MXPA01006897A - Tumor necrosis factor antagonists and their use in endometriosis - Google Patents

Abstract

Tumor necrosis factor antagonists are administered in therapeutically effective doses to treat and/or prevent endometriosis. The antagonists of this invention typically are selected among various classes of molecules but preferably are soluble TNF receptors. The antagonists are useful for the regression of endometriotic lesions and, if combined with other active ingredients, for amelioration of related disorders, like infertility.

Description

ANTAGONISTS OF THE TUMOR NECROSIS FACTOR AND ITS USE IN ENDOMETRIOSIS Field of the invention - ~ - Antagonists of tumor necrosis factor are administered in therapeutically effective doses to treat and / or prevent endometriosis. Antagonists of this invention are typically selected from various classes of molecules, but are preferably soluble TNF receptors. Antagonists are useful for the regression of endometriosis lesions and if they are combined with other active ingredients, for the improvement of related disorders, such as infertility.

Background of the Invention Endometriosis is a female genital disease characterized by the presence of endometrial glands and stroma outside the endometrial cavity and uterine musculature. The most frequently affected anatomical sites are the ovaries, uterosacral ligaments, pelvic peritoneum, rectovaginal septum, cervix, vagina, fallopian tubes and vulva. The Endometriosis is generally similar to a deep infiltrate from the rectovaginal septum in the underlying tissues and is not superficially visible. Occasionally the foci of endometriosis can be found in extraovarial sites, such as in the lungs, bladder, skin, pleura and lymph nodes. The endometrial lesions are progressive: they are first observed as clear vesicles, which turn pink and progress towards color black, fibrotic lesions over a period of a few years (MacSween, 1993). Endometriosis is considered a benign disease, but endometriotic lesions occasionally become malignant. As in other types of malignancies, neoplasms derived from endometriosis are due to concurrent events, which involve alterations in growth factors and / or regulation of oncogenes (Cheung, 1996). Endometriosis is among the most common gynecological diseases, prevalent among women of reproductive age: this disease is found in approximately 5-10% of women of reproductive age (Barbieri, 1988). The endometriosis tissue is completely dependent on estrogen for continuous development, also in ectopic sites. Consequently, endometriosis is rare before menarche and after menopause, when women are deficient in estrogen. The hormonal sensitivity to some of the most common symptoms, which are pelvic pain and dysmenorrhea, is originated from the disseminated endometrial cells of the endocrine-like tissue. uterus to other sites, where viable cells can be implanted "and developed. Two possible mechanisms have been proposed to explain the initial cell expansion. Retrograde menstruation, a common phenomenon among women with a menstrual cycle, makes it possible for fragments detached from the endometrium to come out, through the fluid of the menstrual flow, near the structures in the genital tract. Alternatively, to explain the appearance of endometriosis at sites other than the genital structures, endometrial cells can expand through the uterine veins and spread through the lymphatic system (hematogenous or lymphatic spread). Gynecological surgery can also contribute to this expansion (MacSween, 1993). Apart from the dissemination of endometrial cells, other factors, such as genetic predisposition (Malinak et al., 1980), as well as immunological alterations (Ho et al., 1997) can determine the susceptibility of women to endometriosis. Since endometrial cells are frequently observed in the peritoneal fluid in all women at the time of the menses, mammals may have mechanisms, most likely related to the immune system, to prevent the onset of endometriosis. In general, endometrial cells that escape the host immune response and have adequate estrogenic stimulation may proliferate to form microscopically visible lesions, big. Endometriosis is therefore considered a dynamic process where new lesions are being formed continuously while existing lesions can develop or be destroyed by the host's immune response. The inflammatory reaction, normally associated with endometriosis, changes the peritoneal environment, since there is an increased volume of peritoneal fluid and peritoneal macrophages that increase both in number and activity. Therefore, it has been proposed that the monocyte / macrophage system plays a key role in the development of endometriosis. The secretory products of macrophages, including RANTES (Hornung et al., 1997), Interleukin-6 (Harada et al., 1997), Interleukin-8 (Arici et al., 1996a), Tumor Necrosis alpha factor (Overton et al., 1996), Protein- 1 Monocyte chemotaxis (Arici et al., 1997), were found at a higher concentration in the peritoneal fluid of affected women ^^ oTr --- this disease. Immunological changes have been demonstrated in women with endometriosis, but it has not been demonstrated whether these events are responsible for endometriosis or whether they are a result of inflammation caused by endometriosis (Rana et al., 1996). Knowledge about endometriosis, and its relevance to other diseases, is still limited, even at the diagnostic level. Although endometriosis is considered a major cause of infertility, studies on the pathophysiology of the disease are contradictory and not definitive. There is a poor correlation between the degree of pain or infertility and the severity of the disease, since early lesions are more active metabolically. The rate of infertility is higher than the normal population and studies in rabbits have shown that the surgical induction of endometriosis leads to a decrease in fertility from 75% to 25% (Hahn et al., 1986). It has been found that patients with pelvic pain have endometriosis 71% of the time, while 84% of patients with pelvic pain and infertility had diagnosed endometriosis (Koninckx et al., 1991). In general, infertility can be found when endometriosis is so widespread to interrupt normal vaginal structure, while fertility rates are normal when endometriosis is minimal. Endometriosis can also affect fertility in a different way. The messengers of white blood cells, such as Interleukin-6, Interferon and Tumor Necrosis Factor, also increase, adversely affecting the oocyte-sperm interaction. Serum samples obtained from women with endometriosis were found to be embryotoxic in mouse embryo models and to inhibit sperm motility in Vi tro (Halme, 1991), an increased effect when recombinant Tumor Necrosis alpha factor is added ( Eisermann, 1989). Those studies, however, were not addressed to the problem on which cytokines affect the progression of endometriosis but only showed the effects of such molecules on the viability of germ and embryonic cells.

Hormone therapy and surgery are the two therapeutic modalities currently used to treat endometriosis. The current pharmacological therapy for endometriosis requires hormonal suppression of estrogen production, so that the poor hormonal environment blocks the development of ectopic tissue. Regarding the treatment of infertility related to endometriosis, hormone therapy in patients with minimal disease is not of proven benefit, while other studies showed an increase in fertility rates (Arici et al., 1996b). Hormone therapies have included high doses of progestogens, combinations of estrogen and progesterone (using high-dose oral contraceptive pills, or OCPs, in a "pseudopregnancy" regimen), Danazol (an androgen derivative of ethisterone) and more recently GnRH agonists . These hormonal therapies are effective in pelvic pain and induce an objective regression of lesions, but have various warnings to stop treatment. Estrogens can stimulate and cause endometriotic tissue proliferation because they may be unable to respond to progesterone, even at high doses, so that OCPs can offer partial relief to a limited number of patients (Dawood, 1993). Progestational agents can cause irregular bleeding (50%) along with depression, weight gain, and fluid retention. Danazol suppresses endometriosis that evokes various responses, including the reduction of the levels of soluble tumor necrosis factor alpha, interleukin 1-beta and serum CD8 (Matalliotakis, 1997; Mori, 1990), the inhibition of de novo steroidogenesis and the displacement of estradiol from its receptor. Danazol can improve symptoms in approximately 66-100% of patients suffering from pain, but crude recurrence rates after up to 4 years are approximately 40% -50%. Other disadvantages of Danazol therapy are weight gain and androgenic side effects, which can cause up to 80% of patients to abandon this therapy (Barbieri, 1988). GnRH analogues are more potent and act longer than native GnRH, which work by eliminating estrogenic stimuli for the development of all estrogen-sensitive tissues. The side effects of GnRH analogues are mainly secondary to profound hypoestrogenemia, such as decreased bone density, and of recurrence that are up to 50% after 5 years (Waller and Shaw, 1993). Depending on the degree of disease, the surgical intervention may be conservative, if fertility is desired, or it may lead to the removal of the uterus, tubes and ovaries in case of severe disease. In any case, even limited surgical treatment leads to a significant decrease in fertility. The pregnancy rate after surgery in general is in the range of between 35% and 65%, so that patients need the induction of ovulation and intrauterine insemination to achieve normal fertility (Koninckx and Martin, 1994). Clinical reports show that, after laparotomy and resection of endometriosis, up to 40% of patients required reoperation within 5 years. Even after aggressive surgery, the recurrence of pain from endometriosis continued to be a significant problem. Some of the reasons for the failure of surgical therapy may include incomplete resection with lesions, either unrecognized or completely missing. Various lesions are microscopic and may not be able to be visualized despite the amplification provided by laparoscopy. Therefore, surgery alone can not be expected to cure this disease (Revelli et al., 1995). Since many patients with endometriosis suffer from problems of traditional therapies (including consequences of hormonal imbalance, high rate of recurrence and infertility). It is therefore of interest to provide alternative treatments for endometriosis. A possible therapeutic approach may be represented by the use of immunomodulatory molecules that might be able to improve endometriotic lesions and the immune situation. Such an approach has been considered feasible for the treatment of general symptoms (Rana et al., 1996) but there is no experimental evidence to indicate which cytokine, among all with altered levels of expression after endometriosis, could be a preferred target for intervention therapy. As previously stated, one of the various secretory products of macrophages involved in the inflammatory reaction by endometriosis is the Tumor Necrosis Factor (abbreviated, hereinafter, as in TNF). TNF, also defined as Cachectin, is a pleiotropic cytokine released by activated T cells and macrophages. TNF is a member of the Interferon, Interleukin and Cytokine network of the Colony Stimulation Factor, which has a key role in the signaling system with respect to the pathogenesis of many inflammatory diseases and infections by inducing a number of proinflammatory changes , including the production of another cytokine and adhesion molecule (Fiers, 1991). For convenience, the term TNF collectively will mean, in the full text of the present application, the alpha or beta Factor of Tumor Necrosis from animals or humans, together with alleles thereof, of natural origin, TNF-alpha (Pennica et al. , 1984) . TNF-beta, also referred to as lymphotoxin, has a similar activity but is produced by different cell types (lymphocytes and natural killer cells) in response to antigenic or mitogenic stimuli (Gray et al., 1984). TNF is expressed as a mature 17 kDa protein that is active as a trimer. This complex exerts its biological activity through the aggregation of its cell surface receptors, which mediate the specific effects in different organs and tissues. In the endometrium, the expression of TNF is dependent on the site and the menstrual cycle (Hunt et al., 1992), and induces apoptosis in the endometrium of experimental animals (Shalaby et al., 1989). The adhesion of the stromal cells of the endometrium to the mesothelial cells was significantly increased by pretreatment of the mesothelial cells with TNF (Zhang et al., 1993), supporting in this way the idea that TNF could contribute to the initiation and / or development of endometriosis. TNF exerts its activity, which is required for the normal development and function of the immune system, by linking a family of molecules of the receptor bound to the membrane that includes p55 TNF receptor I, also defined in the literature as TNF -RI, and receptor of TNF p75, defined in the literature also TNF-RII (Bazzoni u Beutler, 1996). The domain of TNF-RI and TNF signal transduction is suggested by the ability of specific agonist antibodies to this receptor to mimic most TNF-induced responses (Shalaby et al., 1990). By binding to its membrane binding receptors, TNF triggers the signaling pathway through cytoplasmic mediators such as TRADD and TRAP-1 (for TNF-RI) or TRAF-1 and TRAF-2 (for TNF-RII), leading to different cellular response such as T-cell proliferation, lysis of tumor cells in vitro, dermal necrosis, insulin resistance, apoptosis. The extracellular portions of both TNF receptors can be scattered and these soluble receptors retain the ability to bind TNF, inactivating the activity of TNF through the formation of high affinity complexes, thereby reducing the binding of TNF to the membrane receptors of TNF. target cells (Nophar et al., 1990). In the endometrium, the levels of TNF receptors bound to the membrane are affected by the administration of estradiol and / or progesterone, resulting in cell-specific and temporal expression of TNF-RI in mice (Roby et al., nineteen ninety six) . However, this study, like many other studies conducted in various models related to endometriosis, did not give any indication on the actual effect of TNF and TNF-RI, either membrane-bound or soluble, in the development of the endometriosis foci but only a description of the immunological abnormalities associated with endometriosis.

Brief Description of the Invention The present Patent Application is based on the assumption that a TNF antagonist is capable, through sequestration of circulating TNF, of blocking the progression of endometriotic lesions. This presumptive is confirmed by the finding reported in the example, which shows that a TNF antagonist significantly reduces the size of foci similar to endometriosis in an experimental rat model. As a result of the applicant's finding, a method is provided herein for treating and / or preventing endometriosis in an individual, which comprises the administration of a therapeutically effective antigen of TNF antagonist. In a second embodiment, the invention relates to a method of treatment and / or prevention of conditions related to endometriosis of infertility in an individual, which comprises the administration of a therapeutically effective cAntided of TNF antagonist in combination with other drugs. A further objective of the present invention is the use of a TNF antagonist together with a pharmaceutically acceptable carrier, in the preparation of pharmaceutical compositions for the treatment of endometriosis. In the present invention, the administration of the TNF antagonist may be parenteral or other effective formulations. Any mode of parenteral administration may be suitable, including intravenous, intramuscular administration. and subcutaneous. In addition to the pharmaceutically acceptable carrier, the composition of the invention may also comprise minor additive additives, such as stabilizers, excipients, buffers and preservatives. TNF antagonists useful in the method of the present invention include soluble TNF receptor molecules, anti-TNF antibodies and compounds that prevent and / or inhibit TNF receptor signaling. It is possible to use the TNF antagonist alone or in combination with other TNF antagonists. The combination with one or more pharmaceutically active products is also possible, in particular to improve the conditions of patients suffering from infertility related to endometriosis.

Description of the invention The invention described in the foregoing clearly shows the unexpected result that the sequestration of TNF (which is only one of the various cytokines whose level of expression increases in the peritoneal fluid after endometriosis), '' by means of A TNF antagonist reduces endometriosis-like foci in an experimental rat model.This model also demonstrates that this effect is obtained without significantly affecting the hormonal balance and activity of natural killer cells.The reduction of endometriotic lesions using antagonists of TNF can also improve fertility rates, since the normalization of the genital structure has a positive effect on the rate of implantation. Therefore, the main objective of the present invention is to provide a method for treating and / or preventing endometriosis in an individual, comprising administering a therapeutically effective antigen of TNF antagonist. In a second embodiment, the invention relates to a method for treating conditions of infertility related to endometriosis, in a individual, comprising administering a therapeutically effective antitumor TNF antagonist in combination with other drugs. A further objective of the present invention is the use of a TNF antagonist together with a pharmaceutically acceptable carrier, in the preparation of pharmaceutical compositions for the treatment and / or prevention of endometriosis. Pharmaceutical compositions prepared in this way are also a further objective of the present invention. The active ingredients of the compositions claimed herein are TNF antagonists. The claimed TNF antagonists exert their activity in one of two ways. First, antagonists can bind to or sequester the TNF molecule itself with sufficient affinity and specificity to substantially neutralize the TNF epitope responsible for binding to the TNF receptor (hereinafter referred to as "sequester antagonists"). Alternatively, TNF antagonists can inhibit the TNF signaling pathway activated by the cell surface receptor after TNF binding (hereinafter referred to as "signaling antagonists"). Both groups of antagonists are useful, either alone or together, in the therapy of endometriosis, according to the present invention. The TNF antagonists are ... easily identified and quantified by routine screening of candidates for their effect on the activity of native TNF on susceptible cell lines in vitro, for example human B cells, in which TNF causes proliferation and the secretion of Ig. The assay contains TNF formulation at varying dilutions of candidate antagonist, for example from 0.1 to 100 times the molar amount of TNF used in the assay, and controls without TNF or only antagonist (Tucci et al., 1992). The sequestering antagonists are the preferred TNF antagonists according to the present invention. Among the sequestering antagonists, those polypeptides that bind to TNF with high affinity and possess low immunogenicity are preferred. Soluble TNF receptor molecules and neutralizing antibodies to TNF are particularly preferred. For example, TNF-RI and TNF-RII are useful in the present invention. The truncated forms of these receptors, which comprise the extracellular domains of the receptors or the functional portions thereof, are antagonists more particularly preferred according to the present invention. The truncated forms of TNF-receptors are soluble and have been detected in urine and serum as TNF inhibitory binding proteins, 30 kDa and 40 kDa, which were originally, respectively called TBPI and TBPII (Engelmann et al. , 1990). The derivatives, fragments, regions and biologically active portions of the receptor molecules functionally resemble the receptor molecules that can be used in the present invention. Such equivalents or biologically active derivatives of the receptor molecule refer to the portion of the polypeptide, or to the sequence encoding the receptor molecule, which is of sufficient size and capable of binding to TNF with an affinity such that interaction with the receptor TNF bound to the membrane is inhibited or blocked. In a preferred embodiment, human soluble TNF-RI is the TNF antagonist to be administered to patients. Soluble, recombinant and natural TNF receptor molecules and methods of their production have been described in European Patent Applications EP 308,378, EP 398,327 and EP 433,900. Multimeric TNF receptor molecules and TNF immunoreceptor fusion molecules, and the derivatives or derivatives thereof are additional examples of receptor molecules useful in the methods of the present invention. The multimeric TNF receptor molecules useful in the present invention comprise all or a functional portion of the extracellular domain of two or more TNF receptors linked via one or more polypeptide linkers. The multimeric molecules may further comprise a signal peptide of a secreted protein to direct the expression of the multimeric molecule. These multimeric molecules and the methods of their production have been described in the European Patent Application EP 526905 Immunoreceptor TNF fusion molecules useful in the methods of the present invention comprise at least a portion of one or more immunoglobulin molecules and all or a functional portion of one or more TNF receptors. These immunoreceptor fusion molecules can be assembled as monomers, or hetero- or homo-multimers. The immunoreceptor fusion molecules can also be monovalent or multivalent. The fusion molecules of the TNF immunoreceptor and the methods for its production have been described in the Application for European Patent EP 620,739, corresponding to PCT Patent Application WO 94/06476. Another class of sequestering antagonists useful in the method of the present invention is represented by anti-TNF antibodies, including recombinant humanized, chimeric, monoclonal antibodies and fragments thereof which are characterized by high binding affinity to TNF in vivo. and due to low toxicity. The antibodies that can be used in the invention are characterized by their ability to treat patients for a sufficient period to have good to excellent regression of endometriotic lesions, improvement of symptoms and low toxicity. Neutralization antibodies are easily produced in animals such as rabbits or mice by immunization with TNF. Immunized mice are particularly useful for the provision of B cell sources for the manufacture of hybridomas, which in turn are cultured to produce large numbers of monoclonal anti-TNF antibodies. Chimeric antibodies are immunoglobulin molecules characterized by two or more segments or portions derived from different animal species. In general, the variable region of the chimeric antibody is derived of a non-human mammalian antibody, such as a murine monoclonal antibody, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. Preferably, both regions and the combination have low immunogenicity as is routinely determined (Elliott et al., 1994). Humanized antibodies are immunoglobulin molecules created by genetic engineering techniques in which the murine constant regions are replaced by human counterparts, while the murine antigen binding regions are retained. The resulting human-mouse chimeric antibody could have reduced immunogenicity and improved pharmacokinetics in humans (Knight et al., 1993). Preferred examples of high affinity monoclonal antibodies and chimeric derivatives thereof, useful in the methods of the present invention, are described in European Patent Application EP 186,833 and PCT Patent Application WO 92/16553. The TNF antagonist can be administered to an individual in a variety of ways. Routes of administration include the intradermal, transdermal (e.g. in slow release formulations), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural, topical, and intranasal Any other therapeutically effective route of administration can be used, for example absorption through epithelial or endothelial tissues or by gene therapy, wherein a DNA molecule encoding the TNF antagonist is administered to the patient (eg, - a vector) which causes the TNF antagonist to be expressed and secreted in vi vo. In addition, the TNF antagonist can be administered together with other components of biologically active agents such as pharmaceutically acceptable surfactants, excipients, diluents or any other carrier. The definition of "pharmaceutically acceptable" means that it encompasses any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered. For example, for parenteral administration, the TNF antagonist can be formulated in a unit dose form for injection into vehicles such as saline, dextrose solution, serum albumin and Ringer's solution. For parenteral administration (for example intravenous, subcutaneous, intramuscular), TNF antagonists can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle (eg water, saline, dextrose solution) and additives that maintain isotonicity (eg mannitol) or chemical stability (eg preservatives and buffers) . The formulation is sterilized by commonly used techniques. --- The bioavailability of TNF antagonists can also be improved by the use of conjugation procedures, which increase the half-life of the molecule in the human body, for example the binding of the molecule to polyethylene glycol, as described in PCT Patent Application WO 92/13095. Therapeutically effective antagonist TNF antagonists will be a function of many variables, including the type of antagonist, the affinity of the antagonist for TNF, any residual cytotoxic activity exhibited by the antagonists, the route of administration, the patient's clinical condition (including the desire to maintain a non-toxic level of endogenous TNF activity), the presence of multiple sites that combine TNF in sequestering agents, for example antibodies.

A "therapeutically effective cost" is such that when administered, the TNF antagonist results in the inhibition of the biological activity of TNF. The dose administered, either single or in multiple doses, to an individual will vary depending on a multiplicity of factors, including the pharmacokinetic properties of the TNF- antagonist, the route of administration, the conditions and characteristics of the patient (sex, age, body weight, health, height), degree of symptoms, concurrent treatments, frequency of treatment and the desired effect. The adjustment and manipulation of the established dosage intervals will be within the skill of those skilled in the art, as well as the in vitro and in vivo methods for determining the inhibition of TNF in an individual. Since the maximum tolerated dose of TNF in clinical trials in humans has been in the range of up to approximately 25 micrograms / m2 body surface / 24 hours, the antagonist content administered in general needs not to exceed a dose calculated to neutralize this cAntided by TNF. Accordingly, the molar dose of the TNF antagonist will vary from about 0.001 to 10 times the maximum tolerated molar dose of TNF, although as noted previously these will be subject to a high therapeutic discretion. . - In addition, the data obtained in clinical studies, where the increase in the concentration of TNF in the peritoneal fluid in women with endometriosis, was demonstrated using various protocols (Eisermann et al., 1988; Halme, 1991 -, - Overton et al. ., 1996), can also be useful in determining the effective dose of the TNF antagonist, to be administered. Usually, a daily dose of the active ingredient may be from about 0.01 to 100 milligrams per kilogram of body weight. Ordinarily from 1 to 40 milligrams per kilogram per day given in divided doses or in a sustained release form, it is effective to obtain the desired results. The second or subsequent administrations may be performed at a dose that is the same, less than or greater than the initial or previous dose administered to the individual. A second or subsequent administration may be administered during or before relapse of endometriosis or related symptoms. The terms "relapse" or "reoccurrence" are defined as encompassing the appearance of one or more of the symptoms of endometriosis. The TNF antagonist can be administered prophylactically or therapeutically to an individual before, simultaneously or sequentially with other regimens or therapeutic agents (e.g. multiple drug regimens), in a therapeutically effective cAntide, in particular for the treatment of infertility. TNF antagonists that are administered simultaneously with other therapeutic agents can be administered in the same or in different compositions. In particular, when infertility is the disorder associated with the endometriosis that is intended to be cured, biologically active human chorionic gonadotropin (hCG), luteinizing hormone (LH) or follicle stimulating hormone (FSH), either in a natural form, can be administered. highly purified or in a recombinant form. Such molecules and methods of their production have been described in European Patent Applications EP 160,699, EP 211,894 and EP 322,438. The present invention will now be illustrated by the example, which is not intended to be limiting in any way, and refers to the following figures.

Description of the Figures Figure 1 shows the effects of Antide (2 mg / kg, subcutaneously given every 3 days), recombinant soluble TNF-RI (10 mg / kg, given subcutaneously in two daily doses in a period of 1 week) on the size of grafts in rats with experimental endometriosis, 2 days and 9 days before the last treatment. These data, which were obtained using 6 animals / group for the first point at the time of sacrifice and 5 animals / group for the second point of time of sacrifice, represents the mean percentage of inhibition ± SEM. Figure 2 shows the effects of recombinant soluble TNF-RI (10 mg / kg subcutaneously given in two daily doses in a period of 1 week) and Antide (2 mg / kg subcutaneously given every 3 days) on NK activity in rat of splenic cells from rat against YAC cells (51CR release) 2 days (panel A) and 9 days after the last treatment (panel B). The data represent the average percentage of lysis ± SEM. Figure 3 shows the effects of recombinant soluble TNF-RI (10 mg / kg subcutaneously given in two daily doses in a period of one week) compared to the control and Antie (2 mg / kg, subcutaneously given every 3 days) on the serum levels of estradiol-17β on experimental endometriosis in rats. The data represent the mean concentration of estradiol-17β ± SEM.

Example Materials and methods Animals . Female rats Sprague-Dawley (250-275 g) were purchased from Charles River Italy (Calco, Lecco, Italy). The animals were housed under the following experimental conditions: temperature 22 ± 2 ° C, relative humidity 55 + 10%, ventilation air 15 ± 3 changes per hour filtered on 99,997% HEPA filters and artificial light with a 12-hour circadian cycle light (7:00 - 7:00 p.m.) Prior to the experiments, the animals were allowed to acclimate to these conditions for a period of at least a week. Animals were fed add l ibi tum for a standard diet of pellets.

Drugs of es tuudi o. Antide was prepared and provided by Bache, (California, United States of America). The recombinant soluble TNF-RI molecule, human, used in the example has a corresponding sequence of 20-180 of human TNF-RI - (Nophar et al., 1990) and was prepared in CHO cells and was provided by Interpharm Laboratories Ltd. (Israel) under the name of rh TBP-1. "~~" ~ Material . The general cell culture material was purchased from Gibco BRL, Life Technologies (Paisley, UK). The RIA team of estradiol-17ß was purchased from DPC (Los Angeles, California, United States of America). Inoketam was purchased from Virbac (Carros, France). Sodium chromate [51 Cr] was purchased from NEN Dupont (Boston, MA, United States of America). Rompun was acquired from Bayer AG (Leverkusen, Germany). The silk suture 7.0 was purchased from Ethicon (Pomezia, Italy).

Experimental model of endometriosis in ra ta. To explore the effects of recombinant soluble TNF-RI on endometriosis, a previously described experimental model was used (Jones, 1987), with minor modifications. Under anesthesia of Inoketam / Rompun, an autologous fragment of endometrial tissue (1 cm in length) was excised from the right uterine horn and placed in PBS at 37 ° C. The segment The uterus was opened by a longitudinal incision, and a section of • 5 x 5 mm was transplanted, without removing the myometrium, on the inner surface of the abdominal wall using a nonabsorbable silk suture at four corners.

Exploration of the effects of the drug under study in the experimental model of endometriosis. Experimental endometriosis was induced surgically in anesthetized rats as previously reported. In addition, another group of rats similarly had a fragment of a uterine horn removed, but a 5 x 5 mm square portion of fat surrounding the uterus (pseudo-operated or false group) was transplanted. An additional group of rats, which did not undergo any surgical procedure, remained as a normal control group. Three weeks after the induction of endometriosis, the animals underwent a second laparatomy (pre-treatment laparatomy) to evaluate the treatment and viability of the ectopic endometrial tissue. The surface area (length x width) was measured using a calibrator and recorded. The animals that showed viable grafts were assigned to the treatment groups designed as reported in Table I, so that at the end of the experiment, six animals / group were obtained for the first sacrifice time point and five animals / group for the second sacrifice time point. The treatments were initiated after a recovery period of 1 week. The control groups received saline alone; another group received three subcutaneous injections of 2 mg / kg of Antide every 3 days with a regimen previously shown to suppress ovarian and hypothalamic activity (Sharpe et al., 1990). An additional group received 10 mg / kg of soluble recombinant TNF-RI, divided into two daily doses over a period of 1 week. Table I (x) From the day of the surgical graft (day 1).

At the designated slaughter time points (2 and 9 days after the last treatment, for example 36 and 43 days after surgical grafting), the animals were anesthetized; Blood samples were collected from the abdominal aorta, the sera were separated and stored at -20 ° C until analyzed for the determination of estradiol-17β level. Spleens were removed for the measurement of the activity of natural killer cells (NK). The surface area of the similar foci of endometriosis was measured at each sacrifice time point, in order to normalize the data, the percentage variation versus the value of laparatomy treatment was calculated using the formula: (X - Xo) x 100 Xo where X0 is the size at the time of the pre-treatment laparatomy and X is the size at the time of sacrifice. The average value of the percentage variation in each group was then computed.

Termination of the activity of NK. The degree of NK activity was determined using the Cr51 release assay. The murine lymphoma YAC-1 cells were harvested during the exponential development phase and washed once with medium (RPMI 1640 containing penicillin / streptomycin, L-glutamine and 10% heat inactivated calf fetal serum). The pellet or cell button was incubated with 100 μCi of [Cr51] -sodium chromate at 37 ° C, 5% C02 for 2 hours. The cells were then washed 3 times with 10 ml of assay medium, resuspended to the desired concentration and added to the test plate in the presence of rat splenocytes. These were suspended in assay medium at the desired concentration (2xl06 / ml) and serial dilutions were carried out in triplicate in the assay medium, in wells of a 96-well U-shaped bottom plate, before the addition of target cells labeled with Cr51. Target cells labeled with Cr51 (5xl03) were added to each well of the assay plate and three effector-to-target ratios (200: 1, 100: 1 and 50: 1) were evaluated for each sample. The plate containing the effector-to-target cell sample was centrifuged at 200xg for 4 minutes and then incubated at 37 ° C, 5% C02 for 4 hours. after one Additional centrifugation of the plate at 200 x g for 4 minutes, 20 μl of the supernatant from each well was transferred to a glass fiber filter and the associated radioactivity was evaluated with a β-counter. The percentage of lysis was calculated as follows: pp? m_estra - pmSp? nt cpmtotal - cpm3pont where: cpmmuestra = the average release of Cr51 in the presence of the effector cells cpmspont = the average release of Cr51 from target cells in the presence of the culture medium cpmtoai = the average release of Cr51 from target cells in the presence of 1% Triton XlOO Determination of is tradi ol - 17ß. Serum estradiol-17β concentrations were determined using commercially available equipment to quantify estradiol in serum without extraction step (DPC, Los Angeles, California, United States of America). In summary, estradiol labeled with 1 5I competes with estradiol in the serum sample for the antibody sites. After the incubation, the separation of the bond from the free estradiol was achieved by decantation. The tube was then counted in a gamma counter (LKB-Pharmacia Wallak), the counts were inversely related to the amount of estradiol present in the serum sample. The amount of estradiol in the samples was determined by comparing the accounts a. a calibration curve. The anti-serum is highly specific for estradiol, with a relatively low cross-reactivity to other steroids of natural origin. Samples from the same experimental session were analyzed in a simple trial.

Statistic analysis . The statistical significance of the differences observed within the treatment groups was evaluated using the ANOVA present in the Statgraphics Plus® software (Version 1.4). Tukey's multiple range test was performed (P <0.05).

Results Exploration of the effects of TNF-RI Recombinant Soluble in experimental endometriosis The successful growth and development of surgically transplanted endometrial tissue in rats provided a search model that has been used to study some aspects of endometriosis that can not be adequately investigated in humans (Dudley et al., 1992). Previous studies in experimental rat endometriosis indicate that Antide works properly as a positive control (Sharpe et al., 1990). In the present example, the effect of Antide was compared, in terms of graft size dimension, before and after treatment, with those obtained using soluble recombinant TNF-RI, as summarized in Table II.

Table II The results are expressed in Figure 1 as the average percent inhibition of the grafted endometrium fragments (calculated as described above). Antide was effective in reducing the size of the endometriosis-like foci (Figure 1), inducing an almost complete (94% and 88% in comparison to the original dimension, respectively) and statistically significant remission (p <0.05, ANDEVA and Turkey test) in both observation times after stopping treatment. Treatment of 1 week with human recombinant soluble TNF-RI (10 mg / kg, two daily doses) resulted in significant size reductions (33% and 64% compared to the original dimension, respectively) of foci similar to endometriosis at both observation time points, but statistically significant (p < 0.05; ANdDEVA and Turkey test) only on day 9. No grafts were observed in the pseudo-operated animals at any time.

Evaluation of cell activities NK The activity of NK cells was evaluated by in vitro tests with splenic cells against YAC cells that showed no difference between the groups (Figure 2), similar to what was observed in baboons, where no difference was found in the cytotoxity. antiendometrial and in the activity of NK cells in animals with or without endometriosis (D'Hoogue et al., 1995). This finding found is in contrast to "Human data where decreased NK activity in patients with endometriosis has been reported with a significant correlation between reduced NK peritional activity and the severity of endometriosis (Osterlynck et al., 1992).

Evaluation of estradiol-l lß in serum.

The serum concentrations of estradiol-17β were measured by radioimmunoassay at different observation time points. A significant difference was observed in the groups found with Antide as a comparison to the untreated controls at the second observation time point. No statistically significant differences were observed for recombinant soluble TNF-RI, when compared to controls (Figure 3; p <0.05, ANDEVA and Turkey test).

Conclusions In the experimental model of rat endometriosis, the administration of a TNF antagonist, the soluble form of TNF-RI, provides, at the same time, a clear evidence of the potential effectiveness, of-1 treatment not related to hormones, - based on cytokine, of this pathological condition. Thus, TNF antagonists represent an alternative to existing medical treatments, in terms of reduced side effects. These studies evaluated the use of TNF antagonists in the treatment of infertility related to endometriosis. Those skilled in the art will know, or will be able to discern, using no more than routine experimentation, many equivalents to the specific embodiments of the inventions described herein. These and other equivalents are intended to be encompassed by the following claims.

List of references Arici, A., Tazuke, S.L., Attar, E., Liman, H.J. and Olive, D.L., 1996a, Mol. Hum. Reprod. 2, 40-45.

Arici, A., Oral, E., Bukulmez, O., Duleba, A., Olive, D.L., and Jones, E.E., 1996b, Fertile. Steril. 65, 603-607.

Arici, A., Oral, E., Attar, E., Tazuke, S.I., and Olive, D.L., 1997, Fertile. Sterile. 67, 1065-1072.

Barbieri, R.L., 1988, N. Engl. J. med. 318, 512-514.

Bazzoni, F. v Beutler, B., 1996, N. Engl. J. Med. 334, 1717-1725.

Cheung, A.N. , 1996, Curr .Opin. Obstert. Gynecol. 8, 46-51 D'Hooghe, T.M., Scheerlinck, J.P., Koninckx, P.R., Hill, J.A., and Bambra, C.S., 1995, Hum. Reprod. 10, 558-562.

Dawood, M.Y., 1993, Int. J. Gynaecol. Obstert. 40 (Suppl.), S29-42 Dudley, D.J., Hatasaka, H.H., Branch, D.W., Hammond, E., and Mitchel, M.D., 1992, Am. J. Obstet. Gynecol. 167, 1774-1780.

Eisermann, J., Gast, M.J., Pineda, J., Odem, R.R., and Collins J.L., 1988, Fertile. Steril. 50, 573-579.

Elliot, M.J., Maini, R.N., Feldmann, M., Long-Fox, A., Charles, P., Bijl, H., and Woody, J. N., 1994, Lancet 344, 1125-1127.

Engelmann, H., Novick, D., and Wallach, D., 1990, J. Biol. Chem. 265, 1531-1536.

Fiers, W., 1991, FEBS Lett, 285, 199-212.

Gray, PW, Aggarwal, BB, Benton, CV, Bringman, TS, Henzel, WJ, Jarrett, JA, Leung, DW, Moffat, B., Ng, P., and Svedersky, LP, 1984, Nature 312, 721- 724 Hahn, D.W., Carraher, R.P., Foldesy, R.G., and McGuire, J.L., 1986, Am.J.Obster. Gynecol. 155, 1109-1113.

Halme, J., 1991, Ann .N. And .Acad. Sci. 622, 266-274.

Harada, T., Yoshioka, H., Yoshida, S., Iwabe, T., Onohara, Y., Tanikawa, M., and Terakawa, N., 1997, Am. J. Obstet. Gynecol. 176, 593-597.

Ho, H.N., Wu, M.Y. and Yang, Y.S., 1997, Am. J. Reprod. Immunol. 38, 400-412.

Hormung, D., Ryan, I.P., Chao, V.A., Vigne, J.L., Schriock, E.D., and Taylor, R.N., 1997, J. Clin. Endocrinol .Metab. 82, 1621-1628.

Hunt, J.S., Chen, H.L., Hu, X.L., and Tabibzadeh, S., 1992, Biol. Reprod. 47, 141-147.

Jones, R.C., 1987, Acta Endocrinol. (Copen) 114, 379-382.

Knight, DM, Trinh, H., Le, J., Siegel, S., Shealy, D., Mcdonough, M., Scallon, B., Moore, MA, Vilcek, J., and Daddona, P., 1993 , Mol. Immunol. 30, 1443-1453.

Koninckx, P.R., Meuleman, C, Demeyere, S., Lesaffre, E., and Cornillie, F.J., 1991, Fertile. Steril. 55, 759-765.

MacSween, R.N.M., 1993, Muir's Texbook of pathology, 13a. ed. (Whaley K., ISBN 0-340-55145-3), 1024-1025.

Malinak, L.R., Buttram, V.C.J., Elias, S., and Simpson, J.L., 1980, Am. J. Obstet. Gynecol. 137, 332-337.

Matalliotakis, I .; Neonaki, M.M, Zolindaki, A., Hassan, E., Georgoulias, V., and Koumantakis, E., 1997, Int .J. Fertile .Womens .med. 42, 211-214.

Mori, H., Nakagawa, M., Itoh, N., Wada, K., and Tamaya, T., 1990, Am. J. Reprod. Immunol. 24, 45-50.

Nophar, Y., Kemper, 0., Brakebusch, C, Englemann, H., Zwang, R., Aderka, D., Holtmann, H., and Wallach, D., 1990, EMBOJ. 9, 3269-3278.

Oosterlynck, D.J., 'Meuleman, C, Waer, M., Vandeputte, M., and Koninckx, P.R., 1992, Fertile. Steril. 58, 290-295.

Overton, C, Fernandez-Shaw, S., Hicks, B., Barlow, D., and Starkey, P., 1996, HumReprod. 11, 380-386.

Pennica, D., Nedwin, G.E., Hayflick, J.S., Seeburg, P.H., Derynck, R., Palladino, M.A., Kohr, W.J., Aggarwal, B.B., and Goeddel, D.V., 1984, Nature 312, 724-729.

Rana, N., Braun, D.P., House, R., Gebel, H., Rotman, C, and Dmowski, W.P., 1996, Fertile. Steril. 65, 925-930.

Revelli, a., Modotti, M., Ansaldi, C, and Massobrio, M., 1995, Obset. Gynecol. Surv. 50, 747-754.

Roby, K.F., Laham, N., and Hunt, J.S., 1996, J. Reprod. Fertile 106, 285-290.

Shalaby, M.R. Laegreid, W.W., Ammann, A.J., and Liggitt, H.D., 1989, Lab. Invest. 61, 564-570.

Shalaby, M.R., Sundan, A., Loetscher, H., Borckhaus, M., Lesslauer, W., and Espevik, T., 1990, J. Exp. Med. 172, 1517-1520.

Sharpe, K.L., Bertero, M.C., and Venon, M.W., 1990, Prog. Clin. Biol. Res. 323, 449-58.

Tucci, A., James, H., Chicheportiche, R., Bonnefoy, J.Y., Dayer, J.M., and Zubler, R.H., 1992, J. Immunol. 148, 2778-2784.

Waller, K.G. and Shaw, R.W., 1993, Fertile -Steril. 59, 511-515.

Zhang, R.J., Wild, R.A., and Ojago, J.M., 1993, Fertil-Steril. 59, 1196-1201.

Claims (17)

1. The use of a TNF antagonist together with a pharmaceutically acceptable carrier, in the preparation of a pharmaceutical composition for the treatment and / or prevention of endometriosis.

2. The use according to claim 1, wherein the TNF antagonist is a polypeptide capable of binding to a specific epitope of TNF in a manner such that TNF is no longer capable of binding to a TNF receptor bound to the membrane.

3. The use according to claim 2, wherein the TNF antagonist is a receptor molecule, derivative or a fragment thereof, which binds to TNF.

4. The use according to claim 3, wherein the receptor molecule is selected from the group consisting of: TNF-RI and TNF-RII.

5. The use according to claim 3, wherein the receptor molecule is the extracellular domain of TNF-RI.

6. The use according to claim 3, wherein the receptor molecule is soluble, human recombinant TNF-RI.

7. The use according to claim 3, wherein the receptor molecule is a multimeric TNF receptor molecule or a functional portion thereof.

8. The use according to claim 7, wherein the TNF receptor multimeric molecule comprises all or a functional portion of two or more extracellular domains of TNF receptors linked via one or more pqlipeptide linkers.

9. The use according to claim 3, wherein the receptor molecule is an immunoreceptor fusion molecule or a functional portion thereof.

10. The use according to claim 9, wherein the immunoreceptor fusion molecule comprises all or a functional portion of a TNF receptor and an immunoglobulin chain.

11. The use according to claim 1, wherein the TNF antagonist is an anti-TNF antibody or a fragment thereof.

12. The use according to claim 11, wherein the monoclonal antibody is selected from the body consisting of: a chimeric monoclonal antibody, a humanized monoclonal antibody or fragment thereof.

13. The use according to claim 1, wherein the TNF antagonist prevents or inhibits the signaling of the TNF receptor.

14. The use of a TNF antagonist together with a pharmaceutically acceptable carrier in the preparation of a pharmaceutical composition, to improve implantation and fertility rate by reducing endometriotic lesions.

15. The pharmaceutical composition containing a TNF antagonist, together with a pharmaceutically acceptable carrier, for the treatment and / or prevention of endometriosis.

16. The pharmaceutical composition containing a TNF antagonist, together with a pharmaceutically acceptable carrier, to improve implantation and fertility rate by reducing endometriotic lesions.

17. The pharmaceutical composition according to claim 15 or 16, wherein the TNF antagonist has the characteristics described in any of claims 1 to 13.