WO2007113682A2

WO2007113682A2 - Tgf-beta pharmaceutical compositions

Info

Publication number: WO2007113682A2
Application number: PCT/IB2007/001530
Authority: WO
Inventors: Lynette Cathrine Kirkwood; Phuong My Tran; Timothy Simon Robinson
Original assignee: Novozymes Gropep Ltd
Priority date: 2006-04-06
Filing date: 2007-03-27
Publication date: 2007-10-11
Also published as: WO2007113682A3

Abstract

This invention relates to pharmaceutical compositions comprising TGFbeta, and in particular to liquid compositions comprising TGFbeta wherein the pH of the composition is at or below 3.7. In one form, the invention is an article of manufacture comprising a vial, cartridge or vaginal applicator and a pharmaceutical composition according to the claims of this application. In another form, the invention is a method of treating an infertility condition by administering to a prospective mother a pharmaceutical composition according to the claims of this application. In another form, the invention is a method of treating an infertility condition, by inducing immune tolerance to a paternal antigen in a prospective mammalian mother lacking said immune tolerance, said method comprising exposing a mucosal surface of said prospective mother to a pharmaceutical composition of this application and semen or an MHC Class I antigen of a prospective father capable of eliciting a Th-1 response.

Description

PHARMACEUTICAL COMPOSITIONS

TECHNICAL FIELD

This invention relates to a pharmaceutical composition comprising a TGFbeta.

BACKGROUND ART

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

TGFbetal , TGFbeta2 and TGFbeta3 are multifunctional cytokines that play an important role in the development and repair of tissue (Massague et al., 1990 Ann N Y Acad ScL 1990;593:59-72; Roberts & Sporn, 1990 Cell Regul. Nov;1 (12):875-82.). They belong to a large family of related factors referred to as the TGFbeta superfamily that share at least 25% sequence homology and affect many cellular processes including cell cycle progression, cell survival, and modulation of immune responses and differentiation during prenatal development and in the adult organism. The TGFbeta superfamily includes over 30 members that are divided into the following groups: the TGFbeta related dimeric proteins (e.g. TGFbeta1-5), Mullerian inhibitory substances (e.g. MIS), bone morphogenetic proteins (e.g. BMP-2-7), inhibins and activins (e.g. inhibin A, inhibin B, activin A and activin AB), growth differentiation factors (e.g. GDF-1), dorsalin-1 (e.g. dsl-1), MIC-1 and Drosophila decapentaplegic gene product (e.g. DPP-C) (Burt, 1992, Biochem Biophys Res Commun. Apr 30;184(2):590-5.). All TGFbeta forms show sequence similarity to the prototype TGFbetal , with a conserved tertiary structure of the cystine knot in each monomer with disulphide linked dimers that includes nine highly conserved cystine residues in the final structure. The highly conserved structure of TGFbeta forms is mirrored in conservation of the metazoan signaling components. The basal components are a type Il ligand-binding receptor, a type I signaling receptor and the cytoplasmic Smad proteins.

The mammalian TGFbeta subfamily consists of five isoforms with similar structure and function, i.e. TGFbetal , TGFbeta2, TGFbeta3, TGFbeta4 and TGFbetaδ. TGFbetal , TGFbeta2 and TGFbeta3 isoforms are expressed in mammalian cells, while TGFbeta4 and TGFbetaδ represent a chicken and a Xenopus laevis TGFbeta isoform, respectively. They are all homodimeric proteins with a molecular weight of approximately 25,000Daltons. The mature peptide sequences of the five isoforms of TGFbeta that have been isolated to date have 64-82% amino acid sequence homology. The TGFbeta subfamily isoforms possess similar biological activities and act through similar signaling cascades that are initiated by TGFbeta binding to heteromeric complexes of type I and type II TGFbeta receptors at the surface of target cells (Wrana et al., 1994 Nature. Aug 4;370(6488):341-7). After initial secretion, the TGFbeta isoforms must become activated to elicit their biological effects (Khalil, 1999 Microbes Infect. Dec; 1 (15): 1255-63).

The three mammalian TGFbeta forms, TGFbetal , TGFbeta2 and TGFbeta3 all possess three major activities in vitro: they inhibit proliferation of most cells but can stimulate growth of some mesenchymal cells; they enhance the formation of extracellular matrix; and they can exert immunosuppressive effects (reviewed by Lawrence, 1996, Eur Cytokine Netw. 1996 Sep;7(3):363-74) and exert immune tolerance induction properties (International Patent Application No. PCT/AU98/00149 (Robertson)). In more complex in vivo systems, the three mammalian TGFbeta forms are involved in wound repair processes and initiating inflammatory reactions and then in resolution of those responses. The latter effects of the TGFbeta forms derive in part from their chemotactic attraction of inflammatory cells and fibroblasts (reviewed by Lawrence, 1996 Eur Cytokine Netw. 1996 Sep;7(3):363-74). These proteins also have pleiotropic and profound effects on the immune system and on hemopoiesis.

The activin subfamily comprises three isoforms of 28kDa polypeptides, namely activin A, activin B and activin AB. Activin binds to the constitutively active Ser/Thr kinase receptor activin type Il (ActRII) or one of four isoforms of activin type NB (ActRIIB). Activin's primary role appears to be as a regulator of the pituitary gonadotropes and in particular as a regulator of follicle stimulating hormone.

It is desirable that pharmaceutical compositions containing any of the TGFbeta forms are stable for long periods of time, retain biological activity and are ready to administer in liquid form rather than a lyophilized form of TGFbeta requiring reconstitution before use. The scientific art has highlighted many problems faced by researchers in developing stable pharmaceutical compositions containing TGFbeta.

Firstly, in liquid formulations under certain conditions, the mammalian TGFbeta forms can aggregate. If aggregation occurs, TGFbeta loses its biological activity and there is an increased risk of adverse immunological effects in vivo.

Secondly, TGFbeta proteins have a high affinity for surfaces of containers and equipment, and may be lost by adsorption when in liquid form, particularly at low concentrations. This is a major problem, because it may result in reduced yields during manufacture of the drug substance and drug product, as the protein will adhere to equipment, thus increasing the difficulty and cost of manufacture. Also, measuring the concentration of the protein in the drug substance and drug product may be compromised by the protein adhering to vials and tubing, resulting in inaccurate determination of drug concentration. This undesired property of TGFbeta proteins may also reduce the actual dose delivered to the patient.

Thirdly, TGFbeta proteins are unstable when in liquid form under conditions currently used in the art and may undergo deamidation and/or oxidation. This is a problem because degradants may have an increased propensity to aggregate, leading to loss of bioactivity and an increased risk of adverse immunological effects.

Fourthly, TGFbeta has a low aqueous solubility in liquid form under non-optimised conditions, for example at physiological pH. If TGFbeta precipitates, then the available bioactivity in solution will be lost Further, if the pharmaceutical composition comprising TGFbeta is adapted for vaginal delivery to the cervix for the treatment of an infertility condition, significant problems arise when the formulation chemist attempts to develop a formulation that does not irritate the vagina.

Finally, if the pharmaceutical composition comprising TGFbeta is adapted for vaginal delivery to the cervix for the treatment of an infertility condition, significant problems arise when the formulation chemist attempts to develop a formulation that is not toxic to sperm thus effecting conception rates.

International Patent Application No. PCT/EPOO/02303 (Arvinte) describes a dry powder pharmaceutical composition comprising TGFbeta and a water soluble salt chosen from calcium chloride and calcium phosphate. PCT/EPOO/02303 proposes that the problems associated with the poor physical and chemical stability and high affinity that TGFbeta has for surfaces of containers can be reduced by the addition of one of these specified water soluble salts.

The pharmaceutical applications of TGFbeta are widely described in the art, however, no pharmaceutical products that contain a TGFbeta have been approved by the FDA or

EMEA. Potential therapeutic applications of TGFbeta described in the art include treatments for oral mucositis, angiogenesis, cancer, tissue repair of bone or cartilage and prevention of scarring.

International Patent Application No. PCT/AU98/00149 (Robertson) describes a method for treating an infertility disorder using TGFbeta wherein the disorder is characterized by a lack of immune tolerance by the prospective mother's immune system to paternal antigens.

This technology has application for the treatment of recurrent miscarriage, pre-eclampsia and intra-uterine growth retardation. The term 'immune tolerance' is taken to mean inhibition of the potentially destructive cell-mediated immune response against specific conceptus antigens, and/or inhibition of synthesis of conceptus antigen-reactive immunoglobulin of complement-fixing isotypes (for example the Th1' compartment of the immune response).

Immune tolerance is antigen specific and is not generalized immunosuppression which is non-antigen specific. Additionally PCT/AU98/00149 discloses that TGFbeta, when administered to the female reproductive tract together with sperm or semen, can elicit tolerance towards male antigens, including paternal MHC class I antigens. This state of immune tolerance is evidenced by inhibition of Th 1 -type immune responses to paternal antigens, including delayed-type hypersensitivity (DTH) responses primed by a previous injection with sperm, production of complement-fixing isotypes of immunoglobulin specific for sperm, and cell-mediated immune rejection of tumor cells bearing the same MHC class I antigens as those contained in the priming sperm inoculum. Example 1 of PCT/AU98/00149 demonstrates that seminal TGFbeta initiates the post-mating inflammatory response in mice and humans. Example 2 of PCT/AU98/00149 demonstrates that seminal vesicle fluid modulates maternal reproductive performance and the maternal immune response to paternal antigens in mice. Example 3 of PCT/AU98/00149 demonstrates that the delivery of paternal antigens in combination with TGFbeta to the female reproductive tract can generate systemic paternal antigen-specific tolerance, specifically by inhibiting the Th1 compartment of the immune response. Example 4 of PCT/AU98/00149 demonstrates that paternal antigen-specific immune deviation improves reproductive performance. The study confirmed that women exposed to semen (containing paternal antigen and natural TGFbeta) around the time of thawed embryo transfer have a reduced risk of early embryonic loss compared to those instructed to abstain from intercourse. Also, BALB/cF1 female mice were immunised by an intra-uterine infusion with CBA sperm with or without 10ng rTGFbetal then were mated naturally with CBA males 2 weeks later. Females were sacrificed on day 17 of pregnancy and the number of viable and resorbing implantation sites, as well as fetal and placental weights of viable conceptuses, were determined. This study showed that TGFbeta improved pregnancy outcome.

Further, studies by another group have demonstrated that the presence of TGFbeta in the female reproductive tract at the time of implantation increases the production of fibronectin, a protein proposed to assist implantation by promoting adhesion of the embryo to the endometrial surface and thus increases the success rate of IVF procedures (PCT/US94/02527 by Feinberg and Kliman).

SUMMARY OF THE INVENTION

The inventors have now surprisingly developed a long term stable liquid pharmaceutical formulation comprising TGFbeta.

In a first aspect, the invention provides a pharmaceutical composition comprising a TGFbeta wherein the pH of the composition is at or below 3.7. Preferably, the pH of the composition is between 3.2 and 3.7. More preferably the pH is between 3.3 and 3.7. For example, the pH of the composition is 3.2, 3.5 or 3.7. In one embodiment the composition is in a liquid form. Preferably the composition does not require reconstitution immediately prior to use. Preferably the composition is in a form that is ready to administer. The composition may be in a form suitable for self-administration by a patient. In a second embodiment the composition comprises a gel polymer. For example, the gel polymer is selected from the group consisting of cellulose based polymers, tragacanth polymers, xanthan gum polymers, acacia polymers, carbomer polymers, gelatin polymers, sodium alginate polymers, poloxomer polymers, polyethylene oxide polymers, polyacrylamide polymers and polyethylene glycol polymers. Preferably the gel polymer is a cellulose-based polymer. More preferably the cellulose-based polymer is selected from the group consisting of hypromellose (also known as hydroxylpropylmethylcellulose), methylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxyethylcellulose and carboxymethylcellulose. Even more preferably the cellulose-based polymer is hypromellose. For example, if hypromellose is chosen, the gel polymer may have a grade of 4000. Preferably, the hypromellose is at a concentration of between 1% to 5% w/w. More preferably the hypromellose is at a concentration of 1.9% w/w. If a cellulose-based gel polymer is used preferably the polymer contains a hydroxyl group and/or a carboxyl group.

In a third embodiment, the composition is devoid of calcium chloride or calcium phosphate. Preferably the composition is devoid of calcium chloride, calcium phosphate, potassium acetate, lithium acetate, ammonium acetate or ammonium bicarbonate. Even more preferably the composition is devoid of calcium chloride, calcium phosphate, potassium acetate, lithium acetate, sodium acetate, ammonium acetate or ammonium bicarbonate.

In a fourth embodiment, the TGFbeta is selected from the group consisting of human TGFbetal , TGFbeta2 and TGFbeta3. Preferably the TGFbeta is human TGFbeta3. Preferably the TGFbeta is substantially purified. Preferably the TGFbeta is recombinant.

In a fifth embodiment, the TGFbeta maintains stability for at least 6 months when stored between 2 and 27⁰C. For example, the TGFbeta maintains high bioactivity, low aggregation, low precipitation, low surface adherence and/or low oxidation and/or low deamidation. Alternatively the TGFbeta maintains stability for at least 6 months when stored between 2 and 8⁰C. Alternatively the TGFbeta maintains stability for at least 12 months when stored between 2 and 27⁰C. Alternatively the TGFbeta maintains stability for at least 12 months when stored between 2 and 8⁰C.

In a sixth embodiment, the composition does not undergo a step of freeze-drying. Preferably, the TGFbeta component does not undergo a step of freeze-drying. In a seventh embodiment, the composition comprises a therapeutically effective amount of TGFbeta. Preferably the concentration of TGFbeta is between 0.01micro-g/ml and 100micro-g/ml. More preferably the concentration of TGFbeta is between 0.2micro-g/ml and 20micro-g/ml. Even more preferably the concentration of TGFbeta is between 0.2micro-g/ml and 2micro-g/ml. For example, the concentration of TGFbeta is 0.2micro-g/ml.

In an eighth embodiment, the viscosity of the composition is between 200 and 30,00OcP and preferably between 500 and 90OcP. For example the viscosity of the composition is 70OcP.

In a ninth embodiment, the composition comprises an osmotic agent. Preferably the osmotic agent is mannitol or glucose. Preferably the mannitol is at a concentration between 2% and 10% w/w, even more preferably the mannitol is at a concentration between 4% and 5% w/w. For example, the mannitol is at a concentration of 4% w/w. In a tenth embodiment the composition comprises a buffering agent. Preferably the buffering agent is glycine. If glycine is chosen as a buffering agent, preferably it is at a concentration between 0.01% and 1 % w/w. More preferably glycine is at a concentration of 0.15% w/w. Alternatively, trisodium citrate or citric acid may be used.

In an eleventh embodiment the composition comprises a preservative agent. Preferably the preservative agent is benzoic acid, methyl paraben, propyl paraben or benzyl alcohol.

In a twelfth embodiment the composition comprises hydrochloric acid, acetic acid or sodium hydroxide.

Preferably the composition comprises water. In a thirteenth embodiment, the composition is adapted for vaginal administration.

In a fourteenth embodiment the composition has:

(1) not more than a total of 10² micro-organisms (aerobic bacteria or fungi) per gram, as tested using standard pour-plate methods described in Appendix XVI B2 of the British Pharmacopoeia 2001; (2) not more than 10¹ enterobacteria and certain other Gram-negative bacteria per gram, as tested using standard pour-plate methods described in Appendix XVI B2 of the British Pharmacopoeia 2001 ;

(3) absence of Pseudomonas aeruginosa, determined on 1g, as tested using standard pour-plate methods described in Appendix XVI B2 of the British Pharmacopoeia 2001 ; and (4) absence of Staphylococcus aureus, determined on 1g, as tested using standard pour-plate methods described in Appendix XVI B2 of the British Pharmacopoeia 2001.

In a second aspect, the invention provides an article of manufacture comprising a vial, cartridge or vaginal applicator and a pharmaceutical composition as described herein. Preferably the article of manufacture is accompanied by, or labelled with, instructions that the article be used in the treatment of an infertility condition. For example the infertility condition is recurrent miscarriage. Preferably the vial, cartridge or vaginal applicator is sealed. Preferably the article of manufacture is accompanied by, or labelled with, instructions that the article should be stored at 27⁰C or below. Alternatively the article of manufacture is accompanied by or labeled with instructions that the article should be stored at temperatures between 2 and 8⁰C. Preferably, the article of manufacture is labelled with a specific expiry date that is between 1 and 24 months after the date of manufacture when stored at temperatures between 2 and 8⁰C. Alternatively the article of manufacture is labeled with a specific expiry date that is between 1 and 24 months after the date of manufacture when stored at temperatures between 2 and 27⁰C. Alternatively the article of manufacture is labeled with a specific expiry date that is between 1 and 24 months after the date of manufacture when stored at room temperature.

In a third aspect, the invention provides a process for preparing an article of manufacture comprising sealing a pharmaceutical composition as described herein in a vial, cartridge or vaginal applicator, from which a therapeutically effective dose of pharmaceutical composition can be administered to a patient in need thereof. Preferably the process further comprises subjecting at least one component of the pharmaceutical composition to filtration, heat or gamma irradiation sterilisation. In a fourth aspect, the invention provides a method of treating an infertility condition comprising administering to a prospective mother a pharmaceutical composition as described herein. In one embodiment of this aspect of the invention, the method comprises the induction of immune tolerance to paternal antigen(s) in a prospective mammalian mother lacking said immune tolerance, the said method comprising exposing a mucosal surface, of said prospective mother, to a pharmaceutical composition as described herein and to semen or an MHC Class I antigen of a prospective father. Preferably the infertility condition is implantation failure or a gestational disorder. More preferably the gestational disorder is recurrent miscarriage, pre-eclampsia and intra-uterine growth retardation. Most preferably the gestational disorder is recurrent miscarriage. In a second embodiment, the prospective mammalian mother lacking said immune tolerance exhibits an immune cell profile indicating lack of immune tolerance to paternal antigen. Preferably the immune cell profile is characterised by low numbers of regulatory T cells or a Th1 cell number higher than the Th2 cell number. Preferably the T regulatory, Th1 and Th2 cell number profiles are derived from cervical brush samples, vaginal wash samples, or peripheral blood. In a third preferred embodiment, the exposure is a multiple exposure. Preferably the pharmaceutical composition is administered prior to, during and/or after intercourse. For example the pharmaceutical composition is administered over a period spanning at least one month before conception. Alternatively the pharmaceutical composition is administered over a period spanning at least one week before conception. Preferably the pharmaceutical composition is administered over a period spanning at least 4 days before conception. In a forth preferred embodiment the MHC Class I antigen is from sperm cells of the prospective father. Preferably the semen or an MHC Class I antigen of the prospective father is in the form of the prospective father's ejaculate. In a fifth embodiment, the mucosal surface is a genital mucosal surface, a respiratory mucosal surface, a gastrointestinal mucosal surface or an oral mucosal surface. Preferably the mucosal surface is a genital mucosal surface. More preferably the pharmaceutical composition is administered in a therapeutically effective amount. In a fifth aspect, the invention provides a method for reducing the adhesion of TGFbeta to the surface of a container and the said method comprises adding a gel polymer to a composition comprising TGFbeta.

In a sixth aspect, the invention provides a method for characterising TGFbeta in a composition where the composition comprising TGFbeta also comprises a gel polymer and the said method comprises evaluating the TGFbeta by means of an analytical method,. Preferably the analytical method is SDS-PAGE analysis. Alternatively the analytical method is reverse phase HPLC. Alternatively the analytical method is a bioassay. Preferably the bioassay is a Mink Lung epithelial (MvI Lu) cell growth inhibition assay. In one embodiment of this aspect, the pH of the composition is at or below pH 3.7. Preferably the pH of the composition is between pH 3.7 and pH 3.2. More preferably the pH of the composition is between pH 3.7 and pH 3.3. Most preferably the composition is a pharmaceutical composition. Preferably the TGFbeta has not undergone a step of freeze-drying.

In a second embodiment, of this aspect of the invention, the method reduces the underestimation of TGFbeta concentration. Preferably the composition is in a liquid form.

Preferably the gel polymer is selected from the group consisting of cellulose based polymers, tragacanth polymers, xanthan gum polymers, acacia polymers, carbomer polymers, gelatin polymers, sodium alginate polymers, poloxomer polymers, polyethylene oxide polymers, polyacrylamide polymers, and polyethylene glycol polymers. More preferably the gel polymer is a cellulose-based polymer. Even more preferably the cellulose-based polymer is selected from the group consisting of hypromellose, methylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxyethylcellulose and carboxymethylcellulose. Most preferably the cellulose-based polymer is hypromellose. In one example the hypromellose has a grade of 4000. Preferably the hypromellose is at a concentration of between 1% to 5% w/w. More preferably the hypromellose is at a concentration of 1.9% w/w. In one example the cellulose-based polymer contains a hydroxyl group and/or a carboxyl group.

In a third embodiment, the composition is void of calcium chloride or calcium phosphate. Preferably the composition is void of calcium chloride, calcium phosphate, potassium acetate, lithium acetate, ammonium acetate or ammonium bicarbonate. More preferably the composition is void of calcium chloride, calcium phosphate, potassium acetate, lithium acetate, sodium acetate, ammonium acetate or ammonium bicarbonate. In a fourth embodiment, the TGFbeta is selected from the group consisting of human TGFbetal , TGFbeta2 and TGFbeta3. Preferably the TGFbeta is human TGFbeta3. Preferably the TGFbeta is substantially purified. Preferably the TGFbeta is recombinant.

In a fifth embodiment, the concentration of TGFbeta is at or below 100micro-g/ml. Preferably the concentration of TGFbeta is at or below 20micro-g/ml. More preferably the concentration of TGFbeta is at or below 4micro-g/ml. Most preferably the concentration of

TGFbeta is at or below 0.5micro-g/ml. Preferably the concentration of TGFbeta is above 0.01 micro-g/ml.

In a seventh aspect, the invention provides an article of manufacture comprising one vial or cartridge comprising a liquid composition comprising a buffering agent in an amount sufficient to maintain the pH of the composition at or below 3.7 and a gel polymer; and a second vial or cartridge comprising a dry powder composition comprising a freeze dried

TGFbeta together with instructions that the liquid composition is added to the dry powder composition prior to use. Preferably the pH of the liquid composition is below 3.7. More preferably the pH of the liquid composition is between 3.2 and 3.7. Even more preferably pH of the liquid composition is between 3.3 and 3.7.

In an eighth aspect, the invention provides an article of manufacture comprising one vial or cartridge comprising a liquid composition comprising a buffering agent in an amount sufficient to maintain the pH of the composition at or below 3.7 and a TGFbeta and a second vial or cartridge comprising a gel polymer together with instructions that the liquid composition is added to the gel polymer composition prior to use. Preferably the pH of the liquid composition is below 3.7. More preferably the pH of the liquid composition is between 3.2 and 3.7. Even more preferably pH of the liquid composition is between 3.3 and 3.7.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Absorbance at 350 nm of 125micro-micro-g/mL TGFbeta3 gels at pH 3.7, 3.8 and 3.9 (minus absorbance of gel vehicle alone)

Figure 2. Results of testing TGFbeta3 gel formulations G, H, I and J at T=O

Figure 3. Results of testing prototype G after storage at 4⁰C for 6 and 12 months

Figure 4. Results of testing prototype H after storage at 4⁰C for 6 and 12 months

Figure 5. Results of testing prototype I after storage at 4⁰C for 6 and 12 months

Figure 6. Results of testing prototype J after storage at 4⁰C for 6 and 12 months Figure 7. Change in viscosity of hypromellose gels with pH 3.0, 3.5 and 5.3 after autoclaving

Figure 8. Viscosity of pH 3.2-5.9 formulations after autoclaving

Figure 9 Resorption rate in abortion-prone CBA/J mice treated with a single dose of 2ng TGFbeta3 or vehicle control

Figure 10 Characteristics of Formulations K to Q

Figure 11 Tests performed at T=O on Prototype Formulations K to Q

Figure 12 Tests performed at T=3 months on Prototype Formulations K to Q

Figure 13 Tests performed at T=6 months on Prototype Formulations K to Q

Figure 14 RP-HPLC results at T=O, 3 and 6 months for (Active) Prototype

Formulations K to Q

Figure 15 UV absorbance results at T=O, 3 and 6 months for (Active) Prototype

Formulations K to Q

Figure 16 UV absorbance results at T=O, 3 and 6 months for (Placebo) Prototype Formulations K to Q

Figure 17 Bioassay results at T=O and 6 months for (Active) Prototype Formulations K to Q

Figure 18 Bioburden results at T=O and 6 months for (Active and Placebo) Prototype

Formulations K to Q

Figure 19 PET results at T=O and 6 months for (Placebo) Prototype Formulations K to

Q

Figure 20 Viscosity results at T=O and 6 months for (Active) Prototype Formulations K to Q Figure 21 Viscosity results at T=O, 3 and 6 months for (Placebo) Prototype Formulations K to Q

Figure 22 Osmolality results at T=O and 6 months for (Active) Prototype Formulations K to Q

Figure 23 Osmolality results at T=O, 3 and 6 months for (Placebo) Prototype Formulations K to Q

Figure 24 pH results at T=O and 6 months for (Active) Prototype Formulations K to Q

Figure 25 pH results at T=O, 3 and 6 months for (Placebo) Prototype Formulations K to Q

DETAILED DESCRIPTION OF THE INVENTION

In a preferred form the protein is a TGFbeta polypeptide. The TGFbeta polypeptide used in the pharmaceutical composition is selected from the group consisting of TGFbetal , TGFbeta2, TGFbeta3, TGFbeta4, TGFbetaδ or activin (including activin A, activin B and activin AB). More preferably the TGFbeta polypeptide is selected from the group consisting of TGFbetal , TGFbeta2, TGFbeta3 or activin (including activin A, activin B and activin AB). Preferably the TGFbeta polypeptide to be used is human TGFbeta however it will be understood that TGFbeta polypeptides from other species may be used. For example bovine TGFbeta polypeptide may be used. Most preferably the TGFbeta is human TGFbeta3. Alternatively, another member of the TGFbeta superfamily may be used including polypeptides selected from the group consisting of Mullerian inhibitory substances (MIS), bone morphogenetic proteins (BMP-2-7), inhibins, growth differentiation factors (GDF-1), dorsalin-1 (dsl-1) and Drosophila decapentaplegic gene product (DPP-C). Methods for assessing other members of the TGFbeta superfamily for suitability for use in the pharmaceutical composition can be addressed by no more than routine experimentation. Preferably the TGFbeta polypeptide member of the TGFbeta family contains an intact cystine knot such that the TGFbeta family member is biologically active.

The TGFbeta may be purified from tissue such as bone, platelets or placenta. Alternatively, recombinant TGFbeta may be used. Methods to manufacture TGFbeta by recombinant methods are well known in the art (Iwata, et al. 1992 MoI Endocrinol. May;6(5):694-702.; ten Dijke et al. 1990 MoI Cell Biol. Sep;10(9):4473-9; Derynck et al., 1988 EMBO J. Dec 1 ;7(12):3737-43; Cerletti, N., 1991 J Protein Chem. 1991 Oct;10(5):565-75; Ogawa et al., 1992 Biochemistry Feb 2;32(4):1164-71).

It will also be understood that various modifications might be made to TGFbetal , TGFbeta2, TGFbeta3, TGFbeta4, TGFbetaδ or activin. Such modified forms of TGFbeta might include substitution, deletion or addition mutants, and might include peptide fragments which may or may not be incorporated into another protein to make a recombinant protein. Alternatively other polypeptide members of the TGFbeta superfamily may also be used or used as a starting point to developing an analogue of the TGFbeta activity, including Mullerian inhibitory substances (MIS), bone morphogenetic proteins (BMP-2-7), growth differentiation factors (GDF-1 ), dorsalin-1 (dsl-1) and Drosophila decapentaplegic gene product (DPP-C).

It is to be clearly understood that the present invention extends to biologically active fragments, functional analogues and derivatives of TGFbeta, i.e. fragments, analogues or derivatives of TGFbeta in which the wild-type TGFbeta sequence contains additions, deletions or substitutions by other amino acids or amino acid analogues, in which the biological activity of the TGFbeta is retained. The methods to identify, manufacture and characterise biologically active fragments, functional analogues or derivatives of TGFbeta are well known to those of ordinary skill in the art, and can be addressed with no more than routine experimentation. Persons skilled in the art will know with a reasonable expectation of success which modifications can be made to the TGFbeta fragment, analogue or derivative to conserve biological activity. In a preferred form, the fragment, functional analogue or derivative of TGFbeta to be used will have substantially the same biological activity as native TGFbeta, e.g. binding to Type I or Type Il TGFbeta receptors, inhibiting Mink Lung cell line CCL64 growth in vitro or stimulating GM-CSF production by murine uterine epithelial cells in vitro. Preferably the modification does not alter the tertiary structure of the cystine knot. Even more preferably the modification does not alter the TGFbeta receptor binding site. In a preferred form, the TGFbeta fragment, functional analogue or derivative has at least 70% amino acid sequence homology with the native TGFbeta amino acid sequence, or preferably at least 90% and more preferably 95%. Methods for assessing the amino acid sequence homology are well known in the art and can be addressed by no more than routine experimentation. For example, a suitable program for determining percentage sequence identity can be found at http://www.ncbi.nlm.nih.gov/blast/b12sea/b12.html (NIH BLAST 2.0 Sequence Comparison). Preferably, the limiting parameters imposed for such a task are the default settings for the program as displayed on this web site. The invention also includes TGFbeta in which the coding sequence for the polypeptide is fused in frame to a polypeptide sequence which aids in expression of the fusion protein in a host cell, for example, a polypeptide leader sequence encoding a fragment of pig growth hormone. Other suitable fusion protein partner leader sequences are known in the art. The inventors have surprisingly discovered certain advantages in maintaining the pH of the liquid pharmaceutical composition at or below pH 3.7 and preferably at pH range of 3.2- 3.7, more preferably 3.3-3.7.

Firstly, no significant TGFbeta aggregation is observed at or below 3.7, however aggregation is observed when the pH increases above 3.7, especially when stored for up to

12 months. Aggregation of TGFbeta is a problem because aggregation causes loss of bioactivity of the pharmaceutical composition and an increased risk of immunogenicity in clinical use.

Secondly, when the pharmaceutical composition is adapted for vaginal administration, irritation is more likely to occur below pH 3.3 and even more likely to occur below pH 3.2. Irritation is a significant problem in the development of formulations adapted for administration to the cervix or uterus via vaginal delivery.

Thirdly, when the pharmaceutical composition is adapted for treatment of an infertility condition administered prior to, during, or after the administration of the prospective father's sperm (for example in the form of the father's ejaculate), a pH compatible with viability of the sperm cells is preferred. Thus the pharmaceutical composition should not be toxic to sperm and adversely effect conception. A reduction of sperm viability is more likely to occur below pH 3.3 and even more likely to occur below pH 3.2. Loss of sperm viability is a significant problem when developing a formulation adapted for vaginal delivery for the treatment of infertility conditions.

Fourthly, if the liquid pharmaceutical composition comprises a gel polymer, any hydrolysis of the gel polymer during heat or radiation sterilization is undesirable as viscosity is altered thus affecting the release profile of the TGFbeta. Loss of viscosity of the gel polymer is significant following heat sterilization when the pH of the pharmaceutical composition is below 3.3 and even more significant when below pH 3.2. Loss of viscosity is not significant following heat sterilization when the pH of the pharmaceutical composition is at or above 3.2 and even less likely at a pH at or above 3.3.

In one embodiment the pharmaceutical composition is in liquid form and not in freeze- dried form. The pharmaceutical composition may be in gel form, comprising a gel polymer. The term 'liquid' includes solutions and gel polymers within its definition as described herein.

In another embodiment the pharmaceutical composition comprises a gel polymer to increase viscosity of the composition thus aiding in delivery of the TGFbeta at the site of therapeutic action. Preferably, the polymer is a gel-like substance suitable for pharmaceutical use. The inventors have surprisingly found that the presence of a gel in the formulation reduces adherence of TGFbeta to surfaces of containers and equipment. Adherence of

TGFbeta to surfaces is a problem for three reasons: (i) it reduces the actual dose delivered to the patient because the TGFbeta is formulated at a low concentration and a large proportion of the TGFbeta will be lost by sticking to the container or delivery device; (ii) it results in low yields during manufacture as the TGFbeta will adhere to equipment and lines, thus increasing the complexity of manufacturing; (iii) measuring the concentration of the TGFbeta in the product by assay is required for product release and will be problematic because the TGFbeta will adhere to vials and instruments thus resulting in an inaccurate determination of TGFbeta concentration.

In one aspect, the presence of a gel in the formulation reduces TGFbeta aggregation. In one specific embodiment, the gel aids in the retention of the TGFbeta at the site of delivery. Further, gels have certain mucoadhesive properties and aid in delivery when administered to a mucosal surface, such as the vaginal, cervical or uterine surface of a prospective human mother. In a preferred form, the gel polymer allows for release of the TGFbeta at the site of delivery thus making the TGFbeta bioavailable.

Suitable gel polymers include cellulose based polymers (dispersible, microcrystalline and derivatives and semi-synthetic), tragacanth (plant derived), xanthan gum (plant derived), acacia (plant derived), carbomer (carbopol) (synthetic acrylic acid polymer), gelatin (derived from animal collagen), sodium alginate (algae extract), poloxomer, polyethylene oxide, polyacrylamide and polyethylene glycol (macrogols). Preferably, the polymer is a cellulose- based gel, such as hydroxypropylmethylcellulose, methylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxyethylcellulose or carboxymethylcellulose. More preferably the cellulose-based polymer is capable of hydrogen bonding, which excludes methylcellulose and ethylcellulose. More preferably the cellulose-based polymer contains a hydroxyl group, which includes hydroxypropylcellulose, hydroxyethylcellulose and hydroxypropylmethylcellulose. Alternatively the cellulose-based polymer contains a carboxyl group, which includes carboxymethylcellulose. Preferably the gel is methylcellulose with a grade of 4000. Preferably the gel is hydroxyethylcellulose with a grade of 4400. Preferably the gel is hydroxypropyl cellulose with a grade of 4000. More preferably the gel is hydroxypropylmethylcellulose, also known as hypromellose. More preferably the gel is hypromellose with a grade of 4000.

Preferably the hypromellose is at a concentration of between 1 % and 5% w/w. Preferably the hypromellose is at a concentration of 1.9% w/w. Preferably the gel polymer is at a concentration of between 1 % to 5% w/w. Preferably the gel polymer is at a concentration of 1.9% w/w.

In a preferred form, the pharmaceutical composition is sterile as described in the British Pharmacopoeia 2001.

In another preferred form, the pharmaceutical composition has a low bioburden, especially when adapted for topical delivery, including vaginal delivery.

Preferably, the pharmaceutical composition is adapted for topical delivery, including vaginal delivery and has: (a) Not more than a total of 10² micro-organisms (aerobic bacteria or fungi) per gram, as tested using standard pour-plate methods described in Appendix XVI B2 of the British Pharmacopoeia 2001.

(b) Not more than 10¹ enterobacteria and certain other Gram-negative bacteria per gram, as tested using standard pour-plate methods described in Appendix XVI B2 of the

British Pharmacopoeia 2001.

(c) Absence of Pseudomonas aeruginosa, determined on 1g, as tested using standard pour-plate methods described in Appendix XVI B2 of the British Pharmacopoeia 2001.

(d) Absence of Staphylococcus aureus, determined on 1g, as tested using standard pour- plate methods described in Appendix XVI B2 of the British Pharmacopoeia 2001.

This low bioburden profile can be achieved by subjecting the pharmaceutical composition or components thereof to heat sterilization (such as use of an autoclave), gamma irradiation, or micro-filtration. Following sterilization, the composition is sealed in a pharmaceutically acceptable container such as a vial, sachet, cartridge or applicator. Preferably, if the pharmaceutical composition is heat sterilized, the TGFbeta is not present but added to the composition following heat sterilization.

The viscosity of the pharmaceutical composition is preferably between 200 and 3O₁OOOcP. More preferably the viscosity is between 500 and 90OcP. More preferably the viscosity is 60OcP or 70OcP. The pharmaceutical composition can comprise an osmotic agent, such as mannitol, lactose, glucose, glycerol, propylene glycol, sodium lactate or sodium citrate. Preferably, the osmotic agent is mannitol.

Preferably, the osmotic agent is at a concentration between 2% and 10% w/w. More preferably the osmotic agents is at a concentration of 4% or 5% w/w. Preferably, if mannitol is used, the concentration is 4% or 5% w/w. Preferably if lactose is used the concentration is 10% w/w. Preferably if glucose is used the concentration is 5% w/w. Preferably if glycerol is used the concentration is 2.5% w/w. Preferably if propylene glycol is used the concentration is 2% w/w. Preferably if sodium lactate is used the concentration is 4.5% w/w. Preferably if sodium citrate is used the concentration is 3% w/w. The pharmaceutical composition can comprise a buffering agent, such as glycine.

Preferably, the glycine is at a concentration of between 0.01% and 1% w/w. More preferably the glycine is at a concentration of 0.15% w/w. Alternatively, the buffering system contains acetic acid. The composition may comprise a salt such as magnesium hydroxide.

Preferably the composition is free of calcium chloride, calcium phosphate, sodium acetate, potassium acetate, lithium acetate, ammonium acetate or ammonium bicarbonate. Most preferably the composition is free of calcium chloride, calcium phosphate, potassium acetate, lithium acetate, ammonium acetate or ammonium bicarbonate. Even more preferably the composition is free of calcium chloride and calcium phosphate. Preferably calcium chloride, calcium phosphate, sodium acetate, potassium acetate, lithium acetate, ammonium acetate or ammonium bicarbonate is not added to the composition at any time during its manufacture. More preferably calcium chloride, calcium phosphate, potassium acetate, lithium acetate, ammonium acetate or ammonium bicarbonate is not added to the composition at any time during its manufacture. Most preferably calcium chloride or calcium phosphate is not added to the composition at any time during its manufacture.

The pharmaceutical composition can comprise a preservative agent, such as benzoic acid, methyl paraben, propyl paraben or benzyl alcohol. Preferably, if benzoic acid is used, the concentration is 0.01 and 1.0% w/w. Preferably, if methyl paraben is used, the concentration is between 0.01% and 1.0% w/w. Preferably, if propyl paraben is used, the concentration is between 0.01% and 1.0% w/w. Preferably, if benzyl alcohol is used, the concentration is between 0.01% and 3% w/w. More preferably, if benzoic acid is used, the concentration is 0.1% w/w. More preferably, if a combination of methyl paraben and propyl paraben is used, the concentration of methyl paraben is 0.07% w/w and the concentration of propyl paraben is 0.03% w/w. More preferably, if benzyl alcohol is used, the concentration is 1.5% w/w or 2% w/w.

The pharmaceutical composition can comprise hydrochloric acid, acetic acid or sodium hydroxide for pH adjustment. The pharmaceutical composition can comprise water to bring the difference in concentration to 100% w/w.

In one aspect of the invention, the TGFbeta formulated in the pharmaceutical composition has not been subjected to a freeze-drying process at any stage of its manufacture or preparation for pharmaceutical use. In another aspect of the invention, the TGFbeta formulated into the pharmaceutical composition has been subject to a freeze-drying process at least one stage of its manufacture or preparation for pharmaceutical use.

The pharmaceutical composition can be stored at temperatures between 2⁰C and 27⁰C (at room temperature or refrigerated). Preferably the pharmaceutical composition is stored between 2⁰C and 8⁰C. Preferably the pharmaceutical composition is stable for 12 or more months when stored at room temperature. Preferably the pharmaceutical composition is stable for 12 or more months when stored between 2⁰C and 8⁰C. Preferably the pharmaceutical composition is stable for at least 6 months when stored at room temperature. Preferably the pharmaceutical composition is stable for 6 months when stored between 2⁰C and 8⁰C.

The invention further comprises a vaginal applicator comprising the pharmaceutical composition. In a preferred form the applicator is sealed such that the pharmaceutical composition retains its low bioburden profile thus maintaining suitability for clinical use. In a preferred form the applicator is labeled, or accompanied by, instructions for use of the device and pharmaceutical composition for the treatment of recurrent miscarriage, pre-eclampsia, intra-uterine growth retardation or implantation failure. Preferably the instructions are for the treatment of recurrent miscarriage. Suitable vaginal applicators are well known in the art. A suitable vaginal applicator, to administer the pharmaceutical composition to the cervix can be purchased from HTI Plastics (http://www.htiplastic.com/). manufactured using Huntsman P4C6N-041 polypropylene (for cap material), Huntsman P4C6N-041 polypropylene (for barrel material) and Santoprene 8281-55 rubber (for piston material).

The invention also comprises a pharmaceutically acceptable vial or container. In a preferred form, the vial or container is sealed such that the pharmaceutical composition retains its low bioburden properties thus maintaining suitability for clinical use. In a preferred form the vial or container is labeled, or accompanied by, instructions for use of the device and pharmaceutical composition for the treatment of recurrent miscarriage.

Preferably the vial or container is constructed from plastic. Preferably the vial or container is constructed from glass, such as silicon-coated glass.

Preferably the vial or container is accompanied by or labeled with instructions that the pharmaceutical composition, the contents therein, has a specific expiry date that is between 1 and 24 months after the date of manufacture when stored at room temperature.

Preferably the vial or container is accompanied by or labeled with instructions that the pharmaceutical composition, the contents therein, has a specific expiry date that is between 1 and 24 months after the date of manufacture when stored between 2⁰C and 8⁰C.

Preferably the vial or container is accompanied by or labeled with instructions that the pharmaceutical composition, the contents therein, has a specific expiry date that is between 1 and 24 months after the date of manufacture when stored between 2⁰C and 27⁰C. A number of assay methods can be used for the characterisation (including quantitation) of TGFbeta in the pharmaceutical composition. Such assays are used routinely for the testing drug products. Methods used for quantitation of TGFbeta include reverse phase HPLC. Preferably the sample of TGFbeta is compared with the profile of a reference standard of TGFbeta to determine concentration. Further, HPLC can be used to determine presence of aggregates and degradation products of TGFbeta. Alternatively the Mink Lung Epithelial (MvI Lu) cell bioassay can be used for TGFbeta quantitation as well as determining the bioactivity of TGFbeta. SDS-PAGE analysis can be used to quantitate aggregates and degradants TGFbeta. The inventors have surprisingly found that the presence of a gel inhibits the adherence of TGFbeta to surfaces of containers, including containers and equipment lines used in assaying the TGFbeta, thus enhancing the accuracy of the assay method used for quantitation of TGFbeta and aggregates and degradants. The loss of TGFbeta onto surfaces of containers and equipment is reduced by the presence of a gel.

Preferably the gel is a cellulose-based polymer, and more preferably the gel is hypromellose. Preferably the TGFbeta to be assayed is below a concentration of 100micro-g/ml and more preferably below, 20micro-g/ml, even more preferably below 4micro-g/ml.

In an alternative form of the invention, the invention is a method for treating an infertility condition in a human patient by the administration of the pharmaceutical composition herein described. Infertility condition includes implantation failure and gestational disorders, which further includes recurrent miscarriage, pre-eclampsia and intrauterine growth retardation.

In an alternative form of the invention, the invention is a method for treating implantation caused by lack of fibronectin production by the conceptus. In an alternative form of the invention, the invention is a method for treating a gestational disorder in a patient by inducing immune tolerance to paternal antigen(s) by the administration of the pharmaceutical composition herein described. A gestational disorder includes recurrent miscarriage, pre-eclampsia and intra-uterine growth retardation. The gestational disorder is preferably a disorder characterized by a lack of immune tolerance to paternal antigen(s) and is not a disorder characterized by a lack of fibronectin production by the trophoblast.

Preferably the patient is a mammal. Preferably the patient is a human.

Methods used to treat such a gestational disorder by the administration of TGFbeta are described in International Patent Application No. PCT/AU98/00149 (Robertson) and are incorporated herein by reference.

The method comprises exposing a mucosal surface of a prospective mother who lacks immune tolerance to paternal antigen (in semen or MHC Class I antigen on the sperm of a prospective father) and a TGFbeta.

Whilst a mucosal exposure may be preferred because it is likely to give rise to a transient tolerant immune reaction, it may also be feasible to provide for another route of exposure. Thus the pharmaceutical composition may be injected for systemic contact.

The mucosal surface can be a genital mucosal surface, a respiratory mucosal surface, a gastrointestinal mucosal surface or an oral mucosal surface. The mucosal surface is preferably the cervical mucosal surface. Methods to identify and select patients with gestational disorders caused by a lack of immune tolerance are widely available. It is expected that the patient would have tested positive to a pregnancy test (hCG test), but then tested negative, thus indicating successful implantation and then pregnancy loss, together with a immune profile characterised by presence of low numbers and/or function of T regulatory cells and/or a Th1 dominant compartment, thus indicating lack of immune tolerance. Further, the patient to be treated has a diminished T regulatory cell response (either diminished number or function of T regulatory cells) and/or a dominant Th1 response to paternal antigen exposure and the method of the invention induces T regulatory cell expansion and/or switches the immune response to a Th2 response indicative of immune tolerance to the paternal antigen. Assays to determine T regulatory cell number and function, and Th1 and Th2 responses in the patient are well known to those of skill in the art (see Schust and Hill, 1996, J. Soc. Gynecol. Investig. 3:259- 61 ; Xing et al., 2001 , Chin. Med. J. 114:921-4; Raghupathy et al., 1999, Cell Immunol. 196:122-30; Mauri et al., 1996, J. Immunol. 26:1511-8; Doncorli et al., 1997, Eur. J. Imm. 27:1451-8; Raziuddin, 1998, J. Rheumatol. 25:329-33; Moverare et al., 2000, Allergy 55:171- 5; Arruvito L et al., J Immunol 2007; 178:2572-8). Hence, a diagnostic assay of interest is one that determines whether the T regulatory cell number and/or activity is diminished in the prospective mother. Another assay of interest is one that determines whether Th1 cell number or cell activity is enhanced. Another assay of interest is one that determines whether Th2 cell number or activity is decreased. Yet another assay of interest is one that determines a higher ratio of Th1 cells to Th2 cells, or Th1 cell activity to Th2 cell activity, thus indicating presence of a gestational disorder caused by lack of immune tolerance to paternal antigen. A number of known assays, for example, immunoassays or bioassays, can be used to make such determinations. For example, T regulatory cells express of CD4, CD35 and Foxp3 and secrete IL-10. Thus assays for such cell-specific markers can provide the basis to conclude a low numbers of T regulatory cells. Interferon gamma, tumor necrosis factor alpha and IL-1 , IL-2 are cytokine markers of Th1 cells. Thus assays for one or more such cell-specific markers can provide the basis to conclude a higher than normal Th1 status. The cytokines IL-4, IL-5, IL-6, IL-10 and IL-13 are known markers of that Th2 cells. In a preferred embodiment, the Th1 cell is a tumor necrosis factor alpha expressing CD3+/CD4+ T cell. In another preferred embodiment, the Th2 cell is an IL-4 expressing CD3+/CD8+ T cell. Suitable methods to determine Th1 levels to diagnose a patient with a gestational disorder caused by a lack of immune tolerance to paternal antigen are described in International Patent Application No. PCT/US2003/027204.

Thus, assays for one or more of such cell-specific markers can provide the basis to conclude an unfavourable immunological status.

In a preferred form the patients who have the gestational disorders caused by a lack of immune tolerance do not have problems associated with lack of fibronectin production by trophoblasts.

The pharmaceutical composition may be administered before or during attempted conception and after conception has been achieved. The paternal antigen may be administered before or during attempted conception and after conception has been achieved.

It may be desirable to deliver the pharmaceutical composition and the antigen together, for example, where the two are combined in a gel, or spray. It is also possible to have a delay between the delivery of the pharmaceutical composition and the paternal antigen. Thus an alternative would be to deposit the antigen first perhaps as ejaculate and then deliver the TGFbeta as a pessary after intercourse. Suitable paternal antigens include those that are particularly antigenic and prominent either on the sperm, or on the conceptus. The most likely candidates are MHC antigens, and more preferably MHC class I. The most efficient manner to present these antigens is in the form that they are naturally present, that is, on any appropriate cell of the intended father that expresses them and those cells would include sperm cells and may include leukocytes. The antigens may also be presented in biological fluids such as seminal plasma which is known to carry certain male antigens. This use of cells other than sperm cells will be pertinent where the sperm count of the prospective father is somewhat low. The use of cells other than sperm cells may be preferred where a non-genital route is used. Alternatively the antigens may be presented in purified or semi-purified form, which may or may not be presented on inert or adjuvant carriers, thus for example it may be presented in the carriers known as ISCOMS. This latter approach however is likely to be more technically complex and expensive. It is additionally possible that the antigens may be encoded within sperm cells in the form of mRNA (or other nucleic acid) and this RNA message is then expressed by maternal genital tract cells. It may be that TGFbeta therefore plays a role in promoting the events leading to presentation of paternal antigen to maternal lymphocytes through activating genital tract antigen presenting cells to take up and translate sperm mRNA.

The level of exposure to paternal antigens may vary, in a preferred form the exposure will be to the prospective mother's genital tract in the form of the prospective father's ejaculate, and the level of exposure will be determined by the cell count and antigenic density on the surface of such cells. Where cells are administered other than in the above manner, a similar number of cells might be used, however, the most effective manner may be determined empirically. It is thought that an exposure of leukocytes in the order of 10⁷- 10⁹ cells might be the appropriate level of exposure to a mucosal surface. The exposure is preferably a multiple exposure. The multiple exposure is preferably performed over a period of at least three months, with the mucosal surface being exposed to the pharmaceutical composition during each exposure to the prospective father's antigens. The multiple exposure can be performed over a period of at least one month before attempted conception. This period of time could however be somewhat reduced, and it may be possible to achieve improvement with one exposure but as a minimum it is anticipated that exposure would be at least one week before conception is attempted. It may also be preferred that non-barrier contraceptive measures be taken prior to the planned conception, where the antigens are associated with sperm cells and these are administered to the genital tract, so that there is some certainty of a period of exposure to the prospective father's antigens before conception. This is particularly the case where the fertility condition is of the type where conception takes place but either spontaneous abortion or pre-eclampsia occurs after conception. For example, suitable non-barrier contraceptive measures include hormonal contraceptives, such as; 1) combined estrogen/progestin contraceptives administered by injection such as Cyclofem and Mesigyna which are administered once a month; 2) combined estrogen/progestin contraceptives administered orally such as Eugynon and Ovral which are administered daily; 3) progestin-only injectable contraceptives such as Depo- Provera and Noristerat which are administered every 3 months; 4) progestin-only oral contraceptives (also known as minipills) such as levonorgestrel (morning after pill), norgestrel which are taken daily; 5) progestin-only implants such as Norplant which are inserted once and can be effective for up to five years; 6) intrauterine devices (IUDs) such as Copper T 380A (copper releasing); or 7) progestin-releasing IUDs such as Progestasert. Preferably the non-barrier method used does not inhibit the effectiveness of the TGFbeta treatment. For example, preferably, IUDs which are known to cause pelvic inflammatory diseases in some patients are not used. Preferably contraceptive methods which have long term residual effects on fertility following termination of use are not to be used. For example, preferably, combined estrogen/progestin contraceptives administered by injection, progestin-only injectable contraceptives and progestin-only implants which are known to exhibit long lasting residual on fertility effects are not to be used. Most preferably, combined oral estrogen/progestin contraceptives and progestin-only oral contraceptives are used.

Suitable non-barrier contraceptive measures also include male contraceptives. For example suitable male contraceptives include methods which suppress sperm production such as Gonadotropin releasing hormone or inhibit the ability of the sperm to fertilise such as Nifedipine.

Alternatively it may also be preferred that barrier contraceptive measures are used where the antigens are administered via mucosal surfaces other than to the genital tract such as the respiratory or gastrointestinal tract. For example the antigen may be presented as an oral, anal or nasal spray or gel. Alternatively it might be desired to take the TGFbeta and the surface antigen in a form that gives exposure to the small and perhaps large intestines, such as perhaps contained in a gelatin capsule. Suitable barrier contraceptive measures include male and female condoms, diaphragms and spermicides. The present invention may be used in conjunction with IVF treatment, whereby the transient tolerant immune response is elicited before transfer of the conceptus or gametes is attempted. It is expected, however, that where the gestational disorder is caused as a result of reduced TGFbeta level in semen, or capacity to activate TGFbeta, it is likely that the trauma of IVF treatment may not be needed and that a 'natural¹ conception may be possible in its place.

The results presented in Example 7 show that the addition of rTGFbetal, rTGFbeta2 and rTGFbeta3 to human cervical keratinocytes in vitro increased GM-CSF production from reproductive tract tissues in women 12 and 24 hours after treatment. The results presented in Example 8 show that a single dose of TGFbeta3 administered to female mice lacking immune tolerance to paternal antigen, either before attempted conception or after attempted conception, actually prevents miscarriage and has no detrimental effect on pregnancy. Thus both these studies provide reasonable support that a single dose of TGFbetal, TGFbeta2 or TG Fbeta3 administered to a prospective human mother lacking immune tolerance to paternal antigen, either before attempted conception or after attempted conception, would prevent miscarriage and have no detrimental effect on pregnancy. It is thus contemplated that the administration of the pharmaceutical compositions described herein to a patient in need thereof provides a suitable treatment of recurrent miscarriage. The treatment of recurrent miscarriage by the administration of the pharmaceutical compositions described herein is within the scope of the current invention.

In accordance with this invention, the TGFbeta is administered in therapeutically effective amounts. The term "therapeutically effective amount" as used herein means that amount necessary at least partly to attain the desired effect, i.e. the elicitation of tolerance towards male antigens. Such amounts will depend on the particular infertility condition being treated, the severity of the condition, and the characteristics of the individual subject, including age, physical conditions, size, weight and other concurrent treatments, and will be at the discretion of the prescribing physician. These factors are well known to those of ordinary skill in the art, and can be addressed with no more than routine experimentation. It is generally preferred that a minimum effective dose be determined according to sound medical judgment. It will be understood by those of ordinary skill in the art that a higher dose may be administered for medical, psychological or other reasons.

The level of TGFbeta in the pharmaceutical composition may be varied. The concentration of TGFbeta will preferably be between 0.01micro-g/ml and 125micro-g/ml, more preferably between 0.1 micro-g/ml and 100micro-g/ml. Preferably, the concentration is between 0.2micro-g/ml and 20micro-g/ml. More preferably the concentration is between 0.2micro-g/ml and 2micro-g/ml. More preferably the concentration is 0.2micro-g/ml. Alternatively, the concentration is between 0.2micro-g/ml and 20micro-g/ml, wherein the total dose is between 0.6ug and 60ug. More preferably the concentration will be between 0.2micro-g/ml and 2micro-g/ml, wherein the total dose is between 0.6ug and 6ug. More preferably the concentration is 0.2micro-g/ml, wherein the total dose is 0.6ug. For example the concentration may be 0.5, 4 or 20micro-g/ml and administered to the patient in a 3ml volume providing total doses of 1.5, 12 or 60ug. For example the concentration may be 0.5, 2 or 20micro-g/ml and administered to the patient in a 3ml volume providing total doses of 1.5, 6 or 60ug. In a preferred form the pharmaceutical composition is administered to the reproductive tract of a patient with a gestational disorder caused by lack of immune tolerance to paternal antigen using a vaginal applicator. Alternatively the pharmaceutical composition is delivered by oral administration (including buccal patch), topical application, topical administration to a mucosal surface, or subcutaneous or intramuscular delivery.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The present invention will now be more fully described with reference to the accompanying non-limiting examples. It should be understood that the following description is illustrative only, and should not be taken in any way as a restriction on the generality of the invention.

ABBREVIATIONS

CaCI₂ Calcium chloride cP centiPoise

DMEM Dulbecco's Modified Eagle's Medium

EC50 Concentration producing 50% of maximal effect

ECM-FCS Ectocervical Culture Medium with 5% Fetal Calf Serum

HBSS Hanks' Balanced Salt Solution

IC50 50% Inhibitory concentration

MC . Methylcellulose mPa.s milliPascal. second

MvI Lu Mink Lung Epithelial Cell n/a Not applicable

ND Not determined

OD optical density

PBS Phosphate Buffered Saline

PET Preservative efficacy testing

RP-HPLC Reversed Phase High Performance Liquid Chromatography

RT Room temperature rTGF Recombinant Transforming Growth Factor

T Time point

TFA Trifluoracetic acid

TGF Transforming Growth Factor

UV Ultraviolet v/w volume per weight w/w weight per weight EXAMPLE 1. TGFbeta3 aggregation in gel formulations at pH 3.5. 3.7. 3.8, 3.9 and 5.4

The inventors studied the stability of TGFbeta3 in solution over the pH range of 3.5- 5.4.

The long-term stability of TGFbeta3 in hypromellose gel formulations at pH 3.5 and 5.4 was examined. Polyacrylimide gel electrophoresis (PAGE) was used to measure the presence of monomer and covalent aggregates in pH 3.5 and pH 5.4 formulations after storage for 14 months at 4⁰C. Samples of 125micro-micro-g/ml_ TGFbeta3 gel were prepared by dissolving lyophilized TGFbeta3 in hypromellose gei at pH 3.5 (hypromellose 1.8%, mannitol 3.5%, glycine 0.15%, hydrochloric acid to pH 3.5, in water; autoclaved at 121⁰C for 15 minutes; viscosity 106OmPa. s) and pH 5.4 (hypromellose 1.8%, mannitol 3.5%, glycine 0.15% in water; autoclaved at 121⁰C for 15 minutes; viscosity 1940mPa.s). Presence of monomer and covalent aggregates of TGFbeta3 in gel samples was measured after storage for 14 months at 4⁰C by the following method. Gel samples were desalted by HPLC on a Vydac C4 5micro-microm 300A, 4.6 x 250mm reversed phase column (Grace Vydac, Hesperia, CA, USA), at 4O⁰C using a 35-100% acetonitrile gradient with 0.08% trifluoroacetic acid over 14 minutes at 1 mL/minute. Gel formulations were diluted 1 part in 6 with 0.1% trifluoroacetic acid then a diluted gel sample containing 10micro-micro-g of TGFbeta3 was loaded and the 2.7mL eluate fraction from 6.9-9.6 minutes was collected. 10 mg of mannitol was added to stabilize the protein during lyophilization. This solution was dried to a white pellet under vacuum at room temperature. The pellet was reconstituted in 40micro-microL of 1x NuPAGE® LDS Sample Buffer (Invitrogen, San Diego, CA, USA). 2micro-microL of the reconstituted sample was withdrawn and diluted with 30micro-microL of 1x Sample Buffer to allow protein content in the sample to be determined from a standard curve. The remaining 38micro-microL (nominal 9.5micro-micro-g) of the test sample and 32micromicro-L (nominal 0.5micro-micro-g) of the diluted test sample were loaded onto a 10 x 1mm well NuPAGE® 4-12% Bis-Tris Pre-Cast gel (Invitrogen, San Diego, CA, USA). 10micro-micro-g Reference Standard (recombinant TGFbeta3) was prepared in an identical manner to the test sample by dilution in 1/6 gel then desalting as described above. The reconstituted Reference Standard was then loaded on to the gel at nominal loads of 9.5micro-micro-g and 0.5micro-micro-g. These reference samples were used to determine the formation of aggregates and monomer in Reference Standard due to the desalting procedure. Untreated Reference Standard samples were also prepared in Sample Buffer and loaded onto the gel at 0.1 , 0.3, 0.5 and 0.8micro-micro-g to allow quantitation of bands from a standard curve over this range. All test and reference samples were heated for 10 minutes at 7O⁰C before loading. One lane was used for Mark 12™ molecular weight markers (Invitrogen, San Diego, CA, USA).

Gels were run in an Xcell II™ Mini-Cell vertical electrophoresis chamber (Invitrogen, San Diego, CA, USA) with a Bio-Rad PowerPac 300 (Bio-Rad Laboratories, Hercules, CA, USA) at 200 V for 35 minutes in NuPAGE® MES SDS running buffer (Invitrogen, San Diego, CA₁ USA). Gels were fixed for 15 minutes in 50% methanol, 7% acetic acid then rinsed (3 x 5 minute rinses with ultrapure water) before staining with GelCode® Blue Stain Reagent (Pierce, Rockford, IL, USA) overnight. Gels were destained for 2 hours using several changes of ultrapure water. Gels were analysed using a Kodak EDAS 120 Digital Camera with Kodak™ Digital Science 1 D Image Analysis v3.0 software.

Protein content of test sample bands was determined from band intensities compared to a standard curve on each gel derived from band intensities of Reference Standard samples over the range of 0.1-0.8micro-micro-g. Aggregate content in test samples was corrected for the background content of aggregates in desalted Reference Standard by subtracting the impurity content in Reference Standard from the impurity content in test samples. Impurities are expressed as a % of the actual total protein content in the sample after HPLC desalting which is determined from quantitation of protein in the test sample loaded at a nominal 0.5micro-micro-g against the standard curve. The quantitation limit for the assay was 0.1micro-micro-g of protein. 1% aggregates was present in pH 3.5 gel compared to 6% in pH 5.4 gel after 14 months storage at 4⁰C. The presence of insoluble aggregates was also evident from the cloudy appearance of the pH 5.4 gel compared to the clear, colourless pH 3.5 gel.

We then used UV spectrophotometry to assess aggregation in different pH gels. Absorbance at 350nm due to light scattering is a sensitive method for monitoring formation of insoluble aggregates in TGFbeta3 solutions (Pellaud et a/, 1999 J Biol Chem. 1999 Mar 19;274(12):7699-704). We compared the absorbance spectrum of different pH gel formulations of TGFbeta3 and gel vehicle alone using a Carey 50 UV-vis scanning spectrophotometer (Varian, Zug, Switzerland). The difference in absorbance at 350nm between TGFbeta3 gel and matching gel vehicle alone was determined for each formulation as a measure of insoluble aggregate content in three different pH formulations.

Gel formulations were prepared as described above but pH was adjusted to 3.7, 3.8 and 3.9. TGFbeta3 gels were prepared by dissolving lyophilized TGFbeta3 in the different pH gels to yield a concentration of 125micro-micro-g/mL. These gels were degassed then absorbance was measured after 1 hour. The absorbance at 350nm of TGFbeta3 gel formulation at pH 3.7 was equal to the gel vehicle alone. This formulation was observed to be clear and not distinguishable from gel vehicle. The TGFbeta3 gel formulation at pH 3.8 had an absorbance of 0.014 AU at 350nm compared to the gel vehicle blank. This formulation was very slightly cloudy and just discemable as different from the gel vehicle blank. The TGFbeta3 gel formulation at pH 3.9 had an absorbance of 0.046 AU at 350nm compared to the gel vehicle. Cloudiness of this formulation was visible to the eye. These results are shown graphically in Figure 1.

These experiments demonstrate that controlling the pH of solution formulations of TGFbeta3 is critical for maintaining stability of the protein. Aggregation was observed when the pH is above 3.7. Over the pH range tested, a pH of 3.7 or below 3.7 is necessary to minimize aggregation.

EXAMPLE 2. Manufacture and testing of TG Fbeta3 pharmaceutical formulations, prototypes G, H, I and J

Four different hypromellose gel formulations of TGFbeta3 were manufactured with the aim of identifying potential stable pharmaceutical formulations of the protein. All candidate formulations had a pH within the range of 3.3-3.7. Hypromellose gel vehicles were manufactured using the "hot-cold" method. Gels were packed in bulk then sterilized by autoclaving at 121⁰C for 15 minutes. TGFbeta3 was added to the sterile gels to produce gel formulations containing 125micro-micro-g/mL TGFbeta3 which were then packed into 10mL glass vials.

The four gel vehicles were prepared as follows. Gel vehicle for Prototype G was manufactured as follows. Glycine buffer was prepared by combining 5Og mannitol, 1.5g glycine, 1g benzoic acid, 55Og purified water and 1.6mL of 10% hydrochloric acid solution. This solution was chilled to approximately 4⁰C. 19g Hypromellose 4000 was dispersed in 382g hot purified water in a 1 L beaker. The cold buffer was added to the hot slurry of hypromellose while stirring to form the gel. Gel vehicle for Prototype H was manufactured as follows. Acetic acid buffer was prepared by combining 4Og mannitol, 71Og purified water and 2.286mL glacial acetic acid and adjusting to pH 3.5 with sodium hydroxide. This solution was chilled to approximately 4⁰C. 19g Hypromellose 4000 was dispersed in 229g of hot purified water in a 1 L beaker. The cold buffer was added to the hot slurry of hypromellose while stirring to form the gel. Gel vehicle for Prototype I was manufactured as follows. Glycine buffer was prepared by combining 40g mannitol, 1.5g glycine, 0.7g methyl paraben, 0.3g propyl paraben, 708g purified water and 1.6mL of 10% hydrochloric acid solution. This solution was chilled to approximately 4⁰C. 19g Hypromellose 4000 was dispersed in 229g hot purified water in a 1 L beaker. The cold buffer was added to the hot slurry of hypromellose while stirring to form the gel.

Gel vehicle for Prototype J was manufactured as follows. Glycine buffer was prepared by combining 40g mannitol, 1.5g glycine, 15mL benzyl alcohol, 695g purified water and

1.6mL of 10% hydrochloric acid solution. This solution was chilled to approximately 4⁰C. 18g Hypromellose 4000 was dispersed in 229g hot purified water in a 1 L beaker. The cold buffer was added to the hot slurry of hypromellose while stirring to form the gel.

For each of the gel vehicles, Prototypes G, H, I and J, 10OmL glass vials were filled with 8Og gel per vial then sealed using bungs and crimp seals. Vials were sterilized by autoclaving at 121⁰C for 15 minutes.

TGFbeta3 gel formulations were prepared from each of the gel vehicles, Prototypes G, H, I and J, by weighing approximately 47OmL of gel into a 25OmL bottle. TGFbeta3 drug substance solution was added to produce a final TGFbeta3 concentration of 125micro-micro- g/mL The solution was mixed by slowly rotating the bottle for 30 minutes. The gel was packed into 1OmL glass vials (approximately 5g per vial). Vials were stored at 2-8⁰C, protected from light.

The TGFbeta3 gel formulations were assessed for appearance (clarity, colour and homogeneous consistency, by visual inspection), pH (by potentiometry), viscosity (using a Brookfield Dial Viscometer), osmolality (by freezing point depression using a Hermann Roebling Automatic Micro-Osmometer), presence of TGFbeta3 covalent aggregates and monomer (using reversed phase HPLC to desalt samples followed by polyacrylimide gel electrophoresis, as described in detail below), TGFbeta3 concentration (by HPLC on a Vydac C4 3OθA, 5micro-micro-m, 4.6 x 250mm reversed phase column (Grace Vydac, Hesperia, CA, USA), at 4O⁰C using a 20-80% acetonitrile gradient with 0.08% trifluoroacetic acid over 14 minutes at 1ml_/minute and UV detection at 215nm) and TGFbeta3 bioactivity (based on inhibition of mink lung epithelial cell growth, according to the method of Absher M, Baldor L, Kelley J. A rapid colorimetric bioassay for transforming growth factor beta (TGFbeta) using a microwell plate reader. J Immunol Methods 1991;138(2):301-1).

Gel formulations were assayed for TGFbeta3 covalent aggregates and monomer as follows. Gel samples were desalted by HPLC on a Vydac C4 5micro-micro-m 3OθA, 4.6 x 250mm reversed phase column (Grace Vydac, Hesperia, CA, USA), at 4O⁰C using a 35- 100% acetonitrile gradient with 0.08% trifluoroacetic acid over 14 minutes at 1mL/minute. Gel formulations were diluted 1 part in 6 in 0.1% trifluoroacetic acid then a diluted gel sample containing 10micro-micro-g of TGFbeta3 was loaded and the 2.7mL eluate fraction from 6.9- 9.6 minutes was collected. 10mg mannitol was added to stabilize the protein during lyophilization. This solution was dried to a white pellet under vacuum at room temperature. The pellet was reconstituted in 40micro-micro-L of 1x NuPAGE® LDS Sample Buffer. 2micro-micro-L of the reconstituted sample was withdrawn and diluted with 30micro-micro-L of 1x Sample Buffer to allow protein content in the sample to be determined from a standard curve. The remaining 38micro-micro-L (nominal 9.5micro-micro-g) of the test sample and 32micro-micro-L (nominal 0.5micro-micro-g) of the diluted test sample were loaded onto a 10 x 1mm well NuPAGE® 4-12% Bis-Tris Pre-Cast gel (Invitrogen, San Diego, CA, USA). 10micro-micro-g Reference Standard was prepared in an identical manner to the test sample by dilution in 1/6 gel then desalting as described above. The reconstituted Reference Standard was then loaded on to the gel at nominal loads of 9.5micro-g and 0.5micro-g. These reference samples were used to determine the formation of aggregates and monomer in Reference Standard due to the desalting procedure. Undesalted Reference Standard samples were also prepared in Sample Buffer and loaded on to the gel at 0.1 , 0.3, 0.5 and 0.8micro-g to allow quantitation of bands from a standard curve over this range. All test and reference samples were heated for 10 minutes at 7O⁰C before loading. One lane was used for SeeBlue® molecular weight markers (Invitrogen, San Diego, CA, USA). Gels were run in an Xcell II™ Mini-Cell with a BioRad PowerPac 300 at 200V for 35 minutes in NuPAGE® MES SDS running buffer (Invitrogen, San Diego, CA₁ USA). Gels were fixed for 15 minutes in 50% methanol, 7% acetic acid then rinsed (3 x 5 minute rinses in ultrapure water) before staining with GelCode® Blue Stain Reagent (Pierce, Rockford, IL, USA) overnight. Gels were destained for 2 hours using several changes of ultrapure water. Gels were analysed using a Kodak EDAS 120 Digital Camera with Kodak™ Digital Science 1 D Image Analysis v3.0 software.

Protein content of test sample bands was determined from band intensities compared to a standard curve on each gel derived from band intensities of Reference Standard samples over the range of 0.1-0.8micro-g. Aggregate content in test samples was corrected for the background content of aggregates in desalted Reference Standard by subtracting the impurity content in Reference Standard from the impurity content in test samples. Impurities are expressed as a percentage of the actual total protein content in the sample after HPLC desalting which is determined from quantitation of protein in the test sample loaded at a nominal 0.5micro-g against the standard curve. The quantitation limit for the assay was 1% (0.1micro-g) and detection limit was 0.5% (0.05micro-g)

Results of release testing of TGFbeta3 gel formulations G, H, I and J, are shown in Figure 2.

All four formulations were acceptable with respect to physical properties, chemical tests and biological activity. All had concentrations of TGFbeta3 within 10% of the nominal concentration. No aggregates were detected in any of the formulations. A small amount of monomer was detected in all formulations as a result of the presence of 0.5-1 % monomer in the drug substance. TGFbeta3 bioactivity recovered from all formulations had equivalent potency to the biological activity of NIBSC TGFbeta3 reference standard.

These candidate formulations were stored at 4⁰C and stability was monitored over 12 months.

EXAMPLE 3. Stability of TGFbeta3 gel formulations The stability of TGFbeta3 in gel formulations was monitored over 12 months when stored at 4⁰C. Formulations were assessed for appearance, pH, viscosity, osmolality, turbidity indicating presence of insoluble aggregates, presence of monomer and covalent aggregates of TGFbeta3, TGFbeta3 concentration and TGFbeta3 bioactivity after 6 and 12 months using the methods described above.

Results of testing TGFbeta3 gel formulations G₁ H, I and J after storage at 4⁰C for 6 and 12 months are shown in Figures 3, 4, 5 and 6.

This 12 month stability study of four candidate gel formulations of TGFbeta3 showed that all formulations were physically and chemically stable and retained biological activity throughout the storage period at 4⁰C. The concentration of TGFbeta3 remained within 10% of nominal; aggregates remained less than 2% and monomer remained less than 1% for all formulations (except the 6 month test of prototype I that was 3% but when tested at 12 months was <0.5%). The TGFbeta3 biological activity of all four formulations remained equivalent to the bioactivity of NIBSC reference standard at all testing time points throughout the 12 month stability study.

EXAMPLE 4. The influence of pH on gel viscosity following heat sterilization

The effect of heat sterilization on hypromellose gel formulations was examined to investigate the potential for preparing a preservative-free unit dose hypromellose gel-based product.

The stability of hypromellose gels to steam sterilization was examined. Samples of hypromellose gel at pH 3.0, 3.5 and 5.3 were prepared using the "hot-cold" method. Glycine buffer was prepared by combining 3.5g mannitol, 0.15g glycine and 7Og purified water. 10% hydrochloric acid solution was added dropwise to pH 3.0 or to pH 3.5. Hydrochloric acid was not added to the buffer for the pH 5.3 gel. The buffer solution was chilled to approximately 4⁰C. 1.9g Hypromellose 4000 was dispersed in 23g hot purified water in a 1 L beaker. The cold buffer was added to the hot slurry of hypromellose while stirring to form the gel. 1OmL glass vials were filled with 9g gel per vial then sealed using bungs and crimp seals. Vials were sterilized by autoclaving at 121⁰C for 15 minutes in a Labec autoclave (Marrickville, NSW, Australia) using the British Pharmacopoeia method for steam sterilization (British Pharmacopoeia, 2004).

There was minimal loss of viscosity seen in pH 5.3 gel. There was a marked decrease in viscosity of pH 3.5 gel to 38% of the pre-autoclaved viscosity and an even greater loss of viscosity of the pH 3.0 gel to 28% of the pre-autoclaved viscosity. These results are illustrated in Figure 7. We then prepared hypromellose gels over a range of pHs to define the pH range that maintained gel viscosities within an acceptable range for the product after autoclaving (1100 ± 300mPa.s).

Samples of hypromellose gel over the pH range of 3.2-5.9 were prepared using the "hot-cold" method. Glycine buffer was prepared by combining 3.5g mannitol, 0.15g glycine and 7Og purified water. 10% hydrochloric acid solution was added dropwise to reduce the pH for pH 3.2-4.8 gels. No pH adjustment was required for the pH 5.4-5.9 gels. The buffer solution was chilled to approximately 4⁰C. 1.8g Hypromellose 4000 was dispersed in 23g hot purified water in a 1 L beaker. The cold buffer was added to the hot slurry of hypromellose while stirring to form the gel. 1OmL glass vials were filled with 9g gel per vial then sealed using bungs and crimp seals. Vials were sterilized by autoclaving at 121⁰C for 15 minutes in a Thackray Hart autoclave (Thackray Hart, Canning Vale, WA, Australia) using the British

Pharmacopoeia method for steam sterilization (British Pharmacopoeia, 2004).

On the basis of these results (Figure 8), the pH to produce a product with appropriate viscosity must be at or above pH 3.2 and preferably at or above pH 3.3.

EXAMPLE 5. Release of TGFbeta from hvpromellose.

The following experiment was performed to show that TGF-beta is released from a hypromellose gel formulation.

TGF-beta3 20micro-g/mL gel was prepared by mixing TGF-beta3 in hypromellose gel (hypromellose 1.8%, mannitol 3.5%, glycine 0.15%, hydrochloric acid to pH 3.5, in water; autoclaved at 121⁰C for 15 minutes; viscosity 870mPa.s).

2.5mL TGF-beta3 gel was placed in a 30mm Millicell® insert with 0.4micro-m hydrophilic polycarbonate membrane (Millipore, Billerica, MA, USA). The Millicell® donor well was suspended in 6mL of solution containing 5% mannitol (for isotonicity with the donor phase), 10% hypromellose gel (as a carrier to reduce adsorptive losses of TGF-beta3) in 0.1% acetic acid in a receiver cell (6-well Millcell® culture plate). The receiver phase was stirred continuously for 18 hours then the unit was disassembled and TGF-beta3 content was measured in the donor phase, receiver phase and in 1 mL wash solution of 25% acetonitrile, 0.08% trifluoroacetic acid in water used to recover TGF-beta3 from the membrane. TGF- beta3 concentration was measured using HPLC on a Vydac C4 300A, 5micro-m, 4.6 x 250mm reversed phase column, at 40⁰C using a 20-80% acetonitrile gradient with 0.08% trifluoroacetic acid over 14 minutes at 1mL/minute and UV detection at 215nm. 54% of the initial TGF-beta3 loaded into the donor cell remained in the donor phase after 18 hours. 14% was present in the receiver phase and 15% was recovered from the membrane. There was an apparent loss of 17% of the initial amount of TGF-beta3 loaded into the donor cell that was likely to be due to adsorption onto the polystyrene surfaces of the device.

This experiment demonstrates that TGF-beta3 is passively released from hypromellose gel in vitro and suggests that it would be bioavailable when administered in vivo.

EXAMPLE 6. Hvpromellose reduces the adherence of TGFbeta to surfaces

Adsorption of proteins onto glass and plastics is a well known phenomenon that can result in protein losses during manufacture, storage and analysis of protein pharmaceuticals (Gibson M. Product optimization. In: Pharmaceutical preformulation and formulation. A practical guide from candidate drug selection to commercial dosage form. Gibson M, Ed. Boca Ratan: CRC Press 2001 , pp. 296-303).

This can pose significant challenges for the formulation of proteins at low concentrations. Low adhesion materials such as silicon or Teflon can be used to minimize losses from protein solutions.

We compared the recovery of TGFbeta3 from a 2.5micro-g/mL solution prepared in hypromellose gel (containing 0.4% hypromellose 4000 in 0.08% trifluoroacetic acid in water) with a 2.5micro-g/mL solution prepared in 0.1% trifluoroacetic acid in water. Both solutions were prepared in silicon-coated glass vials then transferred to polypropylene HPLC vials for analysis. TGFbeta3 recovery was determined by HPLC on a Vydac C4 3OθA, 5micro-m, 4.6 x 250mm reversed phase column (Grace Vydac, Hesperia, CA, USA), at 4O⁰C using a 20- 80% acetonitrile gradient with 0.08% trifluoroacetic acid over 14 minutes at 1 mL/minute and UV detection at 215nm. Twice the amount of TGFbeta3 was recovered from the solution containing hypromellose gel compared to the non-gel containing sample.

This experiment showed that hypromellose gel reduced TGF-beta3 loss from a 2.5micro-g/mL solution that was prepared in a silicon-coated glass vial and transferred to a polypropylene vial for assay. These experiments show that the presence of hypromellose gel reduces adsorptive losses of TGFbeta3 onto silicon-coated glass. We further investigated the benefit of hypromellose gel in reducing loss of TGFbeta3 onto polypropylene. A stock solution of 6.25micro-g/mL TGFbeta3 in 0.1% trifluoroacetic acid in water (8mL) was prepared in a silicon-coated glass vial. Two 0.5mL aliquots were diluted to 1.25micro-g/mL with 0.1% trifluoroacetic acid in water in silicon-coated glass vials; these samples were retained as reference samples for assay. The remaining stock solution was transferred to a 10mL polypropylene tube and vortex mixed briefly. The transfer and vortex mixing of the solution was repeated a further 9 times using a new polypropylene tube on each occasion. A second set of samples was prepared as follows. A stock solution of 6.25micro-g/mL TGFbeta3 in hypromellose gel containing 0.4% hypromellose 4000 and 0.1% trifluoroacetic acid in water (8mL) was prepared in a silicon-coated glass vial. Two 0.5ml_ aliquots were diluted to a concentration of 1.25micro-g/mL with hypromellose gel containing 0.4% hypromellose 4000 in 0.1% trifluoroacetic acid in water. These samples were prepared in silicon-coated glass vials and were retained as reference samples for assay. The remaining stock solution was transferred to a 1OmL polypropylene tube and vortex-mixed briefly. The transfer and vortex mixing of the solution was repeated a further 9 times using a new polypropylene tube on each occasion. Following transfer to multiple polypropylene tubes, there was no detectable TGFbeta3 in the solution prepared in 0.1% trifluoroacetic acid. In contrast, in two independent samples prepared with hypromellose gel, 87% and 101% of the TGFbeta3 present in the initial dilution was recovered in the samples following 10 transfers to polypropylene tubes.

These experiments show that the presence of hypromellose gel reduces adsorptive losses of TGFbeta3 onto polypropylene.

EXAMPLE 7. Recombinant human TGFbetal . TGFbeta2 and TGFbeta3 increases GM-CSF production in cultured human cervical keratinocvtes

Previous studies have implicated the surge in epithelial GM-CSF release as a key mediator in the post-mating inflammatory response since injection of recombinant GM-CSF into the estrous uterus of mice is sufficient to produce cellular changes resembling those seen following natural mating (Robertson eif a/ In: Serono Symposium on the Immunobiology of Reproduction 1994 Eds. Hunt & Burnett). International Patent Application No. PCT/AU98/00149 (Robertson) describes a method of eliciting tolerance towards male antigens through the induction of GM-CSF synthesis in uterine epithelial cells by the administration of TGFbeta to the female reproductive tract together with sperm or semen.

Sharkey (Seminal plasma regulation of the post-coital inflammatory response in the human cervix. Ph.D. Thesis, The University of Adelaide, 2005) studied the effect of TGFbeta on GM-CSF production by cells from the female human reproductive tract in vitro. A summary of this study (Example 7) follows.

Human cervical keratinocvtes were cultured using a modification of the technique described by Rheinwald and Green (Cell 1975;6:331-344). Cervical biopsies were obtained from pre-menopausal women undergoing hysterectomy for non-malignant gynaecological indications. No distinction was made regarding stage of menstrual cycle at the time of surgery. The cervical biopsies were placed in ice-cold HBSS for transport to the laboratory, washed twice in antibiotic containing medium then incubated overnight at 4⁰C in DMEM containing 5U dispase (Boehringer Mannheim). Following a 1h incubation at room temperature, sheets of keratinocytes were stripped from the biopsy using sterile forceps. The tissue was disaggregated into single cells by 30 minutes incubation at 37⁰C in DMEM/0.25% trypsin/0.05% collagenase with repeated aspiration using a needle and syringe. Keratinocytes were cultured in ECM-FCS, at a density of 1-2 x 10⁵ cells/ml, over monolayers of murine 3T3 fibroblasts rendered mitogenically inactive by exposure to 4% mitomycin C (Sigma). Keratinocytes were incubated for 5-7 days to enable attachment and displacement of the 3T3 fibroblasts before the media was replaced with fresh ECM-FCS. The supernatant was aspirated 12 hours later (basal sample) then the media was replaced with 50OuI of ECM-FCS containing 10ng of rTGFbetal , rTGFbeta2 and rTGFbeta3 or culture medium only (control). Supernatant was collected 12 hours later and assayed for GM-CSF activity and then replaced with 500micro-l of fresh ECM-FCS. A third sample of supernatant was collected 12 hours later, or 24 hours after treatment with TGFbeta. The GM-CSF content of the basal supernatant was subtracted from the GM-CSF content determined in supernatant samples collected at 12h and 24h after TGFbeta treatment. GM-CSF in the supernatant samples was assayed using a commercially available GM-

CSF specific Enzyme-Linked Immunosorbent Assay (ELISA) (R&D Systems) according to the manufacturer's instructions. The minimum detectable GM-CSF concentration was 1 U/ml and the EC50 was 50U/ml. The assay was standardised against recombinant GM-CSF and the specificity of the assay was confirmed by the use of a GM-CSF-specific neutralising antibody.

This experiment demonstrated that rTGFbetal , rTGFbeta2 and rTGFbeta3 added to cultured human cervical keratinocytes in vitro increased GM-CSF production 12 and 24 hours after treatment (data published in thesis). The ability of TGFbeta to induce GM-CSF production in human cervical keratinocytes shows that TGFbeta can elicit an inflammatory response in cells from the human reproductive tract of the type observed after exposure to semen.

EXAMPLE 8. Effect of TGFbeta3 on spontaneous fetal resorption in a mouse model

CBA/J female mice mated with DBA/2 males (CBA/J x DBA/2) was first proposed as a model for early spontaneous abortion by Clark et al (Cell Immunol 1980;52: 106-118) and has subsequently been established as the most compelling model of immunologically mediated spontaneous fetal loss.

In this example, rTGFbeta3 was administered vaginally to DBA/2-mated CBA/J female mice to test the ability of TGFbeta3 to reduce the high rates of miscarriage normally observed in this model, by inducing a tolerogenic maternal immune response. A single dose of vehicle (10ul of 0.1% BSA in PBS at pH 7.4) or TGFbeta3 (2ng in 10ul of 0.1 % BSA in

PBS at pH 7.4) was administered either before or after mating. Following mating, the female mice were treated with an intra-peritoneal dose of bacterial lipopolysaccharide (LPS) (1ug in 10OuI) to increase the background recurrent miscarriage rate. 13.5 days after mating the animals were killed, and the uteri were removed for assessment of (i) the number of mice with embryos (the pregnancy rate), and (ii) the number of implantations and the number of resorptions (to calculate the resorption rate).

In the vehicle-treated control group, 15 animals were mated; of these 11 animals became pregnant, carrying a total of 97 implantations: 41% of these implantations were nonviable (resorbing). In the TGFbeta3 treated group 14 animals were mated; all animals became pregnant, producing 115 implantations, of which 26% were resorbing. These results are summarized in Figure 9.

TGFbeta3 reduced the resorption rate in this CBA/J x DBA/2 mouse model of recurrent miscarriage. The result was highly statistically significant, by the Chi-squared test (with Yates' correction).

This result shows that a single dose of TGFbeta3 administered to female mice lacking immune tolerance to paternal antigen, either before or after conception, prevents miscarriage and has no detrimental effect on pregnancy.

EXAMPLE 9. Manufacture and testing of TGFbeta3 pharmaceutical formulations, prototypes K, L, M, N, O. P. Q

Seven prototype formulations (K to Q) were manufactured then stored at two different temperature conditions and stability was monitored over a 6 month period.

Stability of the seven prototype formulations K to Q, was assessed using RP-HPLC, UV spectrophotometry, Mv1 Lu biological assay, microbiological assessment, and viscosity, osmolality, pH and preservative efficacy testing.

Manufacture of Prototype Formulations K to Q (refer to Figure 10).

Procedure for the Manufacture of Hypromellose/MC Gel Solvent Formulation (K, L and P) Hypromellose and methylcellulose gels were prepared using the Hot/Cold Technique sourced from "Methocel Cellulose Ethers" Technical Handbook, Dow Chemical Company September 2002. The citric acid and trisodium citrate were weighed and transferred to a 1 L beaker. For formulations K and L, the mannitol powder was weighed and added to the beaker (note that gel formulation P does not include mannitol). The required amount of cold MiIIiQ water was weighed directly into the beaker and the benzyl alcohol was added. The buffer solution was stirred until all ingredients dissolved and all droplets of benzyl alcohol had fully dispersed. The solution was then stored at 2-8°C for at least one hour. About 60OmL of MiIIiQ water was heated to >90°C. The Hypromellose or methylcellulose was weighed into a clean 1 L beaker. The required weight of hot MiIIiQ water was then added to the powder with thorough stirring until a smooth slurry resulted. The beaker containing the cold buffer and preservative was slowly poured into the slurry while stirring. After a few minutes of stirring all gelling agent had dissolved, forming a homogeneous gel. The gel was placed at 2-8°C overnight before adding the active ingredient. See Section 4 below.

Procedure for the Manufacture of Non-gel Liquid Formulation (M, N and O)

Citric acid, trisodium citrate and mannitol were weighed and added to a beaker. CaCI₂ was added to formulation vehicle N. The required amount of MiIIiQ water was weighed directly into the beaker and the benzyl alcohol was added. The buffer solution was stirred until all ingredients dissolved and all droplets of benzyl alcohol had fully dispersed. The final liquid formulation vehicle was cooled to 2-8°C before adding the active ingredient. See Section 4 below.

Procedure for the Manufacture of Poloxomer Gel Formulation Vehicle (Q) The citric acid, trisodium citrate and mannitol were weighed and transferred to a 1L beaker. The required amount of cold MiIIiQ water was weighed directly into the beaker and the benzyl alcohol was added. The buffer solution was stirred until all ingredients dissolved and all droplets of benzyl alcohol had fully dispersed. The beaker was then put in an ice bath. The poloxamer gel powder was weighed and slowly added to the cold buffer solution while stirring. The beaker was sealed and stirred overnight at room temperature. The vehicle was cooled to 2-8°C before adding the active ingredient. See Section 4 below.

Procedure for adding TGFbeta3 to the gel and non-gel vehicle formulations (K, L, M, N, O, P and Q)

The required amounts of vehicle (K to Q) were weighed into labelled 50OmL Teflon bottles. A solution of TGFbeta3 in 20% ethanol/20mM acetic or 20% ethanol/20mM acetic acid solution was added to each of the vehicle formulations to prepare active and placebo products for each test formulation. The bottles were capped and mixed for 60 minutes at ~16rpm. The final TGFbeta3 concentration in all seven active formulations was 20micro-g/ml_.

Filling procedure for prototype formulations K to Q

The gel/solution formulations were poured directly into 1OmL silicon-coated glass vials at a volume of 2mL per vial. The vials were then stored either at room temperature or 2-8⁰C.

Summary of tests performed at T = 0, 3 and 6 months Figure 11 lists the tests performed at release (T = 0) on Formulations K to Q. Figure 12 lists the tests performed at T = 3 months on Formulations K to Q. Figure 13 lists the tests performed at T = 6 months on Formulations K to Q.

Description of Test Methods

The following stability indicating test methods were used in the stability study.

Optical density at 350nm

Samples were analysed on a Cary 50 UVΛ/is spectrophotometer using disposable plastic cuvettes (Ocean Optics). Measurements were performed by adding approximately 1 ml_ of sample to a micro-cell plastic cuvette with a useable range of 220-900nm. For each formulation, absorbance at 350nm was measured for two separate samples and the results averaged. To detect aggregation, the absorbance of the placebo sample was subtracted from the absorbance of the active sample. This assay has been qualified to show that if the difference between the two is >0.006 nm, it is considered that aggregation has occurred.

Reversed Phase-HPLC TGFbeta3 concentration was determined by RP-HPLC on a Phenomenex Jupiter C₅, δmicro- m, 4.6 x 250mm reversed phase column, at 4O⁰C using a 20-80% acetonitrile gradient with 0.08% trifluoroacetic acid over 14 minutes at 1 mL/minute and UV detection at 215nm. The retention time for TGFbeta3 was approximately 10 minutes and the run time was 25 minutes.

Samples

Samples were prepared for analysis as outlined below.

Preparation of the gel formulations for RP-HPLC analysis (Formulations: K, L, P and Q; all test samples contained 20μg/mL TGFbeta3)

Test samples were first diluted 1 in 4 using the same buffer as the gel vehicle but without the gelling agent. Diluted samples were then centrifuged for 30 minutes at 50000gf to precipitate any insoluble aggregates. 1 mL of the 'supernatant' was mixed with 300micro-L of 80% acetonitrile/0.08% TFA (HPLC buffer B), then transferred into an HPLC vial for analysis. Replicate samples were analysed and compared to a TGFbeta3 reference standard. Preparation of the non-gel formulations for RP-HPLC analysis (Formulations: M, N and O; all test samples contained 20μg/mL TGFbeta3)

1.2mL of the test sample was transferred to a low adhesion tube then centrifuged at 5000Og for 30 minutes. 1mL of the 'supernatant' was mixed with 300micro-L of 80% acetonitrile/0.08% TFA (HPLC buffer B), and placed into an HPLC vial for analysis. Replicate samples were analysed and compared to a TGFbeta3 reference standard.

Bioburden testing

The samples were diluted (1/10) in TSTL. Both TSA and SDA pour-plates were prepared using 1mL aliquots of solution. The TSA plates were incubated at 30-35⁰C for 2 days and the SDA plates were incubated at 20-25⁰C for 5 days.

Viscosity testing The viscosity of gel-containing formulations (K, L, P and Q) was measured using a Brookfield Dial Viscometer RVT model. The viscometer was fitted with cone spindle #21 and rotation speed was set to 10rpm. The sample chamber temperature was set at 25⁰C ± 0.8⁰C. All samples were left for 30 minutes to equilibrate to the set temperature before measurement. Formulation Q contained poloxamer, a thermoreversible gelling agent. The test method used was not qualified for measuring viscosity of this formulation due to the thermosensitive nature of the gel and limitations of the instrument.

pH measurement

Samples were tested using a Metrohm pH meter.

Osmolality

Samples were analysed on a Hermann Roebling Automatic Micro Osmometer using a 290mOsmol/kg near serum osmolality reference solution. For each formulation, three separate samples were tested and the average of the three readings was reported.

Bioassay

Samples were analysed for TGFbeta bioactivity (based on inhibition of mink lung epithelial cell growth) according to the method of Asher M, Baldor L, Kelley J. A rapid colorimetric bioassay for transforming growth factor beta (TGFbeta) using a microwell plate reader. J Immunol Methods 1991 ;138 (2):301-1.

Preservative efficacy testing Preservative efficacy was assessed using a method adapted from the British Pharmacopoeia 2005 (Appendix XVIC). For each formulation, two independent samples were inoculated with micro-organisms, S. aureus and A. niger then counts were performed on the samples at 48h, 7d, 14d and 28d. The final counts were <10 per gram for all seven formulations tested.

Results and Discussion

Figures 14-25 provide summaries of the stability results obtained for prototype formulations K to Q over 6 months.

RP-HPLC results for Active and Placebo Formulations K to Q (Figure 14)

After 6 months storage at 2-8⁰C, the TGFbeta3 concentration in all formulations with a pH of 3.4 was 70-88% of the initial (T=O) concentration. There was no difference between stability of TGFbeta3 in formulations containing CaCI₂ and those without CaCI₂.

Formulations at pH 5.2 with or without Hypromellose showed poor recovery (<0.1 %) at T=O (and also after 6 months storage at either room temperature or 2-8⁰C).

Formulation P containing methylcellulose, which is closely related in structure to Hypromellose, showed slightly better stability over the 6 months than the equivalent Hypromellose gel (formulation K).

The highest recovery of TGFbeta3 was observed in Formulation Q samples (pH 3.4 poloxamer gel) stored at 2-8⁰C; 88% of the TGFbeta3 remained soluble after 6 months.

Samples stored at room temperature showed lower recovery of soluble TGFbeta3 compared with samples stored at 2-8⁰C for all formulations. Hence product should be refrigerated at 2- 8°C for optimal long term stability of TGFbeta3 in liquid or gel formulations.

UV absorbance results for Formulations K to Q (Figures 15 and 16)

Only formulations L and O that had a pH of 5.2 showed significant changes in the optical density, indicating aggregation of TGFbeta3. This aggregation occurred immediately after the formulations were prepared then remained constant during storage for 6 months at either room temperature or 2-8⁰C. All other formulations showed no significant change in optical density after 6 months at the preferred storage temperature of 2-8⁰C. The loss of soluble TGFbeta3 from formulations with a pH of 5.2 was shown to be due to-formation of insoluble aggregates using this UV absorbance assay.

Bioassay results for Prototype Formulations K to Q (Figure 17)

All formulations at pH 3.4 showed full biological activity in the MvILu growth inhibition assay, even in the absence of CaCI₂. In contrast, there was significant loss of TGFbeta3 bioactivity in Formulations L and O (pH 5.2) correlating with formation of insoluble aggregates of TGFbeta3 at pH 5.2.

Bioburden results for Active and Placebo Formulations K to Q (Figure 18) All samples were free from microbial contamination after 6 months storage at either 2-8⁰C or RT.

Preservative efficacy testing of Placebo Formulations K to Q (Figure 19)

Benzyl alcohol 2% was shown to be an effective preservative in all candidate formulations.

Viscosity testing results for Formulations K to Q (Figures 20 and 21) There was no significant change in viscosity of Formulations K, L or P (containing Hypromellose or MC) after 6 month storage at RT or 2-8°C. Formulation Q was a non- viscous liquid at 25°C and was a pourable gel at 37°C. A viscosity reading could not be obtained for this formulation because of the thermosensitive nature of the gel and limitations of the instrument.

Osmolality results for Formulations K to Q (Figures 22 and 23)

There was no change in osmolality for any of the formulations after storage for 6 months at

RT or 2-8°C.

pH results for Formulations K to Q (Figures 24 and 25)

The pH of all samples remained unchanged after storage for 6 months at RT or 2-8°C.

Summary and Conclusion

Formulations L and O (at pH 5.2) had poor stability with almost no detectable soluble TGFbeta3 (by RP-HPLC) and high levels of insoluble aggregates (indicated by the high absorbance at 350nm) at T=O and subsequent 3 and 6 month testing. Bioactivity (by MvILu bioassay) was outside the in-house product specification after 6 months storage at either room temperature or 2-8°C for formulation L and formulation O exhibited minimal bioactivity at T=O. Formulations K, M, N, P and Q (at pH 3.4) had superior stability with recoveries (by RP-HPLC) of greater than 70%, undetectable insoluble aggregates (by absorbance at 350nm) and acceptable bioactivity (by Mv1 Lu bioassay) after storage at 2-8°C for 6 months. This study demonstrates that gel formulations of TGFbeta3 at pH 5.2 are unstable but formulations manufactured using poloxamer or cellulose-based gelling agents at pH 3.4 have suitable long-term stability for pharmaceutical use without the addition of CaCI₂.

EXAMPLE 10. Additional formulations included within the scope of the invention

Formulation 1

TGFbeta3 20micro-g/mL, mannitol (4% w/w), hypromellose 4000 (1.9% w/w), glycine (0.15% w/w), hydrochloric acid (to pH 3.2), purified water (to 100% w/w)

Formulation 2

TGFbeta3 20micro-g/mL, mannitol (4% w/w), hypromellose 4000 (1.9% w/w), glycine (0.15% w/w), hydrochloric acid (to pH 3.7), purified water (to 100% w/w)

Formulation 3

TGFbeta3 20micro-g/mL, glucose (4% w/w), hypromellose 4000 (1.9% w/w), glycine (0.15% w/w), hydrochloric acid (to pH 3.5), purified water (to 100% w/w)

Formulation 4

TGFbeta3 20micro-g/mL, mannitol (4% w/w), hypromellose 4000 (1.9% w/w), lactic acid (0.2% v/w), sodium hydroxide (to pH 3.5), purified water (to 100% w/w)

Formulation 5 TGFbeta3 20micro-g/mL, mannitol (4% w/w), methylcellulose 4000 (1.9% w/w), glycine (0.15% w/w), hydrochloric acid (to pH 3.5), purified water (to 100% w/w)

Formulation 6

TGFbeta3 20micro-g/mL, mannitol (4% w/w), hydroxyethyl cellulose 4400 (1.8% w/w), glycine (0.15% w/w), hydrochloric acid (to pH 3.5), purified water (to 100% w/w)

Formulation 7 TGFbeta3 20micro-g/mL, mannitol (4% w/w), hydroxypropyl cellulose 4000 (1.9% w/w), glycine (0.15% w/w), hydrochloric acid (to pH 3.5), purified water (to 100% w/w)

Formulation 8 TGFbeta3 20micro-g/mL, mannitol (4% w/w), carmellose sodium, high viscosity grade

(1.5% w/w), glycine (0.15% w/w), hydrochloric acid (to pH 3.5), purified water (to 100% w/w)

Formulation 9

TGFbeta3 20micro-g/mL, mannitol (4% w/w), tragacanth (2% w/w), glycine (0.15% w/w), hydrochloric acid (to pH 3.5), purified water (to 100% w/w)

Formulation 10

TGFbeta3 20micro-g/ml_, mannitol (4% w/w), carbomer 940 (0.5% w/w), glycine (0.15% w/w), hydrochloric acid (to pH 3.5), purified water (to 100% w/w)

Formulation 11

TGFbeta3 20micro-g/mL, mannitol (4% w/w), poloxamer 407 (25% w/w), glycine (0.15% w/w), hydrochloric acid (to pH 3.5), purified water (to 100% w/w)

It will be apparent to the persons skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

Claims

CLAIMS:

1. A pharmaceutical composition comprising a TGFbeta, wherein the pH of the composition is at or below 3.7

2. A pharmaceutical composition according to claim 1 , wherein the pH of the composition is between 3.2 and 3.7.

3. A pharmaceutical composition according to claims 1 or 2, wherein the pH of the composition is between 3.3 and 3.7.

4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the composition is in a liquid form.

5. A pharmaceutical composition according to any one of claims 1 to 4, wherein the composition does not require reconstitution immediately prior to use.

6. A pharmaceutical composition according to any one of claims 1 to 5, wherein the composition is a ready to administer composition.

7. A pharmaceutical composition according to any one of claims 1 to 6, wherein the composition further comprises a gel polymer.

8. A pharmaceutical composition according to claim 7, wherein the gel polymer is selected from the group consisting of cellulose based polymers, tragacanth polymers, xanthan gum polymers, acacia polymers, carbomer polymers, gelatin polymers, sodium alginate polymers, poloxomer polymers, polyethylene oxide polymers, polyacrylamide polymers, and polyethylene glycol polymers.

9. A pharmaceutical composition according to claims 7 or 8, wherein the gel polymer is a cellulose-based polymer.

10. A pharmaceutical composition according to any one of claims 8 to 9, wherein the cellulose-based polymer is selected from the group consisting of hypromellose, methylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxyethylcellulose and carboxymethylcellulose.

11. A pharmaceutical composition according to any one of claims 8 to 10, wherein the cellulose-based polymer is hypromellose.

12. A pharmaceutical composition according to any one of claims 1 to 11, wherein the composition is void of calcium chloride or calcium phosphate.

13. A pharmaceutical composition according to any one of claims 1 to 12, wherein the composition is void of calcium chloride, calcium phosphate, potassium acetate, lithium acetate, ammonium acetate or ammonium bicarbonate.

14. A pharmaceutical composition according to any one of claims 1 to 13, wherein the composition is void of calcium chloride, calcium phosphate, potassium acetate, lithium acetate, sodium acetate, ammonium acetate or ammonium bicarbonate.

15. A pharmaceutical composition according to any one of claims 1 to 14, wherein the TGFbeta is selected from the group consisting of human TGFbetal, TGFbeta2 and TGFbeta3.

16. A pharmaceutical composition according to any one of claims 1 to 15, wherein the TGFbeta is human TGFbeta3.

17. A pharmaceutical composition according to any one of claims 1 to 16, wherein the TGFbeta maintains stability for at least 6 months when stored at between 2 and 27⁰C.

18. A pharmaceutical composition according to any one of claims 1 to 17, wherein the TGFbeta maintains stability for at least 6 months when stored at between 2 and 8⁰C.

19. A pharmaceutical composition according to any one of claims 1 to 18, wherein the concentration of TGFbeta is between 0.01micro-g/ml and 100micro-g/ml.

20. A pharmaceutical composition according to any one of claims 1 to 19, wherein the concentration of TGFbeta is between 0.2micro-g/ml and 20micro-g/ml.

21. A pharmaceutical composition according to any one of claims 1 to 20, wherein the concentration of TGFbeta is between 0.2micro-g/ml and 2micro-g/ml.

22. A pharmaceutical composition according to any one of claims 1 to 21 , wherein the concentration of TGFbeta is 0.2micro-g/ml.

23. A pharmaceutical composition according to any one of claims 1 to 22, adapted for intra-vaginal administration.

24. A pharmaceutical composition according to any one of claims 1 to 23, wherein the composition has:

(1) not more than a total of 10² micro-organisms (aerobic bacteria or fungi) per gram, as tested using standard pour-plate methods described in Appendix XVI B2 of the British

Pharmacopoeia 2001 ;

(2) not more than 10¹ enterobacteria and certain other Gram-negative bacteria per gram, as tested using standard pour-plate methods described in Appendix XVI B2 of the British Pharmacopoeia 2001 ; (3) absence of Pseudomonas aeruginosa, determined on 1g, as tested using standard pour- plate methods described in Appendix XVI B2 of the British Pharmacopoeia 2001 ; and (4) absence of Staphylococcus aureus, determined on 1g, as tested using standard pour- plate methods described in Appendix XVI B2 of the British Pharmacopoeia 2001.

25. An article of manufacture comprising,

(1) a vial, cartridge or vaginal applicator; and

(2) a pharmaceutical composition according to any one of claims 1 to 24

26. A process for preparing an article of manufacture comprising sealing a pharmaceutical composition according to any one of claims 1 to 24 in a vial, cartridge or vaginal applicator, from which a therapeutically effective dose of pharmaceutical composition can be administered to a patient in need thereof.

27. A method of treating an infertility condition, said method comprising administering to a prospective mother a pharmaceutical composition according to any one of claims 1 to 24.

28. A method of treating an infertility condition, by inducing immune tolerance to a paternal antigen in a prospective mammalian mother lacking said immune tolerance, said method comprising exposing a mucosal surface of said prospective mother to; (1) a pharmaceutical composition according to any one of claims 1 to 24; and

(2) semen or an MHC Class I antigen of a prospective father capable of eliciting a Th-1 response.

29. A method of reducing the adherence of TGFbeta to the surface of a container, said method comprising the addition of a gel polymer to a composition comprising TGFbeta.

30. A method of characterising TGFbeta in a composition, said method comprising evaluating the TGFbeta by means of an analytical method, where the composition comprising TGFbeta also comprises a gel polymer.

31. An article of manufacture comprising,

(1) a first vial or cartridge comprising a liquid composition comprising a buffering agent in an amount sufficient to maintain the pH of the composition at or below 3.7 and a gel polymer; and

(2) a second vial or cartridge comprising a dry powder composition comprising a freeze-dried TGFbeta, together with instructions that the liquid composition is added to the dry powder composition prior to use.