NZ226209A

NZ226209A - Pharmaceutical composition for topical administration comprising therapeutically active protein and a hydrophobic substance for stabilising the protein

Info

Publication number: NZ226209A
Application number: NZ226209A
Authority: NZ
Inventors: Traute Mundorf; Kurt Schnecker
Original assignee: Boehringer Ingelheim Int
Priority date: 1987-09-17
Filing date: 1988-09-16
Publication date: 1991-06-25
Also published as: DK516288D0; EP0307857A1; FI95770B; NO884123L; DK516288A; DE3731255A1; ES2077559T3; KR890004719A; ATE126063T1; DE3854290D1; HU203045B; FI884240A0; PT88539B; NO884123D0; NO179436C; IL87778A; IE882812L; CA1329121C; JPH01102029A; FI95770C

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

<div class="application article clearfix" id="description"> 22 6 2 0 9 Priority Date(s): ITi. 7).'. Complete Specification Filed:' jr.3.', 9»M: . kfeiKsa/ssy Public* Date: 5JU(J WM P.O. Journal. No: Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION STABILISATION OF THERAPEUTICALLY ACTIVE PROTEINS IN A PHARMACEUTICAL COMPOSITION ](/We, BOEHRINGER INGELHEIM INTERNATIONAL GMBH, a body corporate organised under the laws of the Federal Republic of Germany of D-6507 Ingelheim am Rhein, FEDERAL REPUBLIC OF GERMANY hereby declare the invention, for which JC'we pray that a patent may be granted to p^/ns, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 - (followed by page la) — 1 B— 27ES010-568J2 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 for 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. Various substances for stabilising interferons have already been proposed, and for example hydroxyethylcellulose has been used as a carrier substance for the preparation of gels or ointments containing interferon. However, there was some loss of activity of the interferons which could only be reduced by the addition of a protease inhibitor (EP-A-15234 5). 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 (followed by page 2) n -2 5 2 0 9 proteins and polypeptides such as interferons, more particularly IFN-gamma, in parenteral preparations, (EP—A-162332). Japanese Published Patent Application JP-A-61-5 -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, 10 dextrose and buffer substances are proposed as • stabilisers for an ointment comprising the synergistic j combination IFN-beta/9-(1,3-dihydroxy-2-propoxy- methylguanine (DHPG). A stabilising effect to the standard required 15 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 20 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 O composition, especially with respect to good availabi- 25 lity 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. T Various categories of substances have been 30 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 35 effect on various therapeutically active proteins which we believe to be superior to the effect of the substances proposed up till now. This result r> _ , . 220200 is all the more surprising as the pharmaceutical preparations for topical use which belong to the prior art, such as ointments, in which hydrophobic substances are used as carriers in a suitably large 5 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 10 more therapeutically active proteins, conventional excipients, carriers and/or adjuvants, and-one or more physiologically acceptable hydrophobic substances in finely dispersed form in a quantity sufficient to stabilise the protein. 15 Preferably one or more hydrophobic substances comprise one or more paraffin oils and the 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 20 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 25 of the protein after storage at 4-8°C 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 O that an exact pH value is maintained, since the 30 stabilising addition, of one or more hydrophobic substances reduces the vulnerability of the proteins to fluctuations in the pH value. This advantage is of particular importance for applications which require lower pH values, e.g. application in the 35 vaginal area. The pharmaceutical composition according to the invention also has the advantage, when in O - 4 - 22 62 09 the form of a hydrogel, of being extremely pleasant to use. This is because, even after the gel has dried, the presence of the hydrophobic substance ensures that the coating applied is soft to the touch, which is a particular advantage when applied to the lip area. The advantageous stabilising effect of hydrophobic substances on proteins can possibly be put down to hydrophobic interactions which have hitherto been 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 coat. 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 oil/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 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, T> 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 of from 0.1 to 2.0*. based on the total weight of the composition. In order to ensure that the stabiliser is finely dispersed and the distribution is stable, 5 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 viscosity thereof; in general, it is not more than 1%. Suitable emulsifiers include, 10 in particular, non-ionic emulsifiers such as polysor-bates (polyoxyethylene(n)sorbitanmonolaurate, e.g. TweenR 20), nonoxynol (polyoxyethylene (n)-nonylphenyl-ether, e.g. TritonR N101, TritonR Nlll), and poloxamer (polyethylenepolvpropyleneglycol, PluronicR F68). 15 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 20 to the invention is suitable for the administration of human and animal proteins such as those listed below, including their structurally similar bioactive equivalents (i.e. those proteins which have substantially the same biological activity with a different 25 amino acid sequence): cytokines, e.g. interferons such as huIFNalpha, huIFNbeta, huIFNgamma, huIFNomega, hybrid interferons, animal interferons such as EqIFNbeta, EqIFNgamnta, or lymphokines such as inter-leukin-2, TNFbeta, or monokines such as interleukin-1, 30 TNFalpha; growth factors, e.g. epidermal growth factor (EGF); anticoagulants, e.g. vascular anticoagulant proteins (e.g. VAC alpha, VAC beta), antithrombins; fibrinolytics, e.g. tPA, urokinase; proteins with an anti-allergic activity, e.g. IgE 35 binding factor; therapeutically active enzymes, e.g. lysozyme, superoxide dismutases. The proteins used may either be of natural, cjrigin or produced by - 6 - 22 6 2 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 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, methyl-paraben), sorbic acid, chlorhexidine 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, hydroxyethyl-cellulose, 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 22(5203 n - 7 - required. *.vith regard to the fine dispersion of the stabiliser it should he borne in mind that when the gel has a higher viscosity, the stability of the emulsion is under certain circumstances O 5 adequately ensured by the content of hydrogel 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 C 10 and inorganic buffers may be used, e.g. succinate, 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. 15 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 20 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 25 preparing a pharmaceutical composition according to the invention. To ensure very gentle production of a stable emulsion, in which the stability of the fine dispersion of the stabiliser and hence its activity over 30 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/stabiliser/optionally emulsifier, a phase inversion from 35 a W/O emulsion to an 0/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 s phamaceutical composition a pre-e-ulsion is prepared by adding water to said hydrophobic substances and a finely dispersed snulsifier . optionally contained 5 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 10 O/W pre-emulsion so produced is finely dispersed in a buffered solution, which optionally comprises preservatives and other adjuvants, a hydrogel forming agent is introduced and allowed to swell and finally a protein solution is added. 15 The following procedure is particularly pre ferred: 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 20 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 25 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 30 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 35 separate after half a year's storage at room temperature. o 4 4 25 30 35 22 6 2 0 9 - 9 - 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 5 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 10 same 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 15 the therapeutically active protein. These examples are not intended to limit the scope of the invention. Example 1 20 100 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: a) Preparation of the pre-emulsion The phosphates and the preservative, methy1-paraben, were dissolved in hot water at 80°C, with ^ v-,-«.•».* -*»-.c /r,^vyA -~ .*n-. . » 22l>2 _ - r. _ stirrinq, ann the solution was then cooleo 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 dispersed O/W emulsion. b) Preparation of the hydrogel The paraffin oil emulsion was stirred into the sterile-filtered buffer solution and finely dispersed therein. Then microbiologically pure hydroxyethyl-cellulose 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, measured by the ELISA test (the antibodies used bind biologically active proteins for which they are specific), is maintained; the slight drop shown in the diagram is not significant in view 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 destabilising effect of gelatine on IFN-gamma. Consequently, when gelatine is used ^ _ n _ 220200 as a hydrogel forming acent, the addition of an effective stabiliser is absolutely essential. Fig. 3 shows the stability pattern over a period of 15 months (in this diagram, and in Figs. 5 4, 5 and 6, the log. nat. of the concentration of the therapeutically active protein is shown on the y axis). 10 Example 2 100 g of gel contain: IFN gamma 0.1 9 Methylparaben 0.2 g 15 Sodium dihydrogen phosphate monohydrate 0.05 g Dipotassium hydrogen phosphate trihydrate 0.04 g Natrosol 250 HX 1.75 g Pluronic F68 0.1 g Thin liquid paraffin oil 1.0 g 20 Deionised water ad 100 g 96.76 g The phosphates, the preservative methylparaben and the emulsifier Pluronic F68 were dissolved in hot water at 80°C with stirring and the solution 25 was then cooled to ambient temperature and filtered to sterilise it. The paraffin oil was introduced ) and finely dispersed therein by means of an honogeniser. Then the hydroxyethylcellulose was added with stirring rn vacuo. Finally, the IFN-gamma solution, adjusted 30 to 4 mg/ml, was added. The mixture was transferred as described in Example 1. Example 3 35 100 g of gel contain: TNF alpha Methylparaben 0.1 g 0.213 g c 22 6 2 0 9 - 12 - 3 Sodium dihydrogen phosphate monohydrate 0.053 g Dipotassium hydrogen phosphate trihydrate 0.0427 g Natrosol 250 HX 1.87 g ! ° Polysorbate 20 0.107 g 5 Thin liquid paraffin oil 1.07 g Deionised water ad 100 g 96.5443 g The hydrogel was prepared as described in Example 1. 10 Example 4 100 g of gel contain: IFN alpha 0.000 5 g 15 Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g Dipotassium hydrogen phosphate trihydrate 0.04 g Natrosol 250 HX 1.75 g Polysorbate 20 0.1 g 20 Thin liquid paraffin oil 1.0 g Deionised water ad 100 g 96.8595 g The hydrogel was prepared as described in Example 1. 25 Example 5 100 g of gel contain: IFN gamma 0. .100 g 30 Methylparaben 0. ,2 g Sodium dihydrogen phosphate monohydrate 0. .05 g Dipotassium hydrogen phosphate trihydrate 0. .04 g Tauroglycolic acid 0. .01 g Natrosol 250 HX 1. .75 g 35 Polysorbate 20 0. .1 g Thin liquid paraffin oil 1. .0 g Deionised water ad 100 g 96. .75 g i 4 p o Q _ . .. „ 22o200 - 1 3 - The hydrogel was preoared as in Example 1 and tauro-glycolic acid was stirred into the buffer solution as a permeation accelerator. 5 Example 6 100 g of gel contain: ; O IFN gamma 0.05 g 10 Methylparaben 0.2 g > Sodium dihydrogen phosphate monohydrate 0.05 g < Dipotassium hydrogen phosphate trihydrate 0.04 g Natrosol 250 HX 1.75 g Polysorbate 20 0.1 g 15 Thin liquid paraffin oil 0.6 g Thick liquid paraffin oil 0.4 g Deionised water ad 100 g 96.81 g 20 The hydrogel was prepared as described in Example 1. Example 7 100 g of gel contain: 25 30 IFN gamma 0.05 g Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g Disodium hydrogen phosphate trihydrate 0.04 g Natrosol 250 HX 1.75 g Myristyl alcohol 1.0 g Deionised water ad 100 g 96.91 g The hydrogel was prepared as described in Example 2. The myristyl alcohol was finely dispersed in the sterile-35 filtered buffer solution which had been heated to about 60°C. After the buffer solution had cooled, the procedure was TOlntinued; as-denser^bed in Example 2. 25 BAR 1991 - 14 - Example 8 11 6 2 0 9 100 g of gel substance contain: 10 IFN gamma Methylparaben Succinate buffer pH 6.00 Sodium chloride Natrosol 250 HX Polysorbate 20 Thin liquid paraffin oil Deionised water 0.005 g 0.20 g 0.0191 M 0.1435 M 1.75 g 0.0952 g 0.952 g ad 100 g The hydrogel was prepared as described in Example 1. 15 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. 20 Example 9 100 g of gel substance contain: 25 IFN gamma 0.025 g Methylparaben 0.20 g Succinate (buffer pH 6.2) 0.2362 g Sodium chloride 0.8766 g O Natrosol 250 HX 1.75 g 30 Polysorbate 20 0.1 g LABRAFIL 1944 CS 1.0 g Deionised water ad 100 g The hydrogel was prepared as described in Example 1. 35 22 6 2 0 9 - 15 -Example 10 100 g of gel substance contain: 5 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 10 Polysorbate 20 0.1 g LABRAFIL 2735 CS 1.0 g Deionised water ad 100 g 15 The hydrogel was prepared as described in Example 1. Example 11 100 g of gel substance contain: 20 IFN gamma 0.025 g Methylparaben 0.20 g Succinate (buffer pH 6.2) 0.2362 g Sodium chloride 0.90 g O Natrosol 250 HX 1.75 g 25 Polysorbate 20 0.1 g Myristyl alcohol 1.0 g Deionised water ad 100 g The hydrogel was prepared as follows: the myristyl 30 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. 35 ... - 16 - Example 12 100 g of gel substance contain: 22 6 2 10 TNF beta 0.05 g Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g Dipotassium hydrogen phosphate trihydrate 0.04 g Natrosol 250 HX 1.75 g Polysorbate 20 0.2 g Thin liquid paraffin oil 2.0 g Deionised water ad 100 g The hydrogel was prepared as described in Example 1. 15 Example 13 100 g of gel substance contain: 20 Lysozyme 2.4 million units Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g Dipotassium hydrogen phosphate trihydrate 0.04 g 25 Natrosol 250 HX 1.75 g Polysorbate 20 0.2 g Thin liquid paraffin oil 2.0 g Deionised water ad 100 g 30 The hydrogel was prepared as in Example 1. - 17 - 22 6 2 Example 14 100 g of gel substance contain: VAC alpha 0.03 g Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g Dipotassium hydrogen phosphate trihydrate 0.04 g Natrosol 250 HX 1.75 g Polysorbate 20 0.1 g Thin liquid paraffin oil 1.0 g Deionised water ad 100 g The hydrogel was prepared as in Example 1. *sjK-i1wu. ._ ,C. 1 - 18 -WHAT V7E CLAIM IS: 1. A pharmaceutical composition suitable for topical application which comprises one or more 5 therapeutically active proteins, conventional exci-pients, carriers and/or adjuvants, and one or more physiologically acceptable hydrophobic substances in finely dispersed -form ir a quantity sufficient to stabilise the protein. 10 2. A pharmaceutical composition as claimed in claim 1, comprising a natural or recombinant, human or animal protein, selected from interferons, TNFa, TNF8, and t-PA, including the structurally similar 15 bioactive equivalents thereof, individually or in a therapeutically useful mixture, in a therapeutically active quantity. 3. A pharmaceutical composition as claimed in </div>

Claims

<div class="application article clearfix printTableText" id="claims">
20 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 25 of the preceding claims, comprising as said hydrophobic substance paraffin oil or mixtures of paraffin oils in a quantity of from 0.1 to 3%, based on the total weight of the composition. 

30
5. 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. 

35
6. A pharmaceutical composition as claimed in claim 5, wherein said emulsifier is non-ionic. 

o 

220200 

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7. 2) pharmaceutical composition as claimed in any one of the preceding claims, in the form of a hydrogel.
8. A pharmaceutical composition as claimed in 5 claim 7, comprising as hydrogel forming agents, 

cellulose derivatives, gelatine or synthetic polymers such as polyvinyl alcohol either individually or in admixture. 

10
9. A pharmaceutical composition as claimed in claim 8, comprising hydroxyethylcellulose as said hydrogel forming agent.
10. \ pharmaceutical composition as claimed in 

15 any one of the preceding claims, comprising a physiologically acceptable preservative.
11. A pharmaceutical composition as claimed in claim 10 comprising a preservative selected from 

20 p-hydroxybenzoate derivatives, sorbic acid, chlor-hexidine digluconate, benzalkonium chloride and hexadecyltrimethyl ammonium bromide either individually or in admixture. 

25
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. 

30
13. 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. 

35
14. A process of preparing a pharmaceutical composition as claimed in any one of claims 1 - 13, comprising 

226209 

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a first step in which a system comprising one or more hydrophobic substances/water/optionally emulsifier, is phase inverted fran a W/O emulsion to an O/W emulsion and the fine emulsion thus obtained 5 is combined with the majority of the aqueous phase.
15. 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 

10 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 15 of of up to 50% and phase inversion, the fine O/W pre-emulsion so produced is finely dispersed in a buffered solution, which optionally comprises preservatives and other adjuvants, a hydrogel forming agent is introduced and allowed to swell and finally 20 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 

25 pharmaceutical compositions for topical application.
17. Use as claimed in claim 16, wherein the hydrophobic substance is paraffin oil.
30 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 35 in claim 1, substantially as In i ifrhln I 1 I i nlnijfo 

226209 

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20. A pharmaceutical composition substantially as herein described and as illustrated in the examples
21. A process of preparing a pharmaceutical composi-5 tion, as claimed in claim 1, substantially as herein described.
22. A method for the topical administration of a therapeutically active protein to a non-human subject 10 wherein said protein is comprised in a pharamceutical composition according to any cme.of claims 1-13, 19 or 20. 

BOEHRINGER INGELHEIM INTERNATIONAL GMBH 

By Their Attorneys BALDWIN, SON & CAREY 



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