ADENOSINE DIPHOSPHATASE AND ACTIVATORS THEREOF AND THEIR MEDICAL USES

Abstract: The present invention relates to materials and methods for the diagnosis and treatment of pre-eclampsia and related conditions characterised by platelet aggregation. In particular it relates to the use of adenosine diphosphatase (ADPase) and activators of ADPase for the treatment of pre-eclampsia, in order to overcome the inhibition of ADPase by inositol phosphoglycans (IPGs). Also provided is a method for screening for compounds with ADPase-stimulatory activity in the presence or absence of IPGs.

Description: Adenosine Diphosphatase and Activators Thereof and Their Medical Uses Field of the Invention The present invention relates to adenosine diphosphatase (ADPase) and activators thereof, and the medical uses of these substances, in particular in the diagnosis and treatment of pre-eclampsia and other conditions characterised by platelet aggregation. Background of the Invention Pre-eclampsia is a placental disease characterised by insufficiency of the uteroplacental circulation. It affects 10-12% of all pregnancies and is a major factor in the perinatal mortality rate. There is evidence that one or more placentally-derived factors are released into the maternal circulation which either directly or indirectly cause maternal endothelial dysfunction and ensuing maternal problems with activation of the clotting system increased vascular permeability and ischaemia in maternal organs secondary to vasoconstriction. However, while pre-eclampsia is a major complication of human pregnancy, its cause and pathogenesis are poorly understood (Faas et al, 1994). Many of the actions of growth factors on cells are thought to be mediated by a family of inositol phosphoglycan (IPG) second messengers (Rademacher et al, 1994) . It is thought that the source of IPGs is a "free" form of glycosyl phosphatidylinositol (GPI) situated in cell membranes. IPGs are thought to be released by the action of phosphatidylinositol-specific phospholipases following the binding of growth factors to receptors on the cell surface. There is evidence that IPGs mediate the action of a large number of growth factors including insulin, nerve growth factor, hepatocyte growth factor, insulin-like growth factor I (IGF-I), fibroblast growth factor, transforming growth factor β, the action of IL-2 on B-cells and T-cells, ACTH signalling of adrenocortical cells, IgE, FSH and hCG stimulation of granulosa cells, thyrotropin stimulation of thyroid cells, cell proliferation in the early developing ear and rat mammary gland. 098/11116 and 098/11117 (Rademacher Group Limited) disclose the purification, isolation and characterisation of IPG-P and IPG-A from human tissue. Prior to these applications, it had not been possible to isolate single components from the tissue derived IPG fractions, much less in sufficient quantities to allow structural characterisation. WO98/10791 (Rademacher Group Limited) discloses that excess production and release of P-type IPGs from the placenta of pregnant women is one of the causative factors of pre-eclampsia and can be used in the diagnosis of this condition. The application also discloses that IPG-P antagonists can be used to treat pre-eclampsia. Summary of the Invention The present invention is based on the finding that the inhibition of adenosine diphosphatase (ADPase) , in particular by P-type inositolphosphoglycans (IPGs), causes symptoms of pre-eclampsia such as platelet aggregation and glomerulonephritis, thereby defining a new therapeutic target for the diagnosis and treatment of pre-eclampsia or other conditions characterised by these or related symptoms. For example, since ADP is a potent platelet aggregatory agent, and ecto-ADPase is important in the breakdown of circulating ADP, it is envisaged that inhibition of ecto- ADPase will be of significance in clinical conditions where platelet aggregation occurs, so that activation of ecto-ADPase and/or removal of an ecto-ADPase inhibitor represents a therapeutic approach to the treatment of pre-eclampsia and other conditions where platelet aggregation, e.g. causing a reduction in the platelet count, glomerulonephritis or thrombus formation. Examples of such conditions include disseminated intravascular coagulation, localised consumptive coagulopathy and autoimmune diseases such as lupus. In addition, there may be a role in sickle cell anaemia, on the basis of a report by Lutty et al (1996) entitled "A rat model for sickle cell-mediated vascular occlusion in retina". In addition, recent studies have established that an elevated plasma level of total homocysteine is an independent risk factor for cardiovascular disease, and relates to the progression of symptomatic peripheral arterial disease. Homocysteine inter alia inhibits the ecto-ADPase activity of human umbilical vein endothelial cells. The disruption in several different vessel wall- related anticoagulant functions provide plausible mechanisms for the occurrence of thrombosis in hyper- homocysteinemia . At present there is interest in the treatment of patients with hyperhomocysteinemia and coronary heart disease with vitamin supplements (folic acid, B12 and B6) . The present invention further discloses that treatments which promote the removal of circulating ADP - a potent platelet aggregatory factor - can be used as an adjunct to this vitamin therapy, and that normalisation of IPG levels and regulation of ecto- ADPase can play a significant role in a broad spectrum of thrombolytic events, and hence provide a therapeutic route for treating conditions associated with such events . The present invention may also find application in connection with the treatment of septic shock (see Arrieta et al, 1999) and chronic heart failure (see Niebauer et al, 1999) . The above indicated findings in relation to pre-eclampsia suggests that pre-eclampsia and the conditions described above can be treated by administering ADPase or an ADPase activator. The present invention further discloses that the amount or activity of ADPase can be used as a diagnostic indicator to determine whether a patient has or is at risk of developing pre-eclampsia or a related condition as set out above. In a further aspect, the inhibition of ADPase can be employed in assays to screen candidate compounds for activity as ADPase activators, e.g. for use in the treatment of pre-eclampsia. Accordingly, in a first aspect, the present invention provides the use of adenosine diphosphatase or an adenosine diphosphatase activator for the preparation of a medicament for the treatment of a condition characterised by platelet aggregation. Examples of these conditions include pre-eclampsia, disseminated intravascular coagulation, localised consumptive coagulopathy, and autoimmune diseases including lupus, sickle cell anaemia, cardiovascular disease such as thrombosis or chronic heart failure, and septic shock. In a preferred embodiment, the medicament is for the treatment of pre-eclampsia. In this case, advantageously, the ADPase activators are capable of activating ADPase in the presence of placental P-type IPGs. In other conditions characterised by platelet aggregation, the ADPase may be inhibited by other factors and the ADPase activators should preferably work in the presence of these inhibitory factors. In a further aspect, the present invention provides a method of treating a patient who has or at risk of developing pre-eclampsia or a condition characterised by platelet aggregation, such as disseminated intravascular coagulation, localised consumptive coagulopathy, and autoimmune diseases including lupus, sickle cell anaemia, cardiovascular disease such as thrombosis or chronic heart failure, and septic shock, the method comprising administering to the patient a therapeutically effective amount of adenosine diphosphatase or an adenosine diphosphatase activator. In a further aspect, the present invention provides a method of diagnosing a patient who has or at risk of developing a condition characterised by platelet aggregation, and especially diagnosing pre-eclampsia, the method comprising determining the amount of adenosine diphosphatase and/or adenosine diphosphatase activity in a sample obtained from the patient. In one embodiment, the present invention provides a method of diagnosing a patient who has or at risk of developing a condition characterised by platelet aggregation, such as pre-eclampsia, the method comprising the steps of: (a) contacting a sample obtained from the patient with a solid support having immobilised thereon a binding agent having binding sites specific for adenosine diphosphatase; (b) determining the amount of ADPase or the activity of ADPase which binds to the binding agent; and, (c) correlating the value obtained in step (b) with measurements obtained from control subjects to determine whether the patient has or is at risk of developing the condition. In a further aspect, the present invention provides the use of adenosine diphosphatase and one or more inositolphosphoglycans (IPGs) for screening candidate compounds for the property of activating adenosine diphosphatase, e.g. to identify compounds for the treatment of pre-eclampsia. These compounds may be used as lead compounds in pharmaceutical research. ADPase may be used without IPG as an initial screen for activating compounds, but the use of IPGs in combination with ADPase for screening has the advantage that it specifically looks for ADPase activation in the presence of its inhibitor linked to pre-eclampsia; i.e. it is looking for a compound that will reverse the inhibition of ADPase by IPG, preferably IPG-P, from pre-eclamptic placenta. In a further aspect, the present invention provides a method of screening for adenosine diphosphatase activators, the method comprising: (a) providing ADPase and one or more IPGs in a reaction medium and determining the activity of the ADPase; (b) contacting the reaction medium comprising ADPase and IPGs with a candidate compound determining the ADPase activity; (c) comparing that activity with the activity of the ADPase in comparable reaction medium untreated with the candidate compound or compounds; and, (d) selecting candidate compounds which cause activation of ADPase. In the screening aspects of the invention, preferably the IPGs are P-type IPGs. In step (a), the IPGs inhibit ADPase activity and the extent to which the candidate compound can activate ADPase in the presence of the IPGs can be determined. In some embodiments, the method comprises the further step of testing the candidate compounds in cell culture. Preferably, the cell culture comprises placental cells, more preferably, placental cells showing elevated levels of IPGs, and especially IPG-P, characteristic of pre- eclampsia. In one embodiment, this further step includes contacting the cells with the candidate compound, e.g. in a suitable reaction medium, and measuring the ADPase activity before and after addition of the candidate compound. These and other aspects of the invention will be apparent from the foregoing description, and from the following description wherein embodiments of the present invention are described, by way of example and not by limitation, with reference to the accompanying drawings. Brief Description of the Drawings Figure 1 shows the activity of ADPase in different fractions prepared from rat mitochondria. Figure 2 shows the effect of different IPGs on liver mitochondrial ADPase expressed as a % of ADPase activity in the absence of the IPGs. Figure 3 shows ADPase inhibition by P-type IPGs with varying concentrations of IPG. Detailed Description Adenosine Diphosphatase As used herein, the terms "adenosine diphosphatase" and "ADPase" encompass native sequence and variant enzymes having ADPase activity. Polypeptides having ADPase activity can be isolated from a variety of plant and animal sources, or prepared by recombinant or synthetic methods. By way of example, the cloning of potato ADPase from potato tubers is described in Handa & Guidotti (1996) and the isolation of ADPase from rat mitochondria is described in Smith et al (1980) . ADPases have been biochemically characterised, see for example Richardson et al (1982) and Delgado et al (1997). ADPases have been found in a variety of cell types such as such as rat brain synaptosomes, HUVAC cells, smooth muscle cells, cardiac cells, lymphocytes and platelets. As defined herein, "adenosine diphosphatase activity" refers to the activity of the enzyme on adenosine diphosphate (ADP) to produce adenosine monophosphate (AMP) . Adenosine diphosphatase activity can be readily determined by the skilled person using common general knowledge in the art, e.g. using the assays set out in the examples or shown in Richardson et al, 1982. A "native sequence ADPase" comprises a polypeptide having the same amino acid sequence as ADPase derived from nature. Such native sequence ADPase can be isolated from nature or can be produced by recombinant and/or synthetic means. The term "native sequence ADPase" specifically encompasses naturally-occurring truncated or secreted forms (e.g. an extracellular domain sequence), naturally- occurring variant forms (e.g. alternatively spliced forms) and naturally-occurring allelic variants of the ADPase. "ADPase variant" means an active ADPase enzymes as defined above, other than a native sequence ADPase, which include the insertion, substitution and/or deletion of 1, 2, 3, 4, 5, 6, 8, 10, 15 or 20 or more amino acid residues providing a variant ADPase which retains ADPase activity. "Adenosine diphosphatase activators" are substances which increase the activity of ADPase, and in particular as compared to its inhibited state in the presence of IPG-P, e.g. by binding to the ADPase. ADPase activity can be determined using assays such as those described in the examples employing labelled ADP. ADPase activators may act by: (a) increasing the activity of ADPase; or, (b) blocking the action of a specific ADPase inhibitor acting on ADPase, e.g. by blocking an interaction site on ADPase with which the inhibitor interacts thereby to inhibit ADPase activity, e.g. by using an antibody which specifically binds to the interaction site of ADPase. Preferably, the activity of ADPase is elevated by the activating compound by at least about 25%, as compared to the activity in the presence of the IPGs, more preferably at least about 50%, and still more preferably at least about 75%. As shown in the examples, IPGs inhibit ADPase activity by between about 20% to 80% of the activity without the addition of the IPGs. In a preferred embodiment, the activating compound substantially restores the activity of the ADPase to the level observed in the absence of the inhibitory IPGs, e.g. to within a normal range for the patient. Preferably, the term "ADPase activators" excludes the classes of substances disclosed in WO98/10791 for the treatment of pre-eclampsia which discloses that elevated levels of P-type IPGs are a therapeutic target for the treatment of pre-eclampsia, and in particular, IPG-P antagonists which act by: (a) inhibiting the release of IPG-P from placenta; (b) reducing the levels of placenta derived IPG-P using an anti-IPG-P antibody; (c) reducing the effects of placenta derived IPG-P using an IPG-P antagonist. IPGs and IPG Analogues IPG-A mediators modulate the activity of a number of insulin-dependent enzymes such as cAMP dependent protein kinase (inhibits) , adenylate cyclase (inhibits) and cAMP phospho-diesterases (stimulates) . In contrast, IPG-P mediators modulate the activity of insulin-dependent enzymes such as pyruvate dehydrogenase phosphatase (stimulates) and glycogen synthase phosphatase (stimulates) . The IPG-A mediators mimic the lipogenic activity of insulin on adipocytes, whereas the IPG-P mediators mimic the glycogenic activity of insulin on muscle. Both IPG-A and IPG-P mediators are mitogenic when added to fibroblasts in serum free media. The ability of the mediators to stimulate fibroblast proliferation is enhanced if the cells are transfected with the EGF-receptor . IPG-A mediators can stimulate cell proliferation in the chick cochleovestibular ganglia . Soluble IPG fractions having IPG-A and IPG-P activity have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle, brain, adipose, heart) and bovine liver. IPG-A and IPG-P biological activity has also been detected in human liver and placenta, malaria parasitized RBC and mycobacteria . The ability of an anti-inositolglycan antibody to inhibit insulin action on human placental cytotrophoblasts and BC3H1 myocytes or bovine-derived IPG action on rat diaphragm and chick cochleovestibular ganglia suggests cross-species conservation of many structural features. However, it is important to note that although the prior art includes these reports of IPG-A and IPG-P activity in some biological fractions, the purification or characterisation of the agents responsible for the activity is not disclosed. IPG-A substances are cyclitol-containing carbohydrates, also containing Zn 2+ ion and optionally phosphate and having the properties of regulating lipogenic activity and inhibiting cAMP dependent protein kinase. They may also inhibit adenylate cyclase, be mitogenic when added to EGF-transfected fibroblasts in serum free medium, and stimulate lipogenesis in adipocytes. IPG-P substances are cyclitol-containing carbohydrates, also containing Mn 2+ and/or Zn 2+ ions and optionally phosphate and having the properties of regulating glycogen metabolism and activating pyruvate dehydrogenase phosphatase. They may also stimulate the activity of glycogen synthase phosphatase, be mitogenic when added to fibroblasts in serum free medium, and stimulate pyruvate dehydrogenase phosphatase. Methods for obtaining IPG-A and IPG-P and for determining characteristic activities of these substances are set out in Caro et al, 1997, and in W098/11116 and W098/11117. The role of P-type IPGs as one of the causative factors in pre-eclampsia is disclosed in O98/10791. Drug Formulation The enzymes and activators of the invention may be derivatised in various ways. As used herein "derivatives" of the enzymes and activators includes salts, coordination complexes with metal ions such as Mn 2+ and Zn 2+ , esters such as in vivo hydrolysable esters, free acids or bases, hydrates, prodrugs or lipids, coupling partners . Salts of the enzymes and activator compounds of the invention are preferably physiologically well tolerated and non toxic. Many examples of salts are known to those skilled in the art. Compounds having acidic groups, such as phosphates or sulfates, can form salts with alkaline or alkaline earth metals such as Na, K, Mg and Ca, and with organic amines such as triethylamine and Tris (2- hydroxyethyl) amine . Salts can be formed between compounds with basic groups, e.g. amines, with inorganic acids such as hydrochloric acid, phosphoric acid or sulfuric acid, or organic acids such as acetic acid, citric acid, benzoic acid, fumaric acid, or tartaric acid. Compounds having both acidic and basic groups can form internal salts. Esters can be formed between hydroxyl or carboxylic acid groups present in the compound and an appropriate carboxylic acid or alcohol reaction partner, using techniques well known in the art. Derivatives which as prodrugs of the enzyme and activator compounds are convertible in vivo or in vi tro into one of the active enzymes and activators. Typically, at least one of the biological activities of compound will be reduced in the prodrug form of the compound, and can be activated by conversion of the prodrug to release the compound or a metabolite of it. Other derivatives include coupling partners of the compounds in which the compounds is linked to a coupling partner, e.g. by being chemically coupled to the compound or physically associated with it. Examples of coupling partners include a label or reporter molecule, a supporting substrate, a carrier or transport molecule, an effector, a drug, an antibody or an inhibitor. Coupling partners can be covalently linked to compounds of the invention via an appropriate functional group on the compound such as a hydroxyl group, a carboxyl group or an amino group. Other derivatives include formulating the compounds with liposomes. Pharmaceutical Compositions The enzymes and activators of the invention can be formulated in pharmaceutical compositions. These compositions may comprise, in addition to one or more of the ADPases or ADPase activators, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes. Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. The compounds of used according to the present invention are preferably given to an individual in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual, e.g. the amelioration of one of more symptoms of pre-eclampsia, e.g. a reduction in platelet aggregation, a reduction in thrombus formation or a reduction of glomerulonephritis . The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980. A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. By way of example, the treatments disclosed in this application may be combined with the use of P-type IPG antagonists disclosed for the treatment of pre-eclampsia in O98/10791. Examples of conditions that can be treated using ADPase or ADPase activators includes pre-eclampsia, other conditions characterised by platelet aggregation, e.g. leading to a reduction in platelet count, glomerulonephritis or thrombus formation, such as disseminated intravascular coagulation, localised consumptive coagulopathy, and autoimmune diseases including lupus, sickle cell anaemia, cardiovascular disease such as thrombosis or chronic heart failure, and septic shock. Diagnostic Assays Methods for determining ADPase activity or the amount or concentration of this enzyme in biological samples from individuals are well known in the art and can be employed in the context of the present invention to determine whether an individual has or is at risk of developing pre-eclampsia or one of the other conditions discussed herein. The purpose of such analysis may be used for diagnosis or prognosis to assist a physician in determining the severity or likely course of the pre- eclampsia and/or to optimise treatment of it. Preferred diagnostic methods rely on the determination of ADPase activity as the inhibition of this enzyme was found to be associated with pre-eclampsia. In one embodiment, this method comprises determining adenosine diphosphatase activity in a biological sample obtained from the patient, e.g. by measuring the action of ADPase on a labelled substrate such as ADP. Alternatively or additionally, the concentration or amount of ADPase in a sample may be determined. Typically, the methods employ biological samples such as blood, serum, tissue samples (especially placenta) or urine. Depending on the sample, it may be advantageous to carry out a pretreatment step, e.g. to remove cellular debris or unwanted contaminants from the sample. In some embodiments, the assay methods for determining ADPase activity employ a binding agent having binding sites capable of specifically binding to ADPase in preference to other molecules, and especially other molecules likely to be present in the sample. Examples of binding agents include antibodies, receptors and other molecules capable of specifically binding ADPase. Conveniently, the binding agent is immobilised on solid support, e.g. at a defined location, to make it easy to manipulate during the assay. This can be achieved using techniques well known in the art such as physisorption or chemisorption, e.g. employing biotin/avidin or biotin/streptavidin to chemically link the binding agent to the solid support. The sample is generally contacted with a binding agent under appropriate conditions so that ADPase present in the sample can bind to the binding agent. ADPase activity can be determined by employing a suitable substrate such as ADP and monitoring the enzymatic reaction, e.g. by following the amount of substrate or reaction product (s), e.g. AMP, in the assay system. This process can be facilitated by labelling the substrate, and detecting the reduction in the label on the substrate or the increase of the label in a reaction product. In the case of determinations of the amount of ADPase in the sample (rather than its activity) , the fractional occupancy of the binding sites of the binding agent can then be determined using a developing agent or agents. The developing agent can be used in a competitive method in which the developing agent competes with the analyte for occupied binding sites of the binding agent (e.g. using a labelled analogue of the analyte) , or non- competitive method, in which the labelled developing agent binds analyte bound by the binding agent or to occupied binding sites (e.g. using an antibody with appropriate binding specificity) . Both methods provide an indication of the number of the binding sites occupied by the analyte, and hence the concentration of the analyte in the sample, e.g. by comparison with standards obtained using samples containing known concentrations of the analyte. Typically, the substrates or developing agents are tagged with a label or reporter molecule which are directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently . Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule. Any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al, Nature 144:945, 1962; David et al, Biochemistry 13:1014, 1974; Pain et al, J. Immunol. Meth. 40:219, 1981; and Nygren, J Histochem. and Cytochem. 30:407, 1982. One favoured mode is to covalently link of each species of substrate or developing agent with an individual fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, luciferin, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine. Other detectable labels include radioactive isotopic labels, such as 3 H, 14 C, 32 P, 35 S, 126 I, or 99ra Tc, and enzyme labels such as alkaline phosphatase, β-galactosidase or horseradish peroxidase, which catalyze reactions leading to detectable reaction products and can provide amplification of signal. Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyze reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. As mentioned above, the substrates or developing agents are labelled (e.g. with radioactive, fluorescent or enzyme labels) so that they can be detected using techniques well known in the art. Thus, radioactive labels can be detected using a scintillation counter or other radiation counting device, fluorescent labels using a laser and confocal microscope, and enzyme labels by the action of an enzyme label on a substrate, typically to produce a colour change. Methods of Screening for ADPase activators The present invention discloses that adenosine diphosphatase and inositolphosphoglycans (IPGs) interact to inhibit ADPase in pre-eclampsia. This provides a new therapeutic target that can be used for screening candidate compounds for the property of activating adenosine diphosphatase, e.g. to identify compounds for the treatment of pre-eclampsia. These compounds may be used as lead compounds in pharmaceutical research. This use of IPGs in combination with ADPase for screening has the advantage that it specifically looks for ADPase activation in the presence of its inhibitor linked to pre-eclampsia . It is well known that pharmaceutical research leading to the identification of a new drug may involve the screening of very large numbers of candidate substances, both before and even after a lead compound has been found. This is one factor which makes pharmaceutical research very expensive and time-consuming. Means for assisting in the screening process can have considerable commercial importance and utility. A method of screening for candidate compounds which modulate activity of ADPase may include contacting one or more candidate compounds with the ADPase and/or IPGs in a suitable reaction medium, testing the activity of the ADPase and comparing that activity with the activity of the ADPase in comparable reaction medium untreated with the candidate compound or compounds. A difference in activity between the treated and untreated tests is indicative of a modulating effect of the relevant candidate compound or compounds. Combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of ADPase. Such libraries and their use are known in the art. In one embodiment, the present invention provides a method of screening for adenosine diphosphatase activators, the method comprising: (a) providing ADPase and one or more IPGs in a reaction medium and determining the activity of the ADPase; (b) contacting the reaction medium comprising ADPase and IPGs with a candidate compound determining the ADPase activity; (c) comparing that activity with the activity of the ADPase in comparable reaction medium untreated with the candidate compound or compounds; and, (d) selecting candidate compounds which cause activation of ADPase. In the screening aspects of the invention, preferably the IPGs are P-type IPGs. Following identification of a compound from the candidate compounds which modulates or affects polypeptide activity, the compound may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals . Thus, the present invention extends in various aspects not only to a compound identified using the screening methods described herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a compound, a method comprising administration of such a composition to a patient, e.g. for treatment (which may include preventative treatment) of pre-eclampsia, or other conditions characterised by platelet aggregation or glomerulonephritis, use of such a compound in manufacture of a composition for administration, and a method of making a pharmaceutical composition comprising admixing such a compound with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients. A compound identified using the above screening method may be peptide or non-peptide in nature. Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the compound (particularly if a peptide) may be designed for pharmaceutical use. The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property. There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. Alanine scans of peptide are commonly used to refine such peptide motifs. These parts or residues constituting the active region of the compound are known as its "pharmacophore" . Once the pharmacophore has been found, its structure is modelled to according its physical properties, eg stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process . In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic. A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide based, further stability can be achieved by cyclising the peptide, increasing its rigidity. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing. Examples Example 1 : Effect of IPGs on rat liver mitochondrial ADPase Mitochondria were isolated from male Wistar rats weighing 130 g, according to the method of Schnaitman & Greenawalt, 1968. Liver homogenate (1:10) was made in mannitol-sucrose-Hepes buffer containing bovine serum albumin. Mitochondria were isolated by fractional centrifugation and washed twice with the same buffer. The protein content of the final preparation was determined spectrophotometrically and diluted to a concentration of 100 mg/ml. An equal volume of digitonin was added to freshly isolated mitochondria in order to disrupt the outer and inner membrane. Figure 1 shows the activity of ADPase during the enzyme fractionation: Ml - whole homogenate. M2 - crude mitochondria. M3 - final mitochondrial fraction. M4 - final mitochondrial fraction after freezing + thawing. M5 - M3 after digitonin treatment. The figure shows that there is a linear response when using double amount of extract (Figure A) and a good agreement when results are expressed per 100 mg of protein (Figure B) . Digitonin treatment (M5) markedly reduces ADPase activity while slight increase in the activity, was found after freezing and thawing (M4). Figure 2 shows the effect of IPGs extracted from liver and liver mitochondrial ADPase activity when exposed to blood glucose lowering plants (Sato, Mara) and blood glucose lowering Thai tea. The figure shows that there is a significant decrease in ADPase activity but the response was not linear when double amount of IPG was used (except Liver P, Sato A and Tea P) . Figure 3 shows that ADPase inhibition by IPG type P from normal placenta. The linear response was obtained with 10 and 40 μl of IPG. The results indicate that a four fold change in IPG concentration results in a four fold decrease in ADPase activity and, therefore, ADPase is an enzyme which is sensitive to IPG inhibition. References : The references mentioned herein are incorporated by reference in their entirety. WO98/10791, W098/11116 and W098/11117 (Rademacher Group Limited) . Faas et al, Am. J. Obstet. Gynecol., 171:158-164, 1994 Lutty et al, Microvascular Research (1996) 52:270-280 Arrieta et al, Int. J. Exp. Path. (1999) 80(1):11-16 Niebauer et al, The Lancet (29 May 1999) 353:9167 Handa & Guidotti, Biochem. Biophys. Res. Comm. , 218 : 916- 923, 1996. Richardson et al, Clinica Chimica Acta, 121:87-94, 1982. Delgado et al, Biochem. J., 327:899-907, 1997. Smith et al, Biochem. J. , 192:527-535, 1980. Fiske & Subba-Row, J. Biol. Chem. , 66:375, 1925. Schnaitman & Greenawalt, J. Biol. Chem., 38:158-175, 1968.

Claims: Claims : 1. Use of adenosine diphosphatase or an adenosine diphosphatase activator for the preparation of a medicament for the treatment of a condition characterised by platelet aggregation. 2. The use of claim 1, wherein the condition characterised by platelet aggregation is developing pre- eclampsia . 3. The use of claim 2, wherein the ADPase ro ADPase activators are capable of treating pre-eclampsia in the presence of placental IPG-P. 4. The use of claim 1, wherein the conditions characterised by platelet aggregation is disseminated intravascular coagulation, localised consumptive coagulopathy, autoimmune diseases such as lupus, sickle cell anaemia, cardiovascular disease, thrombosis or chronic heart failure or septic shock. 5. A method of diagnosing a patient who has or at risk of developing a condition characterised by platelet aggregation, the method comprising determining the amount of adenosine diphosphatase and/or adenosine diphosphatase activity in a sample obtained from the patient. 6. The method of claim 5 which comprises the steps of: (a) contacting a sample obtained from the patient with a solid support having immobilised thereon a binding agent having binding sites specific for adenosine diphosphatase (ADPase) ; (b) determining the amount of ADPase or the activity of ADPase which binds to the binding agent; and, (c) correlating the value obtained in step (b) with measurements obtained from control subjects to determine whether the patient has or is at risk of developing the condition. 7. The method of claim 5 or claim 6 wherein the condition is pre-eclampsia. 8. The method of claim 5 or claim 6, wherein the conditions characterised by platelet aggregation is disseminated intravascular coagulation, localised consumptive coagulopathy, autoimmune diseases such as lupus, sickle cell anaemia, cardiovascular disease, thrombosis or chronic heart failure or septic shock. 9. Use of adenosine diphosphatase for screening candidate compounds for the property of activating adenosine diphosphatase thereby to identify compounds for the treatment of pre-eclampsia or conditions characterised by platelet aggregation. 10. The use of claim 9 which employs one or more inositolphosphoglycans (IPGs) in combination with ADPase for screening for compounds which can activate ADPase in the presence of IPGs. 11. The use of claim 10, wherein the IPGs are P-type IPGs from pre-eclamptic placenta. 12. A method of screening for adenosine diphosphatase activators, the method comprising: (a) providing ADPase and one or more IPGs in a reaction medium and determining the activity of the ADPase; (b) contacting the reaction medium comprising ADPase and IPGs with a candidate compound determining the ADPase activity; (c) comparing that activity with the activity of the ADPase in comparable reaction medium untreated with the candidate compound or compounds; and, (d) selecting candidate compounds which cause activation of ADPase. 13. The method of claim 13, wherein the IPGs are IPG-P. 14. The method of claim 12 or claim 13, wherein the method comprises the further step of testing the candidate compounds in cell culture. 15. The method of claim 14, wherein the cell culture comprises placental cells. 16. The method of claim 15, wherein the placental cells have elevated levels of IPG-P characteristic of pre- eclampsia. 17. The method of any one of claim 12 to 16, wherein the further step includes contacting the cells with the candidate compound in a suitable reaction medium and measuring the ADPase activity before and after addition of the candidate compound. 18. A method which comprises, having identified a candidate compound by the method of any one of claims 12 to 17, the further step of manufacturing the compound in bulk and formulating it in a pharmaceutical composition.

Inventors: GREENBAUM LESLIE, MCLEAN PATRICIA, RADEMACHER THOMAS WILLIAM, GREENBAUM, LESLIE, MCLEAN, PATRICIA, RADEMACHER, THOMAS, WILLIAM
Applicants: GREENBAUM LESLIE, MCLEAN PATRICIA, RADEMACHER GROUP LTD, RADEMACHER THOMAS WILLIAM, GREENBAUM, LESLIE, MCLEAN, PATRICIA, RADEMACHER GROUP LIMITED, RADEMACHER, THOMAS, WILLIAM
Application year: 2000
Main classification: A61K38
IPC codes: A61K38, A61K45, A61P7, A61P9, A61P15, A61P17, A61P31, A61P37, A61P43, C12Q1, G01N33