AU615473B2 - Stabilisation of therapeutically active proteins in a pharmaceutical composition - Google Patents

Abstract

The pharmaceutical preparations are for topical use, contain one or more stabilised therapeutically active proteins and, where appropriate, customary auxiliaries, vehicles and additives, and are prepared as described using physiologically tolerated hydrophobic substances for stabilising proteins.

Description

615473 COMMONWEALTH OF A 3TRALTA PATENTS ACT 1952 COMPLETE SPECIICATION NAME ADDRESS OF APPLICANT: Boehringer Ingelheim International GmbH D-6507 Ingelheim am Rhein Federal Republic of Germany NAME(S) OF INVENTOR(S): Traute MUNDORF Iurt SCHNECKER ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: Stabilisation of therapeutically active proteins in a pharmaceutical composition The following statement is a full description of this invention, including the best method of performing it known to me/us:- 53283/000.313 Stabilisation of therapeutically active proteins in a pharmaceutical composition This invention relates to a pharmaceutical composition suitable for topical application comprising one or more stabilised, therapeutically active proteins.

The invention also relates to a process fo.' preparing such a pharmaceutical composition and the use of physiologically acceptable hydrophobic substances for the stabilisation of proteins.

An essential requirement in the topical application of therapeutically active proteins is their stability in the pharmaceutical formulation. The stability must be ensured for a sufficiently long period of time during storage under refrigeration, at ambient temperature and also at body temperature when it is "in situ" for several hours. Hitherto, no entirely satisfactory solutions have been found to meet this requirement.

SVarious substances for stabilising interferons have already been proposed, and f-r example hydroxyethylcellulose has been used as a carrier substance for the preparation of gels or ointments containing interferon. However, there was some loss of S activity of the interferons which could only be reduced S by the addition of a protease inhibitor (EP-A-152345).

It has also been proposed to stabilise interferons in gels, ointments, etc. by the use of various sugar alcohols, optionally in combination with sugar acids or the salts thereof, mild reducing agents, anionic surfactants or combinations of these substances (EP-A-80879).

It has been proposed to use a physically and chemically modified gelatin, particularly as a replacement for human serum albumin, to stabilise proteins and polypeptides such as interferons, more 2 particularly IFN-gamma, in parenteral preparations, (EP-A-162332).

Japanese Published Patent Application JP-A-61-277633 discloses stabilising interferons in solution with certain surface-active substances.

EP-A-135171 mentions human serum albumin as a suitable stabiliser for oil/water microemulsions.

According to US Patent No. 4606917 albumin, dextrose and buffer substances are proposed as stabilisers for an ointment comprising the synergistic combination IFN-beta/9-(1,3-dihydroxy-2-propoxymethylguanine (DHPG).

A stabilising effect to the standard required has not yet been achieved with the substances proposed hitherto for stabilising therapeutically active proteins, particularly in hydrogels.

It is among the objects of this invention to provide a stabiliser for one or more therapeutically active proteins in a pharmaceutical composition for topical use, particularly in hydrogels, which in addition to being physiologically acceptable satisfies other requirements imposed on such a composition, especially with respect to good availability of the active substance, the full development of its activity and a mild method of preparation which takes account of the vulnerability of the proteins to shear forces.

Various oategories of substances have been investigated with respect to their suitability for stabilising proteins. It was found, surprisingly, that even small amounts of hydrophobic substances used as additives in very finely divided form, particularly paraffin oils, have a stabilising effect on various therapeutically active proteins which we believe to be superior to the effect of the substances proposed up till now. This result is all the more surprising as the pharmaceutical preparations for topical use which belong to the prior art, such as ointments, in which

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*99 99 9* *9 99 9 3 hydrophobic substances are used as carriers in a sulitably large proportion require the separate addition of a stabiliser for the protein.

Accordingly, a first aspect of the invention provides a pharmaceutical composition suitable for topical application which comprises one or more therapeutically active proteins, together with any one or more conventional excipients, carriers or adjuvants, and one or more physiologically acceptable hydrophobic substances in finely dispersed form in a quantity sufficient to stabilise the protein'.

Preferably one ur more hydrophobic substances comprise one or more paraffin oils and thk composition is in the form of a hydrogel. With the addition of a stabilising quantity of one or more hydrophobic substances in finely dispersed form, a pharmaceutical composition is obtained which, under the conditions of use, make the active substance available in active form over a long period of time. For instance, we have prepared pharmaceutical compositions according to the invention wherein, the level of activity of the protein after storage at 4-80C over a period of at least 12 months is substantially unchanged. A further advantage of compositions according to the invention is that there is less need to ensure that an exact pH value is maintained, since the stabilising addition of one or more hydrophobic substances reduce*$ the vulnerability of the proteins to fluctuations in the pH value. Thls advantage is of particular importance for applicatiorns which require lower pH values, e.g. application in the vaginal area.

The pharmaceutical composition according to the invention also has Ahe advantage, when in the form of a hydrogel, of being extremely pleasant to Use. This is because, even after the gel has driedt the presence of the hydrophobic substance ensures that the coating applied is soft to the touch, which is a particular 4 advantage when applied to the lip area.

The advantageous stabilising effect of hydrophobic substances on proteins can possibly be p~t down to hydrophobic interactions which have hithertQ bee scarcely noticed if at all. Stabilising proteins according to the prior art appears to rely on two principles: a) stabilising by complex binding of the substance to the protein and hence steric fixing of the protein molecule; and b) binding of free bulk water by polar substances and hence stabilising the protein by influencing its hydrate (.oat. The hydrophobic interactions which presumably come into play in the present invention and which also occur in micellar structures, appear to bring about stabilisation by virtue of the fact that the hydrophobic regions of the protein which are created by the spatial distribution of the hydrophobic and hydrophilic amino acid groups are fixed to the Dil/water phase interface, so that the hydrophobic regions project into the oil droplet and the hydrophilic parts project into the polar phase.

suitable hydrophobic substances include, in addition to the preferred paraffin oils, higher fatty 9.

acids such as linoleic acid and palmitic acid, or higher alcohols such as myristyl alcohol, or fatty acid esters such as triglycerides, or modified, e.g.

polyoxyethylenated and glycosylated, glycerides (Labrafil individually or in admixture., Of the paraffin oils, liquid, thin-liquid or thick-liquid paraffin oil according to Ph. Eur. and USP or mixtures thereof are suitable. The hydrophobic substances are preferably present in an amount cf from 0.1 to based on the total weight of the composition.

In order to ensure that the stabiliser is finely dispersed and the distribution is stable, emulsifiers may be added. The quantity used will depend particularly on the nature and quantity of stabiliser, the carrier used and, in the case of hydrogels, the 5 viscosity thereof; in general, it is not more than 1%.

Suitable emulsifiers include, in particular, non-ionic emulsifiers such as polysorbates (polyoxyethylene(n)sorbitanmonolaurate, e.g. TweenR nonoxynol (polyoxyethylene(n)- nonylphenyl-ether, e.g.

TritonR N101, TritonR N111), and poloxamer (polyethylenepolypropyleneglycol, PluronicR F68). If the pharmaceutical preparation is in the form of a hydrogel, the emulsifiers will not only bring about a fine dispersion of the stabiliser but will also improve the spreading of the gels.

The pharmaceutical composition according to the invention is suitable for the administration of human and animal proteins such as those listed below, S including their structurally similar bioactive Ss equivalents those proteins which have Ssubstantially the same biological activity with a S different amino acid sequence): cytokines, e.g.

S interferons such as huIFNalpha, hulFNbeta, huIFNgamma, huIFNomega, hybrid interferons, animal interferons such as EqIFNbeta, EqIFNgamma, or lymphokines such as interleukin-2, TNFbeta, or monokines such as interleukin-l, TNFalpha; growth factors, e.g. epidermal growth factor (EGF); anticoagulants, e.g. vascular anti-coagulant proteins VAC alpha, VAC beta), antithrombins; fibrinolytics, e.g. tPA, urokinase; proteins with an anti-allergic activity, e.g. IgE binding factor; therapeutically active enzymes, e.g. lysozyme, superoxide dismutases. The proteins used may either be of natural origin or produced by a recombinant method.

The range of indications will depend on the biological activity of the protein which is to be applied; within the specific spectrum for each protein, any application is possible which requires topical administration of the active substance. The content of therapeutically active protein in the pharmaceutical composition will naturally depend on the activity of the protein, the needs of the j /y "1 j S6 particular indication and the type of composition used.

It may span a wide range of quantities.

Suitable forms for administration include, in particular, hydrogels, suppositories and forms for vaginal use.

The use of excipients, carriers and additives will depend on the particular application selected, whilst care should be taken to ensure that they do not affect the stability of the protein by the type and quantity used.

The pharmaceutical composition according to the invention may contain, preservatives such as p-hydrobenzoate derivatives (nipa esters, methylparaben), sorbic acid, chlo,'hexidine digluconate, benzalkonium chloride and hexadecyltrimethyl ammonium bromide.

In order to accelerate the absorption of the active substance through the skin, permeation accelerators such as dimethylsulphoxide or taurogly-colic acid may be added to the pharmaceutical composition.

Hydrogel forming agents which may be used include gelatine and cellulose derivatives such as methylcellulose, hydroxypropylcellulose and, in a particularly preferred embodiment, hydroxyethylcellulose, as well as synthetic polymers such as polyvinyl alcohol. The nature and quantity of the hydrogel forming agent used or the mixtures thereof will depend on the particular viscosity required. With regard to the fine dispersion of the stabiliser it should be borne in mind that when the gel has a higher viscosity, the stability of the emulsion is under certain circumstances adequately ensured by the content of bydrogel forming agent and therefore there is no need to add an emulsifier. Buffer systems used are selected according to the optimum pH for the particular protein and matched to the particular application; both organic s and inorganic buffers may be used, e.g. succinate, 7 acetate and phosphate buffers.

The carrier used will depend on the form of administration; when the pharmaceutical preparation takes the form of a hydrogel the carrier is water.

Moisture-retaining substances such as glycerol, sorbitol, 1,2-propyleneglycol, butyleneglycol and polyols may be present in compositions according to the invention.

Hydrogel compositions according to the invention are so-called "low-filled" emulsions, because of their low oil content. Such compositions tend to break down easily, as is well known, so particular importance must be given to their stability.

The invention also provides a process for preparing pharmaceutical composition according to the invention.

To ensure very gentle production of a stable emulsion, in which the stability of the fine •e distribution of the stabiliser and hence its activity over a long period of time is ensured, a two-step "process is preferably used in the manufacture of a composition according to the invention, particularly a hydrogel.

In the first step, in a system of water/stabil-iser/ optionally emulsifier, a phase inversion from a W/o emulsion to an O/W emulsion is brought about and the fine pre-emulsion thus obtained is combined with the majority of the aqueous phase.

Thus in this preferred process of preparing a pharmaceutical composition a pre-emulsion is prepared by adding water to said hydrophobic substances and a finely dispersed emulsifier optionally contained therein, with stirring, until a coarse W/0 emulsion is obtained, stirring is then stopped until the emulsion has sedimented, and then resumed with the addition of more water to give a content of of up to about 50%, and phase inversion, the fine O/W pre-emulsion so produced is finely dispersed in a buffered solution, which 8optionally comprises preservatives and other adjuvants, a hydrogel forming agent is introduced and allowed to swell and finally a protein solution is added.

The following procedure is particularly preferred: first of all, a pre-emulsion is produced by the so-called "continental" method: the emulsifier is distributed in the paraffin oil and water is slowly added until a very coarse W/O emulsion is formed. At this stage, which is reached when the water content is about 20-40%, according to our experiments, the mixing process is stopped and the emulsion is briefly allowed to settle. When mixing is subsequently resumed and water is added up to a content of about 50%, the emulsion is inverted to form a fine O/W emulsion.

During the second step of the process the pre-emulsion obtained is stirred into the buffer solution and dispersed, after which the hydrogel forming agent is added and allowed to swell. The time at which the protein solution is added is not critical; this is preferably the final step of the process. Using the preferred process according to the invention, extremely stable emulsions are obtained which show no tendency to separate after half a year's stor&ge at room temperature.

In the case of smaller quantities or when technically more complicated homogenisers such as nozzle homogenisers are available, an O/W emulsion may also be produced in a single step without the preparation of a pre-emulsion; however, the process which is preferred according to the invention provides a method of manufacture which not only produces a stable emulsion but is also simple, requires little energy or complex technology and is at the sane time subjects the active protein to little shear.

The examples which follow illustrate the invention with reference to hydrogel formulations containing IFN alpha, IFN gamma and TNF alpha as the therapeutically I -4o *I Yy 3 ,P K~fi 9 active protein. These examples are not intended to limit the scope of the invention.

Example 1 100 grams of gel contain: IFN gamma 0.1 g Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g dipotassium hydrogen phosphate trihydrate 0.04 g Natrosol 250 HX (hydroxyethylcellulose) 1.75 g Polysorbate 20 0.1 g Thin-liquid paraffin oil 1.0 g Deionised water ad 100 g 96.76 g The hydrogel was produced by the preferred two-step method: S" a) Preparation of the pre-emulsion The phosphates and the preservative, methylparaben, were dissolved in hot water at 80°C, with stirring, and the solution was then cooled to ambient temperature and then filtered to sterilise it. The emulsifier polysorbate 20 was distributed in the paraffin oil using a fast-rotating homogeniser.

Sufficient water was added slowly, with stirring, to produce an approximately 30% coarse W/O emulsion. This emulsion was briefly left to stand, whereupon it separated. After the stirrer was switched on again the emulsion was brought to the point of phase inversion, to produce a very finely cispersed O/W emulsion.

b) Preparation of the hydrogel The paraffin oil emulsion was stirred into the i sterile-filtered buffer solution and finely dispersed therein. Then microbiologically pure hydroxyethylcellulose was sprinkled into the emulsion and distributed therein with stirring. To obtain total swelling, the gel was left to swell for 10-15 hours under laminar flow. Finally, the IFN gamma solution, adjusted to 4 mg/ml, was slowly stirred in. This mixture was transferred into sterile tubes under laminar air flow conditions.

The course of the storage experiments is shown in Fig. 1. As can be seen from the diagram, the addition of paraffin oil ensures that the activity of IFN-gamma, S measured by the ELISA test (the anti-bodies used bind biologically active proteins for which they are specific), is maintained; the slight drop shown in the diagram is not significant in viow of the test distribution.

Fig. 2 shows a comparison test with gelatine as a constituent of a hydrogel formulation without the separate addition of a stabiliser, showing the clearly S' destabilising effect of gelatine on IFN-gamma.

Consequently, when gelatine is used as a hydrogel forming agent, the addition of an effective stabiliser is absolutely essential.

Fig. 3 shows the stability pattern over a period of months (in this diagram, and in Figs. 4, 5 and 6, the log. nat. of the concentration of the therapeutically active protein is shown on the y axis).

Example 2 100 g of gel contain: IFN gamma 0.1 g Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g Dipotassium hydrogen phosphate trihydrate 0.04 g 11 Natrosol 250 HX 1.75 g Pluronic F68 0.1 g Thin liquid paraffin oil 1.0 g Deionised water ad 100 g 96.76 g The phosphates, the preservative methylparaben and the emulsifier Pluronic F68 were dissolved in hot water at 800C with stirring and the solution was then cooled to ambient temperature and filtered to sterilise it.

The paraffin oil was introduced and finely dispersed therein by means of an homogeniser. Then the hydroxyethyl-cellulose was added with stirring in vacuo.

Finally, the IFN-gamma solution, adjusted to 4 mg/ml, was added. The mixture was transferred as described in Example 1.

Example 3 100 g of gel contain: trll TNF alpha 01 g M ethylparaben 0.213 g 7" Sodium dihydrogen phosphate monohydrate 0,033 g Dipotassium hydrogen phosphate trihydrate 0.0427 g Natrosol 250 HX 1.87 g Polysorbate 20 0.107 g Thin liquid paraffin oil 1. 07 g Deionised water ad 100 g 96.5443 g The hydroge was prepared as described in Example 1.

Example 4 00 g of gel contain: tFN alpha 0.0005 g I A4K)i 12 Methylparabe'n 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g Dipotassium hydrogen phosphate trihydrate 0.04 g Natrosoi 250 HX 1.75 g Polysorbate 20 0.1 g Thin liquid paraffin oil 1.0 g Deionised water ad 100 g 96.8595 g The hydrogel was prepared as described in Example 1.

Examnle 100 9 of gel contain: IFN gamma 0.100 g Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g Dipotassium hydrogen phosphate trihydrate 0.04 9 Tauroglyaolic acid 0.01 g Natrosol 250 UX 1,75g Poly8Qrbate 20 g Thd~n liquid paratffin oil 3..0 g Deionised water ad 100 g 96.75' g The hydrogel was prepared as in Example 1. and tauroglycolic acid was stirred into the buffer solution as a permeation accelerator.

Exaimple -6 100 g of gel conitain: TFN gamma 0.05i g lMethylparaben 0.2 g Sodium dihydrogen phosphate monohydrato 0.05 g Dipotassiumn hydrogen phosphata trihydrato 0.04 g Natrosol 250 R-X 1.75 g -13 Polysorbate 20 0.1 g Thin liquid 1j uzaff in oil 0.6 g Thick liquid paraffin oil 0.4 g Deionifed water ad 100 g 96.81 g The hydrogel was prepared as described in Example 1.

Example 7 100 g of gel contain: IFN gjamma 0.05 g Methylparaben 0.2 g Sodium dihycIogen phosphate monohydrate 0.05 g Disodium hydrogen phosphate trihydrate 0.04 g NatrosQl 250 liX 1.75 g Myr3styl alcohol 1.0 g rDeionised water ad 100 g 69 g The hydrogel was prepareO, as described in Example 2. The myristyl alco1r.'' was. dispersed in the sterile- filtered 1,)uffer solut~om which had been heate1. to about After the buffer, solution had coolecd, the procedure. was continued as described in Example 2.

Example 8 100 g of gel substance contain: IFN j~amma 0.005 g Methylparaben 0.20 g Succinate buffer pH- 6.00 0.0191 M Sodium chloride 0.1435 M Natrosol 250 HX 1.75 g Polysorbate 2Z0 0.0952 g Thin liquid paraffin oil 0.952 q Deionised water ad 100 g -14 The hydrogel was prepared as described in Example 1. The stability curve is shown in Fig. 4. Fig. 5 shows the curve of a comparison test in which the same formulation was used without any added paraffin oil. The results of the comparison test show a drop in stability shortly after manufacture.

Example 9 100 g of gel substance contain: IFN gamma 0.025 g Methylparaben 0.20 g Succinate (buffer pH 6.2) 0.2362 g Sodium chloride 0.8766 g Natrosol 250 HX 1.75 g Polysorbate 20 0.1 g LABRAFIL 1944 CS 1.0 g Deionised water ad 100 g 4 1 o' The hydrogel was prepared as described in Example 1.

Example a a o0 100 g of gel substance contain: IFN gamma 0.025 g Methylparaben 0.20 g Succinate (buffer pH 6.2) 0.2362 g Sodium chloride 0.8766 g Natrosol 250 HX 1.75 g Polysorbate 20 0.1 g LABRAFIL 2735 CS 1.0 g Deionised water ad 100 g The hydrogel was prepared as described in Example 1.

i. -i 15 Example 11 100 g of gel substance contain: IFN gamma Methylparaben Succinate (buffer pH 6.2) Sodium chloride Natrosol 250 HX Polysorbate 20 Myristyl alcohol Deionised water 0.025 g 0.20 g 0.2362 g 0.90 g 1.75 g 0.1 g 1.0 -g ad 100 g t

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S-

.4 *5f4 The hydrogel was prepared as follows: the myristyl alcohol was melted at 50-60°C and then the pre- emulsion was prepared as described in Example 1 but at 50-60°C.

The rest of the method was as in Example 1. The stability curve is shown in Fig. 6.

Example 12 100 g of gel substance contain: TNF beta Methylparaben Sodium dihydrogen phosphate monohydrate Dipotassium hydrogen phosphate trihydrate Natrosol 250 HX Polysorbate .0 Thin liquid paraffin oil Deionised water 0.05 g 0.2 g 0.05 g 0.04 g 1.75 g 0.2 g 2.0 g ad 100 g The hydrogel was prepared as described in Exampi, i.

Example 13 100 g of gel substance contain: 16 Lysozyme mnill ion 2.4 4 4 *449*4 4.

.4 4 4 .4,9 9* 449 4 44 9.

*9 .444 4 Methylparaben Sodium dihydrogen phosphate inonohydrate Dipotassium hydrogen phosphate trihydrate Natrosol 250 liX Polysorbate 20 r!,Ikin liquid1 paraffin oil Deionised water The hydrogel was prepared as in Example 1.

Exampl1e 14 100 g of gel substance contain: VAC alpha Methylparaben Sodium d hydrogen phosphate monohydrate Dipotassiun hydrogen phosphate trihydrate Natrosol 250 HX Polysorbate 20 Thin 1.,quid paraffin oil Deionised water The hydrogel was prepared as in Example 1.

units 0.2 g 0.05 g 0.04 g 1.75 g 0.2 g 2.0 g ad 100 g 0.03 g 0.2 g 0,05 g 0.04 g 1.75 g 0.1 g 1.0 g ad 100 g

Claims (22)

1. A pharmaceutical composition suitable for topical application which comprises one or more therapeutically active proteins, together with any one or more conventional excipients, carriers or adjuvants, and one or more physiologically acceptable hydrophobic substances in finely dispersed form in a quantity sufficient to stabilise the protein.

2. A pharmaceutical composition as claimed in claim 1, wherein the one or more therapeutically active proteins is/are selected from a natural or recombinant, human or S animal protein, selected from interferons, TNFa, TNFB, and t-PA, including the structurally similar bioactive equivalents thereof, individually or in a therapeutically useful mixture, in a therapeutically active quantity.

3. A pharmaceutical composition as claimed in claim 1 or 2 wherein said physiologically acceptable hydrophobic substances comprise one or more paraffin oils.

4. A pharmaceutical composition as claimed in any one of the preceding claims, comprising as said hydrophobic substance paraffin oil or mixtures of paraffin oils in a quantity of from 0.1 to based on the total weight of the composition.

A pharmaceutical composition as claimed in any one of the preceding claims, comprising an emulsifier which serves to produce a fine dispersion of the hydrophobic substance.

6. A pharmaceutical composition as claimed in claim wherein said emulsifier is non-ionic.

7-R* r 0 18 7. A pharmaceutical composition as claimed in any one of the preceding claims, in the form of a hydrogel.

8. A pharmaceutical composition as claimed in claim 7, comprising as hydrogel forming agents, cellulose derivatives, gelatine or synthetic polymers such as polyvinyl alcohol either individually or in admixture.

9. A pharmaceutical composition as claimed in claim 8, comprising hydroxyethylcellulose as said hydrogel forming agent.

A pharmaceutical composition as claimed in any one of the preceding claims, comprising a physiologically acceptable preservative.

11. A pharmaceutical composition as claimed in claim comprising a preservative selected from p-hydroxybenzoate derivatives, sorbic acid, chlorhexidine digluconate, benzalkonium chloride and hexadecyltrimethyl ammonium bromide either individually or in admixture. 'T

12. A pharmaceutical composition as claimed in any one of the preceding claims, comprising as adjuvants, permeation accelerators and/or moisture retaining agents and/or a buffer system.

S13. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the level of activity of the protein after storage at 4-8"C over a period of at least 12 months is substantially unchanged.

14. A process of preparing a pharmaceutical composition as claimed in any one of claims 1 13, comprising a first step in which a system comprising one or more hydrophobic substances/water/optionally emulsifier, is I p'^ 19 phase inverted from a W/O emulsion to an O/W emulsion and the fine pre-emulsion thus obtained is combined with the majority of the aqueous phase.

A process of preparing a pharmaceutical composition as claimed in claim 14, in which a pre-emulsion is prepared by adding water to said hydrophobic substances and a finely dispersed emulsifier optionally contained therein, with stirring, until a coarse W/O emulsion is obtained, stirring is then stopped until the emulsion has sedimented, and then resumed with the addition of more water to give a content of of up to about 50% and S phase inversion, the fine O/W pre-emulsion so produced is finely dispersed in a buffered solution, which E' optionally comprises preservatives and other adjuvants, S a hydrogel forming agent is introduced and allowed to swell and finally a protein solution is added.

16. Use of one or more physiologically acceptable hydrophobic substances in finely dispersed form for stabilising therapeutically active proteins in pharmaceutical compositions for topical application.

S17. Use as claimed in claim 16, wherein the hydrophobic substance is paraffin oil.

18. Use as claimed in claim 16 and 17 wherein the pharmaceutical composition is in the form of a hydrogel.

19. A pharmaceutical composition, as claimed in claim 1, substantially as hereinbefore described.

A pharmaceutical composition substantially as herein described and as illustrated in the examples.

21. A process of preparing a pharmaceutical composition, as claimed in claim 1, substantially as f 20 herein described.

22. A method for the topical administration of and therapeutically active protain wherein said protein is comprised in a pharmaceutical composition according to any one of claims 1-13, 19 or DATED this 17th day of July, 1991 BOEHRINGER INGELdIEIM INTERNATIONAL GmbH By its Patent Attorneys DAVIES COLLISON 0S S S S S St *S S #4 S St t~ S. S S $9 .4 S 4. S. Sk 5*44S* S A