MXPA99002312A - Materials and methods relating to the diagnosis and treatment of pre-eclampsia and diabetes - Google Patents

Abstract

The present invention relates to materials and methods for the diagnosis and treatment of pre-eclampsia, and more particularly to the role of P-type inositolphosphoglycans (IPGs) in the occurrence of pre-eclampsia. Methods of diagnosing pre-eclampsia by determining the level of P-type IPGs and uses of antagonists of P-type IPGs in the treatment of pre-eclampsia are disclosed, together with a method for screening for P-type IPG antagonists.

Description

MATERIALS AND METHODS RELATED TO THE DIAGNOSIS AND TREATMENT OF PRE - ECLAMPSIA AND DIABETES Field of the Invention The present invention relates to materials and methods for the diagnosis and treatment of pre-eclampsia psia and diabetes and more particularly to the role of inositol fos foglicanos P (IPG) in the occurrence of pre-eclampsia, a methods of diagnosis of pre-eclampsia and the use of P-type IPG antagonists in the treatment of pre-eclampsia.

Background of the Invention The pre-ecl ampsia is a placental disease [1] characterized by inadequate uteroplacental circulation [2], which affects 10-12% of all pregnancies and which is a major factor in the perinatal mortality rate. There is evidence that one or more placentally derived factors are released in the maternal circular that cause either directly or REF.:29728 indirectly maternal endothelial dysfunction and presenting maternal problems with the activation of vascular permeability increased by the coagulation system and ischemia in maternal organs collateral to vasoconstriction [3].

Brief Description of the Invention The present invention originates from the investigations to determine if there is a correlation between pre-eclampsia and its degree of severity and the profile of the foglicanos inosi tolfos (IPG) in pre-ecliptic subjects and their normal controls, corresponding to age and parity. In order to obtain information on the meaning of the disordered metabolism of carbohydrates in the placenta in pre-eclampsia, as previously revealed by the massive increase in glycogen accumulation [4], a comparison was made with pregnant, diabetic women in which placental accumulation of glycogen is also a prominent feature [4,5], although it was not accompanied by the same degree of life-threatening sequelae of pre-eclampsia.

Accordingly, in a first aspect, the present invention provides the use of an inositol phosphoglycan (IPG) type P antagonist in the preparation of medicament for the treatment of pre-eclampsia. In a further aspect, the present invention provides a method for treating pre-eclampsia in a patient, the method comprising administering a therapeutically effective amount of an IPG, P-type antagonist to a patient. In a further aspect, the present invention provides a pharmaceutical composition comprising a P-type antagonist in combination with a pharmaceutically acceptable carrier. The P-type IPGs and the method for isolating them from human tissue are described below. This in turn allows those skilled in the art to prepare P-type IPG antagonists. In the present invention, "P-type IPG antagonists" includes substances having one or more of the following properties. (a) inhibit the placental release of P-type IPGs; (b) reducing the P-type IPG levels, derived from the placenta, via an IPG-binding substance (eg, an antibody or a specific binding protein) against the IPGs derived from the placenta; and / or (c) reduce the effects of type P IPGs derived from the placenta. In a further aspect, the present invention provides a method for detecting IPG type P antagonists, the method comprising: (a) contacting a candidate antagonist and an IPG type P in an assay for a biological property of the IPG type P under conditions in which the P-type IPG and the candidate antagonist can compete; (b) measure the biological property of the IPG type P; and (c) selecting the candidate antagonists that reduce the biological activity of the P-type IPG. Some of the biological properties of the P-type IPGs and tests to determine these properties that can be used in the indoor detection method are set forth in the description later. The techniques of combinatorial chemistry are particularly suitable for the production of large numbers of synthetic candidate antagonists, which can be detected for the activity in the previous method. The particular emphasis placed on the determination of IPG production in pregnant women, pre-eclectic and diabetic women, is related to the known fundamental importance of the class of compounds in key regulatory sites in metabolic pathways, that, in different tissues, result in the direction of carbohydrates towards the synthesis of glycogen and oxidation in the case of the IPG type P, or towards lipogenesis in the case of the IPG type A; This regulation is related to both organs and inter-organ [6, 9]. In co-pending applications claiming the priority of GB-A-9618934.5, the urinary content of the IPG in diabetic patients was reported, from which evidence has been alleged for a critical role of the altered profiles of the fogl ical inosyphi in relation to the parameters linked to syndrome X, such as insulin resistance, obesity and high blood pressure. There are similarities between metabolic changes in pre-eclampsia and syndrome X [10].

The results of these investigations described below indicate the following: (a) 24-hour production of P-type IPG in urine in pre-eclamptic women is significantly higher (2 to 3 times) than in control subjects with pregnancy, corresponded to age, parity and gestational status. (b) Diabetic pregnant women do not show any significant change in the urinary production of the IPG type P in relation to control subjects with pregnancy correlated to age, parity and gestation stage. (c) Pregnancy itself is associated with an increased production in the urine of the IPG type P in relation to controls without pregnancy correlated to age. (d) No significant changes were found in the daily production of type A IPG in pre-ecliptic or diabetic groups, with the exception of an increase in type A IPG in the pre-ecclastic group when the results were expressed as units per mmol of creatinine. (e) The urinary excretion of IPG type P was correlated with markers of the severity of pre-eclampsia, plasma alanine-aspartate-transaase, the degree of proteinuria and with platelet counts. (f) The human placenta contained very high concentrations of IPG type P, somewhat 100 X greater than human or rat liver. It also appears to contain an inhibitor of IPG type P activity as evidenced by a calculated decrease in activity when the volume of the same preparation tested in the PDH-phosphatase system is increased (Figure 7). The pre-ecplastic placenta contains approximately twice as much IPG type -P as the placenta of normal subjects with pregnancy. IPG type A (fraction of pH 1.3) isolated from the placenta showed no activity when tested for its ability to stimulate lipogenesis in rat adipocytes. (g) Evidence has been found that suggests that the use of contraceptive pills can be related to an increase in P-type IPG in the urine of normal women. (5 values only in each group). (h) After storage for 10 months at -80 ° C, the urine of the pre-eclamptic women showed increased P-type activity (Figure 8A) indicating that the urine initially contained a labile inhibitor). The production of IPG type A isolated from the same urine decreased in activity (Figure 8B). (i) Significant differences were found between the IPG type P and the IPG "type A" ratios in the non-pregnant women and in normal male subjects, while the P-type IPG was similar in both groups, and the IPG type A was 5 to 6 times more in women. (j) There is an increase of 2.7 times in the IPG type P in the urine of pre-eclamptic women, compared to normal subjects with pregnancy. There is a 2.7-fold increase in P-mediators derived from the placenta of pre-eclamptic women compared to normal subjects with pregnancy (see Table 5). (k) The high urinary excretion of IPG type P in pre-eclampsia reflects high placental and circulation levels and is directly related to the accumulation of glycogen in the placenta in this condition, because the P-type IPG activates the glycogen- synthase phosphase. (1) The concentration of P-type mediators in the urine of pre-eclamptic women returns to the baseline in the post-natal sample (see Figure 6) confirming that the source of the P-mediator relevant in pre-eclamptic women It's the placenta. (m) A high level of P-type IPG circulation that originates in the placenta may have paracrine effects, for example, in the stimulation of other endocrine glands, and / or by affecting endothelial cells that may contribute to the pathogenesis of the syndrome of pre-eclampsia. The present invention provides, inter alia, a therapeutic treatment of pre-eclampsia: (1) to inhibit the release of the P-mediator of the placenta; (2) to reduce the levels of the IPG type .P derivative of placenta via the antibody against the IPG derived from placenta; (3) to reduce the effects of the P-type IPG derived by placenta via the P-type antagonist. The substance can be administered as the sole active substance, or as an adjunct to other forms of treatment. Due to their small molecular weight and thermal and acid stability, the IPG must be suitable for oral administration, but other forms of administration are also contemplated. In the case of antibodies, or other proteins or substances that are not suitable for oral administration, other methods such as parenteral administration can be used. The antibodies for administration are preferably human or "humanized" according to known techniques. This is discussed further below. The invention also contemplates the measurement of P-type IPG in blood or urine as a diagnosis for pre-eclampsia. Thus, in a further aspect, the present invention provides a method of diagnosing pre-eclampsia in a patient, the method comprising determining the level of P-type IPG in a biological sample obtained by the patient. In this way, a diagnosis can then be made by correlating this level with known levels of the P-type IPG. In one embodiment, the method comprises the steps of: (a) contacting a biological sample obtained from the patient with a solid support having immobilized therein a binding agent having specific binding sites for one or more of the P-type IPGs; (b) contacting the solid support with a labeled developing agent, capable of binding to the unoccupied binding sites, the bound P-type IPGs or the unoccupied binding sites; and (c) detecting the label of the developing agent that specifically binds in step (b) to obtain a value representative of the level of the P-type IPGs in the sample. As discussed below, in this aspect of the invention, the level of the P-type IPG can be further confirmed by using a marker that correlates with the level of the P-type IPGs. Now, the present invention will be described by way of example and no limitation with reference to the attached drawings.

Brief Description of the Drawings Figure 1 shows (A, B) individual values for the P-type IPG concentration (units / mmol of creatine) in the urine of pregnant women in the pre-ecclastic group, diabetic group, in their normal, corresponding, respective pregnancy groups, and in non-pregnant women, and (C, D) individual values for the concentration of IPG type P (units / mmol of creatine) in the urine of pregnant women in the pre-ecliptic group, diabetic group, and their normal, corresponding, respective pregnancy groups, and in the non-pregnant woman .

Figures 2A, B show the effects of the contraceptive pill in the urinary production of IPG type P and IPG type A in control subjects without pregnancy. Figures 3A, B show the relationship between type P and the gestation stage at which the samples were collected from the groups of pregnant women who were in control, pre-eclamptic, diabetic and reciprocated.

Figure 4 shows (A) the relationship between the P-type IPG of the urine and the protein in the urine in pre-eclampsia, (B) the relationship between the P-type IPG of the urine and the elevated plasma levels of alanine -spartate-transaminase, and (C) the relationship between the P-type IPG of the urine and the platelet counts in pre-eclampsia.

Figure 5 shows (A) and (B) the concentration of P-type IPG in the urine in the ante-natal and post-natal samples of pre-eclampsia, and (C) volumes of urine in ante-natal and post-natal samples, pre-eclampsia.

Figure 6 shows a scheme that exposes the factors that regulate the placental metabolism of glycogen in pre-eclampsia and diabetes.

Figure 7 shows the effect of tissue weight and volume used in the assay in the estimation of the P-type IPG in the placenta of normal subjects.

Figures 8A, B show evidence of an unstable P-type IPG inhibitor in the urine of pregnant women, both in normal and pre-eclattic subjects, and the effect of storage of urine samples for 10 months at -80 ° C in the IPG activity type P and type A.

Figures 9 and 10 show the relationship between the production of the IPG type P and the weight of the placental tissue extracted in normal and pre-ecliptic subjects.

Detailed description of the invention IPG Studies have shown that type A mediators modulate the number of insulin-dependent enzyme activity such as cAMP-dependent protein kinase (inhibits), adenylate cyclase (inhibits) and cAMP-phospho-diestearases (stimulates). In contrast, P-mediators modulate the activity of insulin-dependent enzymes such as pyruvate-dehydrogenase-phosphatase (stimulates) and glycogen-synthase-phosphatase (stimulates). Type A mediators mimic the lipogenic activity of insulin in adipocytes, whereas P-type mediators mimic the glycogenic activity of insulin in muscle. Mediators of both type A and type B are mitogenic when added to the fibroblasts in the serum-free medium. The ability of mediators to stimulate fibroblast proliferation is improved if the cells are transfected with the EGF receptor. Type A mediators can stimulate cell proliferation in the cochleovestibular ganglia of chicks. The soluble fractions of IPG having both type A and type P activity have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle, brain, adipose, heart, placenta) and bovine liver. The biological activity of IPG type A and type B has also been detected in the human liver and placenta, RBC infested with malaria and microbacteria. The ability of an anti- iosinophil antibody to inhibit the action of insulin in human placental trophoblast coughs and BC3H1 myocytes or the action of bovine-derived IPG on the rat diaphragm and chick ganglia suggest conservation interspecies of many structural characteristics. However, it is important to point out that although the previous technique includes reports of activity of IPG type A and type B in some biological fractions, the purification or characterization of the agents responsible for the activity is not described.

In co-pending patent applications claiming the priority of GB-A-9618930.3 and GB-A-9618929.5, isolation and characterization of type P and type A IPGs have been described. Type A substances are carbohydrates containing ciclitol, which also contain Zn2 + and optionally phosphate and which have the properties of regulating lipogenic activity and of inhibiting cAMP-dependent protein kinase. They can also inhibit adenylate cyclase, be mitogenic when added to transfected fibroblasts with EGF in the serum-free medium, and stimulate lipogenesis in adipocytes. P-type substances are carbohydrates containing cyclitol, which also contain Mn2 +, and / or Zn2 + ions and optionally phosphate and which has the properties of regulating glycogen metabolism and activating pyruvate-dehydrogenase-phosphatase. They can also stimulate the activity of glycogen-s intaphosphatase fatase, be mitogenic when added to fibroblasts in serum-free medium, and stimulate pyruvate-dehydrogenase-phosphatase. It was also found that type A and type B substances have the following properties: 1. Migrate near the origin in descending paper chromatography using butanol / ethanol / water 4/1/1 as a solvent. 2. The substances contain phosphate that is directly related to the activity. 3. Free GPI precursors are resistant to cleavage by GPI-PLC. 4. Unite in Dowex AG50 cation exchange resin (H +). 5. Unite in an AG3A anion exchange resin. 6. The activity is resistant to pronase. 7. Detected using a Dionex chromatography system. 8. The P-type substance is partially retained in the C-18 affinity resin. The substances type A and type P can be obtained from the liver or human placenta by: (a) making an extract by the thermal and acid treatment of a liver homogenate, the homogenate that is processed from the tissue immediately frozen in liquid nitrogen; (b) after centrifugation and carbon treatment allow the resulting solution to interact overnight with the anion exchange resin AG1-X8 (formate form); (c) collecting a fraction having IPG type A activity obtained by eluting the column with 50 mM HCl, or a fraction having IPG type P activity obtained by eluting the column with 10 mM HCl; (d) neutralize to pH 4 (do not exceed pH 7.8) and lyophilize the fraction to isolate the substance. (e) descending on descending paper chromatography using butanol / ethanol / water 4/1/1 as solvent. (f) purification using high voltage paper electrophoresis in pyridine / acetic acid / water. (g) purification using Dionex anion exchange chromatography or purification and isolation using Vydac 301 PLX575 HPLC chromatography. More details of the methods for obtaining these IPGs in the patent applications are provided, the contents of which are incorporated herein by reference.

Antagonists As mentioned above, antagonists of P-type activity, whether occurring naturally or synthetically, include substances having one or more of the following properties: (a) substances capable of inhibiting the release of the P-type mediator the placenta; (b) substances capable of reducing the levels of the P-type IPG derived from placenta via an IPG-binding substance (eg, an antibody or specific binding protein) against the IPG derived from placenta; and / or (c) substances capable of reducing the effects of the P-type IPG derived from placenta. In one embodiment, IPG antagonists are specific binding proteins. The binding proteins, specific, that occur naturally can be obtained by selecting biological samples for proteins that bind to the IPG. In a further embodiment, the antagonists are antibodies. The production of polyclonal and monoclonal antibodies is well established in the art, and example protocols are set forth in the following examples. Monoclonal antibodies can be subjected to recombinant DNA technology techniques to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. These techniques may comprise the introduction of the DNA encoding the immunoglobulin variable region, or the regions of complementarity determination (CDR), of an antibody to the constant regions, or constant regions plus the framework regions, of a different immunoglobulin. For example, see EP-A-184187, GB-A-2188638 or EP-A-239400. A hybridoma that produces a monoclonal antibody can be subjected to genetic mutation or other changes, which may or may not alter the binding specificity of the antibodies produced. Antibodies can be obtained using techniques that are normal in science. Methods for producing antibodies include immunization of a mammal (e.g., mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies can be obtained from animals immunized using any of a variety of techniques known in the art, and detected, preferably using the binding of the antibody to the antigen of interest. For example, Western blotting or immunoprecipitation techniques can be used (Armitage et al., Nature, 357: 80-82, 1992). The isolation of the antibodies and / or the cells that produce antibodies from an animal can be achieved by a step of sacrificing the animal. As an alternative or supplement to the immunization of a mammal with a peptide, a protein-specific antibody can be obtained from a recombinantly produced library of the immunoglobulin variable domains, expressed, for example, using the lambda bacteriophage or the filamentous bacteriophage that exhibits immunoglobulin binding domains, functional or their surfaces; for example, see WO 92/01047. The library can be native, which is constructed from sequences obtained from an organism that has not been immunized with any of the proteins (or fragment) or can be constructed using sequences obtained from an organism that has been exposed to antigen of interest. The antibodies according to the present invention can be modified in a number of ways. Certainly, the term "antibody" should be considered as covering any binding substance that has a binding domain with the required specificity. In this manner, the invention covers antibody fragments, derivatives, functional equivalents and antibody homologs, including synthetic molecules and molecules whose shape mimics that of an antibody that allows binding to an antigen or epitope. Exemplary antibody fragments, capable of binding to an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CH1 domains; the Fd fragment consisting of the VH and CH1 domains, the Fv fragment consisting of the VL and VH domains of an individual arm of an antibody; the dAb fragment consisting of a VH domain; the isolated CDR regions and the F (ab ') 2 fragments, a bivalent fragment including two Fab fragments linked by a disulfide bridge in the region of articulation. Individual Fv fragments are also included. ' Humanized antibodies in which CDRs from a non-human source are grafted onto human framework regions, typically with alteration of some of the amino acid residues of structure, to provide antibodies that are less immunogenic than the non-human antibodies of origin, they are also included within the present invention. A hybridoma that produces a monoclonal antibody according to the present invention can be subjected to genetic mutation or other changes. Furthermore, it will be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technologies to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. These techniques may comprise the introduction of the DNA encoding the immunoglobulin variable region, or regions of complementarity determination (CDR), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for example, EP-A-184187, GB-A-2188638 or EP-A-0239400. The cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023. producing the antibody with the desired binding characteristics are within the scope of the present invention, such as host, eukaryotic or prokaryotic cells, which contain the nucleic acid encoding the antibodies (including antibody fragments) and capable of their expression. The invention also provides methods for the production of antibodies that include culturing a cell capable of producing the antibody under conditions in which it is produced the antibody, and it is preferentially secreted. The antibodies described above can also be used in the diagnostic aspects of the invention by identifying them with a brand or reporter molecule that can generate, directly or indirectly, detectable signals, preferably measurable. The linkage of the indicator molecules can be directly or indirectly, or covalently, for example, via a peptide or non-covalent bond. The linkage via a peptide linkage can be as a result of the recombinant expression of a gene fusion encoding the antibody and the reporter molecule. A favorable mode is by the covalent binding of each antibody with a laser, phosphorus or individual fluorochrome dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine. Other indicators include colloidal acromolecular particles or particulate material such as latex beads that are colored, are magnetic and paramagnetic, and biologically or chemically active agents that can directly or indirectly cause signals to be observed visually, are detected electronically or are recorded in another way. These molecules can be enzymes that catalyze reactions that reveal or change colors or cause changes in electrical properties, as an example. They can be molecularly excitable, such that the electronic transitions between the states of energy result in characteristic spectral emissions or absorption. They can include chemical entities used in conjunction with biosensors. Biotin / avidin or biotin / streptavidin and alkaline phosphatase detection systems may be employed. In a further embodiment, the IPG antagonists are synthetic compounds. They can be produced by conventional chemical techniques or using the combination chemistry, and then selected for the IPG antagonist activity. These compounds may be useful in themselves or may be used in the design of mimics, which provide candidate guide compounds to be developed as pharmaceuticals. Synthetic compounds may be desirable where it is easy to synthesize them in a comparative manner or where they have properties that make them suitable for administration as pharmaceutical products, for example, the antagonists which are unsuitable active agents for oral compositions if they are degraded by proteases in the alimentary canal. The mimetic design, the synthesis and the test is used in general to avoid randomly selecting a large number of molecules for an obj ective property.

Production of Monoclonal Antibodies Inositol fos foglicane (IPG) purified from rat liver by sequential thin layer chromatography (TLC) was used to immunize New Zealand rabbits and Balb / c mice using conventional procedures. After immunization, monoclonal antibodies were prepared using the mouse splenocyte fusion approach (5 x 10 6 cells / ml) with mutant myeloma cells (10 6 cells / ml). The myeloma cell lines used were those that lack hypoxant ina-guanine-fos foribasyl-trans ferase. The hybridoma cell detection method was based on a non-competitive, solid-phase enzyme immunoassay in which the antigen (IPG) was immobilized on a solid phase. The supernatant of the culture was added and the positive hybridoma cells were selected. An individual cell cloning was done by limiting the dilution. Hybridomas were selected for three monoclonal antibodies (2D1, 5HG and 2P7). All monoclonal antibodies were determined to be I gM using an EK-5050 kit (Hyclone). In order to prove that all monoclonal antibodies recognized IPG, a non-competitive, solid-phase enzyme immunoassay was used. Funs Polysorp Nunc-Immuno plates were used for the assay. Polysorp surface is recommended for tests where certain antigens are immobilized. The immobilized antigen (IPG) diluted to 1: 800 captured the monoclonal antibody from the tissue culture supernatant, ascitic acid, and when the purified monoclonal antibody was used. The detection method used a biotinylated complete antibody, anti-mouse IgM (from goat) and a biotinylated horseradish peroxidase complex with streptavidin (Amersham), ABTS and buffer for ABTS (Boehringer Mannheim). The same immunoassay was used to evaluate the monoclonal antibody. In this assay, the detection method employed an anti-rabbit Ig, a specific, biotinylated (donkey) specific antibody. The antibodies can be purified using the following method. Liquid, rapid protein chromatography (Pharmacia FPLC system) with a gradient programmer GP-250 Plus and a high-precision pump P-500 was used in order to purify an IPG antibody, polyclonal. A protein A affinity column was used HiTrap for the purification of polyclonal IPG from rabbit serum. Protein quantification was done using a Micro BCA Protein assay reagent kit (Pierce). . Monoclonal IgM antibodies were purified in two steps. Precipitation with ammonium sulfate was the method chosen as a first step. The tissue culture supernatant was treated with ammonium sulfate (50% saturation). The sediment diluted in PBS was transferred to the dialysis pipe before the second step. Since precipitation with ammonium sulfate is not suie for individual pass purification, it was followed by gel filtration chromatography of the antibody solution in PBS run on a Sepharose 4B column from Pharmacia. Protein quantification was done by reading the absorbance at 220-280 nm in a Perkin-Elmer lambda 2 UV / VIS spectrophotometer.

Protocol for the Intercalation ELISA The protocol then discloses an indirect, non-competitive, solid phase enzyme immunoassay (intercalation ELISA) for the quantification of inosy-tolfosfoglycans (IPG) in biological fluids, such as human serum.

In the assay, monoclonal IgM antibodies are immobilized on a solid phase. The tissue culture supernatant, the ascitic fluid from mice with a peritoneal tumor induced by injecting hybridoma cells into the peritoneum and the purified monoclonal antibody have been used for the immunoassay. F96 Maxisorp Nunc-Immuno plates were used for these assays. The Maxisorp surface is recommended where proteins, specifically glycoproteins such as antibodies, they join the plastic. The immobilized antibody captures the antigen from the test sample (human serum or IPG used as a positive control). It is necessary that a binding antibody (a polyclonal IPG antibody, purified from rabbit) binds to the anti-biotinylated antibody to the antigen. The detection method employs a biotinylated, species-specific incomplete antibody, anti-rabbit Ig (donkey) and a radish peroxidase complex, biotinylated with streptavidin (Amersham), ABTS and buffer for ABTS (Boehringer Mannheim). The ELISA test can be carried out as follows: 1. Add ifjul / well in all steps. 2. Add the monoclonal antibody diluted 1: 100 in PBS on a F96 Maxisorp Nunc-Immuno plate. Incubate at least 2 days at 4 ° C. '3. Wash with PBS three times. 4. Add a blocking reagent for ELISA (Boehringer Mannheim) in distilled water (1: 9) 2 hours at room temperature. 5. Wash with PBS-Tween 20 (0.1%) three times. 6 Add a purified monoclonal antibody (diluted 1: 100 in PBS) overnight at 4 ° C. 7. Wash with PBS-Tween 20 (0.1%) three times. 8. Add a specific antibody specific to the species, biotinylated, anti-rabbit Ig (Amersham) diluted 1: 1000 in PBS, 1 hr at 30 min at room temperature. 9. Wash with PBS-Tween 20 (0.1%) three times. 10. Add a complex of horseradish peroxidase, biotinylated with streptavidin (Amersham) diluted 1: 500 in PBS, 1 hr at 30 min at room temperature. 11. Wash with PBS three times. 12. Add crystals of the salt of 2,2-azino-di- (3-ethylbenzolinezoline (6)) diammonium sulphonate (ABTS) (Boehringer Mannheim) to buffer for ABTS (BM); the buffer for ABTS is added to distilled water (1: 1 v / v). 1 mg of ABTS is added to 1 ml of diluted buffer for ABTS. 13. Read the absorbance in a Multiscan Plus P 2.01 using a 405 mm filter in 5-15 min.

Pharmaceutical compositions Antagonists of the invention can be formulated in pharmaceutical compositions. These compositions may comprise, in addition to one or more of the P-type antagonists, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials known to those skilled in the art. These materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, for example, the intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal route.

The pharmaceutical compositions for oral administration may be in the form of a tablet, capsule, powder or liquid. A tablet can include a solid carrier such as gelatin or an adjuvant. The liquid pharmaceutical compositions may generally include a liquid carrier such as water, petroleum, vegetable or animal oils, mineral oil or synthetic oil. Physiological saline, dextrose or other solution of saccharides or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous, cutaneous or subcutaneous injection, or injection at the affliction site, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is free of pyrogens and has a suitable pH, adequate isotonicity and stability. Those of skill in the art are able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactate Ringer's injection, preservatives, stabilizers, buffers, antioxidants and / or other additives. may include, as required.

If it is a polypeptide, antibody, peptide, small molecule or other pharmaceutically useful compound according to the present invention to be given to an individual, the administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" ( as the case may be, although prophylaxis may be the therapy considered), this is enough to show benefit to the individual. The actual amount administered, and the speed and time course of administration, will depend on the nature and severity of the treatment. The prescription of treatment, for example, decisions on dosage, etc., is within the responsibility of general practitioners and other medical doctors, and typically taking into account the disorder in question, the condition of the individual patient, the site of distribution, the method of administration and other factors known to practitioners. Examples of protocol techniques mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.), 1980.

A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition being treated.

Diagnostic methods Methods for determining the concentration of analytes from biological les of individuals are well known in the art and can be employed in the context of the present invention to determine whether an individual has an elevated level of P-type IPG, and thus whether he has or is at risk of pre-eclampsia. The purpose of this analysis can be used for diagnosis or prognosis to assist a physician in determining the severity or probable course of pre-eclampsia and / or to optimize the treatment of pre-eclampsia. Examples of diagnostic methods are described in the subsequent experimental section. The preferred diagnostic methods depend on the detection of P-type IPGs, an elevated level of which is found to be associated with pre-eclampsia. The methods may employ biological samples such as blood, serum, tissue samples (especially placenta), or urine. Test methods for determining the concentration of P-type IPGs typically employ a binding agent that has binding sites capable of specifically binding to one or more of the P-type IPGs in preference to other molecules. Examples of binding agents include antibodies, receptors and other molecules capable of specifically binding to P-type IPGs. Conveniently, the binding agent is immobilized on solid support, for example, at a defined location, to make it easy to handle during the test. The sample is generally contacted with a binding partner under appropriate conditions so that the P-type IPG present in the sample can bind to the binding agent. The fractional occupancy of the binding agent binding sites can then be determined using a developing agent or agents. Typically, the developing agents are labeled (e.g., with radioactive, fluorescent or enzyme labels) so that they can be detected using techniques well known in the art. In this way, radioactive labels can be detected using a scintillation counter or other radiation counting device, fluorescent labels using a laser or confocal microscope, and enzymatic labels by the action of an enzymatic label on a substrate, typically to produce a color change. The developing agent can be used in a competitive method in which the developing agent competes with the analyte (IPG type P) for the binding sites occupied by the binding agent, or a non-competitive method, in which the agent of developed, labeled is bound to the analyte bound by the binding agent or to the binding sites occupied. Both methods provide an indication of the number of binding sites occupied by the analyte and therefore, the concentration of the analyte in the sample, for example, by comparison with standards obtained using samples containing known concentrations of the analyte.

Experimental description A. Experimental 1. Test for IPG type A and IPG type P The activity of IPG type A and type P in urine and placental extracts was studied using specific bioassay procedures. IPG type P was determined using PDH-phosphatase activation [11]. The complex of PDH and PDH-phosphatase (metal-dependent form) were prepared from the heart of beef as described by Lilley and colleagues [11] and the phosphatase activation assay was performed by the spectrophotometric variant of the two-system stages described by these authors. This test is considered to be a characteristic feature of the IPG type P (see Larner et al [12]). The IPG type A was determined by the stimulation of lipogenesis as measured by the incorporation of [U14C] -glucose in lipid adipocytes isolated from the epididymal fat pads by the Rodbell method [13]. A high degree of specificity for the IPG type A was found for this bioassay. A straight line relationship was obtained between the added IPG and the stimulation of PDH-fos-fatase activity (IPG type P) and lipogenesis in the intact adipocytes (IPG type a); this relationship was maintained at least until a stimulation of +250%. These observations provided a basis for a unit that is defined and used for the purpose of comparing the IPG performances of different tissues and urine samples. The linearity between the added IPG and the percentage of change in the response has been observed by others (see Lilley et al [11] and Newman et al. [14)]), although Asplin et al [15] did not show linearity in their study in the IPG in human urine of normal and diabetic subjects, an effect that was marked particularly with the IPG type A (fraction of pH 1.3). 2. Extraction of IPG type P and IPG type A of urine Urines were extracted as described by Asplin et al [15]. The final fractions were freeze-dried and stored at -20 ° C. For use, the IPG fractions were redispersed in water, immediately before the test, so that 10 μl of the redissolved IPG corresponded to 10 ml of urine. In view of the possibility that high amounts, and variables, of IPG could be excreted in the different groups of subjects with pregnancy and pre-eclampsia, and in order to ensure that the capacity of the resin is well in excess of the applied load. , preliminary test runs were made to determine the optimum ratio of the resin to the volume of urine start. The linearity of the recovery was obtained up to 100 ml of urine per 18 g of resin. In the present study, the ratio of 80 ml of urine to 18 g of resin was maintained to allow for variation in the IPG content. 3. Preparation of the placenta In preliminary studies, normal placentas were obtained and treated as follows: (i) The first was collected within 40 minutes of the distribution and the tissue samples were fixed frozen and stored and transported in solid C02. (ii) The second placenta was collected at an estimated 30 minutes after distribution and a 15 g sample was frozen immediately. The rest was divided into two, one half was placed at room temperature and the other half stored on ice. Samples of 10 g of each of these halves were removed after 1, 3 and 5 hours, they were held frozen and treated as before. These samples were stored at -80 ° C until they were extracted. 4. Extraction of inosi tolfosfoglicanos from the placenta The extraction processing comprised the spraying of the frozen tissue (5 g) under liquid nitrogen and then the extraction with 50 mM formic acid at 100 ° C for 3 minutes. The supernatant fraction, after centrifugation, was treated with carbon (10 mg / ml) and centrifuged again. The supernatant of carbon was passed through a Millipore filter, it was diluted 5 times with water and then it was set at pH 6 with ammonia. After centrifugation, the extract was added to 15 g of AG1-X8 and put overnight at 0 ° C. The resin was then transferred to columns and washed with 40 ml of water followed by 40 ml of HCl, pH, 3. IPG type P was eluted with 100 ml of HCl, pH 2.0, and IPG type A with 100 ml of HCl, pH 1.3. The extracts were placed at pH 4 with ammonia and rotary evaporated to about 5 ml before they were transferred to smaller tubes and lyophilized. They were stored in this stage at -20 ° C until they were used. This extraction procedure was based on that described by Nestler and colleagues [16]. The wide variations in the content of inositol fos foglicanos of different tissues [9] indicated the preliminary examination of: (i) the optimal weight of the placental tissue in relation to the weight of resin to be used in the isolation and separation of the IPG type P and IPG type A, and (ii) the amount of the isolated portions to be used in the bioassay systems to ensure that the assays fall within the linear portion of the dose response curve.

It was established that, using a column containing 18 ml of resin, the maximum activity of the IPG type P was recovered when the placenta samples were less than 0.3 g (Figures 9 and 10) and that the overall test was more reliable when the IPG Type P thus obtained was redispersed in 100 μl of water, and of which 1 or 2 μl was used for the assay in the PDH-fos-fatase activation system. Under these conditions, a stimulation of up to +300% was obtained and the response was linear within the given parameters. When placental quantities greater than 0.3% were used, the P-type IPG yield (calculated pro gram extracted) dropped abruptly, either due to the co-extraction of a potent inhibitor or the presence of competing materials for sites of union available in the resin. No activity of IPG type A was detected, as evidenced by the stimulation of lipogenesis in rat adipocytes. Six separate extracts of pH 1.3 prepared from placenta; 4 separate extractions were tested, all in triplicate. The parallel extractions of the rat liver, carried out at the same time, produced values of 1.92 units / g and an insulin standard valued at the same time gave a stimulation above the baseline of +258%.

Expression of results An IPG unit is defined as the amount that causes a 50% activation at the basal level of the test system. The performance of IPG in urine is based on three different bases: (i) percentage of stimulation of the test system by 10 μl of final urine extract (Col 1), allowing direct comparison with the data of Asplin and colleagues [ 15], (ii) Units of IPG per 1 mmol of creatinine. (iii) IPG units found in a sample of a 24-hour urine collection; that is, the total daily production at that stage of gestation. The results are given as mean ± SEM and are evaluated on the basis of the paired sample, corresponding, the selection of the subjects that corresponded to the gestation stage, parity and age, and based on the Mann classification test. -Whitney for non-parametric data. 6. Experiment design The ten pre-eclamptic and pregnant diabetic women studied each corresponded with a normal control subject, with pregnancy for the gestation stage (± 13 days), parity (0, 1-3, 4 +) and age (± 4) years) . The gestation interval was from 26 to 37 weeks for the pre-ecliptic group and from 31 to 38 weeks for the diabetic group with pregnancy. Normal non-pregnant women of reproductive age were included to allow evaluation of the effect of pregnancy per se on the urinary excretion of inositol fos foglicans. Changes in inositol fos foglicans in pre-eclampsia correlated with the severity of the condition. Urine: Creatinine, urea, Na *, K +, Ca +, protein and volume / 24 hours. Blood: Creatinine, asparatato-transaminase (liver enzyme marker), platelet count.

Biodata: Age, gestational age, parity, blood pressure, birth weight, placental weight.

B. Results 1. Stability of the inosi tolfosfoglicanos isolated from the placenta The material of the first placenta, subjected to freezing, immediately at the reception of the distribution, gave exceedingly high yields of IPG type P, no IPG activity type A was found. The activity of the immediate sample of the second placenta was appreciably lower , but, nevertheless, it contained approximately 7 units of activity / g. Samples of this placenta stored at room temperature 1, 3 or 5 hours were all devoid of P-type IPG activity. The sample stored on ice for 1 hour had lost approximately half of its activity compared to the immediate sample subjected to freezing. , while those stored for 3 to 5 hours had produced less than one unit of activity / g. It is concluded that IPG are highly unstable in untreated tissue, even at 0 ° C. 2. Inosi tol osfoglicanos in placenta distributed to term The values for the tissue units / g of the IP-G type P and IPG type A extracted are shown in Table 1 for the human placenta, human liver and rat liver. The exceptionally high value for the P-type IPG in the placenta is apparent. The occurrence of a very high P-type IPG in the placenta is in agreement with the known role of this putative insulin mediator in steroidogenesis in this tissue, and with the reported action of P-type IPG in glycogen synthase-phosphatase activation [16, 17]. In addition, as a first approximation, it can be argued that an increase in the urinary P-type IPG in pregnancy, whether in diabetic or pre-ecliptic subjects, normal, with pregnancy, originates in the placenta. In this way, measurements of the concentration and daily 24-hour excretion of the P-type IPG can be an indicator of the placental production of this mediator. A comparative study of planetary tissues, pre-ecliptic and normal, shows a 2.7-fold increase in the P-type IPG in the pre-ecliptic placenta (see Table 5). The inability of the pH 1.3 fractions of the six placentas studied to stimulate lipogenesis in rat adipocytes may indicate a high degree of tissue specificity for the IPG type A, placental, and a very specialized function for the IPG type A, Placental 3. Inosi tolfosfoglicanos in urine in pre-eclampsia and diabetes: The concentration and daily production, total of the foglicanos inositolfos in the urine of diabetic and control subjects, pre-ecliptic or with pregnancy, are given in Table 2. The results are given as the stimulation of the bioassay system produced by μl of the P-type IPG fractions (pH 2.0) or from the IPG type A fractions (pH 1.3) to allow comparison with the data of Asplin et al [15]. The results are also shown as units / mmol of creatinine and as the daily production of 24 hours in units. The most notable difference was seen in the P-type IPG in the pre-ecliptic group that was two or three times higher than the corresponding control group. Diabetes did not result in a significant difference in inositolophosphates in urine relative to its control group at the same stage of pregnancy. The individual values that show the range of volumes for pre-eclámpticos, diabetic and control groups are presented in Figures 1 A, B. A significant difference lies in the present observation that the control group not with pregnancy has a lower concentration of IPG type P and total daily excretion than control groups with pregnancy for pre-ecliptic and diabetic subjects (see Table 2). These differences were statistically significant. The remarkable finding that the pre-ecclastic group only showed a significant increase of type P among pregnant women, and that the P-type IPG was lower in the non-pregnancy group, provided evidence for a link between pre-eclampsia and the production of this inositolfosfoglicano. Thus, it is strongly suggested that the increased urinary P-type IPG originates in "the placenta, by the present observation that all subjects with pregnancy have an increased value of P-type IPG in relation to controls not with pregnancy and that the placenta itself has an exceedingly high endogenous concentration of the P-type IPG (see Table 5) .If the highest extraction rate of the P-type IPG in pre-ecliptic subjects and their corresponding controls in comparison to normal subjects not with pregnancy is equals the contribution of the placenta to the IPG type P urinary (Table 2), then the effect of pre-eclampsia is even more strictly underlined with pre-ecliptic values that are somewhat 5 to 6 greater than the pre-ecliptic control values "control for the 'pre-ecliptic group' for all modes of expression (Table 3). This interpretation is strengthened when a similar calculation is made for the IPG type A. In this case, there is no "excess" of IPG type A that can be attributed to the presence of the placenta, a finding with the present inability to demonstrate the presence of type A in this tissue (see Table 1). Also, the values fall post-natally, as shown in Figure 6. The only significant difference in the IPG type A * urinary recorded in Table 2 and Figure 1 C, D was the increased value in the pre-subjects -elastic when the results were expressed as units / mmol of creatinine. No difference was observed in the 24-hour production of IPG type A in this condition. 4. Inosi tol osfoglicanos in non-pregnant subjects The ten subjects without pregnancy in this study included five in the antico'nceptive pill and in order to determine if the altered hormonal antecedent could have an influence on the profile of inosi tol fos foglicanos, these two subsets were considered separately. These data are shown in the Figure 2 from which it can be seen that there is some evidence for a higher production of type P in the group that takes contraceptive pills. The number of subjects in each subset is too small in which it is based on firm conclusions, but nevertheless, the results suggest that it deserves an extension of this inspection. In a separate project, in the changes in urinary IPGs in diabetic, male subjects who are treated in the clinic as non-hospitalized patients in the Middlesex Hospital, a group of normal men was included. The comparison of the data of this study with those of the present study of non-pregnant normal women revealed the interesting finding that while the IPG type P / mmol of creatinine was similar in both groups, the IPG type A was significantly higher in the samples of urine of normal women for 5 to 6 times (Table 4). There was a marked difference in the IPG type P / IPG type A ratio, which can be 0.6 for women and 3.1 for men.

. Inosy tolfos oglicanos and stage of gestation In view of the evidence of a progressive decrease in the activity of a number of placental enzymes comprised in glucose metabolism and in the placental content of glycogen towards the end of pregnancy [5, 18-20], the present data in the urinary inosi tolfosfoglicanos were examined with respect to the stage of gestation in which the samples were taken; this varied between 26-37 weeks. While these data should be interpreted with caution in view of the small number of samples in the early stages of gestation and the possible weight for a remarkably high value at 26 weeks, certain trends are apparent. There was a significant correlation between the gestation stage and the P-type IPG in the urine of the pre-ecliptic subjects (r = 0.609, P <0.05) (Figure 3A), the highest P-type IPG values found at At the beginning of the third trimester, the values of pre-ecliptic control groups correlated in age that closely approximate in the period of 35-37 weeks. This correlation with normal diabetic control groups was not seen (see Figure 3B). In addition, no correlation was found between the type A IPG in the urine and the gestation stage. In this current study, individual 24-hour urine collections were made at different stages in the 26-37 week period of pregnancy. Speculation opens if the differences between the P-type IPG in the group's urine, pre-ecliptic and their corresponding controls reflect a relative immaturity of the cells in the pre-ecliptic subjects [4, 21], and a delay in a decline that occurs naturally in the production of IPG type P towards the end of gestation, or if the significantly increased values of the P type IPG in the pre-ecliptic group is a specific marker for the degree of severity of the pre-ecliptic group. eclampsia. The question of possible changes in the IPG type P in the urine at different stages of pregnancy remains to be answered by the measures of urinary IPG in the same subject at regulated intervals throughout the pregnancy. 6. Inosi tolfosfoglicanos urinalis and markers for pre-eclampsia: 6. 1 IPG type P and markers of pre-eclampsia: The IPG type P excreted was examined in relation to markers of pre-eclampsia, which include protein in the urine, the activity of alanine-aspartate-transaminase in plasma and blood pressure, which is known to increase in the pre-eclampsia, and the platelet count that decreases. The correlations between these different markers and the IPG type P / mmol of creatinine in pre-ecliptic subjects is shown in Figures 4 A-C. In summary, these results show that: (i) The protein in the urine correlates positively with IPG type P, P < 0.01. (ii) alanine-aspartate transaminase correlates positively, P < 0.05. (iii) Platelet count negatively correlates with the IPG type P, P < 0.05. 6. 2 IPG type A and markers of pre-eclampsia: It was noted, in Table 2, that IPG type A showed an upward trend in pre-ecliptic subjects although this was only significant based on the IPG type A / mmol of creatinine and was less marked than the P-type IPG. The present relatively small differences in a sample of ten subjects makes only tentative conclusions drawn from these results, it was found that there was a positive correlation between the increased IPG type A and the systolic blood pressure (P <0.05). This observation is, perhaps, strengthened by the parallel correlation found in a separate study of 31 diabetic male subjects in which there was a positive, clear correlation between the IPG type A and the increased blood pressure. However, it is likely that rigorous control of blood pressure is maintained in all pre-eclámpticos subjects, masking possible underlying links between the IPG and blood pressure in the present study.

C. Discussion The possibility that inositolphosphoglycans may be particularly important as parts of the signal transduction system in the placenta in the regulation of glucose and glycogen metabolism and steroidogenesis results from the evidence that: (i) Insulin mediators of this class can be isolate from placental plasmatic membranes [22] and placental tissue, intact, subjected to freezing (Table 1); (ii) IPG type P activates glycogen synthase-phosphatase and pyruvate-dehydrogenase-phosphatase [6,7,11,17]; (iii) Foglican inositolfos have been shown to be a system of signal transduction in the regulation of placental, human teroidogenesis [16, 23]. The present results clearly show that the human placenta is a particularly rich source of P-type IPG and the differences between the groups with pregnancy and not with pregnancy provides strong evidence that the increase in urinary content in normal and pre-pregnancy Eclampsia originates from the placenta. This was confirmed by direct analysis of the placenta samples. The highly significant increase of two to three times in the IPG type P in the urine of the pre-ecclastic group, in excess of the corresponding control group, a difference that is even more significant in the early stages in pregnancy (Figure 3A), it has been shown to correlate with the markers of pre-eclampsia (Figure 4A-C).

In addition, high P-type IPG provides an explanation for the marked accumulation of glycogen in the placenta in pre-eclampsia (Scheme 1).

It is postulated that different mechanisms are involved in the accumulation of glycogen in the placenta in pre-eclampsia and diabetes (see Scheme 1). In contrast to pre-eclampsia, there is no evidence of an increase in P-type IPG in urine in diabetic subjects with pregnancy in relation to their corresponding controls (Table 2) and in this condition the increased accumulation of glycogen can be related to increases in glucose and glucose 6-phosphate in this tissue as shown by Shafrir et al [5, 24]. As noted by these authors, both glucose and glucose 6-phosphate are activators of glycogen synthase, the increased accumulation of glycogen is related in this way to maternal hyperglycemia. In this regard, the placenta in diabetes shows some parallelism to the response of the kidney in diabetes, the latter has an increased content of glucose and glucose 6-phosphate and also accumulates glycogen. In this way, both tissues exhibit characteristics of glucose over-utilization in diabetes [25]. While the present results show a close association between the markers of pre-eclampsia and the concentration and daily excretion of the P-type IPG it is not known if this is "cause" or "effect". It is likely that the plasma concentration of P-type IPG also increases in pre-eclampsia. The experimental data should confirm this assumption, the question can then be asked as if the increased level of a mediator in circulation, which can activate both the paracrine and autocrine signaling systems, can affect the functions of other tissues and endocrine organs. Exposure of endothelial cells, widely thought to be dysfunctional in pre-eclampsia [3], at increased levels of P-type IPG, could be critical in the link between the production of IPG type P in the placenta in systemic effects . Funglican inosyphodies appear to have autocrine and paracrine regulatory functions affecting steroidogenesis is placental [16], the synthesis of insulin-dependent progesterone in granulosa cells of pig ovary [23], the stimulation of FSH and HSG cells of granulosa [26]. ACTH signaling of adrenal, bovine cells [27], TSH stimulation of thyroid cells [28], IGF1 stimulation of BALB / C3T3 granulosa cells [29], transforming growth factor B into chondrocytes [30], and activation of human platelets [31]. A particularly interesting facet of the present study is the apparent absence of IPG type A extractable from six different placentas and multiple samples of an individual placenta (Table 1). You can advance a number of explanations. The IPG type A may decrease markedly in the third trimester, which is less stable in the placenta after birth and before freezing or extraction, which has a high degree of tissue specificity and is not active in the test system. of adipocytes, or more interestingly, which is certainly markedly under the placenta. Contrary to the IPG type P, the enzymes affected by the IPG type A include a number that focuses on the regulation of protein phosphorylation, these include: inhibition of adenylate cyclase and cAMP-dependent protein kinase and activation of the membrane bound to a cAMP-phosphodiesterase of low Km [6, 7]. It could be argued that the placenta is essentially a unidirectional system, which transfers nutrients from the maternal to the fetal circulation, and that acts as a buffer system for glucose in the storage and release of glycogen. The on / off and biosynthesis / degradation cycles regulated by the protein phosphorylation cycles seen in the liver (glycolysis / gluconeogenesis) and adipose tissue (lipogenesis / lipolysis) do not seem to play an important role in placental function [18-20]. Additional work is needed in this aspect of regulation of the placental metabolism to better define the regulation of the synthesis and the role of the inositolophosphagiants. The new findings of excretion increased the P-type IPG in the urine, and the possibility that originates in the placenta in pre-eclampsia, suggests a mechanism for the accumulation of glycogen in the trophoblast syncytia of pre-pregnancy pregnancies. eclámpticos. The correlation of these changes in the P-type IPG with the markers of the severity of the pre-eclampsia syndrome also suggests that at least part of this dysfunction may come from excessive levels of the signaling system.

Table 1 Yields of IPG type P and IPG type A of placenta and liver Weight Units Weight Units Units taken (g) Calculated / (g) taken (g) Calculated / g Calculated / g IPG type P IPG type P IPG type A (pH 2.0) (pH 2.0) (pH 1.3) HUMAN PLACENTA CONTROL PRE-ECLAMTIC? Placenta No. Placenta No.

PE 1JB 0.18 581 With 8CA 0.34 266 ND 0.26 522 0.49 159 ND 0.47 242 1.05 47 ND 1.03 81 Human Liver 1.82 1.60 (n = 2) Rat Liver 2.60 ± 0.22 2.6 ± 0.12 (n = 13) ND- not detected Table 2. The concentration and total daily production of inositolfosfoglicanos in the urine of pre-eclámpticos control subjects, or diabetics, with pregnancy, and of control corresponded and a control group not with pregnancy Stimulation Units Production Stimulation Production Units Creatinine 10 μl of IPG daily ti of 10 μl of IPG daily rate of (mmol / L) urine po P / mmol 24 hours urine P / mmol of 24 hours of creation (units) creatinine (units) tinine PRE-ECLAMPTICO GROUP CONTROLS 94.4 30.5 316 81.4 25.9 251 6.94 (n = 10) ± 11.1 ± 8.94 ± 62 ± 8.4 ± 3.48 ± 42 ± 0.90 PRE- 20.5 96.4 854 102 48.5 407 4.79 ECLAMSIA ± 43 ± 29.2 ± 318 ± 8.4 ± 6.98 ± 78 ± 0.58 (n = 10) ** ** ** ** DIABETIC GROUP WITH PREGNANCY CONTROLS 129 38.5 374 84.5 29.2 294 6.41 (n - 10) ± 26 ± 5.12 ± 47 ± 13.5 ± 5.5 ± 57 ± 0.53 DIABETICS 89.8 37.3 275 82.1 34.6 253 5.94 (n = 10) ± 11.0 ± 7.64 ± 48 ± 9.4 ± 8.12 ± 45 ± 0.76 GROUP NOT WITH PREGNANCY (n = 10) 38.2 18.8 187 97.8 32.7 346 7, 11 ± 12.3 ± 1.98 ± 25 ± 11.8 ± 6.02 ± 75 + 0.81 The results are given as Mean ± SEM for 10 subjects in each group . The control subjects for the pre-ecliptic and diabetic groups corresponded to the stage of gestation, parity and age; A normal group is shown not with pregnancy. The data are given as percentage of stimulation of pyruvate-dehydrogenase-phosphatase (IPG type P) or percentage of stimulation of lipogenesis (IPG type A) (i) per unit volume of urine (10 μl = 10 ml) of urine); (ii) as units of activity of IPG / mol of creatinine or (iii) as total production of 24 hours of units of IPG activity. A unit is defined as the amount of IPG that causes a 50% increase in the bioassay system. The statistical test was assessed by the Mann-Whitney test *, P, < 0.05: **, P < 0.01.

Table 3 Calculation of the placental contribution to the amount of IPG type P found in the urine of pregnant women, including groups of diabetics, pre-ecliptic (PET) and their corresponding control groups GROUP IPG type P DIFFERENCES BETWEEN GROUPS WITH PREGNANCY AND NOT WITH PREGNANCY (units / mmol of creatinine) Group 18.8 ± 1.98 without pregnancy Groups with pregnancy Diabetic group Controls 38.5 ± 5.12 + 19.7 ± 5.12 Diabetics 37.3 ± 7.64 + 18.5 ± 7.64 D / C ratio 0.97 0.94 Pre-ecliptic group Controls 30.5 ± 8.94 + 11.7 ± 8.94 Pre-ec1amps ia 96.4 ± 29.2 + 77.6 ± 29.2 PET / C ratio 3.2 6.6 Calculated data in Table 2. Each group contained 10 values; the results are given as mean ± SEM Table 4 The content of IPG type P and IPG type A of urine of normal female subjects not with pregnancy and of normal male subjects IPG type IPG type A IPG P P IPA A (Units / mmol of creatinine) Feminine (10) 1 '± 1.9! 32.7 ± 6.02 0.57 No treatment 16.1 ± 2.14 29.5 ± 6.02 (5) 21.5 ± 3.01 43.4 ± 10.5 Men (27) 19.3 ± 1.8 6.30 ± 0.78 3.06 P value, NS < 0.001 female value V The values for male are taken from a separate survey of IPA A - and IPG type P in the urine of male and diabetic control subjects.

Table 5 IPG type P in placenta and human urine in subjects with pre-ecplastic and normal pregnancies Pre-eclastic Control PE / C Placenta [1] Units / g Units / g IPG type P 81 ± 11 (5) 30 ± (4) ** 2.7 Urine [2] Units / 24 h units / 24 h IPG type P 54 ± 318 (10) 316 ± 62 (10) 2.7 Table 6 Placental glycogen content and glycogen synthase activity [3] Pre- ecliptic PE / C control Chorionic hair-glycogen content ug / g of tissue) 1300 570 2.3 Microvesicles STB-glycogen content (ug / mg protein) 223 25 9.7 STB-glycogen synthase microvesicles (units / mg of proteins) 1323 83 16 symbols from the table: [1] Unrequited samples of placenta [2] Corresponded urine samples for gestational age and parity [3] Data from Arkwright, Rolemaker, Dwek, Redman '1993) J. Clin Invest 91: 2744-2753 IPG type P activates pyruvate-dehydrogenase-phosphat-asa and glycogen-synthase-phosphatase. Unit A of IPG activity type P is defined as the amount that produces a stimulation of 50 % of PDH-phosphatase. The values are the mean ± SEM; the P values of Fisher shown by ** P < 0.01 Table 7 IPG CONTENT OF PRE-ECLAMIC URINAL TREATMENTS POST-NATAL Cretatinin status Units / L Units / mmol Total mmol / L urine excretion of creatinine IPG TYPE P Ante- 6.58 ± 0.86 632 ± 112 98.9 ± 12.1 850 ± 105 Post- 5.78 ± 0.64 406 ± 51 70.8 ± 12.2 747 ± 127 Pairing (t) NS NS ** ** IPG TYPE A Ante- 149 ± 23.2 26.2 ± 5.6 234 ± 60 Post- 148 ± 19.2 30.4 ± 6.5 286 ± 66 Matched (t) NS NS NS n = 9 References The references mentioned herein are all expressly incorporated by reference. 1. Redman, C. W. G., 1 991. Current topic: pre-eclampsia and the placenta. Placenta, 12: 301-308. 2. Robertson, W. B., Brosens, I. A. and Dixon ,. H. G. 1967. The pathological response of the vessels of the placental bed to hypertensive pregnancy. J. Path. Bacteriol. 93; 581-5923. Roberts, J.M., Taylor, R.N., Musci, T.J., Rodgers, G.N., Hubel, C.A. and McLaughlin, M.K. 1989. Pre-eclampsia,: and endothelial cell disorder. Am. J. Obstet. Gynecol. 161: 1200-1204. 4. Arkwright, P.D. Rademacher, T. W., Dwek, R. A. and Redman, C. W. 1993. Pre-eclampsia is associated with and increase in trophoblast glycogen content and glycogen synthase activity similar to that found in hydatiform mole. J. Clin. Invest. 91 2744-2753.

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It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property:

Claims (16)

1. The use of an IPG type P antagonist in the preparation of a medication for the treatment of pre-eclampsia.

2. The use according to claim 1, wherein the P-type IPG antagonist has the biological property of: (a) inhibiting the release of the P-type IPG from the placenta; (b) reduce levels of P-type IPG, derived from placenta; and / or (c) reduce the effects of the P-type IPG derived from the placenta.

3. The use according to claim 1 or claim 2, wherein the P type IPG antagonist is an anti-IPG type P antibody or binding protein.

4. The use according to any of claims 1 to 3, wherein the medicament is administered to patients having a high level of IPG type P compared to control subjects.

5. The use according to any of the preceding claims, wherein the high level of the P-type IPG is greater than about 2 times the level in the control subjects.

6. A pharmaceutical composition, characterized in that it comprises a monoclonal antibody which is an IPG type P antagonist, in combination with a pharmaceutically acceptable carrier.

7. A method for the diagnosis of pre-eclampsia in a patient, the method is characterized in that it comprises determining the level of the P-type IPG in a biological sample obtained from the patient.

8. The method according to claim 7, characterized in that the level of the P-type IPG is determined using an assay for the biological activity of the P-type IPG.

9. The method according to claim 8, characterized in that the level of IPG type P is determined in an assay that measures the activation of pyruvate-dehydrogenase-phosphatase by IPG type P.

10. The method according to claim 7, characterized in that the level of the P-type IPG is determined using a binding agent capable of specifically binding to the P-type IPGs.

11. The method according to claim 10, the method is characterized in that it comprises the steps of: (a) contacting a biological sample obtained from a patient with a solid support having immobilized therein the binding agent having sites of specific binding for one or more of the P-type IPGs; (b) contacting the solid support with a developing, labeling agent capable of binding to the unoccupied binding sites, the P-type IPGs bound or the binding sites occupied; and (c) detecting the label of the developing agent that specifically binds in step (b) to have a value representative of the level of the P-type IPGs in the sample.

12. The method according to claim 11, the method is characterized in that it comprises the additional step of: (d) correlating the value obtained in step (c) with the P-type IPG levels in the control subjects to determine whether the patient has a high level of IPG type P.

13. The method according to any of claims 10 to 12, characterized in that the binding agent is an anti-IPG type P antibody.

14. The method according to any of claims 7 to 13, characterized in that a high level of the P-type IPG is greater than about twice the level in the control subjects.

15. The method according to any of claims 7 to 14, characterized in that the sample is a blood, serum, tissue or urine sample.

16. A method for detecting P-type IPG antagonists, the method is characterized in that it comprises: (a) contacting a candidate antagonist and an IPG type P in an assay for a biological property of the IPG type P under conditions in which the IPG type P and the candidate antagonist can compete; (b) measure the biological property of the IPG type P; (c) select the candidate antagonists that reduce the biological activity of the IPG type P.