EP0517902A1 - Proteine ayant un effet inhibiteur sur la choriogonadotropine humaine - Google Patents
Proteine ayant un effet inhibiteur sur la choriogonadotropine humaineInfo
- Publication number
- EP0517902A1 EP0517902A1 EP92903686A EP92903686A EP0517902A1 EP 0517902 A1 EP0517902 A1 EP 0517902A1 EP 92903686 A EP92903686 A EP 92903686A EP 92903686 A EP92903686 A EP 92903686A EP 0517902 A1 EP0517902 A1 EP 0517902A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- hcg
- production
- human
- substance
- vitro
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
Classifications
-
- 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
- C07K14/575—Hormones
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to a decidual inhibitory protein (DIP) having an inhibitory effect on the produc ⁇ tion of human choriogonadotropin (hCG) . More particu ⁇ larly, this invention relates to a protein isolated from 5 human decidual cells with a preliminary amino acid analysis and a molecular weight of about 7,000 to about 10,000 daltons (as measured by ultrafiltration and gel chromatography) which may be used to inhibit hCG produc ⁇ tion in vivo (to induce abortion) or _in vitro. measured 10 .in vivo or in vitro to diagnose the cause of hCG inhibi ⁇ tion as an indication of potential miscarriage, or used in the treatment of hCG-secreting tumors.
- DIP decidual inhibitory protein
- the fetoplacental unit plays an essential role in the
- Human choriogonadotropin a hormone secreted by the trophoblast cells of the placenta, is present in the blood and urine during pregnancy.
- the primitive trophoblast begins producing hCG soon after
- hCG 20 fertilization and concentrations of hCG rise to peak values at 8-12 weeks of pregnancy, then decrease to a plateau level that is maintained during the remainder of the pregnancy.
- hCG may also be produced by certain trophoblastic or ectopic neoplasms.
- hCG stimulates the sustained secretion of progesterone by the corpus luteum. which is necessary for the growth of the uterine endometriu .
- hCG also is a stimulus for the production of fetal testicular androgens.
- hCG decreased production of hCG can result in spontaneous abortion. Many abnormal pregnancies are associated with decreased levels of hCG as compared to normal pregnancies of the same duration. Most of these pregnancies end in spontaneous abortions. (B. Saxena, in Endocrinology of Pregnancy, Fuchs et al. Eds, Harper & Row, Philadelphia (1983) pp. 50-72) .
- the decidualized endometrium of the uterus also pro ⁇ quizges substances necessary for gestation.
- the decidual ⁇ ized endometrium and the placental trophoblast are con ⁇ tiguous tissues, and prior studies have investigated the possibility that each may influence the function of the other. Studies performed over a decade ago showed that hCG secretion from placental explants rose over the initial 72 hour period, leading researchers to suggest that loss of an endogenous hCG inhibitory factor (lost as it was removed from culture during changes of growth media) was responsible. (A. Golander et al., Endo ⁇ crinology 102:597-605 (1978)).
- Characterization of an endometrial hCG inhibitor was heretofore unknown. Potential uses for such a substance include as an agent to induce therapeutic abortions, as a basis to develop methods of evaluating hCG inhibition in vivo, or as a treatment of hCG-secreting tumors.
- the present inventors noted the inhibitory effect of a sub ⁇ stance produced by decidual cells, and searched for the active material.
- a protein of a preliminary amino acid analysis and a molecular weight of about 7,000 to about 10,000 daltons is produced by the decidua and has an inhibitory effect on hCG production by the trophoblast and by JEG-3 choriocarcinoma cells.
- the present invention is directed to a composition of matter which inhibits hCG production by human trophoblast cells and JEG-3 choriocarcinoma cells.
- the present inven- tion is also directed to a method for inhibiting hCG release comprising contacting hCG producing tissue with an effective amount of the composition of the invention.
- the present invention is also directed to methods of evaluat ⁇ ing hCG inhibition .in vivo and .in vitro, comprising guan- tifying the amount of the composition of the invention produced in vivo or in vitro.
- the protein of the present invention may also be of use in the treatment of hCG-secreting tumors. Since the placental trophoblast is the normal site of synthesis of hCG, it is understandable that both gestational and non- gestational trophoblastic tumors synthesize and secrete hCG. Indeed, hCG measurements have been quite useful for the diagnosis of these tumors, staging the tumors, and for monitoring the effects of therapy. In addition, some nontrophoblastic tumors may produce hCG ectopically. hCG may act as a growth factor for some tumors (Melmed S. and BraunaLei ⁇ GD: Human chorionic gonadotropin stimulates proliferation of Nb 2 rat lymphoma cells. J. Clin.
- the present composition comprises a substantially- pure polypeptide of a preliminary amino acid analysis and a molecular weight of about 7,000 to about 10,000 daltons (as measured by ultrafiltration and gel chromatography) , wherein said polypeptide inhibits the production of hCG by human trophoblast and JEG-3 choriocarcinoma cells.
- This polypeptide can be isolated from cultures of human deci- dual cells by cultivation and centrifugation of the cul ⁇ ture medium.
- substantially pure is intended to mean that DIP has been isolated from the decidual cells.
- Figure 1 is a graph showing the relationship between hCG release by trophoblast cells and initial decidual cell concentration in conculture of trophoblast cells (lxlO 6 / well) and decidual cells during 120 hours of incubation.
- Figure 4 is a graph showing the dose-response rela ⁇ tionship between DCM and hCG release. JEG-3 cells and trophoblast cells were exposed to different concentrations of DCM for 72 hours. Each bar represents mean + SEM of hCG in six wells. DCM - Decidual conditioned medium.
- Term placenta and associated membranes were obtained from 10 healthy pregnant women undergoing Cesarean sec ⁇ tion.
- Cytotrophoblasts were isolated from villus tissue using the method of H.J. Kliman et al., (Endocrinology 118:1567-1582 (1986)). Briefly, washed villus tissue was minced and then digested with 0.125% trypsin (Sigma, St. Louis, MO) and 0.2 g/L DNase (Sigma) at 37°C for 30 in. Dispersed tissue was applied to a 5-70% percoll (Sigma) gradient. After centrifugation at 1200 X g, the middle layer containing cytotrophoblasts was collected and washed.
- Decidual cells were isolated from decidual tissue dissected from placental membranes by the method of M.B. Braverman et al., (J. Clin. Endocrinol. Metab. 58:521-525 (1984)). This procedure involved the digestion of deci ⁇ dual tissue with 0.25% coliagenase A (Mannhem, Indiana ⁇ polis, IN) at 37"C for 2 hours, followed by centrifugation through a 20-60% percoll gradient. Cell viability was greater than 90% using trypan blue exclusion. Human choriocarcinoma cell line JEG-3 was purchased from American Type Culture Collection (Rockville, MD) .
- trophoblast lxlO 6 cells/ ml/well
- decidual cells (0.83-7.5xl0 5 cells/ml/well)
- trophoblast plus decidual cells (sum of both cell numbers/ 2ml/well) were cultured in 24-multiwell culture plates with 16 mm well diameter (Costar, Cambridge, MA) contain ⁇ ing CMRL-1066 medium (GIBCO, Grand Island, NY) supple- mented with 10% fetal bovine serum (GIBCO) , 25 mM HEPES (Sigma) , 1 U/ml insulin (Nordisk, Gentofte, Denmark) , 10 mM glutamine (Irving, Santa Ana, CA) , 100 U/ml penicil ⁇ lin and 100 ug/ l streptomycin (GIBCO) in an atmosphere of 5% C0 2 -95% air at 37°C for 120 h. The medium was removed and replaced each 24 h.
- the trophoblast (lxlO 6 cells/2ml/ well) or JEG-3 (lxlO 5 cells/2ml/well) were precultured for 24 hours. After removing the precultured medium, the cells were cultured in control medium or medium containing test substances. In all experiments, the media contained the same volume of CMRL-1066. In addition, in some trophoblast experiments and all of the JEG-3 cell experi ⁇ ments, the control medium contained the same volume of 0.01M phosphate buffered saline (PBS) (pH 7.4) as was used to dissolve the test substance in the experimental media. In the time course studies, the medium was removed and replaced with control or experimental medium each 24 hours during 72-96 hour incubation period. In other experi ⁇ ments, the medium in both control and experimental wells were removed at termination of the experiments after 72 hours' incubation.
- PBS phosphate buffered saline
- Decidual conditioned medium was prepared by incubating decidual cells (5-7.5xl0 6 /lu ⁇ _l/flask) in 75cm 2 cell culture flasks (Costar, Cambridge, MA) containing CMRL-1066 medium for 24-72 hours. In the time course experiments, DCM was removed each 24 hours and used for the next 24-hour treatment of trophoblast cells derived from the same placenta as were the decidual cells. In other studies, DCM was removed during the first 36-48 hours of incubation.
- the effect of proteolytic enzyme treatment on DCM was evaluated by incubating DCM for 16 hours at 37 " C with trypsin or chymotrypsinogen-A (Sigma, 40 ug enzyme/mg DCM protein) , followed by the addition of 5 ug ⁇ -l-antitryp- sin(Sigma)/ug trypsin to the treated DCM with incubation at 37°C for an additional 4 hours.
- the same weight of bovine serum albumin (BSA, Sigma) as DCM was added to CMRL-1066 medium, which served as a control, and the medium was treated as described above (60 ug enzyme/mg BSA). DCM was also tested after heating to 100°C for 30 minutes in a water bath.
- the approximate molecular size of the active fraction of DCM was evaluated through ultrafiltration and gel chromatography.
- DCM was ultrafiltered through an Amicon Diaflo Apparatus (Amicon Corp., Lexington, MA) with a YM-10 membrane which has a molecular exclusion limit of 10,000. The greater than i ⁇ ,U00 and the less than 10,000 Dalton fractions were tested.
- 20ml DCM was concentrated by ultrafiltration using an Amicon YM-2 membrane with a cut off 1,000 Daltons, followed by lyophilization of the concentrated DCM.
- AV-3 cells 5xl0 6 cells/lOml/flask
- RPMI-1788 cells 5xl0 6 cells/lOml/flask
- 0.5mM 8-bromo-cAMP Sigma was used to stimulate the JEG or trophoblast cells.
- Another positive control for the system was composed of incubation of decidual cells for 96 hours in medium derived from trophoblast culture, with measurement of prolactin (PRL) and placental protein 12 (PP12; insulin-like growth factor-I-binding protein) as indices of decidual protein production.
- PRL prolactin
- PP12 placental protein 12
- Sensitivity of the assays were 1 ng/ml for hCG and hPL, 1.7 ng/ml for PRL,and 7.8 ng/ml for PP12.
- the protein content of the cells and media were measured by the method of M.M. Bradford in Anal. Biochem. 72:248-254 (1976), using bovine serum albumin as a standard and Bio-Rad protein reagents (Bio-Rad Laboratories, Richmond, CA) . Concentrations of the hormones in the media were expressed as ng/mg protein or ng/well.
- decidual choriogonadotropin inhibitory protein was purified by ultrafiltration, Sepha- dex G50 column chromatography, dialysis of the fractions containing the inhibitory protein, followed by concentra ⁇ tion by lyophilization, and application onto a low molecu ⁇ lar weight SDS polyacrylamide gel.
- the 8,000-10,000 Dalton band was electrically transblotted to a PVDF mem ⁇ brane (Trans-Blot Transfer Medium; BioRad Labs, Richmond, CA 94804) and subjected to amino acid analysis by Pico Tag chemistry using pre-column reaction with PITC (Phenyl iso- thiocyanate) using the method of Matsudaira (Matsudaira, PT: A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press. 1989: pp. 5-7) .
- This method does not measure free asparagine, glutamine, cysteine, tryptophan, and may under represent tyrosine.
- the amino acid analysis was run on a fee-for-service basis by Dr. Audree Fowler, at UCLA School of Medicine, Department of Biological Chemistry, Los Angeles, CA 90024-9842; 213-825-9842).
- Trophoblast cell (or decidual cell) exposed for 96 hours to 50% DCM (or trophoblast culture medium) .
- DCM trophoblast culture medium.
- Each hormone level in the medium is mean ⁇ SEM of 20 wells from three different placental experiments. *p ⁇ 0.005, **p ⁇ 0.001 compared to control.
- DCM Decidual conditioned medium.
- JEG-Cells Cultured in DCM-Containing Medium JEG-3 cells exposed for 72 hours to 25% DCM also had a significant reduction in hCG secretion into the medium compared to control cultures, but there was neither stimu- lation nor inhibition of hPL release.
- the hCG value in media is meaniSEM of six wells from at least two experiments.
- DCM Decidual conditioned medium. *p ⁇ 0.05 compared to control. 4. Dose-Dependent Inhibition of hCG Release
- the results of our study indicate that the human decidual cell secretes a factor that inhibits hCG release by placental trophoblasts and JEG-3 cells.
- the inhibition was DCM dose-dependent.
- the inhibition was also specific for hCG, as DCM (1) did not inhibit total protein synthe ⁇ sis by JEG-3 cells or placental trophoblasts, and (2) stimulated hPL release from primary trophoblast cultures and did not alter hPL secretion by JEG-3 cells (failure of DCM to stimulate hPL release from JEG-3 cells may be due to the paucity of hPL production by these cells) .
- the specificity of this effect is supported further by the absence of hCG inhibition in JEG-3 cells incubated with negative control media.
- the preliminary characterization of the inhibitory factor indicates that it is a protein with a preliminary amino acid analysis and a molecular weight between about 7,000 and about 10,000 Daltons determined by ultrafiltra ⁇ tion and gel chromatography. (A molecular weight up to about 15,000 daltons was measured by SDS polyacrylamide gel electrophoresis. Garfin, D.E. "1-Dimensiona1 Gel Electro.” MT-Deutch. ed. , Guide to Protein Purification- Acad. Press 1989 p. 425-441.) Yuen and associates have previously suggested that PRL inhibits hCG production in vitro and in vivo.
- PRL has a molecular weight of 22,500 Daltons whereas our inhibitory factor has a molecular weight of less than about 10,000 Daltons (or 15,000 Daltons determined as described above) .
- the average concentration of immunoreactive PRL in our DCM was approximately 200 ng/ml, which did not inhibit hCG release from trophoblast cells in our studies (Ren and Braunstein, unpublished observations) .
- Progesterone has also been found by some ban ⁇ gators to inhibit hCG production by placental tissue in vitro. (T. Maruo et al..
- progesterone is synthesized by the trophoblast rather than the decidual cells. Since the active hCG inhibitory factor in DCM is destroyed by heat and proteo- lytic enzymes it is unlikely that progesterone (a steroid hormone) is responsible for the hCG inhibition.
- Trophoblast hPL secretion was stimulated by DCM, a finding that is consistent with those of T. Fay et al.,
- Trophoblast cell culture medium did not, however, change PP12 release from decidual cells indicating that decidual PRL and PP12 regulation are under different control.
- hCG production of hCG by trophoblast and JEG-3 cells is inhibited in a dose-dependent fashion by a protein of about 7,000 to about 10,000 daltons produced by decidual cells.
- This protein will be useful for sake ⁇ gating abnormalities of human pregnancy, and may provide the basis for diagnostic agents to detect such abnormali ⁇ ties, and for therapeutic agents to induce abortion by decreasing hCG production.
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Substance ayant un effet inhibiteur sur la production de choriogonadotropine humaine (hCG), comprenant une protéine isolée à partir de cellules déciduales humaines ayant une analyse d'acide aminé préliminaire ainsi qu'une masse moléculaire comprise entre 7000 et environ 10000 daltons, et utilisée pour inhiber la production de hCG in vivo ou in vitro, dont la mesure fournit une indication d'une cause d'inhibition de hCG, ou pouvant être utilisée dans le traitement de tumeurs secrétant de la hCG.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US07/639,249 US5140100A (en) | 1990-12-28 | 1990-12-28 | Protein that inhibits production of human choriogonadotropin |
US639249 | 1990-12-28 | ||
US80691491A | 1991-12-13 | 1991-12-13 | |
US806914 | 1991-12-13 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0517902A1 true EP0517902A1 (fr) | 1992-12-16 |
EP0517902A4 EP0517902A4 (fr) | 1994-02-02 |
Family
ID=27093292
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP92903686A Withdrawn EP0517902A1 (fr) | 1990-12-28 | 1991-12-19 | Proteine ayant un effet inhibiteur sur la choriogonadotropine humaine |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0517902A1 (fr) |
AU (1) | AU641835B2 (fr) |
CA (1) | CA2075981A1 (fr) |
PT (1) | PT99953A (fr) |
WO (1) | WO1992012178A1 (fr) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6319504B1 (en) | 1996-06-24 | 2001-11-20 | University Of Maryland Biotechnology Institute | Treatment and prevention of HIV infection by administration of derivatives of human chorionic gonadotropin |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3862002A (en) * | 1962-05-08 | 1975-01-21 | Sanfar Lab Inc | Production of physiologically active placental substances |
US4489166A (en) * | 1982-05-27 | 1984-12-18 | Research Corporation | Monitoring of human endometrial function by radioimmunoassay of PEP |
US4734398A (en) * | 1983-03-15 | 1988-03-29 | University Of Southern California | Follicular regulating protein |
-
1991
- 1991-12-19 EP EP92903686A patent/EP0517902A1/fr not_active Withdrawn
- 1991-12-19 WO PCT/US1991/009642 patent/WO1992012178A1/fr not_active Application Discontinuation
- 1991-12-19 CA CA 2075981 patent/CA2075981A1/fr not_active Abandoned
- 1991-12-19 AU AU91648/91A patent/AU641835B2/en not_active Ceased
- 1991-12-27 PT PT9995391A patent/PT99953A/pt not_active Application Discontinuation
Non-Patent Citations (5)
Title |
---|
CLINICAL RESEARCH vol. 38, no. 1 , January 1990 , THOROFARE,N.J.,USA page 148A REN ET AL 'A DECIDUAL PROTEIN INHIBITS CHORIOGONADOTROPIN RELEASE FROM HUMAN TROPHOBLASTS' * |
CLINICAL RESEARCH vol. 38, no. 2 , April 1990 , THOROFARE,N.J.,USA page 298A REN ET AL 'THE DECIDUA PRODUCES A PROTEIN THAT INHIBITS CHORIOGONADOTROPIN RELEASE FROM HUMAN TROPHOBLASTS' * |
PLACENTA vol. 9, no. 2 , 1988 , EASTBOURNE,ENGLAND pages 109 - 115 VICOVAC ET AL 'COINCUBATION-AN EXPERIMENTAL APPROACH TO THE STUDY OF DECIDUAL-TROPHOBLAST INTERACTION' * |
See also references of WO9212178A1 * |
THE JOURNAL OF CLINICAL INVESTIGATION vol. 87, no. 1 , January 1991 , NEW YORK,USA pages 326 - 330 REN ET AL 'DECIDUA PRODUCES A PROTEIN THAT INHIBITS CHORIOGONADOTROPHIN RELEASE FROM HUMAN TROPHOBLASTS' * |
Also Published As
Publication number | Publication date |
---|---|
EP0517902A4 (fr) | 1994-02-02 |
AU641835B2 (en) | 1993-09-30 |
AU9164891A (en) | 1992-08-17 |
CA2075981A1 (fr) | 1992-06-29 |
WO1992012178A1 (fr) | 1992-07-23 |
PT99953A (pt) | 1993-01-29 |
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