CA1329121C

CA1329121C - Stabilisation of therapeutically active proteins in pharmaceutical preparations

Info

Publication number: CA1329121C
Application number: CA000577614A
Authority: CA
Inventors: Traute Mundorf; Kurt Schnecker
Original assignee: Boehringer Ingelheim International GmbH
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 1987-09-17
Filing date: 1988-09-16
Publication date: 1994-05-03
Anticipated expiration: 2011-05-03
Also published as: AU615473B2; DK516288D0; RU2093145C1; IE882812L; NO884123L; ATE126063T1; DE3731255A1; PT88539B; IL87778A0; HU203045B; IL87778A; JPH01102029A; KR890004719A; EP0307857A1; ZA886903B; FI884240A0; FI884240A; JP2783552B2; ES2077559T3; NO179436B

Abstract

Abstract Stabilisation of therapeutically active proteins in a pharmaceutical composition A pharmaceutical composition suitable for topical application which comprises one or more therapeutically active proteins, conventional exci-pients, carriers and/or adjuvants, and one or more physiologically acceptable hydrophobic substances in finely divided form in a quantity sufficient to stabilise the protein, e.g. paraffin oil in an amount of e.g. 0.1 to 3% by weight.

Description

- ~32~2 11 Stabilisation of therapeutical~y active proteins in a pharmaceutical com~osition 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 applica-tion of therapeutically active proteins is their stability in the pharmaceutical formulation. The stability must be ensured for a sufEiciently 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-142345~.
It has also been proposed to stabilise inter-ferons 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 ,~ ~

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proteins and polypeptides such as interferons, more particularly IFN-gamma, in parenteral prepara-tions, (EP-~-162332). ~
Japanese Published Patent Application JP-~-61--277633 discloses stabilising interferons in solution with certain surface-active substances.
EP-A-135171 mentions human serum albumin as a suitahle 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-propoxy-methylguanine (DHPG).
~ stabilising effect to the standard required has not yet been achieved with the substances proposed hitherto ~or 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 eor 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 availabi-lity of the active substance, the full developmentof its activity and a mild method of preparation which takes account of the vulnerability of the proteins to shear forces.
Various categories of substances have been 3n 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 ~ ~ ' i . : .
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~329121 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 proportion require the separate addition of a stabi-liser for the protein.
Accordingly, a first aspect of the invention provides a pharmaceutical composition suitable for topical application which comprises one or more therapeutically act;ve proteins, conventional exciplents, carriers and~or ad~uvants, and one or more physiologically acceptable hydrophobic substances in finely divided ~orm in a quantity sufficient to stabilise the protein.
Preferably one or more hydrophobic substances comprise one or more parafEin oils and the composition is in the Eorm oE a hydrogel. ~ith the addition Oe a stabilising quantity of one or more hydrophobic substances in finely divided form, a pharmaceutical composition is obtained which, under the conditions of use, make the active substance available in active fo~m 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 4-8C 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 ~ 30 stabilising addition of one or more hydrophobic i 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 vaginal area.
The pharmaceutical composition according to the invention also has the advantage, when in , , .": , , . . .
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` 132~21 the form o 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 i5 so~t to the touch, which is a particular advantage when applied to the lip area.
The advantageous stabilising effect of hydro-phobic substances on proteins can possibly be put down to hydrophobic interactions ~hich have hitherto been scarcely noticed if at all. Stabilising proteins according to the prior ark appears to rely on two principles: a) stabilising by complex binding of the substance to the protein and hence steric ~ixing of the protein molecule; and b) binding of free bul~ water by polar substances and hence stabilising the protein by influencing its hyc~rate 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 interace, so that the hydrophobic regions project into the oil droplet and the hydrophi~
lic parts project into the polar phase.
Suitable hydrophobic substances include, in addition to the preferred paraffin Oilsr 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, glycerides ~LabrafilR)~ individually or in admixture.
Qf the paraffin oils, liquid, thin-liquid or thick-liquid paraffin oil according to Ph. Eur. and USPor mixtures thereof are suitable. The hydrophobic substances are preferably present in an amount ., ~ , .:

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of from O.l to 3.0% based on the total weight of the composition.
In order to ensure that the stabiliser is finely divided 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 viscosity thereof; in general r it is not more than l~. Suitable emulsifiers include, in particular, non-ionic emulsifiers such as polysor-bates (polyoxyethylene(n)sorbitanmonolaurate, e.g.
TweenR 20), nonoxynol (polyoxyethylene(n)-nonylphenyl-ether, e.g. TritonR NL01, ~ritonR Nlll), and poloxamer (polyethylenepolypropyleneglycol, PluronicR F68).
If the pharmaceutical preparation is in the ~orm of a hydrogel, the emulsifiers will not only bring about a fine distribution of the stabiliser but will also im~rove the spreading of the gels.
The pharmaceutical composition according to the invention is suitable or the administration o~ human and animal proteins such as those listed below, including their structurally similar bioactive equivalents (i.e. those proteins which have substan-tially the same biological activity with a different amino acid sequence1: cytokines, e.g. interferons such as huIFNalpha, huIFNbeta, huIFNgamma, huIFNomega, hybrid interferons, animal interferons such as ; EqIFNbeta, EqIFNgamma, or lymphokines such as inter-leukin-2r TNFbeta, or monokines such as interleukin-l, TNFalpha; growth factors, e.g. epidermal growth factor (EGF); anticoagulants, e.g. vascular anti-coagulant prGteins (e.g. VAC alphal VAC beta~, antithrombins; fibrinolytics, e.g. tPA, urokinase;
proteins with an anti-allergic activity, e~g. IgE
3S binding factor; therapeutically active enzymes, e.g. lysozyme, superoxide dismutases~ The proteins used may either be of natural origin or produced by J

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- ~32~121 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 ~articular 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, careiers and additives will depend on the particular application selected, whilst care should be taken to ensure that they do not af~ect 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 a~monium 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 composi-tion.
Hydrogel forming agents which may be used include qelatine 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 , :~
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1~%~7 required. With regard to the fine distribution 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 hydrogel forming agent and therefore there is no need to add an emulsifier. BufEer systems used are selected according to the o~timum pH for the particular protein and matched to the particular application; both organic 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 preparatlon takes the orm of a hydrogel the carrier is water.
~5 Moisture-retaining substances such as ~lycerol, sorbitol, l,2-propyleneglycol, butylene~lycol and polyols may be present in compositions according to the invention.
~ ydrogel 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.
~he invention also provides a process for preparing a pharmaceutical composition according to the invention.
To ensure very gentle production of a stable emulsion, in which the stability of the fine distribu-tion 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/stabili-ser/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.

~32~21 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 divided 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 phase inversion, the fine O/W pre-emulsion so produced is finely divided 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.
The following procedure is particularly pre-ferred: ~irst of all, a pre-emulsion is produced by the so-called "continental" method: the emulsiEier 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 sto~ped 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. ~uring 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 cri~ical; 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 storage at room tempera-~: ture.

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. . g 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 provdes 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 same time su~jects the active protein to little shear.
The examples which follow illustrate the invention with reference to hydrogel formulations containing IFN alphaj IF~ gamma and TNF alpha as the therapeutically active protein. These examples are not intended to limit the scope o~ the inventlon.

Example 1 100 grams o gel contain:

IFN gamma 0.1 g Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0O05 g 25 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 ~ethod:
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a) Preparation of the pre-emulsion The phosphates and the preservative, methyl-paraben, were dissolved in hot water at 80C, with ~. . . ~, :

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stirring, and the solution was then cooled to ambient temperature and then ~iltered 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. ~fter the stirrer was switched on again the ~mulsion was brought to the point of phase inversion, to produce a very finely divided O/W emulsion.

b) Preparation of the~hydrogel rrhe paraffin oil emulsion was stirred into the sterile-filtered bueer solution and finely divicled therein. Then microbiologically pure hydroxyethyl-cellulose was sprinkled into the emulsion and distri-buted 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, ~easured 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 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 ,, . :
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1329~21 as a hydrogel forming agent, 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.
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 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 80C with stirring and the solution 25 was then cooled to ambient temperature and filtered to sterilise it. The paraffin oil was introduced and distributed therein by means of an homogeniser.
Then the hydroxyethylcellulose was added with stirring ; in vacuo. Finally, the IFN-gamma solution, adjusted , 30 to 4 mg/ml, was added. The mixture was transferred as described in Example 1.

I Example 3 m 35 100 g of gel contain:

TNF alpha 0.1 g Methylparaben 0,213 g :, :
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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 gDeionised water ad lO0 g 96.5443 g The hydrogel was prepared as described in Example 1.

~xample 4 lO0 g of gel contain:

IFN alpha 0-0005 g 15 Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 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 lO0 9 96.8595 g The hydrogel was prepared as described in Example 1.

_ample 5 lO0 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 9 Natrosol 250 HX 1.75 g 35 Polysorbate 20 Ool g - . Thin liquid paraffin oil 1.0 g Deionised water ad lO0 9 96.75 g .
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--" 132~21 ~ 13 -The hydrogel was prepared as in Example 1 and tauro-glycolic acid was stirred into the buffer solution as a permeation accelerator.

Example 6 100 g of gel contain:

IFN gamma 0.05 g 10 Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g Dipotassium hydrogen phosphate tr;hydrate 0.04 g Natrosol 250 HX 1.75 g Polysorbate 20 0.1 g 15 Thin liquid parafin oil 0.6 g Thick liquid paraEin oil 0.4 g Deionised water ad lOO g 96.81 g The hydrogel was prepared as described in Example 1.
~ 20 Example 7 100 g of gel contain:

25 IFN gamma 0.05 g ' Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g ~ Disodium hydrogen phosphate trihydra~e 0.04 g ',: Natrosol 250 HX 1.75 g ~ 30 Myristyl alcohol 1.0 g .~ ~eionised water ad 100 g 96.91 g The hydrogel was prepared as described in Example 2.
The myristyl alcohol was distributed in the sterile-, 35 filtered buffer solution which had been heated to about 60C. After the buffer solution had cooled, the procedure was continued as described in Example 2.

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Example 8 100 g of gel substance contain:

5 IFN gamma 0.005 9 Methylparaben 0.20 g Succinate buffer pH 6.00 0.0191 M
Sodium chloride 0.1435 M
Natrosol 250 HX 1.75 g lO Polysorbate 20 0.0952 g Thin liquid paraffin oil 0.952 g Deionised water ad lO0 g The hydrogel was prepared as described in Example l.
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:

25 IFN gamma 0.025 g Methylparaben 0.20 g Succinate (buffer pH 6.2) 0.2362 g Sodium chloride 0.8766 g Natrosol 250 ~X 1.75 g 30 Polysorbate 20 0.1 g LABRAFIL 1944 CS l.0 g ~eionised water ad lO0 g The hydrogel was prepared as described in Example l.

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~32~21 Example 10 100 g of gel substance contain:

5 IFN gamma 0.025 g Methylparaben 0.20 g Succinate lbuffer 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 The hydrogel was prepared as described in ~xample 1.

FJxample 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 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 alcohol was melted at 50-60C and then the pre-emulsion was prepared as described in Example 1 but at 50-60C. The rest of the method was as in Example 1. The stability curve is shown in Fig. 6.

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Example 12 lO0 g oE gel substance contain:

5 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 10 Polysorbate 20 0.2 g Thin liquid paraffin oil 2.0 g Deionised water ad lO0 g The hydrogel was prepared as described in Example 1.

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 j Polysorbate 2Q 0.2 g Thin liquid paraffin oil 2.0 g Deionised water ad 100 g The hydrogel was prepared as in Exarple l.

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Example 14 100 g of gel substance contain:

5 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 10 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.

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Claims

1. A pharmaceutical composition suitable for topical application which comprises at least one therapeutically active protein, an excipient, carrier or adjuvant, and at least one physiologically acceptable hydrophobic substance in finely divided form in a quantity sufficient to stabilise the protein.

2. A pharmaceutical composition as claimed in claim 1, comprising as a therapeutically active protein, a therapeutically active quantity of a natural or recombinant, human or animal protein, selected from an interferon, TNF.alpha., TNF.beta. and t-PA, or a structurally similar bioactive equivalent thereof.

3. A pharmaceutical composition as claimed in claim 1 wherein said hydrophobic substance comprises one or more paraffin oils.

4. A pharmaceutical composition as claimed in claim 1 or 2 wherein said hydrophobic substance comprises a paraffin oil or a mixture of paraffin oils in a quantity of from 0.1 to 3%, based on the total weight of the composition.

5. A pharmaceutical composition as claimed in claim 1, comprising an emulsifier to produce a fine distribution of the hydrophobic substance.

6. A pharmaceutical composition as claimed in claim 1, 2 or 3, comprising a non-ionic emulsifier to produce a fine dis-tribution of the hydrophobic substance.

7. A pharmaceutical composition as claimed in claim 1, 3 or 5, in the form of a hydrogel.

8. A pharmaceutical composition as claimed in claim 1, 3 or 5, comprising as a hydrogel forming agent, a cellulose deri-vative, gelatine or a synthetic polymer.

9. A pharmaceutical composition as claimed in claim 1, 3 or 5 comprising polyvinyl alcohol as a hydrogel forming agent.

10. A pharmaceutical composition as claimed in claim 1, 3 or 5, comprising hydroxyethylcellulose as a hydrogel forming agent.

11. A pharmaceutical composition as claimed in claim 1, 3 or 5, comprising a physiologically acceptable preservative.

12. A pharmaceutical composition as claimed in claim 1, 3 or 5 comprising a physiologically acceptable preservative selected from p-hydroxybenzoate derivatives, sorbic acid, chlorhexidine digluconate, benzalkonium chloride and hexadecyltrimethyl ammonium bromide either individually or in admixture.

13. A pharmaceutical composition as claimed in claim 1, 3 or 5, comprising an adjuvant, permeation accelerator or moisture retaining agent or a buffer system.

14. A pharmaceutical composition as claimed in claim 1, 3 or 5, 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.

15. A process of preparing a pharmaceutical composition as claimed in claim 3 or 5, which process comprises phase inverting a system comprising one or more hydrophobic substances/water/
optionally emulsifier, from a W/O emulsion to an O/W emulsion to obtain a fine emulsion and then combining with the majority of the aqueous phase.

16. A process for preparing a pharmaceutical composition as claimed in claim 1, 3 or 5 which process comprises preparing a pre-emulsion by adding water to a hydrophobic substance and a finely divided emulsifier optionally contained therein, with stirring, until a coarse W/O emulsion is obtained: then allowing the emulsion to sediment; subsequently resuming stirring with the addition of more water to give a content of up to about 50%
and allowing phase inversion to take place to thereby yield a fine O/W pre-emulsion which is finely divided in a buffered solu-tion, which buffered solution optionally comprises preservative and an adjuvant; subsequently introducing a hydrogel forming agent and allowing said hydrogel forming agent to swell and finally adding a protein solution.