were then visualized by electrophoretic run on 1 % agarose gel containing ethidium bromide.

Primers were designed by using Primer3Plus software. The sequences are as follows: for MISRII receptor (1) Forward-5'CCC TGC TAC AGC GAA AGA AC 3'; Reverse- 5'ATG GCA ACC AGT TTT CCT TG 3'. For MISRII receptor (2) Forward- 5'AAC TGG CCT ATG AGG CAG AA 3'; Reverse- 5'GGT CTG CAT CCC AAC AGT CT 3'. For GAPDH gene Forward- 5'GGT GTC AAC GGA TTT GGT CG 3'; Reverse- 5'CTT CCC GTT CTC AGC CTT GA 3'.

In figure 3, agarose gel visualization of gene amplification products for MISRII and for GAPDH, used as control, in endometriotic cells is reported.

REFERENCES

1. Giudice LC , and Kao LC: Endometriosis. The Lancet, 364: 1789-1799, 2004.

2. Houston DE: Evidence for the risk of pelvic endometriosis by age, race, and socioeconomic status. Epidemiol Rev, 6: 167-191 , 1984.

3. Von Rokitansky C: Ueber uterusdrusen-neubildung in uterus and ovarilsarcomen. Z Ges Aertze Wein, 37: 577-593, 1860.

4. La Marca A et al.: Anti-Mullerian hormone (AMH): what do we still need to know? Hum Reproduct, 24: 2264-2275, 2009.

5. Lee MM et al.: Mullerian inhibiting substance in humans: normal levels from infancy to adulthood. J Clin Endocrinol Metab, 81 : 571-576, 1996.

6. La Marca A., Volpe A: The anti-Mullerian Hormone and ovarian cancer. Hum Reproduct., 13: 265-273, 2007.

7. Masiakos PT, MacLaughlin DT, Maheswaran S, Teixeira J, Fuller AF Jr, Shah PC, et al. Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) type II receptor, bind, and are responsive to MIS. Clin Cancer Res 1999; 5: 3488-99

Claims

1.Antimullerian hormone or biologically active form thereof for use in the treatment and/or slowing of endometriosis in a subject in need thereof, wherein said hormone or said biologically active form thereof is administered by oral, transdermal or transvaginal route.

2. Antimullerian hormone or biologically active form thereof according to claim 1 , wherein

said oral administration is at a dosage comprised between 0.3 mg and 6 mg per day;

said transdermal administration is at a dosage of 0.25 mg to 4 mg per day; said vaginal administration is at a dosage of 0.5 mg to 6 mg per day.

3. Antimullerian hormone or biologically active form thereof according to any one of claims 1-2, wherein said form comprises the C-terminal portion of said hormone.

4. Antimullerian hormone or biologically active form thereof according to any one of claims 1-3, which is of human origin.

5. Pharmaceutical composition comprising antimullerian hormone and/or biologically active form thereof and at least one pharmaceutically acceptable excipient for use in the treatment and/or slowing of endometriosis in a subject in need thereof wherein said composition is administered by oral, transdermal or transvaginal route.

6. Composition according to claim 5, wherein

7. Composition according to claim 4 or 5, wherein said form comprises the C- terminal portion of said hormone.

8. Composition according to any one of claims 4-7, further comprising at least one active ingredient useful in the treatment of endometriosis.

9. Composition according to claim 8, wherein said at least one active ingredient is selected from the group comprising: non-steroidal or steroidal antiphlogistics; folliculoid or estrogens hormones and inhibitors thereof; luteoid or progestin hormones and inhibitors thereof; associations of estro-progestins; testicular hormone and inhibitors thereof; agonists and antagonists of GHRH, GH-IH, CRH, PIH, PRH, MIH, MRH, LH- RH, FSH-RH; agonists and antagonists of STH, ACTH, TRH, FSH, LH, vasopressin, oxytocin; aromatase inhibitors; antineoplastic drugs, antibiotics; coagulants and anticoagulants; antianemic and anemic agents; platelet genetic agents; platelet lowering agents; vasoconstrictors, anti-hypertensive-hypotensive; diuretics and antidiuretics; organic and inorganic substances used as tracers in the diagnosis.

10. Kit comprising aliquots of the composition according to any one of the claims 4-

9, for sequential or concomitant use of said antimullerian hormone or said biologically active form and said at least one active ingredient for use in the treatment and/or slowing of endometriosis.