WO1995002411A1

WO1995002411A1 - Stabilised polypeptide growth factor formulation at low ph

Info

Publication number: WO1995002411A1
Application number: PCT/AU1994/000391
Authority: WO
Inventors: Stanley Shepherd; Robert Moore
Original assignee: Mallinckrodt Veterinary Limited
Priority date: 1993-07-16
Filing date: 1994-07-14
Publication date: 1995-01-26

Abstract

The invention provides formulations comprising a polypeptide growth factor and a pharmaceutically-acceptable carrier, which provide long-term biochemical stability of the growth factor at 4 °C. Preferably the growth factor is epidermal growth factor.

Description

STABILISEDPOLYPEPTIDEGROWTHFACTORFORMULATIONATLOWPH

This invention relates to pharmaceutical compositions suitable for administration to humans and animals, and in particular to formulations of biologically- active proteins, which compositions have superior stability and shelf life. The compositions of the invention are particularly suitable for use with polypeptide growth factors, and in a preferred embodiment, the invention provides stable compositions of epidermal growth factor.

Background of the Invention

Many polypeptides and proteins are useful as pharmaceutical agents, growth stimulators, etc., in human and veterinary medicine. They are administered by a variety of routes, depending upon whether a local or a systemic effect is desired. Because in many cases proteins or polypeptides are susceptible to digestion in the stomach, parenteral administration is frequently necessary, although in some situations topical administration is necessary or advantageous. Thus proteins or polypeptides for administration to man or animals are typically delivered by parenteral injection. In order to minimise trauma at the injection site and to avoid the persistence of residues, aqueous solutions or suspensions are preferred. However, aqueous solutions of protein tend to be unstable, with the proteins deteriorating more or less rapidly as a result of factors such as denaturation, chemical change or the activity of traces of contaminant proteolytic enzymes.

These problems can be avoided by distributing the protein as a dry powder or lyophilizate, which is reconstituted with a suitable diluent prior to use. In some cases, however, this procedure is inconvenient to the user or commercially impracticable for cost reasons. In some cases, delivery of proteins is most effective if sustained over a period of time or if given as a series of pulsed releases. Considerable research effort is devoted to achieving sustained or pulsed release from a single administration.

Formulations of biologically-active proteins will only be practicable and commercially attractive if they meet certain basic criteria:

1. The formulation must preserve the biological activity of the protein.

2. The formulation must present the protein to the recipient in a manner which achieves the desired effect.

3. The formulation must not elicit injection site reactions or other side effects which are unacceptable in degree.

4. The formulation must exhibit sufficient stability, in terms of the physical and chemical properties of its components and of the formulation as a whole, to permit practical use and meet criteria for regulatory approval.

Instability of proteins is a familiar problem in protein chemistry. One of the standard texts (Methods in

Enzymology Vol. 182 Guide to Protein Purification ed. M.P. Deutscher Academic Press 1990) lists the typical causes of loss of protein activity and alteration of structure as: dilution, change in solution conditions, degradative enzymes, oxygen, heavy metals, freezing and thawing, and inactivation at surfaces, (see Ch. 8 Maintaining Protein Stability, Page 84) .

Polypeptide growth factors are polypeptides of relatively low molecular weight, which have the ability to regulate the growth of normal cells. Many such factors of human and animal origin have been identified, and their amino acid sequences have been determined. A number of factors have also been cloned, using recombinant DNA technology. Growth factors within the scope of this invention include, but are not limited to epidermal growth factor (EGF), nerve growth factor (NGF) , acidic and basic fibroblast growth factor (FGF) , transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF) , insulin-like growth factors (IFG-I and IFG-II), somatomedin (SM-A, SM-B, SM-C) , vaccinia growth factor and angiogenin.

The invention is particularly described by reference to epidermal growth factor. Human EGF is a polypeptide of 53 amino acids, which has mitogenic activity for a number of cell types, including cells of epithelial and mesenchymal origin. Naturally-occurring variants of EGF have been described, such as γ-urogastrone, which has 52 amino acids, and muteins and chemically-modified derivatives of EGF. In addition to its growth-promoting activity, EGF has been described as having angiogenic activity, and the ability to promote epidermal growth and to inhibit gastric acid secretion. It has therefore been intensively investigated for treatment of gastric ulcers and for use in promotion of wound healing.

EGF also affects hair growth, and its use in the defleecing of sheep and other hair- or wool-producing animals, as an alternative to mechanical shearing, has been described. See for example Australian Patent No. 546354 by Commonwealth Scientific and Industrial Research Organisation. A variety of formulations of EGF has been described, principally for applications such as treatment of gastric ulcers, for topical application to promote wound healing and for preservation of ophthalmic samples such as corneal tissue. For most, if not all, of these applications, low pH is contra-indicated. A simple aqueous solution of urogastrone is disclosed in British Patent No. 1,394,846 by Imperial Chemical Industries Limited and solutions in isotonic saline or isotonic dextrose, optionally buffered to pH from 5 to 9, are disclosed in British Patent No. 1,417,776 by Imperial Chemical

Industries Limited. Neither of these specifications gives any indication as to the stability of the solution. Oral formulations of urogastrone are described in U.S. Patent No. 4,820,690 by Gregory and McCullagh, but this specification does not given any indication as to the pH of the composition. European Patent Application No. EP-Al-308238 by Ethicon, Inc. discloses a stable lyophilised formulation containing a growth factor, a water-soluble extender for lyophilisation, and a polymer capable of increasing the viscosity of the reconstituted solution. The diluent for reconstitution optionally contains a buffer of pH between 5 and 8, preferably 7 to 8. There is no information as to the effect of pH on the stability of the reconstituted solution. Australian Patent Application No. 80641/87 by Ethicon, Inc. discloses a method for stabilising growth factor-containing compositions against loss of biological activity in the presence of moisture; the composition comprises a water- soluble polysaccharide in an effective amount. The preferred polysaccharides are cellulose derivatives, and the pH of the solution is again between 5 and 8, preferably 7 to 8. Gel formulations containing growth factors are disclosed in Australian Patent Application No. 22235/88 by Ethicon, Inc.; these formulations are described as having pH between 6 and 10, preferably between 6 and 7.5. The formulations are described as being suitable for promotion of wound healing. A method of stabilisation of methionine- containing polypeptides, such as EGF and TGF, in liquid or semi-solid formulations is disclosed in International Patent Application No. WO 92/15614 (PCT/US92/01167) by Chiron Ophthalmics, Inc.; this utilises methionine in an amount sufficient to inhibit oxidation of methionine residues in the polypeptide. All of the aforementioned specifications, other than the two British patents, are described with specific reference to EGF, but state that the methods disclosed therein are applicable to other polypeptide growth factors.

Stabilisation of growth factors by direct chemical modification of the polypeptide has also been described. U.S. Patent No. 5,070,118 by Njieha and Shalaby describes acylated derivatives of EGF, which are stabilised against enzymic attack. Stabilisation by other modifications such as phosphorylation and esterification of reactive amino acid residues or cycloalkylation of the guanidinium group of arginine is also suggested. International Patent Application No. WO 93/03757 (PCT/US92/06870) by Chiron Corporation describes muteins of EGF in which the histidine at position 16 is replaced with a neutral or acidic amino acid. These muteins are stated to be active at pHs lower than the native form of EGF, showing activity at pHs as low as 4, whereas the wild type EGF shows substantially reduced receptor binding at pH less than 6.5. Modifications at other sites in the molecule may be made in order to modify receptor affinity and/or mitogenicity. Although the stability at pH between 4 and 6.5 is not stated, it is implied that stability for up to three months would be useful.

Gregory et al in Int. J. Peptide Protein Res., 1977 9. 107-118 describe urogastrone as being able to be maintained in aqueous solution at pH 1 to 11 for at least 20 hours without loss of activity, and that up to six of the amino acids at the C-terminal may be removed without adversely affecting the biological activity of the polypeptide.

Surprisingly, we have now found that aqueous solutions of EGF of low pH have excellent stability, suitable to provide a commercially practicable shelf-life, and also meet the other basic criteria for efficacy and acceptability, whereas formulations at physiological and higher pH are unstable during long-term storage.

Similar formulations provide advantages for the delivery of proteins related to EGF, other growth factors, and pharmacologically useful proteins generally. Summary of the Invention

In one aspect, the invention provides a formulation comprising a pharmaceutically effective amount of a polypeptide growth factor, said composition having a pH of 2.8 to 3.8, whereby biochemical stability of the growth factor is maintained for at least 20 weeks at 4°C. The formulation of the invention may optionally comprise a pharmaceutically-acceptable polymeric material for providing viscosity, said viscosity preferably being between 20 and 200 centipoise.

Where the formulation is to be used for injection, there is a lesser risk of local reaction at the injection site at pH above 3, with the risk of local reaction decreasing as the pH increases. Therefore in preferred embodiments of the invention the pH is preferably between 3.0 and 3.8, more preferably between 3.5 and 3.8, and most preferably between 3.6 and 3.7. However, we have found that EGF is stable even at pH 2.0.

Preferred viscosity-providing agents suitable for use in the invention include, but are not limited to, methylcellulose and hydroxypropyl methylcellulose. Suitable concentrations of the viscosity providing agent will preferably be between 1 and 5%, depending on the molecular weight of the agent. A large variety of suitable thickening agents are known in the art, and it is considered to be a matter of mere trial and error experiment for a person skilled in the art to test whether a given agent is compatible with conditions of low pH, and to establish the concentration necessary to provide a suitable viscosity. Such a person will appreciate that it will be necessary to establish a suitable pharmacokinetic profile for the desired purpose, while still providing a solution which is sufficiently fluid to enable easy handling and injection. A suitable viscosity will usually be between 20 and 200 centipoise, most commonly 50 centipoise. Where the formulation is to be used for topical application, such as preparations for wound healing, or for ophthalmic applications, the viscosity may be much higher. Again the skilled person will readily be able to establish suitable conditions using trial and error experimentation. Provided that the pH remains below 3.8, the solutions of the invention do not require to be buffered. However, it will frequently be found convenient to incorporate a suitable buffer in the solution. A skilled person will be aware of many suitable physiologically compatible buffer systems, which will preferably be organic acids and their salts. For example acetic acid/sodium acetate and citric acid/sodium citrate are suitable. Phosphoric acid may be used to lower the pH. In general, the buffering capacity and tonicity of the solution will preferably be fairly low, in order to minimise disturbance to the local milieu on injection or topical application.

The solutions will preferably also comprise one or more preservative agents in order to prevent microbial degradation; many such preservatives are well-known in the art, such as sodium methyl hydroxybenzoate, or sodium propyl hydroxybenzoate. Again these may be tested by routine methods in order to determine whether they are compatible with low pH. Similarly other excipients or carriers may be tested at low pH.

Preferably the polypeptide growth factor will be selected from the group consisting of epidermal growth factor, acidic or basic fibroblast growth factor, platelet derived growth factor, transforming growth factor-α, transforming growth factor-β, insulin-like growth factor I or II, and nerve growth factor, angiogenin, somatomedin (SM-A,SM-B, SM-C, MSA), platelet-derived growth factor, and vaccinia growth factor.

More preferably the growth factor will be selected from the group consisting of epidermal growth factor, acidic or basic fibroblast growth factor, platelet derived growth factor, transforming growth factor-α, transforming growth factor-β, insulin-like growth factor I or II, and nerve growth factor. Most preferably the growth factor will be epidermal growth factor, preferably EGF. The growth factors will preferably be of human, bovine, porcine, rat or mouse origin, and may be natural, synthetic or recombinant.

Cytokines such as erythropoietin and colony stimulating factor, and hormones such as growth hormone, may also be suitable for use in this invention. It will be clearly understood that where a growth factor or other polypeptide or protein is referred to, biologically-active analogues, derivatives and fragments thereof, such as muteins, are also considered to be within the scope of the invention. Mixtures of one or more polypeptides or proteins may also be used in the formulations of this invention.

The formulations of the invention do not require lyophilised polypeptide to be used. The formulations may optionally comprise other stabilisers of a growth factor, such as methionine, described in WO 92/15614 by Chiron

Ophthalmics, Inc., a surfactant as described in Japanese Patent Publication No. 4159215-A by Earth Seiyaku Kabushiki Kaisha, or zinc ions as described in Australian Patent Application No. 54968/90 by Ethicon, Inc. Modified forms of the growth factor, such as the muteins of EGF or the

EDTA-chelated forms of EGF referred to above may be used. Furthermore, a second therapeutic agent, such as an anti- tumour agent as described in U.S. Patent No. 4,863,902 by Wakunaga Seiyaku Kabushiki Kaisha may be used; again it is a matter of routine to determine whether such a second agent retains its activity at the pH of the formulations of the invention.

In preferred embodiments, the formulation of the invention is selected from the group consisting of an injectable formulation, a gel formulation for topical formulation, an ophthalmic solution, a cream, ointment or lotion, and a tissue storage medium. It is contemplated that the formulations according to the invention will be useful for a variety of applications, such as medical and veterinary treatment in promotion of wound healing and therapy of gastroenterological conditions, promotion of gut maturation in premature animals, and promotion of early weaning in full term animals, and for defleecing of wool- or hair- bearing mammals such as sheep and goats.

In a preferred embodiment, this invention is concerned specifically with the delivery of epidermal growth factor, and of biologically-active analogues or derivatives of EGF, to sheep in order to achieve a defleecing effect.

Brief Description of the Figures Figure 1 compares the HPLC profiles of the

"original formulation" before storage (Figure la) and after storage at 25°C for 6 weeks, at 25°C for 6 months, and at 50°C for seven days (Figures la, lb and lc respectively) . Figure 2 compares the HPLC profiles of a blank Nil erm Injection formulation before (Figure 2a) and after storage for one month at 4°C and 30°C (Figures 2b and 2c respectively) .

Figure 3 shows HPLC profiles of samples of met- EGF formulation of pH 3.55 before and after storage at 4°C for 55 weeks (Figures 3a and 3b respectively) .

Figure 4 compares HPLC profiles of samples of a met-EGF formulation of pH 3.73, buffered with sodium acetate/acetic acid, before and after storage at 4°C for 34 weeks (Figures 4a and 4b respectively) . Figure 5 depicts HPLC profiles of a thickened met-EGF formulation of pH 3.55, before and after storage at 4°C for 55 weeks (Figures 5a and 5b respectively) .

Figure 6 shows HPLC profiles of a thickened formulation of met-EGF, pH 3.65, before and after storage at 4°C for 34 weeks (Figures 6a and 6b respectively) . Description of the Invention

The present invention arose from studies on the development of formulations of mouse epidermal growth factor to be used for the defleecing of sheep. It was recognised at an early stage that a single parenteral injection of aqueous EGF solution would not generally achieve defleecing, but that infusion over a period of 12 hours or more was highly effective.

Following studies on a variety of compositions, we developed a simple formulation in which the addition of a thickening agent resulted in the release of EGF from the injection site into the bloodstream over an appropriately prolonged period, and consequently achieved the defleecing effect. Preliminary experiments showed that this formulation was unstable. The most significant changes observed were those in the profile of EGF as displayed by high performance liquid chromatography. This profile showed that whereas freshly purified EGF appeared as a predominantly single peak on HPLC, the material became distributed through a multiplicity of subsidiary peaks when the formulation was stored for a period of time.

These subsidiary peaks are believed to represent altered conformations of EGF, amino acid side-chain modifications and perhaps partial degradation products.

While it has not been possible to show precise correlations between HPLC profile and biological activity, these complex changes preclude accurate quantitation of active material and therefore also preclude establishment of a practically useful shelf life.

Similar instability has been observed with reσombinant analogues of EGF including methionyl-mouse EGF ("met-EGF"), on which most of our development work has been done. Freeze-dried EGF has been shown to be highly stable. However, commercial distribution of freeze-dried material and reconstitution by the user immediately before use is not a practicable option in the context of veterinary use or use as a defleecing agent. In particular, the most suitable product for use by farmers is a pack from which multiple doses can be taken over a substantial period of time.

In the case of EGF defleecing formulations, freezing and thawing is desirably avoided for economic reasons, while chelation of heavy metals is readily achieved by incorporation of ethylenediamine tetraacetate (EDTA), which also inhibits some degradative enzymes. Other enzymes cannot practicably be inhibited in formulations because of the cost and potential toxicity of most inhibitors, but can be removed as far as possible during manufacture. International Patent Application No.

PCT/US92/01167 (WO 92/15614) by Chiron Ophthalmics Inc) identifies oxidation of methionine residues as a factor in the stability of EGF preparations. The first suspected cause of instability was oxidation. The choice of commercially useful antioxidants compatible with aqueous formulations is limited. Sodium ascorbate was tried, but did not affect met-EGF stability. De-oxygenation of the water by boiling or by sparging with nitrogen was also ineffective. Impurities in, or direct effects of, the thickening agent were considered as possible causes of instability of met-EGF in the formulation. On the other hand, additives such as glycerol and some macromolecules, including thickening agents, can have a stabilising effect on protein structure during storage. A range of alternative thickeners was examined, but no thickener was found to have any significant effect on met-EGF stability as determined by HPLC.

The pH of the formulation was considered to be a possible significant variable. According to Deutscher ( op . cit . at p.84 and 85), "protein solutions should not be exposed to extremes of pH" and "the theoretical goal is to reproduce the cellular milieu as closely as possible. This would mean....close to neutral pH...". Most proteins exist in their normal conformation at around physiological pH, and tend to lose normal biological activity at substantially higher or lower values. Such changes may be reversible or irreversible. The physiological pH for EGF would be expected to be about pH 7.2, as it exists in the intercellular spaces as a hormone conveying stimuli from cell to cell. It is known from earlier purification protocols for EGF that this protein survives transitory exposure to low pH solutions (e.g. pH 3.0); however, the protocols specify subsequent re-establishment of neutral or physiological pH. See for example Gregory et al 1977 (op. cit.) . Natural EGF loses activity below pH 6.5.

Formulations buffered to pH 10.0, 7.0 and 4.0 were prepared. Met-EGF formulations at pH 10.0 and 7.0 were found to be unstable, and the met-EGF was insoluble at pH 4.0, which is not unexpected as the isoelectric point of EGF is about pH 4.6.

However, in our initial HPLC studies, in which the mobile phase contained 0.1% trifluoroacetic acid (pH approximately 2.0), the samples were routinely diluted in this mobile phase. Surprisingly, some samples which were held for a considerable time before analysis were observed to have undergone much less change than the parent formulation over the same period. This observation suggested that a low pH might help to stabilise met-EGF in aqueous solutions. Met-EGF was then formulated at both low

(approximately 3.5) and high (approximately 9.5) pH values and stabilities compared with that of the initial formulation (approximately pH 8.0). HPLC traces showed that the low pH formulation only was highly stable with respect to met-EGF profile. Similar formulations without thickening agent were also tested, and again the low pH formulation only was found to be stable. Extreme pH in injectable formulations would be expected to cause increased irritancy and tissue damage, and hence potentially interfere with dispersion from the injection site as well as reducing acceptability in use. On the other hand, at least one commercially- available injectable formulation (Nilverm Injection) which has a low pH (pH 3.3) is widely used in sheep and cattle without causing significant adverse responses by the animal, or causing significant injection site lesions. However, this formulation disperses rapidly from the injection site. A thickened low pH formulation might be expected to cause a greater degree of irritation or tissue damage if the rate of dispersion of the acidic components is retarded. The efficacy (i.e. retention of structure and defleecing activity of the met-EGF) and acceptability of several formulations was determined by a defleecing trial in sheep. The low pH formulations performed satisfactorily in terms of both efficacy in defleecing and acceptability of injection-site reactions and animal responses.

In summary, the surprising features of the low pH formulations are:-

1. The high degree of stability shown by EGF in aqueous solution, when the pH is between 2.5 and 3.8, during prolonged storage at temperatures of 4°C and higher.

2. The low degree of injection site reactions and animals responses following subcutaneous injection of either thickened or unthickened low pH formulation into sheep. This minor reaction is regarded as being veterinarily acceptable.

3. The retention of full biological activity of EGF in thickened or unthickened low pH formulations, as measured by efficacy of defleecing of sheep. This could be a result of met-EGF retaining this activity at low pH, or more likely complete recovery of activity as the pH of its environment reverts to physiological values following injection into sheep. 4. The increased efficacy of thickened or unthickened low pH formulations by comparison with the corresponding thickened or unthickened medium to high pH formulations respectively, as measured by defleecing efficacy. This difference may be related to the slightly but detectably prolonged release of EGF into the bloodstream, as measured by serum EGF analyses, following a low pH injection. 5. The comparable efficacies of the unthickened low pH formulation and of the standard thickened medium to high pH formulation. This suggests that the use of low pH may permit the omission of the thickening agent, which would reduce the cost of materials and production. The invention will now be described in detail by way of reference only to the following non-limiting examples.

Example 1 The "Original Formulation"

Satisfactory biological efficacy (defleecing of sheep) was obtained when authentic mouse EGF was formulated as follows:

Methyl Cellulose A15 37.5 g/L Benzyl alcohol 20.0 g/L

EGF 7.5 g/L and administered to sheep by subcutaneous injection at a dose of EGF of 250 μg per kg liveweight.

This formulation is unbuffered, but has a pH of approximately 8.0. The benzyl alcohol is present as a biocidal preservative. The methyl cellulose acts as a thickener, increasing the viscosity of the preparation. It has the effect of retarding the dispersion of the EGF from the injection site into the bloodstream, but the mechanism of this effect has not been established. It has been found that this prolonged release of EGF was necessary to achieve a reliable defleecing effect (Australian Patent No. 546354 by Commonwealth Scientific and Industrial Research Organisation) . This original formulation is not satisfactory as the basis for a useful product. This is because EGF in this formulation undergoes chemical changes which are readily detected by analysis using high performance liquid chromatography. Recombinant analogues of EGF, such as methionyl mouse EGF (metEGF) , formulated in the same way, possess the same biological activity and also display the same instability.

Figure 1 shows HPLC traces of formulated met-EGF taken before and after storage at 25°C, or for 6 weeks or 6 months at 25° or at 50°C for 7 days. The major met-EGF peak, eluting at approximately 18 minutes, diminishes markedly during storage, the material being redistributed among other minor peaks of the trace. The major met-EGF peak, eluting at approximately 18 minutes, represents 16% of the total met-EGF in the top chromatograph but only 25.3% in the bottom chromatograph. This demonstrates that there is significant change on storage.

Storage at temperatures down to 4°C elicits similar changes, occurring at slower rates.

Example 2 Alternative Thickening Agents

One of the first approaches to improving the stability of met-EGF formulations was to examine alternative thickening agents. A number of such agents were compared with Methocel A15, the aim being to find candidates with comparable properties and good physical stability, and then to determine whether the stability of met-EGF was enhanced by the use of such alternative thickeners.

No thickener was identified which was both an appropriate alternative to Methocel A15 and had any effect in improving met-EGF formulations.

Example 3 Low pH Formulations

During our initial investigation of formulation stability by HPLC, samples were prepared and diluted with buffers containing tri luoroacetic acid and were stored for extended periods of time. These samples were held at pH 2.0, and it was observed that the major EGF fraction remained unchanged during such storage, whereas the formulated starting material displayed instability as described above.

Accordingly the possibility of developing an acceptable low pH formulation was investigated.

"Nilverm Injection" is a commercial formulation of the anti-helminthic agent levamisole, which is used widely in cattle and sheep. Its specified pH is 3.3-3.5, which has proved acceptable in use and does not elicit significant animal responses or injection site lesions. This formulation, with the active ingredient omitted, was therefore used as the starting point for experimental met- EGF formulations. A blank "Nilverm Injection" formulation was prepared as follows:-

Methyl Hydroxybenzoate 0.50 g/L Propyl Hydroxybenzoate 0.05 g/L Ethylenediaminetetraacetate (EDTA) tetrasodium salt 2.0 g/L

Sodium metabisulphite 2.0 g/L Sodium citrate (anhydrous) 12.2 g/L Phosphoric acid B.P (85%) 3.73 mL/L Water to 1 L 3.73 mL phosphoric acid was mixed with 700 mL of water and the weighed ingredients added while stirring. When all ingredients had dissolved the volume was made up to 1 L with water.

16.3 mg met-EGF was then added to a 1 mL portion of the blank formulation, but failed to dissolve despite extensive sonication and dilution to 1 mL with water.

However, when the mixture was acidified with 0.1 mL glacial acetic acid the met-EGF dissolved readily on shaking. The final pH was estimated at approximately 3.2.

Samples of this formulated material were analysed by HPLC before and after storage for 1 month.at 4°C and 30°C. The HPLC trace was essentially unchanged during these periods of storage, as shown in Figure 2. Figure 2a is before storage, Figure 2b is after 1 month at 4°C, and Figure 2c is after 1 month at 30°C.

Example 4

The formulation was then modified by reducing the total salt concentration by halving the concentrations of sodium citrate, sodium metabisulphite and EDTA, and by reducing the amount of phosphoric acid and increasing the amount of acetic acid. These changes were designed to increase the solubility of met-EGF, and to facilitate adjustment of pH to a value within the specification range of Nilverm Injection.

Composition: 1. Methylhydroxybenzoate 0.50 g/L

2. Propylhydroxybenzoate 0.05 g/L

3. EDTA, tetrasodium salt 1.0 g/L 4. Sodium metabisulphite 1.0 g/L 5. Sodium citrate 6.1 g/L 6. Phosphoric acid (85%) 2.0 mL/L

7. Acetic acid (glacial) 50.0 mL/L

8. Water to 1 L

Components 1 and 2 were dissolved in 100 mL of water heated to 80°C. Components 3, 4 and 5 were dissolved in 750 mL water at ambient temperature. The two solutions were combined, and components 6 and 7 added while stirring.

Met-EGF was then added to portions of the formulation at a concentration of 8 mg/mL. The met-EGF dissolved readily with sonication.

Example 5

The formulation was further simplified by omission of sodium citrate, sodium metabisulphite and phosphoric acid.

Vehicle: Item Description g (or mL) per litre

1 Milli-Q water - 800 mL 2 Methyl hydrobenzoate BP 0.5002 g 3 Propyl hydroxybenzoate BP 0.0501 g 4 Ethylenediaminetetra acetic acid (tetra sodium salt) USP 2.003 g Milli-Q water q.s to 1 L

Met-EGF formulation: Item Description g (or μL) per 25 mL

6 Met-EGF, Batch 9 0.2007 g

7 Glacial Acetic Acid (99.0%) BP 200 μL 8 Glacial Acetic Acid (99.0%) BP 200 μL

Preparation of Vehicle:

The Milli-Q water (Item 1) was heated to 80°C in a 1 L beaker.

Methyl hydroxybenzoate (Item 2) and Propyl hydroxybenzoate (Item 3) were dispensed and added with continuous stirring until dissolved. The solution was allowed to cool to room temperature. EDTA (Item 4) was added with stirring when the solution was cool. 4. The solution was transferred to a 1 L calibrated vessel and diluted to volume with Milli-Q water (Item 5) .

Preparation of formulation: 5. A 25 mL aliquot of the vehicle from the calibrated vessel was transferred to a 50 mL beaker.

6. Met-EGF (Item 6) was added to the beaker and dissolved by stirring. The pH was measured and recorded. pH = 9.51.

7. Glacial Acetic Acid (Item 7) was added with stirring until the solution was clear (approximately 15 minutes) . The pH was measured and recorded. pH = 3.81. 8. The pH was adjusted to 3.4 - 3.8 with Glacial

Acetic Acid (Item 8). The final pH was 3.55. The total volume of Acetic Acid added was 0.400 mL. 9. The formulation was transferred to commercial vaccine containers (Vaxipak) , and sealed with rubber bungs and aluminium overseals. 25 mL in a 50 mL Vaxipak was stored at 4°C for chemical analysis and pH measurement.

Concentration: The final met-EGF concentration in the pH adjusted formulation was calculated as follows:

0.2007 x 1000 = 7.90 mg/xn

25.40

This formulation, packaged in Vaxipaks, was stored at 4°C for periods of 0, 5, 8, 37 and 55 weeks. After each of these time periods, one of the containers was taken and the contents examined for the following parameters: Appearance PH

Total Met-EGF content, as determined by HPLC (the sum of all HPLC peaks related to Met-EGF) . Percentage of Met-EGF in major HPLC peak.

Appearance of new HPLC peaks indicating degradation of Met-EGF.

This analysis, summarised in Table 2, shows excellent stability of this formulation on prolonged storage at 4°C.

Figure 3 shows the HPLC traces of samples before and after storage of this formulation at 4°C for 55 weeks. Very small differences are detectable amongst the minor peaks.

Table 2: Stability at 4°C of nan-thickened low pH formulation containing 7.9 mg/mL Met-EGF.

Test Release Expiry Results after storage time Speci ication Speci ication

Initial 5 weeks 8 weeks 37 weeks 55 weeks

Appearance A clear, straw A clear, straw Clear, straw Clear, straw Clear, straw Clear, straw Clear, straw coloured liquid coloured liquid coloured coloured coloured coloured coloured liquid liquid liquid liquid liquid

PH 3.4 - 3.8 3.4 - 3.8 3.55 3.62 3.71 NT 3.66

I

Potency Met- 7.125-7.875 mg/mL 6.75-8.25 mg/mL 7.90 mg/mL NT NT 7.81 mg/mL 7.69 mg/mL t EGF (mg/mL) (95 - 105%) (90 - 100%) (100.0%) (98.9%) (97.3%)

(% of Initial assay)

Major Met-EGF 70% of total 60% of total 80.0% NT NT 81.0% 79.4% peak content peak area count peak area count

% degradation 1% For predictive 0% NT NT < 1.5% < 3.0% peaks purposes only

Note: NT = Not Tested

Example 6

In a further modification, sodium acetate was added to the formulation to improve the buffering capacity.

Vehicle:

Item Description g (or mL) per litre

1 Sodium methyl hydroxybenzoate BP 0.5002 g

2 Sodium propyl hydroxybenzoate BP 0.5000 g 3 Milli-Q water ~ 700 mL

4 Sodium acetate (anhydrous) ϋSP 1.0000 g (tetra sodium salt) ϋSP

5 Ethylenediaminetetra acetic acid 1.0001 g (tetra sodium salt) DSP . 6 Milli-Q water q.s. to 1 L

Met-EGF formulation:

Item Description g (or mL) per

250 mL 7 Met-EGF, Batch 21 1.9342 g

8 Glacial Acetic Acid (99.0%) BP 3.0 mL

9 Glacial Acetic Acid (99.0%) BP 3.0 L

Preparation of vehicle: 1. Sodium Methyl Hydroxybenzoate (Item 1) and Sodium

Propyl Hydroxybenzoate (Item 2) were dispensed and added to the Milli-Q water (Item 3) in a 1 L beaker and stirred with a laboratory stirrer until dissolved. 2. Sodium Acetate (Item 4) and EDTA (Item 5) were added to the beaker and stirred until dissolved. 3. The solution was then transferred to a 1 L calibrated vessel and diluted to volume with

Milli-Q water (Item 6) . Preparation of formulation:-

4. 250 mL of the vehicle was transferred from the calibrated vessel into a 300 mL beaker.

5. Met-EGF (Item 7) was added to the beaker and dissolved by stirring. The pH was measured and recorded. pH = 9.63.

6. Glacial Acetic Acid (Item 8) was added with stirring until the solution was clear (approximately 15 minutes) . The pH was measured and recorded. pH = 3.84.

7. The pH was adjusted to between 3.4 and 3.8 with Glacial Acetic Acid (Item 10) and the final pH was recorded. pH = 3.73. The total volume of Acetic Acid added was 6.0 mL. 8. The formulation was transferred to Vaxipak containers and sealed with rubber bungs and aluminium overseals. 25 mL in a 50 mL Vaxipak container was stored at 4°C for chemical analysis. 25 mL in a 50 mL Vaxipak container was stored at 4°C for pH measurement.

Concentration:

The final Met-EGF concentration in the formulation, after pH adjustment, was calculated as follows:

1.9342 x 1000 = 7. 74 mςr/mL

256. 0

This formulation was packaged into Vaxipaks and stored at 4°C for periods of 0, 6, 15, 27 and 34 weeks. After each time period, a container was opened and the contents examined as in Example 5. The analysis, summarised in Table 3, shows excellent stability of this formulation on prolonged storage at 4°C.

Figures 4 shows the HPLC traces of samples before and after storage of the formulation at 4°C for 34 weeks. Very small differences are detectable amongst the minor peaks.

Table 3: Stability at 4°C of non-thickened low pH formulation containing 7.5 mg/mL Met-EGF.

Test Release Expiry Results after storage time Specification Specification

Initial 6 weeks 15 weeks 27 weeks 34 weeks

pH 3.4 - 3.8 3.4 - 3.8 3.73 3.62 3.62 3.69 3.57

Potency Met¬ 7.125-7.875 mg/mL 6.75-8.25 mg/mL 7.74 mg/mL 7.74 mg/mL 7.71 mg/mL 7.69 mg/mL 7.69 mg/mL EGF (mg/mL) (95 - 105%) (90 - 100%) (100%) (100%) (99.6%) (99.5%) (99.6%) (% of Initial in assay)

Major Met-EGF 70% of total 60% of total 76 . 3% 75 .4% 74 .0% 74 .4% 73 .3% peak content peak area count peak area count

% degradation 1% For predictive 0% 0% < 0.5% < 1.0% < 1.0% peaks purposes only

Example 7 Thickened low pH formulations:

Since it was shown that low pH aqueous solutions of met-EGF were stable, further formulations were prepared which included thickening agents selected to achieve appropriate rates of dispersion of met-EGF following subcutaneous injection.

The objective was approached in two ways, firstly by acidification of the "original formulation" containing

Methocel A15 and secondly by modifying the low pH formulation through the addition of various thickening agents.

The "original formulation" was prepared and the pH adjusted to 3.55 by the addition of glacial acetic acid:-

7.5 mg/mL Met-EGF thickened low pH formulation.

Vehicle:

Item Description g (or mL) per litre

1 Milli-Q water - 600 mL

2 Methyl Cellulose 15 Cps DSP Premium 37.5 g

3 Benzyl Alcohol BP 10.0 mL

4 Milli-Q water q.s. to 1 L

Met-EGF formulation:

Item Description g (or μL) per

25 mL

5 Met-EGF, Batch 9 0.1999 g 6 Glacial Acetic Acid (99.0%) BP 150 μL

7 Glacial Acetic Acid (99.0%) BP 75 μL

Preparation of Vehicle:

1. Milli-Q water (Item 1) was placed at ambient temperature into a 1 L beaker.

2. Methyl Cellulose (Item 2), was slowly added to the beaker and stirred with a laboratory stirrer until dissolved.

3. Benzyl Alcohol (Item 3) was added and dissolved with stirring. . The solution was then transferred to a 1 L calibrated vessel and placed into an ultra sonic bath to remove any air bubbles.

5. The solution was then diluted to volume with Milli-Q water (Item 4) . The vehicle was stored overnight at 4°C to fully hydrate the methyl cellulose.

Preparation of formulation:

6. A 25 mL aliquot of the vehicle was transferred from the calibrated vessel into a 50 mL beaker. 7. Met-EGF (Item 5) was dispensed and added into the beaker and dissolved by stirring. The pH was measured and recorded. pH = 7.40.

8. Glacial acetic acid (Item 6) was added using Transferpette with stirring until the solution was clear (approximately 15 minutes) . The pH was measured and recorded. pH = 3.66.

9. The pH was adjusted to 3.4 - 3.8 with Glacial Acetic Acid (Item 7) and the final pH was recorded. pH = 3.55. The total volume of Acetic Acid added was 0.225 mL.

10. The formulation was transferred to Vaxipak containers and sealed with rubber bungs and aluminium overseals. 25 mL in a 50 mL Vaxipak was stored at 4°C for chemical analysis and pH measurement.

11. The viscosity of the vehicle was measured using a Brookfield Viscometer LVT, (Spindle 1, Speed 30, Factor 2). Release limits 45 - 75 cps. Viscosity = 49.0 cps. Concentration:

The final Met-EGF concentration in the formulation, after pH adjustment was calculated as follows:- 0 .1999 x 1000 = 7. 92 mg/mL

25.225

This formulation was then packaged, stored and analysed as in Examples 5 and 6. Table 4 presents the results of this stability analysis and shows the thickened low pH formulation to be highly stable with respect to pH, total met-EGF, proportion of met-EGF in major HPLC peak and met-EGF degradation. A significant increase in viscosity after 8 weeks storage is without practical importance.

Figure 5 illustrates the HPLC trace before and after storage at 4°C for 55 weeks. Very small changes are detectable amongst the minor peaks.

Table 4: Stability at 4°C of non-thickened low pH formulation containing 7.5 mg/mL Met-EGF.

Test Release Expiry Results after storage time Specification Specification

Initial 5 weeks 8 weeks 37 weeks 55 weeks

PH 3.4 - 3.8 3.4 - 3.8 3.55 3.47 3.55 NT 3.66

1 viscosity 45 - 75 cps 45 - 75 cps 49 cps 48 cps 55 cps NT 56 cps

Potency Met¬ 7.125-7.875 mg/mL 6.75-8.25 mg/mL 7.92 mg/mL NT NT 7.82 mg/mL 7.73 mg/mL EGF (mg/mL) (95 - 105%) (90 - 100%) (100%) (98.7%) (97.6%) 1 (% of Initial assay)

Major Met-EGF 70% of total 60% of total 79.9% NT NT 81.0% 78.7% peak content peak area count peak area count

% degradation 1% For predictive 0% NT NT < 1.5% < 2.5% peaks purposes only

Note: Viscosity measured on vehicle only. NT = Not Tested

Example 8

A low pH formulation, similar to that used in Example 6, was prepared with the addition of Methyl Cellulose A15 at 37.5 g/L, and a final pH of 3.65 was obtained by addition of glacial acetic acid.

Thickened low pH formulation containing 7.5 mg/mL Met-EGF.

Vehicle: Item Description g (or mL) per litre

1 Sodium methyl hydroxybenzoate BP 0.5003 g

2 Sodium propyl hydroxybenzoate BP 0.5000 g

3 Milli-Q water ~ 700 mL 4 Sodium acetate (anhydrous) DSP 1.0008 g

5 Ethylenediaminetetra acetic acid 1.0001 g (tetra sodium salt) ϋSP

6 Methyl Cellulose 15000 USP 36.8 g

7 Milli-Q water q.s. to 1 L

Met-EGF formulation:

Item Description g (or mL) per

400 mL

8 Met-EGF, Batch 21 3.0940 g 9 Glacial Acetic Acid (99.0%) BP 6.0 mL

10 Glacial Acetic Acid (99.0%) BP 4.0 mL

Preparation of vehicle:

1. Sodium Methyl Hydroxybenzoate (Item 1) and Sodium Propyl hydroxybenzoate (Item 2) were dispensed and added to the Milli-Q water (Item 3) in a 1 L beaker and stirred with a laboratory stirrer until dissolved.

2. Sodium Acetate (Item 4) and EDTA (Item 5) were added to the beaker and stirred until dissolved.

3. Methyl Cellulose (Item 6) was slowly added, with continuous stirring until dissolved. 4. The solution was then transferred to a 1 L calibrated vessel, and diluted to volume with Milli-Q water (Item 7). The vehicle was stored overnight at 4°C to fully hydrate the methyl cellulose.

Preparation of formulation:

5. 400 mL of the vehicle was transferred from the calibrated vessel into a 500 mL beaker. 6. Met-EGF (Item 8) was added to the beaker with stirring and dissolved by stirring. The pH was measured and recorded. pH = 9.44.

7. Glacial Acetic Acid (Item 9) was added with stirring until the solution was clear (approximately 15 minutes) . The pH was measured and recorded. pH = 3.87.

8. The pH was adjusted to between 3.4 and 3.8 with Glacial Acetic Acid (Item 10) and the final pH was recorded. pH = 3.65. The total volume of Acetic Acid added was 10 mL.

9. The formulation was transferred to Vaxipak containers and sealed with rubber bungs and aluminium overseals. 25 mL in a 50 mL Vaxipak container was stored at 4°C for chemical analysis. 150 mL in a 200 mL Vaxipak container was stored at 4°C for viscosity and pH measurement. 10. The viscosity of the formulation was measured using a Brookfield Viscometer LVT (Spindle 1, Speed 30, Factor 2). Release limits 45-75 cps.

Viscosity = 56 cps.

Concentration:

The final met-EGF concentration in the formulation, after pH adjustment, was calculated as follows:- 3 . 094 x 1000 = 7. 546 mg/mL

410. 0

This formulation was packaged, stored and analysed as in Examples 5, 6 and 7.

Table 5 summarises the stability data of this formulation. The thickened low pH formulation was shown to be highly stable with respect to pH, total met-EGF, proportion of met-EGF in major HPLC peal and met-EGF degradation.

Figure 6 shows the HPLC trace before and after storage at 4°C for 34 weeks. Very small changes are detectable amongst the minor peaks.

Table 5: Stability at 4°C of Thickened low pH formulation containing 7.5 mg/mL Met-EGF.

Test Release Expiry Results after storage time Specification Specification

Initial 6 weeks 15 weeks 27 weeks 34 weeks

PH 3.4 - 3.8 3.4 - 3.8 3.65 3.69 3.69 3.69 3.61

Viscosity 45 - 75 cps 45 - 75 cps 56 cps 61 cps 70 cps 51 cps 50 cps to

Potency Met- 7.125-7.875 mg/mL 6.75-8.25 mg/mL 7.55 mg/mL 7.53 mg/mL 7.53 mg/mL 7.49 mg/mL 7.49 mg/mL EGF (mg/mL) (95 - 105%) (90 - 100%) (100%) (99.7%) (99.7%) (99.2%) (99.2%)

(% of Initial assay)

Major Met-EGF 70% of total 60% of total 75 .9% 76 .5% 73 .5% 74 .4% 71 .8% peak content peak area count peak area count

% degradation 1% For predictive 0% 0% < 0.5% < 1.0% < 1.0% peaks purposes only

EFFICACY AND ACCEPTABILITY OF LOW pH EGF FORMULATIONS IN SHEEP

When the high stability of low pH formulations had been demonstrated, they were then tested in sheep to determine efficacy (in terms of defleecing) and acceptability (in use in terms of animal responses to injection and subsequent injection-site tissue reactions) .

Example 9 100 Merino sheep (5-6 year old wethers, 21 micron wool) were used in this trial. The trial was carried out in deep-litter pens indoors, the sheep were maintained using normal husbandry methods, and were fed a standard pellet diet based on lucerne meal and oats. Met-EGF treatments were administered on a individual weight basis at 250 μg/kg, injected subcutaneously in the inguinal region.

Formulation details :

Treatment Group No . Description Buffered, "Original low pH, thickened

Item

Met-EGF 7.5 9 7.5 g

Methyl cellulose 36.8 g 36.8 g

Benzyl alcohol 10.0 •ml". 10.0 mL

Sodium acetate (anhydrous) 1.0 Q -

Glacial acetic acid 26.0 mL -

Water (Milli-Q) to 1 L to 1 L

pH (adjusted with glacial acetic acid) 3.5 8.6 Viscosity (25°C) CPS 50 50 Treatment Group No.

Description Buffered, Buffered, low pH, low pH, thickened

Item

Met-EGF 7.5 g 7.5 g

Methyl hydroxybenzoate 0.5 g 0.5 g

Propyl hydroxybenzoate 0.05 g 0.05 g Sodium acetate (anhydrous) 1.0 g 1.0 g

EDTA (Tetrasodium salt) 1.0 g 1.0 g

Hydroxypropyl methyl cellulose - 8.0 g

Glacial acetic acid 26.0 mL 26.0 mL

Water (Milli-Q) to 1 L to 1 L pH (adjusted with glacial acetic acid) 3.6 3.6

Viscosity (25°C) CPS 1.0 115

(Final concentration of met-EGF in Groups 1, 2 and 3 was 7.28 g/L as consequence of pH adjustment).

Formulation 1 corresponds to that of Example 5 except for use of benzyl alcohol in place of the methyl and propylhydroxybenzoate.

Formulation 2 corresponds to that of Example 6.

Formulation 3 corresponds to that of Example 8 except for use of Hydroxypropylmethyl Cellulose in place of Methyl Cellulose.

Blood samples were collected at intervals up to 48 hours after injection, and met-EGF levels in the serum were measured by means of a sandwich ELISA immunoassay, to provide an estimate of the rate of release of EGF from the injection site.

The defleecing efficacy of the treatments was assessed by inspecting the sheep at 6 weeks after treatment. The sheep were classified into four categories according to the degree of break in the fleece.

1. Tight: Cannot remove fleece by hand.

2. Slight weakness: Impractical to remove fleece by hand, conventional shearing necessary. 3. Weakness: Practicable to remove fleece by hand, conventional shearing not necessary.

4. Clean break: Wool readily comes away in hand.

All sheep were carefully inspected at 3 and 4 weeks after met-EGF injection, to assess irritation, lesions or any other adverse injection site reactions. The results are summarized in Table .

Table 6

Results:

Group Defleecing results- Site reaction

No. of sheep

(n/20 ?)

1. low pH, Methyl Cellulose 5 15 10/20 CO

2. low pH, not thickened 3 17 1 100//2200 ^

3. low pH, Hydroxy Methyl Cellulose 3 17 14/20

4. "Original" 6 14 8/20

Successful defleecing requires essentially all sheep to show a category 3 or 4 effect. In this trial, all three low pH formulations resulted in defleecing at least as effective as with the "original" formulation. It was surprising that the unthickened low pH formulation was fully effective. Previous data have always shown non-thickened subcutaneous injection formulations of EGF to be largely ineffective. The measurements of met-EGF in serum showed that the time course of dispersion and elimination for this group of sheep was very similar to that in the other groups.

All the low pH formulations showed a greater incidence of injection site reactions, with quantitatively more intense reactions, than did the original formulation. However, these reactions, which resulted in the formation of loose fibrous lumps between skin and muscle, are not serious and resolve fully over a period of several weeks.

Example 10 180 Merino sheep (3-5 year old wethers, 21-22 micron wool) were used in this trial, in which sheep were kept as in Example 2.

Met-EGF treatments were administered on a individual weight basis at the dose specified below, injected subcutaneously in the inguinal region. The treatment groups are summarized in Table 7.

Table 7: Treatment Groups

Group CFL Description Dose Blood No. of

No. No. Rate sample sheep

(μg/kg)

1 2504 Methyl Cellulose, pH 8.4, 50 CPS 250 Yes 20

2 2504 Methyl Cellulose, pH 8.4, 50 CPS 125 Yes 20

3 2507 Methyl Cellulose, pH 3.6, 50 CPS 250 No 20

4 2507 Methyl Cellulose, pH 3.6, 50 CPS 125 Yes 20

5 2508 Thickened, buffered, pH 3.6, 1 CPS 250 No 20 > vo

6 2508 Thickened, buffered, pH 3.6, 1 CPS 125 Yes 20

7 2505 Thickened, buffered, pH 8.7, 1 CPS 125 Yes 20

8 2506 Placebo saline 2 mL No 20

2510 Placebo pH 3.5 2 mL No

9 2509 Placebo Methyl Cellulose, pH 3.5 2 mL No 20

The details of the fozmulation are given in Table 8.

Table 8: Formulation details

Treatment Group No. 1 and 2 7 8

Description "Original" Non thickened, Saline

High pH, Placebo Item

Met-EGF 7.75 g 3.875 g -

Methyl Cellulose 36.8 g - -

Benzyl alcohol 10.0 mL - -

Sodium chloride - - 9.0 g

Water (Milli-Q) to 1 L to 1 L to 1 L

PH 8.6 8.6 6.45

Viscosity (25°C) 50 CPS 1 CPS 1 CPS

Table 8 (continued)

Treatment Group No. 3 and 4 5 and 6

Description Thickened, Buffered, Placebo, Placebo, Buffered, Low pH, Thickened Low pH Non-thickened,

Item

Met-EGF 4.79 g 4.79 g - -

Methyl hydroxybenzoate 0.5 g 0.5 g 0.5 g 0.5 g

(Sodium salt)

Propyl Hydroxybenzoate 0.05 g 0.05 g 0.05 g 0.05 g 1

Sodium acetate 1.0 g 1.0 g 1.0 g 1.0 g y

EDTA (Tetrasodium salt) 1.0 g 1.0 g 1.0 g 1.0 g 1

Methyl cellulose 36.8 g - 36.8 g -

Glacial acetic acid 26.0 mL 26.0 mL 26.0 mL 26.0 mL

Water (Milli-Q) to 1 L to 1 L to 1 L to 1 L

PH 3.6 3.6 3.5 3.5

Viscosity (25°C) 50 CPS 1 CPS 50 CPS 1 CPS

Blood samples were collected at intervals up to 36 hours after injection from Groups 1, 2, 4, 6 and 7, and met-EGF levels in the serum were measured by means of a Sandwich ELISA immunoassay, to provide an estimate of the rate of release of EGF from the injection site.

The defleecing efficacy of the treatments was assessed by inspection of the sheep at 6 weeks after treatment. The sheep were classified into five categories according to the degree of break in the fleece:-

1. Tight: Cannot remove by hand.

2. Slight weakness: Impractical to remove fleece by hand, conventional shearing necessary.

3a. Weakness: Can remove fleece by hand, with large effort.

3b. Weakness: Can remove fleece by hand, with small effort.

4. Clean break: Wool readily comes away in hand.

All sheep were carefully inspected at 5 weeks after met-EGF injection, to assess irritation, lesions or any other adverse injection site reactions. The results are summarized in Table 9.

Table 9

Group Dose Defleecing results Site reactions (μg/kg) No. of sheep (n/20)

Category 3a 3b

1. Original 250 - - 1 19 11/20

2. Original 125 10 3 3 4 3/20

3. low pH, thickened 250 - - - 20 14/20

4. low pH, thickened 125 1 2 3 14 14/20 |

5. low pH, unthickened 250 - 1 3 16 8/20 .t* eo

6. low pH, unthickened 125 5 4 7 4 2/20 |

7. high pH, unthickened 125 5 8 5 2 0/20

8. Saline placebo - NA - - 0/20

9. low pH placebo - NA - - 8/20

10. low pH, thickened placebo NA 15/20

Successful defleecing requires essentially all sheep to show a category 3b or 4 effect. In this trial, at the higher dose, the original and the thickened low pH formulations achieve this, and the unthickened low pH formulation comes very close.

At the lower dose (half the established standard dose rate), the thickened low pH formulation is clearly more effective than the original formulation, while the low pH unthickened formulation performs at least as well as the original (thickened) formulation.

All these groups showed significant met-EGF levels in serum at 12 hours post treatment.

Injection site reactions were most marked and most frequent in the groups treated with low pH and particularly with low pH thickened formulation, with or without met-EGF. However, these reactions were not so severe as to be of practical concern, and they resolve over a period of 2 months and do not affect the carcass after slaughter and skinning. This trial confirmed that low pH formulation of met-EGF, besides being of satisfactory stability, are fully effective and acceptable in defleecing sheep. Examples 9 and 10 both suggest that the low pH may enhance efficacy in defleecing in comparison with material of higher pH.

Example 11 Field Trial of Low pH EGF Formulations

148 Merino sheep (2-year-old wethers, 20.5 micron wool) were used in a field trial carried out during summer (November/December) . 77 sheep were treated with a thickened low pH formulation and 71 with an unthickened low pH formulation, by subcutaneous injection in the inguinal region. The formulations were as follows:-

Treatment Group Formulation Thickened, Low pH, Low pH unthickened

met-EGF 6.75 g 6.75 g

Methyl hydroxybenzoate 0.5 g 0.5 g (Sodium salt)

Propyl hydroxybenzoate 0.05 g 0.05 g (Sodium salt)

Sodium acetate 1.0 g 1.0 g

EDTA (tetrasodium salt) 1.0 g 1.0 g

Methyl Cellulose 15 CPS 36.8 g -

Glacial Acetic acid 20.0 ml 20.0 ml

Water (Milli-Q) to 1 litre to 1 litre

PH 3.7 3.7

Viscosity (25°C) CPS 50.0 1.0

met-GF doses:

Dose range (μg/kg) 239 - 342 221 - 297 Mean Dose (μg/kg) 290 265

The sheep were injected with 2ml of formulated material. Because of the variation in live weight, the dose of met-EGF administered also varied, as indicated in the table. 5 weeks after treatment, the defleecing efficacy was determined by inspecting and categorising the sheep as in Example 10. The results are summarized in Table 10.

Table 10

Treatment Group Defleecing Efficacy Total Category

1 2 3a 3b 4 nd Sheep

Thickened 0 0 1 8 67 1 77 unthickened 0 0 0 13 58 0 71 Both low pH treatments were fully effective under field conditions.

Minor injection site reactions were observed in both groups of sheep. There was no evidence of irritation or discomfort for the sheep; the reaction is a loose fibrous lump which generally resolves and disappears, but which, if still present in sheep at slaughter, is removed from the carcass by the process of skinning.

It will be clearly understood that the invention in its general aspects is not limited to the specific details referred to herein above.

Claims

1. A formulation comprising a pharmaceutically- effective amount of a polypeptide growth factor together with a pharmaceutically-acceptable carrier, said composition having a pH of 2.8 to 3.8, whereby biochemical stability of the growth factor is maintained for at least 20 weeks at 4°C.

2. A formulation according to Claim 1, wherein the pH of the composition is 3.0 to 3.8.

3. A composition according to Claim 2, wherein the pH of the composition is 3.5 to 3.8.

4. A composition according to Claim 3, wherein the pH of the composition is 3.6 to 3.7.

5. A composition according to any one of Claims 1 to 4, which additionally comprises a pharmaceutically- acceptable polymeric material for providing viscosity.

6. A composition according to Claim 5, wherein the viscosity is 20 to 100 centipoise.

7. A composition according to Claim 5 or Claim 6, wherein the viscosity is 50 centipoise.

8. A composition according to any one of Claims 5 to 7, wherein the viscosity-providing agent is present at a concentration of 1 to 5%.

9. A composition according to any one of claims 5 to 8, wherein the viscosity-providing agent is methylcellulose or hydroxymethylcellulose.

10. A composition according to any one of the preceding claims, wherein the polypeptide growth factor is selected from the group consisting of epidermal growth factor, acidic or basic fibroblast growth factor, platelet derived growth factor, transforming growth factor-α, transforming growth factor-β, insulin-like growth factor I, insulin-like growth factor II, nerve growth factor, angiogenin, somatomedin (SM-A, SM-B, SM-C, MSA), platelet- derived growth factor, and vaccinia growth factor.

11. A composition according to Claim 10, wherein the growth factor is selected from the group consisting of epidermal growth factor, acidic or basic fibroblast growth factor, platelet derived growth factor, transforming growth factor-α, transforming growth factor-β, insulin-like growth factor I, insulin-like growth factor II, and nerve growth factor.

12. A composition according to Claim 11, wherein the growth factor is epidermal growth factor.

13. A composition according to any one of the preceding claims, selected from the group consisting of an injectable formulation, a gel formulation for topical application, an ophthalmic solution, a cream, an ointment, a lotion, and a tissue storage medium.

14. A method of defleecing a wool- or hair-bearing mammal, comprising the step of administering to said mammal a defleecing effective amount of a composition according to Claim 12.