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WO2009052119A1 - Procédé de traitement de l'endométriose par administration d'hormone anti-müllérienne - Google Patents

Procédé de traitement de l'endométriose par administration d'hormone anti-müllérienne Download PDF

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Publication number
WO2009052119A1
WO2009052119A1 PCT/US2008/079890 US2008079890W WO2009052119A1 WO 2009052119 A1 WO2009052119 A1 WO 2009052119A1 US 2008079890 W US2008079890 W US 2008079890W WO 2009052119 A1 WO2009052119 A1 WO 2009052119A1
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WIPO (PCT)
Prior art keywords
mis
endometriosis
inhibiting substance
human
mullerian inhibiting
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PCT/US2008/079890
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English (en)
Inventor
Jeff Wang
Drew Tortoriello
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Columbia University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones

Definitions

  • the invention is in the field of treatment and prevention of endometriosis.
  • Endometriosis is a common medical condition characterized by growth beyond or outside the uterus of tissue resembling endometrium, the tissue that normally lines the uterus. Endometriosis is associated with a lowered fertility and is the second leading cause of infertility in females that ovulate normally. Extrauterine endometrial growths typically respond to the varying levels of estrogen associated with the menstrual cycle. Thus, endometrial growths proceed through a cycle of proliferation and breakdown. Unlike the uterine lining, however, the body is unable to shed the extrauterine endometrial growths, and breakdown of this tissue results in internal bleeding, inflammation of the surrounding area and formation of scar tissue.
  • endometriosis occurs in roughly 5% to 10% of women. Endometriosis can also occur in postmenopausal women, with an estimated 2% to 4% of all endometriosis cases being diagnosed in the postmenopausal phase of a woman's life. Endometriosis in postmenopausal women is an extremely aggressive form of this disease. Endometriosis most commonly exists in the lower region of the female pelvis.
  • the most common site of disease is the ovary (approximately half of the cases); less commonly lesions can be found on the bladder, intestines, ureters, bowel and diaphragm Very rarely endometriosis is found distant from the pelvis, in sites such as the lung, brain, and kidney.
  • Treatment options are available based on the patient's desire for future fertility, symptoms, the stage of disease, and to some extent, age. Possible treatment options include analgesic treatments, such as nonsteroidal anti-inflammatory agents and prostaglandin synthetase-inhibiting drugs, and hormonal therapy.
  • analgesic treatments such as nonsteroidal anti-inflammatory agents and prostaglandin synthetase-inhibiting drugs, and hormonal therapy.
  • Common hormonal therapies include oral contraceptive pills; progestational agents, which cause decidualization in the endometriotic tissue; danazol, a weak androgen that is the isoxazole derivative of 17-alpha-ethinyl testosterone (ethisterone); and gonadotropin-releasing hormone (GNRH) agonists, which are analogues of the 10- amino-acid polypeptide hormone GnRH and act via the suppression of gonadotropin secretion, resulting in elimination of ovarian steroidogenesis and suppression of endometrial implants.
  • progestational agents which cause decidualization in the endometriotic tissue
  • danazol a weak androgen that is the isoxazole derivative of 17-alpha-ethinyl testosterone (ethisterone)
  • GNRH gonadotropin-releasing hormone
  • Certain embodiments of the invention are directed to a method for treating endometriosis in a female animal, preferably a human, by administering a therapeutically effective amount of Mullerian inhibiting substance (MIS), preferably human recombinant MIS, or a biologically active fragment or variant thereof.
  • MIS Mullerian inhibiting substance
  • the MIS is administered locally to the site of the endometriosis.
  • the therapeutically effective amount of Mullerian inhibiting substance for treating endometriosis is from about 50 to 1,000 ng/ml, more preferably from about 100 to about 500 ng/ml.
  • the therapeutically effective amount of Mullerian inhibiting substance is an amount that decreases the proliferation of endome trio tic cells.
  • Another embodiment is directed to a method of preventing a recurrence of endometriosis in a female, preferably a human, by administering a therapeutically effective amount of Mullerian inhibiting substance, or a biologically active fragment or variant thereof, preferably human recombinant MIS.
  • Another embodiment is directed to a method of preventing endometriosis in a female, preferably a human, who is at risk of developing endometriosis by administering a therapeutically effective amount of Mullerian inhibiting substance, or a biologically active fragment or variant thereof, preferably recombinant human Mullerian inhibiting substance.
  • Other embodiments are directed to pharmaceutical formulations of MIS, preferably human recombinant Mullerian inhibiting substance, in an amount of from about 50 to 1,000 ng/ml, more preferably from about 100 to about 500 ng/ml.
  • FIG. 1 MIS signalling genes are present in human endometrium.
  • cDNA derived from a representative proliferative endometrium sample was PCR amplified for the following genes and conditions: HPRT(Lane 1), HPRT (no cDNA control (Lane 2), HPRT (no reverse transcriptase control( Lane 3), ALK3 ( Lane 4), MISRII ( Lane 5), SMAD 1 ( Lane 6), MIS ( Lane 7) and SMAD ( Lane 9).
  • FIG. 2 MIS and MISRII protein are expressed in human endometrium. Flixed sections of human proliferative (a, d) and secretory (b, e) endometrium stained for MIS (a, d) and MISRII (b,e) revealed strong immunoreactivity for both proetins in the glandular cells. Stromal cell immunoreactivity for these proteins appeared to be increased in the secretory phase (b, e). Utilization of secretory antibody only as a negative control revealed no immunoreactivity (c, f).
  • FIG. 3 MIS and MISRII protein are present in mitosing cultured human endometirial cells.
  • Cultured primary human endometrial glandular and stromal cells manifested robust cytoplasmic expression of MIS (a) and MISRII (b) protein in those cells undergoing mitosis (4OX magnification. )FIG. 4.
  • Estradiol increases endometrial stromal cell secretion of MIS.
  • Sensitivive ELISA reveals that primary human ESCs secrete MIS protein. After 72 hours in culture with estradiol 1 micromolar, there was a two-fold increase in the MIS concentration in the conditioned media.
  • FIG. 5 MIS decreases endometrialstromal cell viability in vitro. Treatment of human endometrial cells with 100 ng/mL recombinant human MIS results in a 15% inhibition in cell viability compared to vehicle after 72 hours, p ⁇ 0.05 compared to vehicle control.
  • FIG. 6 Effect of increasing the endogenous production of MIS in human ESCs. Transient transfection of human ESCs with an MIS expression plasmid (100 ng/well) induced a 30-fold increase in the amount of MIS secreted into the culture media.
  • Transient transfect ion of both plasmids (50 ng of each plasmid for a combined total of 100 ng/well) further increased the caspase 3/7 activity (c). p ⁇ 0.05 compared to pCMV (empty vector) control.
  • MIS Mullerian Inhibiting Substance
  • a therapeutically effective amount of human MIS or a biologically active fragment or variant thereof is administered to a patient having endometriosis, or at risk of developing or having a reoccurrence of endometriosis.
  • the therapeutically effective amount of MIS is an amount that decreases the proliferation of endometriotic cells, an amount that our results show is significantly higher than the normal circulating levels of endogenous MIS.
  • the subject is human and MIS is recombinant human MIS or a biologically active fragment or variant thereof, that is preferably administered locally to one or more of the sites of the endometriosis.
  • the therapeutically effective amount of MIS ranges from about 50-1000 ng/ml, more preferably from about 100-500 ng/ml.
  • Certain other embodiments are directed to a pharmaceutical composition for treating endometriosis that contains from about 50-1,000 ng/ml, more preferably from 100-500 ng/ml MIS, preferably recombinant human MIS.
  • the embryonic reproductive system of both sexes is initially indistinguishable, consisting of indifferent gonads and the anlagen of the male and female reproductive tract.
  • the Mullerian duct forms from the coelomic epithelium and develops into the fallopian tubes, uterus, cervix, upper vagina, and ovarian surface epithelium.
  • regression of the Mullerian duct is essential for correct sexual differentiation. This process is induced by Mullerian Inhibiting Substance (MIS), a 140 kDa homodimeric glycoprotein which belongs to the TGF- ⁇ superfamily, is secreted by Sertoli cells in the embryonic testes.
  • MIS Mullerian Inhibiting Substance
  • Mullerian Inhibiting Substance is also known as Anti-Mullerian hormone (AMH), Mullerian inhibiting factor (MIF), and Mullerian inhibiting hormone (M1H).
  • AMH Anti-Mullerian hormone
  • MIF Mullerian inhibiting factor
  • M1H Mullerian inhibiting hormone
  • GenBank number for the cDNA for human MIS is K03474, and the amino acid sequence is AC005263.1. The significance of MIS in the postnatal period has not been firmly established.
  • mice with null mutations in either MIS or MISRII have Leydig cell hyperplasia and higher expression levels of cytochrome P450C 17-20 hydroxylase/lyase which catalyzes the committed step in testosterone synthesis [6- 8].
  • MIS ovarian granulosa cells begin to secrete low levels of MIS postnatally. Levels surge at the time of puberty to approximately 5-8 ng/ml, but then gradually decline throughout reproductive life until they become undetectable by menopause [2, 9]. Analogous to the suppressive effect MIS exerts on Leydig cell differentiation and testosterone synthesis in the testes, in vitro experiments suggest that MIS partially inhibits the initial recruitment of primordial follicles [10] and antagonizes subsequent FSH-dependent follicular growth [H].
  • MIS knockout mice exhibit increased recruitment of primordial follicles during the prepubertal period, resulting in the premature exhaustion of the follicle pool and earlier cessation of ovulation [12].
  • MIS appears to modulate ovarian follicular recruitment in ovulatory women, its role in the physiology of adult endometrium, another key M ⁇ llerian duct derivative, has never been explored.
  • MISRII has been shown in human endometrium [13] and rat cervical tissue [14] by immunohistochemistry, functional analyses to investigate the effect of MIS on these tissues have not been undertaken.
  • ELISA reveals that MIS is actively secreted by human endometrial stromal cells in vitro, and this process is significantly increased by estradiol treatment; and 5) Increasing local MIS concentration in cultured human endometrial stromal cells either by exogenous administration or transient transfection significantly decreases the number of viable cells and increases their rate of apoptosis.
  • MIS type II receptor a transmembrane serine-threonine kinase
  • MISRII a transmembrane serine-threonine kinase
  • MISRII a transmembrane serine-threonine kinase
  • MISRII recruits and phosphorylates a type I receptor.
  • ALK2 ALK2
  • BMPRlA ALK3
  • ALK6 bone morphogenetic protein
  • MIS is expressed in both proliferative and secretory human endometrium
  • mRNA for MIS, MISRII, ALK3, Smad I, and Smad9 FIG. 1.
  • Human endometrium and cultured human endometrial cells produce both MIS and MISRII protein.
  • cultured human endometrial cells are a good model for studying endometriosis, particularly abnormal proliferation.
  • Fixed cross-sections of proliferative and secretory human endometrium demonstrated strong immunoreactivity for both MIS and MISRII in the glandular epithelium.
  • MIS protein expression in ESCs is increased by the administration of estradiol. Estradiol has been reported to activate the MIS promoter in vitro [22]. Based on this and our immunocytochemistry findings, we hypothesized that estradiol would affect the in vitro expression of MIS in our ESCs (ESCs).
  • estradiol treatment of ESCs for 72 hours significantly increased MIS secretion by 57% (P ⁇ 0.0001) compared to that of the vehicle control group using Students t-test (FIG. 4).
  • This observation shows that endometrial cell function may be modulated by an interplay between estradiol and MIS.
  • rhMIS Recombinant Human MIS Induces Growth Inhibition in ESCs derived from human endometrium.
  • endometriosis can be treated or prevented by administering a therapeutically effective amount of MIS or a biologically active fragment or variant thereof.
  • the therapeutically effective amount is an amount that reduces or ameliorates one or more symptoms of endometriosis, which is typically an amount that decreases the proliferation of abnormal endometriotic cells.
  • human MIS is administered to human subjects, preferably as recombinant human MIS.
  • MIS is administered in an amount of between 75 ng/ml to about 1,000 ng/ml, most preferably from about 50-100 ng/ml.
  • MIS is administered locally to areas with endometriosis at time of surgery such as the bed of an ovarian endometrosiosis cyst after it has been removal to kill any remaining abnormal cells and to prevent recurrence. Any method known in the art for delivering MIS to the endometrial site can be used. As is explained below, the therapeutically effective amounts of MIS will vary depending on the severity of the condition, route of administration, pharmaceutical formulation, frequency of administration and other factors known to a person of skill in the art. Where the patient does not require surgery, MIS can be administered locally by injection, or can be delivered by implanting a pump or slow release preparation such as a hydrogel containing MIS.
  • compositions that include MIS, preferably human recombinant MIS, or a biologically active fragment or variant thereof, preferably in an amount of from about 75 ng/ml to about 1,000 ng/ml.
  • MIS is administered by vaginal suppository.
  • the endometrium is a highly specialized tissue capable of dramatically remodeling itself on a monthly basis. This process requires cellular proliferation, differentiation, breakdown and repair. Although this cyclic activity is broadly orchestrated by the ovarian sex steroids estrogen and progesterone, autocrine/paracrine mechanisms at the level of the endometrium are crucial for fine-tuning these processes. Indeed, a role for the local modulation of endometrial physiology has already been described for a myriad of growth factors, cytokines, proteases, and immunomodulatory agents [23-28].
  • TGF- ⁇ superfamily such as TGF ⁇ l, TGF ⁇ 2, TGF ⁇ 3, activin A, macrophage inhibitory cytokine (MIC)-I, BMP-2, BMP-7, BMP-4, and BMP-8 are dynamically expressed during different phases of the menstrual cycle, consistent with their critical roles in cell proliferation, differentiation, immunomodulation, apoptosis, and tissue remodeling [29].
  • MIC macrophage inhibitory cytokine
  • BMP-2, BMP-7, BMP-4, and BMP-8 are dynamically expressed during different phases of the menstrual cycle, consistent with their critical roles in cell proliferation, differentiation, immunomodulation, apoptosis, and tissue remodeling [29].
  • cyclic fluctuations in the expression of their receptors also have been demonstrated.
  • TGF- ⁇ Rl and -R2 expression are increased 3-4 fold in the endometrial stroma during the secretory phase of the menstrual cycle, coinciding with the upregulation of TGF- ⁇ l expression [25].
  • MIS another member of the TGF- ⁇ superfamily, also plays a role in the modulation of cellular functions involved in endometrial tissue remodeling.
  • MIS production in the adult female has been heretofore presumed to be primarily restricted to ovarian granulose cells, a concept largely attributable to the finding that a woman's serum MIS level gradually diminishes over the course of her reproductive lifespan until it becomes undetectable at menopause [31].
  • MIS has also been shown to inhibit the proliferation of several human cancer cell lines, including those originating from the cervix [14], endometrium [13], ovarian epithelium [32, 34], and breast [38].
  • MIS-mediated inhibition of cancer cell proliferation correlates with the upregulation of pl6INK4a protein, an inhibitor of the cyclin/CDK complex kinase activity [32].
  • Certain embodiments of the invention are directed to pharmaceutical compositions that include MIS, preferably the human recombinant form, or a biologically active fragment or variant thereof, in an amount that decreases the proliferation of abnormal endometriotic cells.
  • the compounds of this invention can be formulated and administered to prevent or treat endometriosis by any means that produces contact of the active ingredient with the agent's site of action.
  • the human MIS is administered locally to the site of the endometriotic cells, however, MIS can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as an individual therapeutic active ingredient or in a combination of therapeutic active ingredients.
  • MIS can be administered alone, but it is generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • the dosage administered will be a therapeutically effective amount of the compound sufficient to reduce or ameliorate one or more symptoms of the endometriosis.
  • the therapeutically effective amount of MIS is an amount that significantly reduces endometrial cell proliferation. Significantly lower or significantly higher means that the difference is statistically significant.
  • Our results show that the amount of MIS needed to reduce ESC proliferation was significantly higher than the typical circulating levels of endogenous MIS. Therefore in a preferred embodiment, MIS is administered locally to the site of endometriosis instead of systemically to treat endometriosis in order to achieve a high enough local concentration.
  • the therapeutically effective amount of MIS for local administration ranges from about 50-1000 ng/ml, more preferably from about 100-500 ng/ml.
  • MIS is administered locally as a hydrogel. 0.000001 mg/kg body weight to about 200 mg/kg body weight.
  • Local delivery can be accomplished by applying or installing the formulation into body cavities, by parenteral introduction, peritoneal, subcutaneous, or intradermal administration.
  • a particular route can provide a more immediate and more effective reaction than another route, depending on the circumstances.
  • Local delivery can be accomplished by any method known in the art. [0037]
  • the formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art.
  • Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient or the reduction of symptoms. Persons of ordinary skill can determine optimum dosages, dosing methodologies and repetition rates. Persons of ordinary skill in the art can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. The actual effective amount of a peptide also varies according to its size, biodegradability, bioactivity and bioavailability.
  • Optimum dosages can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease, preferably using lower amounts than were required to treat the initial endometriosis.
  • Factors that may influence the dosage required to effectively treat a subject including but not limited to the severity of the condition, previous treatments, the general health and/or age of the subject, and other disorders or diseases present.
  • Treatment of a subject with a therapeutically effective amount of a protein or polypeptide can include a single treatment or, preferably, can include a series of treatments. Appropriate doses also depend upon the potency of the therapeutic agent with respect to the expression or activity to be modulated. A physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the therapeutic agent MIS and biologically active fragments or variants thereof can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human. It is understood however, that administration can also be to cells in vitro as well as to in vivo model systems such as non-human transgenic animals.
  • Formulations of MIS proteins or peptides may contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. [0041] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • MIS can be modified as described below to increase efficacy or stability or other desirable properties.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds or therapeutic agents can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous.
  • parenteral e.g., intravenous, intradermal, subcutaneous.
  • a person of skill in the art will know which methods will optimize delivery of the active therapeutic agents to the targeted site of endometriosis.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diamante tetra acetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • composition In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., protein, peptide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • MIS may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection or using a pump.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • sustained and/or timed release formulations may be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference.
  • control release preparations can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylene vinylacetate, methyl cellulose, carboxymethyl cellulose, hydrogels, poly (lactic acid) or protamine sulfate.
  • concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release.
  • these agents can also be used to trap the compound in microcapsules.
  • compositions of the present invention can be formulated using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein may be readily selected for use with the pharmaceutical compositions of the invention.
  • Variants of MIS or biologically active fragments thereof include proteins and peptides that are substantially homologous to MIS that are produced by chemical synthesis or by recombinant methods.
  • two proteins are substantially homologous when the amino acid sequences are at least about 70-75%, typically at least about 80-85%, and most typically at least about 90-95%, 97%, 98% or 99% or more homologous.
  • Variants include conservative Amino Acid Substitutions: Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine.
  • a variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these.
  • Variant polypeptides can be fully functional or can lack function in one or more activities.
  • Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids, which results in no change or an insignificant change in function. Variants of MIS include those that reduce endometrial cell proliferation. [0052] As indicated, variants can be naturally-occurring or can be made by recombinant means of chemical synthesis to provide useful and novel characteristics of the desired protein.
  • Substantial homology can be to the entire amino acid sequence or to fragments of these sequences.
  • Biologically active fragments can be derived from the full naturally occurring amino acid sequence.
  • the invention also encompasses fragments of the variants of MIS as described herein. Accordingly, a fragment can comprise any length that retains one or more of the desired biological activities of the protein. Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide.
  • Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally-occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in polypeptides are described below.
  • the polypeptides also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification of the mature polypeptide or a pro-protein sequence.
  • a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included
  • the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification of the mature polypeptide or a pro-protein sequence.
  • MIS and biologically active analogs, derivatives, fragments and variants for use in the present invention can be modified according to known methods in medicinal chemistry to increase its stability, half-life, uptake or efficacy. Certain known modifications are described below.
  • polypeptides are not always entirely linear.
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of post-translation events, including natural processing events and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non- translational natural processes and by synthetic methods.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. Blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally-occurring and synthetic polypeptides. For instance, the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N- formylmethionine.
  • the modifications can be a function of how the protein is made.
  • the modifications will be determined by the host cell posttranslational modification capacity and the modification signals in the polypeptide amino acid sequence. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells, and, for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins having native patterns of glycosylation. Similar considerations apply to other modifications.
  • the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain more than one type of modification.
  • RNAlater® (Ambion, Austin, TX) and then frozen at - 80°C. When a sufficient number of samples were obtained, they were thawed, removed from the RNAlater®, and then dounced in Trizol® reagent (Invitrogen, Carlsbad, CA). RNA was then extracted according to manufacturer's instructions. Approximately 500ng of total RNA as assessed by spectrophotometry were then reversed transcribed into cDNA using the Sensiscript RT kit (Qiagen, Valencia, CA) according to manufacturer's instructions. cDNA was then purified using QIAEX II kit (Qiagen).
  • PCR was then carried out on a Bio-Rad thermocycler (Hercules, CA) using LA-Taq (Takara Bio USA, Madison WI) and functionally prevalidated primers for MIS, MISRII, ALK3 (BMPRIA), SMADl, SMAD9, and HPRT (Qiagen). PCR conditions were 95°C for 10 minutes, followed by 40 cycles of 30 seconds each of 94°C, 55°C, 72°C.
  • Endometrial Cell Culture At the time of specimen acquisition, a small portion of endometrium was placed into DMEM/F12 media. This tissue was then mechanically cleared of clots and debris and minced into small pieces ( ⁇ 1 mm3) under a dissecting microscope. The minced tissue was then incubated in culture media with 0.5% collagenase and deoxyribonuclease.
  • Endometrial cells were collected by centrifugation (500 x g, 10 min), resuspended in "complete" DMEM/F12 culture media (10% fetal calf serum, 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin, 0.25 ⁇ g/ml amphotericin, ImM sodium pyruvate) and plated in culture flasks at 37°C in a 5% CO2 atmosphere. Endometrial stromal cells (ESCs), which were not adherent after 30 minutes, were aspirated from the flask and replated, leaving the adherent epithelial (glandular) cells.
  • ESCs Endometrial stromal cells
  • Immunostaining was performed by incubating coverslips with the following antibodies overnight at 4°C: 1) rabbit anti-human MISRII (Abgent, San Diego, CA), 2) goat anti-human MIS (R&D Systems, Minneapolis, MN), 3) mouse antihuman ER- ⁇ (ABR-Affinity BioReagents, Golden CO), 4) mouse anti-human progesterone receptor (R&D Systems), 5) rabbit anti -human vimentin (ABR-Affinity BioReagents), and 6) goat anti-human cytokeratin (ABR-Affinity BioReagents).
  • Diaminobenzidine tetrachloride (Vector Laboratories, Burlingame, CA) was used to colorize the tissue after incubation with appropriate HRP-conjugated secondary antibodies. Some freshly obtained endometrial tissue was placed directly into Z-fix for immunohistochemistry using the MIS and MISRII antibodies. Slides hybridized only to secondary antibodies were also prepared to serve as negative controls.
  • Transient transfection The full-length coding sequence of the human MIS gene (GenBank No. KQ ⁇ -474) and the MISRII genes were individually ligated between two Notl recognition sites within the multiple cloning region of the mammalian expression vector pCMV-XL6 (Origene, Rockville, MD). Purified plasmid preparations were made (WizardPlus SV minipreps, Promega, Madison, WI) from overnight LB-ampicillin broths containing a single transformed colony of competent E. coli cells (ToplO cells, Invitrogen, Carlsbad, CA). Presence and orientation of inserted sequences were validated by PCR and restriction digestion with Notl.
  • MIS ELISA Prior to all MIS assays, human endometrial cells were twice washed in PBS and then cultured in serum-free media in order to prevent potential confounding from any MIS present in the fetal calf serum.
  • ESCs were grown to 80% confluence, trypsinized, and then transferred into 96- well plates at a density of 2000 cells in 100 ⁇ L of serum-free DMEM per well. After 16 hours, the media was aspirated, the cells were washed in PBS, and lOOuL of fresh serum-free, phenol red-free DMEM media containing either vehicle or 100 ng/mL recombinant human MIS (R&D Systems, Minneapolis, MN) was placed into each well. After 72 hours in culture, 20 uL of a resazurin containing reagent (CellTiter-Blue, Promega) was then added directly to each well.
  • a resazurin containing reagent CellTiter-Blue, Promega
  • the fluorescence per well (560Ex/590Em) produced by the conversion of resazurin to resorufin, which is directly proportional to viable cell number, was quantified.
  • the caspase activity within these same wells was then measured by adding 120 ⁇ L of Apo-ONE Homogeneous Caspase-3/7 Assay reagent (Promega) to each well. Cells were incubated for an additional hour at ambient temperature prior to recording fluorescence (485Ex/527Em).

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Abstract

Cette invention concerne la découverte selon laquelle l'hormone anti-Müllérienne (AMH) peut être utilisée de manière thérapeutique pour traiter ou prévenir l'endométriose chez le mammifère, en particulier chez l'homme. Dans un mode de réalisation préféré, une quantité thérapeutiquement efficace d'AMH humaine ou d'un fragment ou d'un variant biologiquement actif de celle-ci est administrée pour traiter un patient atteint d'endométriose, ou pour prévenir l'endométriose chez un patient à risque de développer une endométriose ou prévenir une récidive d'endométriose. La quantité thérapeutiquement efficace d'AMH est une quantité qui réduit la prolifération des cellules de l'endomètre, une quantité que nos résultats montrent significativement supérieure aux taux normaux circulants de l'AMH endogène.
PCT/US2008/079890 2007-10-14 2008-10-14 Procédé de traitement de l'endométriose par administration d'hormone anti-müllérienne WO2009052119A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7807620B2 (en) 2002-01-31 2010-10-05 The Trustees Of Columbia University In The City Of New York Long-acting follicle stimulating hormone analogues and uses thereof
WO2011080660A1 (fr) 2009-12-28 2011-07-07 Koninklijke Philips Electronics N.V. Robot de cuisine comprenant un dispositif de pesée
ITRM20120285A1 (it) * 2012-06-18 2013-12-19 Alfonso Baldi Ormone anti-mulleriano.
ITRM20130455A1 (it) * 2013-08-05 2015-02-06 Alfonso Baldi Ligandi dell'ormone anti-mulleriano
WO2016030901A1 (fr) * 2014-08-31 2016-03-03 Tel Hashomer Medical Research Infrastructure And Services Ltd. Procédés pour empêcher l'activation des follicules prématurée
US10478473B2 (en) 2015-03-04 2019-11-19 The Center For Human Reproduction (Chr) Compositions and methods of using anti-mullerian hormone for treatment of infertility

Citations (4)

* Cited by examiner, † Cited by third party
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US6245896B1 (en) * 1993-01-12 2001-06-12 The Johns Hopkins University School Of Medicine Antibodies that binds growth differentiation factor 5
US6265393B1 (en) * 1998-08-07 2001-07-24 Heinrichs William Leroy Prevention of endometriosis signs or symptons
US20040258685A1 (en) * 2002-11-21 2004-12-23 Genentech, Inc. Therapy of non-malignant diseases or disorders with anti-ErbB2 antibodies
US20070128203A1 (en) * 2000-08-07 2007-06-07 Jill Giles-Komar Anti-integrin antibodies, compositions, methods and uses

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6245896B1 (en) * 1993-01-12 2001-06-12 The Johns Hopkins University School Of Medicine Antibodies that binds growth differentiation factor 5
US6265393B1 (en) * 1998-08-07 2001-07-24 Heinrichs William Leroy Prevention of endometriosis signs or symptons
US20070128203A1 (en) * 2000-08-07 2007-06-07 Jill Giles-Komar Anti-integrin antibodies, compositions, methods and uses
US20040258685A1 (en) * 2002-11-21 2004-12-23 Genentech, Inc. Therapy of non-malignant diseases or disorders with anti-ErbB2 antibodies

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7807620B2 (en) 2002-01-31 2010-10-05 The Trustees Of Columbia University In The City Of New York Long-acting follicle stimulating hormone analogues and uses thereof
WO2011080660A1 (fr) 2009-12-28 2011-07-07 Koninklijke Philips Electronics N.V. Robot de cuisine comprenant un dispositif de pesée
ITRM20120285A1 (it) * 2012-06-18 2013-12-19 Alfonso Baldi Ormone anti-mulleriano.
WO2013190443A1 (fr) * 2012-06-18 2013-12-27 Signorile Pietro Giulio Hormone antimullérienne
ITRM20130455A1 (it) * 2013-08-05 2015-02-06 Alfonso Baldi Ligandi dell'ormone anti-mulleriano
WO2015019269A1 (fr) * 2013-08-05 2015-02-12 Signorile Pietro Giulio Ligands marqués d'hormone antimüllérienne pour le diagnostic de l'endométriose
US9861711B2 (en) 2013-08-05 2018-01-09 Pietro Giulio Signorile Labeled ligands of anti-Mullerian hormone for diagnosis of endometriosis
WO2016030901A1 (fr) * 2014-08-31 2016-03-03 Tel Hashomer Medical Research Infrastructure And Services Ltd. Procédés pour empêcher l'activation des follicules prématurée
CN107106657A (zh) * 2014-08-31 2017-08-29 堤乐哈修门医学研究基础建设及服务有限公司 用于预防过早的卵泡活化的方法
US10478473B2 (en) 2015-03-04 2019-11-19 The Center For Human Reproduction (Chr) Compositions and methods of using anti-mullerian hormone for treatment of infertility

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