AU2003204933A1 - EG-VEGF/prokineticin 2-receptor antagonists - Google Patents

Abstract

A composition that contains, as active agent (A), an endocrine gland vascular endothelial growth factor (EG-VEGF) nucleic acid, polypeptide or antisense nucleic acid, antibody against EG-VEGF or its receptor (EG-VEGF-R), or EG-VEGF-R antisense nucleic acid, is useful for the treatment or prevention of endometrial diseases : Independent claims are also included for the following: (1) method for detecting uterine receptivity by determining the amount of EG-VEGF polypeptide and/or nucleic acid, using the new composition; or (2) test system for identifying antagonists (B) of EG-VEGF-R. ACTIVITY : Cytostatic; Gynecological. No details of tests for these activities are given. MECHANISM OF ACTION : Inhibiting activity or expression of endocrine gland vascular endothelial growth factor (EG-VEGF) , which is overexpressed in patients with endometriosis; gene therapy.

Description

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AUSTRALIA

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ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Schering AG Mullerstrasse 178 13353 Berlin Germany Address for Service: DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Invention Title: EG-VEGF/prokineticin 2-receptor antagonists The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 EG-VEGF/Prokineticin 2-Receptor Antagonists The invention relates to EG-VEGF-/prokineticin 2-receptor antagonists and their use as pharmaceutical agents for the treatment of diseases of the endometrium, such as, endometriosis, endometrial carcinoma and dysfunctional bleeding.

Endometriosis is one of the most common gynecological diseases by which roughly 2-10% of all women of reproductive age are affected (Gazvani and Templeton 2002, Reproduction 123, 217-226; Smith 2001, Trends Endocrinol. Metab. 12, 147-151).

The cause is the ectopic implantation of endometrial tissue mainly in the peritoneal cavity. In addition to pain and numerous other symptoms, many endometriosis patients are sterile. Reasons for the sterility are largely unknown (Ryan and Taylor 1997, Obstet.

Gynecol. Surv. 52, 365-371; Haney 1997, Reprod. Med. Rev. 6, 145-161).

No causal therapy is available for treating endometriosis at this time. The treatment of endometriosis is based on the concept of the withdrawal of estrogen. After an invasive intervention, in which endometriosis foci are removed or obliterated during a laparoscopy or laparotomy, a medicinal treatment normally follows, which by the induction of a hypoestrogenicity results in an at least partial inhibition of the proliferation of endometriosis foci that remain but cannot be detected (Sillem 1998, Programmed® 23, Suppl. 1, 1-28). The medications that are used have numerous side effects partly because of their androgenicity, and they result in breakthrough bleeding, menopausal symptoms and osteopenia. Although the medicinal treatment has proven effective for an initial treatment of endometriosis, to date no reduction in the high rate of recurrence has been achieved.

Endometrial carcinoma is the fourth most common form of cancer in Western countries. Based on the stage of the diagnosis, the survival rate after 5 years is 27% to 88%. A primary cause seems to be an elevated estrogen level (Akhmedkhanov et al.

2001, Ann. N. Y. Acad. Sci. 943, 296-315). This results in increased proliferation of cells of the endometrium, which can cause errors in DNA replication and somatic mutations and can ultimately result in a malignant tumor. As in other tumors, angiogenesis plays an essential role in the clinical picture of endometrial carcinoma.

Several studies have noted an increased vascularization and an overexpression of the endothelial growth factor VEGF (vascular endothelial growth factor) and its receptors (Abulafia et al. 1999, Gynecol. Oncol. 72, 220-231).

At present, several therapies are used to treat endometrial carcinoma.

Antiestrogens show good effects, but an additional therapy with cytostatic agents or radiation must often also be performed.

About 10% of women regularly suffer from strong bleeding during menstruation caused by changes in the blood vessels of the endometrium. An elevated proliferation rate of the endothelial cells was detected in the endometrium of such women (Kooy 1996, Hum. Reprod. 11, 1067-1072). In addition, structural differences in the blood vessels were observed, such as, for example, the incomplete build-up of the smooth muscle cell layer (Abberton et al. 1999, Hum. Reprod. 14, 1072-1079). Since the blood loss during menstruation is partly controlled by the constriction of blood vessels, it suggests that the defects of the smooth muscles contribute significantly to the bleeding.

In addition to operative intervention (hysterectomy), treatment can also be done medicinally. Steroid hormones are also used here. In addition, non-steroidal antiinflammatory substances are used. Said substances are very effective but have many side effects, so that they can be used only for a relatively short time (Irvine and Cameron 2000, Baillieres Best Pract Res Clin Obstet Gynaecol. 13, 189-202).

The urgent need exists to find new treatment methods and substances that make possible a specific treatment of the diseases of the endometrium. This invention solves the problem by a new therapy method, in which EG-VEGF, prokineticin 2 and the corresponding receptors are used as target substances for new medications.

EG-VEGF (endocrine gland derived vascular endothelial growth factor), also referred to as prokineticin 1, was already described as a specific growth factor for endothelial cells from the adrenergic cortex of the adrenal glands. Its expression was detected in the ovary, the testis, the placenta and in the adrenal glands (LeCouter et al.

2001, Nature 412, 877-884). In addition, contractile effects on the smooth muscles of the gastrointestinal tract were detected (Li et al. 2001, Mol. Pharmacol. 59, 692-698). In WO 02/00711 (Genentech, Inc), the nucleic acid sequence and the corresponding amino acid sequence of the EG-VEGF polypeptide are disclosed, as well as their use in the treatment of diseases that are associated with a hormone-producing tissue ovary). EG-VEGF and prokineticin 2 (also named Bv8) belong to the same family: they have a 60% identity and are 75% similar. Both proteins have a preserved area that comprises 10 cysteine radicals (Wechselberger et al., 1999, FEBS Lett. 462, 177-181). Expression of prokineticin 2 was detected almost exclusively in the testis and in the brain (Wechselberger et al., 1999, FEBS Lett. 462, 177-181; LeCouter et al., 2003, Proc. Natl.

Acad. Sci. USA 100, 2685-2690). Prokineticin 2 promotes angiogenesis in the testis (LeCouter et al., 2003, Proc. Natl. Acad. Sci. USA 100, 2685-2690), contraction of the smooth muscles of the gastrointestinal tract (Li et al. 2001, Mol. Pharmacol. 59, 692-698) and mediates the day/night cycle of the suprachiasmatic nucleus of the brain (Cheng et al.

2002, Nature 417, 405-410). The meaning of EG-VEGF or prokineticin 2 in the endometrium is not considered in detail. The role of EG-VEGF or prokineticin 2 in diseases of the endometrium was not previously known.

In this invention, it was possible to show that EG-VEGF is expressed in the endometrium and is ramped up in the so-called implantation window of the cycle (Figures 3 and By means of semi-quantitative and quantitative determinations, the mRNA from the human endometrium was compared to three clinical study groups. The result of these studies indicated that the mRNA of EG-VEGF in comparison to control group a (before the implantation window is opened) is ramped up in group b (time of the open implantation window) in healthy women. Also, in group c (endometriosis patients), an increased amount of EG-VEGF-mRNA was noted.

The binding of EG-VEGF or prokineticin 2 to two related G-protein-coupled receptors (GPR73a and GPR73b) results in the activation of the receptor. These physiological binding partners of the EG-VEGF polypeptide and the prokineticin 2 polypeptide were only recently identified and characterized (Lin et al. 2002, J. Biol.

Chem. 277, 19276-19280). The effects that are mediated by the receptors are responsible for the biological effects of EG-VEGF and prokineticin 2, which in turn are responsible for the pathological diseases of the endometrium.

In this invention, it was possible to show that the expression of the EG-VEGF, the prokineticin 2 and the EG-VEGF receptor GPR73a in lesions that originate from different patients affected by endometriosis is significantly higher than in the healthy tissue.

(Figure In addition, it was possible to identify a new human splice-variant of GPR73a (Fig. 2c). The RNA of this variant contains an additional exon, which, however, contains a stopcodon and thus results in a shortened protein. The shortened receptor only goes up to the third transmembrane domain and should thus not be able to mediate the signal transduction. The receptor could be a dominant-negative inhibitor of GPR73a or GPR73b that recovers ligands or binds to heterodimers.

An inhibition of the binding of growth factor EG-VEGF or prokineticin 2 to the EG-VEGF receptors, on the one hand, or a direct inhibition of the EG-VEGF polypeptide or prokineticin 2 polypeptide suppresses the biological function of EG-VEGF or prokineticin 2. This inhibition makes possible the specific treatment of the above-cited diseases. The invention provides pharmaceutical agents for this specific treatment.

The invention therefore relates to the use of a pharmaceutical composition that as an active component contains a substance that is selected from the group of: an EG- VEGF nucleic acid or a prokineticin 2 nucleic acid, an EG-VEGF polypeptide or a prokineticin 2 polypeptide, an antibody that is directed against EG-VEGF or prokineticin 2, an EG-VEGF-antisense nucleic acid or a prokineticin 2-antisense nucleic acid, an EG- VEGF/prokineticin 2 receptor, an EG-VEGF/prokineticin 2-receptor-antisense nucleic acid and an antibody that is directed against an EG-VEGF/prokineticin 2 receptor, for the production of a medication for treating or preventing diseases of the endometrium, especially endometriosis, endometrial carcinoma or dysfunctional bleeding.

EG-VEGF nucleic acid comprises DNA, cDNA and RNA, or parts thereof. The DNA and protein sequences are shown in Fig. 1. The EG-VEGF nucleic acid is preferably a DNA.

Prokineticin 2 nucleic acid comprises DNA, cDNA and RNA, or parts thereof.

TheDNA and protein sequences are desribed in the GeneBank data base under accession no. NM_021935. The prokineticin 2 nucleic acid is preferably a DNA.

EG-VEGF/prokineticin 2-receptor-nucleic acid comprises DNA, cDNA and RNA, or parts thereof. The DNA and protein sequences are shown in Figures 2a and 2b.

The DNA and protein sequences of the new splice variants are shown in Figure 2c. The EG-VEGF/prokineticin 2-receptor-nucleic acid is preferably a DNA.

EG-VEGF polypeptide is defined as the entire sequence as well as parts thereof.

Prokineticin 2 polypeptide is defined as the entire sequence as well as parts thereof.

EG-VEGF-antisense nucleic acid is a DNA and/or RNA that is complementary to an EG-VEGF mRNA. It can comprise the entire complementary sequence or partial sequences.

Prokineticin 2-antisense nucleic acid is a DNA and/or RNA, which is complementary to a prokineticin 2 mRNA. It can comprise the entire complementary sequence or partial sequences.

EG-VEGF/prokineticin 2-receptor-antisense nucleic acid is a DNA and/or RNA that is complementary to an EG-VEGF/prokineticin 2-receptor mRNA. It can comprise the entire complementary sequence or partial sequences.

An antibody against EG-VEGF, prokineticin 2, or EG-VEGF/prokineticin 2receptors can be monoclonal or polyclonal. It can be directed against EG-VEGF, prokineticin 2, or EG-VEGF/prokineticin 2-receptors or against fragments thereof. Such an antibody can be obtained according to standard methods by immunization of test animals.

The pharmaceutical compositions of the invention are produced with commonly used solid or liquid vehicles or diluents and commonly used pharmaceutical and technical adjuvants according to the desired type of administration with a suitable dosage in a way that is known in the art. Tablets can be obtained, for example, by mixing the active ingredient with known adjuvants, for example inert diluents such as dextrose, sugar, sorbitol, mannitol, polyvinyl pyrrolidone, explosives such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as carboxypolymethylene, carboxymethyl cellulose, cellulose acetate phthalate or polyvinyl acetate. Capsules that contain active ingredient can be produced, for example, by the active ingredient being mixed with an inert vehicle such as lactose or sorbitol and encapsulated in gelatin capsules.

The pharmaceutical compositions according to the invention can also be used in suitable solutions, such as, for example, physiological common salt solution.

For parenteral administration, especially oily solutions, such as, for example, solutions in sesame oil, castor oil and cottonseed oil, are suitable. To increase solubility, solubilizers, such as, for example, benzyl benzoate or benzyl alcohol, can be added.

The pharmaceutical composition according to the invention can also be used to diagnose endometriosis and endometrial carcinoma. In this case, the amounts of EGr VEGF or prokineticin 2 are determined, for example, by an ELISA test or a protein chip.

An elevated expression of the EG-VEGF or the prokineticin 2 shows the presence of an endometriosis or an endometrial carcinoma.

In addition, this invention can be employed by use of the pharmaceutical composition as an agent for gene therapy. In this connection, the effects of EG-VEGF or prokineticin 2 are blocked by the use of gene therapy. In this case, a vector that contains an EG-VEGF-antisense sequence or a prokineticin 2-antisense sequence, or an EG- VEGF/prokineticin 2-receptor-antisense sequence is designed and applied. Examples are vectors that are derived from adenovirus, adenovirus-associated virus, Herpes simplex virus or SV40. The gene therapy can be performed as described (Gomez-Navarro et al.

1999, Eur. J. Cancer, 35, 867-885). The administration can be done locally, directly into the uterus or systemically, via the blood circulation. This results in a blocking of the expression of EG-VEGF, prokineticin 2, or the EG-VEGF/prokineticin 2 receptor in the endometrium. As a result, the biological function of the EG-VEGF or the prokineticin 2 is inhibited.

Another use of gene therapy consists in that a vector that contains the sequence of the GPR73a-splice variant is designed and applied. Examples are vectors that are derived from adenovirus, adenovirus-associated virus, Herpes simplex virus or SV40. The gene therapy can be performed as described (Gomez-Navarro et al. 1999, Eur. J. Cancer, 867-885). The administration can be done locally, directly into the uterus, or systemically, via the blood circulation. This results in a blocking of the function of the EG-VEGF/prokineticin 2 receptor GPR73a and/or GPR73b in the endometrium. As a r result, the signal transduction that is induced by EG-VEGF or prokineticin 2 is stopped prematurely.

This invention also relates to the use of a pharmaceutical composition according to the invention for detecting uterine receptivity. The uterine receptivity is a requirement for a successful nidation ofblastocysts. This deposition can be carried out only during a short time span, the so-called implantation window, which is opened in humans duringthe middle secretory phase, thus about 7-9 days after ovulation. Since this invention shows that the EG-VEGF expression is ramped up at the time of the open implantation window, the suitable time for a transfer of the ovocytes that are fertilized by means of an in vitro fertilization can be determined by measuring the amount of EG-VEGF mRNA or EG-VEGF polypeptide. The measurement is made by a Northern Blot, a PCR or chip hybridization or by an immune test.

In addition, the invention relates to a method for detecting the uterine receptivity, whereby the amount of EG-VEGF polypeptide and/or the amount of EG-VEGF nucleic acid is determined with the aid of a pharmaceutical composition according to the invention. First, a blood or endometrium sample is taken from the woman, and then the amount of EG-VEGF mRNA or EG-VEGF polypeptide is determined.

This invention relates to the use of the pharmaceutical composition according to the invention for identifying substances that influence the effects of EG-VEGF or prokineticin 2. Effectors are substances that influence the biological activity of the EG- VEGF polypeptide or prokineticin 2 polypeptide, either by binding to the EG-VEGF polypeptide or prokineticin 2 polypeptide or by competition with the EG-VEGF polypeptide or prokineticin 2 polypeptide with its binding to its physiological binding partner. Effectors can also be substances that preferably bind to the receptor splice variants and thus modulate or inhibit the function of GPR73a or GPR73b. Effectors can be low-molecular substances or else peptides or proteins, such as, antibodies.

Nucleic acids such as spiegelmers or aptamers that can bind the target molecule specifically because of their spatial structuring and thus can prevent an interaction of a ligand with its receptor represent another possibility. To identify such effectors, a cell that expresses an EG-VEGF/prokineticin 2 receptor is treated with EG-VEGF or prokineticin 2. This results in a change of the intracellular calcium level, which is measured in turn. Antagonists are substances that can inhibit the calcium inflow. In addition, the receptor splice variants together with GPR73a or GPR73b can be coexpressed to search for substances that selectively cause the heterodimerization and thus block the EG-VEGF- or prokineticin 2-induced signals. Other detection systems for the screening of EG-VEGF/prokineticin 2 antagonists can also be used.

Description of the Figures Fig. 1 shows the DNA and protein sequence of the human EG-VEGF.

Fig. 2 shows the DNA and protein sequence of the human EG-VEGF/prokineticin 2 receptors GPR73a GPR73b (2b) and the new splice variant of GPR73a (2c).

Fig. 3 shows the expression of RNA from EG-VEGF in the endometrium that is detected by the RT-PCR method. A ramping up of the human EG-VEGF gene in group b (LH+7-9/fertile) in comparison to group a (LH+2-4) is clear. Specific "primers" for the EG-VEGF were used. As an internal control, an antisense-S9 probe was amplified.

After the PCR amplification, the products were separated onto an agarose gel and stained with ethidium bromide. The intensity of the bands was quantified. Group a: before the opening of the implantation window; group b: time of the opened implantation window in healthy women; LH: luteinizing hormone; 2-4 or 7-9: period in days.

Fig. 4 shows the expression of RNA from EG-VEGF in the endometrium that is detected by the TaqMan method. A ramping up of the human EG-VEGF gene in groups b (LH+7-9/fertile) and c (LH+7-9/endometriosis), in comparison to group a is clear. Specific "primers" for the EG-VEGF were used. In addition, the amounts of LIF and calcitonin mRNA, two genes that are ramped up in a known way at the time of the implantation window, were determined. The amounts of PCR products were determined by staining with SyberGreen. "All amounts of mRNA were adjusted to the amount of actin mRNA. Group a: before the opening of the implantation window; group b: time of the opened implantation window in healthy women; group c: time of the opened implantation window in endometriosis patients; LH: luteinizing hormone; 2-4 or 7-9: period in days.

Fig. 5 shows the expression of the RNA of EG-VEGF and EG-VEGF receptor GPR73a (here named EG-VEGF Rl) in lesions that originate from patients affected by endometriosis and that are detected by the TaqMan method. The relative mRNA expression was adjusted to the amount of cyclophilin-mRNA. In female patients DF3 Ihl, DF33hl and DF26hl, this is eutopic tissue of healthy control patients (white bars); the samples of endometriosis patients AV103, AV 104, FC108 and DF51 originate from the endometrium (eutopic, hi; gray bars) or from ectopic lesions (el or e3; black bars). A ramping up of the human gene for the GPR73a (EG-VEGF R1) in different lesions in comparison to healthy tissue is clear. Specific "primers" for the GPR73a (EG- VEGF R1) were used. As a control, the GPR73a (EG-VEGF R1)-RNA amounts in the testis and in the endometrium were measured. The amounts of PCR products were determined by staining with SyberGreen.

Examples Example 1: Determining the EG-VEGF-RNA amounts in endometrial samples by semiquantitative RT-PCR analysis 1. Group a (LH plus 2-4 days): 2-4 days after the ovulation is triggered by the release of the luteinizing hormone the endometrium is still not receptive for an embryo. This group consisted of female patients who participated in an IVF program because of a tubal obstruction or because the man was sterile and who had a normal endometrium.

2. Group b (LH plus 7-9 days): At this time, the "implantation window" is open, and the endometrium is receptive to the deposition of the blastocysts. Through the opening of the "implantation window," the expression or inhibition of genes is expected, whose protein products play an essential role in the nidation of the embryo.

3. Group c (LH plus 7-9 days plus endometriosis): At this time, the implantation window in healthy women is open. The patients of this group were sterile for unknown reasons, however, and suffered from endometriosis.

Endometrium was removed from patients who belong to the two groups LH+2-4/fertile and LH+7-9/fertile and was quick-frozen in liquid nitrogen.

Total-RNA with the RNeasy Kit (Qiagen) was isolated therefrom. Then, a DNase I-digestion (invitrogen) was made. Beginning with 5.7 pg oftotal-RNA, a firststrand synthesis was then performed with the ProSTAR First-Strand RT-PCR kit of stratagene. For the semi-quantitative determination of the EG-VEGF amounts, ptl of first-strand DNA and specific primer were used for EG-VEGF TCAATCATGCTCCTCCTAGTAACTGTG-3' and AAGTCCATGGAGCAGCGGTAC-3'). The PCR conditions were: 2 minutes, 94C; 20 seconds, 94 0 C; 20 seconds, 65 0 C; 40 seconds, 72°C (40 cycles); minutes, 72'C. As an internal control, S9-RNA was amplified (primer: AGGACCCACGGCGTCTGTTCG-3' and ATCCAGCAGCCCAGGAGGGAC-3'). The PCR conditions were: 2 minutes, 94°C; 20 seconds, 9.4 0 C; 20 seconds, 60 0 C; 40 seconds, 72 0 C (24 cycles); minutes, 72 0 After the reaction, the amplification products were separated onto a 2% agarose gel and stained with ethidium bromide. The intensity of the bands was determined with the GelDoc device. (Fig. 3) Example 2: Determining the EG-VEGF-RNA amounts in endometrial samples by real-time quantitative RT-PCR analysis Endometrium was removed from patients who belong to the three groups LH+2-4/fertile LH+7-9/fertile and LH+7-9/endometriosis and was quick-frozen in liquid nitrogen. Total-RNA was isolated by means of TRIZOL (invitrogen) from the tissues that were pulverized by "mortars" under liquid nitrogen. Starting from 5 p.g of total-RNA, first a DNase I-digestion (invitrogen) and then a first-strand synthesis were performed with use of the SUPER-SCRIPT II First-Strand Synthesis System for RT-PCR (invitrogen). For the amplification of the transcripts for relative quantification, 0.125 p.1 of first-strand DNA was used. With use of the specific primer pairs (EG-VEGF: CTTCTTCAGGAAACGCAAGCA-3' and 5'-CGGGAACCTGGAGCACAG-3'; calcitonin: 5'-CAACTTTGTGCCCACCAATGT-3' and GAGTCATTCAGCTGCTCAGGC-3'; leukemia inhibitory factor (LIF): CTAGTTCCCCACCTCAATCCC-3' and and the SYBR Green PCR Master Mix (PE Biosystems), the amplification was performed under the following PCR conditions: 10 minutes, 95 0 C; seconds, 95 0 C; 1 minute, 60 0 C (40 cycles). As an internal control, the RNA was amplified for subunit 1A of the cytochromoxidase (primer: CGTCACAGCCCATGCATTTG-3' and The measurement of the fluorescence as a yardstick for the increase of amplification products was carried out online by means of an ABI Prism 7700 Sequence Detector (PE Biosystems). The purity of the amplification products was examined by plotting melt curves. (Fig. 4) Example 3: Determining the EG-VEGF, prokineticin 2, and EG-VEGF-receptor (GPR73a) RNA amounts in lesions of female patients.

After removal, the tissue was quick-frozen in liquid nitrogen. Total-RNA was isolated by means of TRIZOL (invitrogen) from the tissues that were pulverized by "mortars" under liquid nitrogen. A concentration of the polyA RNA was carried out in a batch process with use of oligo-dT cellulose (Pharmacia). Starting from 100-400 ng ofpolyA+-RNA, a first-strand synthesis was performed with use of the SUPER-SCRIPT II First-Strand Synthesis System

I

for RT-PCR (invitrogen). For the amplification of the transcripts for relative quantification, 0.25 pl1 of first-strand DNA was used. With use of the specific primer pairs (EG-VEGF: 5'-CTTCTTCAGGAAACGCAAGCA-3' and CGGGAACCTGGAGCACAG-3'; EG-VEGF recptor GPR73a: ATTGACAGGTATCTGGCTATTGTCC-3' and CCAAGGCAATCAGGCCAGT-3'); corresponding TaqMan samples (EG- VEGF: 5'-TET-ACACCTGTCCTTGCTTGCCCAACC-TAMRA-3'); EG- VEGF receptor GPR73a: CCAAACA-TAMRA-3'), and the TaqMan PCR Master Mix (PE Biosystems), the amplification as a muliplex-PCR was performed under the following PCR conditions: 2 minutes, 50 0 C; 10 minutes, 95 0 C; 15 seconds, 95 0 C; 1 minute, 62 0

C

cycles). As an internal control, RNA was amplified for cyclophilin (primer: 5'-GAAGTTGGCCGCATGAAGA-3' and 5'-GCCTAAAGTTCTCGGCCGT-3'; sample: 5'-VIC-CGAGCTCTTTGCAGACGTTGTGCCT-TAMRA-3'). The measurement of the fluorescence as a yardstick for the increase of amplification products was carried out online by means of ABI prism 7700 Sequence Detector (PE Biosystems).

For the relative quantification of the prokineticin 2-RNA, 0.5 pl of firststrand DNA was used, starting from polyA -RNA. With use of specific primer pairs (5'-CTCCTGTCATGGCACGGAA-3' and AGCCCAACAGCAGAGCTGAA-3') and the SYBR Green PCR Master Mix (Eurogentec), the amplification was performed under the following PCR conditions: 2 minutes, 50 0 C; 10 minutes, 95'C; 15 seconds, 95 0 C; and 1 minute, (40 cycles). As am internal control, in turn the RNA was amplified for cyclophilin (Fig. Example 4: Test system for tracing receptor antagonists of the EG- VEGF/prokineticin 2 receptor A cell line CHO or HEK) is established that expresses the EG- VEGF/prokineticin 2 receptors GPR73a, GPR73b or the splice variant of GPR73a in a transient or stable manner. After a ligand is added, a substance data base is checked to find substances that displace the ligand binding (binding assay). As an alternative, a functional assay is performed. The calcium release that is caused by the ligands is measured, for example, by an aequorin-based luminescence assay.

Here, an expression plasmid for aequorin in the cells is co-transfixed. Aequorin is bonded by the released calcium, and this results in increasing the luminescence.

The inhibition of this activity by a receptor antagonist is used as a measurement variable for the tests.

Throughout this specification and the claims which follow, unless the context requires otherwise the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as an acknowledgment or any form of suggestion that, that prior art forms part of the common general knowledge in Australia.

Claims (9)

1. Use of a pharmaceutical composition that as an active component contains a substance that is selected from the group of an EG-VEGF nucleic acid, an EG-VEGF polypeptide, an antibody that is directed against EG-VEGF, an EG-VEGF-antisense nucleic acid, a prokineticin 2 nucleic acid, a prokineticin 2 polypeptide, an antibody that is directed against prokineticin 2, a prokineticin 2-antisense nucleic acid, an EG-VEGF/prokineticin 2 receptor, an EG-VEGF/prokineticin 2- receptor-antisense nucleic acid and an antibody that is directed against an EG-VEGF/prokineticin 2 receptor, for the production of a medication for treating or preventing diseases of the endometrium.

2. Use of a pharmaceutical composition according to claim 1, whereby the disease is endometriosis.

3. Use of a pharmaceutical composition according to claim 1, whereby the disease is endometrial carcinoma.

4. Use of a pharmaceutical composition according to claim 1, whereby the disease is dysfunctional bleeding.

Use of a pharmaceutical composition according to claim 1 for the diagnosis of endometriosis or endometrial carcinoma.

6. Use of a pharmaceutical composition according to claim 1 as an agent for gene therapy, whereby the active component is an EG-VEGF or prokineticin 2 nucleic acid.

7. Use of a pharmaceutical composition according to claim 1 for detecting uterine receptivity.

8. Method for detecting uterine receptivity, characterized in that the amount of EG-VEGF polypeptide and/or the amount of EG-VEGF nucleic acid is determined with the aid of a composition according to claim 1.

9. Test system for tracing EG-VEGF/prokineticin 2-receptor antagonists, whereby the following components are brought into contact with one another: a cell line that expresses the EG-VEGF/prokineticin 2- receptor GPR73a or GPR73b or the splice variant of GPR73a in a transient or stable manner; a ligand that binds these receptors; and a substance data base that contains potential antagonists. The tracing of antagonists can be carried out by a binding test or via a functional test, such as, for example, the measurement of the calcium release. Use of an EG-VEGF/prokineticin 2-receptor antagonist for the production of a medication for treating endometriosis, whereby the above-mentioned EG-VEGF/prokineticin 2-receptor antagonist in a cell that expresses an EG-VEGF/prokineticin 2 receptor inhibits by activation the intracellular calcium-ion concentration that is released by a ligand by at least DATED this 2 6 th day of June, 2003 SCHERING AG By Their Patent Attorneys DAVIES COLLISON CAVE