WO2001019387A1 - Utilisation de la substance d'inhibition des canaux de muller pour le traitement des etats pathologiques caracterises par un exces d'androgenes - Google Patents

Utilisation de la substance d'inhibition des canaux de muller pour le traitement des etats pathologiques caracterises par un exces d'androgenes Download PDF

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WO2001019387A1
WO2001019387A1 PCT/US2000/025094 US0025094W WO0119387A1 WO 2001019387 A1 WO2001019387 A1 WO 2001019387A1 US 0025094 W US0025094 W US 0025094W WO 0119387 A1 WO0119387 A1 WO 0119387A1
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Prior art keywords
mis
cells
kda
androgens
testosterone
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PCT/US2000/025094
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English (en)
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Patricia K. Donahoe
Jose Teixeira
Eric Fynn-Thompson
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The General Hospital Corporation
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Priority to CA002384991A priority Critical patent/CA2384991A1/fr
Priority to JP2001523019A priority patent/JP2003509377A/ja
Priority to EP00963414A priority patent/EP1218025A4/fr
Priority to AU74830/00A priority patent/AU7483000A/en
Publication of WO2001019387A1 publication Critical patent/WO2001019387A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/28—Antiandrogens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This application is directed generally to methods of treating prostate cancer, polycystic ovarian disease, benign prostatic hypertrophy, and precocious puberty.
  • MIS Mullerian inhibiting substance
  • TGF ⁇ transforming growth factor- ⁇ family of growth and differentiation factors.
  • SRY Sertoli cells of the fetal testis begin procuring MIS, which is a phenotypic hallmark of testis development (Swain et al, Nature
  • MIS also known as anti-Mullerian hormone (Jossa et al, Recent Prog. Horm. Res. 48 1-59 (1993)), is absolutely required for normal male reproductive tract development because it affects the regression of the Mullerian duct of the bipotential urogenital ridge, which, is left undisturbed, would give rise to female reproductive tract structures such as the uterus, Fallopian tubes, and upper vagina (Jost, A , Arch. Anat. Microsc. Morphol. Exp. 36 271-315 (1947),
  • the present invention provides a method of treating a condition or disease characterized by an excess of one or more androgens, the method comprising administering an effective amount of MIS to a patient
  • the present invention also provides a method of treating a condition or disease characterized by an excess of one or more androgens, the method comprising administering an effective amount of a nucleic acid encoding MIS to a patient
  • the present invention also provides a method of decreasing the plasma level of one or more androgens, the method comprising administering to a patient an effective amount of MIS, wherein the amount of MIS is sufficient to decrease the plasma level of the one or more androgens below the normal level for the one or more androgens
  • the present invention also provides a method of decreasing the plasma level of one or more androgens, the method comprising administering to a patient an effective amount of nucleic acid encoding MIS, wherein the amount of MIS is sufficient to decrease the plasma level of the one or more androgens below the normal level for the one or more androgens
  • the methods of the present invention are particularly well-suited for the treatment of prostate cancer, polycystic ovarian disease, benign prostatic hypertrophy, and precocious puberty
  • FIG 1 depicts the pathways of testosterone biosynthesis 21 -Carbon testosterone is synthesized from the 27-carbon cholesterol molecule by the activities of P450-scc (cytochrome P450 side-chain cleavage), P450cl7 (cytochrome P450cl7 hydroxylase/lyase), 3 ⁇ HSD, and 17KSR (17-ketosteroid reductase)
  • FIG 2 depicts results of a study of MIS type II receptor mRNA expression in rodent Leydig cells Total RNA was isolated from the indicated sources, denatured, electrophoresed, bloated to a nylon membrane, and probed with a radio-labeled MIS type II receptor riboprobe The blot was exposed to x-ray film to detect the migration of hybridized mRNA The mol wt marker shown indicates the migration of the 2-kb band of a ⁇ Hindlll digest
  • FIGS 3A and 3B depict the results of an analysis of steroid production in MA- 10 cells
  • Culture medium was collected and assayed by RLA for total accumulated progesterone (FIG 3 A) and testosterone (FIG 3B), shown are levels after 1 or 2 days of treatment with 105 nM MIS in presence or absence of 50 ⁇ M cAMP
  • Cross morphology was indistinguishable between MIS-treated and untreated cells
  • Error bars represent the SEM *, P ⁇ 0 05, **, ⁇ 0 001, *** 3 ⁇ 0 0001 Significance was measured using Student's t test
  • FIGS 4A-C depict a Northern analysis of the expression of RNAs for steroidogenic enzymes Northern analysis of the steady state levels of mRNAs from the indicated cells for steroidogenic enzymes was performed as described in
  • FIGS 5A-C depict the results of a study of MIS regulation of the
  • P450cl7 promoter A promoter/reported mini-gene system employing the luciferase reporter gene was used to characterize the promoter of the P450cl7 ⁇ gene
  • FIG 5 A MA- 10 cells were incubated, 24 h post-transfection, with vehicle control or 1 4, 7, 35, 70, 105 and 175 nM MIS for 18 or 29 h Cell extracts were assayed for firefly luciferase and renilla luciferase activity, and the results are shown as firefly/renilla values normalized to vehicle control values at 1000 relative light units
  • FIG 5B Cells were incubated with vehicle control or 105 nM MIS for the indicated periods of time starting 24 h after transfection Cells were also incubated with an inactive L9 noncleavable mutant MIS for 18 h (shown with an asterisk) Cell extracts were assayed for luciferase activity, and the results are shown as firefly/renilla values normalized to
  • FIG 6 depicts the results of a study of MIS regulation of testosterone concentration iv vivo
  • Conditions or diseases characterized by an excess of one or more androgens include, but are not limited to, a condition or disease selected from the group consisting of polycystic ovarian disease or syndrome, precocious puberty, and McCune- Albright syndrome Whether androgen levels are excessive can be readily diagnosed by one of ordinary skill in the art, using clinical chemisty, including urinalysis, and blood, serum, or plasma analysis
  • testosterone includes testosterone and its metabolites, such as dihydrotestosterone, androsterone, estradiol, and etiocholanolone
  • testosterone and its metabolites such as dihydrotestosterone, androsterone, estradiol, and etiocholanolone
  • the normal plasma level of testosterone is about 10-35 nmol/L (about 3-10 ng/mL) See Harrison's Principles of Internal Medicine, 14* Edition, Fauci, A S et al , Eds , McGaw-Hill, New York (1998), at page 2040, Table 332-2, the content of which is incorporated by reference
  • a patient has a condition or disease characterized by an excess of one or more androgens, and particularly polycystic ovarian disease or syndrome or precocious puberty, can be readily diagnosed by one of ordinary skill in the art, based upon physical examination, clinical chemistry analysis, family history, and patient interviews S ee Harrison 's Principles of Internal Medicine, 14 th Edition, Fauci, A S et al , Eds , McGaw-Hill, New York (1998) In patients with benign prostatic hypertrophy, to whom MIS, or nucleic acid encoding MIS, is administered, the extent of prostatic hypertrophy is diminished or decreased The extent of prostatic hypertrophy, and changes therein, can be readily determined by one of ordinary skill in the art
  • MIS Mullerian Inhibiting Substance
  • the protein migrates on gel electrophoresis at an apparent molecular weight of 70 kDa
  • the protein can be proteolytically cleaved by exogenous plasmin into two distinct fragments that migrate electrophoretically as 57 kDa and 12 5 kDa moieties with cleavage at residue 427 of the intact 535 amino acid monomer (Pepinsky, et al, J. Biol Chem. 263 18961-4 (1988))
  • carboxy-terminal (C-terminal) fragment of MIS is intended to include compounds and materials structurally similar to the about 12 5 kDa (about 25 kDa under non-reducing conditions) C-terminal fragment of MIS resulting from proteolytic (e g , plasmin) cleavage at residue 427 of the intact 535 amino acid human MIS monomer
  • proteolytic (e g , plasmin) cleavage site is at residue 443 of the 551 amino acid bovine MIS molecule
  • “carboxy- terminal (C-terminal) fragment of MIS” is intended to include the about 25 kDa homodimeric C-terminal fragment of MIS
  • N-terminal fragment of MIS is intended the about 57 kDa fragment resulting from the above-noted cleavage at residue 427 of the intact 535 amino acid human MIS monomer (residue 443 of the 551 amino acid bovine MIS)
  • the methods of the present invention can be practiced using mutant forms of the C-terminal fragment of MIS which have substantially the same biological activity as the C-terminal fragment of MIS
  • mutant forms would be C-terminal fragment of MIS molecules carrying a deletion, insertion, or alteration of amino acid sequence
  • the C-terminal fragment of MIS can be modified to increase its half-life in vivo
  • addition of one or more amino acids or other chemical agents to the amino and/or carboxyl end of the C-terminal fragment can be used to increase the fragment's stability
  • the C-terminal fragment of MIS can be obtained from a mammalian source or through the use of recombinant DNA technology, or from chemical synthesis of the C-terminal polypeptide
  • a gene is said to be a "recombinant” gene if it results from the application of Recombinant DNA Techniques
  • recombinant DNA techniques include cloning, mutagenesis, transformation, etc Recombinant DNA Techniques are disclosed in Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N Y (1982, 1989) "Recombinant MIS” refers to MIS polypeptide, or a fragment thereof, and particularly the C-terminal fragment, that is prepared using recombinant means
  • Recombinant MIS can be expressed in a protein expression system
  • prokaryotic and eukaryotic expression systems are well understood by those of ordinary skill in the art
  • bacterial e g , E. co
  • fungi e g , yeast
  • mammalian cells e g , CHO cells, COS cells
  • insect cells e g , baculovirus cells
  • the C-terminal fragment human or bovine
  • the C-terminal fragment can be readily produced by the recombinant DNA techniques described in U S Patent No 5,047,336, which is fully incorporated by reference herein
  • Of particular interest is expression of the C-terminal fragment in E. coli and other bacteria, since the C-terminal fragment is not glycosylated
  • various sites may be selected for insertion of the gene coding for MIS or C-terminal fragment of MIS These sites are usually designated by the restriction endonuclease which cuts them and are well recognized by those of skill in the art Various methods for inserting
  • DNA sequences into these sites to form recombinant DNA molecules are also well known These include, for example, dG-dC or dA-dT tailing, direct ligation, synthetic linkers, exonuclease and polymerase-linked repair reactions followed by ligation, or extension of the DNA strand with DNA polymerase and an appropriate single-stranded template followed by ligation It is, of course, to be understood that a cloning or expression vehicle useful in this invention need not have a restriction endonuclease site for insertion of the chosen DNA fragment Instead, the vehicle could be joined to the fragment by alternative means
  • expression control sequences may also be chosen to effect the expression of recombinant DNA sequences
  • These expression control sequences include, for example, the lac system, the ⁇ -lactamase system, the trp system, the tac system, the trc system, the major operator and promoter regions of phase ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e g , Pho5, the promoters of the yeast ⁇ -mating factors, promoters for mammalian cells such as the S V40 early promoter, adenovirus late promoter and metallothionine promoter, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses and various combinations thereof
  • it is additionally possible to amplify the expression units by linking the gene to that for dihydrofolate reductase and applying a selection to host Chinese
  • these DNA sequences are operatively-linked to one or more of the above-described expression control sequences in the expression vector
  • Such operative linking which may be effected before or after the MIS or C-terminal fragment of MIS DNA sequence is inserted into a cloning vehicle, enables the expression control sequences to control and promote the expression of the DNA sequence
  • the vector or expression vehicle, and in particular the sites chosen therein for insertion of the selected DNA fragment and the expression control sequence employed in this invention, is determined by a variety of factors, e g , number of sites susceptible to a particular restriction enzyme, size of the protein to be expressed, expression characteristics such as start and stop codons relative to the vector sequences, and other factors recognized by those of skill in the art
  • the choice of a vector, expression control sequence, and insertion site for the MIS or C-terminal fragment of MIS DNA sequence is determined by a balance of these factors, not all selections being equally effective for a given case
  • the DNA sequences coding for MIS or the C-terminal fragment of MIS that are inserted at the selected site of a cloning or expression vehicle may include nucleotides which are not part of the actual gene coding for MIS or the C-terminal fragment of MIS or may include only a fragment of the actual gene It is only required that whatever DNA sequence is employed, a transformed host will produce MIS or the C-terminal fragment of MIS
  • the MIS DNA sequences of this invention may be fused in the same reading frame in an expression vector of this invention to at least a portion of a DNA sequence coding for at least one eukaryotic or prokaryotic signal sequence, or combinations thereof
  • Such constructions enable the production of, for example, a methionyl or other peptidyl-MIS polypeptide, that is part of this invention This N-terminal methionine or peptide may either then be cleaved intra- or extra-cellularly by a variety of known processes or the MIS polypeptide with the
  • the MIS polypeptide may include polypeptides in the form of fused proteins (e g , linked to prokaryotic, eukaryotic or combination N-terminal segment to direct excretion, improve stability, improve purification or improve possible cleavage at amino acid residue 443 to release an active C-terminal fragment), in the form of a precursor of MIS (e g , starting with all or parts of a MIS signal sequence of other eukaryotic or prokaryotic signal sequences), in the form of a mature MIS polypeptide, or in the form of an fmet-MIS polypeptide
  • the present invention also encompasses substituting codons for those of the MIS or C-terminal fragment of MIS nucleotide sequences These substituted codons may code for amino acids identical to those coded for by the codons replaced but result in higher yield of the polypeptide Alternatively, the replacement of one or a combination of codons leading to amino acid replacement or
  • MIS polypeptide, or a fragment thereof, and MIS-encoding nucleic acid can be administered in the form of a pharmaceutical compositions
  • the pharmaceutical composition can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby MIS or the C-terminal fragment of MIS or their functional derivatives are combined in admixture with a pharmaceutically acceptable carrier vehicle Suitable vehicles and their formulation, inclusive of other human proteins, i e , human serum albumin, are described for example in Remington's Pharmaceutical Sciences, 18th edition, A R Gennaro, Ed , Mack Publ , Easton, PA (1990)
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of MIS or the C-terminal fragment of MIS, or their functional derivatives, together with a suitable amount of carrier vehicle
  • an "effective amount" of MIS is one which is sufficient to inhibit the progression of and/or reduce the severity of polycystic ovarian disease or syndrome, or to slow and/or halt the
  • an "effective amount" of MIS is one which is sufficient to decrease the plasma level of one or more androgens, including testosterone and/or its metabolites, such as dihydrotestosterone, androsterone, estradiol, and etiocholanolone, below the normal level
  • the normal plasma level of testosterone below about 10-35 nmol/L (about 3-10 ng/mL) See Harrison's Principles of Internal Medicine, 14 th Edition, Fauci, A S et al , Eds , McGaw-Hill, New York (1998), at page 2040, Table 332-2, the content of which is incorporated by reference
  • an effective amount of MIS is one which is sufficient to decrease the plasma level of testosterone below about 10-35 nmol/L (about 3-10 ng/mL)
  • an effective amount of MIS is one which is sufficient to decrease the plasma level of testosterone below about 15-30 nmol/L
  • the effective amount may vary depending upon criteria such as the age, weight, physical condition, past medical history, and sensitivity of the recipient
  • the effective amount will also vary depending on whether administration is oral, intravenous, intramuscular, subcutaneous, local, or by direct application to the tumor In the case of direct tumor application, it is preferable that a final serum concentration of at least 0 1 nM, preferably about 0 1-1 0 nM, of MIS be achieved Likewise, for direct tumor application of the C-terminal fragment of MIS, it is preferable that a final serum concentration of at least 0 1 nM, preferably about 0 1-1 0 nM, of the C-terminal fragment of MIS be achieved.
  • Effective individual dosage through the additionally named means of administration can be readily determined by methods well known to those of ordinary skill in the art For example, using the size ratio calculation as detailed above, one of ordinary skill in the art can determine optimal dosage levels for any means of administration In treating a patient, it is preferable to achieve a serum level of at least 10 ng/ml of MIS In treating a patient with the C-terminal fragment of MIS, it is preferable to achieve a serum level
  • a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient
  • Such an agent is said to be physiologically significant if its presence results in a detectable change in the physiology of a recipient patient
  • compositions containing MIS or the C-terminal fragment of MIS or their functional derivatives may be administered orally, intravenously, intramuscularly, subcutaneously, or locally Additional pharmaceutical methods may be employed to control the duration of action
  • Controlled release preparations may be achieved by the use of polymers to complex or adsorb MIS or the C-terminal fragment of MIS or their functional derivatives
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, and protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release
  • MIS or the C-terminal fragment of MIS into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers
  • a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsion
  • a “functional derivative” of MIS is a compound which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of MIS
  • the term “functional derivatives” is intended to include the “fragments,” “variants,” “analogs,” or “chemical derivatives” of a molecule
  • a “fragment” of a molecule such as either MIS is meant to refer to any polypeptide subset of the molecule Fragments of MIS which has activity and which are soluble (i e not membrane bound) are especially preferred
  • a “variant” of a molecule such MIS is meant to refer to a molecule substantially similar in structure and function to either the entire molecule, or to a fragment thereof
  • a molecule is said to be " substantially similar” to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity
  • two molecules possess a similar activity they are considered variants as that term is used herein even if the structure of one of the molecules not found in the other, or if the
  • MIS (or its functional derivatives, agonists, or antagonists) may be administered to patients intravenously, intramuscularly, subcutaneously, enterally, or parenterally
  • the administration may be by continuous infusion, or by single or multiple boluses
  • MIS molecules (and their functional derivatives, agonists and antagonists can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle Suitable vehicles and their formulation, inclusive of other human proteins, e g , human serum albumin, are described, for example, mRemingto 's Pharmaceutical Sciences, 18th edition, A R Gennaro, Ed , Mack Publ , Easton, PA (1990) In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of MIS molecule, or its functional derivatives, agonists, or antagonists, together with a suitable amount of carrier vehicle
  • Control release preparations may be achieved through the use of polymers to complex or absorb MIS or its functional derivatives, agonists, or antagonists
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release
  • Another possible method to control the duration of action by controlled release preparations is to incorporate the MIS molecule, or its functional derivatives, agonists, or antagonists, into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers
  • kits Preferably, such kits will contain two or more containers which are specially adapted to receive MIS or one of its functional derivatives, and an agonist of MIS
  • patient is intended to include animal patients More preferably, “patient” is intended to include mammalian patients, most preferably, human patients
  • protein fragment is meant to include both synthetic and naturally-occurring amino acid sequences derivable from the naturally occurring amino acid sequence of MIS
  • the protein is said to be "derivable from the naturally-occurring amino acid sequence of MIS” if it can be obtained by fragmenting the naturally-occurring chosen sequence of MIS, or if it can be synthesized based upon a knowledge of the sequence of the naturally occurring amino acid sequence or of the genetic material (DNA or RNA) which encodes this sequence
  • an "effective amount" of nucleic acid encoding MIS is one which is sufficient to inhibit the progression of and/or reduce the severity of polycystic ovarian disease or syndrome, or to slow and/or halt the progression of precocious puberty
  • an "effective amount” of nucleic acid encoding the C-terminal fragment of MIS is one which is sufficient to inhibit the progression of and/or reduce the severity of polycystic ovarian disease or syndrome, or to slow and/or halt the progression of precocious puberty
  • an "effective amount" of nucleic acid encoding MIS is one which is sufficient to decrease the plasma level of one or more androgens. including testosterone and/or its metabolites, such as dihydrotestosterone, androsterone, estradiol, and etiocholanolone, below the normal level
  • an effective amount of MIS is one which is sufficient to decrease the plasma level of testosterone below about 10-35 nmol/L (about 3-10 ng/mL)
  • an effective amount of MIS is one which is sufficient to decrease the plasma level of testosterone below about 15-30 nmol/L
  • an effective amount of MIS is one which is sufficient to decrease the plasma level of testosterone below about 20-25 nmol/L
  • a vector contains a gene capable of expressing an "effective amount of MIS” or an "effective amount of the C-terminal fragment of MIS” can be determined following the protocols set forth in Example 4 in U S Patent No 5,661,126, which is hereby incorporated by reference in its entirety
  • the gene therapy methods relate to the introduction of nucleic acid (DNA,
  • RNA and antisense DNA or RNA sequences into an animal to achieve expression of the MIS polypeptide of the present invention
  • This method requires a polynucleotide which codes for an MIS polypeptide operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue
  • gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to an MIS polynucleotide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide
  • a polynucleotide DNA or RNA
  • RNA polynucleotide
  • the engineered cells then being provided to a patient to be treated with the polypeptide
  • Such methods are well-known in the art For example, see Belldegrun, A , et al, J. Natl. Cancer Inst. 85 207-216 (1993), Ferrantini, M et al. , Cancer Research 53 1107-1112 (1993), Ferrantini, M etal, J. Immunology 153 4604-4615 (1994), Kaido, T , et al, Int. J. Cancer 60 221- 229 (1995), Ogura, H , et al, Cancer Research 50 5102
  • the cells which are engineered are arterial cells
  • the arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection
  • the MIS polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like)
  • the MIS polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier
  • the MIS polynucleotide is delivered free of any delivery vehicle that acts to assist, promote or facilitate entry into the cell In another embodiment, the MIS polynucleotide is delivered free of viral sequences
  • the MIS polynucleotide is delivered free of viral particles In another embodiment, the MIS polynucleotide is delivered free of liposome formulations In another embodiment, the MIS polynucleotide is delivered free of lipofectin In another embodiment, the MIS polynucleotide is delivered free of precipitating agents
  • the MIS polynucleotides can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art Such methods are described, for example, in U S Patent Nos 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference
  • the MIS polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene, p
  • Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter, or heterologous promoters, such as the cytomegalovirus (CMV) promoter, the respiratory syncytial virus (RS V) promoter, inducible promoters, such as the MMT promoter, the metallothionein promoter, heat shock promoters, the albumin promoter, the ApoAI promoter, human globin promoters, viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter, retroviral LTRs, the b-actin promoter, and human growth hormone promoters
  • the promoter also may be the native promoter for MIS
  • one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months
  • the MIS polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue
  • Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below They may be conveniently delivered by injection into the tissues comprising these cells They are preferably delivered to and expressed in persistent, non-
  • an effective dosage amount of DNA or RNA will be in the range of from about 0 05 mg/kg body weight to about 50 mg/kg body weight
  • the dosage will be from about 0 005 mg/kg to about 20 mg/kg and more preferably from about 0 05 mg/kg to about 5 mg/kg
  • this dosage will vary according to the tissue site of injection
  • the appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration
  • the preferred route of administration is by the parenteral route of injection into the interstitial space of tissues
  • parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose
  • naked MIS DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure
  • the naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns” These delivery methods are known in the art
  • the constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc Such methods of delivery are known in the art
  • the MIS polynucleotide constructs are complexed in a liposome preparation
  • Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations
  • cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl Acad. Sci.
  • Cationic liposomes are readily available for example, N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO
  • liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer)
  • DOTAP l,2-bis(oleoyloxy)-3-(trimethylammonio)propane liposomes
  • DOTMA liposomes Preparation of DOTMA liposomes is explained in the literature, see, e g , P Feigner et al., Proc. Natl. Acad. Sci. USA 84 7413-7417, which is herein incorporated by reference Similar methods can be used to prepare liposomes from
  • anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala ), or can be easily prepared using readily available materials
  • Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others
  • DOPC dioleoylphosphatidyl glycerol
  • DOPE dioleoylphoshatidyl ethanolamine
  • DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC
  • negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar
  • MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes
  • the material to be entrapped is added to a suspension of preformed MLVs and then sonicated
  • liposomes containing cationic lipids the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA
  • the liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA SUVs find use with small nucleic acid fragment
  • the ratio of DNA to liposomes will be from about 10 1 to about
  • the ration will be from about 5 1 to about 1 5 More preferably, the ratio will be about 3 1 to about 1 3 Still more preferably, the ratio will be about 1 1
  • cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding MIS
  • Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines Examples of packaging cells which may be transfected include, but are not limited to, the PE501.
  • the vector may transduce the packaging cells through any means known in the art Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO 4 precipitation
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host
  • the producer cell line generates infectious retroviral vector particles which include polynucleotide encoding MIS
  • retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo
  • the transduced eukaryotic cells will express MIS
  • cells are engineered, ex vivo or in vivo, with MIS polynucleotide contained in an adenovirus vector
  • Adenovirus can be manipulated such that it encodes and expresses MIS, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis
  • adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz, A R et al. (1974) Am. Rev. Respir.
  • Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel 3 499-503
  • the adenovirus vector Ad2 is useful and can be grown in human 293 cells These cells contain the El region of adenovirus and constitutively express Ela and Elb, which complement the defective adenoviruses by providing the products of the genes deleted from the vector
  • Ad2 other varieties of adenovirus (e.g , Ad3, Ad5, and Ad7) are also useful in the present invention.
  • the adenoviruses used in the present invention are replication deficient Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles.
  • the resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells
  • Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes. Ela, Elb, E3, E4, E2a, or LI through L5
  • the cells are engineered, ex vivo or in v vo, using an adeno-associated virus (AAV)
  • AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol Immunol. 158 97 (1992)).
  • Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4 5 kb
  • Methods for producing and using such AAVs are known in the art See, for example, U S Patent Nos 5,139,941, 5,173,414, 5,354,678, 5,436, 146, 5,474,935, 5,478,745, and 5,589,377.
  • an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration
  • the MIS polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press (1989)
  • the recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc.
  • helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses
  • helper viruses Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the MIS polynucleotide construct These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo
  • the transduced cells will contain the MIS polynucleotide construct integrated into its genome, and will express MIS
  • Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e g encoding MIS) via homologous recombination (see, e g , U S Patent No 5,641,670, issued June 24, 1997, International Publication No WO 96/29411, published September 26, 1996, International Publication No WO 94/12650, published August 4, ⁇ 994, Ko ⁇ e ⁇ etal, Proc. Natl Acad. Sci.
  • Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter Suitable promoters are described herein
  • the targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence
  • the targeting sequence will be sufficiently near the 5' end of the MIS desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination
  • the promoter and the targeting sequences can be amplified using PCR
  • the amplified promoter contains distinct restriction enzyme sites on the 5' and 3' ends
  • the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction site as the 3' end of the amplified promoter
  • the amplified promoter and targeting sequences are digested and ligated together
  • the promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc , described in more detail above
  • transfection-facilitating agents such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc , described in more detail above
  • the P promoter- targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc The methods are described in more detail below
  • the promoter-targeting sequence construct is taken up by cells Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous MIS sequence is placed under the control of the promoter
  • the promoter then drives the expression of the endogenous MIS sequence
  • the polynucleotide encoding MIS contains a secretory signal sequence that facilitates secretion of the protein
  • the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5' end of the coding region
  • the signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected Additionally, the signal sequence may be chemically synthesized using methods known in the art
  • any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect
  • direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al, Science 243 375 (1989))
  • a preferred method of local administration is by direct injection
  • a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries
  • Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries
  • Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound
  • a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound
  • Therapeutic compositions useful in systemic administration include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention
  • Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site
  • Preferred methods of systemic administration include intravenous injection, aerosol, oral and percutaneous (topical) delivery Intravenous
  • Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal
  • a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal
  • examples of such carriers include plastic capsules or tablets, such as those known in the art
  • Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e g , DMSO) that is capable of passing into the skin
  • Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration
  • the frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject
  • the precise amount, number of doses, and timing of doses will be determined by the attending physician Phenotypes of genetically altered adult mice have yielded several clues for possible role for MIS in the adult, including regulation of steroidogenesis Mice chronically overexpressing a human MIS transgene develop varying degrees of gonadal abnormalities in the adult (Behringer et al.
  • MIS overexpression might interfere with androgen biosynthesis in Leydig cells
  • homologous recombination in mice so that they no longer expressed either the MIS ligand (Behringer et al, Cell 79 415-425 (1994)) or the MIS type II receptor (Mishina et al, Genes Dev. 10 2577-2587 (1996)) also resulted in gonadal abnormalities consisting of Leydig cell hyperplasia and focal atrophy of the germinal epithelium
  • MIS appears to have a role in maintaining steroid hormone balance in both male and femal gonads after birth
  • Leydig cells or intestinal cells are found in the testes surrounding the seminiferous tubules Their major function is to produce testosterone, which is essential for the normal male phenotype Testosterone is synthesized from cholesterol in five steps by the activity of four enzymes (FIG 1), three of which the present inventors have studied P450scc, P450cl7, and 3 ⁇ -hydroxysteroid dehydrogenate/ ⁇ 5 - ⁇ 4 -isomerase (3 ⁇ HSD)(Payne and Youngblood, Biol. Reprod.
  • P450cc cytochrome P450-side chain cleavage, also known as CYPl 1 A
  • CYPl 1 A cytochrome P450-side chain cleavage, also known as CYPl 1 A
  • CYPl 1 A cytochrome P450-side chain cleavage, also known as CYPl 1 A
  • CYPl 1 A cytochrome P450-side chain cleavage, also known as CYPl 1 A
  • CYPl 1 A cytochrome P450-side chain cleavage
  • MIS-overexpressing transgenic mice as was the level of serum testosterone and estradiol (Racine et al, Proc. Natl Acad. Sci. USA 95:594-599 (1998), Rouiller- Fabre et ⁇ /., Endocrinology 139 1213-1220 (1998))
  • Correlative PT-PCR results showed that the MIS type II receptor mRNA was present in purified Leydig cells, suggesting that the MIS exterted its observed Leydig cell effects directly via the
  • Ligand specificity within the family is determined by the type II receptor, which, in turn, recruits and phosphorylates the appropriate type I receptor for subsequent downstream signaling via subsets of ligand-specific Smads (Kretzschmar and Massague, Curr. Opm. Genet. Dev.
  • MIS ligand binding to its receptor we are dissecting the role that MIS plays in Leydig cell function and steroidogenesis
  • R2C and MA- 10 we have established a system for studying MIS signal transduction and have been able to show that MIS regulates steroidogenesis at the transcriptional level
  • FCS fetal calf serum
  • R2C a rat Leydig tumor cell line (American Type Culture Collection, Manassas, VA), was maintained in F- 10 medium containing 15% horse serum and 2 5% female FCS (Aires Scientific/Biologos)
  • the mouse MA- 10 Leydig rumor cell line a gift from Dr Mario Ascoli (University of Iowa, Ames, IA), was maintained in Waymouth's MB 752/1 medium (Life Technologies, Inc ) modified to contain 20 mm HEPES (pH 7 4), 50 ⁇ g/ml gentamicin, and 15% horse serum
  • R2C and MA- 10 cells were incubated in a humidified atmosphere with 5% CO 2 at 37 C C and fed every other day until reaching 80-90% confluence
  • Recombinant human MIS was prepared from Chinese hamster ovary (CHO) cells stable transected with a linear construct of the human MIS gene (Cate et al, Cell 45 685-698 (1986)) Active secreted protein in the growth medium was then passed over an immunoaffinity column prepared with the monoclonal MIS antibody, 6E1 1, conjugated to Affi-Gel-10 (Ragin, et al, Protein Expr.
  • Bound MIS was then eluted off the column with 3M ammonium thiocyanate solution, pH 7 4, or 1M acetic acid, pH 3 0 Protein concentrations were determined by Bradford assays after pH neutralization and desalting by centrifugal filtration (Bradford, M M , AnalBwchem. 72 248-254 (1976)) The bioactivity of the MIS was then verified using an established organ culture assay, which grades the regression of the 14 5-day gestation rate urogential ridge (Donohoe et al , J. Surg. Res. 23 141- 148 (1977)) Vehicle control or 105 nM MIS in the presence or absence of
  • the P450cl7 promoter fragment of 1018 bp was amplified by PCR from mouse genomic DNA using 5'-GAGCTCGAGTATTGGCATTGCGTCCC and
  • Sacl/Xhol PGL3B vector was digested with Sacl and Xhol and ligated to the P450cl7 promoter fragment, generating P450c-17- wc
  • the constructs were purified by cesium chloride ultracentrifugation MA- 10 cells were plated at 2xl0 5 in triplicate and were transected with P450c-17- ⁇ -Z «c using the FuGene 6 lipid protocol (Roche Molecular Biochemicals) PGL3B, the promoterless patent plasmid was used as a negative control pRL-TK encoding Ren ⁇ la luciferase (Promega Corp ) under the control of a HSV thymidine kinase promoter was cotransfected as an internal control for transfection efficiency and steroid pathway specificity Cell lysates were made and assayed for luciferase activity using a Berthold Automatic luminometer (Mallac, Inc , Turku, Finland)
  • the MIS ligand binds to the MIS type II receptor to initiate downstream signaling Northern analysis (see FIG 2) was performed to determine whether there was requisite expression of the MIS type II receptor in total RNA isolated from rat testes, R2C cells, MA-10 cells, and purified primary Leydig cells
  • MA- 10 are a mouse Leydig cell tumor line that, in addition, have functional gonadotropin receptors resulting in enhanced steroid production in response to cAMP or LH/hCG, thus mimicking the physiological state of Leydig cells in vivo (Payne and Youngblood, Biol Reprod. 52 217-225 (1995), Ascoli, M , Endocrinology 108 88-95 (1981))
  • FIG 3 A shows that incubation of MA-10 cells with MIS for 2 days resulted in a modest, but significant, 40% reduction in progesterone secretion in both the cAMP-stimulated and unstimulated states
  • FIG 3B shows the concentration of testosterone secreted by MA-10 cells incubated with MIS over a 2-day time course was 10-fold lower than that of cells not treated with MIS
  • the level and time course of steroid hormone reduction were similar to those seen when human follicular cells, harvested at the time of in vivo fertilization, were incubated with recombinant MIS (Kim et al, J. Chn. Endocrinol. Metab. 75 911-
  • FIG 5B shows that MIS incubation on the day after transfection has a slight, but significant, effect on luciferase after 3 h and becomes pronounced after 18 h
  • MA-10 cells when incubated with L9, an inactive form of the MIS ligand that has been mutated so that pro-MIS could not be cleaved to generate the bioactive C-terminal portion of the hormone did not affect luciferase activity as did bioactive, cleavable MIS at the same concentration and time (shown with an asterisk in FIG 5B)
  • L9 is produced in CHO cells and purified in the same manner as wild-type MIS, we can conclude that the effect on P450cl7 transcription is due to MIS and not to a possible copurified contaminant
  • Luciferase expression appears maximal after overnight transfection, as luciferase activity was not significantly different in the control MA-10 cells with 3- to 18-h additional incubation (FIG 5B)
  • FIG 5C 18h after transfection, we observed a 4-fold decrease in luciferase activity with added MIS, which is considerably greater than that observed when MIS was added 1 day later This observation suggests that MIS can more effectively repress expression of the Cyp ⁇ 7 promoter-driver reporter before it is fully activated
  • MIS regulation of steroidogenesis in Leydig cells by transcrptional control of Cyp 17 exposes a conundrum in MIS-mediated suppression of testosterone synthesis that may indicate is developmentally regulated Fetal Leydig cells proliferate and show increased androgen synthesis, which is required for male phenotypic development, independent of gonadotropin stimulation in the rodent (O'Shaughnessy et al , Endocrinology 739:1141-1146 (1998), Kendall et al, Genes Dev. 9 2007-2019 (1995)) We would have expected that these fetal
  • Leydig cells must be refractory to MIS-mediated inhibition of androgen synthesis, because the level of MIS is also high at this time.
  • MIS-overexpressing mice Behringer et al, Nature 345 167-170 (1990)
  • impairment of the testosterone-regulated differentiation of the fetal Wolfian duct which is the precursor of the vas deferens, epididymides, and seminal vesicles, was observed
  • incubation of fetal rat Leydig cells with MIS in vitro results in suppression of testosterone synthesis (Rouiller-Fabre et al.
  • MIS levels reach their nadir (Lee et al, J. Chn. Endocrinol Metab. 81 571-576 (1996), Hudson et al, J. Chn. Endocrinol Metab. 70 16-22 (1990)), and begin producing androgens after stimulation with LH (Saez, J M , Endocr. Rev. 15 574- 626 (1994))
  • LH LH
  • MlS-specific checkpoints in the MIS-mediated down-regulation of testosterone synthesis could be activated and used to lower endogenous testosterone in such clinical settings as prostatic cancer and benign prostatic hypertrophy or to lower elevated steroids in precocious puberty syndromes
  • Current treatments with GnRH long acting analogs to down-regulate the GnRH receptor have been successful in the treatment of central precocious puberty (Hoffman and Crowley,
  • Example 5 MIS Regulates Testosterone In Vivo
  • serum was collected from each of the animals and tested for testosterone concentration using a radioimmunoassay The results are shown in FIG 6 The mean values in each group are shown, and error bars represent SEM, p ⁇ 0 05 These data show that serum testosterone is lowered by MIS administration.
  • Example 6 Acute Administration of MIS Regulates Testosterone In Vivo
  • LH or hCG is administered to animals by intraperitoneal injection.
  • animals are injected intraperitoneally with LH (25 IU) with or without MIS in a single injection.
  • LH 25 IU
  • testes are harvested for preparation of RNA and for histology mRNA expression of steroidogenic enzymes (SCC, 3 ⁇ HSD, Cyp 17, etc.) is analyzed by Northern analysis rhMIS suppresses serum testosterone concentration.
  • LH or hCG is administered to animals via Alzet osmotic minipumps
  • MIS is administered to animals via Alzet osmotic minipumps
  • the effects of chronic MIS exposure is analyzed daily for one week LH levels are measured, since low testosterone will elevate LH, which in turn could increase the testosterone concentration, to compensate for and possibly obscure an MIS effect
  • LH or cAMP and subsequently high testosterone levels may be required to observe the MIS inhibitory effects, as with the cAMP stimulation of MA- 10 cells in vitro If LH is found to compensate for the MIS effect in vivo, the studies are repeated, in either hypophysectomized rats or animals physiologically clamped with a long acting LHRH analog, with subsequent carefully controlled hCG or LH delivery prior to MIS treatment rhMIS suppresses serum testosterone concentration It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.

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Abstract

La présente invention concerne une méthode de traitement d'un état pathologique ou d'une maladie caractérisé par un excès de un ou plusieurs androgènes. Cette méthode consiste à administrer à un patient une dose efficace d'une protéine d'inhibition des canaux de Müller (MIS) ou d'un acide nucléique codant pour ladite protéine MIS. La présente invention concerne également une méthode permettant de faire passer le niveau d'un ou de plusieurs androgènes en deçà du niveau normal, ladite méthode consistant à administrer à un patient une dose efficace d'une protéine MIS ou d'un acide nucléique codant pour ladite protéine MIS. Les méthodes selon la présente invention sont particulièrement indiquées pour le traitement du cancer de la prostate, des ovaires polykystiques, de l'hypertrophie bénigne de la prostate, et de la puberté précoce.
PCT/US2000/025094 1999-09-14 2000-09-14 Utilisation de la substance d'inhibition des canaux de muller pour le traitement des etats pathologiques caracterises par un exces d'androgenes WO2001019387A1 (fr)

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JP2001523019A JP2003509377A (ja) 1999-09-14 2000-09-14 過剰なアンドロゲン状態を処置するためのミューラー阻害物質の使用
EP00963414A EP1218025A4 (fr) 1999-09-14 2000-09-14 Utilisation de la substance d'inhibition des canaux de muller pour le traitement des etats pathologiques caracterises par un exces d'androgenes
AU74830/00A AU7483000A (en) 1999-09-14 2000-09-14 Use of mullerian inhibiting substance for treating excess androgen states

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WO2014164981A1 (fr) * 2013-03-12 2014-10-09 The General Hospital Corporation Protéines de substance d'inhibition mullerienne (mis) modifiées et leurs utilisations pour le traitement de maladies
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US10258668B2 (en) 2013-09-20 2019-04-16 The General Hospital Corporation Viral vectors for expressing a modified mullerian inhibiting substance (MIS) protein
US11135269B2 (en) 2013-12-11 2021-10-05 The General Hospital Corporation Use of mullerian inhibiting substance (MIS) proteins for contraception and ovarian reserve preservation
US11998590B2 (en) 2013-12-11 2024-06-04 The General Hospital Corporation Use of Mullerian inhibiting substance (MIS) proteins for contraception and ovarian reserve preservation
US11518793B2 (en) 2016-12-14 2022-12-06 The General Hospital Corporation Mullerian inhibiting substance (MIS) proteins for ovarian and uterine oncoprotection, and ovarian reserve and uterine preservation

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AU7483000A (en) 2001-04-17
EP1218025A4 (fr) 2005-06-01
CA2384991A1 (fr) 2001-03-22
EP1218025A1 (fr) 2002-07-03
JP2003509377A (ja) 2003-03-11

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