WO2020044298A1 - A novel use of the anti-müllerian hormone - Google Patents

Abstract

The present invention relates to the anti-Müllerian hormone, i.e. AMH, or a composition comprising said AMH for use in the prevention and/or treatment and/or follow-up of benign tumors of the uterus.

Description

DESCRIPTION

TITLE

A novel use of the anti-Miillerian hormone Technical Field

The present invention relates to the anti-Mullerian hormone, i.e. AMH, or a composition comprising said AMH, for use in the prevention and/or treatment and/or follow-up of benign tumors of the uterus.

State of the Art

Benign tumors are characterized by the growth of some cells that develop more than they should, giving rise to masses that can also take on considerable sizes. However, unlike malignant tumor cells, these benign masses remain well delimited, retain the characteristics of the tissue from which they originate and do not tend to invade surrounding organs or produce metastases.

Among the most common benign tumors in the world, those of the uterus account for a significant percentage. In fact, it is estimated that one out of ten women is affected by a benign tumor of the uterus.

Benign tumors of the uterus are distinguished into myomas, polyps and endometrial hyperplasias.

A myoma, also called leiomyoma or uterine fibroid, is a benign gynecological tumor due to an exaggerated development of the smooth muscle cells of the uterus which make up the myometrium. A myoma is a monoclonal myometrial tumor originating from the Mullerian ducts and is the most common tumor of the female reproductive system.

Although myomas are well known in terms of their clinical manifestations, their etiopathogenesis still remains unclear, even if estrogens seem to be implicated in the multiplication of the cells of the myoma itself. Not coincidentally, myomas are very unlikely to appear before puberty, whereas after menopause, when the production of estrogens ceases, myomas can become reduced in size until disappearing altogether. During pregnancy, by contrast, when the estrogen level is high, a myoma, if present, can increase considerably in volume with an increase in the risk for the pregnancy.

The growth of myomas mainly derives from ovarian steroids (estrogens and progesterone) which act through the receptors present both on myometrial cells and on connective tissue cells and it is probable that the lack of control of growth is partly due to an abnormality in apoptotic processes.

Today, different courses of therapy are available which take account of the patient’s age, the position and number of myomas, the presence of concomitant pathologies, previous therapeutic failures and the desire for motherhood, where present.

Although myomas are a benign gynecological pathology, the symptoms resulting from them have considerable repercussions in terms of the quality of life of women affected by them. The current medical therapies include progestins, selective estrogen receptor modulators (SERMs), aromatase inhibitors, gonadotropin-releasing hormone (GnRh) analogues and selective progesterone receptor modulators (SPRMs). However, none of them are free of side effects, which sometimes make it necessary to suspend the treatment itself.

Furthermore, if a myoma does not respond to pharmacological therapy or if it is large in size, in most cases a surgical intervention is resorted to.

This, also in the light of epidemiologic data, should lead to a search for increasingly effective and individualized therapies, in order to be able to expect the preset results while reducing adverse events to a minimum. Given the widespread nature of uterine fibromatosis, most of the times associated with very severe, debilitating pain, there is always a greatly felt need for novel therapeutic approaches capable of curing a myoma while avoiding a surgical intervention.

In particular, clinical research in this sector has always been very focused on identifying new therapies of a conservative type, for example pharmacological protocols that ensure treatment of the myoma while avoiding any surgical intervention.

A particular role in cell differentiation and in the control of cell growth is played by the transforming growth factor beta (TGF- b) superfamily, which includes activins, inhibins, the anti-Mullerian hormone (AMH) and the bone morphogenetic protein (BMP).

The roles performed by TGF-b signaling include that of controlling proliferation, differentiation and other functions in the majority of cells.

In particular, AMFI, also known as anti-Mullerian Flormone (AMFI) Mullerian-inhibiting-factor (MIF) or Mullerian-inhibiting-substance (MIS) or Mullerian-inhibiting-hormone (M IH), is a dimeric glycoprotein, whose gene in humans is located on the short arm of chromosome 19, band 19p13.3 (Cohen-Flaguenauer et al., 1987), and it is divided into five exons. One of the main roles of AMFI is that of promoting the development of the Wolffian duct in humans, blocking the development of the Mullerian duct and favoring the growth of the male genital apparatus.

In women, AMFI is produced exclusively in the ovary by granulosa cells (gonadal somatic cells) and is involved in the regulation of follicle growth and development (La Marca and Volpe, 2006a).

The serum levels of AMFI in women decrease with age, until becoming undetectable in the post-menopausal period (La Marca et al. 2010). Similarly, in patients with primary ovarian insufficiency (POI), AMFI is undetectable or is very low, based on the number of residual follicles in the ovary. In contrast, the levels of AMFI increase in conditions such as polycystic ovary syndrome (PCOS), where the ovary is rich in follicles (La Marca et al., 2009).

Summary of the invention

A first aspect of the present invention relates to the anti-Mullerian hormone, i.e. AMFI, or a composition comprising AMFI, for use in the prevention and/or treatment and/or follow-up of benign tumors of the uterus, in particular of myomas, polyps and endometrial hyperplasias.

In fact, the Applicant has surprisingly found that: the receptor AMHR2 is more expressed in tissue and in cell cultures deriving from a myoma than in tissue and cultures deriving from healthy myometrium;

AMHR2 is biologically active, above all in cells deriving from a myoma, since it participates in the regulation of SMAD3 and SMAD5 genes, typically involved in the signal transduction pathway; and

the administration of AMH induces the death by apoptosis of cells deriving from a myoma by binding with its receptor, upregulating the proapoptotic genes PARP-1 and caspase 3 and downregulating the antiapoptotic gene BCL-2.

A second aspect of the present invention relates to a method for the treatment of benign tumors of the uterus, preferably a myoma or leiomyoma or fibroid, said method comprising a step of administering an effective amount of AMH or of the composition comprising said AMH to a subject who is affected or suspected of being affected by said tumor, or said subject having been surgically treated for removal of said tumor.

Brief description of the drawings

The present invention is described below in detail by way of illustration without any limitation, also with reference to the appended Figures as briefly described below.

- Figure 1 shows the result of the electrophoretic run of the products of RT-PCR reactions performed for the AMHR2 gene and the normalizer thereof b-actin. M = myometrium; L = leiomyoma; (-) = negative control (H20).

- Figure 2 shows a semi-quantification of the PCR reaction products for the AMHR2 gene of Figure 1. The light intensity generated by the bands in the gel, directly proportional to the expression of the AMR2 gene, was calculated by densitometric analysis, applying the formula optical density x band area using Quantity One (Biorad) software.

- Figure 3 shows the presence of the receptor AMHR2 in tissue or cell cultures originating from healthy myometrium or a leiomyoma semi- quantified by conversion of the fluorescence signal to grayscale and expressed as arbitrary units of fluorescence. For each sample, three fields were analyzed, expressed as the mean value ± standard deviation.

- Figure 4 shows the expression of the SMAD3 and SMAD5 genes after 24 hours of incubation with increasing amounts of AMFI resulting from the RT- qPCR reactions. The results refer to the mean expression calculated in cell cultures deriving from a myoma or healthy myometrium of 4 different patients. The significance values were calculated with the method called Student’s t-test considering the values with a probability p<0.005 as significant.

- Figure 5 shows the expression of the PARP-1 , caspase-3 and BCL-2 genes after 48 and 96 hours of incubation with AMFI (100 ng/ml) resulting from the RT-qPCR reactions. The results refer to the mean expression calculated in cell cultures deriving from a myoma or healthy myometrium of 4 different patients. The significance values were calculated with the method called Student’s t-test considering the values with a probability p<0.005 as significant.

- Figure 6 shows the expression of the PARP-1 , BCL-2 and caspase-3 proteins induced by the treatment of cell cultures deriving from a myoma (left) or healthy myometrium (right) with AMFI (100 ng/ml).

- Figure 7 shows: panel A, the percentage of cells positive to the anti- PARP-1 reaction. The graphs show the mean of three cell counts in fields of 1 ,000 cells each and expressed as the mean ± standard deviation; Panel B, the fluorescent signal (green, semi-quantification) converted to grayscale and expressed as arbitrary units of fluorescence. For every sample, three fields expressed as the mean value ± standard deviation were analyzed.

- Figure 8 shows the percentage of cells positive to the TUNEL assay (fragmentation of nuclei, index of apoptosis). The cells were counted in three fields of 100 cells with the double-blind method and expressed as the mean ± standard deviation. - Figure 9 shows the expression of SMAD5 and FAS following incubation with AMH25-560 or AMFI₄₅₃-56o (100 ng/ml) for 24 hours, dissolved in the incubation medium. The letter a indicates a significant difference between the two AMFIs tested and the control (p<0.05); b indicates a significant difference between the two AMHs used (p<0.05). The significance values were calculated with the method called Student’s t-test.

- Figure 10 shows the expression of SMAD5 and FAS following intramyoma injection of 50 mI of AMH25-560 or AMH₄₅₃-56o (100 ng/ml) analyzed after 24 hours. The letter a indicates a significant difference between the two AMHs tested and the control (p<0.05); b indicates a significant difference between the two AMHs used (p<0.05). The significance values were calculated with the method called Student’s t- test.

- Figure 1 1 shows the expression of SMAD5 and FAS in primary cultures of myoma cells treated with 100 ng/ml of AMH25-560 or AMH₄₅₃-56o for 24 hours. The letter a indicates a significant difference between the two AMHs tested and the control (p<0.05); b indicates a significant difference between the two AMHs used (p<0.05). The significance values were calculated with the method called Student’s t-test.

Definitions

In this context, the term “anti-Mullerian hormone”, also called “AMH”, refers to the protein identified with the accession number NCBI: P03971 encoded by the gene located on chromosome 19, NCBI Gene ID: 268. In the context of the present invention, the cDNA of AMH is characterized by the sequence essentially corresponding to SEQ ID NO: 1 shown in Table 1 . The AMH protein is characterized by the sequence corresponding to SEQ ID NO. 2. The amino acid sequence of AMH₄₅₃-56o is characterized by the sequence corresponding to SEQ ID NO. 3. The AMH gene is characterized by the sequence essentially corresponding to SEQ ID NO: 4.

In this context, the term “agonist” means every chemical substance, a fragment or a derivative of the AMH protein (peptide/oligopeptide or the modified AMH protein), capable of binding and activating the AMH receptor.

In this context, the term“antagonist” means every chemical substance, a fragment or a derivative of the AMH protein (peptide/oligopeptide or the modified AMH protein), capable of antagonizing the bond between AMH and the receptor thereof to prevent transduction of the signal of the AMH receptor after it has been activated

In this context, the term“antiMIR” or anti-miRNA means a molecule of nucleic acid, complementary DNA or RNA, preferably according to the Watson and Crick rules, and thus capable of binding, in whole or in part, a micro-RNA or miRNA. In particular, in this context the target of the antiMIRs is the miRNA(s) that silence the gene and/or the AMH messenger. Therefore, in this context the antiMIRs recognize and bind the miRNAs, which, on recognizing and binding the AMH messenger, induce the silencing and/or the degradation thereof.

Detailed description of preferred embodiments of the invention

The present invention relates to the anti-Mullerian hormone, i.e. AMH, or a composition comprising said AMH, for use in the prevention and/or treatment and/or follow-up of benign tumors of the uterus.

In the context of the present invention, benign tumor of the uterus means a pathological condition caused by an abnormal growth of uterine cells which give rise to masses that remain well delimited in the uterus and do not invade surrounding tissues.

In one embodiment, the benign tumors are preferably selected from: myomas, adenomyomas, polyps and endometrial hyperplasias.

In this context, AMH also means any combination of an AMH agonist or an AMH antagonist or an AMH antiMIR.

Said AMH is preferably human.

Said AMH is preferably used as a recombinant or purified/isolated protein. In this context, reference is preferably made to the use of the entire AMH protein, or to homologues, analogues, variants, derivatives or fragments of the AMH protein provided that the activity of AMH is maintained.

In one embodiment, said AMH is a biologically active homologue of AMH. Said AMH is preferably encoded by the nucleotide sequence essentially corresponding to SEQ ID NO: 1.

Said AMH is preferably encoded by the nucleotide sequence corresponding to SEQ ID NO: 1.

The protein sequence of the pre-protein of said human AMH is preferably shown in Table 1 as SEQ ID NO: 2. The 1 -24 region of said SEQ ID NO: 2 refers to the signal peptide. The cleavage site is located in position 457 of SEQ ID NO: 2. The encoded pre-protein will be proteolytically processed so as to give rise to two subunits, the N- and the C-terminal, which, by homodimerizing, will form the biologically active molecule.

In a preferred embodiment of the invention, said AMH is a portion of the entire AMH protein, essentially corresponding to SEQ ID NO: 3 in Table 1 , i.e. the 453-560 region of the entire AMH protein.

In a preferred embodiment of the invention, said AMH corresponds to SEQ ID NO: 3 in Table 1.

Said human AMH is preferably encoded by the gene characterized by the sequence essentially corresponding to SEQ ID NO: 4 shown in Table 1 . Said human AMH is preferably encoded by the gene characterized by the sequence corresponding to SEQ ID NO: 4 shown in Table 1 .

Table 1

In this context, the protein and/or nucleotide variants of AMH characterized by a degree of similarity and/or identity of 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96% 98% and 99% to the amino acid and/or nucleotide sequences shown above are understood as described and thus included for the uses claimed herein.

In one embodiment of the invention, the protein variants of AMH to which reference is made have modifications in the N-terminal and/or C-terminal region, for example adapted to increase the activity of AMH. Said modifications are preferably selected from among deletions, additions, alterations of amino acids and combinations thereof. Alternatively, said AMH can be modified, preferably in the primary structure thereof, by acetylation, carboxylation, glycosylation, phosphorylation and combinations thereof.

In a further embodiment, said AMH is conjugated/bound to a molecule, a metal, or a marker, for example proteins, for the preparation of fusion proteins. In a further embodiment of the invention, said AMH is modified by means of molecular biology techniques to improve its resistance to proteolytic degradation and/or to optimize its solubility or improve its pharmacokinetic characteristics. In a further embodiment, said AMH, preferably the protein form, is conjugated to at least one molecule capable of improving its stability and/or half-life and/or water solubility and/or immunological characteristics. Said molecule is preferably a polyethylenic polymer; solely by way of example, polyethylene glycol (PEG).

In a further embodiment of the invention, said AMH protein is synthesized by means of conventional protein synthesis techniques known to the person skilled in the art. For example, the protein can be synthesized by chemical synthesis using solid phase peptide synthesis.

In a further embodiment of the invention, the AMH protein is isolated or purified with methods known to the person skilled in the art. For example, AMH can be purified with chromatographic methods (gel filtration, ion exchanges and immunoaffinity), by high performance liquid chromatography (HPLC, RP-HPLC, ion exchange HPLC, size-exclusion HPLC) or by precipitation (immunoprecipitation). Alternatively, AMH can be produced with recombinant DNA techniques known to the person skilled in the art.

In one embodiment, said AMH is used for the medical purposes described above in an amount preferably comprised between 0.1 mg/kg and 10 mg/kg a day, more preferably between 0.3 and 3 mg/kg a day.

In one embodiment said AMH is used for the medical purposes described above in an amount such as to maintain plasma concentrations preferably between 50 and 1000ng/ml, more preferably between 100 and 500 ng/ml. For the medical purposes described here, said AMH or composition comprising said AMH is preferably administered by any route usable for this purpose; preferably the administration is selected from: parenteral, more preferably by the intravenous, intra-arterial or intramuscular route, or by the enteral route, more preferably by the topical, oral, sublingual transdermal, intravaginal, intrauterine, intramyoma or intramyometrial route.

In a preferred embodiment of the invention, the administration of said AMH or of the composition comprising said AMH is selected from: intravaginal, intrauterine, intramyoma and intramyometrial.

According to a preferred embodiment of the invention, the composition is formulated for parenteral administration. In particular, the composition is formulated in liquid form, preferably in the form of a sterile solution, emulsion or suspension.

In one embodiment, the composition is in lyophilized form in order to be reconstituted to obtain a liquid formulation.

Said AMH or the composition comprising said AMH is preferably administered directly into the uterus by means of an intrauterine device.

In one embodiment of the invention, said AMH or said composition comprising said AMH is formulated for enteral administration, preferably for oral administration. In particular, the composition is formulated in solid form, preferably in the form of lozenges, capsules, tablets granular powder, hard-shelled capsules, orally dissolving granules, sachets or pills. In a further embodiment, said AMH or said composition comprising said AMH is formulated for topical administration, preferably for vaginal, intravaginal and perivaginal administration. In particular, the composition is formulated in the form of vaginal suppositories, ointments, creams, tampons, gels, foams and/or sprays.

In a further embodiment, said AMH or the composition comprising said AMH comprises salts, buffers, surfactants, excipients, carriers, preservatives and/or combinations thereof which are accepted for the preparation of pharmaceutical products.

For the purposes of the medical uses described above, the anti-Mullerian hormone, or a composition comprising said AMH, can be taken once a day. In a preferred embodiment, the AMH or a composition comprising said AMH is taken in a single administration or in a number of administrations daily, in a continuous manner or according to need.

In one embodiment, the AMH or the composition comprising said AMH can also be administered in combination with drugs used in therapy for the treatment of benign tumors, in particular for the treatment of myomas. Said drugs are for example: GNRH analogues, hormonal contraceptives, progestins, SPRMs, antiestrogens, androgens and combinations thereof.

In one embodiment, the AMH or the composition comprising said AMH can be administered in association and/or in combination with a surgical treatment, preferably for the treatment of myomas, more preferably before or after an intervention of partial or total myomectomy.

A further aspect of the present invention relates to a method for the treatment of benign tumors of the uterus. Said method comprises at least a step of administering an effective amount of AMH, or of a composition that comprises said AMH as described above in detail, to a subject (individual) affected by or suspected of being affected by a benign tumor of the uterus.

In the context of the present invention, benign tumor of the uterus means a pathological condition caused by an abnormal growth of uterine cells which give rise to masses that remain well delimited in the uterus and do not invade surrounding tissues.

In one embodiment, the benign tumors are selected from: myomas, adenomyomas, polyps and endometrial hyperplasias.

In a preferred embodiment of the invention, the benign tumor of the uterus is a myoma or leiomyoma or fibroid. Therefore, the present invention relates to a composition comprising AMH, as described above, for use in the treatment and/or prevention and/or for the follow-up of myomas. In the specific case the myomas can be subserous, intramural, submucous or intraligamentary and/or can have sizes preferably ranging from less than one centimeter to many centimeters.

In fact, as demonstrated in the example, the administration of AMH or of a composition comprising said AMH is capable of inducing death by apoptosis of the myoma cells, without interfering with the viability of healthy myometrial cells. Therefore, the AMH, or a composition comprising said AMH is also useful for inducing cell death, preferably by apoptosis. Said cells preferably express the receptor AMHR2, preferably the biologically active form of the receptor AMHR2.

In a preferred embodiment of the invention, said AMH or said composition comprising said AMH is capable of inducing apoptosis in myoma cells through the binding and/or the activation of the molecular network or pathway of the receptor AMHR2.

Said binding preferably induces the modulation, preferably the activation and/or an increase in the expression of genes encoding the family of proteins defined as SMAD“adaptors”, preferably SMAD3 and/or SMAD5. Therefore, the present invention also relates to an AMH or a composition comprising said AMH with the purpose of modulating, preferably activating and/or increasing the expression of genes encoding the family of proteins defined as SMAD “adaptors”, preferably SMAD3 and/or SMAD5. This activation is preferably correlated to and/or induces the apoptosis of the cells, preferably benign tumor cells, more preferably myoma cells. Cell death is preferably induced by and/or following the binding of said AMH with the receptor AMHR2.

Furthermore, said AMH or said composition comprising said AMH is capable of modulating the expression of genes and/or proteins that regulate the apoptosis in cells, preferably of a myoma.

In a preferred embodiment of the invention, said AMH or the composition comprising said AMH:

- modulates, preferably increases, the expression of the mRNA and the proteins of the proapoptotic factors PARP-1 and/or caspase-3; and/or

- modulates, preferably, decreases (reduces) the expression of the antiapoptotic factor BCL-2 in cells which preferably express AMHR2, preferably a biologically active form of AMHR2. Said cells are preferably benign tumor cells, preferably selected from: myoma, leiomyoma and fibroid.

A further aspect of the present invention relates to a method for the treatment of benign tumors of the uterus, preferably as defined above, more preferably a myoma or leiomyoma or fibroid, said method comprising a step of administering an effective amount of AMH or the composition comprising said AMH as described in detail above, to a subject who is affected or suspected of being affected by said tumor, or said subject having been surgically treated for removal of said tumor.

EXAMPLE

Assessment of the apoptotic activity of AMH in vitro

Tissue sampling

The myoma and myometrium samples were taken from the uterus of 9 female patients with a regular menstrual cycle who underwent surgical treatment for a uterine myoma. The patients used in this study had a mean age of 42 ± 8 years and had not followed any hormonal therapy in the 6 months preceding the intervention.

Cell cultures

The nine samples of uterine myoma and the nine samples of normal myometrium were taken from uterine tissue removed by hysterectomy for uterine fibromatosis. The samples were obtained during the proliferative or secretory phase of the menstrual cycle. Various types of primary cultures were prepared and used for the different experiments. The cells deriving from the myoma and from healthy myometrium were obtained following a standard protocol, based on washes with 1 X PBS. Then the individual samples were placed in a sterile Petri dish with the addition of 4 drops of collagenase, type II [0.1 %, or 10 mg in 10 ml] or type VIII [10 mg in 12 ml], diluted in DMEM with 1 % penicillin/streptomycin and 50 mg gentamycin/liter without FBS (serum). After removal of the thin outer layer the sample was divided into pieces as small as possible. The tissue thus fragmented was placed in a 50 ml test tube and covered with the collagenase solution to 8 ml. The samples were then incubated at 37°C in a temperature-controlled bath for 90 minutes and gently stirred every 20 minutes, avoiding the formation of foam, until complete digestion of the tissue (the process lasts at least 4 hours). The digested samples were filtered through a membrane filter with a pore size of 250 pm and the lysates thus obtained were centrifuged at 1200 rpm for 12 minutes. Removal of the supernatant and resuspension of the cells accumulated on the bottom of the tube (about 1 ml) with the addition of 3 ml of FBS was followed by incubation at 37°C in a controlled atmosphere with 5% CO2 for 20 minutes. The cell suspension was distributed in flasks and incubated at 37°C with 5% CO2 for 24 hours, then underwent further centrifugation at 460 xg for 5 minutes followed by three washes with PBS 1 X containing 1 % antibiotic. The treatment with the collagenase makes it possible to obtain a “pure” smooth muscle cell population, i.e. one that is free of contamination by other types of cells, such as stromal cells or glandular epithelial cells. The cells isolated from the myoma and from the myometrium with the above-described method were plated at an approximate density of 106 cells/well on a culture plate with a surface area of 10-cm², 4x10⁴ cells/well in a chamber with a two-well slide and 1 x10⁴ cells/well in a 96-well plate specific for tissue cultures.

The cells isolated from the myoma and the normal myometrial cells on the culture plates and the two-well plate were incubated at 37°C for 120 minutes in a humid atmosphere with 5% CO2 and 95% air, in the culture medium DMEM (without phenol red) supplemented with 10% FBS (vol/vol; Invitrogen Life Technologies, Inc., Grand Island, NY).

The cells isolated from the myoma and the normal myometrial cells plated on the 96-well plate were instead incubated for an additional 72 hours under the culture conditions described above.

The cells were maintained under culture until reaching 70% confluence and then incubated with incremental doses of recombinant human AMFI (rhAMH, R&D, England). The doses of rhAMH used in the experiments were the following: 10 ng/ml, 20 ng/ml, 100 ng/ml dissolved in DMEM without phenol red supplemented with 10% FBS for an additional 48 or 96 hours.

Immunocvtochemical staining

The cells deriving from the myoma and from the healthy myometrium cultured on the two-well plate were washed three times with PBS 1X, fixed with methanol at 4°C for 20 minutes and further washed three times with PBS 1 X. The fixed cells were used in immunofluorescence methods and analyzed by two investigators with the double-blind method.

After treatment with 3% purified bovine serum albumin (BSA) in PBS 1 X for 30 minutes at room temperature, both cell cultures were incubated with the primary antibody (mouse anti-Cleaved PARP-1 , Cell Signalling; USA) diluted 1 :25 in PBS containing 3% BSA for 1 hour at room temperature. After washing with 1 X PBS, the samples were incubated for 1 hour at room temperature with the secondary antibody diluted 1 :20 in 1 X PBS containing 3% BSA (sheep anti-mouse FITC conjugated). After one wash with 1 X PBS and one with water, the samples were counterstained with 1 pg/ml of DAPI in FI20 and mounted using an anti-fading medium (0.21 M DABCO and 90% glycerol in 0.02 M Tris, pH 8.0). The negative controls were incubated with the incubation mixture without the primary antibody. Images were acquired with a Leica TCS SP2 AOBS confocal laser scanning microscope. The samples that bound the DAPI (blue stain of the DNA contained in the nucleus) and the FITC bound to the secondary antibody were excited at the wavelength of -405nm/25mW - generated by the blue laser diode and at the wavelength of 488-nm/20mW generated by the argon laser.

The excitation and detection of the fluorescence of the samples were carried out sequentially, avoiding the superimposition of signals. The sections were scanned with the laser intensity, confocal aperture and gain adjustments with the grayscales set on the constant. The optical sections were obtained at increments of 0.3 pm along the z- axis and were digitalized with a scanning mode format of 512 x 512 or 1024 x 1024 pixels and 256 gray levels. The serial sections examined under the confocal microscope were analyzed with Leica LCS software to obtain three-dimensional projections. The format suitable for publication was obtained using Adobe Photoshop software.

Assessment of the immunofluorescence of the cleaved form of PARP-1 The original images obtained in green with the confocal microscope were converted to grayscales by applying a filter called median. An intensity value ranging between 0 (black) and 255 (white) was assigned to every pixel. The background fluorescence (background noise) was subtracted from the analysis and the intensity of the immunofluorescence was calculated as the mean intensity for every area selected. The frequency of positivity of cells positive for PARP-1 was determined by observing more than 1 ,000 nuclei per experimental sample.

Western blot

The proteins were extracted from the cultured cells deriving from the myoma and from the healthy myometrium. The cells were lysed at 4°C for 20 minutes with the lysis buffer (50 mM Tris-CI, pH 7.8, containing 1 % Nonidet P40, 140 mM NaCI, 0.1 % SDS, 0.1 % Na deoxycholate, 1 mM Na3V04, 1 X protease inhibitor cocktail). The lysates were then centrifuged for 15 minutes in a refrigerated centrifuge at the maximum speed of 16000 xg and immediately boiled in sodium dodecyl sulfate (SDS) buffer for the loading of the samples. The extracted proteins were quantified with the Bradford method. About 40 pg of proteins per sample was subjected to vertical electrophoresis under SDS-PAGE denaturing and reducing conditions and the proteins thus fractionated were transferred onto a nitrocellulose membrane.

The protocol of the method followed, called Western Blot, is the one described by Sambrook et al., 1989. Briefly, the membrane was blocked with 3% whole milk powder and 2% BSA in Tris buffered saline - Tween 20 (TBS-T) and the following antibodies diluted 1 :1000 overnight at 4°C under stirring: anti-mouse cleaved PARP-1 (Cell Signalling), Caspase-3 active (SIGMA) and anti-mouse Bcl-2 (BD Transduction Laboratories). After 3 washes with TBS-T to remove the excess of unbound primary antibody, the membrane was incubated with the following secondary antibodies conjugated to horseradish peroxidase (HRP): goat anti-rabbit IgG antibody (1 :10000) or HRP-conjugated sheep anti-mouse IgG antibody (1 :3000) for 30 minutes at room temperature. The immunoreactive proteins were detected with ECL (Amersham). After a first acquisition, the membranes were treated in order to remove the primary antibody used (membrane stripping) so as to enable a further incubation with a different primary antibody, namely, anti-mouse b-tubulin (SIGMA) as a control on the amounts of protein loaded.

gPCR

About 1 X10⁵ cells deriving from the cell cultures of myoma or non-myoma myometrial cells, untreated or treated with increasing doses of human recombinant AMH, were lysed in the commercial product Tri-reagent for the extraction of total RNA according to the protocol provided by the manufacturer.

After the quantification and determination of the degree of purity of the RNA extracted, evaluated by spectrophotometry, about 1 pg of total RNA was reverse transcribed to cDNA (RTmaxima, Biorad, USA). 1 mI of cDNA deriving from each sample was used as a template for the PCR reaction using the primers specific for every gene analyzed and the enzyme SYBRgreen (Biorad). The expression of the genes analyzed (PARP-1 ; Bcl-2; Caspase 3; AMH receptor; SMAD-3; SMAD-5) was normalized using the constitutively expressed b-actin gene. The specificity of the amplified products thus obtained was checked both by analyzing the melting curve and after fractionation of the aforesaid products in a run on 1 % agarose gel in a tris-acetate-EDTA buffer (TAE) 1 X.

Trypan Blue cell viability test The cells of the two cell populations (myoma and healthy myometrium) were incubated with a solution of 5% Trypan blue in 1 X PBS for five minutes at room temperature. The cells positive to the stain (dead) were counted by two operators with the double-blind method using a Burker chamber under an inverted microscope (40X objective) and expressed as a percentage.

TUNEL test to assess apoptosis

The fragmentation of the nuclear DNA of apoptotic cells was assessed by means of the TUNEL technique (terminal deoxynucleotidyl transferase dUTP nick end labeling) using the commercial kit In Situ Cell Death Detection Kit, AP (ROCHE) and following the instructions present in the kit for the application of the method with cells fixed on slides. The population of apoptotic cells was calculated by counting at least 5 fields of 200 cells on the slide and expressed as a percentage in relation to the negative cells (not-apoptotic).

The AMH type-2 receptor (AMHR2) is more highly expressed in the tissue and cell cultures deriving from a myoma than in the tissue and cultures deriving from healthy myometrium

The presence of the gene coding for AMHR2 was first verified by means of the RT-PCR method followed by fractionation of the products by means of an electrophoretic run on an agarose gel (Fig. 1 ). The volumes of cDNA to be used in the reactions were standardized thanks to the use of the stably expressed b-actin gene. The presence of bands in every sample confirms the actual presence of AMHR2 in every sample.

As may be inferred from the data related to the semi-quantification of the luminosity of the bands by densitometry and shown in Figure 2, the expression of AMHR2 is about 5 times higher in the myoma than in the healthy myometrium, irrespective of whether tissue or cell cultures are considered.

The presence of AMHR2 as a produced protein was demonstrated, finally, by immunohistochemistry using an anti-AMHR2 primary antibody. The images shown in Fig. 3 not only demonstrate the actual presence and location of the receptor both in tissues and in cell cultures deriving from a myoma and healthy myometrium, but also and above all they confirm the data obtained previously with molecular biology. The red fluorescence, which indicates the cells that express AMH on their cell membrane, is decidedly more marked in samples deriving from the myoma than in those deriving from the myometrium. In fact, using a software to calculate the intensity of the fluorescence signal, it was empirically established that in the samples deriving from the myoma, be it tissue or a cell culture, there is an approximately 50% increase in cells expressing AMHR2 compared to the corresponding healthy myometrial counterparts (Fig.3).

AMFIR2 is biologically active, in that it participates in the regulation of SMAD 3 and SMAD5 genes typically involved in its signal transduction pathway.

AMFIR2 present on the membrane of cells deriving from a myoma or from healthy myometrium, when activated by binding with its AMFI hormone- ligand, is capable of triggering a signal transduction pathway, which, through conformational changes involving the intracellular part of the receptor, leads to an increase in the expression of genes coding for a family of proteins defined as SMAD“adaptors”. The most important among them are SMAD3 and SMAD5. In the experimental system used, increasing doses of AMFI (range 0-100 ng/ml) are capable of increasing the expression of SMAD3 and SMAD5, thus confirming the functionality of AMFIR2 in the system. The gene expression data shown in Figure 4 were obtained with the RT-qPCR method, which enables quantification of the genic expression of a gene of interest in relation to the basal expression calculated in the control samples. Although all of the tested concentrations of AMFI induce a general dose-response effect, the cultures deriving from a myoma seem to be more responsive than the cell cultures derived from healthy myometrium. In particular, the maximum concentration of AMFI (100 ng/ml) is the only one to have generated statistically significant gene inductions. Therefore, this concentration was selected as the optimal one for conducting the subsequent experiments.

AMH brings about apoptosis by binding with its receptor, upregulating the proapoptotic genes PARP-1 and caspase 3 and downregulating the antiapoptotic gene BCL-2

In order to analyze any differences in terms of apoptosis generated by the treatment with AMH, cell cultures deriving from a myoma and healthy myometrium were treated with AMH (100 ng/ml) for 48 and 96 hours and analyzed with the RT-qPCR method. As may be inferred from the data shown in Figure, 5 AMH is capable of controlling, in a selective and significant manner, the genic expression of the genes involved in apoptotic phenomena solely in cell cultures deriving from a myoma.

The control, in vitro, of AMH over the genes involved in apoptotic phenomena is also confirmed at the protein level.

With the aim of confirming the data relating to the genic expression of the apoptotic genes obtained by RT-qPCR, thus based on the expression of messenger RNAs (mRNAs), samples of cell cultures deriving from a myoma and healthy myometrium were treated for 48 or 96 hours with AMH (100 ng/ml) and finally analyzed with the immunoblot method. A series of primary antibodies was used to investigate the apoptotic process and simultaneously avoid false positives due to the use of a single antibody. The data presented in Figure 6 clearly demonstrate that the amount of proteins involved in apoptosis produced by the myometrial cells stimulated with AMH is wholly comparable to the basal amount of each protein present in control cells not treated with AMH. By contrast, cells deriving from a myoma respond to treatment with AMH with increases or decreases in expression which reflect the gene regulation previously shown in RTqPCR. In particular, as regards PARP-1 , a single intense band with the expected molecular weight of 89 KDa is evident in the samples treated with AMH versus the controls. A situation similar to that of PARP-1 is also found in the case of caspase-3 (confirmation of the proapoptotic role of AMH), whereas the production of BCL-2 proves to be greatly diminished after 48 hours of treatment with AMH until becoming barely detectable after 96 hours of incubation with AMH (Fig. 6).

In the cell cultures deriving from myometrium, by contrast, no differences in production were found between the control samples and samples with AMH (100 ng/ml) for any of the proteins examined (Fig. 6).

AMH selectively induces apoptosis in myoma cells while preserving the cellular viability of healthy myometrial cells.

As a last phase of study, after the expression of different actors in the apoptotic process had been analyzed, the apoptotic process in itself was investigated by means of immunohistochemical methods enabling the percentage of apoptotic cells to be calculated.

The percentage of apoptotic cells was determined using the anti-PARP-1 cleaved form antibody specific for the detection of the endogenous levels of the large fragment (89 KDa) of the human protein resulting from the cleavage of the native protein. The antibody used does not recognize the full-length PARP-1 or the other isoforms thereof, but only the bioactive portion of the protein. Some of the most representative images (staining patterns) are shown in Figure 7 (J-O), where there is an evident positive staining at the site of the apoptotic nucleus of the myoma cell, as revealed by co-immunostaining with DAPI/FITC.

Epithelial cells positive to PARP-1 were found in all samples of cells deriving from the myoma treated with AMH (100 ng/ml) (Fig.7 J-O), whereas the number of positive cells was very low in the cultures of untreated myoma cells and in those deriving from myometrium (Fig.7) both untreated and treated with AMH. The percentage of positive cells was 20% ± 0.40 (SD) in the cells deriving from the myoma treated with 100 ng/ml of AMH for 48 or 96 hours, whereas the percentage counted for the other cell cultures was 3.0% ± 0.37 (SD).

Furthermore, cell viability tests conducted with the Trypan blue method on cells deriving from a myoma and from healthy myometrium not treated or treated with 100 ng/ml of rhAMH for 48 and 96 hours showed an approximately 20% increase in the percentage of dead cells, thus effectively confirming the results obtained with the immunohistochemical methods. The limit of this technique, however, is the impossibility of distinguishing the cells dying as a result of the activation of the programmed process of cell death (apoptosis) from necrosis (a disorganized, energy-wasting process of cell death that is not due to a precise cellular programming).

With the aim of distinguishing viable cells from necrotic cells and apoptotic cells, the method for analyzing nuclear DNA fragmentation known as TUNEL was carried out (Fig. 8). The data obtained with the TUNEL method show that approximately 20% of the“dead” cells generated by the rhAMH treatment at the concentration of 100 ng/ml is actually ascribable to the activation of programmed cell death by apoptosis and is not attributable to necrosis. The treatment with rhAMH at concentrations below 100 ng/ml showed to be ineffective both on cell viability and on myoma cells, as well as on normal myometrial cells as verified with all of the above-mentioned techniques.

AMH exerts inhibitory effects on cultured myoma cells, increasing apoptosis without interfering with the normal viability of normal myometrial cells taken from the same uterus. The effects of rhAMH are dose- dependent but not time-dependent, since no significant difference was found in the parameters analyzed after 48 hours of treatment when compared with the data obtained after 96 hours of incubation.

Comparison between different isoforms of AMH in inducing apoptosis in myoma cells and tissues

With the aim of assessing a possible different therapeutic effect in the administration of AMH25-560 and AMH₄53-56o (SEQ ID NO: 3), various experimental models were set up, as described below. Assessment of the induction of apoptosis in cells

Biopsies of myoma tissue were obtained from 7 patients affected by uterine myoma. Initially, the myoma biopsy sample was washed 3 times with 1X PBS. The initial biopsy sample, 1.5 cm³ in volume, was divided into 7 smaller pieces of approximately the same size.

For the first part of the study 3 of the aforesaid 7 pieces were individually transferred into 3 wells of a 12-well multi-well plate and immersed for 24 hours, under controlled atmosphere (5% C02) and temperature (37 °C) conditions in the incubation medium (DMEM supplemented with 0.5% FBS) alone (control) or containing 100 ng/ml of AMFI 25-560 or AMFI453- 560. At the end of the incubation (24 hours), the samples of myoma were lysed in 1 ml of the commercial product TRI-FAST (Euroclone, U.K.), which enables the extraction of total RNA from the samples. After analyses performed with a spectrophotometer to determine concentration and purity, about 1 pg of total RNA was reverse transcribed to cDNA. The cDNAs thus obtained (1 mI) were used as a template in RT-qPCR reactions carried out in triplicate. The primers used amplify a junction region between two exons present in the mRNAs of the genes evaluated: SMAD5 and FS-7-associated surface antigen (FAS).

FAS codes for the FAS transmembrane receptor belonging to the death receptor family, i.e. it is capable of promoting apoptosis. The role of the proapoptotic gene FAS in the physiology of the endometrium has mainly been studied for its involvement in phenomena of remodeling of the uterine mucosa due to the progression of the menstrual cycle and its implications in pathologies such as endometriosis. The relative expression of the two genes was normalized against the constitutively expressed b- actin gene according to the method called 2^_DDa (Livak and Schmittgen, 2001 ).

As may be inferred from the data shown in Figure 9, both forms of AMFI, the tested concentration (100 ng/ml) and duration of treatment (24 hours) being equal, are capable of inducing the expression of SMAD5 and FAS in a statistically significant manner compared to the control. Though both were effective, AMH₄53-56o induced levels of gene expression that were significantly higher also compared to AMH25-560.

Assessment of the induction of apoptosis in tissues

The second part of the study was dedicated to studying the effects of AMH on cell viability when AMH was injected inside a myoma biopsy sample ( intramyoma injection). For these experiments 3 of the initial 7 pieces of tissue were individually placed in 3 wells of a 6-well multiwell plate and injected by means of a syringe with a (30 Gauge) needle with 50 mI each of AMH (100 ng/ml) or with saline solution (0.9% NaCI) in the case of the control. The injected myoma biopsy samples were kept in an incubator for 24 hours before being processed for the extraction of total RNA and analyzed with the RT-qPCR method as previously described.

The data presented in Figure 10 demonstrate that, as in the case of incubation with AMHs dissolved in the medium, both isoforms of AMH administered by injection upregulate the expression of the SMAD5 and FAS genes in a significant manner compared to the control. The inductions of the expression of both genes analyzed, stimulated by the isoform AMH₄₅₃-56o, are moreover significantly greater at the levels induced by AMH25-560. Based on a comparison with the previous graph shown in Figure 9, it may be inferred that the levels of induction generated by both AMHs administered by intramyoma injection are significantly higher (approximately 20-30% higher) than those obtained by incubating myomas in the culture medium in which the AMHs were dissolved. It remains to be determined, however, whether the greater induction found in the injected samples, in particular with regard to the expression of the proapoptotic gene FAS, is ascribable to a better delivery and dissemination of the hormone in the myoma tissue (from the inside to the outside in the case of injection, the opposite in the case of incubation with a medium supplemented with AMH) or to the contribution to apoptosis resulting from the tissue damage generated by the injection or a combination of both these factors.

In order to obviate the possible contributions to apoptosis due to hypoxia in the case of stimulation with AMHs dissolved in the incubation medium or the tissue damage generated by the injection, the last part of this study was dedicated to assessing the effects of AMH on the viability of myoma cells purified according to the protocol described in Ciarmela et al., 201 1. The seventh piece of myoma out of the 7 initially prepared ones was in fact subjected to enzymatic digestion by incubation with collagenase type IV (Sigma, St. Louis, Mo, USA) for 3 hours at 37°C in order to recover individual myoma cells. The cells thus obtained were seeded at the concentration of 20x10³ cells/well in a 12-well plate. The cells were maintained in the culture medium (DMEM supplemented with 10% FBS and 2 mM L-glutamine) in an incubator until reaching confluence (about 80% of the available surface of the occupied well) before the administration of AMH. The percentage of live myoma cells calculated in the confluent wells by incubation with Trypan Blue nonviable stain (Sigma, St. Louis, MO, USA) by two different operators with the double-blind method was always greater than or equal to 95% of the population (data not shown). The confluent myoma cells were then incubated in a stimulation medium (DMEM with 0.5% FBS) supplemented with AMH25-560 or AMH₄53-56o (100 ng/ml), or with saline solution in the case of the controls, for 24 hours and finally processed as previously described.

Like in the previous two sets of experiments, in this case as well (Fig. 1 1 ) both AMH-based preparations, the concentration and incubation time being equal, are capable of significantly inducing the expression of the SMAD5 and FAS genes compared to the control. As in the previous experiments, in this one, too, AMH₄₅₃-56o demonstrated to be statistically more effective than AMH25-560 in upregulating the expression of the genes analyzed. Incubation with AMH generates, in primary cultures of myoma cells, profiles of expression for the two genes considered which are similar to those obtained with an intramyoma injection, which thus suggests that the contribution to apoptosis of the tissue damage caused by the penetration of the needle into the tissue is minimal.

In vivo AMH treatment of uterine leiomyomas xenotransplanted in a mouse model

A first part of the study was dedicated to developing a mouse model xenotransplanted with human-derived leiomyoma tissue. The use of “nude” mice as experimental models for studying the physiology of and/or pathologies tied to endometrium has been reported in various publications [Grummer et al., 2006, peer review]. For this study, use was made of nude mice of the NU/J strain ( Jackson Laboratory), as carriers of a genetic mutation (deletion of the FOXN1 gene) that causes inhibition of the immune system; therefore, these mice can be transplanted with different types of tissues (xenotransplantation) without triggering rejection phenomena.

In the second part of the study we tested the effects exerted by the injection, inside the transplanted myoma, of different concentrations of AMFI at different incubation times. The control samples in our experiments were transplanted mice injected with saline solution rather than AMFI or non-injected transplanted mice in order to assess the effect of the local inflammation due to the injection.

Obtainment of the leiomyoma

The leiomyoma samples were taken from the uterus of patients in reproductive age who underwent surgical treatment for uterine leiomyoma. The patients enrolled in this study had not undergone any hormonal therapy in the 6 months preceding the intervention.

Immunocompromised mouse model

For this study use was made of female nude mice of the NU/J strain ( Jackson Laboratory) upon their reaching ten weeks of age. During these 10 weeks the mice were fed and maintained under sterile conditions in the animal unit of the university of Modena in order to prevent infections, as these mice are“nude”, i.e. immunocompromised.

The ovaries were removed one week before the transplant so as to eliminate a possible source of endogenous AMH, since mouse AMH is about 70% homologous with human AMH.

Tissue sampling

Initially, the myoma biopsy sample was recovered after the surgical intervention and transferred under sterile conditions in 1 X PBS. Upon arriving in the laboratory, the biopsy sample was washed 3 more times with 1 X PBS and processed under a laminar flow hood in sterile conditions. From the initial biopsy sample with a volume of 1.5 - 2 cm³, pieces of about 0.3 cm³ were removed using scalpels and immediately fixed in 4% paraformaldehyde buffered in PBS for the subsequent histological assessments. In the next part of the study use was made only of myomas which, based on the analysis of this phase, showed a normal morphology and histology.

The remaining myomas were transferred individually into 12 wells of a 12- well multi-well plate and immersed for 24 hours, under controlled atmosphere (5% C02) and temperature (37 °C) conditions in a maintenance medium (DMEM supplemented with 0.5% FBS and 1 mM of antibiotics, i.e. penicillin/streptomycin) in order to enable the tissue to recover from the surgical intervention.

Preparation of the myoma and xenotransplantation

After one day in the maintenance medium, the myomas were incubated with 8 pmol/l of carboxyfluorescein diacetate, succinimidyl ester (CFDA SE, Molecular Probes, USA) in 1 X PBS for 15 minutes in a temperature- controlled bath at 37 °C. This nonspecific marking (it stains all the cells of the myoma) is necessary in order to be able to distinguish, on slides, the transplanted tissue from the mouse tissue under a fluorescence microscope.

The mice were transplanted with human myomas according to the method described in Nisolle et al., 2000 and described briefly here. After the skin of the mouse had been sterilized with 95% ethanol, an incision was made at the epidermal level in order to enable subcutaneous implantation of the marked human myoma. The myoma-transplanted mouse was then sutured with a single stitch using a 3-0 Vicryl® suture. The mice were anesthetized by inhalation of isoflurane before and during the entire procedure.

Treatment of the myoma with AMH

After three days in the animal unit, three animals received 2 daily injections of 100 pg AMH for five days, in accordance with the concentration tested by Segev et al. (2001 ), directly inside the transplanted myoma. Three animals were treated with a dose of 10 pg AMH according to what had already been tested in a series of our preliminary experiments. Three animals were treated with an injection of saline solution in the myoma transplantation site and were used as the control group. The remaining 3 myoma-transplanted animals received no injection in order to act as a control for a possible inflammatory effect due to the injection.

At the end of the fifth day of treatment the mice were sacrificed by inhalation of CO2 followed by cervical dislocation and the transplanted myoma samples were excised and processed according to the methods described below.

Histology and immunocvtochemistrv

Pieces deriving from the original biopsies of the patients and pieces recovered from the transplanted myomas were fixed overnight in 4% paraformaldehyde buffered in 1X PBS, dehydrated with an ascending scale of ethanol, clarified in xylol and finally embedded in paraffin. The slides were prepared with 5 pm-thick slices of tissue obtained with a microtome.

The slides deriving from the original pre-transplantation biopsies were subjected to staining with hematoxylin and eosin in order to confirm the correct morphology of the tissue.

The slides deriving from transplanted myomas were used in immunofluorescence methods for the identification of the cleaved form of PARP-1 and analyzed by two different investigators with the double-blind method. After the slices had been deparaffinized and rehydrated on the slides, the samples were incubated with the primary antibody (human anti cleaved PARP-1 , Cell Signalling; USA) diluted 1 :25 in 1 X PBS containing 3% BSA for 1 hour at room temperature. After washing with 1 X PBS, the samples were incubated for 1 hour at room temperature with the secondary antibody diluted 1 :20 (rabbit anti-human rhodamine conjugated). After one wash with 1 X PBS and one with water, the samples were counterstained with 1 pg/ml of DAPI in H20 and mounted using an anti-fading medium (0.21 M DABCO and 90% glycerol in 0.02 M Tris, pH 8.0). The negative controls were incubated with the incubation mixture without the primary antibody.

Images were acquired with a Leica TCS SP2 AOBS confocal laser scanning microscope. The samples that bound the DAPI (blue stain of the DNA contained in the nucleus) and the rhodamine bound to the secondary antibody, were excited at the wavelength of 405 nm generated by the blue laser diode and at the wavelength of 470 generated by the argon laser for the rhodamine. The excitation and detection of the fluorescence of the samples were carried out sequentially, avoiding the superimposition of signals. The sections were scanned with the laser intensity, confocal aperture and gain adjustments with the grayscales set on the constant.

The serial sections examined under the confocal microscope were analyzed with Leica LCS software to obtain three-dimensional projections. The frequency of positivity of cells positive for PARP-1 was determined by observing more than 1 ,000 nuclei per experimental sample.

TUNEL test to assess apoptosis

The fragmentation of the nuclear DNA of apoptotic cells was assessed by means of the TUNEL technique using the commercial kit In Situ Cell Death Detection Kit, AP (Roche) and following the instructions present in the kit for the application of the method with cells fixed on slides. The population of apoptotic cells was calculated by counting at least 5 fields of 200 cells on the slide and expressed as a percentage in relation to the negative cells (non-apoptotic).

Western blot

The proteins associated with each transplanted myoma were extracted by lysis of the tissues at 4°C for 20 minutes with a lysis buffer (50 mM Tris-CI, pH 7.8, containing 1 % Nonidet P40, 140 mM NaCI, 0.1 % SDS, 0.1 % Na deoxycholate, 1 mM Na3V04, 1 X protease inhibitor cocktail). After centrifugation (15 minutes at 4 °C at the maximum speed of 16000 x g), sodium dodecyl sulfate and b-mercaptoethanol were added to the samples and heated to 99 °C to denature the proteins, linearizing them prior to pre- loading of the samples in the acrylamide gel. The extracted proteins were quantified with the Bradford method before being loaded into the gel [Bradford et al., 1976]. About 40 pg of proteins per sample were subjected to vertical electrophoresis under SDS-PAGE denaturing and reducing conditions and the proteins thus fractionated were transferred onto a nitrocellulose membrane. After saturation of the nonspecific binding sites on the membrane obtained with 3% whole milk powder and 2% BSA in Tris buffered saline - Tween 20 (TBS-T), the membrane was incubated with the following primary antibodies diluted 1 :1000 at 4°C under stirring overnight: anti-human cleaved PARP-1 (Cell Signalling), Caspase-3 active (Sigma) and anti-human Bcl-2 (BD Transduction Laboratories). After 3 washes with TBS-T to remove the excess of unbound primary antibody, the membrane was incubated with the following secondary antibodies conjugated to horseradish peroxidase HRP: rabbit anti-human IgG antibody (1 :10000) or H PR-conjugated sheep anti-human IgG antibody (1 :3000) for 30 minutes at room temperature. The immunoreactive proteins were detected with ECL (Amersham). After the first acquisition, the membranes were treated in order to remove the primary antibody used (membrane stripping) and enable a further incubation with a different primary antibody, namely, anti-human b-tubulin (Sigma) as a control on the amounts of protein loaded.

RT-gPCR

Pieces of transplanted myoma (treated with AMH or the control) were lysed in the commercial product Tri-Fast (NEB; U.K.) for the extraction of total RNA according to the protocol provided by the manufacturer. After the quantification and determination of the degree of purity of the extracted RNA evaluated by spectrophotometry, about 1 pg of total RNA was reverse transcribed to cDNA (RTmaxima, Biorad, USA). 1 mI of cDNA deriving from each sample was used as a template for the PCR reaction using primers specific for every gene analyzed and the enzyme SYBRgreen (Biorad). The expression of the genes analyzed (PARP-1 ; Bcl-2; Caspase 3; AMH receptor II; smad-3; smad-5) was normalized using the constitutively expressed b-actin gene according to the method called 2^°* [Livak et al. 2001 ]

The specificity of the amplified products thus obtained was checked both by analyzing the melting curve and after fractionation of the aforesaid amplified products in a run on 1.5% agarose gel in tris-acetate-EDTA (TAE) 1 X buffer.

Results

The following study demonstrates the effectiveness of exogenous AMH in inducing apoptosis in myomas in an in vivo model where the myoma is transplanted into immunocompromised mice. Not only does AMH induce apoptosis in leiomyoma tissue (whereas the healthy myometrial tissue is not affected by the treatment), but the route of delivery of the drug can also influence this phenomenon. In fact, by injecting AMH directly into the myoma, it would be possible to reach higher levels of apoptosis than with an “external” administration. This could be ascribable both to a better delivery and dissemination of the hormone in myoma tissue (from the inside to the outside in the case of injection, the opposite in the case of “external” incubation of AMH) or to the contribution to apoptosis of the tissue damage generated by the injection or a combination of both of these factors.

If confirmed, the effectiveness of AMH in inducing cell death could point to a future use of AMH as an alternative therapeutic approach to in vivo surgical intervention, strictly localized in the myoma as the site of injection in order to prevent possible side effects on healthy adjacent tissues.

Claims

1. Anti-Mullerian hormone (AMH) or a composition comprising said AMH for use in the prevention and/or treatment and/or follow-up of benign tumors of the uterus, wherein said AMH is human, encoded by a gene characterized by a sequence essentially corresponding to SEQ ID NO: 4 and/or a cDNA characterized by a sequence essentially corresponding to SEQ ID NO: 1.

2. The AMH or the composition for use according to claim 1 , wherein said AMH is characterized by a protein sequence essentially corresponding to SEQ ID NO: 3.

3. The AMH or the composition for use according to claim 1 or 2, wherein the benign tumors of the uterus are selected from: myomas, adenomyomas, polyps, endometrial hyperplasias and combinations thereof.

4. The AMH or the composition for use according to any one of claims 1 -3, wherein the cells of said benign tumor, preferably of said myoma, express the AMH receptor (AMHR2), preferably the biologically active form of AMHR2.

5. The AMH or the composition for use according to any one of claims 1 -4, wherein said AMH is conjugated with at least one polyethylenic polymer, preferably said polyethylenic polymer is polyethylene glycol (PEG).

6. The AMH or the composition for use according to any one of claims 1 -5, wherein said AMH is administered, preferably daily, in an amount of between 0.1 mg/kg and 10 mg/kg, preferably between 0.3 and 3 mg/kg.

7. The AMH or the composition for use according to any one of claims 1 -6, wherein the route of administration of said AMH or of the composition comprising said AMH is selected from: intravaginal, intrauterine, intramyoma injection and intramyometrial injection.

8. The AMH or the composition for use according to any one of claims 1 -6, wherein the route of administration of said AMH or of the composition comprising said AMH is selected from: parenteral, more preferably intravenous, intra-arterial, intramuscular, subcutaneous, or enteral, more preferably topical, oral, sublingual and transdermal.

9. The AMH or the composition for use according to any one of claims 1 -8 formulated as an emulsion, sterile solution, sterile suspension, lyophilized powder, lozenges, capsules, tablets, granular powder, hard-shelled capsules, orally dissolving granules, sachets or pills, vaginal suppositories, ointments, creams, tampons, gels, foams or sprays.

10. The AMH or the composition for use according to any one of claims 1 -9 in combination with at least one molecule used in therapy for the treatment of benign tumors, preferably for the treatment of myomas, said molecule being preferably selected from: GNRH analogues, hormonal contraceptives, progestins, SPRMs, antiestrogens, androgens and combinations thereof.

1 1. The AMH or the composition for use according to any one of claims

1 -10 in association and/or in combination with a surgical treatment, preferably before or after partial or total myomectomy.