HUMAN DISEASE CONDITIONS

The present invention relates generally to a diagnostic assay for ovarian hyperstimulation syndrome and to a method of preventing or treating same. The present invention also relates to the assay and treatment of tumours and cancers which require enhanced vascular permeability for growth.

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

Ovarian hyperstimulation syndrome (hereinafter referred to as "OHSS") is characterised by the ovaries becoming rapidly enlarged and by increased capillary permeability within the abdominal and to a lesser extent, thoracic cavities. OHSS appears to result from an over stimulation of normal ovarian physiological processes. Although the exact cause of OHSS is apparently unknown, it only develops in women undergoing ovulation induction. The likelihood and severity of OHSS appears to be related to the level of ovarian response to stimulation with fertility drugs such as clomiphene citrate, human chorionic gonadotropin (hCG), human menopausal gonadotropin, follicle stimulating hormone (FSH) and lutienising hormone releasing hormone (LHRH). The syndrome is a potentially life-threatening, major complication of ovarian induction therapy.

Women undergoing ovulation therapy such as ovarian stimulation in assisted reproduction or in vitro fertilization (IVF), are assessed for OHSS by a variety of techniques such as ultrasound to determine ovarian size, the observance of symptoms and measuring the extent of fluid accumulation. Although these techniques provide indirect evidence for OHSS, they are necessarily subjective and frequently a putative diagnosis occurs too late in the ovulation protocol to take preventative measures. There is a need, therefore, for

a diagnostic assay for OHSS so that women can be accurately assessed during ovulation therapy.

In work leading up to the present invention, the inventors discovered that women with OHSS have increased levels of the hormone Vascular Permeability Factor (hereinafter referred to as "VPF") [VPF is also known as Vascular Endothelial Growth Factor or "VEGF". For convenience, VPF is used herein to encompass the abbreviation of VEGF]. VPF is, therefore, indicated for the first time as a possible cause of OHSS and as a possible marker ofthe disease. VPF is also now implicated in the growth of tumours and cancers due to their requirement for enhanced vascular permeability for growth, such tumours and cancers include ovarian cancer amongst others.

Accordingly, one aspect of the present invention contemplates a method for assaying for a disease condition in a human subject wherein said disease condition results from or is otherwise promoted by enhanced vascular permeability, said method comprising screening for and/or quantitating the presence of VPF over time in a biological sample of said human subject wherein an elevation of VPF in said biological sample relative to a normal control is indicative of or a propensity to develop said disease condition.

The present invention relates generally to any human disease condition requiring enhanced vascular permeability for its development or at least its maintenance. Examples of such disease conditions include OHSS following ovulation therapy or a variety of tumours or cancers such as ovarian cancer.

A particularly important aspect of the present invention is the ability to detect OHSS as soon as possible during ovulation therapy. The detection of OHSS or a propensity to develop OHSS at an early stage may assist the clinician in reducing further development of the condition by, for example, stopping or reducing ovarian stimulation, delaying embryo transfer or initiating a therapeutic protocol to reduce OHSS development.

According to this aspect of the present invention there is contemplated a method for assaying for OHSS in a human subject, said method comprising screening for and/or quantitating the presence of VPF over time in a biological sample of said human subject wherein an elevation of VPF in said biological sample relative to a normal control is indicative of OHSS or a propensity to develop OHSS.

In accordance with this aspect of the present invention, an increase in VPF over time is indicative of the development of or at least potential development of OHSS. The results over time are preferably quantitative although the subject method is also adaptable for qualitative analysis. By a "normal control" is meant the levels of VPF in a reproductive age female not undergoing ovarian stimulation. An "elevation" is considered to mean a statistically significant increase in the amount of VPF.

The biological sample used in accordance with the present invention is any suitable and conveniently obtained fluid containing or potentially containing VPF. Preferably, the fluid is selected from urine, serum, ovarian follicular fluid, saliva, peritoneal fluid, pleural fluid and pericardial fluid. Most preferably, the fluid is urine or serum. Preferably, the VPF is measured prior to administration of an ovulating hormone (e.g. hCG lutienizing hormone or recombinant forms thereof) or prior to egg pick up. If elevated levels of VPF cannot be measured prior to administration of ovulating hormone, then preferably VPF is measured within an approximately 48 hour period and more preferably within an approximately 30 hour period following ovarian hormone administration.

In a particularly preferred embodiment, there is provided a method for assessing the development or potential development of OHSS in a human subject undergoing ovarian stimulation in assisted reproduction, said method comprising determining the levels of VPF in a biological sample from said human subject period either before administration of an ovulating hormone or prior to embryo transfer and comparing said levels to a normal control wherein an increase over the normal control is indicative or predictive of the development of OHSS.

The assay for VPF is most conveniently conducted using antibodies specific to VPF or to an antibody binding fragment, part, derivative, homologue or analogue thereof. Alternatively, antibodies cross reactive with VPF may also be used with appropriate controls. Although the antibodies may be monoclonal or polyclonal, monoclonal antibodies are preferred particularly because of the ability to produce them in large quantities and due to the homogeneity of the product. The antibodies may be prepared in the laboratory as described hereinafter. The antibodies used in the experiments ofthe present invention were obtained from Monsanto Company, St. Louis, Missouri, USA.

Methods for obtaining antibodies are well known in the art. Polyclonal sera are easily prepared by injection of a suitable laboratory animal with an effective amount of VPF or antigenic fragments, parts, derivatives, homologues, analogues or immunological relatives thereof. Native or recombinant human VPF is the preferred antigen. Polyclonal antibodies are conveniently isolated and purified using, for example, immunoadsorbant techniques.

Monoclonal antibodies to VPF and like molecules are prepared from hybridoma cell lines derived by fusing an immortal cell line and lymphocytes sensitised against VPF. Techniques for preparing monoclonal antibodies are well known in the art and reference can conveniently be made to Douillard and Hoffman, "Basic Facts About Hybridomas" in Compendium of Immunology Vol II ed by Schwarts; Kohler and Milstein Nature 256: 495-499, 1975; European Journal of Immunology 6: 511-519, 1976; and in US Patent No. 5,036,003 (Monsanto Company).

The presence of VPF in a patient's biological fluid (as hereinbefore defined) can be detected using a wide range of immunoassay techniques such as those described in US Patent Nos. 4,016,043, 4,424,279 and 4,018,653. This includes both single-site and two- site, or "sandwich", assays of the non-competitive types, as well as in the traditional competitive binding assays. Sandwich assays are particularly useful and include ELISA dip-stick assays and Immunofluorometric assays (IFA). Competitive binding assays include radioimmunoassays (RIA). Western blot assays may also be used. The choice of assay will depend on the sensitivity required, materials available and the biological

sample employed.

A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in one assay, an unlabelled antibody capable of binding VPF is immobilised onto a solid substrate and the biological sample to be tested for VPF brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody- VPF primary complex, a VPF specific antibody, labelled with a reporter molecule capable of producing a detectable signal, is then added and incubated, allowing time sufficient for the formation of a secondary complex of antibody- VPF-labelled antibody. Any unreacted material is washed away, and the presence of the VPF is determined by observation of a signal produced by the reporter molecule on the second antibody. The results may either be qualitative, by simple observation of the visible signal or may be quantitated by comparing with a control sample containing known amounts of VPF. Variations of this assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and then added simultaneously to the bound antibody. Furthermore, the second antibody may be unlabelled and a labelled anti-immunoglobulin antibody used to detect bound second antibody. These techniques are well known to those skilled in the art, and the possibility of minor variations will be readily apparent. The antibodies used above may be monoclonal or polyclonal.

In this assay, a first antibody having specificity for VPF or antigenic parts thereof is either covalently or passively bound to a solid surface. The solid substrate is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microtitre plates, or any other surface suitable for conducting an immunoassay such as a dipstick. The binding processes are well-known in the art and generally consist of cross-linking, covalently binding or physically adsorbing the molecule to the insoluble carrier.

By "reporter molecule", as used in the present specification, is meant a molecule which, by its chemical nature, produces an analytically identifiable signal which allows the detection of VPF-bound antibody. The most commonly used reporter molecules in this type of assay include enzymes, fluorophores and radionuclide containing molecules (i.e. radioisotopes). In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production,upon hydrolysis by the corresponding enzyme, of a detectable colour change.

Alternatively, fluorescent compounds, such as fluorescein, anthanide such as europium and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in an enzyme immunoassay (EIA), the fluorescent labelled antibody is allowed to bind to VPF in the first antibody- VPF complex. After washing off the unbound reagent, the remaining ternary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent molecules or bioluminescent molecules may also be employed. It will be readily apparent to the skilled technician how to vary the procedure to suit the required purpose. It will also be apparent that VPF in a sample could be captured by other than antibodies.

Accordingly, one aspect ofthe present invention contemplates a method of detecting VPF in a biological sample, said method comprising the steps of contacting said biological sample with an immobilised antibody to VPF for a time and under conditions sufficient for a VPF-antibody complex to form and subjecting said complex to a detecting means.

The latter complex may be detected by, for example, the addition of a second antibody labelled with a reporter molecule and specific for said VPF.

Alternatively, a competitive immunoassay may be used. The most convenient assay of this type is a radioimmunoassay (RIA). In this assay, reagents required generally include an antiserum raised to VPF (e.g. recombinant human VPF), a labelled VPF (e.g. iodinated recombinant human VPF) and VPF as standard (e.g. recombinant human VPF). In one particularly preferred RIA, the standard VPF or biological sample to be tested is incubated with an appropriate dilution of antisera. Incubation may range from minutes or hours to days. Labelled (e.g. iodinated) VPF is then added and the test tubes incubated for a further period ranging from minutes or hours to days. An anti-IgG serum is then added to precipitate the VPF-antibody complex. The levels of VPF in the sample are determined using standard data management systems.

The biological samples may be used directly or first concentrated or diluted. When urine is used, it is preferable to first remove small molecular weight components by, for example, gel filtration to ensure these components do not interfere with the assay. Additionally, when urine is the biological sample, it is particularly useful to measure VPF as a ratio with creatinine.

The present invention is also directed to a kit for assaying for VPF in a biological sample. The arrangement ofthe kit will vary depending on the type of detection system employed. For example, in one embodiment, the kit comprises in compartmental form, a first container adapted to receive a biological sample. Generally, the first container contains an antibody specific to VPF immobilised to all or a part of its inner surface. The kit also comprises a second container adapted to contain an antibody specific to VPF and labelled with a reporter molecule. The kit may also contain other containers adapted to contain reagents, diluents and/or substrates to facilitate the assay.

Alternatively, the kit may comprise a first container adapted to contain a biological sample, a second container adapted to contain VPF and a third container adapted to contain a labelled VPF (e.g. iodinated VPF). Such a kit would be particularly useful for

an RIA assay.

The correlation between OHSS and VPF provides an opportunity to develop an effective therapeutic protocol to prevent development of or monitoring the progression of or to ameliorate the symptoms of OHSS. One therapeutic protocol is based on withholding administration of native or recombinant hCG (or other ovulation stimulating compound such as lutienizing hormone) or withholding embryo transfer or modifying other aspects of treatment which may promote OHSS. In addition or alternatively, the protocol may use antagonists of VPF.

According to one aspect of the present invention there is provided a method for preventing or ameliorating the effects of OHSS in a female patient undergoing ovulation therapy wherein said method comprises screening for and/or quantitating the presence of VPF over time in a biological sample of said human subject wherein an elevation of VPF in said biological sample relative to a normal control is indicative of or a propensity to develop OHSS and thereafter modifying the ovulation therapy to prevent development of OHSS. Such a modification would include: (i) withholding administration of ovulating hormone (e.g. hCG); (ii) withholding embryo transfer; (iii) surgical intervention; and/or

(iv) administering an antagonist of VPF.

According to this latteraspect ofthe present invention, there is contemplated a method for preventing or ameliorating the effects of OHSS in a female patient, said method comprising administering to said patient, a VPF inhibiting effective amount of an antagonist to VPF for a time and under conditions sufficient to inhibit, reduce or otherwise ameliorate the effects of the symptoms of OHSS.

The present invention, therefore, also contemplates a pharmaceutical composition comprising an effective amount of a VPF antagonist and one or more pharmaceutically acceptable carriers and/or diluents. Any of a range of VPF antagonists may be used such as antibodies specific to VPF or fragments, parts or peptide components of VPF or

receptor antagonists of VPF. All such molecules are encompassed by the term "VPF antagonist". When a non-human antibody is employed as an antagonist, it may first need to be "humanised" meaning that parts ofthe antibody are substituted with parts of human proteins to reduce or minimise the risk of an immune response against the antibody.

The active ingredients of the pharmaceutical composition comprising the VPF antagonist are contemplated to exhibit excellent therapeutic activity, for example, in the prevention or amelioration of OHSS when administered in an amount which depends on the particular case. For example, from about 0.5 μg to about 20 mg per kilogram of body weight per day may be administered. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.

The active compound may be administered in any convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slow release molecules). Depending on the route of administration, the active ingredients which comprise one or more antagonists may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, low lipophilicity of the antagonists may allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis.

In order to administer the composition by other than parenteral administration, the antagonists will be coated by, or administered with, a material to prevent their inactivation. For example, the antagonists may be administered in an adjuvant, co- administered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.

The active compound may also be administered in dispersions prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size ^' in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thormerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by, for example, the use in the compositions of agents delaying absorption.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient(s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder ofthe active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the antagonists are suitably protected as described above, the composition may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.01% by weight and more preferably at least 0.1-1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% ofthe weight ofthe unit. The amount of active compound in the pharmaceutical compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.5 μg and 200 mg and more preferably lOμg and 20 mg of active compound.

The tablets, pills, capsules and the like may also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

As used herein, pharmaceutically acceptable carriers and diluents include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutc effect in association with the required pharmaceutical carrier and/or diluent. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics ofthe active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseases condition in which bodily health is impaired as herein disclosed in detail.

In further accordance with the present invention, there may also be a correlation between VPF and tumours and cancers which require for growth enhanced vascular permeability such as, but not limited to, ovarian cancer. The methods of assay, kits, methods of treatment and pharmaceutical compositions herein described, therefore, also extend to the detection, monitoring and treatment of these tumours and cancers such as ovarian cancer. For convenience and by way of short hand notation, reference to "ovarian cancer" includes reference to tumours and cancers requiring for growth enhanced vascular permeability.

Accordingly, another aspect ofthe present invention contemplates a method for assaying for ovarian cancer in a human subject, said method comprising monitoring the presence of VPF in a biological sample of said human subject wherein a substantial elevation of

VPF in said biological sample relative to a normal control is indicative of ovarian cancer or a propensity to develop ovarian cancer. A normal control is a female without OHSS or ovarian cancer.

A further aspect of the instant invention contemplates a method for preventing or ameliorating the effects of ovarian cancer said method comprising administering to said human subject a VPF inhibiting effective amount of an antagonist to VPF for a time and under conditions sufficient to inhibit, reduce or otherwise ameliorate the symptoms of ovarian cancer.

According to these and related aspects of the present invention, VPF can be used as a marker for early stages of ovarian cancer or for recurrence of ovarian cancer. It also provides a marker to correlate with ovarian ultrasound appearances and colour cloppler assessment of ovarian blood flow. The measurement of VPF additionally provides a convenient marker to monitor ovarian cytotoxic chemotherapy and other treatments employed in ovarian cancer such as oophoarectomy. VPF may also be a suitable marker for pre-malignant ovarian diseases.

In a further embodiment, the present invention contemplates the use of a ribozyme to VPF mRNA to reduce levels of VPF in a human subject. According to this embodiment, there is provided a genetic construct comprising a ribozyme having a hybridising region and a catalytic region wherein said hybridising region is capable of hybridising under in vivo conditions to all or a portion of VPF mRNA wherein said catalytic region is capable of cleaving said mRNA. Such a ribozyme would be useful in the treatment of OHSS and/or ovarian cancer.

In an alternative embodiment, genetic therapy in the form of antisense molecules capable of targetting VPF mRNA may be ued. The antisense molecule may be a molecule capable of hybridising to the full length VPF mRNA or to a part or portion thereof. For example, the antisense molecules may be from 7 to 200 nucleotides in length or from 10 to 100 nucleotides in length or from 15 to 150 nucleotides in length. Antisense molecules are conveniently designed from knowledge of the VPF cDNA sequence.

Still yet a further aspect of the present invention include an agent comprising an antagonist of VPF useful for preventing or ameliorating the effects of OHSS or ovarian cancer and the use of an antagonist of VPF in the manufacture of a medicament for the treatment of OHSS or ovarian cancer.

The present invention is further described by the following non-limiting figures and/or examples.

In the Figures:

Figure 1 is a schematic diagram of one protocol used for ovarian stimulation in assisted reproduction.

Figure 2 is a graphical representation showing chromatographic fractionation of ascites fluid on an ion exchange column. The vasodilatory activity was detected by the Miles assay.

(A) Pooled ascites fluid from women with OHSS;

(B) Pooled ascites fluid from subjects with chronic liver failure;

(C) Pooled ascites fluid from subjects with chronic liver failure to which recombinant human VPF had been added prior to chromatography.

Figure 3 is a graphical representation showing the vasodilatory activity (as detected by the Miles assay) in OHSS ascites and liver ascites with recombinant human VPF when preincubated with and without control goat serum and serum from goats immunised with recombinant human VPF. The OHSS ascites and liver ascites with recombinant human VPF were initially fractionated by ion exchange chromatography (Figure 2) and two pools (I and II) prepared for assay.

Figure 4 is a graphical representation showing dose response curves of a urine sample and VPF standard in the VPF radioimmunoassay. The data has been logit log dose transformed to linearise the response lines in order to assess for parallelism between sample and standard.

Figure 5 is a graphical representation showing validation of VPF assay, (a) The urinary VPF profile of VPF as measured by RIA following fractionation on reverse phase (RP)- high pressure liquid chromatography (HPLC). (b) As for (a) but spiked with human recombinant VPF.

Figure 6 is a graphical representation of urinary VPF levels expressed as VPF/creatinine ratios from urine collections over the day from 8 normal women in the reproductive age.

Figure 7 is a graphical representation showing the relationship between VPF levels (expressed as VPF/creatinine ratios) in urine collected early morning and the VPF levels in 24 hour urine collections.

Figure 8 is a graphical representation of urinary VPF levels in two individual women undergoing IVF (expressed as VPF/creatinine ratios) who do not show OHSS.

Figure 9 is a graphical representation of urinary VPF levels in 10 women undergoing IVF (expressed as VPF/creatinine ratios) who do not show OHSS from 10 days before until 20 days after egg pick up. Values at each day represent the mean ± standard error. The division in the graph at 8 days after egg pick up correspond to cycles which were pregnant. Values in parenthesis are numbers of samples/day. The shaded area corresponds to the days -2 to +4 around the day of egg pick up.

Figure 10 is a graphical representation showing the urinary VPF levels in 14 women undergoing IVF (expressed as VFP/creatinine ratios) who do not show OHSS and two women who had moderate OHSS between days -2 to +4 around the day of egg pick up.

Figure 11 is a graphical representation showing the urinary VPF levels in three women undergoing IVF (expressed as VPF/creatinine ratios) who showed severe signs of OHSS. Paracentesis (draining of ascites fluid) is designated by arrows. These samples were provided by Dr Haning, Brown University, Womans and Infants Hospital Providence, Rhode Island, USA.

Figure 12 is a graphical representation showing the urinary VFP levels in women with a) no reproductive complaints, i.e. normal postmenopausal women, normal women in the reproductive age (see Example 4) women in the reproductive age undergoing IVF (average subject VPF/creatinine values from Example 8) and therefore are considered as controls, (b) benign ovarian tumours, (c) borderline mucinous carcinomas and (d) malignant tumours. The borderline mucinous carcinomas represent a premalignant condition and possibly represents more the benign type of tumour.

EXAMPLE 1

Figure 1 is a schematic representation of one protocol used for ovarian stimulation in assisted reproduction.. Fertility drugs such as FSH and gonadotrophin releasing hormone (GnRH) agonist are administered prior to hCG injection. Eggs are collected at day 0. Embryo transfer occurs at around day 1 to 4 or more preferably around 1 to 2.5. OHSS becomes clinically evidence after hCG administration.

EXAMPLE 2 Identification of VPF in OHSS Ascites Fluid

OHSS ascites fluid was collected at therapeutic paracentesis, twice from three patients with severe OHSS and once from three patients with non-malignant chronic liver failure. Liver failure ascites, as an obstructive back-pressure ascites, acted as a control for the OHSS ascites. Three 5ml pools were prepared: one from the OHSS samples and two from the liver failure samples. Recombinant human VPF (52μg, Monsanto Company, St Louis, Missouri, USA) was added to one of the liver ascites pools. Ion exchange chromatography was performed (see Figure 2) and the fractions tested for capillary permeability in the Miles assay (see below). The ion exchange chromatographic conditions were as follows. Five ml ascites fluid was initially gel filtered (Sephadex G25 columns, Pharmacia, Uppsala, Sweden) in 20mM sodium acetate pH5.0 and fractionated on a column (1.6cm x 50cm) of S-Sepharose Fast Flow Gel (Pharmacia) in a 120 min 0-1 M NaCl gradient in 20mM sodium acetate buffer pH5.0 at 1.5ml min with 4ml fractions. Protein was detected by UV absorbance at 280nm. The fractions were gel filtered in

50mM ammonium acetate pH7, lyophilised and dissolved in 20mM phosphate buffer pH7 containing 0.154M NaCl and 1% w/v bovine serum albumin prior to assay for capillary permeability in the Miles assay (Miles, A A & Miles, E M Journal of Physiology 118: 28-257, 1952).

In the Miles assay, samples were injected intradermally, under inhalation anaesthesia, in a randomised design to 36 injection sites on the dipilated back of a guinea pig which had received an intracardiac injection of Evans Blue dye 5 minutes earlier. After 25 minutes, the animals were killed, skinned and the capillary leakage of dye at each injection site quantified using the Cue-2 version 4.0 image analysis system (Galai Production, 10500 Migdal Haemak, Israel) to produce an Integrated Opitical Density (IOD) score for each point.

Vascular permeability activity was detected in two regions of the ion exchange chromatograph (designated Pool I and II, see Figure 2) ofthe OHSS ascites with little or no detectable activity observed in the liver failure ascites fluid. The addition of recombinant human VPF to the liver ascites fluid resulted in a similar pattern of activity as OHSS ascites. No activity was detected in tubes 1 to 40. Pools I and II from the ion exchange chromatography of OHSS and liver ascites plus recombinant human VPF were pooled and tested in the Miles assay alone, with VPF antiserum (raised against recombinant human VPF in a goat; Monsanto Company) to block any VPF activity present or with control goat serum (Animal House, Monash University, Melbourne,

Victoria, Australia). In the presence of VPF antiserum, capillary permeability activity was suppressed by 49% and 50% (p<0.05) for the two liver ascites plus recombinant human VPF pools and by 79% and 65% (ρ<0.01) for the two OHSS pools (Figure 3).

By contrast, control serum suppressed activity by 17% and 0% and 24% and 27% (not significant), respectively.

These results show that the vascular permeability in OHSS ascites migrates in a similar pattern as to recombinant human VPF and is markedly neutralised by preincubation with antisera raised to recombinant human VPF. No similar activity was observed in liver failure ascites indicating that VPF is specifically found in OHSS ascites.

EXAMPLE 3 Measurement of VPF in serum and urine

An assay method was developed consisting of a radioimmunoassay utilising a goat antiserum raised to recombinant human VPF (rhVPF, 165 amino acid form), iodinated rhVPF (iodinated by the chloramine T procedure) and rhVPF as standard. The antiserum and rhVPF were provided by Monsanto Company. The assay consisted of the initial incubation of standard or sample with an appropriate dilution of antisera for one day at room temperature. Iodinated rhVPF was added on day 2 and the tubes maintained a further day at room temperature. A goat anti-rabbit IgG serum was added to precipitate the VPF-antisera complex. The levels of VPF in the sample were determined using standard data management systems.

Urine samples were either assayed (a) directly or (b) concentrated (10-20 times) by microconcentrators (e.g. Beverly, MA, USA) and assayed directly or (c) gel filtered on columns in a volitile buffer such as ammonium acetate (e.g. PD10 columns, Pharmacia, Uppsala, Sweden) and lyophilised prior to assay. In these initial experiments the use of gel filtration was routinely used in order to ensure that small molecular weight components in urine did not interfere in the assay. Serum was assayed directly.

The assay working range was 0.8-6.5ng rhVPF/ml of sample using this assay, VPF was readily detected in serum and urine samples.

EXAMPLE 4

VPF concentrations in urine

VPF levels were determined in urine from women during their reproductive life, during pregnancy and after menopause and the results are shown in Table 1. The VPF levels ranged from non-detectable to 0.4 ng/ml with an average of 0.24 ng/ml. Urine from 2 women with moderate OHSS gave values of 2.5 and 2.7 ng/ml, ten times the values found in women without OHSS. With one woman who had a past history of moderate

OHSS and was undergoing gonadrotrophin treatment for IVF showed elevated levels of urinary VPF without any clinical manifestations of OHSS indicating that the measurement of urinary VPF may be useful to predict OHSS.

TABLE 1

Condition Number of VPF levels in urine

Women (ng/ml)

Fertile age 2 0.20, 0.29

Using contraceptives 2 0, 0.31

Pregnant 2 0.26, 0.34

Peri, post-menopausal 2 0.16, 0.35

Ovarian hyperstimulation syndrome

- slight OHSS 1 0.8

- moderate OHSS 2 2.5, 2.7

- past history of moderate OHSS, no clinical manifestations 1 1.24 during this course of treatment

EXAMPLE 5 The VPF concentrations in serum

The VPF concentrations in serum of women collected in a reproduction endocrine clinic and who represent both fertile and infertile women showed a narrow range of values (Table 2). One woman with OHSS who was monitored over a period of time prior to the observed clinical symptoms of OHSS showed a 50% increase over this period. The increase in both serum and urinary VPF in women with OHSS provides a basis for a diagnostic of OHSS.

TABLE 2

Condition Days following VPF levels in serum taking of first sample ng/ml

Women without OHSS ¹ range 1.4-2.2

Woman with OHSS ² 0 2.05 (0%) ³

3 2.33 (14%)

5 2.47 (20%)

18 2.72 (32%)

33 (hospitalised) 3.1 (51%)

¹ The number of women tested was 100. The number of women tested was 1.

The figures in parentheses represent the percentage increase relative to the amount at day 0.

EXAMPLE 6 Antagonist to VPF

The results in Example 2 showing 65-79% suppression of capillary permeability in the presence of VPF antiserum identify anti-VPF antibodies as effective antagonists to VPF.

EXAMPLE 7

One aspect of the validation of the VPF immunoassay was to assess for parallelism between VPF in a sample and VPF in standards. Levels of VPF over a range of dilutions were determined in two urine samples (S.D. #1 and S.D. #2) and in VPF standards by radioimmunoassay. The data are shown in Figure 4. The data have been linearised by logit log-dose transformation. These results affirm the validity of the VPF levels

determined in biological samples in the other Examples.

EXAMPLE 8

The VPF assay was further validated by determining the VPF profile in urine by radioimmunoassay by fractionation on RP-HPLC followed by fractionation of an aliquot of the same urine sample which had been spiked with human recombinant VPF as control. Concentrated urine was divided into two fractions, to one fraction recombinant human VPF was added. These two preparations were fractionated by RP-HPLC on a Aquapore Butyl 10 micron 100 x 100 mm column (Brownlee, San Jose, CA, USA) with 0-50% v/v acetonitrile gradient in 0.1% v/v trifluoracetic acid at 1 ml min and 1 ml/fraction. These samples were lyophilised in the presence of carrier protein (bovine serum albumin), dissolved in assay buffer prior to VPF radioimmunoassay. The results in Figure 5 show substantial similarity in elution profiles between purified VPF and urinary VPF validating the VPF assay.

EXAMPLE 9

Urinary VPF was determined as VPF/creatinine ratios from urine collections over the day from 8 normal women of reproductive age. The levels are depicted in Figure 6 and show that there is no variation in urinary VPF throughout the day. Figure 7 shows the relationship between urinary VPF levels (expressed as VPF/creatinine ratios) collected early morning and VPF levels in 24 hour urine collections. The significant correlation indicates that VPF/creatinine ratios of early morning urine collections provides a good index of daily secretion of VPF and, thus, is a reasonable means of monitoring daily urinary VPF excretion.

EXAMPLE 10

The urinary VPF levels in 10 women undergoing IVF (expressed as VPF/creatinine ratios) who do not show OHSS from 10 days before until 20 days after egg pick up are shown in Figure 8. Note the increase in urinary VPF/creatinine ratios in days after hCG administration and before days +2. The clinical protocol is as described in Example 1. Values at each day represent the mean ± standard error. The division in the graph at day 8 after egg pick up corresponds to cycles which were pregnant and not pregnant. The division was made at this day as it is believed that this is the earliest time that the implanted embryo will exert an effect on the uterus. Values in parenthesis are numbers of samples/day. The shaded area corresponds to the days -2 to +4 around the day of egg pick up and is examined in more detail and with more subjects in Example 11.

EXAMPLE 11

The urinary VPF levels in 14 women undergoing IVF (expressed as VFP/creatinine ratios) who do not show OHSS and two women who had moderate OHSS between days -2 to +4 around the day of egg pick up are shown in Figure 9. There is a significant increase in urinary VPF levels between days -1 and +2 (p=0.011) in the women without OHSS. The two women with OHSS show a similar but magnified response.

EXAMPLE 12

The urinary VPF levels in three women undergoing IVF (expressed as VPF/creatinine ratios) who showed severe signs of OHSS are shown in Figure 10. Note the elevated levels with VPF/creatinine ratios reaching values of 1.5 - 4.5. In one and possibly two subjects where there is overlap, the VPF/creatinine ratios are elevated compared to pregnant women without OHSS. Note the decline in urinary VPF following paracentesis suggesting that the source of urinary VPF in these women with severe OHSS is likely to be the ascites fluid.

EXAMPLE 13

Figure 11 shows the urinary VFP levels in women with a) no reproductive complaints, i.e. normal postmenopausal women, normal women in the reproductive age (see Example 9) women in the reproductive age undergoing IVF (average subject VPF/creatinine values from Example 11) and therefore are considered as controls, (b) benign ovarian tumours, (c) borderline mucinous carcinomas and (d) malignant tumours. The borderline mucinous carcinomas represent a premalignant condition and possibly represents more the benign type of tumour. In assessing the data, only 2 VPF/creatinine values out of 31 controls (group a) above or 6% had a VPF/creatinine ratio >0.056 while 7/18 values for the combined ovarian tumour group or 39% were >0.056. These data suggest that VPF levels are higher in the ovarian tumour groups although the distinction between types of cancers is not possible on this small data set.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.