WO1994000766A1

WO1994000766A1 - Gravidin: assays, dna fragments or genes and uses thereof

Info

Publication number: WO1994000766A1
Application number: PCT/NZ1993/000051
Authority: WO
Inventors: Theresa Wilson; Graham Collingwood Liggins
Original assignee: Auckland Uniservices Limited
Priority date: 1992-06-23
Filing date: 1993-06-22
Publication date: 1994-01-06
Also published as: AU4361493A

Abstract

Gravidin is a protein described as: inhibiting biosynthesis of arachidonic acid (acting on phospholipase A2), occurring in amniotic fluid, blood and particularly pre-term chorionic tissues, having an MW = 80 Kilodaltons and a pI of 8.6, having an N-terminal sequence of K-S-P-I-F-G-P-E-E-V and being homologous to Secretory Component. Discovery of the role of gravidin in pregnancy has led to: bioassays and immunoassays for serum gravidin, providing an improved method for predicting pregnancy-induced hypertension (PIH) in humans and animals. Excess gravidin appears to raise synthesis rates of the vasoconstrictor thromboxane A2 while lowering the vasodilator prostacyclin (PGII2). Methods for making assay reagents and pharmaceuticals, such as purified gravidin (or biologically active fragments of gravidin) and anti-gravidin antibodies have been developed. Gravidin may be harvested from tissue, milk, or other fluids, or manufactured by biotechnology after gene manipulation. Medical treatment with fragments or complete molecules of gravidin, or antagonists of gravidin includes anti-inflammatory actions, anti-PIH actions, and the prevention and treatment of cancer are provided.

Description

TITLE: GRAVIDIN: ASSAYS, DNA FRAGMENTS OR GENES AND USES THEREOF

TECHNICAL FIELD

This invention relates to one or more chemical compounds which can inhibit the activity of the enzyme phospholipase A₂ in vivo and to assays, preferably immunoassays for those compounds.

BACKGROUND ART

Pregnancy induced hypertension

Pregnancy induced hypertension (PIH) with or without proteinuria is a major cause of maternal mortality and morbidity, of perinatal mortality and of long-term physical and intellectual handicap resulting from pre-term delivery. PIH is also known as GH (gestational hypertension) or HOP (hypertension of pregnancy).

PTH develops in 10-20% of those first pregnancies reaching the last third of pregnancy. In its mild form, the disorder is characterised by hypertension only and in severe form by hypertension, proteinuria, convulsions (eclampsia), renal failure, liver failure, coagulopathy and various cardiovascular complications. The pathogenesis of PIH remains uncertain. There is evidence that prostanoids are involved in that the disorder is associated with a reduction in the synthesis of prostacyclin (PGI₂) which is a vasodilator and an increase in the synthesis of thromboxane A₂ (TXA₂) which is a vasoconstrictor.

Agents such as aspirin that selectively inhibit the synthesis of TXA₂ prevent the development of PIH provided that treatment precedes the onset of the disorder. A necessary component of a regimen aimed at prevention of PIH is a reliable and simple test to identify those pregnant women at risk of developing PIH. Available methods for assessment of the risk of PIH in pregnant women generally comprise (a) hospitalisation, (b) administration of angiotensin II, and (c) recording of the rise in blood pressure that may occur as a result, which is not generally feasible as a population screening test. Gynaecology

The premature termination of pregnancy by preterm labour (often induced in order to treat the above problem, PIH) is a major medical problem in that ensuring the survival of premature infants is an intensive and costly business (estimated at a thousand NZ dollars per day for each baby); a number of babies either do not survive or emerge with impaired functions, and the stress and suffering imposed on the parents of premature babies should be avoided if at all possible.

Alleviation of inflammation

Many anti-inflammatory pharmaceuticals act by at least partially inhibiting the synthesis of prostaglandins (as facilitated by phospholipase A₂). Some of these compounds have side-effects (such as gastric mucosal damage by the aspirin/salicylates family of anti-inflammatory drugs). There is a need for alternatives especially as few compounds in current use are naturally occurring in the mammalian body.

Prevention of Cancer and Heart Disease

Recent reports indicate that persons taking low doses of aspirin for the prevention of coronary thrombosis have a substantially reduced incidence of bowel and other cancers. Since this mode of action of aspirin in regard to cancer is highly likely to be mediated by the inhibitors of prostanoid synthesis, it follows by analogy that other inhibitors such as an inhibitor of arachidonic acid synthesis may also be active in preventing cancer, as well as in preventing heart disease, particularly coronary thrombosis.

Treatment of Cancer

Neoplasia is estimated to cause the death of one in three people in the developed (Western) world. Chemotherapy depends on a differential drug sensitivity of neoplastic or altered cells as compared to normal cells, and there are indications that compounds according to this invention have substantially no effect on several tumour cell lines (in terms of the inhibition of phospholipase activity); whereas human lymphocytes (as well as placental tissues) are significantly affected. STATEMENT OF INVENTION

In one aspect, the invention provides an assay for the confirmation or prediction of pregnancy-induced hypertension (PIH) characterised in that the assay comprises a first step of taking one or more samples of blood or other fluid from a subject, and a second step of measuring the concentration of gravidin in the or each sample.

In a second aspect, the invention provides a gene or fragment of DNA characterised in that it comprises a genetic sequence coding for the protein component of the molecule of gravidin or a part thereof.

In a third aspect, the invention provides a fragment of DNA characterised in that it comprises a genetic sequence coding for suppressor or promoter sequences capable of controlling the expression of a gene carrying the code for gravidin or a part thereof.

In a fourth aspect, the invention provides a cell line characterised in that it has an altered genome causing the expression of substantially raised amounts of gravidin or a part thereof within at least some cells or into their medium.

In a fifth aspect, the invention provides a strain of transgenically modified animals characterised in that it has an altered genome causing the expression of substantially raised amounts of gravidin or a part thereof in extracellular fluids.

In a sixth aspect, the invention provides a synthetic gravidin, or biologically active fragments of the gravidin molecule characterised in that it is prepared by genetic engineering means.

In a seventh aspect, the invention provides a method for treatment of aberrant prostaglandin metabolism particularly at the enzyme phospholipase A^ characterised in that it comprises at least the parenteral administration of gravidin.

In an eighth aspect, the invention provides a method for the treatment of pregnancy- induced-hypertension and symptoms thereof characterised in that it comprises the administration of a gravidin antagonist. In another aspect the invention provides a method for assaying the concentration of gravidin (or gravidin-like chemicals) in serum samples.

In another aspect the invention provides a method for the synthesis of gravidin or at least a compound, such as an artificially modified protein, other chemical, or peptide, which retains the biological activity of the naturally found gravidin, substantially possessing its biological activity by one of a number of biotechnological means such as monoclonal techniques or genetic engineering.

In another aspect the invention provides for the use of Secretory Component for diagnostic or therapeutic use as a phospholipase inhibitor or the like, in a manner analogous to that of gravidin, when recovered from milk, colostrum, or saliva by known isolation and purification procedures.

In another aspect the invention provides a method for determining whether a given pregnancy - human or animal (as one example: equine) - is likely to suffer premature termination, based on determining the serum concentration of gravidin (or gravidin-like chemicals).

In another aspect this invention consists of a method for testing the levels of gravidin (or gravidin-like chemicals) in serum samples repeatedly taken from pregnant women (or animals) known to have subnormal levels, in order to monitor the effectiveness of therapy designed to maintain pregnancy.

In another aspect the invention consists of a method for testing for the presence or absence of pregnancy.

In another aspect the invention consists of an antibody to the protein gravidin or the like for use in specific test procedures.

In another aspect this invention consists of the application of the naturally found protein gravidin, gravidin-like compounds or derivatives based on an understanding of its structure and function as an inhibitor of prostaglandin synthesis in medical conditions where an inflammatory or neoplastic process requires to be subdued. These and other aspects of this invention which should be considered in all its novel aspects will become apparent from the following description with reference to the preferred embodiments and to the more general claims at the end of this specification.

DEFINITION OF GRAVIDIN

This patent is based on the appreciation of the physiological and pathophysiological role played by an apparently novel protein, which we have called "gravidin". We define gravidin according to our present understanding of its nature as: having inhibitory activity on the biosynthesis of arachidonic acid, being found in amniotic fluid and in blood, being found in significantly greater quantity in pre-term chorionic tissues than in tissues taken during or after labour, having a molecular weight of 80 Kilodaltons, having a pi = 8.6 (SDS-PAGE electrophoresis), having relatively low glycosylation, (associated carbohydrates), and having the amino acid a sequence at its N-terminal of (in code) K-S-P-I-F-G-P-

E-E-V, or lys - ser - pro - ile - phe - gly - pro - glu - (glu) - (val); the last two are uncertain as only a small sample was available.

Thus gravidin resembles the known protein Secretory Component (SC) associated with the transfer of immunoglobulin A (IgA) - in molecular weight, chemical affinity behaviour (see protocols below), and in the N-terminal sequence given above. Antibodies have been raised which react with gravidin and not with SC. Known functions of SC include the transport of immunoglobulin IgA (or IgM) across endothelial cells. A function of phospholipase inhibition has not previously been described for SC. Further evidence for the relationship between gravidin and SC includes an analogous inhibitory effect of purified SC on lymphocytes, and inhibition of the inhibitory activity by monoclonal antibody to secretory compound.

In the interim, awaiting full sequencing data from gravidin, we assume that SC and gravidin are homologous proteins with similar structures and in vitro properties but are distinguished from each other especially with respect to functions. On the one hand SC is related to immunoglobulin A (IgA) release while on the other hand gravidin acts as an inhibitor and possibly as a regulator of prostaglandin synthesis. Gravidin is known to be difficult to separate from the protein lactoferrin, which has also been confused with SC in previous publications. Nevertheless it has been found convenient to use methodology established for SC in the harvesting of gravidin from fluids (eg see protocols 3, 6 and 7), in part relying on cross-reaction of gravidin with a Sigma antibody to SC.

Biochemistry

The original work on which this invention is based is the discovery that a release of prostaglandins from uterine tissues occurs at the onset of parturition. A necessary part of the intrinsic prostaglandin synthesis process is the formation of arachidonic acid, promoted by the enzyme phospholipase A_j.

The protein 'gravidin' which is an example of the compounds covered by this patent was discovered in the course of a biochemical study of prostaglandin synthesis, wherein gravidin was identified as a protein which inhibits at least one pathway (promoted by the enzyme phospholipase A₂ for prostaglandin synthesis within living tissues.

Known contemporary publications in the field that deal with phospholipase A₂are restricted to studies of factors contributing to premature birth by accelerating prostaglandin synthesis involving phospholipase A. none looked at natural inhibitors of that enzyme. However other publications have considered inhibitors of prostaglandin synthesis and specifically the enzyme PG synthetase, also named cyclooxygenase.

PROTOCOL 1 - BIOPOTENCY TEST METHOD

The following preferred embodiment describes an in vitro assay for enzyme-inhibiting activity suitable for estimations of the biopotency of material containing gravidin. A preferred method for detecting and quantifying substances (herein called "gravidin") capable of inhibiting phospholipase A₂ is as follows;

1. Decidual cells scraped from the chorionic membrane of a human placenta

(selected as they are active in synthesising arachidonic acid) are dispersed with collagenase 1% in G199 medium containing Hank's salts and lmg/ml BSA.

The cells are washed and re-suspended in the same medium without collagenase. An aliquot is counted in a Coulter cell counter.

2. The cells are incubated with ³H arachidonic acid for lh and then 10⁵ cells are pipetted onto a "Millipore" filter (pore size 0.45 μm, diameter 2.5 cm).

3. The cells are perfused with G199 medium containing either no additive, a standard concentration of gravidin, or a standard concentration of gravidin plus varying concentrations of a putative gravidin inhibitor.

4. Radioactivity in 200 μl fractions is counted until a stable baseline is reached after about lh. Histamine 10^"5M is added to the perfusate and the collection of fractions continued for a further 20min.

5. The release of ³H arachidonic acid is stimulated by histamine (100-400%). Stimulated release is inhibited by gravidin (60% at 10-lOM). The presence of a gravidin inhibitor is indicated by loss of the inhibitory effects of gravidin on stimulated release of arachidonic acid.

A porcine pancreas bioassay has also been used, based on inhibition of phospholipase A*

PROTOCOL 2

Gravidin can also be assayed by antibody reaction methods, preferably using the ELISA (Enzyme-Linked ImmunoSorbent Assay) test, two versions of which may be conducted as follows.

1. 100 μl of serum is put onto a Sephadex G-75 column (0.5 x 5 cm) equilibrated in Tris HC1 at pH = 7.4, 50 mM, in which benzamidine (1 mM) and EGTA (1 mM) is dissolved. This is buffer 'A'. (EGTA is a preferred calcium chelator).

2. The protein is eluted, collected and pui through a DE32 column (0.5 x 4 cm) equilibrated in Buffer A. The flowthrough is collected.

The protein solution is diluted 1: 100,000 and 100 μl aliquots are pipetted into wells in a 96-well flatbottomed ELISA plate.

4. The plate is incubated overnight and then blocked in 1% "Marvel" (a milk protein preparation) in Buffer B (Tris HC1 10 mM, pH = 7.4 with 150 mM

NaCl) by incubation for 30 min. Specific antibody (diluted 1:1000 in 1% Marvel in buffer B) is added.

5. A further incubation takes place for 30 minutes at room temperature. After washing 3 times in buffer B a second antibody to the first (such as anti- rabbit IgG peroxidase -linked) is added. A further incubation at room temperature for 30 min takes place.

6. After washing again for 3 times in buffer B the plates are incubated in a solution of OPD (ortho-phenylene diamine) in citrate buffer at pH = 5.0 with a trace of hydrogen peroxide. A further incubation for 30 min is carried out and then the reaction is stopped with 50 μl of 1M HC1.

7. Finally the optical densities are read in an ELISA plate reader at 490 nm wavelength.

PROTOCOL 3

A further method for ELISA measurement of gravidin, as used during clinical protocols, is as follows: (a modification of the SC assay described by Kvale and Brandtzaeg).

<*.

1. Nunc Maxisorb 96-well plates were coated with monoclonal antibody to secretory component (Sigma 16635). 2 Antibody was diluted 1:2500 in carbonate buffer (pH 9.6) and 100 μl pipetted into each well.

3 The plates were incubated overnight at room temperature, washed with

10 mM Tris buffer pH 7.4 containing 150mM NaCl (Buffer A) and then

"blocked" with Buffer A containing 1% fish gelatin for fifteen minutes. 4 A volume of 100 μl serum diluted 1:50 with Buffer A was placed in each well. 5 Duplicate dilutions were made and triplicates of each were assayed.

6 Diluted serum samples were incubated at room temperature overnight.

7 The plates were washed (once in Buffer A containing 0.05% Tween 20 and then twice with Buffer A) and incubated for one hour with polyclonal antibody to SC(1:2000 dilution) raised in goats (Sigma S1640).

8 The plates were washed again and incubated with swine antigoat antibody, (peroxidase-linked, diluted 1:3000; Caltag G50007).

9 After further washing, bound peroxidase was detected with ortho phenylene diamine. Antibodies except the primary antibody were diluted in blocking buffer.

10 Plates were read at 490-405 nm with a Biotek ELA autoreader model EL 310.

Interassay variation was less than 10%. Intra-assay variation was less than 6%. Both antibodies to gravidin bound to free and bound gravidin and did not bind to free IgE, IgG IgM or monomeric IgA (signal less than 1% of that for slgA).

PROTOCOL 4

Purification of gravidin for calibration of slgA standards

This was performed substantially as described above. Lyophilised human amniotic fluid (4g) was reconstituted and dialysed against buffer (Tris-HCl, 50 mM pH 7.4, containing 1 mM benzamidine and 1 mM EGTA at 4^βC and was passed through a DE32 column (10 cm x 2 cm) in Buffer A. The flowthrough was precipitated (100% ammonium sulphate) and eluted proteins separated on a calibrated Sephadex G100 column (in Buffer A). The 80KDa fraction was fractionated with a Mono-S FPLC column equilibrated in 0.1 mM HEPES (pH 7.0), washed with 0.1 mM HEPES and protein eluted with a gradient of 1 mM NaCl in 0.1 mM HEPES (pH 7.0). The gravidin peak was collected and stored at -20°C in 50% glycerol. Purity was ascertained from a single band obtained by Coomassie Blue staining of a Laemmli gel.

Validation of the assay

Secretory IgA in a volume of 2 μl was diluted into control human serum (minus IgA and IgM - Sigma 5393) (1:50 dilution) to give a final concentration of from 2 to 100 ng/100 μl. The SC content by weight of commercially available slgA was in the order of one third although there was considerable variation between batches when assayed against a gravidin standard as prepared above. The 1:50 dilution of the test serum always fell within the range of the standards. A linear curve was obtained in the assay from the addition of slgA (2-100 ng/lOOμl) to the diluted control serum. Standards and blanks (1:50 dilution of control serum) were run on each ELISA plate. The minimum detectable concentration of gravidin was 1.25 nM; this gave a value greater than two standard deviations above the blank value. Although there is some disagreement in the literature as to the molecular weight of this form of secretory component a weight of 80 KDa was assumed. This assay assumes cross-reaction between SC and gravidin antibodies.

RECOVERY OF GRAVIDIN FROM FLUIDS OR TISSUES

BOUND GRAVIDIN

To extract gravidin from suitable tissues a preferred method, published by Mestecky & Kilian in Methods in Enzymology, vol 116, 1985 for the recovery of Secretory Component - which is believed to have substantially the same properties during separation procedures as those shown by gravidin is as follows.

PROTOCOL 5

Isolation of IgA-Bound SC

Background: The majority of SC is disulphide-linked to the Fc portion of a single 7 S subunit of polymeric S-IgA. Therefore, the cleavage of disulphide bonds is required for its release. However, a small proportion of SC in human S-IgA molecules is not linked to the α chain by disulphide bridges and can be released from S-IgA by 5 M guanidine- HC1. S-IgA, dissolved in 5 M guanidine-HCl is applied to a column of Sephadex G- 200 equilibrated in the same solution. Under these conditions, SC and small amounts of L chain dimers are eluted after the principal peak of S-IgA. SC can be released from reduced (0.2 Λ 2 mercaptoethanol in 0.5 M Tris-HCl buffer, pH 8.2, 1 hr, at room temperature) and alkylated (0.33 M iodoacetamide, for 30 min at room temperature) or partially S-sulphonated (see above) S-IgA. This material is applied to a Sephadex G- 200 column equilibrated either in Tris- HC1 buffer, pH 8.0 containing 1 M NaCl or 1% NH₄HCO₃. Most of the SC that is, however, contaminated by free H and L chains is eluted after IgA molecules. Sequential immunosorption with insolubilized anti-IgA and anti-human serum can be used to remove such impurities. To avoid immunosorption with insolubilized antibodies, cleavage of S-IgA with CNBr before or after reduction and alkylation or oxidative sulphitolysis has been used. As described above, S-IgA with cleaved disulphide bonds resolves into H and L fractions upon gel-filtration on Sephadex G-200 in 5 M guanidine-HCl. However, SC coelutes with H chains on this gel. Pretreatment with CNBr results in cleavage of the α chain at the methionine residues with a considerable decrease in the molecular weight; the molecular weight and the elution position of SC, however, remains unchanged due to the absence of methionine in SC.

Materials:

Partially S-sulphonated lyophilized S-IgA 70% formic acid

CNBr (Eastman Kodak, Rochester, N.Y.) Sephadex G-200 column in 5 M guanidine-HCl Sephadex G-25 column in 1% NH₄HCO₃

Procedure:

1. S-sulphonated lyophilized S-IgA is dissolved in ice-cooled 70% formic acid at a proportion of 20 mg protein/ml formic acid. 2. A 2-fold amount of solid CNBr (w/w) is added and the mixture is incubated overnight at 4\ Steps (1) and (2) must be performed in a chemical hood with a strong external exhaust. After the addition of CNBr the vessel is closed with a glass stopper and placed overnight in a refrigerator. 3. This solution is then diluted with 10 volumes of distilled water and lyophilized. 4. Lyophilized protein is applied on a column of Sephadex G-200 equilibrated in 5 Λf guanidine-HCl. SC is present in fraction 2 while cleaved α chains are in fraction 3. To obtain immunochemically pure SC this gel filtration step must be repeated to achieve a complete separation of SC from cleaved α chains. Guanidine-HCl can be removed from SC by desalting on Sephadex G-25 or dialysis. The solubility of SC in nondissociating solvents such as PBS is low due to the denaturation of SC. This preparation is, however, suitable for chemical analysis of SC or production of anti-SC antibodies in rabbits and goats.

J PROTOCOL 6

Isolation of Free GRAVIDIN / SC

Owing to their high concentrations of SC, human colostrum and milk are more convenient sources than saliva, which also contains free SC. Isolation procedures are

10 based either on the physicochemical properties of SC or its unique ability to combine in vitro with polymeric IgA or IgM. Because SC is sensitive to proteolysis the isolation should be performed in the presence of protease inhibitors. Several procedures of SC purification have been proposed, which include gel filtration and ion-exchange chromatography as principal methods. The most common contaminants of free SC are

15 lactoferrin and trace amounts of monomeric IgA and IgG. The method described is a composite of techniques used in several laboratories with a final step of affinity chromatography on heparin columns to remove lactoferrin.

Materials 20 Human colostrum or early milk is processed as described under Purification of S-IgA except for the buffer used. Instead of PBS with 0.1% NaN₃, 0.1 M Tris-HCl buffer, pH

7.6 with 0.5 M NaCl, 1 mM PMSF, 50 mM eta amino n caproic acid, and 0.1% NaN₃ should be used. PMSF is prepared as 100 mM solution in isopropanol and is added to the Tris-HCl buffer in a proportion of 1:100. 25

Sephadex G-200

Saturated solution of (NH₄)₂S0₄

DEAE-cellulose in 0.01 M phosphate buffer, pH 7.4-7.6

0.01 M phosphate buffer pH 7.4-7.6 30 Heparin affinity column (see Purification of S-IgA)

0.05 M Tris-HCl buffer, pH 8.0 containing 0.2 M NaCl

Procedure

35 1. Initial steps of free SC isolation from human colostrum are described under Purification of S-IgA. To prevent cleavage of free SC during the preparation, fresh colostrum is diluted in 0.1 M Tris-HCl buffer pH 7.6 with protease inhibitors. This buffer is also used during gel-filtration of Sephadex G-200. Defatted colostrum from which casein has been removed by acidification and centrifugation is precipitated by saturated (NH₄)₂SO₄ to a final saturation of

70%. At this saturation, both S-IgA and SC are precipitated and can be used for subsequent isolation of free SC and S-IgA. Free SC is separated on a Sephadex

G-200 column as described.

2. The second fraction from a Sephadex G-200 column (Fig. 1) contains SC which is precipitated with (NH₄)₂SO₄ at 70% saturation at 4" overnight. The precipitate is collected by centrifugation at 10,000 g for 20-30 min and subsequently dissolved and dialyzed overnight at 4"C against 0.01 M phosphate buffer, pH 7.6. Alternatively, the SC-containing solution can be desalted on a Sephadex G-25 column in 0.01 M phosphate buffer, pH 7.4-7.6. 3. This solution is applied on a DEAE-column equilibrated in the same buffer. Under these conditions SC is not retained and is eluted by washing the column in this buffer. SC present in the eluate is concentrated by negative pressure dialysis or precipitation with (NH₄)₂SO₄ to 70% final saturation. Further steps depend upon requirement for SC purity. To remove small amounts of serum and colostral proteins, Brandtzaeg used the insolubilized gamma-globulin fraction of anti-normal human serum and anti-human colostrum (nonreactive with SC) reagents. Additional steps in other purification procedures include preparative acrylamide electrophoresis or zone electrophoresis.

The most prevalent contaminant is lactoferrin which can be removed by adsorbtion on a column of polymerized albumin. In our laboratory we prefer heparin affinity chromatography, which selectively removes lactoferrin, and can also be used for a single step purification of lactoferrin from human milk. The SC solution is dialyzed against 0.05 M Tris-HCl buffer, pH 8 with 0.2 M NaCl and applied on a heparin-affinity column equilibrated in the same buffer. SC is not retained under these conditions and is eluted. An additional step of Sephadex G-200 gel filtration may be necessary to obtain immunochemically pure SC for immunization purposes. PROTOCOL 7

Isolation of Free SC by Affinity Chromatography on Insolubilized Polymeric IgA or IgM

The affinity of free SC for polymeric IgA or IgM is amply documented. As demonstrated by Brandtzaeg SC binding is dependent on the J chain content of the polymeric IgA or IgM molecules. Because various preparations of polymeric IgA myeloma or macroglobulinemic IgM proteins may contain variable amounts of J chain and therefore differ in their SC binding capacity, it is advisable to examine several IgA or IgM preparations by a simple immunoelectrophoretic procedure for SC binding ability.

Materials Polymeric myeloma IgA (purified according to known techniques for the Purification of

Serum IgA) or macroglobulinemic IgM

CNBr-activated Sepharose 4B (Pharmacia)

Defatted and decaseinated colostral whey or pooled second peak from Sephadex G-200 fractionated colostrum (see Purification of S-IgA) PBS

0.15 M NaCl acidified to pH 2.5 with 1 acetic acid

O.l M NaOH

0.01 M citrate, 0.02 M phosphate buffer pH 5N6.8

Fe(NH₄)₂SO₄ 1 M KCNS

Sephadex G-200

Procedtt-re 1. Follow manufacturer's procedure for covalent binding of polymeric IgA or IgM to CNBr-activated Sepharose 4B. Diluted colostral whey (1:10 in PBS) is applied on a column of Sepharose 4B with covalently linked polymeric IgA.

The column is thoroughly washed with PBS and bound SC is eluted with 0.15

M NaCl acidified to pH 2.5 with acetic acid. 2. SC-containing solution is immediately neutralized with 0.1 M NaOH and concentrated by precipitation with saturated (NH₄)₂SO₄ to 70% final saturation. The precipitate is collected by centrifugation (10,000 g for 20-30 min) dissolved and dialyzed in 0.05 M Tris-HCl buffer pH 8 containing 0.2 M NaCl. Lactoferrin, which also binds to IgA, is removed on a heparin column as described above (Purification of Free SC). Lactoferrin can also be removed by passage through a column of absorbed on an insolubilized anti-lactoferrin reagent.

SC purification by IgM-Sepharose 4B affinity chromatography used 0.2-0.5 ml of Sepharose 4B-IgM conjugate/1 ml of colostral whey diluted 1:10 in 0.01 M citrate, 0.02 M phosphate pH 8.6 containing 0.25 mg Fe(NH₄)₂SO₄ per ml of diluted whey. After thorough washing with PBS, SC is eluted with 1 M KCNS in 0.01 M phosphate buffer pH 6.8-7.0. The eluate is dialyzed against PBS and passed through a Sephadex G-200 column. The second fraction contains pure SC as ascertained by immunoelectrophoresis and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The theoretical yield of SC is approximately 70% as compared to 2-10% yield of SC isolated by the above described chromatographic methods.

OCCURRENCE OF GRAVIDIN IN HUMAN TISSUES

Since the first report that prostaglandin F2a (PGF2a) plays a role in the control of labour, as reviewed by Liggins in 1981 (Liggins, GC: "Endocrinology of Parturition" in "Fetal Endocrinology" eds M J Novey & J A Resko, Academic Press, New York), it has become accepted that the onset of labour at term in women is due to the increased release of PGF2a from uterine tissues. The mechanism controlling prostaglandin synthesis has remained undefined. In most tissues, the rate of synthesis is determined by the rate of release of arachidonic acid by deacylation at the sn-2-position of phospholipids. Thus a major rate-limiting step in the synthesis of uterine prostaglandins is the activity of phospholipase A₂. We have partially purified two proteins from human amniotic fluid which specifically inhibit phospholipase A^. The smaller protein has now been purified and further characterised. Activity of the protein in media from incubated chorion obtained before labour is much greater than after the onset of labour. We conclude that the protein gravidin could contribute to the control of the onset of labour in women. The major source of the protein inhibitor of phospholipase A₂ isolated from human amniotic fluid which we have called gravidin was sought in tissues in close proximity to this fluid. Amnion, chorion and decidua were taken from placentae delivered at elective caesarean section and cultured in Earles's 199 medium for 48h at 37°C. Gravidin was purified from the medium and assayed for inhibition of porcine pancreatic phospholipase A₂ activity. The most active extracts were those from cultures of chorion (Fig. 1).

The amount of gravidin in cultures of chorion from women undergoing caesarean section at term before the onset of labour (not-in-labour, NIL) was compared with that from chorion from women delivered after spontaneous labour (in-labour, IL) either vaginally or by caesarean section. After purification, gravidin was assayed by two methods; (i) a bioassay system measuring stimulated release of labelled arachidonic acid from dispersed human decidual cells (Fig 2 a,b), and (ii) inhibition of activity of porcine pancreas phospholipase A^ (Fig 3). In both assays, cultures of chorion cultured after the onset of labour contained significantly less activity when compared to that obtained before the onset of labour. The mean percentage inhibition (± SEM) in the bioassay by NIL and IL samples respectively was 30.4 ± 4.6 vs -4.0 ± 10.8 (stimulation by histamine; p<0.02) and 30.7 ± 2.4 vs 0.4 ± 3.9 (stimulation by calcium ionophore; p<0.001). In the porcine pancreas phospholipase A₂ assay (Fig. 3) the NIL and IL values were 35.8 ± 14.5 and 1.3 ± 1.6 (ρ<0.005) respectively.

On SDS PAGE, identical bands were seen when material from the two types of chorion was compared, but the IL material stained less intensely. Quantification of the purified protein revealed that recovery from the IL chorion was significantly less (3.10 ± 1.4 μg/ml medium, mean + SEM, n=6) compared to the NIL chorion (9.00 + 2.05 μg/ml, n=6). When media from NIL chorion and IL chorion were pooled before purification no reduction in the expected recovery was found, indicating that the lesser amount of protein in the IL preparation did not result from its destruction during the recovery process.

Gravidin was found to exist as a doublet with molecular weight bands of 80 KDa. There was no reduction of molecular weight under reducing conditions. The pi for both bands was calculated as 8.6 by interpolation from Sigma pi standards. Gravidin fulfills three basic requirements for a significant role in the control of human parturition. First, it inhibits the release of arachidonic acid from phospholipid which is the rate-limiting process in prostaglandin synthesis (4). Second, its inhibitory activity is significantly reduced with the onset of labour. Third, it is released by chorion, a tissue that is contiguous with decidua which is considered to be the major source of uterine prostaglandin F_2α .

Endogenous inhibitors of prostaglandin synthesis that could be involved in human parturition have been described by others but all inhibit cyclooxygenase rather than phospholipase A₂ and none has been purified. Lipocortins, the mediators of glucocorticoid action are the only endogenous inhibitors of phospholipase to have been purified and characterised but their role in reproductive tissues has not been studied.

The therapeutic potential of phospholipase A₂ inhibitors is of considerable current interest in relation to a variety of disorders resulting from the release of abnormal amounts of arachidonic acid metabolites.

Tests for phospholipase A₂ inhibition were carried out on several types of non- reproductive cells. Although gravidin was clearly inhibitory (in terms of the amounts of tritiated arachidonic acid released) for human lymphocytes, there was no inhibitory activity for mouse mastocytoma cells, nor on human tumour cell lines. This absence of inhibition on tumour lines, of cells which in general have high phospholipase activity, may be of importance in chemotherapy.

Gravidin has been shown to inhibit phospholipase A₂ activity in human lymphocytes, decidual cells, endometrial cells, and fibroblasts.

CLINICAL MEASUREMENTS

Patients

A single serum sample was obtained with informed consent from normotensive (NT) nulliparous women booking in to a large maternity hospital at 9-34 weeks of pregnancy. Serum (1ml) was stored below 20^βC for up to eight years before assay. Subjects described as normal in this study were normotensive throughout pregnancy, delivered between 38-43 weeks inclusive and had no reported medical problems during pregnancy. The patients allocated to the PIH group were normotensive before 20 weeks of pregnancy, but had a resting diastolic blood pressure above 90 mmHg on two separate occasions after twenty weeks gestation with or without proteinuria. Patients with medical problems such as chronic hypertension or diabetes were excluded from the study. Serum samples were obtained before diagnosis of PIH, so patients were not being treated at the time the serum was drawn. The mean time of storage for PIH and NT serum samples was not significantly different and the samples were treated without distinction. The average age of the patients was 26.3 ± 3.4 years for the PIH group and 27.8 ± 2.4 years (mean ± sem) for the normotensive group. No difference between the PIH and NT groups was found with regard to ethnicity. The mean time of sampling serum was 19.4 ± 1.1 weeks gestation for PIH patients (n=39) and 19.9 ± 0.6 weeks for normotensive patients (n=103).

The method used for these ELISA (Enzyme-Linked ImmunoSorbent Assay) tests are described above (Protocol 3)

Purification of gravidin for calibration of slgA standards was according to Protocol 4 above. Validation of the assay was also according to Protocol 4.

Statistical Analysis

The data were normally distributed (skewness < 0.63, kurtosis < ±1.2) for all analysed groups so parametric tests were used. Analysis of variance (ANOVA) was used for multiple comparisons and Student's T-test was used for comparisons between two groups. A 2-way ANOVA was performed to determine whether the difference between the PIH and NT groups depended on the stage in pregnancy at which the serum samples were taken.

A list of preferred forms of treatment for threatened premature labour includes: Progesterone therapy, bed rest and reduced physical activity, treatment with synthetic gravidin, treatment with purified human gravidin, treatment with recombinant gravidin, treatment with a gravidin subunit treatment with "Ventolin", treatment with indomemacin, treatment with "Berotec", or treatment with nifedipine.

ILLUSTRATIONS

Fig. 1: is an illustration that demonstrates the inhibition of porcine phospholipase A_j by gravidin recovered from placental tissues.

Fig. 2a: shows the effect of histamine as a stimulent for the release of arachidonic acid.

Fig. 2b: shows the effect of calcium ionophore instead of histamine.

Fig. 3: is an illustration of the effect of gravidin, obtained from before and after labour, on the stimulated release of tritiated arachidonic acid from perfused human decidual cells.

Fig. 4: is an illustration of the effect of gravidin on the activity of phospholipase in cells of human tumour lines.

Fig. 5: is a graphical illustration of the serum gravidin concentrations in pregnancies which remained normal or became hypertensive.

Fig. 6: is a graphical illustration relating the concentration of gravidin and weeks of gestation at delivery.

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Fig. 7: is a graphical illustration relating the concentration of serum gravidin and severity of PIH.

Fig. l

Inhibition of porcine phospholipase A₂ activity by gravidin recovered from placental tissues.

(Amnion - shaded bars. Chorion - hatched bars. Decidua - unfilled bars.) These tissues were dissected from placentae obtained by elective caesarean section (NIL). Pieces weighing 2.44g ± 0.39, (amnion) 2.16g ± 0.48 (chorion) and 4.07g ± 0.88 (decidua), were washed three times in sterile Hank's 199 medium, and incubated in 20 ml Earle's 199 medium for 48h in an atmosphere of 5% ^^5% air at 37^*C.

Precipitate from 70-100% ammonium sulphate at 4^*C was further purified by Sephacryl S200 gel filtration. The fraction eluting at 55-85 KDa was lyophilised and HPLC performed on C14 radial pak column with a gradient of 0.1% TEA into 78% acetonitrile in 0.1% TFA at a flow rate of lml/min. The fraction eluting between 28 and 30 min was collected and lyophilised. Purified protein was recovered from 1 ml incubation medium and redissolved in 1 ml buffer and used for triplicate assay determinations. The final concentration in the chorion extract was between 1 and 2 x 10^*8M.

A and B refer to tissues from individual patients. In C the tissues of three patients were incubated separately and the media pooled. Phospholipase determinations were performed by the method of Ballou and Cheung and modified as described previously.

Fig.2a & Fig.2b Effect of gravidin from chorion obtained before and after labour on the stimulated release of [³H]-arachidonic acid from perfused human decidual cells. Fresh placentae were obtained after vaginal delivery following spontaneous labour (IL) and elective caesarean section (NIL).

Two placentae were from caesarean sections performed after the onset of labour. Decidual tissue was scraped from the outer surface of the chorionic membrane. The tissue was washed three times in Hanks's 199 medium, cut into 1x2 mm pieces and digested for 30 min with stirring at 37^*C in a solution containing 130mM NaCl, 3 mM Na_jPO,,, 2mM MgS0₄, 12 mM NaHC0₃, 1 mM CaCl_j, 2 mg ml glucose, 0.25 ng/ml collagenase, 0.12 mg/ml trypsin. The cell suspension was incubated overnight in the presence of 10% fetal calf serum and viability determined by trypan blue exclusion.

Cells were labelled with [³H] - arachidonic acid and perfused as previously described.

Gravidin was prepared from both NIL and IL chorion as described in the Legend to Fig. 1. Histamine (Fig. 2a) or calcium ionophore (Fig. 2b) was used to stimulate release of arachidonic acid. The concentration of gravidin recovered from the NIL extracts was between 1 and 2xlO-10M in d e perfusion medium.

Fig.3. Effect of gravidin from human chorion obtained before and after labour on the activity of porcine pancreatic phospholipase .A-.

Gravidin from chorionic tissues of individual patients was prepared and assayed for inhibitory activity against phospholipase A^ as described in the legend to Fig. 1. One quarter of the protein recovered from a 200μ HPLC application was used in each of triplicate determinations.

Fig.4

An illustration of the inhibitory effect of gravidin on the activity of phospholipase in cells of human tumour lines. In this illustration the normal cells are represented by the black bar, the tumour cells by the hatched bar. 'Basal', A₂3187', and 'Histamine' refer to the use (or not, for 'basal') of phospholipase stimulating factors. Note that in all cases the apparent phospholipase activity of normal cells as shown by arachidonic acid release is substantially reduced; whereas that of the tumour cells is not. These graphs include die accumulated results from trials on a number of separate cell lines.

Fig.5

This shows serum gravidin concentrations in pregnancies which remained normal or became hypertensive. Serum obtained from women at different times in pregnancy was assayed by ELISA for gravidin content. Values shown are means ± SEM. (Numbers of samples are in brackets). Intermittently hatched (left side) bars represent data from normotensive (NT) pregnancies. Regularly hatched bars (the right bar of each pair) represent data from patients who were showing signs of PIH or had developed PIH.

The difference between NT and PIH was highly significant (p < 0.001) when analysed by 2-way ANOVA.

Fig.6

This shows a relationship between the concentration of gravidin and weeks of gestation at delivery. Serum obtained from pregnant women was assayed by ELISA for gravidin content. The data was analysed according to gestational age at delivery. Values are means ± SEM. Numbers in brackets are sample numbers. Data was analysed by ANOVA and Duncan's multiple range test. The column marked with a double asterisk (**) is significantly different from those marked with one (p<.05) or no asterisks (p<0.01).

Fig.7

This figure shows a relationship between the concentration of serum gravidin and d e severity of PIH. Serum obtained from pregnant women was assayed by ELISA for gravidin content. The data was analysed according to whether the women developed PIH widi or without accompanying proteinuria. Values shown are means ± SEM. The number of samples per group is shown in brackets. PU = proteinuria.

Significant differences in the action of the compounds covered by this patent occur between normal human cells and cell lines derived from tumours.

RESULTS

Serum Measurements Serum gravidin was measured in serum taken at different stages of pregnancy. Levels from patients who had developed or subsequently developed PIH were raised compared to NT patients at all times investigated in pregnancy (Fig. 6). The difference between PIH and NT gravidin levels did not appear to depend on time of gestation as the interaction between groups was not significant as determined by 2-way ANOVA.

Severity of disease and serum gravidin

Patients who developed PIH were categorised according to the presence or absence of accompanying proteinuria and according to gestational age at delivery. Although serum gravidin measurements were higher in patients with proteinuria, the difference was not significant when a Student's t-test was performed (Fig. 7). However, when patients were analysed according to length of gestation, those delivering before 33 weeks witii PIH were significantly different from those with PIH who delivered later (after 33 weeks, p<0.05) and also from the control normotensive patients (p < 0.001) when analysed by ANOVA and Duncan's Multiple range test. The mean gravidin concentration for induced PIH patients was 10.69 nM ± 0.77 (n=17) compared to 8.33nM for noninduced PIH patients (±0.49, n=22, p<0.01 as analysed by Student's t- test; mean ± sem).

DISCUSSION

This study shows that raised serum gravidin levels in pregnancy are associated with development of PIH. The abnormality is evident early in pregnancy indicating that a predisposition to PIH exists before clinical symptoms appear. This observation is consistent with a previous report that women who develop PIH have impaired trophoblast invasion of the spiral vessels.

We have found that incubation of gravidin with its monoclonal antibody inhibits the anti-phospholipase A₂ effect. Gtravidin may bind to IgA or IgM may thereby reducing its activity in patients. As IgM and IgA levels are reported to be significantly decreased in sera of patients with PIH it is possible that more free (active) gravidin circulates in PIH than in NT patients.

The source of the high circulating levels of gravidin in normal pregnancy and PIH is unknown and currently under investigation. Possible fetal sources are the amnion and the developing lung in which gravidin has been detected histologically at 8 weeks. If gravidin crosses the placenta, fetal production could account for the raised serum gravidin in pregnancy but it seems likely that there is also a maternal contribution.

Fig. 7 shows a relationship between high serum gravidin levels and the severity of PIH. Although we cannot yet define the mechanism whereby gravidin could cause hypertension, it is relevant that decreased prostacyclin synthesis has been observed in PIH. As gravidin is a phospholipase inhibitor and serum levels are elevated during pregnancy it seems possible that high circulating gravidin levels might inhibit prostacyclin production and so contribute to the pathology. Elevated thromboxane synthesis is also associated with PIH suggesting that gravidin may be selective with respect to its target tissues. The possibility of a selective effect of gravidin on platelets and endothelial cell prostaglandin synthesis is currently under investigation.

In summary, elevated serum gravidin appears to contribute to the development of PIH. Serum gravidin concentrations are raised during gestation and reach a peak towards term. In subjects who later develop PIH, serum gravidin concentrations are significantly raised above d e normal pregnant level and d e increased is related to the severity of the disease. Therefore, an assay of serum gravidin provides a useful predictive test for PIH, and it has been found to be as accurate as angiotensin II challenges, even when a single blood sample for gravidin assay is taken.

ADVANTAGES

We have found tiiat the concentration of gravidin in serum measured by ELISA predicts both the development of PIH and its severity; the higher the levels of gravidin compared to normal pregnant women, die more severe PIH is likely to be. The test is comparable in predictive worth to the angiotensin II test yet requires no hospitalisation nor stress.

Practical benefits foreseen apply to clinical medicine and in particular the field of obstetrics and include (1) the prediction of likely premature births in mammals and in particular humans by testing for the chemical compound of this invention in its naturally occurring form, (2) a method for testing medication designed to alleviate premature birth, (3) a chemical compound of tiiis invention having generally medically useful anti-inflammatory properties and (4) further uses in various preventative and chemomerapeutic strategies in oncology.

Gynaecology: The premature termination of pregnancy by pre term labour (often induced in order to treat PIH) is a major medical problem in that ensuring the survival of premature infants is an intensive and costly business (estimated at a thousand NZ dollars per day for each baby); a number of babies either do not survive or emerge with impaired functions, and me stress and suffering imposed on the parents of premature babies should be avoided if at all possible.

Alleviation of inflammation: The chemical compounds which form the basis of this invention act to at least partially inhibit the synthesis of prostaglandins (as faciUtated by phospholipase A₂) and thus should be a useful alternative to the aspirin/salicylates family of anti-inflammatory drugs.

Prevention of Cancer and Heart Disease: Recent reports indicate that persons taking low doses of aspirin for the prevention of coronary thrombosis have a substantially reduced incidence of bowel and other cancers. Since this mode of action of aspirin in regard to cancer is highly likely to be mediated by the inhibitors of prostanoid synthesis, it follows at gravidin, a part thereof, or an analogue may also be active in preventing cancer. The efficacy of aspirin in preventing heart disease, particularly coronary thrombosis, suggest that gravidin, a part thereof or an analogue may be effective in preventing heart disease.

Cancer treatment: Neoplasia is estimated to cause the death of one in three people in the developed (Western) world. Indications are that gravidin may provide a tool in chemotherapy in that several tumour cell lines tested so far for inhibition of phospholipase activity are not affected; whereas human lymphocytes (as well as placental tissues) are affected. Significant differences in the action of the compounds covered by this patent occur between normal human cells and cell lines derived from tumours. Finally, various other alterations and modifications may be made to the foregoing without departing from the spirit or scope of this invention.

GENETIC ENGINEERING PROCEDURES for the MANUFACTURE OF GRAVIDIN

Given knowledge of die amino acid sequence of at least part of the protein of gravidin, it is possible to apply the principles of gene manipulation in order to produce quantities of the protein gravidin or derivatives thereof in a reactor. It must be realised that the following steps are also applicable to the synthesis of active portions of the entire molecule.

The manufacturing process comprises at least some of the following steps. 1. Establishing the sequence of gravidin.

Alternative 1:

2A. Manufacturing a novel artificial gene having the correct triplet coding to prescribe gravidin, bearing in mind that extra peptide chains may be deleted during the steps of intracellular protein maturation including folding of the peptide chain, and carbohydrate addition. 3A. Manufacturing novel sequences of DNA having a coding sequence suitable for activating a natural or an artificially introduced gene diat prescribes the structure of gravidin. 4A. Optionally assembling a novel gene that codes for an enzyme capable of synthesising the carbohydrate portion of gravidin. 5 A. Introducing the above artificial genes into cells by one or more of the known techniques, preferably cells of mammalian cell lines, and providing conditions capable of enhancing the expression of related RNA and hence production of gravidin.

Alternative 2:

2B. using sequenced portions of an artificial gene to locate the gene for gravidin on die chromosome array of an animal cell. 3B. Walking along the chromosome and examining it for die presence of associated suppressor or promoter genes. 4B. Introducing novel genetic material capable of acting on the genetic regulatory mechanism in order to raise the intrinsic level of production of gravidin. 5. (for either routes A or B above)

Supplying novel lines of cells having their genetic complement altered as above with nutrients in a reactor vessel and collecting, purifying, and storing the gravidin (or derivatives tiiereof) so produced.

6 (Alternative 3)

Creating novel strains of transgenic animals, preferably having an altered genome made according to the principles above, such that at least some females secrete raised amounts of the protein gravidin (or fragments thereof) into milk. Sheep or goats may be preferred species of animal.

THERAPEUTIC ADMINISTRATION OF GRAVIDIN OR ITS FRAGMENTS

There is considerable scope for die use of the protein gravidin (or fragments thereof having similar activity) as an anti-inflammatory substance, as gravidin inhibits at least one step of the biosynthetic pathway for prostaglandins, mediators of the inflammatory response. It has the advantage that it is substantially different in structure and physiological actions to the conventional and widely used anti-inflammatory substances such as aspirin. Uses of gravidin (or fragments thereof) as outlined above include control of hypertension in pregnancy, the prevention or treatment of cancer, and die prevention of heart disease.

Therapeutic administration of gravidin may be applicable to humans, in order to minimise die above syndromes, and related applications in veterinary medicine include assisting with breeding of endangered species in zoos, and the particular problem of maintenance of pregnancy in brood mares, many of which have a distressingly low proportion of successful pregnancies.

As large proteins cannot generally be introduced into the body by an oral route, gravidin may be used eitiier by a parenteral route at dose rates sufficient to cause the serum concentration of gravidin to rise to an effective level, or sufficiendy small, yet active fragments may be able to be introduced by effectively non-invasive means such as transdermal electrophoresis.

Antagonists to gravidin may also be used. Such antagonists may include antibodies to gravidin, less specific molecules having an avidity for gravidin, molecules which block its receptor site on the target enzyme, or molecules which block the synthesis of gravidin.

Claims

CLAIMS:

1. An assay for the confirmation or prediction of pregnancy-induced hypertension (PIH) characterised in that the assay comprises a first step of taking one or more samples of blood or other fluid from a subject, and a second step of measuring the concentration of gravidin in die or each sample.

2. An assay as claimed in claim 1 characterised in that the assay is based on immunological reactions.

3. An assay as claimed in claim 1 characterised in that the assay is based on measuring die metabolic effect of gravidin on die enzyme phospholipase A2.

4. An assay as claimed in claim 2 for the confirmation or prediction of PIH, characterised in that the method comprises a first step of taking one or more samples of blood or otiier fluid from a subject, and a second step of measuring the concentration of gravidin in the or each sample using reagents having immunological activity against secretory component (SC).

5. A gene or fragment of DNA characterised in that it comprises a genetic sequence coding for die protein component of the molecule of gravidin or a part thereof.

6. A fragment of DNA characterised in that it comprises a genetic sequence coding for suppressor or promotor sequences capable of controlling the expression of a gene carrying the code for gravidin or a part thereof.

7. A cell line characterised in that it has an altered genome causing the expression of substantially raised amounts of gravidin or a part tiiereof witiiin at least some cells or into their medium.

8. A strain of transgenically modified animals characterised in that it has an altered genome causing the expression of substantially raised amounts of gravidin or a part thereof in extracellular fluids.

9. A synthetic gravidin, or biologically active fragments of the gravidin molecule characterised in mat it is prepared by genetic engineering means.

10. A method for treatment of aberrant prostaglandin metabolism particularly at the enzyme phospholipase A₂ characterised in that it comprises at least the parenteral administration of gravidin.

11. A metiiod as in claim 10 characterised in mat it comprises die administration of one or more biologically active fragments of gravidin.

12. A method as in claim 10 characterised in mat it comprises the administration of one or more biologically active antagonists of gravidin, capable of blocking the effects of gravidin on prostaglandin syndiesis within the body.

13. A method for the treatment of pregnancy-induced-hypertension and symptoms thereof characterised in that it comprises the administration of a gravidin antagonist.