WO1995010298A1 - Method of contraception - Google Patents

Method of contraception Download PDF

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Publication number
WO1995010298A1
WO1995010298A1 PCT/US1994/011588 US9411588W WO9510298A1 WO 1995010298 A1 WO1995010298 A1 WO 1995010298A1 US 9411588 W US9411588 W US 9411588W WO 9510298 A1 WO9510298 A1 WO 9510298A1
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WO
WIPO (PCT)
Prior art keywords
lra
receptor
implantation
compound
antagonist
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PCT/US1994/011588
Other languages
French (fr)
Inventor
Mary Lake Polan
Carlos Simon
Original Assignee
Mary Lake Polan
Carlos Simon
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mary Lake Polan, Carlos Simon filed Critical Mary Lake Polan
Priority to AU80160/94A priority Critical patent/AU8016094A/en
Publication of WO1995010298A1 publication Critical patent/WO1995010298A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/545—IL-1
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour

Definitions

  • the present invention relates to methods and devices interfering with implantation of embryonic stage organisms in the maternal endometrium, and for preventing ovulation.
  • IUD intrauterine device
  • Mifepristone (RU- 486) is an antiprogestin which, when given during the first trimester of pregnancy, stimulates release of prostaglandins in the uterus, stimulates uterine motility and effects detachment from the uterus to cause abortion of the fetus.
  • This compound though an effective abortifactant, causes severe discomfort and may induce uterine bleeding requiring further medical management.
  • RU-486 it has not yet been approved for medical use in the United States.
  • Diethylstilbestrol the so-called “morning after pill” is a synthetic nonsteroidal compound having estrogen agonistic properties.
  • This compound has the disadvantage that if gestation occurs, teratogenic effects such as vaginal adenocarcinoma and uterine malformations may occur in female fetuses. Also, a significant incidence of nausea and vomiting leads some patients to discontinue the medication in the middle of therapy.
  • the invention includes a composition for use in preventing ovulation and implantation of an embryo in a uterus of a mammalian subject, comprising an IL-1 receptor antagonist.
  • the interleukin-1 receptor antagonist may be a peptide, such as interleukin receptor antagonist IL- lra (SEQ ID NO: 2) or icIL-lra (SEQ ID NO: 4), or an oligopeptide selected from a combinatorial library.
  • the compound is selected from a combinatorial library of compounds, by the steps of measuring (i) agonist activity, and (ii) antagonist activity of a library of compounds, in a functional IL-1 receptor assay, and selecting a specific compound in the library if it has (i) substantially no agonist activity, and (ii) substantial antagonist activity.
  • as compound may be selected by (i) reacting compounds from a library with cells having an IL-1 cell-surface receptor, in the presence of a reporter-labeled IL-1, (ii) assaying the ability of library compounds to displace labeled IL-1 from such cell-surface receptors, and (iii) selecting a compound from the library effective to displace labeled IL-1 from the cells.
  • a contraceptive device for use in preventing ovulation and implantation of an embryo or in dislodging an embryo in a mammalian uterus.
  • the device includes a vaginal insert or a cervical cap, and compound release means in said insert or cap for releasing an IL-1 receptor antagonist compound at a dose effective to prevent or disrupt implantation of an embryo in the uterus.
  • the invention includes a method of identifying a compound for use in preventing ovulation and implantation of an embryo or in dislodging an embryo in a mammalian uterus.
  • the method includes measuring (i) agonist activity, and (ii) antagonist activity of a library of compounds, in a functional IL-1 receptor assay, and selecting a specific compound in the library if it has (i) substantially no agonist activity, and (ii) substantial antagonist activity.
  • the method may further include measuring further includes measuring the binding affinity of the compound in an IL-1R tl IL-1 ligand competitive displacement binding assay, and said selecting further includes selecting the compound if its affinity in such binding assay is at least within two orders of magnitude of the binding affinity of an IL-1 receptor antagonist selected from the group consisting of IL-lra and icIL-lra.
  • IL-receptor antagonist compounds used in the methods and compositions described herein have several advantages over known contraceptive methods. They act locally, rather than systemically, resulting in few if any systemic side effects. Moreover, they are effective after the fertilization event and can be used as an alternative to currently practiced post-fertilization means of terminating pregnancy.
  • Figures IA and IB show Northern blots (IA) of IL-1R ti mRNA in cultured human endometrial stromal cells (ESC) , where the migration positions of 18S and 28S ribosomal RNA markers are indicated to the left, and corresponding quantitation of the blots by densitometric analysis (IB) ;
  • Figures 2A and 2B show Northern blots (2A) of IL-1R TI MRNA in cultured human endometrial epithelial cells (EEC) , where marker positions are indicated as in Figure IA, and show corresponding quantitation of the blots by densitometric analysis (2B);
  • Figure 3 (A-E) shows indirect immunofluorescence immunolocalization of mouse IL-1R TI in mouse uterus (3A-3D) and of IL-1/3 (3E) in the mouse embryo;
  • Figure 4 (A-E) shows micrographs of sections of mouse uteri taken at various post-fertilization stages of untreated mice (4A, 4C, 4E) and of mice treated with hr IL-lra (4B, 4D, 4F) ;
  • Figure 5 is a temporal depiction of the human female menstrual cycle, indicating appropriate times in the cycle at which intervention with IL-lra receptor blockade will prevent embryonic implantation;
  • Figure 6 shows DNA and amino acid sequences of IL-lra as SEQ ID NO: 1 and SEQ ID NO: 2, respectively;
  • Figure 7 shows the DNA and amino acid sequences of iCIL-lra as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • endometrium refers to the glandular inner layer of the uterus, overlying a muscular myometrial layer.
  • endometrial stromal cell refers to a specific endometrial cellular component underlying epithelial cells in the uterus.
  • endometrial epithelial cells refers to a specific cellular component of the uterine endometrium. These cells are in contact with the lumen of the uterus.
  • oocyte refers to the immature female egg cell present in the ovaries and having a full diploid complement of chromosomes.
  • ovum refers to the mature female haploid egg cell which has matured from an oocyte by undergoing meiosis. The ovum is released at the time of ovulation from the follicles of the ovaries.
  • ovulation refers to the release by the follicles of one or more ova. Ovulation is characterized in humans by specific hormonal changes; in particular, it occurs just subsequent to a spike in luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels and a decrease in level of estrogen.
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • embryo refers to a fertilized ovum.
  • blastocyst refers to a specific stage of embryo, characterized by the presence of a central cavity surrounded by dividing cells.
  • peri-implantation period refers to a time period shortly before, during and after uterine implantation of the embryo, as described in Section V herein.
  • IL-1 receptor antagonist refers to a compound that blocks the effects of endogenous or exogenously added IL-1. IL-1 receptor antagonists may act competitively or non-competitively at the receptor level.
  • IL-1 inhibitor refers to a compound that blocks IL-1 activity. IL-1 inhibitors include IL-1 receptor antagonists, but also include agents that act by decreasing an amount of endogenous IL-1 available or released.
  • Interleukin-1 receptor antagonist compounds are characterized by an ability to inhibit binding and biological effects of IL-l ⁇ and IL-13, as described in Section III, below.
  • Receptor antagonist compounds include, in addition to competitive inhibitors of receptor binding, other macromolecules, such as antibodies or exogenously added IL-1 binding proteins that interfere with IL-1 binding to its receptor.
  • IL-1 receptor antagonist compounds that are particularly useful in practicing the present invention are those which interfere with the interaction between IL-1 and its receptor(s) in the uterus.
  • Particularly useful compounds include IL- lra, a specific peptide antagonist and derivatives thereof, collectively termed IL-lra peptides. It is the discovery of the present invention that blocking the interaction between embryonic IL-1 and uterine IL-1 receptors, particularly type I receptors, in the mammalian uterus results in disruption of implantation of the embryo in the uterus.
  • IL-l receptor antagonist compounds have been identified and isolated from biological sources including serum, synovial exudates, and urine. Some of these compounds are not specific to the IL-l receptor, since they also inhibit binding and/or activity of other cytokines, notably IL-2. While such non-specific inhibitors may be used in practicing the methods of the invention described herein, antagonist compounds that are specific to the IL-l receptor, and particularly the IL-l type I receptor (IL-1R TI) are generally preferred for use in the invention.
  • IL-1R TI IL-l type I receptor
  • IL-lra is IL-l receptor antagonist that can be used in practicing the invention.
  • IL-lra is a 22-25 kDA peptide which is found in the urine of febrile human patients suffering from monocytic leukemia (Seckinger) , but has also been identified in other tissues, including human monocytes (Eisenberg) .
  • This compound is an antagonist of binding of IL-l ⁇ and IL-1/3 at the IL-l receptor, particularly at the IL-l Type I receptor.
  • IL-lra selectively inhibits binding of IL-l ( ⁇ or ⁇ ) to the
  • IL-l receptor The selectivity of this compound is evidenced by the fact that it does not inhibit biological effects of IL-2 or other cytokines.
  • IL-lra cDNA has been identified and cloned from a monocyte library (Eisenberg) .
  • the complete DNA sequence is shown in Figure 7 a ⁇ SEQ ID NO: 1.
  • an amino acid sequence has been deduced and verified. This sequence is shown in Figure 7 as SEQ ID NO: 2.
  • the cDNA shown encodes a 25 amino acid secretory peptide leader sequence followed by a 152 amino acid polypeptide.
  • IL-lra is generally isolated from natural sources or produced by recombinant methods known in the art (Seckinger; Ausubel) and described in the sections which follow.
  • IL-lra Derived from the same gene, but having an alternate splicing pattern, is an intracellular form of IL-lra, termed icIL-lra (Haskill) .
  • This IL-lra peptide lacks the N-terminal 21 amino acids the 25 residue signal sequence of IL-lra. In their place are three different amino acids.
  • the DNA and peptide sequences are shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively, in Figure 7.
  • IL-lra has been isolated from a number of biological sources, using standard biochemical purification procedures known in the art.
  • One such procedure that has been published in the medical literature by Carter, et al. involves isolation of the peptide from culture supematants of differentiated U937 (myelomonocytic) cells. This method includes ultrafiltration through molecular weight cut-off filters, Superose 12 FPLC, followed by two rounds of TSK Biosil 125 HPLC and C4 RP HPLC (Carter) .
  • This method summarized in Example 6 can be used to obtain IL-lra of sufficient purity for use in the inventive methods described herein.
  • the purification method described by Hannu et al. incorporated herein by reference, can be used to purify IL-lra for use in the methods and compositions of the present invention.
  • IL-lra Human recombinant IL-lra (hr IL-lra) can be expressed in bacterial or mammalian expression systems, according to methods known in the art. In one such method, used to produce IL-lra for the studies reported herein, and detailed in Example 5, IL-lra is expressed as a fusion protein using a pMAL vector (New England Biolabs, Beverly, MA) . This vector is constructed to contain the DNA clone shown as SEQ ID NO: 1 in Figure 6. Alternatively or in addition, the method described by Hannum et al. in U.S. Patent No. 5,075,222, incorporated herein by reference, is used to produce IL-lra
  • the expressed fusion protein is purified from the periplasm of E. coli by Amylose affinity chromatography (New England Biolabs) .
  • the isolated fusion protein is cleaved by Factor Xa treatment to produce hr IL-lra and the carrier maltose binding protein. Homogeneity of hlL-lra is achieved with further purification using, for example, Q-sepharose and Sephacryl S-300 gel filtration chromatographies (Pharmacia) , according to methods known in the art.
  • the purified protein has a molecular mass (Mr) of 17,000. Specific activity of recombinant IL-lra can measured by any of a number of biological assays for ability to block IL-l activity.
  • One such assay is a mouse D10 cell proliferation assay (Polan, et al . ) , detailed in Example 8 and described in Section III below.
  • IL-lra The intracellular form of IL-lra (icIL-lra) is produced by recombinant expression methods, such as the COS cell expression method described in the reference by Haskill, et al. (incorporated herein by reference) .
  • COS cells are transfected with a SR/ ⁇ vector containing a Pst I fragment containing the sequence shown in Figure 7 as SEQ ID NO: 3.
  • Cell lysates are then used for purification of the icIL-lra peptide, essentially as described for IL-lra, above.
  • derivatives of IL-lra can be constructed by making conservative amino acid substitutions into the parent IL-lra primary structure SEQ ID NO: 2, or into the parent icIL-lra structure SEQ ID NO: 4.
  • conservative amino acid substitutions are known in the art to encompass such substitutions as serine for alanine and the like. For example, according to a standard Dayhoff frequency exchange matrix, twenty naturally occurring amino acids are placed in six categories, based on substitutions in nature (Schwartz) . Conservative substitutions can be made between amino acids present in any single class.
  • the six classes are as follows: Class I, CYS; Class II, Ser, Thr, Ala, Gly, representing small aliphatic side chains and OH group side chains; Class III, Asn, Asp, Glu, Gin, representing neutral and negatively charged side chains capable of forming hydrogen bonds; Class IV, His, Arg, Lys, representing basic polar side chains; Class V, lie, Val, Leu, representing branched aliphatic side chains, and Met; Class VI, Phe, Tyr, Trp, representing aromatic side chains.
  • Proline and 4- hydroxyproline are members of Class II; however, substitution of these amino acids for other Class II residues may introduce new secondary structure into the polypeptide backbone.
  • Proline and 4- hydroxyproline may be considered to comprise an additional class, for purposes of conservative substitution.
  • each class may include certain related amino acid analogs, such as ornithine, homoarginine, N-methyl lysine, dimethyl lysine (Class IV) and halogenated tyrosine in Group VI.
  • IL-lra derivatives suitable for use in the present invention will have an IL-l antagonist profile that is substantially like that exhibited by
  • IL-lra as described in Section III, below. That is such derivatives will have very little or no IL-l agonist activity and will have activity profiles that approximate those of IL-lra.
  • approximately is meant that such compounds will have a potency in an IL-l antagonist assay which is minimally within about 2-3 orders of magnitude of the potency of IL- lra or icIL-lra.
  • IL-l receptor antagonist compounds for use in the invention IL-lra derivatives as described above, a practitioner will be guided by the present invention to construct such derivatives according to the conservative substitution guidelines discussed above using methods known in the art, such as by site-specific oligonucleotide-directed mutagenesis techniques (Ausubel et al.; Maniatis et al.) . Available from commercial sources are kits for site-directed mutagenesis (Stratagene, La Jolla, CA) , that can be used in constructing IL-lra peptide derivatives.
  • IL-lra peptide derivatives formed according to one or more of the above techniques are then tested for binding to the IL-l receptor (IL-IR tl) and/or for inhibition of IL-l activity in vitro, according to the methods set forth in Section III, below.
  • Derivative compounds having binding and inhibitory activities, or minimally, functional inhibitory activity, approximating a range defined by the activity of IL-lra and icIL-lra, described below, are also utilizable in the methods and compositions of the present invention.
  • interference with uterine implantation may also be accomplished by other compounds that diminish the biological effects of IL-l in vivo, such as by reducing production of IL-l by the embryo or by stimulating endogenous production of IL-lra.
  • a compound such as zymosan (Tenidap®) , that reduces secretion of endogenous IL-l, may also find use in the present invention.
  • Monoclonal antibodies raised against the mouse type I IL-l receptor have been shown to block immune and inflammatory responses of IL-l at the receptor (Mclntyre) .
  • monoclonal antibodies having such binding and inhibitory properties with respect to the human IL-IR tl can be used in the methods and compositions of the present invention.
  • monoclonal antibodies will be raised against the human IL-IR tl, according to methods well known in the art (Mishell) .
  • Immunogen material for preparing such antibodies will be prepared by biochemical or recombinant techniques as described in Section II, then used to immunize a suitable host animal, including, but not restricted to, mouse, rat, goat, or rabbit. Mice are generally considered preferable for such purposes.
  • Host animals are inoculated with an appropriate amount of a pure or partially pure human IL-IR tl.
  • the amount will depend on the animal used. For mice, an amount of immunogen corresponding to about 1-100 ⁇ q protein will be sufficient for such purposes.
  • Sera are then tested for presence of antibodies, such as in an ELISA test according to standard methods known in the art. Spleens are then removed from animals exhibiting evidence of immunoreactivity with human IL-IR tl. Dispersed spleen cells are then fused with an appropriate fusion partner myeloma cell, using hybridoma production techniques known in the art (Harlow) .
  • Cultures are further tested for presence of immunoglobulin and anti-IL-lR tl activities by ELISA and/or receptor binding assays, such as the receptor binding assay described herein.
  • such antibodies will be "humanized” according to recombinant methods.
  • mouse-derived monoclonal antibody variable regions are combined with human constant regions to produce "humanized” mouse monoclonal antibodies (Taylor) .
  • Such humanized antibodies may be preferred for use in humans, due to their reduced potential for production of non-specific immune reactions.
  • the present invention defines a method for selecting or screening compounds for use as contraceptives.
  • compounds having IL-l receptor antagonism activity that is substantially similar to such activity exhibited by IL-lra will be useful in the methods and compositions of the invention.
  • candidate compounds include, but are not limited to, IL-lra peptide derivative compounds, described above.
  • Other preferred candidate compounds include peptide fragments of IL-lra, as well peptides and other compounds generated by combinatorial libraries, as described below, or a random-sequence peptide library, such as a library using filamentous phage fUSE5 as a vector (Scott; Cwirla) .
  • Combinatorial libraries of oligomers may be formed by a variety of solution-phase or solid-phase methods in which mixtures of different subunits are added stepwise to growing oligomers or parent compound, until a desired oligomer size is reached.
  • a library of increasing complexity can be formed in this manner, for example, by pooling multiple choices of reagents with each additional subunit step (Houghten, 1991) .
  • the library may be formed by solid-phase synthetic methods in which beads containing different-sequence oligomers that form the library are alternately mixed and separated, with one of a selected number of subunits being added to each group of separated beads at each step (Furka, 1991; Lam, 1991, 1993; Zuckermann; Sebestyen) .
  • Still another approach that has been proposed involves the synthesis of a combinatorial library on spatially segregated arrays (Fodor) .
  • the library is a combinatorial library of hexapeptides or heptapeptides, containing all or some defined subset of permutations of amino acids.
  • the approached generally follows published method (Houghten, 1991) .
  • One screening assay for identifying IL-l receptor antagonists from a combinatorial library is based on inhibiting IL-l activity in a physiological setting. Such activities can be assessed in certain in vitro assays known in the art. Described below are representative in vitro assays useful in measuring IL-l activity and blockade thereof. It is appreciated that any of a number of known IL-l activity assays can be used to assess inhibition of IL-l. Such assays may be substituted for the particular assays described below without deviating from the form of the present invention. Alternatively, or in addition, IL-l receptor antagonist compounds in a combinatorial library can be identified on the basis of their ability to displace IL-l from its normal receptor sites on a cell surface.
  • the library compounds may screened for their ability to displace reporter- labeled, e.g., radiolabeled, IL-l ⁇ or IL-23 from the surface of cells having a suitable IL-l surface receptor, such as the EL-4 cells described in Example 7.
  • the assay generally follows the procedure disclosed in this example, for displacement of IL-l ⁇ or IL-1/3.
  • the identity of library compound ⁇ with observed inhibitory and/or binding displacement activity can be determined by conventional means, such a ⁇ iterative synthesis methods in which sublibraries containing known residues in one subunit position only are identified as containing active compounds.
  • IL-l receptor antagonist ⁇ that are u ⁇ eful in the pre ⁇ ent invention are not necessarily competitive antagonists of IL-l at the IL-IR tl, compounds, such a ⁇ IL-lra, which bind competitively are preferred in at lea ⁇ t one embodiment of the invention.
  • a determination that binding i ⁇ competitive, as opposed to non-competitive, may be asses ⁇ ed u ⁇ ing a number of pharmacological tool ⁇ , including, for example, Schild plot regre ⁇ sion analysis of dose-re ⁇ pon ⁇ e curve ⁇ generated from functional a ⁇ ay ⁇ (Kenakin) .
  • a competitive displacement binding as ⁇ ay (Mizel) a competitive displacement binding as ⁇ ay (Mizel) a competitive displacement binding as ⁇ ay (Mizel) .
  • a competitive displacement binding as ⁇ ay (Mizel) a competitive displacement binding as ⁇ ay (Mizel) .
  • intact cells are incubated with 125 I-IL-l ⁇ , and binding to the cells is determined following separation from the cells of unbound 125 I-IL-l ⁇ .
  • competitive binding is evidenced by competitive displacement of bound 125 I-IL-l ⁇ from the cells when the cells are incubated with a test binding compound.
  • IL-lra ha ⁇ approximately the same affinity as does IL-l in binding to EL-4 cells, as well to 3T3 (fibroblast ⁇ ) and CHO cell ⁇ (Mclntyre) .
  • IL-l receptor antagonists preferred for use in the present invention will have affinities for the IL-l binding site, and more specifically, for IL-IR tl, which are sub ⁇ tantially that of IL-lra for the IL- lRti.
  • preferred antagonists will exhibit affinities that are higher than or only ⁇ lightly lower than (K ; within about 2-3 log unit ⁇ ) the affinity of IL-lra for IL-IR tl, when compared in the ⁇ ame binding assay.
  • K a receptor antagonist preferred for use in the present invention
  • a receptor antagonist preferred for use in the present invention will have a K ; that is at least about 10 "7 M.
  • Functional or biological assays provide means of asses ⁇ ing whether a compound ha ⁇ agonist or antagonist activity at the IL-IR tl.
  • Such asses ⁇ ment i ⁇ generally carried out by measuring activity of a known agonist compound in the a ⁇ ay, and comparing to thi ⁇ activity the activity of the agoni ⁇ t in the pre ⁇ ence of te ⁇ t compound.
  • the te ⁇ t compound i ⁇ al ⁇ o evaluated in the functional assay in the absence of the standard known agonist to determine whether it posses ⁇ e ⁇ agoni ⁇ t activity a ⁇ well.
  • preferred antagonists will exhibit little or no agonist activity, relative to antagonist activity.
  • their antagonistic activity will be substantially similar to that of IL-lra, as discussed below.
  • Mouse DIP cellular Proliferation Assay Example 8 details a method for assessing IL-l receptor antagonist activity in an a ⁇ ay in which IL-l ⁇ timulates proliferation of mouse T-helper cells.
  • Human IL-l ⁇ and human IL-l ⁇ stimulate proliferation of cells, mea ⁇ ured by uptake of tritiated thy idine, in ⁇ uch an a ⁇ ay.
  • 30 nM IL-lra wa ⁇ effective to completely inhibit the effect ⁇ of IL-l ⁇ or IL-1/3 when the agoni ⁇ t ⁇ were present at concentrations ranging from about 0.1 pM to approximately 50 nM.
  • IL-l ⁇ or IL-10 is added to the assay at a half-maximal stimulatory concentration (EC50) .
  • concentration of IL-lra or test compound is varied from about 10 "12 M to 10 "* ⁇ , to determine a K t value for the compound in the assay, according to standard procedures (Kenakin) .
  • Compound ⁇ preferred for use in the invention will have a K_ that i ⁇ substantially the same as a K. determined for IL-lra or icIL-lra in the as ⁇ ay. That i ⁇ , they will have a K, that i ⁇ less than about 2-3 log units higher than the K t determined for IL- Ira or icIL-lra in the a ⁇ ay.
  • Certain paracrine cytokine ⁇ are known to be involved in interaction ⁇ between the bla ⁇ tocy ⁇ t and endometrium. For example, secretion by the maternal uterus of the leukemia inhibitory factor (LIF) has been demon ⁇ trated to be required for ⁇ uccessful implantation of the fertilized ovum.
  • Other paracrine cytokines which have been implicated in implantation include maternal colony ⁇ timulating factor-1 (CSF-1) , mutation ⁇ of which have been ⁇ hown to compromi ⁇ e implantation.
  • disruption prevention or dislodgement of embryonic implantation can be effected in vivo by blockade in the maternal endometrium of the endogenou ⁇ cytokine IL-l receptor type I (IL-IR ti) .
  • the interleukin-1 (IL-l) system includes IL-l ⁇ , IL-1/3, IL-l receptor antagonist (IL-lra) and the IL- 1 receptor (IL-l R) .
  • Two IL-l receptor subtypes have been identified. They are termed IL-IR type I (IL-IR tl (Sims)) and IL-IR type II (IL-IR tl (Horuk) ) .
  • Both IL-l ⁇ and IL-1/8 bind to IL-IR tl (Dower) , and their effect on thi ⁇ receptor is antagonized by IL-lra.
  • IL-l and its receptor have both been localized to endometrial cells in mice and humans.
  • IL-l ⁇ and IL-l ⁇ have been shown to be secreted by the human embryo prior to implantation.
  • the agonist IL-l has been used to induce pre-term parturition in mice, and the antagoni ⁇ t IL- lra inhibit ⁇ thi ⁇ abortifactant effect of IL-l (Romero) .
  • an important aspect of the present invention is the discovery that antagonism of IL-lr tl by receptor antagonists is effective to prevent implantation of a fertilized ovum, when IL- lra is present in the uterus during the implantation event, as well as to dislodge such implantation after it has occurred. More generally, according to these finding ⁇ , it can be stated that IL-l receptor antagonism is effective to provide contraception during the peri-implantation period. This period is a time during which the fate of the embryo can be manipulated by hormonal or biochemical mean ⁇ related to the implantation proce ⁇ s.
  • thi ⁇ time period refer ⁇ to the time period during which admini ⁇ tration of IL-lra to the subject can disrupt, that is, prevent or reverse implantation, as described in Section V below.
  • ⁇ tudie ⁇ carried out in ⁇ upport of the pre ⁇ ent invention indicate that IL-l receptor antagonism is effective to prevent ovulation in animals stimulated to ovulate, as described in sub- ⁇ ection D, below.
  • FIGS. 1 and 2 show results of experiments in which human uterine endometrial stromal cells (ESC) and endometrial epithelial cells (EEC) , isolated from ovulatory women in the luteal pha ⁇ e, were cultured and grown in basal conditions in steroid-free media, a ⁇ detailed in Example 1.
  • ESC human uterine endometrial stromal cells
  • EEC endometrial epithelial cells
  • the re ⁇ ulting monolayer ⁇ were maintained for 6 day ⁇ under the growth conditions prescribed by an established model for in vitro decidualization (Irwin) , as detailed in Example 1.
  • one of the following embryonic product ⁇ was added to the endometrial culture medium: human recombinant IL-lb (rIL-lb) , Human Chorionic Gonadotropin (HCG) , Platelet activation factor (PAF) , or conditioned media from human blastocy ⁇ t (HBCM; prepared a ⁇ described in Example 1) .
  • rIL-lb human recombinant IL-lb
  • HCG Human Chorionic Gonadotropin
  • PAF Platelet activation factor
  • HBCM conditioned media from human blastocy ⁇ t
  • Cells were subjected to RNA analysis by Northern blot with human IL-IR tl cRNA probe, according to established procedures, a ⁇ de ⁇ cribed in Example 1.
  • Northern blot data were normalized to a ⁇ t
  • IL-IR tl mRNA and immunoreactive IL-IR tl were found in both human ESC and EEC.
  • human ESC cultured under basal condition ⁇ in the pre ⁇ ence of proge ⁇ terone (P) and epidermal growth factor (EGF) only IL-IR tl mRNA expression was upregulated 2.6-fold after 8 days in culture.
  • P proge ⁇ terone
  • EGF epidermal growth factor
  • IL-IR tl mRNA constitutively expressed at higher at levels than those observed in ESCa.
  • IL-IR tl mRNA was not significantly regulated by any of the embryonic products ob ⁇ erved to increa ⁇ e receptor expre ⁇ ion in ESC ( Figures 2A and 2B) .
  • mice model was u ⁇ ed to study implantation.
  • the validity of the mouse model to the ⁇ tudy of implantation in hu an ⁇ was asses ⁇ ed by determining (a) the pre ⁇ ence in the mouse uterus endometrium of immunoreactive IL-IR tl following pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) administration, and (b) the presence of IL-IR tl in the epithelial region surrounding the bla ⁇ tocyst during the peri- implantation period, specifically at day 4 of pregnancy, since these conditions have been observed in human uterine tissue ⁇ .
  • PMSG pregnant mare's serum gonadotropin
  • hCG human chorionic gonadotropin
  • IL-l ⁇ and IL-1/3 mRNA ⁇ and 11-1 bioactivity in the peri- implantation uteru ⁇ are known to increa ⁇ e from day 3 and peak between day ⁇ 4 and 5, po ⁇ t-fertilization.
  • Bla ⁇ tocyst implantation generally occurs late on post-fertilization day 4 in mice, but may be delayed until about day 8, due to the phenomenon of "delayed implantation" which is apparently inherent to mice.
  • Figure 3 show ⁇ the results of experiments in which immunoreactivity of IL-IR tl and IL-13 were measured in the mouse maternal endometrium and in the mouse placenta, respectively, following fertilization, as detailed in Example 2.
  • IL-IR tl and IL-13 were measured in the mouse maternal endometrium and in the mouse placenta, respectively, following fertilization, as detailed in Example 2.
  • mou ⁇ e IL-IR tl i ⁇ localized in the apical region of the lumenal epithelium and in ⁇ cattered areas of the stroma.
  • Increased IL-IR tl immunoreactivity wa ⁇ observed in the epithelial tis ⁇ ue ⁇ urrounding the blastocyst, as indicated by the arrow in Figure 3D.
  • the female reproductive system comprise ⁇ the ovaries, Fallopian tubes or oviducts, uterus and vagina. Ova mature in the ovarian follicles from oocytes contained within. Mouse and humans ovulate (relea ⁇ e mature ova from the follicles) in respon ⁇ e to approximately the same hormonal stimuli, which include a periodic spike of luteinizing hormone (LH) produced in the pituitary and measurable in the serum. Ovulation in mice occurs spontaneously about every 4 day ⁇ ; in human ⁇ , ovulation occur ⁇ about every 28 days.
  • LH luteinizing hormone
  • the ovum is taken into an oviduct, where it is thought to remain fertile for 10-24 hours in humans and for about 12 hours in mice. Fertilization, if it occurs, generally occurs in the ampulla of the fallopian tube in humans, also known as the oviduct in mice.
  • capacitation usually occurs about 1 hour following ejaculation.
  • Mouse ⁇ perm generally remain viable in the female reproductive tract for about 6 hour ⁇ after ejaculation.
  • Human ⁇ perm generally remain viable for at lea ⁇ t about 48 hours after ejaculation.
  • coitu ⁇ ⁇ hould occur no more than about 48 hour ⁇ before or 24 hours after ovulation.
  • the maturing embryo migrates to the uterus.
  • the embryo undergoe ⁇ maturation through cell divi ⁇ ion.
  • the embryo generally about 2.5-3 day ⁇ po ⁇ t-fertilization in mice and about 4 day ⁇ po ⁇ t-fertilization in human ⁇ , it ha ⁇ progressed through the morula stage to the blastocy ⁇ t ⁇ tage.
  • the embryo generally in morula or blastocy ⁇ t stage, attaches to the uterine wall in a proces ⁇ known a ⁇ implantation.
  • implantation occurs between days about 4 to 7, and particularly 5.5 to 6 after ovulation.
  • mice usually takes place between day 4.5 and day 5 following ovulation, though it can be delayed until about day 8, due to the delayed implantation phenomenon discussed above.
  • a morphologic longitudinal study wa ⁇ carried out to monitor embryonic implantation in the mouse from pregnancy day 4 to day 9 in control, untreated animal ⁇ compared with hr IL-lra treated animals, as described above.
  • Fig. 4 show ⁇ micro ⁇ copic sections of mice uteri made at various days post- fertilization.
  • Figure 4A showing a longitudinal section of the uterus at day 4.
  • the free blastocyst is located within the lumen of the uterus.
  • Figure 4C and Figure 4F showing section ⁇ taken at day ⁇ 7 and 9 of normal pregnancy, re ⁇ pectively, the mouse embryo i ⁇ observed to be completely implanted in the stro a.
  • the ⁇ tudie ⁇ de ⁇ cribed above indicate that embryonic IL-l binding to maternal IL-IR tl i ⁇ required for ⁇ uccessful implantation of the embryo in the uterus, since a specific antagonist of this binding ' prevents the implantation proces ⁇ .
  • the ⁇ e ⁇ tudie ⁇ al ⁇ o provide ba ⁇ i ⁇ for the contention that plentiful di ⁇ tribution of IL-IR tl throughout the lumenal epithelium i ⁇ required for initiation of receptor-ligand interaction initiated wherever the embryo attache ⁇ .
  • IL-IR tl Once the embryo has traversed the epithelium and begins ⁇ tro al invasion, it ⁇ own ⁇ ecretion of IL-l, and perhap ⁇ other paracrine factor ⁇ , induce ⁇ IL-IR tl in the ⁇ urrounding ⁇ troma, allowing implantation to proceed. However, as shown herein, blockage of the IL-IR tl by an antagonist is sufficient to prevent implantation.
  • IL-lra Recombinant human IL-lra was further tested for possible effects on later developmental stages of the mouse embryo, including bla ⁇ tocy ⁇ t hatching, trophobla ⁇ t outgrowth and migration, a ⁇ detailed in Example 4B.
  • Bla ⁇ tocysts were cultured 5 day ⁇ on fibronectin-coated plate ⁇ in the presence of absence of Il-lra, as described in Example 4B. Hatching, outgrowth and migration were found to be similar in the pre ⁇ ence or absence of rh IL-lra.
  • an IL-IR tl antagonist compound will preferably be administered during the post- implantation period, which extend ⁇ from the implantation period to at lea ⁇ t several days following the initial implantation event, as indicated in Figure 5.
  • a further ob ⁇ ervation of the present invention is that IL-l receptor antagonism is effective to inhibit or prevent ovulation in animals stimulated to ovulate.
  • IL-l receptor antagonism is effective to inhibit or prevent ovulation in animals stimulated to ovulate.
  • female rats were primed with gonadotropin. Prior to stimulation with hCG, the animals IL-lra wa ⁇ admini ⁇ tered to the animals, and ovulation wa ⁇ ⁇ ignificantly inhibited.
  • the invention include ⁇ in one a ⁇ pect a method of contraception by prevention of implantation of the fertilized ovum or embryo.
  • Thi ⁇ a ⁇ pect of the invention may al ⁇ o include prevention of ovulation.
  • an IL-l receptor antagoni ⁇ t is delivered to the uteru ⁇ during the time period between coitu ⁇ and the implantation period; that i ⁇ , during a period 1-2 day ⁇ before ovulation or 3-7 day ⁇ after ovulation.
  • the ⁇ ignificance of the ⁇ e time period ⁇ will become more apparent with reference to the temporal event ⁇ depicted in Figure 5, a ⁇ de ⁇ cribed below.
  • IL-l receptor antagoni ⁇ t ⁇ for u ⁇ e in preventing ovulation and embryonic implantation are ⁇ elected a ⁇ described in Sections II and III, above. That is, suitable IL-l receptor antagonist compounds are selected for their ability to (i) bind to the IL-l tl receptor, and/or (ii) antagonize the effects of IL-l ⁇ or IL-13 at the receptor. Such compounds will exhibit a pharmacological profile that is substantially similar to that of IL-lra or icIL-lra, and will have very little or no IL-l agonist activity.
  • IL-lra and derivative ⁇ thereof may be u ⁇ ed in the method of treatment, in accordance with the working example ⁇ de ⁇ cribed in Section IV, above.
  • Figure 5 shows a temporal depiction of the human men ⁇ trual cycle, in which day 1 indicate ⁇ initiation of menstrual flow.
  • ovulation occurs at about day 14 of the cycle, a ⁇ depicted; however, thi ⁇ may vary con ⁇ iderably in and among individual females.
  • it i ⁇ nece ⁇ ary to refer to the day of ovulation such a depiction must be taken as a convenient guideline, rather than a standard.
  • the reference point is preferably the day of ovulation.
  • ovulation occurs ju ⁇ t ⁇ ubsequent to a spike of LH and FSH levels in serum.
  • LH and FSH levels in serum.
  • these and other hormonal or physiological measure ⁇ of ovulation may be measured according to standard methods known in the art.
  • the day of coitus may be used as a reference point, in which case the pre- implantation period will extend to the outer limits shown in Figure 5, as de ⁇ cribed below.
  • the ovum is viable for about 24 hours following ovulation.
  • a fertilization period of 3 day ⁇ (0-3 days post-coitu ⁇ (p.c) ; days 12-15 in the idealized menstrual cycle) i ⁇ indicated in Figure 5.
  • thi ⁇ time will be from about day 3 to about day 11 p.c, a ⁇ uming that coitus may have occurred at any time between day 12 and day 15 of the idealized cycle.
  • Thi ⁇ implantation period i ⁇ indicated a ⁇ day ⁇ 18-22 in the men ⁇ trual cycle.
  • prevention of implantation will be effected by admini ⁇ tration of an IL-l receptor antagoni ⁇ t between about day 3 post-ovulation and about day 7 post-ovulation.
  • day of ovulation is uncertain, it will be a ⁇ sumed that it spans the three day fertilization period shown in Figure 5.
  • compound will preferably be administered between about day 1 and about day 9 p.c. More preferably, the compound will be administered between about day 3 and day 7 p.c.
  • an IL-l receptor antagonist will also be effective to inhibit ovulation, in the case where ovulation has not occurred prior to intercourse, such as during the period 1-2 days before ovulation (days 12-14 in the idealized cycle illustrated in Figure 5) .
  • the compound will be effective if administered to the subject between about day 0 and day 9 post-coitus.
  • compositions The dosage of IL-l receptor antagonist required to disrupt implantation will depend on the specific compound and the route of administration, as described below. Generally, an effective do ⁇ age will be a dosage that provides in the uterus a concentration that is equivalent to a concentration of IL-lra of about 1-50 ⁇ g/ml. In human ⁇ , ⁇ eru concentration ⁇ of about 30 ⁇ g/ml have been achieved by administering an intravenous dose of 10 mg/kg IL- lra (Granowitz) .
  • any mode of administration may be used, so long as it provides sufficient concentration of compound to the uterus, it can be appreciated that certain modes of administration will be preferred in the treatment method.
  • intravenous administration is possible; alternatively, for convenient self- admini ⁇ tration by the patient, other mode ⁇ of admini ⁇ tration, such as nasal insufflation or intravaginal insertion may be preferable.
  • the relative phar acokinetics of ⁇ uch mode ⁇ of admini ⁇ tration are known in the art.
  • the IL-l receptor antagonist will be formulated in an intravaginal suppo ⁇ itory in ⁇ ert having slow release properties, such as are afforded by the formulation described in U.S. Patent No. 5,116,619, incorporated herein by reference.
  • This formulation which includes, in addition to the active ingredient, about 65-85% lactose, 2-4% starch pa ⁇ te, 2-4% corn ⁇ tarch and 0.4-0.6% magne ⁇ ium ⁇ tearate, allow ⁇ for prolonged release of active ingredient over 48-72 hours. It can be appreciated that such a suppository formulation can be dispensed for alternate night self-administration, during the peri-implantation period determined according to the practitioner's understanding of the particular patient' ⁇ ovulatory cycle and the principle ⁇ taught herein.
  • Cervical caps such as the cervical cap described in U.S. Patent No. 4,961,436 incorporated herein by reference, are known in the art, and may be used to deliver over the peri-implantation period, a medicament containing an IL-l receptor antagonist effective to produce in the uterus a concentration of the antagonist equivalent to about 1-50 ⁇ g/ml IL-lra.
  • IL-l receptor antagonist peptides may be administered by transdermal delivery, as through "patch” application.
  • Example 4C describe ⁇ a ⁇ y ⁇ tem ⁇ uitable for transdermal delivery of a peptide formulation suitable for use in the methods of the pre ⁇ ent invention.
  • EEC Endometrial tis ⁇ ue ⁇ samples were minced into small pieces of less than 1 mm, and subjected to mild collagenase dige ⁇ tion a ⁇ follow ⁇ : Tissue pieces were incubated with shaking for 2 hour ⁇ at 37°C, in DMEM (Gibco, Grand Island, NY) containing 0.2% collagenase Type I and 0.005% deoxyribonuclease Type I (Worthington, Freehold, NJ) .
  • ESC and EEC were i ⁇ olated from the mixture by centrifugation, then cultured, and grown to confluence in 75% Dulbecco' ⁇ Modified Eagle' ⁇ Medium (Gibco, Grand I ⁇ land, NY) and 25% MCDB-105 (Sigma, St. Louis, MO) , containing antibiotics, supplemented with 10% serum (Charcoal-Dextran treated FBS, Hyclone, Logan, Utah) and 5 ⁇ g/ml insulin (Sigma, St. Louis, MO) . ESC and EEC were grown in steroid free medium until they were confluent monolayers.
  • Dulbecco' ⁇ Modified Eagle' ⁇ Medium Gibco, Grand I ⁇ land, NY
  • MCDB-105 Sigma, St. Louis, MO
  • ESC and EEC were grown in steroid free medium until they were confluent monolayers.
  • the homogeneity of culture ⁇ wa ⁇ determined by morphological characteristics and verified by immunocytochemical localization of cytokeratin, vimentin, and CD68 antigen as markers for epithelial cells, stromal cells, and human macrophages, respectively.
  • EEC culture ⁇ contained le ⁇ than 3% of ESC and le ⁇ than 0.1% of macrophage ⁇ . After cells were confluent (1-2 weeks) , the media was supplemented with 5 ⁇ g/ml ascorbic acid, 10 ⁇ g/ml transferrin and hormones, as indicated below.
  • Monolayers were cultured for 6 days with progesterone (P) (10 nM) and epidermal growth factor (EGF) (3.3 nM) , according to an established model for in vitro decidualization (Irwin) .
  • P progesterone
  • EGF epidermal growth factor
  • HCG human chorionic gonadotropin
  • PAF platelet- activating factor
  • HBCM conditioned media from human blastocyst
  • HBCM is conditioned media (500-1000 ml) from human embryo ⁇ cultured in vitro until bla ⁇ tocyst ⁇ tage.
  • embryo ⁇ were cultured in Human Tubal Fluid medium (HTF Medium, Irvine Scientific, Santa Ana, CA) containing penicillin (0.5 mg/ml) , streptomycin (0.5 mg/ml) and 1% human serum albumin (Fraction V, Irvine Scientific) for approximately 4 to 5 days.
  • HEF Medium Human Tubal Fluid medium
  • streptomycin 0.5 mg/ml
  • human serum albumin Fraction V, Irvine Scientific
  • RNA from cultured endometrial cell ⁇ wa ⁇ analyzed by Northern analy ⁇ is with human IL-IR tl cRNA probe.
  • a 28S rRNA cDNA probe was used to normalize the data a ⁇ according to standard methods
  • the filter ⁇ were hybridized with a cRNA probe for human IL-IR tl (Simm ⁇ ) .
  • IL-IR tl mRNA level ⁇ were normalized to 28S rRNA level ⁇ and expre ⁇ sed as an arbitrary unit, the IL-IR tl mRNA/28S rRNA level at day 0 of treatment. Densitometric analysis of Northern blots of IL-l R tl mRNA from different experiment ⁇ were combined and expre ⁇ ed a ⁇ the mean ⁇ standard deviation (sd) . Protein studies were performed by immunocytochemistry u ⁇ ing both indirect immunofluore ⁇ cence and avidin-biotin method ⁇ (Simon) .
  • FIG. IA and IB shows Northern blots and a bar graph of quantitation of IL-IR tl mRNA in cultured human endometrial stromal cells (ESC) .
  • ESC human endometrial stromal cells
  • Thi ⁇ lane serves as control.
  • IL-IR tl mRNA levels of ESC treated with progesterone and epidermal growth factor (P+EGF) for 6 and 8 days are ⁇ hown in lane ⁇ 2 and 3, respectively.
  • Lanes 4-7 how blots from ESC treated with P+EGF for 6 days, then, treated for 2 additional day ⁇ (until day 8) with media from human blastocy ⁇ t (1 ml total, Lane 4), 200 nM PAF (Lane 5) , IL-lb 10 IU/ml (Lane 6) , or hCG 10 IU/ml (Lane V) .
  • Figure ⁇ 2A and 2B show the same representations described above for Figure ⁇ IA and IB for endometrial epithelial cells (EEC) .
  • EEC cultured until confluence in steroid free-conditions were treated a ⁇ described for ESC, above.
  • Treatments and lanes correspond to tho ⁇ e de ⁇ cribed above for ESC treatment.
  • Example 2 Immunodetection of IL-IR tl and IL-l ⁇
  • PMSG pregnant mare serum gonadotropin
  • Figure 3 show ⁇ the re ⁇ ult ⁇ of ⁇ tudie ⁇ u ⁇ ing indirect immunofluore ⁇ cence to localize of immunoreactive endometrial mouse IL-IR tl (A-D) and immunoreactive IL-l ⁇ (E) in the mouse embryo.
  • FIG. 3 (A-E) ⁇ how ⁇ results
  • A Mouse uterus obtained 24 h. after pregnant mare serum gonadotropin (PMSG) injection (5 IU) ; mouse IL-IR tl is localized in the apical region of the lumenal epithelium and in scattered areas of the stroma;
  • PMSG pregnant mare serum gonadotropin
  • Figure 3E show ⁇ immunolocalization of mou ⁇ e embryonic IL-lb, at day 9 of pregnancy with twin embryo ⁇ (indicated at "e” in the figure) immunoreactive IL-lb i ⁇ pre ⁇ ent in the mouse placenta (indicated at "p” in the figure) with increased staining at the interface with the maternal endometrium (indicated at "m” in the figure) ; (x400) .
  • Monoclonal antibodie ⁇ again ⁇ t mou ⁇ e IL-lb and the extracellular domain of the mou ⁇ e IL-IR tl were acquired from Genzyme (Cambridge, MA) .
  • Example 3 Inhibition of Embryonic Implantation in vivo
  • PMSG Pregnant Mare' ⁇ Serum Gonadotropin
  • hCG human chorionic gonadotropin
  • Females with vaginal plugs (de ⁇ ignated pregnancy day 1) were randomly allocated to three group ⁇ : A, Control non-injected; B, Buffer injected animals and C, animals injected i.p. with 20 ⁇ g of recombinant human IL-lra (rh IL-lra) every 12 hours beginning on pregnancy day 3 and ending on day 9. Intraperitoneal injections were carefully directed away from the pregnant uterine horns. Injections were continued until day 9 to avoid the pos ⁇ ibility of delayed implantation, and animals were sacrificed 12 hours after the last injection. Animals appeared healthy throughout the experiment. Implantation site ⁇ in both uterine horn ⁇ in IL-lra injected animals were counted and compared with control animals receiving buffer or no injection.
  • Re ⁇ ult ⁇ are shown in Figure 4 (A-F) .
  • Micrographs from untreated animals are shown in Figures 4A, 4C, and 4E.
  • Human recombinant IL-lra treated animals are ⁇ hown in figure ⁇ 4B, 4D, and 4F.
  • Figure 4A ⁇ how ⁇ day 4 normal pregnancy; longitudinal ⁇ ection of a free blastocyst surrounded by enlarged uterine lumenal epithelial cell ⁇ [He atoxylin/Eo ⁇ in (H & E) , original magnification x400] .
  • Figure 4B shows Day 4 pregnant animals injected with IL-lra; Cross-section of a free blastocy ⁇ t.
  • Figure 4E show ⁇ the uteru ⁇ of day 9 normal pregnant mouse; longitudinal section through embryo and decidual mass (H & E x200) .
  • Figure 4F show ⁇ the uteru ⁇ of day 9 of pregnancy, IL-lra injected mouse; stromal decidual reaction (arrows) with intact glandular epithelium and degenerated trophoblastic cells in the uterine lumen (arrowhead) can be ob ⁇ erved, but implantation did not occur in thi ⁇ animal (H & E x800) .
  • Thi ⁇ is a section from the single IL-lra injected mouse described in Table 1 that was macro ⁇ copically pregnant.
  • mice preferably hairles ⁇ guinea pig ⁇ or mice (subjected to a depilatory agent) are mated, and examined for evidence of pregnancy as described in Part A, above. Animals are then fitted with a transdermal patch sy ⁇ tem, according to method ⁇ described by Kumar, et al. (J. Controlled Release 18 . : 213-220, 1992) , incorporated herein by reference. Briefly, the patch consi ⁇ t ⁇ of a die cut re ⁇ ervoir ring of clo ⁇ ed cell polyethylene foam having contact adhesive on one side. The ring is fitted one side with a Millipore GVWP hydrophilic filter.
  • the ring and the filter join to form an anode reservoir, into which is placed a gel containing IL-l receptor antagonist.
  • the gel con ⁇ i ⁇ t ⁇ of Polyjel HV (polyglyceryl citrate + hydroxypropyl methyl cellulose; Guardian Chemical, Hauppauge, NY) mixed with acetate buffer (0.05 M, pH 5.8) into which is mixed the peptide at about 0.1% (wt/wt) .
  • the reservoir is clo ⁇ ed with a tran ⁇ parent polyethylene film having contact adhe ⁇ ive on one side, into which is embedded a stainle ⁇ ⁇ teel ⁇ nap button electrode.
  • a cathode patch consi ⁇ ting of a karaya gumpad (Iomed, Inc., Salt Lake City, UT) i ⁇ used.
  • the patch is connected to a pulsed voltage output power source with a current limiting feature.
  • Animals are treated using a current limit of 0.17 mA/cm 2 at 50 KHz and 50% duty cycle or 0.85 mA for a total area of 5 cm 2 , for 5 hours. Blood i ⁇ sampled for presence of the peptide, to determine the delivered dose. Control animals are given the carrier gel, without IL-l receptor antagonist compound.
  • Example 5 Recombinant Expression of IL-lra Plasmids containing the human IL-l receptor antagonist (hlL-lra) gene were obtained Immunex Corporation, Seattle, WA.
  • the gene sequence of the insert is shown as SEQ ID NO: 1 in Figure 6 herein.
  • the gene can be synthesized using an oligonucleotide synthesizer, or obtained by the isolation/synthetic method described in the reference by Carter, et al., incorporated herein by reference, and verified, using the sequence shown as SEQ ID NO: 1.
  • primers ba ⁇ ed on thi ⁇ ⁇ equence can be u ⁇ ed for preparation of the gene sequence, according to standard procedures (Ausubel) .
  • E. coli human IL-lra wa ⁇ expre ⁇ ed a ⁇ a fu ⁇ ion protein u ⁇ ing the pMAL vector (New England Biolab ⁇ ) .
  • the expre ⁇ ed fu ⁇ ion protein wa ⁇ detected and purified from the peripla ⁇ m of E . coli by Amylo ⁇ e affinity chromatography (New England Biolab ⁇ ) .
  • the amylose eluate pool was dialyzed into 50 mM Tris-HCL, 100 mM NaCl pH 7.5 and cleaved with Factor Xa (Enzyme Research Labs) at a concentration of 10 ng to 50 ⁇ g of fusion protein.
  • Example 6 Purification of IL-lra IL-lra i ⁇ purified from cellular sources as described by Carter, et al. as follow ⁇ : U937 cell culture ⁇ are obtained from the American Type Culture Collection, Rockville, MD. Cell ⁇ are cultured at 37°C for 48 h at 5 x 10 5 cells ml "1 in RPMI 1640 medium (GIBCO) , 7% fetal bovine serum (FBS, GIBCO) , 2mM L-glutamine, 100 U ml "1 penicillin, 100 ⁇ g ml "1 streptomycin, 20mM HEPES buffer, and 100 nM phorbol myristate acetate (PMA) (Sigma, St. Loui ⁇ , MO) .
  • RPMI 1640 medium fetal bovine serum
  • PMA phorbol myristate acetate
  • the medium i ⁇ removed and the cell ⁇ , now adherent, are gently wa ⁇ hed once with a ⁇ mall volume of Dulbecco' ⁇ PBS.
  • Cell ⁇ are then cultured for an additional 48 h in RPMI 1640 containing 1% low-endotoxin FBS (HyClone, Logan, UT) , L-glutamine, penicillin, streptomycin, and rhGM-CSF (75 U ml "1 ; Amgen, Torrance, CA) .
  • Cell-free supernatant ⁇ are collected, pooled and frozen at - 20°C After thawing, phenylmethyl ⁇ ulphonyl fluoride is added to 0.2 mM final concentration.
  • the solution ⁇ are concentrated by ultrafiltration with YM-5 and YM-10 filter ⁇ (Amicon) .
  • Protein mea ⁇ urement ⁇ are made u ⁇ ing Bradford assay (Bio-Rad Kit) using BSA a ⁇ the ⁇ tandard protein.
  • TSK Bio- Sil 125 HPLC-1 Pooled samples are then subjected to TSK Bio- Sil 125 HPLC-1, as follows: in 20 repetitive cycle ⁇ , 50 ⁇ l of fraction 3 are injected onto a TSK-Bio-Sil- 125 HPLC column (7.5 x 600 mm), u ⁇ ing the ⁇ ame buffer ⁇ y ⁇ tem a ⁇ described above. Fractions of 200 ⁇ l are collected. Appropriate fractions are pooled and concentrated by Centricon-10 filter unit ⁇ and finally by Speed Vac centrifugation a ⁇ de ⁇ cribed above.
  • Proteins which remain bound to the matrix following an extensive wash with 0.1% trifluoroacetic acid (TFA) are desorbed using a linear gradient from 28 to 32% acetonitrile in 0.1% TFA over a period of 44 min at a flow rate of 1 ml min "1 (room temperature) .
  • Representative aliquots of each fraction or pool are removed, taken to complete drynes ⁇ by Speed Vac centrifugation, and then re-solubilized in tis ⁇ ue culture medium in preparation for assay.
  • Radioiodinated IL-l ⁇ and IL-1/3 are obtained from commercial ⁇ ource ⁇ (DuPont NEN Research
  • EL-4 cells (6xl0 6 /ml; mouse lymphoma cells; ATCC TIB 39, American Type Culture Collection, Rockville, Maryland) are su ⁇ pended in binding medium (RPMI 1640 containing 5% fetal calf serum and 25 mM HEPES, pH 7.2). Aliquots (100 ⁇ l) are placed in 12 x 75 mm polypropylene tubes. *2S I-labelled IL-l is added at a concentration of about 1 xlO" 10 M. Incubation is carried out for 3 hours at 4°C in the pre ⁇ ence of te ⁇ t compound, a ⁇ de ⁇ cribed below.
  • Non ⁇ pecific binding i ⁇ determined by include ⁇ ion of 1 xlO 8 M unlabeled IL-l. Unbound 125 I-IL-1 is separated from cell ⁇ by centrifugation through 200 ⁇ l of a ⁇ ilicone oil mixture in a Beckman microfuge.
  • Di ⁇ placement of binding by te ⁇ t compound i ⁇ determined by adding to the above incubation mixture, varying concentrations of unlabeled test compound ranging from about 10 "12 -10 "8 M compound.
  • Mouse T helper D10.G4.1 cell ⁇ (American Type Culture Collection, Rockland, MD; ATCC TIB 224) were ⁇ u ⁇ pended in culture medium (RPMI 1640 containing 5% fetal calf serum, 5 x 10 "8 M 2-mercaptoethanol, 8 ⁇ g/ml gentamicin, 2 mM L-glutamine, and 2.5 ⁇ g/ml concanavalin A) . Cells were ⁇ u ⁇ pended to produce a cell concentration of about 1-2 x lO ⁇ g/ml. IL-lra wa ⁇ added to triplicate culture ⁇ , 1 hour prior to addition of IL-l ⁇ or IL-10 to the culture ⁇ .
  • hIL-l ⁇ or hIL-lj ⁇ was added in increasing concentrations to the a ⁇ ay, the EC 50 of hIL-l ⁇ wa ⁇ 7 pM and the EC 50 of hIL-1/3 wa ⁇ 450 pM.
  • hlL-lra completely inhibited stimulation of proliferation by hIL-l ⁇ to a concentration of 100 pM and by hIL-l ⁇ to a concentration of about 100 nM.
  • the thymocytes are resu ⁇ pended in RPMI 1640 ti ⁇ ue culture medium (Gibco) containing 50 ⁇ M 2-mercaptoethanol (Fi ⁇ her) , 2 mM glutamine (Gibco) , 1 mM pyruvate, non-essential amino acid solution, penicillin (100 U/ml) streptomycin (lOO ⁇ g/ml) solution, 10% heat inactivated fetal bovine serum and Phytohemaglutinin (PHA, Pharmacia, final concentration 10 ⁇ g/ml) .
  • RPMI 1640 ti ⁇ ue culture medium Gibco
  • PHA Phytohemaglutinin
  • Cells are cultured in round-bottom 96 well microtiter tis ⁇ ue culture plate ⁇ , 6xl0 5 cell ⁇ per well in a volume of 100 ⁇ l. Te ⁇ t compound i ⁇ diluted in tissue culture medium and added to the wells in the presence and ab ⁇ ence of IL-l (recombinant human IL-l, R&D Sy ⁇ tem ⁇ catalog # 201-LB, 0.1 ng/ml) . Total volume is 150 ⁇ l per well. Plates are incubated for 72 hour ⁇ (95% air/5% C0 2 , 37°C) . During the last four hours of incubation, tritiated thymidine (Amersham, 49 Ci/mM) is added (0.5 ⁇ Ci per well).
  • Untreated cell ⁇ ⁇ how minimal DNA ⁇ ynthe ⁇ i ⁇ (thymidine incorporation 80 cpm/well) .
  • PHA alone produce ⁇ a 2-3 fold stimulation of cell proliferation.
  • Treatment with 0.1 ng/ml IL-l in the presence of PHA results in 60 fold ⁇ timulation.
  • mice were given IL-l are injected with IL-lra (5 mg/kg, ⁇ ubcutaneou ⁇ ly, ( ⁇ .c. ) ) or te ⁇ t receptor antagonist. Mice are immediately given IL-l ⁇ (5 ⁇ g/kg ⁇ .c) . Blood samples from the mice are taken 3 hours later.
  • Serum levels of IL-6 are determined using a modification of the B9 hybridoma cell as ⁇ ay de ⁇ cribed by Aarden et al. and incorporated herein by reference.
  • B9 cell ⁇ are treated with twofold serial dilutions of the test sera in 96-well microtiter plates. After a 3-day incubation at 37° in a humidified atmo ⁇ phere of 5% C0 2 and 95% air, the wells are pulsed with 0.5 ⁇ Ci of [ 3 H]thymidine ([ 3 H]TdR) and incubated for an additional 18 hours. The cells are then harvested onto glass fiber filters and the level of [ 3 H]TdR incorporation determined in a liquid scintillation counter.
  • IL-6 units are defined as the inverse of the serum dilution that produces half-maximal [ 3 H]TdR incorporation compared with a reference ⁇ tandard.

Abstract

Method of contraception utilizing IL-1 receptor antagonists are disclosed. In one embodiment, the method includes preventing ovulation and implantation of an embryo by administering to a subject a pharmacologically effective dose of an interleukin-1 (IL-1) receptor antagonist during the pre-implantation period. In a related method, the IL-1 receptor antagonist dislodges the implanted embryo, when it is administered during the post-implantation period. Also disclosed are contraceptive compositions, including IL-1 receptor antagonists, for use in the methods of the invention. Further disclosed is a method of selecting compounds for use in the contraceptive methods of the invention.

Description

METHOD OF CONTRACEPTION
Field of the Invention
The present invention relates to methods and devices interfering with implantation of embryonic stage organisms in the maternal endometrium, and for preventing ovulation.
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Background of the Invention
The search for convenient and reliable means of birth control is one which occupies a significant portion of the U.S. pharmaceutical industry. Public reluctance to accept abortion as a means of eliminating unwanted pregnancies has resulted in intensified efforts to provide alternative methods of post-coital birth control. Currently the most widely used methods of birth control are those which prevent fertilization of the ovum. Known preventive methods currently in use include those which prevent conception by physical means (condoms) , combined physical and chemical barriers (e.g., diaphragms and spermicides) or estrogen-based contraceptive formulations. Prevention of pregnancy may also be effected by insertion in the uterus of an intrauterine device (IUD) , which may include a chemical component, such as the hormone progesterone. Although the foregoing methods of birth control are generally reliable when used correctly, they suffer from the disadvantage that a certain amount of planning must be taken on the part of at least one of the sexual partners prior to sexual intercourse.
As stated above, public attitudes in the U.S. have deterred widespread use of post-coital forms of birth control, or abortifactants. Mifepristone (RU- 486) is an antiprogestin which, when given during the first trimester of pregnancy, stimulates release of prostaglandins in the uterus, stimulates uterine motility and effects detachment from the uterus to cause abortion of the fetus. This compound, though an effective abortifactant, causes severe discomfort and may induce uterine bleeding requiring further medical management. Moreover, RU-486 it has not yet been approved for medical use in the United States.
Currently, there is only one formulation that is approved for prevention of pregnancy in those women who have had unprotected sexual intercourse.
Diethylstilbestrol, the so-called "morning after pill", is a synthetic nonsteroidal compound having estrogen agonistic properties. This compound has the disadvantage that if gestation occurs, teratogenic effects such as vaginal adenocarcinoma and uterine malformations may occur in female fetuses. Also, a significant incidence of nausea and vomiting leads some patients to discontinue the medication in the middle of therapy.
Summary of the Invention In one aspect, the invention includes a composition for use in preventing ovulation and implantation of an embryo in a uterus of a mammalian subject, comprising an IL-1 receptor antagonist.
The interleukin-1 receptor antagonist may be a peptide, such as interleukin receptor antagonist IL- lra (SEQ ID NO: 2) or icIL-lra (SEQ ID NO: 4), or an oligopeptide selected from a combinatorial library. In the latter case, the compound is selected from a combinatorial library of compounds, by the steps of measuring (i) agonist activity, and (ii) antagonist activity of a library of compounds, in a functional IL-1 receptor assay, and selecting a specific compound in the library if it has (i) substantially no agonist activity, and (ii) substantial antagonist activity. Alternatively, as compound may be selected by (i) reacting compounds from a library with cells having an IL-1 cell-surface receptor, in the presence of a reporter-labeled IL-1, (ii) assaying the ability of library compounds to displace labeled IL-1 from such cell-surface receptors, and (iii) selecting a compound from the library effective to displace labeled IL-1 from the cells.
Also forming part of the invention is a contraceptive device for use in preventing ovulation and implantation of an embryo or in dislodging an embryo in a mammalian uterus. The device includes a vaginal insert or a cervical cap, and compound release means in said insert or cap for releasing an IL-1 receptor antagonist compound at a dose effective to prevent or disrupt implantation of an embryo in the uterus.
In another aspect, the invention includes a method of identifying a compound for use in preventing ovulation and implantation of an embryo or in dislodging an embryo in a mammalian uterus. The method includes measuring (i) agonist activity, and (ii) antagonist activity of a library of compounds, in a functional IL-1 receptor assay, and selecting a specific compound in the library if it has (i) substantially no agonist activity, and (ii) substantial antagonist activity. The method may further include measuring further includes measuring the binding affinity of the compound in an IL-1R tl IL-1 ligand competitive displacement binding assay, and said selecting further includes selecting the compound if its affinity in such binding assay is at least within two orders of magnitude of the binding affinity of an IL-1 receptor antagonist selected from the group consisting of IL-lra and icIL-lra.
IL-receptor antagonist compounds used in the methods and compositions described herein have several advantages over known contraceptive methods. They act locally, rather than systemically, resulting in few if any systemic side effects. Moreover, they are effective after the fertilization event and can be used as an alternative to currently practiced post-fertilization means of terminating pregnancy.
Brief Description of the Figures Figures IA and IB show Northern blots (IA) of IL-1R ti mRNA in cultured human endometrial stromal cells (ESC) , where the migration positions of 18S and 28S ribosomal RNA markers are indicated to the left, and corresponding quantitation of the blots by densitometric analysis (IB) ;
Figures 2A and 2B show Northern blots (2A) of IL-1R TI MRNA in cultured human endometrial epithelial cells (EEC) , where marker positions are indicated as in Figure IA, and show corresponding quantitation of the blots by densitometric analysis (2B); Figure 3 (A-E) shows indirect immunofluorescence immunolocalization of mouse IL-1R TI in mouse uterus (3A-3D) and of IL-1/3 (3E) in the mouse embryo; Figure 4 (A-E) shows micrographs of sections of mouse uteri taken at various post-fertilization stages of untreated mice (4A, 4C, 4E) and of mice treated with hr IL-lra (4B, 4D, 4F) ;
Figure 5 is a temporal depiction of the human female menstrual cycle, indicating appropriate times in the cycle at which intervention with IL-lra receptor blockade will prevent embryonic implantation; and
Figure 6 shows DNA and amino acid sequences of IL-lra as SEQ ID NO: 1 and SEQ ID NO: 2, respectively; and
Figure 7 shows the DNA and amino acid sequences of iCIL-lra as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
Detailed Description of the Invention I. Definitions
The term "endometrium", as used herein refers to the glandular inner layer of the uterus, overlying a muscular myometrial layer.
The term "endometrial stromal cell" (abbreviated "ESC") refers to a specific endometrial cellular component underlying epithelial cells in the uterus. The term "endometrial epithelial cells" (abbreviated "EEC") , refers to a specific cellular component of the uterine endometrium. These cells are in contact with the lumen of the uterus.
The term "oocyte" refers to the immature female egg cell present in the ovaries and having a full diploid complement of chromosomes. The term "ovum" refers to the mature female haploid egg cell which has matured from an oocyte by undergoing meiosis. The ovum is released at the time of ovulation from the follicles of the ovaries. The term "ovulation" refers to the release by the follicles of one or more ova. Ovulation is characterized in humans by specific hormonal changes; in particular, it occurs just subsequent to a spike in luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels and a decrease in level of estrogen.
The term "embryo" refers to a fertilized ovum.
The term "blastocyst" refers to a specific stage of embryo, characterized by the presence of a central cavity surrounded by dividing cells.
The term "peri-implantation" period, refers to a time period shortly before, during and after uterine implantation of the embryo, as described in Section V herein. The term "IL-1 receptor antagonist" refers to a compound that blocks the effects of endogenous or exogenously added IL-1. IL-1 receptor antagonists may act competitively or non-competitively at the receptor level. The term "IL-1 inhibitor" refers to a compound that blocks IL-1 activity. IL-1 inhibitors include IL-1 receptor antagonists, but also include agents that act by decreasing an amount of endogenous IL-1 available or released.
II. Interleukin-1 Receptor Antagonists
Interleukin-1 receptor antagonist compounds are characterized by an ability to inhibit binding and biological effects of IL-lα and IL-13, as described in Section III, below. Receptor antagonist compounds include, in addition to competitive inhibitors of receptor binding, other macromolecules, such as antibodies or exogenously added IL-1 binding proteins that interfere with IL-1 binding to its receptor.
IL-1 receptor antagonist compounds that are particularly useful in practicing the present invention are those which interfere with the interaction between IL-1 and its receptor(s) in the uterus. Particularly useful compounds include IL- lra, a specific peptide antagonist and derivatives thereof, collectively termed IL-lra peptides. It is the discovery of the present invention that blocking the interaction between embryonic IL-1 and uterine IL-1 receptors, particularly type I receptors, in the mammalian uterus results in disruption of implantation of the embryo in the uterus.
A. IL-l Receptor Antagonists
Several naturally occurring IL-l receptor antagonist compounds have been identified and isolated from biological sources including serum, synovial exudates, and urine. Some of these compounds are not specific to the IL-l receptor, since they also inhibit binding and/or activity of other cytokines, notably IL-2. While such non- specific inhibitors may be used in practicing the methods of the invention described herein, antagonist compounds that are specific to the IL-l receptor, and particularly the IL-l type I receptor (IL-1R TI) are generally preferred for use in the invention.
B. IL-lra peptides
IL-lra is IL-l receptor antagonist that can be used in practicing the invention. IL-lra is a 22-25 kDA peptide which is found in the urine of febrile human patients suffering from monocytic leukemia (Seckinger) , but has also been identified in other tissues, including human monocytes (Eisenberg) . This compound is an antagonist of binding of IL-lα and IL-1/3 at the IL-l receptor, particularly at the IL-l Type I receptor.
As is described in Section III, below, IL-lra selectively inhibits binding of IL-l (α or β) to the
IL-l receptor. The selectivity of this compound is evidenced by the fact that it does not inhibit biological effects of IL-2 or other cytokines.
IL-lra cDNA has been identified and cloned from a monocyte library (Eisenberg) . The complete DNA sequence is shown in Figure 7 aε SEQ ID NO: 1.
Based on the cDNA sequence, an amino acid sequence has been deduced and verified. This sequence is shown in Figure 7 as SEQ ID NO: 2. The cDNA shown encodes a 25 amino acid secretory peptide leader sequence followed by a 152 amino acid polypeptide.
For use in the present invention, IL-lra is generally isolated from natural sources or produced by recombinant methods known in the art (Seckinger; Ausubel) and described in the sections which follow.
Derived from the same gene, but having an alternate splicing pattern, is an intracellular form of IL-lra, termed icIL-lra (Haskill) . This IL-lra peptide lacks the N-terminal 21 amino acids the 25 residue signal sequence of IL-lra. In their place are three different amino acids. The DNA and peptide sequences are shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively, in Figure 7.
1. Isolation of IL-lra peptides a. Purification of IL-lra from Biological Sources.
IL-lra has been isolated from a number of biological sources, using standard biochemical purification procedures known in the art. One such procedure that has been published in the medical literature by Carter, et al. involves isolation of the peptide from culture supematants of differentiated U937 (myelomonocytic) cells. This method includes ultrafiltration through molecular weight cut-off filters, Superose 12 FPLC, followed by two rounds of TSK Biosil 125 HPLC and C4 RP HPLC (Carter) . This method, summarized in Example 6) can be used to obtain IL-lra of sufficient purity for use in the inventive methods described herein. Alternatively, the purification method described by Hannu et al., incorporated herein by reference, can be used to purify IL-lra for use in the methods and compositions of the present invention.
b . Recombinant Production of IL-lra. Human recombinant IL-lra (hr IL-lra) can be expressed in bacterial or mammalian expression systems, according to methods known in the art. In one such method, used to produce IL-lra for the studies reported herein, and detailed in Example 5, IL-lra is expressed as a fusion protein using a pMAL vector (New England Biolabs, Beverly, MA) . This vector is constructed to contain the DNA clone shown as SEQ ID NO: 1 in Figure 6. Alternatively or in addition, the method described by Hannum et al. in U.S. Patent No. 5,075,222, incorporated herein by reference, is used to produce IL-lra
The expressed fusion protein is purified from the periplasm of E. coli by Amylose affinity chromatography (New England Biolabs) . The isolated fusion protein is cleaved by Factor Xa treatment to produce hr IL-lra and the carrier maltose binding protein. Homogeneity of hlL-lra is achieved with further purification using, for example, Q-sepharose and Sephacryl S-300 gel filtration chromatographies (Pharmacia) , according to methods known in the art. The purified protein has a molecular mass (Mr) of 17,000. Specific activity of recombinant IL-lra can measured by any of a number of biological assays for ability to block IL-l activity. One such assay is a mouse D10 cell proliferation assay (Polan, et al . ) , detailed in Example 8 and described in Section III below.
2. Isolation of icIL-lra
The intracellular form of IL-lra (icIL-lra) is produced by recombinant expression methods, such as the COS cell expression method described in the reference by Haskill, et al. (incorporated herein by reference) . According to this method, COS cells are transfected with a SR/α vector containing a Pst I fragment containing the sequence shown in Figure 7 as SEQ ID NO: 3. Cell lysates are then used for purification of the icIL-lra peptide, essentially as described for IL-lra, above.
C Derivatives of IL-lra Peptides
In accordance with known principles of conservative amino acid substitutions in a protein, it can be appreciated that derivatives of IL-lra can be constructed by making conservative amino acid substitutions into the parent IL-lra primary structure SEQ ID NO: 2, or into the parent icIL-lra structure SEQ ID NO: 4. Such conservative amino acid substitutions are known in the art to encompass such substitutions as serine for alanine and the like. For example, according to a standard Dayhoff frequency exchange matrix, twenty naturally occurring amino acids are placed in six categories, based on substitutions in nature (Schwartz) . Conservative substitutions can be made between amino acids present in any single class. According to this scheme, the six classes are as follows: Class I, CYS; Class II, Ser, Thr, Ala, Gly, representing small aliphatic side chains and OH group side chains; Class III, Asn, Asp, Glu, Gin, representing neutral and negatively charged side chains capable of forming hydrogen bonds; Class IV, His, Arg, Lys, representing basic polar side chains; Class V, lie, Val, Leu, representing branched aliphatic side chains, and Met; Class VI, Phe, Tyr, Trp, representing aromatic side chains. Proline and 4- hydroxyproline are members of Class II; however, substitution of these amino acids for other Class II residues may introduce new secondary structure into the polypeptide backbone. Proline and 4- hydroxyproline may be considered to comprise an additional class, for purposes of conservative substitution. Furthermore, each class may include certain related amino acid analogs, such as ornithine, homoarginine, N-methyl lysine, dimethyl lysine (Class IV) and halogenated tyrosine in Group VI.
IL-lra derivatives suitable for use in the present invention will have an IL-l antagonist profile that is substantially like that exhibited by
IL-lra, as described in Section III, below. That is such derivatives will have very little or no IL-l agonist activity and will have activity profiles that approximate those of IL-lra. By "approximate" is meant that such compounds will have a potency in an IL-l antagonist assay which is minimally within about 2-3 orders of magnitude of the potency of IL- lra or icIL-lra.
Thus in selecting as IL-l receptor antagonist compounds for use in the invention IL-lra derivatives as described above, a practitioner will be guided by the present invention to construct such derivatives according to the conservative substitution guidelines discussed above using methods known in the art, such as by site-specific oligonucleotide-directed mutagenesis techniques (Ausubel et al.; Maniatis et al.) . Available from commercial sources are kits for site-directed mutagenesis (Stratagene, La Jolla, CA) , that can be used in constructing IL-lra peptide derivatives.
In accordance with the invention, IL-lra peptide derivatives formed according to one or more of the above techniques are then tested for binding to the IL-l receptor (IL-IR tl) and/or for inhibition of IL-l activity in vitro, according to the methods set forth in Section III, below. Derivative compounds having binding and inhibitory activities, or minimally, functional inhibitory activity, approximating a range defined by the activity of IL-lra and icIL-lra, described below, are also utilizable in the methods and compositions of the present invention.
It is also appreciated that in accordance with the discovery of the present invention interference with uterine implantation may also be accomplished by other compounds that diminish the biological effects of IL-l in vivo, such as by reducing production of IL-l by the embryo or by stimulating endogenous production of IL-lra. Thus, a compound such as zymosan (Tenidap®) , that reduces secretion of endogenous IL-l, may also find use in the present invention.
D. Monoclonal antibodies against IL-l Receptor
Monoclonal antibodies raised against the mouse type I IL-l receptor have been shown to block immune and inflammatory responses of IL-l at the receptor (Mclntyre) . In accordance with the present invention, monoclonal antibodies having such binding and inhibitory properties with respect to the human IL-IR tl, can be used in the methods and compositions of the present invention.
Preferably, monoclonal antibodies will be raised against the human IL-IR tl, according to methods well known in the art (Mishell) . Immunogen material for preparing such antibodies will be prepared by biochemical or recombinant techniques as described in Section II, then used to immunize a suitable host animal, including, but not restricted to, mouse, rat, goat, or rabbit. Mice are generally considered preferable for such purposes.
Host animals are inoculated with an appropriate amount of a pure or partially pure human IL-IR tl. The amount will depend on the animal used. For mice, an amount of immunogen corresponding to about 1-100 μq protein will be sufficient for such purposes. Sera are then tested for presence of antibodies, such as in an ELISA test according to standard methods known in the art. Spleens are then removed from animals exhibiting evidence of immunoreactivity with human IL-IR tl. Dispersed spleen cells are then fused with an appropriate fusion partner myeloma cell, using hybridoma production techniques known in the art (Harlow) . Cultures are further tested for presence of immunoglobulin and anti-IL-lR tl activities by ELISA and/or receptor binding assays, such as the receptor binding assay described herein. Preferably, for use in humans, such antibodies will be "humanized" according to recombinant methods. For example, according to one such method, mouse-derived monoclonal antibody variable regions are combined with human constant regions to produce "humanized" mouse monoclonal antibodies (Taylor) . Such humanized antibodies may be preferred for use in humans, due to their reduced potential for production of non-specific immune reactions.
III. Method of Screening for IL-l Receptor Antagonists
In accordance with the discoveries described herein, it can be appreciated that the present invention defines a method for selecting or screening compounds for use as contraceptives. In this regard, compounds having IL-l receptor antagonism activity that is substantially similar to such activity exhibited by IL-lra will be useful in the methods and compositions of the invention.
It can be further appreciated that candidate compounds include, but are not limited to, IL-lra peptide derivative compounds, described above. Other preferred candidate compounds include peptide fragments of IL-lra, as well peptides and other compounds generated by combinatorial libraries, as described below, or a random-sequence peptide library, such as a library using filamentous phage fUSE5 as a vector (Scott; Cwirla) .
A. Combinatorial Libraries
A variety of combinatorial libraries of random- sequence oligonucleotideε, polypeptides, or synthetic oligomers have been proposed (Kramer;
Houghten, 1985, 1986, 1991, 1992; Ohlmeyer; Dooley, 1993a-1993b; Eichler; Pinella, 1992, 1993; Ecker; and Barbas) . A number of small-molecule libraries have also been developed (e.g., Ellman) . Combinatorial libraries of oligomers may be formed by a variety of solution-phase or solid-phase methods in which mixtures of different subunits are added stepwise to growing oligomers or parent compound, until a desired oligomer size is reached. A library of increasing complexity can be formed in this manner, for example, by pooling multiple choices of reagents with each additional subunit step (Houghten, 1991) .
Alternatively, the library may be formed by solid-phase synthetic methods in which beads containing different-sequence oligomers that form the library are alternately mixed and separated, with one of a selected number of subunits being added to each group of separated beads at each step (Furka, 1991; Lam, 1991, 1993; Zuckermann; Sebestyen) . Still another approach that has been proposed involves the synthesis of a combinatorial library on spatially segregated arrays (Fodor) .
In one preferred embodiment, the library is a combinatorial library of hexapeptides or heptapeptides, containing all or some defined subset of permutations of amino acids. The approached generally follows published method (Houghten, 1991) .
One screening assay for identifying IL-l receptor antagonists from a combinatorial library is based on inhibiting IL-l activity in a physiological setting. Such activities can be assessed in certain in vitro assays known in the art. Described below are representative in vitro assays useful in measuring IL-l activity and blockade thereof. It is appreciated that any of a number of known IL-l activity assays can be used to assess inhibition of IL-l. Such assays may be substituted for the particular assays described below without deviating from the form of the present invention. Alternatively, or in addition, IL-l receptor antagonist compounds in a combinatorial library can be identified on the basis of their ability to displace IL-l from its normal receptor sites on a cell surface. For example, the library compounds may screened for their ability to displace reporter- labeled, e.g., radiolabeled, IL-lα or IL-23 from the surface of cells having a suitable IL-l surface receptor, such as the EL-4 cells described in Example 7. The assay generally follows the procedure disclosed in this example, for displacement of IL-lα or IL-1/3. The identity of library compoundε with observed inhibitory and/or binding displacement activity can be determined by conventional means, such aε iterative synthesis methods in which sublibraries containing known residues in one subunit position only are identified as containing active compounds.
B. Binding to IL-IR tl Although IL-l receptor antagonistε that are uεeful in the preεent invention are not necessarily competitive antagonists of IL-l at the IL-IR tl, compounds, such aε IL-lra, which bind competitively are preferred in at leaεt one embodiment of the invention. A determination that binding iε competitive, as opposed to non-competitive, may be assesεed uεing a number of pharmacological toolε, including, for example, Schild plot regreεsion analysis of dose-reεponεe curveε generated from functional aεεayε (Kenakin) .
One aεsay for determining whether a compound binds competitively to an IL-l receptor, iε a competitive displacement binding asεay (Mizel) . In this assay, as in the one detailed in Example 7, intact cells are incubated with 125I-IL-lα, and binding to the cells is determined following separation from the cells of unbound 125I-IL-lα. In such an aεεay, competitive binding is evidenced by competitive displacement of bound 125I-IL-lα from the cells when the cells are incubated with a test binding compound.
According to resultε of competitive diεplacement aεεayε, εuch aε that reported above, IL-lra haε approximately the same affinity as does IL-l in binding to EL-4 cells, as well to 3T3 (fibroblastε) and CHO cellε (Mclntyre) . IL-l receptor antagonists preferred for use in the present invention will have affinities for the IL-l binding site, and more specifically, for IL-IR tl, which are subεtantially that of IL-lra for the IL- lRti. That iε, preferred antagonists will exhibit affinities that are higher than or only εlightly lower than (K; within about 2-3 log unitε) the affinity of IL-lra for IL-IR tl, when compared in the εame binding assay. In one cell binding asεay that employε YTNCl cellε, IL-lra had a K; of about 0.25 nM. Accordingly, when measured in this εyεtem, it is anticipated that a receptor antagonist preferred for use in the present invention will have a K; that is at least about 10"7 M.
It is also appreciated that activity of a compound in a competitive displacement binding assay does not generally predict whether the teεt compound iε an agonist or antagonist. Therefore, competitive displacement activity is not generally considered definitive of competitive antagonism. Compounds shown to bind competitively to the receptor are tested in one or more functional assays, such aε one or more of those described below, to determine whether binding to the receptor results in agonist or antagonist activity at the receptor.
C Functional Aεεavs for IL-l Receptor Antagonism Functional or biological assays provide means of assesεing whether a compound haε agonist or antagonist activity at the IL-IR tl. Such assesεment iε generally carried out by measuring activity of a known agonist compound in the aεεay, and comparing to thiε activity the activity of the agoniεt in the preεence of teεt compound. In addition, the teεt compound iε alεo evaluated in the functional assay in the absence of the standard known agonist, to determine whether it possesεeε agoniεt activity aε well. In general, preferred antagonists will exhibit little or no agonist activity, relative to antagonist activity. Moreover, their antagonistic activity will be substantially similar to that of IL-lra, as discussed below.
1. Mouse DIP cellular Proliferation Assay Example 8 details a method for assessing IL-l receptor antagonist activity in an aεεay in which IL-l εtimulates proliferation of mouse T-helper cells. Human IL-lα and human IL-lβ stimulate proliferation of cells, meaεured by uptake of tritiated thy idine, in εuch an aεεay. In thiε aεεay, 30 nM IL-lra waε effective to completely inhibit the effectε of IL-lα or IL-1/3 when the agoniεtε were present at concentrations ranging from about 0.1 pM to approximately 50 nM.
More generally, to aεsess relative antagonist potency in such an assay, IL-lα or IL-10 is added to the assay at a half-maximal stimulatory concentration (EC50) . The concentration of IL-lra or test compound is varied from about 10"12M to 10"*^, to determine a Kt value for the compound in the assay, according to standard procedures (Kenakin) . Compoundε preferred for use in the invention will have a K_ that iε substantially the same as a K. determined for IL-lra or icIL-lra in the asεay. That iε, they will have a K, that iε less than about 2-3 log units higher than the Kt determined for IL- Ira or icIL-lra in the aεεay.
2. Inhibition of serum IL-6 production A pharmacological effect of IL-l is stimulation of IL-6 production in serum. IL-lra inhibits εuch production, when given to mice in addition to IL-l
(Mclntyre) . Example 9 detailε a method by which IL-
1 receptor antagoniε iε assesεed in mice according to the ability of a compound to block εuch εtimulation. In thiε aεεay, aε in the proliferation aεεay, it iε anticipated that preferred IL-l antagonist compounds will behave subεtantially like IL-lra or icIL-lra, as described for the proliferation assay in sub-part 1 above.
IV. IL-l Receptor Antagonist-mediated Contraception
A. Involvement of IL-l in Intrauterine Function
Certain paracrine cytokineε are known to be involved in interactionε between the blaεtocyεt and endometrium. For example, secretion by the maternal uterus of the leukemia inhibitory factor (LIF) has been demonεtrated to be required for εuccessful implantation of the fertilized ovum. Other paracrine cytokines which have been implicated in implantation include maternal colony εtimulating factor-1 (CSF-1) , mutationε of which have been εhown to compromiεe implantation. It iε a diεcovery of the present invention that disruption (prevention or dislodgement) of embryonic implantation can be effected in vivo by blockade in the maternal endometrium of the endogenouε cytokine IL-l receptor type I (IL-IR ti) .
The interleukin-1 (IL-l) system includes IL-lα, IL-1/3, IL-l receptor antagonist (IL-lra) and the IL- 1 receptor (IL-l R) . Two IL-l receptor subtypes have been identified. They are termed IL-IR type I (IL-IR tl (Sims)) and IL-IR type II (IL-IR tl (Horuk) ) . Both IL-lα and IL-1/8 bind to IL-IR tl (Dower) , and their effect on thiε receptor is antagonized by IL-lra.
IL-l and its receptor have both been localized to endometrial cells in mice and humans. In addition, IL-lα and IL-lβ have been shown to be secreted by the human embryo prior to implantation.
The IL-l receptor (IL-IR tl) iε alεo expreεεed in the human endometrium throughout the menstrual cycle. The agonist IL-l has been used to induce pre-term parturition in mice, and the antagoniεt IL- lra inhibitε thiε abortifactant effect of IL-l (Romero) . In contrast to the anti-abortive effects of IL-lra described above, as described in the following sections, an important aspect of the present invention is the discovery that antagonism of IL-lr tl by receptor antagonists is effective to prevent implantation of a fertilized ovum, when IL- lra is present in the uterus during the implantation event, as well as to dislodge such implantation after it has occurred. More generally, according to these findingε, it can be stated that IL-l receptor antagonism is effective to provide contraception during the peri-implantation period. This period is a time during which the fate of the embryo can be manipulated by hormonal or biochemical meanε related to the implantation proceεs. In the context of the present invention, thiε time period referε to the time period during which adminiεtration of IL-lra to the subject can disrupt, that is, prevent or reverse implantation, as described in Section V below. Additionally, εtudieε carried out in εupport of the preεent invention indicate that IL-l receptor antagonism is effective to prevent ovulation in animals stimulated to ovulate, as described in sub- εection D, below.
B. Embryonic Regulation of Expreεεion of IL-l Receptor in Uterine Endothelial Cells
Experiments carried out in support of the present invention show that fertilized ova secrete εubεtanceε into their surroundings which, when brought into contact with uterine endometrial cells, stimulate the cells to express increased levels of the IL-l receptor type I (IL-IR tl) . Figures 1 and 2 show results of experiments in which human uterine endometrial stromal cells (ESC) and endometrial epithelial cells (EEC) , isolated from ovulatory women in the luteal phaεe, were cultured and grown in basal conditions in steroid-free media, aε detailed in Example 1. The reεulting monolayerε were maintained for 6 dayε under the growth conditions prescribed by an established model for in vitro decidualization (Irwin) , as detailed in Example 1. At that time, one of the following embryonic productε was added to the endometrial culture medium: human recombinant IL-lb (rIL-lb) , Human Chorionic Gonadotropin (HCG) , Platelet activation factor (PAF) , or conditioned media from human blastocyεt (HBCM; prepared aε described in Example 1) . Cells were subjected to RNA analysis by Northern blot with human IL-IR tl cRNA probe, according to established procedures, aε deεcribed in Example 1. For quantitation of results, Northern blot data were normalized to a εtandard 28S rRNA cDNA probe εtandard, according to eεtabliεhed methodε (Simon) .
Aε εhown in Figures 1 and 2, IL-IR tl mRNA and immunoreactive IL-IR tl were found in both human ESC and EEC. In human ESC, cultured under basal conditionε in the preεence of progeεterone (P) and epidermal growth factor (EGF) only IL-IR tl mRNA expression was upregulated 2.6-fold after 8 days in culture. Addition of embryonic productε to the cellε for 2 dayε (from day 6 to 8) produced a further up-regulation of IL-IR tl as follows: conditioned media from human blastocyst (7.5-fold increase), PAF (5.7-fold increase) , IL-lb (4.2-fold increase), hCG (3.5-fold increase) (Figures IA and IB) .
In EEC, IL-IR tl mRNA constitutively expressed at higher at levels than those observed in ESCa. However, in these cells, IL-IR tl mRNA was not significantly regulated by any of the embryonic products obεerved to increaεe receptor expreεεion in ESC (Figures 2A and 2B) .
C Preventing Embryonic Implantation
!• Murine Model of Embryonic Implantation. A mouse model was uεed to study implantation. The validity of the mouse model to the εtudy of implantation in hu anε was assesεed by determining (a) the preεence in the mouse uterus endometrium of immunoreactive IL-IR tl following pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) administration, and (b) the presence of IL-IR tl in the epithelial region surrounding the blaεtocyst during the peri- implantation period, specifically at day 4 of pregnancy, since these conditions have been observed in human uterine tissueε. Other indicatorε of the validity of the murine model to human conditionε are known in the art (Clark) . Specifically, IL-lα and IL-1/3 mRNAε and 11-1 bioactivity in the peri- implantation uteruε are known to increaεe from day 3 and peak between dayε 4 and 5, poεt-fertilization. Blaεtocyst implantation generally occurs late on post-fertilization day 4 in mice, but may be delayed until about day 8, due to the phenomenon of "delayed implantation" which is apparently inherent to mice. Figure 3 showε the results of experiments in which immunoreactivity of IL-IR tl and IL-13 were measured in the mouse maternal endometrium and in the mouse placenta, respectively, following fertilization, as detailed in Example 2. As εhown, mouεe IL-IR tl iε localized in the apical region of the lumenal epithelium and in εcattered areas of the stroma (Figures 3A and 3B) . Increased IL-IR tl immunoreactivity waε observed in the epithelial tisεue εurrounding the blastocyst, as indicated by the arrow in Figure 3D. As shown in Figure 3E, at day 9 of pregnancy, immunoreactive IL-1/3 was preεent in the mouse placenta (p) with increased staining at the interface with the maternal endometrium (m) . These results serve as biochemical validation of the mouse model for human implantation, with respect to the IL-l cytokine syεtem, aε described above. Although gestational length differε considerably between mouse and human, since certain common developmental stages can be identified, comparable timepoints can be establiεhed between the two εpecieε. Extrapolation of critical geεtational timepointε from mice to humans is known in the art to be within the skill of the practitioner; however, when appropriate, such extrapolationε are also provided herein.
2. Time Course of Embryonic Implantation
For the purposeε of the preεent diεcuεεion, the female reproductive system compriseε the ovaries, Fallopian tubes or oviducts, uterus and vagina. Ova mature in the ovarian follicles from oocytes contained within. Mouse and humans ovulate (releaεe mature ova from the follicles) in responεe to approximately the same hormonal stimuli, which include a periodic spike of luteinizing hormone (LH) produced in the pituitary and measurable in the serum. Ovulation in mice occurs spontaneously about every 4 dayε; in humanε, ovulation occurε about every 28 days.
Following ovulation, the ovum is taken into an oviduct, where it is thought to remain fertile for 10-24 hours in humans and for about 12 hours in mice. Fertilization, if it occurs, generally occurs in the ampulla of the fallopian tube in humans, also known as the oviduct in mice.
In the mouse, following the releaεe of εperm into the female reproductive tract, the sperm undergo capacitation, a process that renders them competent for fertilization. Capacitation usually occurs about 1 hour following ejaculation. Mouse εperm generally remain viable in the female reproductive tract for about 6 hourε after ejaculation. Human εperm generally remain viable for at leaεt about 48 hours after ejaculation. Generally, according to these timepoints indicate it is thought that for fertilization to occur in humanε, coituε εhould occur no more than about 48 hourε before or 24 hours after ovulation.
Following fertilization, the maturing embryo migrates to the uterus. During thiε migration, the embryo undergoeε maturation through cell diviεion. By the time the embryo reacheε the uteruε, generally about 2.5-3 dayε poεt-fertilization in mice and about 4 dayε poεt-fertilization in humanε, it haε progressed through the morula stage to the blastocyεt εtage. At the uterus, the embryo, generally in morula or blastocyεt stage, attaches to the uterine wall in a procesε known aε implantation. In humans, implantation occurs between days about 4 to 7, and particularly 5.5 to 6 after ovulation. In mice, implantation usually takes place between day 4.5 and day 5 following ovulation, though it can be delayed until about day 8, due to the delayed implantation phenomenon discussed above.
3. Inhibition of Embryonic Implantation by Blockade of IL-l Receptors It is one diεcovery of the preεent invention that blockade of the IL-l receptor can prevent embryonic implantation, thereby interfering with pregnancy. In experiments carried out in support of the invention, Il-lra waε ad iniεtered by intraperitoneal injection to female mice previously mated with males of the same strain and age, and outcome was assessed by measuring the number of succeεεful implantationε, aε detailed in Example 3. Briefly, 12-week-old B6C3F-1 female mice were super- εtimulated to ovulate by administration PMSG/hCG. Stimulated females were mated with maleε of the εame εtrain and age. Coituε was asεeεsed by detection of vaginal εperm plug 24 hourε after hCG injection. At day 3 of pregnancy, mice were divided in 3 groupε: control non-infected, control buffer injected, and mice injected with IL-lra. Reεultε of the mouεe implantation studies are shown in Table 1. As indicated, treatment of female mice with IL-lra reεulted in a εignificant decrease in the pregnancy rate (p=.000115 compared to control buffer injected and non-injected animals (Fisher exact test) . These results indicate that post- fertilization administration of IL-lra is effective to interfere with pregnancy.
Table 1
Effect of rh IL-lra Administration on the Reproductive Outcome of Pregnant Mice
Figure imgf000029_0001
In further support of the invention, a morphologic longitudinal study waε carried out to monitor embryonic implantation in the mouse from pregnancy day 4 to day 9 in control, untreated animalε compared with hr IL-lra treated animals, as described above. Fig. 4 showε microεcopic sections of mice uteri made at various days post- fertilization. With reference to Figure 4A, showing a longitudinal section of the uterus at day 4. Here, the free blastocyst is located within the lumen of the uterus. With reference to Figure 4C and Figure 4F, showing sectionε taken at dayε 7 and 9 of normal pregnancy, reεpectively, the mouse embryo iε observed to be completely implanted in the stro a.
In the IL-lra treated animalε, the free blaεtocyεt waε alεo found within the uteruε by post- fertilization day 4, shown in Figure 4B. By day 7 the embryo in the IL-lra treated animal had neither attached nor implanted (Fig 4D) . At day 9, no stromally implanted embryoε were obεerved in the IL- lra treated animal; however degenerated trophoblaεt cellε were found in the uterine lumen (Figure 4F) . The εtudieε deεcribed above indicate that embryonic IL-l binding to maternal IL-IR tl iε required for εuccessful implantation of the embryo in the uterus, since a specific antagonist of this binding 'prevents the implantation procesε. Theεe εtudieε alεo provide baεiε for the contention that plentiful diεtribution of IL-IR tl throughout the lumenal epithelium iε required for initiation of receptor-ligand interaction initiated wherever the embryo attacheε. Once the embryo has traversed the epithelium and begins εtro al invasion, itε own εecretion of IL-l, and perhapε other paracrine factorε, induceε IL-IR tl in the εurrounding εtroma, allowing implantation to proceed. However, as shown herein, blockage of the IL-IR tl by an antagonist is sufficient to prevent implantation.
Evidence that the effects observed in the mouse model were specific effects, rather than non- εpecific toxic effectε on the embryo was provided by experiments in which early (2-cell) mouse embryos were flushed from the oviductε of the εame group of mice uεed for in vivo experiments. The embryos were incubated with increasing concentrations of rh IL- lra for three days, as detailed in Example 4A. Assesεment of viability was made, based on the number of embryos that reached blaεtocyεt εtage after 72 hourε in culture. As indicated by the reεultε preεented in Example 4, no εignificant differences in survival were noted between embryos incubated in the presence of concentrations of IL- lra ranging from 0 to 200 μg/ml.
Recombinant human IL-lra was further tested for possible effects on later developmental stages of the mouse embryo, including blaεtocyεt hatching, trophoblaεt outgrowth and migration, aε detailed in Example 4B. Blaεtocysts were cultured 5 dayε on fibronectin-coated plateε in the presence of absence of Il-lra, as described in Example 4B. Hatching, outgrowth and migration were found to be similar in the preεence or absence of rh IL-lra. These experiments demonstrate that rh IL-lra is not toxic to the embryo itself and provide further evidence that the contraceptive effects of IL-lra are specific to the implantation process.
D. Dislodging Implanted Embryos
It is alεo an obεervation of the preεent invention that interference with implantation can occur after the initial implantation event. To be effective, an IL-IR tl antagonist compound will preferably be administered during the post- implantation period, which extendε from the implantation period to at leaεt several days following the initial implantation event, as indicated in Figure 5. E. Prevention of Ovulation
A further obεervation of the present invention is that IL-l receptor antagonism is effective to inhibit or prevent ovulation in animals stimulated to ovulate. In studies carried out in support of the present invention, and described in Example 10, female rats were primed with gonadotropin. Prior to stimulation with hCG, the animals IL-lra waε adminiεtered to the animals, and ovulation waε εignificantly inhibited.
V. Contraceptive Compoεitionε and Methodε of Treatment A. Methodε of Contraception 1. Prevention of Ovulation and Implantation
The invention includeε in one aεpect a method of contraception by prevention of implantation of the fertilized ovum or embryo. Thiε aεpect of the invention may alεo include prevention of ovulation. According to thiε method, an IL-l receptor antagoniεt is delivered to the uteruε during the time period between coituε and the implantation period; that iε, during a period 1-2 dayε before ovulation or 3-7 dayε after ovulation. The εignificance of theεe time periodε will become more apparent with reference to the temporal eventε depicted in Figure 5, aε deεcribed below.
IL-l receptor antagoniεtε for uεe in preventing ovulation and embryonic implantation are εelected aε described in Sections II and III, above. That is, suitable IL-l receptor antagonist compounds are selected for their ability to (i) bind to the IL-l tl receptor, and/or (ii) antagonize the effects of IL-lα or IL-13 at the receptor. Such compounds will exhibit a pharmacological profile that is substantially similar to that of IL-lra or icIL-lra, and will have very little or no IL-l agonist activity. IL-lra and derivativeε thereof may be uεed in the method of treatment, in accordance with the working exampleε deεcribed in Section IV, above. Figure 5 shows a temporal depiction of the human menεtrual cycle, in which day 1 indicateε initiation of menstrual flow. Generally, ovulation occurs at about day 14 of the cycle, aε depicted; however, thiε may vary conεiderably in and among individual females. In determining the pre- implantation period, it iε neceεεary to refer to the day of ovulation, such a depiction must be taken as a convenient guideline, rather than a standard. For the purposeε of determining the peri-implantation period in an individual, the reference point is preferably the day of ovulation. Generally, ovulation occurs juεt εubsequent to a spike of LH and FSH levels in serum. These and other hormonal or physiological measureε of ovulation may be measured according to standard methods known in the art. Alternatively, the day of coitus may be used as a reference point, in which case the pre- implantation period will extend to the outer limits shown in Figure 5, as deεcribed below. A ε deεcribed above, in humanε, sperm are thought to remain viable in the female uteruε and fallopian tubeε for at least 2 and posεibly as long as 5 dayε following coitus. The ovum is viable for about 24 hours following ovulation. Using the 2 day viability period for sperm, a fertilization period of 3 dayε (0-3 days post-coituε (p.c) ; days 12-15 in the idealized menstrual cycle) iε indicated in Figure 5.
Since in humanε, implantation generally takeε place between about 4 and 7 dayε after ovulation, it can be appreciated, with reference to Figure 5, that thiε time will be from about day 3 to about day 11 p.c, aεεuming that coitus may have occurred at any time between day 12 and day 15 of the idealized cycle. Thiε implantation period iε indicated aε dayε 18-22 in the menεtrual cycle. Theεe eventε, coupled with the animal εtudieε reported in Section IV, define a pre-implantation period that rangeε from about day 12 to about day 18 of an idealized menεtrual cycle. Preferably and practically, in accordance with the preεent invention, it will be appreciated that prevention of implantation will be effected by adminiεtration of an IL-l receptor antagoniεt between about day 3 post-ovulation and about day 7 post-ovulation. When the day of ovulation is uncertain, it will be aεsumed that it spans the three day fertilization period shown in Figure 5. In εuch a case, compound will preferably be administered between about day 1 and about day 9 p.c. More preferably, the compound will be administered between about day 3 and day 7 p.c.
In accordance with the invention, it is appreciated that an IL-l receptor antagonist will also be effective to inhibit ovulation, in the case where ovulation has not occurred prior to intercourse, such as during the period 1-2 days before ovulation (days 12-14 in the idealized cycle illustrated in Figure 5) . In εuch a case, if the time of ovulation is uncertain, the compound will be effective if administered to the subject between about day 0 and day 9 post-coitus.
2. Dislodging the Implanted Embryo
With continued reference to Figure 5, it will be appreciated that prevention of pregnancy can also be achieved, when IL-l receptor antagonism interferes with implantation after the initial implantation event. According to the cycle shown in the figure, where implantation occurs between day 18 to day 21 of the cycle, post-implantation interference, or "dislodgment" will occur aε early aε day 18 and aε late as day 28 in the cycle. Preferably, such dislodgement will be effected between about day 4 and day 10 following ovulation.
B. Pharmaceutical Compositions The dosage of IL-l receptor antagonist required to disrupt implantation will depend on the specific compound and the route of administration, as described below. Generally, an effective doεage will be a dosage that provides in the uterus a concentration that is equivalent to a concentration of IL-lra of about 1-50 μg/ml. In humanε, εeru concentrationε of about 30 μg/ml have been achieved by administering an intravenous dose of 10 mg/kg IL- lra (Granowitz) .
Although any mode of administration may be used, so long as it provides sufficient concentration of compound to the uterus, it can be appreciated that certain modes of administration will be preferred in the treatment method. In the case of IL-lra, intravenous administration is possible; alternatively, for convenient self- adminiεtration by the patient, other modeε of adminiεtration, such as nasal insufflation or intravaginal insertion may be preferable. The relative phar acokinetics of εuch modeε of adminiεtration are known in the art.
In one preferred embodiment, the IL-l receptor antagonist will be formulated in an intravaginal suppoεitory inεert having slow release properties, such as are afforded by the formulation described in U.S. Patent No. 5,116,619, incorporated herein by reference. This formulation, which includes, in addition to the active ingredient, about 65-85% lactose, 2-4% starch paεte, 2-4% corn εtarch and 0.4-0.6% magneεium εtearate, allowε for prolonged release of active ingredient over 48-72 hours. It can be appreciated that such a suppository formulation can be dispensed for alternate night self-administration, during the peri-implantation period determined according to the practitioner's understanding of the particular patient'ε ovulatory cycle and the principleε taught herein.
Another preferred administration mode includes the use of a cervical cap to deliver IL-lra to the uterus through the cervix. Cervical caps, such as the cervical cap described in U.S. Patent No. 4,961,436 incorporated herein by reference, are known in the art, and may be used to deliver over the peri-implantation period, a medicament containing an IL-l receptor antagonist effective to produce in the uterus a concentration of the antagonist equivalent to about 1-50 μg/ml IL-lra.
In accordance with current techniques in transdermal iontophoresiε of peptides, it is also appreciated that IL-l receptor antagonist peptides may be administered by transdermal delivery, as through "patch" application. Example 4C describeε a εyεtem εuitable for transdermal delivery of a peptide formulation suitable for use in the methods of the preεent invention.
The following exampleε illustrate, but in no way are intended to limit the present invention.
Materials Interleukin lα (IL-lα) and Interleukin Iβ (IL-
1/3) were obtained from Genzyme (Norwalk, CT) and are also available from Gibco (Grand Island New York) .
All other reagents may be obtained from the supplierε listed in the text or are standard in the field. Example 1
Localization of IL-l and IL-IR tl in Uterine and Embryonic Tiεεueε
A. Culture of Endometrial Cellε Human endometrial samples from ovulatory women undergoing hysterectomy for non malignant indications were obtained in the secretory phase.
Endometrial stromal cellε (ESC) and epithelial cellε
(EEC) were isolated according to standard procedures, cultured, and grown in basal conditions in steroid-free media supplemented with 10% serum until confluent monolayers were formed. Endometrial tisεueε samples were minced into small pieces of less than 1 mm, and subjected to mild collagenase digeεtion aε followε: Tissue pieces were incubated with shaking for 2 hourε at 37°C, in DMEM (Gibco, Grand Island, NY) containing 0.2% collagenase Type I and 0.005% deoxyribonuclease Type I (Worthington, Freehold, NJ) . ESC and EEC were iεolated from the mixture by centrifugation, then cultured, and grown to confluence in 75% Dulbecco'ε Modified Eagle'ε Medium (Gibco, Grand Iεland, NY) and 25% MCDB-105 (Sigma, St. Louis, MO) , containing antibiotics, supplemented with 10% serum (Charcoal-Dextran treated FBS, Hyclone, Logan, Utah) and 5 μg/ml insulin (Sigma, St. Louis, MO) . ESC and EEC were grown in steroid free medium until they were confluent monolayers. The homogeneity of cultureε waε determined by morphological characteristics and verified by immunocytochemical localization of cytokeratin, vimentin, and CD68 antigen as markers for epithelial cells, stromal cells, and human macrophages, respectively. Aε defined by these criteria, ESC monolayers contained lesε than 2% of EEC and leεε than 0.1% of macrophageε. EEC cultureε contained leεε than 3% of ESC and leεε than 0.1% of macrophageε. After cells were confluent (1-2 weeks) , the media was supplemented with 5 μg/ml ascorbic acid, 10 μg/ml transferrin and hormones, as indicated below.
Monolayers were cultured for 6 days with progesterone (P) (10 nM) and epidermal growth factor (EGF) (3.3 nM) , according to an established model for in vitro decidualization (Irwin) . On day 6, human recombinant IL-lb (hrlL-lb) (10 IU/ml) , human chorionic gonadotropin (HCG) (10 IU/ml), platelet- activating factor (PAF) (200 nM) , or conditioned media from human blastocyst (HBCM; 1ml) were added to the media and cells were further cultured for 2 days and compared with control cells cultured only with P and EGF.
HBCM is conditioned media (500-1000 ml) from human embryoε cultured in vitro until blaεtocyst εtage. To prepare HBCM, embryoε were cultured in Human Tubal Fluid medium (HTF Medium, Irvine Scientific, Santa Ana, CA) containing penicillin (0.5 mg/ml) , streptomycin (0.5 mg/ml) and 1% human serum albumin (Fraction V, Irvine Scientific) for approximately 4 to 5 days. The medium covering theεe cells was then collected, frozen, and stored at -70°C degreeε until uεe. Growth medium waε renewed every 2 dayε, and cellε were removed every 2 dayε for RNA analysis.
B. Regulation of IL-lra Expresεion by Embryonic Factors
RNA from cultured endometrial cellε waε analyzed by Northern analyεis with human IL-IR tl cRNA probe. A 28S rRNA cDNA probe was used to normalize the data aε according to standard methods
(13, 14) . Data were expressed as arbitrary units:
IL-IR tl mRNA 285 rRNA
Total RNA was extracted from ESC cells according to methods known in the art (Chomczynski) , then 10 μg εampleε were fractionated by electrophoreεiε according to εtandard methodε (Auεubel) and tranεferred to a nitrocelluloεe membrane. The filterε were hybridized with a cRNA probe for human IL-IR tl (Simmε) . Equal loading of RNA on the filterε waε confirmed by denεitometric analyεiε of 28S rRNA cDNA hybridization signals. IL-IR tl mRNA levelε were normalized to 28S rRNA levelε and expreεsed as an arbitrary unit, the IL-IR tl mRNA/28S rRNA level at day 0 of treatment. Densitometric analysis of Northern blots of IL-l R tl mRNA from different experimentε were combined and expreεεed aε the mean ± standard deviation (sd) . Protein studies were performed by immunocytochemistry uεing both indirect immunofluoreεcence and avidin-biotin methodε (Simon) .
ESC were cultured until confluence in εteroid-free conditionε aε detailed above, RNA extracted, tranεferred and hybridized with IL-IR tl cRNA probe. Figureε IA and IB shows Northern blots and a bar graph of quantitation of IL-IR tl mRNA in cultured human endometrial stromal cells (ESC) . In the bar graphs combine valueε obtained from εeveral experimentε aε indicated by "n" . Valueε are expreεεed aε mean ± standard deviation (sd) . The positionε of 18S and 28S ribosomal RNA are indicated to the left of the Northern blots.
With reference to Figure IA, lane 1 IL-IR tl mRNA expresεion at day 0, prior to any further treatment of cellε. Thiε lane serves as control. IL-IR tl mRNA levels of ESC treated with progesterone and epidermal growth factor (P+EGF) for 6 and 8 days are εhown in laneε 2 and 3, respectively. Lanes 4-7 εhow blots from ESC treated with P+EGF for 6 days, then, treated for 2 additional dayε (until day 8) with media from human blastocyεt (1 ml total, Lane 4), 200 nM PAF (Lane 5) , IL-lb 10 IU/ml (Lane 6) , or hCG 10 IU/ml (Lane V) .
Figureε 2A and 2B show the same representations described above for Figureε IA and IB for endometrial epithelial cells (EEC) . EEC cultured until confluence in steroid free-conditions were treated aε described for ESC, above. Treatments and lanes correspond to thoεe deεcribed above for ESC treatment.
Example 2 Immunodetection of IL-IR tl and IL-lβ A female mouse was given 5 IU pregnant mare serum gonadotropin (PMSG) by I.P. injection. Twenty-four hours later, the uterus was removed and stained with rat monoclonal anti-mouse IL-lr TI antibody, according to standard immunohistochemical methods (Simon, et al . ) (14). Experiments were repeated with at least three different sampleε in each case, unless otherwise indicated herein.
Figure 3 showε the reεultε of εtudieε uεing indirect immunofluoreεcence to localize of immunoreactive endometrial mouse IL-IR tl (A-D) and immunoreactive IL-lβ (E) in the mouse embryo.
Figure 3 (A-E) εhowε results (A) Mouse uterus obtained 24 h. after pregnant mare serum gonadotropin (PMSG) injection (5 IU) ; mouse IL-IR tl is localized in the apical region of the lumenal epithelium and in scattered areas of the stroma;
(original magnification x400) . (B) Mouεe uterus obtained 24 h. after hCG injection (5 IU) ; Stronger IL-IR tl staining throughout the epithelium and in the stroma is observed; (x400) . (C) Negative control by deletion of the primary antibody; (x400) . (D) Mouse uterus obtained at day 4 of pregnancy (single experiment) . Increased staining was observed for IL-IR tl in the epithelium εurrounding the blaεtocyst (arrow) ; (x400) .
Figure 3E showε immunolocalization of mouεe embryonic IL-lb, at day 9 of pregnancy with twin embryoε (indicated at "e" in the figure) immunoreactive IL-lb iε preεent in the mouse placenta (indicated at "p" in the figure) with increased staining at the interface with the maternal endometrium (indicated at "m" in the figure) ; (x400) . Monoclonal antibodieε againεt mouεe IL-lb and the extracellular domain of the mouεe IL-IR tl were acquired from Genzyme (Cambridge, MA) .
Example 3 Inhibition of Embryonic Implantation in vivo A. Periodic Injection of Il-lra Female 12-week-old B6C3F-1 female mice (approximately 20 g) were εtimulated with 5 I.U. of Pregnant Mare'ε Serum Gonadotropin (PMSG; Sigma, St. Louiε, MO) i.p., followed 24 hourε later by 5 I.U. human chorionic gonadotropin (hCG) i.p. adminiεtration. The mice were then mated immediately with males of the same strain and age. Females with vaginal plugs (deεignated pregnancy day 1) were randomly allocated to three groupε: A, Control non-injected; B, Buffer injected animals and C, animals injected i.p. with 20 μg of recombinant human IL-lra (rh IL-lra) every 12 hours beginning on pregnancy day 3 and ending on day 9. Intraperitoneal injections were carefully directed away from the pregnant uterine horns. Injections were continued until day 9 to avoid the posεibility of delayed implantation, and animals were sacrificed 12 hours after the last injection. Animals appeared healthy throughout the experiment. Implantation siteε in both uterine hornε in IL-lra injected animals were counted and compared with control animals receiving buffer or no injection. Two different preparationε of IL-lra (26) and two different bufferε were uεed in four εeparate experimentε. Data were analyzed by Fiεher Exact Test. Resultε of theεe studies are shown in Table 1, in Section IV.C.3, above.
B. Morphological Assessment of Effects of IL- lra on Embryonic Implantation PMSG/hCG stimulated 12-week-old B6C3F-1 femaleε were mated with maleε of the εame εtrain and age. Femaleε with vaginal plugε (designated pregnancy day 1) were randomly allocated to two groups: I, Buffer injected animalε, and II, animalε injected i.p. with 20 μg of recombinant human IL-lra (rh IL-lra) (26) every 12 hourε beginning on pregnancy day 3 until day 9. Starting on day 3, one mouse from each group was sacrificed each day. Uteri were removed and immediately fixed in 10% formalin in phoεphate buffered saline. Fixed tissue were embedded in paraffin, sectioned and mounted on glass slides. Serial sections (4 μm) from each εample were then εtained with hematoxylin-eoεin
(H&E) and viewed with an Olympuε 35 mm camera and an Olympuε BH2 microεcope.
Reεultε are shown in Figure 4 (A-F) . Micrographs from untreated animals are shown in Figures 4A, 4C, and 4E. Human recombinant IL-lra treated animals are εhown in figureε 4B, 4D, and 4F. Figure 4A εhowε day 4 normal pregnancy; longitudinal εection of a free blastocyst surrounded by enlarged uterine lumenal epithelial cellε [He atoxylin/Eoεin (H & E) , original magnification x400] . Figure 4B shows Day 4 pregnant animals injected with IL-lra; Cross-section of a free blastocyεt. In thiε micrograph, the epithelium εurrounding the blaεtocyst iε not enlarged aε obεerved with normal pregnancy (H & E x400) . Figure 4C εhows the uterus of day 7 normal pregnant mouse; longitudinal section through embryo and decidual mass demonstrating complete stromal implantation (H & E x400) . Figure 4D showε the uteruε of day 7 of pregnant mouεe, IL-lra injected. Here the blastocysts are still free. No attachment or invasion is obεerved (H & E x400) . Figure 4E showε the uteruε of day 9 normal pregnant mouse; longitudinal section through embryo and decidual mass (H & E x200) . Figure 4F showε the uteruε of day 9 of pregnancy, IL-lra injected mouse; stromal decidual reaction (arrows) with intact glandular epithelium and degenerated trophoblastic cells in the uterine lumen (arrowhead) can be obεerved, but implantation did not occur in thiε animal (H & E x800) . Thiε is a section from the single IL-lra injected mouse described in Table 1 that was macroεcopically pregnant.
C Tranεdermal delivery of IL-l receptor antagoniεt peptideε
Female rodent subjects, preferably hairlesε guinea pigε or mice (subjected to a depilatory agent) are mated, and examined for evidence of pregnancy as described in Part A, above. Animals are then fitted with a transdermal patch syεtem, according to methodε described by Kumar, et al. (J. Controlled Release 18.: 213-220, 1992) , incorporated herein by reference. Briefly, the patch consiεtε of a die cut reεervoir ring of cloεed cell polyethylene foam having contact adhesive on one side. The ring is fitted one side with a Millipore GVWP hydrophilic filter. The ring and the filter join to form an anode reservoir, into which is placed a gel containing IL-l receptor antagonist. The gel conεiεtε of Polyjel HV (polyglyceryl citrate + hydroxypropyl methyl cellulose; Guardian Chemical, Hauppauge, NY) mixed with acetate buffer (0.05 M, pH 5.8) into which is mixed the peptide at about 0.1% (wt/wt) . The reservoir is cloεed with a tranεparent polyethylene film having contact adheεive on one side, into which is embedded a stainleεε εteel εnap button electrode. Alternatively, for use with acidic molecules, a cathode patch, consiεting of a karaya gumpad (Iomed, Inc., Salt Lake City, UT) iε used.
The patch is connected to a pulsed voltage output power source with a current limiting feature.
Animals are treated using a current limit of 0.17 mA/cm2 at 50 KHz and 50% duty cycle or 0.85 mA for a total area of 5 cm2, for 5 hours. Blood iε sampled for presence of the peptide, to determine the delivered dose. Control animals are given the carrier gel, without IL-l receptor antagonist compound.
After an appropriate gestational period, aε deεcribed in part A above, animalε are then tested for presence implanted embryos, as described in Example 3, part A, above.
Example 4 Effect of Il-lra on Embryonic Development
A. Culture of Early Embryoε with IL-lra 2-cell mouεe embryoε were fluεhed from the oviducts on day 1 of pregnancy from animals impregnated as described in Example 3. Embryoε (n=276) were rinsed and placed in 4-well Nuncon plates containing 500 μl of human tubal fluid medium (HTF Medium, Irvine Scientific Santa Ana, CA) with Oμg/ml (n=91) , lμg/ml (n=36) , 50 μg/ml (n=36) , 100 μg/ml (n=52) , and 200 μg/ml (n= 61) of rh IL-lra. After 72 h in culture the percentage of embryos reaching the blastocyst εtage was 85.7%, 91.6%, 94.4%, 96% and 85.2% respectively . B. Effectε of IL-lra on Blaεtocyεt Development
Effectε of rh IL-lra on blastocyst hatching, trophoblaεt outgrowth and migration, were determined by further culture of the blastocystε for 5 days on fibronectin-coated plates (Upstate Biotechnology,
Inc., Lake Placid, NY, 27). Ten blaεtocyεtε obtained from control wellε (0 μg/ml rh IL-lra) and 10 from wellε grown in the preεence of 200 μg/ml of rh IL-lra were placed in plates containing 200 μg/ml of rh IL-lra. Ten blaεtocyεtε from control and IL-ra containing cultureε were placed in control plateε lacking IL-lra. In both groupε, hatching, outgrowth and migration were documented to be similar in the presence or absence of rh IL-lra.
Example 5 Recombinant Expression of IL-lra Plasmids containing the human IL-l receptor antagonist (hlL-lra) gene were obtained Immunex Corporation, Seattle, WA. The gene sequence of the insert is shown as SEQ ID NO: 1 in Figure 6 herein. Alternatively, the gene can be synthesized using an oligonucleotide synthesizer, or obtained by the isolation/synthetic method described in the reference by Carter, et al., incorporated herein by reference, and verified, using the sequence shown as SEQ ID NO: 1. Alternatively, primers baεed on thiε εequence can be uεed for preparation of the gene sequence, according to standard procedures (Ausubel) .
For use in experiments carried out in support of the preεent invention, recombinant E. coli human IL-lra waε expreεεed aε a fuεion protein uεing the pMAL vector (New England Biolabε) . The expreεεed fuεion protein waε detected and purified from the periplaεm of E . coli by Amyloεe affinity chromatography (New England Biolabε) . The amylose eluate pool was dialyzed into 50 mM Tris-HCL, 100 mM NaCl pH 7.5 and cleaved with Factor Xa (Enzyme Research Labs) at a concentration of 10 ng to 50 μg of fusion protein. SDS-PAGE was run to detect Factor Xa cleavage of the fusion protein. Homogeneity of hlL-lra waε achieved with further purification onto Q-Sepharoεe pH 8.5 and Sephacryl S-300 gel filtration chromatographieε (Pharmacia) . The purified protein haε a molecular maεε (Mr) of 17, 000 dalton. Human Il-lra activity was measured by a mouse D10 cell proliferation assay, protein quantitation by gel laser densitometer (Molecular Dynamics) and endotoxin level was measured by the Limulus aεsay (Whittaker Bioproducts) .
Example 6 Purification of IL-lra IL-lra iε purified from cellular sources as described by Carter, et al. as followε: U937 cell cultureε are obtained from the American Type Culture Collection, Rockville, MD. Cellε are cultured at 37°C for 48 h at 5 x 105 cells ml"1 in RPMI 1640 medium (GIBCO) , 7% fetal bovine serum (FBS, GIBCO) , 2mM L-glutamine, 100 U ml"1 penicillin, 100 μg ml"1 streptomycin, 20mM HEPES buffer, and 100 nM phorbol myristate acetate (PMA) (Sigma, St. Louiε, MO) . After differentiation, the medium iε removed and the cellε, now adherent, are gently waεhed once with a εmall volume of Dulbecco'ε PBS. Cellε are then cultured for an additional 48 h in RPMI 1640 containing 1% low-endotoxin FBS (HyClone, Logan, UT) , L-glutamine, penicillin, streptomycin, and rhGM-CSF (75 U ml"1; Amgen, Torrance, CA) . Cell-free supernatantε are collected, pooled and frozen at - 20°C After thawing, phenylmethylεulphonyl fluoride is added to 0.2 mM final concentration. The solutionε are concentrated by ultrafiltration with YM-5 and YM-10 filterε (Amicon) . Protein meaεurementε are made uεing Bradford assay (Bio-Rad Kit) using BSA aε the εtandard protein.
The preparation iε then εubjected to Superoεe 12 FPLC-1 chromatography in 17 repetitive cycleε. Two-ml aliquots of Fraction 2 concentrate are injected onto a Superose 12 prep grade FPLC column (1.6 x 50 cm) equilibrated with 0.1 M potasεiu phosphate, 1 M NaCl, pH6.0. Fractions of 1 ml are collected at a flow rate of 1 ml min"1. Fractionε are collected and individually concentrated by Centricon-10 units (Amicon) . After analysiε by SDS- PAGE and bioassay for IL-lra activity, appropriate fraction concentrates are pooled and further concentrated to 1 ml by Speed Vac centrifugation (Savant) .
Pooled samples are then subjected to TSK Bio- Sil 125 HPLC-1, as follows: in 20 repetitive cycleε, 50 μl of fraction 3 are injected onto a TSK-Bio-Sil- 125 HPLC column (7.5 x 600 mm), uεing the εame buffer εyεtem aε described above. Fractions of 200 μl are collected. Appropriate fractions are pooled and concentrated by Centricon-10 filter unitε and finally by Speed Vac centrifugation aε deεcribed above.
Pooled sampleε are further purified, uεing TSK Bio-Sil 125 HPL-2, aε followε: in εeven repetitive cycleε, 20 μl of fraction 4 are injected onto the same column as described above. Active fractionε are pooled to give a final volume of 8.5 ml, and are εubjected to C4 reverse phase HPLC, as followε: appropriate fractionε from the TSK Bio-Sil 125 run (fraction 5) are injected directly onto a C4 reverse phase column (4.6 x 150 mm). Proteins which remain bound to the matrix following an extensive wash with 0.1% trifluoroacetic acid (TFA) are desorbed using a linear gradient from 28 to 32% acetonitrile in 0.1% TFA over a period of 44 min at a flow rate of 1 ml min"1 (room temperature) . Representative aliquots of each fraction or pool are removed, taken to complete drynesε by Speed Vac centrifugation, and then re-solubilized in tisεue culture medium in preparation for assay.
Example 7
IL-l receptor binding assayε Radioiodinated IL-lα and IL-1/3 are obtained from commercial εourceε (DuPont NEN Research
Products, Boston, MA) or prepared by chloramine-T radioiodination of unlabeled peptide.
EL-4 cells (6xl06/ml; mouse lymphoma cells; ATCC TIB 39, American Type Culture Collection, Rockville, Maryland) are suεpended in binding medium (RPMI 1640 containing 5% fetal calf serum and 25 mM HEPES, pH 7.2). Aliquots (100 μl) are placed in 12 x 75 mm polypropylene tubes. *2SI-labelled IL-l is added at a concentration of about 1 xlO"10 M. Incubation is carried out for 3 hours at 4°C in the preεence of teεt compound, aε deεcribed below. Nonεpecific binding iε determined by incluεion of 1 xlO8 M unlabeled IL-l. Unbound 125I-IL-1 is separated from cellε by centrifugation through 200 μl of a εilicone oil mixture in a Beckman microfuge.
Diεplacement of binding by teεt compound iε determined by adding to the above incubation mixture, varying concentrations of unlabeled test compound ranging from about 10"12-10"8 M compound.
IC50 and Kj values are calculated for the teεt compound according to methodε well known in the art (Kenakin) . Example 8 Inhibition of Cellular Proliferation
A. Inhibition of IL-l stimulation of mouse DIP cell Mouse T helper D10.G4.1 cellε (American Type Culture Collection, Rockland, MD; ATCC TIB 224) were εuεpended in culture medium (RPMI 1640 containing 5% fetal calf serum, 5 x 10"8 M 2-mercaptoethanol, 8 μg/ml gentamicin, 2 mM L-glutamine, and 2.5 μg/ml concanavalin A) . Cells were εuεpended to produce a cell concentration of about 1-2 x lOμg/ml. IL-lra waε added to triplicate cultureε, 1 hour prior to addition of IL-lα or IL-10 to the cultureε. The plates were then incubated and incorporation of[3H]Thymidine into the cells meaεured according to εtandard techniques. In experiments carried out in support of the present invention, hIL-lα or hIL-ljδ was added in increasing concentrations to the aεεay, the EC50 of hIL-lα waε 7 pM and the EC50 of hIL-1/3 waε 450 pM. At a concentration of 30 nM, hlL-lra completely inhibited stimulation of proliferation by hIL-lα to a concentration of 100 pM and by hIL-lβ to a concentration of about 100 nM.
Determination of IC50 and KD values for IL-l receptor antagonists in these asεayε iε accompliεhed according to methods known in the art.
B. Inhibition of IL-l Stimulation of Thymocytes Mouse thymocytes are prepared, and the action of IL-l, which produceε growth εtimulation, iε measured using standard techniqueε (O'Gara, 1990) . Three to εix week old C3H/HeN mice are obtained from Simonεen Laboratories, Gilroy, California and sacrificed by εtandard procedures. Thymi are immediately removed, separated from adherent non-thymic tissue, homogenized in Hank's balanced salt solution (Gibco) using a glasε homogenizer, and centrifuged at 180 x g for 10 minutes at 15°C. Following an additional wash in HBSS, the thymocytes are resuεpended in RPMI 1640 tiεεue culture medium (Gibco) containing 50μM 2-mercaptoethanol (Fiεher) , 2 mM glutamine (Gibco) , 1 mM pyruvate, non-essential amino acid solution, penicillin (100 U/ml) streptomycin (lOOμg/ml) solution, 10% heat inactivated fetal bovine serum and Phytohemaglutinin (PHA, Pharmacia, final concentration 10 μg/ml) . Cells are cultured in round-bottom 96 well microtiter tisεue culture plateε, 6xl05 cellε per well in a volume of 100 μl. Teεt compound iε diluted in tissue culture medium and added to the wells in the presence and abεence of IL-l (recombinant human IL-l, R&D Syεtemε catalog # 201-LB, 0.1 ng/ml) . Total volume is 150 μl per well. Plates are incubated for 72 hourε (95% air/5% C02, 37°C) . During the last four hours of incubation, tritiated thymidine (Amersham, 49 Ci/mM) is added (0.5 μCi per well). Cells are harveεted onto Whatman 934-AH glass microfiber filters and counted in a Beckman LS 6000 scintillation counter. Resultε are expreεsed as counts per minute well. Cytotoxicity iε meaεured in theεe aεsays using the MTT reduction assay, as deεcribed below.
Untreated cellε εhow minimal DNA εyntheεiε (thymidine incorporation 80 cpm/well) . PHA alone produceε a 2-3 fold stimulation of cell proliferation. Treatment with 0.1 ng/ml IL-l in the presence of PHA results in 60 fold εtimulation. Addition of the IL-lra reεultε in a doεe dependent inhibition of IL-l εtimulation.
Example 9 IL-6 Bioaεεav
Mice were given IL-l are injected with IL-lra (5 mg/kg, εubcutaneouεly, (ε.c. ) ) or teεt receptor antagonist. Mice are immediately given IL-lα (5 μg/kg ε.c) . Blood samples from the mice are taken 3 hours later.
Serum levels of IL-6 are determined using a modification of the B9 hybridoma cell asεay deεcribed by Aarden et al. and incorporated herein by reference. B9 cellε are treated with twofold serial dilutions of the test sera in 96-well microtiter plates. After a 3-day incubation at 37° in a humidified atmoεphere of 5% C02 and 95% air, the wells are pulsed with 0.5 μCi of [3H]thymidine ([3H]TdR) and incubated for an additional 18 hours. The cells are then harvested onto glass fiber filters and the level of [3H]TdR incorporation determined in a liquid scintillation counter. IL-6 units are defined as the inverse of the serum dilution that produces half-maximal [3H]TdR incorporation compared with a reference εtandard.
Example 10
Prevention of Ovulation Female rats were administered PMSG (25 I.U.), then anesthetized with pentobarbital. One horn of the uterus was exposed, and 1 μg of IL-lra waε injected unilaterally directly into the ovarian burεa. Animalε were then εti ulated by adminiεtration of hCG (25 I.U.). Animalε were later examined for presence of ova. Using the contralateral uninjected ovary as control, a 40% reduction in ovulation waε obεerved after IL-lra treatment.
While the invention haε been described with reference to specific methods and embodimentε, it will be appreciated that variouε modificationε and changeε may be made without departing from the invention.

Claims

IT IS CLAIMED:
1. A composition for use in preventing ovulation and implantation of an embryo in a uterus of a mammalian subject, comprising an IL-l receptor antagonist.
2. The composition of claim 1, wherein the interleukin-1 receptor antagonist is a peptide.
3. The composition of claim 2, wherein said interleukin receptor antagonist is selected from the group consiεting of IL-lra (SEQ ID NO: 2) and icIL- lra (SEQ ID NO: 4) .
4. The compoεition of claim 3, wherein said interleukin receptor antagonist is IL-lra (SEQ ID NO: 2) .
5. The composition of claim 1, wherein said compound iε εelected from a combinatorial library of compoundε, by the εtepε of: measuring (i) agonist activity, and (ii) antagonist activity of a library of compounds, in a functional IL-l receptor assay, and selecting a specific compound in the library if it has (i) substantially no agonist activity, and (ii) substantial antagonist activity.
6. A contraceptive device for use in preventing ovulation and implantation of an embryo or in dislodging an embryo in a mammalian uterus, comprising a vaginal insert or a cervical cap, and compound release meanε in εaid inεert or cap for releaεing an IL-l receptor antagoniεt compound Dl
at a dose effective to prevent or disrupt implantation of an embryo in the uterus.
7. The device of claim 6, wherein the interleukin-1 receptor antagonist is a peptide.
8. The device of claim 7, wherein said interleukin receptor antagonist is selected from the group consisting of IL-lra (SEQ ID NO: 2) and icIL- Ira (SEQ ID NO: 4) , and said compound release means is effective to produce a concentration of said peptide at the uterus of about 1-50 μg/ml.
9. The device of claim 6, wherein the said interleukin receptor antagonist is selected from a combinatorial library of compounds, by the steps of: measuring (i) agonist activity, and (ii) antagonist activity of a library of compounds, in a functional IL-l receptor,assay, and selecting a specific compound in the library if it has (i) substantially no agonist activity, and (ii) substantial antagonist activity.
10. A method of identifying a compound for use in preventing ovulation and implantation of an embryo or in dislodging an embryo in a mammalian uterus, comprising measuring (i) agonist activity, and (ii) antagonist activity of a library of compounds, in a functional IL-l receptor assay, and selecting a specific compound in the library if it has (i) substantially no agonist activity, and (ii) substantial antagonist activity.
11. The method of claim ιo, wherein said measuring further includes measuring the binding affinity of the compound in an IL-IR tl IL-l ligand competitive diεplacement binding assay, and said selecting further includes selecting the compound if itε affinity in εuch binding aεεay iε at least within two orders of magnitude of the binding affinity of an IL-l receptor antagonist εelected from the group conεiεting of IL-lra and icIL-lra.
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US10799589B2 (en) 2013-03-13 2020-10-13 Buzzard Pharmaceuticals AB Chimeric cytokine formulations for ocular delivery
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