IE882812L - 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

/ 70908 - 1 - Stabilisation of therapeutically active proteins in a pharmaceutical composition The invention relates to pharmaceutical preparations for topical application containing one or more stabilised, 5 therapeutically active proteins and optionally conventional excipients, carriers and additives. The invention also relates to a process for preparing these pharmaceutical preparations and the use of physiologically acceptable hydrophobic substances for 10 the stabilisation of proteins.

One of the essential requirements in the topical application of therapeutically active proteins is concerned with their stability in the pharmaceutical 15 formulation. The stability must be ensured for a sufficiently long period of time both during storage under refrigeration and at ambient temperature and also at body temperature, as well as "in situ" for several hours. Hitherto, no entirely satisfactory solutions 20 have been found to meet these requirements. 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, under the 25 conditions of use, there was some loss of activity of the interferons which could only be reduced by the addition of a protease inhibitor (EP-A-142345).

For the stabilising of interferons in gels, ointments, 30 etc. it has also been proposed to use various sugar alcohols, optionally together with sugar acids or the salts thereof, mild reducing agents, anionic surfactants or combinations of these substances (EP-A-80879) 35 For stabilising proteins and polypeptides such as interferons, more particularly IFN-gamma, in parenteral preparations, it has been proposed to use a physically f > 70908 - 2 - and chemically modified gelatin, particularly as a replacement for human serum albumin (EP-A-162332) Japanese Published Patent Application JP-A-61-277633 5 discloses the stabilising of interferons in solution with certain surface-active substances.

EP-A-135171 mentions human serum albumin as a suitable stabiliser for oil/water microemulsions. 10 For the topical application of the synergistic combination IFN-beta/9-(1,3-dihydroxy-2-propoxy-methyl) guanine (DHPG) according to US Patent No. 4606917 in the form of an ointment, albumin, dextrose and buffer 15 substances are proposed as stabilisers.

The therapeutic composition according to EP-A-0233629 contains, in addition to an antiviral substance and a substance with anti-tumour activity, amongst others 20 mineral oil or petrolatum as a physiologically acceptable carrier, in the high amounts typical of carrier substances. These carrier substances must ensure that the stability of the active substances is not-affected. 25 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. 30 The aim of this invention was to provide pharmaceutical preparations for topical applications, more particularly hydrogels, which contain a stabiliser for therapeutically active proteins, which in addition to. 35 being physiologically acceptable satisfies all the requirements imposed on formulations of this kind, especially with respect to the optimum availability of - 3 - the active substance and the full development of its activity and with respect to the gentlest possible method of preparation which takes account of the vulnerability of the proteins to shear forces. 5 Starting from this problem, various categories of substances have been investigated with respect to their suitability for solving this problem. It was found, surprisingly, that even small amounts of hydrophobic 10 substances used as additives in very finely divided form, particularly paraffin oils, have a stabilising effect on various therapeutically active proteins which is superior to the effect of the substances proposed up till now. This result is all the more surprising as the 15 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 stabiliser. 20 The invention solves the problem by using physiologically acceptable hydrophobic substances, particularly paraffin oils, for stabilising the therapeutically active proteins in pharmaceutical 25 preparations for topical use, especially hydrogels.

The invention thus relates to pharmaceutical preparations of the type mentioned hereinbefore, containing one or more stabilised, therapeutically 30 active proteins as well as conventional excipients, carriers and additives, which are characterised in that they contain one or more physiologically acceptable hydrophic substances, in finely distributed form, more particularly paraffin oil/s, as stabiliser, in an amount 35 which stabilises the protein and is less than the amount in which the substance has a carrier function. - 4 - With the aid of the addition of a stabilising quantity of hydrophobic substances in finely divided form, pharmaceutical preparations are obtained which, under the conditions of use, make the active substance 5 available in active form over a lengthy period of time. By means of the pharmaceutical preparations according to the invention, the level of activity of the protein after storage at 4-8°C over a period of at least 12 months is substantially unchanged. A further advantage 10 of the formulations according to the invention is that there is less need to ensure that an exact pH value is maintained, since the stabilising addition of hydrophobic substances reduces the vulnerability of the proteins to fluctuations in the pH value. This 15 advantage is of particular importance for applications which require lower pH values, e.g. application in the vaginal area.

The pharmaceutical preparation according to the 20 invention also has the advantage, when present in 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 25 particular advantage for application in the lip area.

The advantageous stabilising effect of hydrophobic substances on proteins can possibly be put down to hydrophobic interactions which have hitherto been 30 noticed scarcely or not at all. In the stabilising of proteins according to the prior art, the following two operating principles were obviously taken as a starting point: a) stabilising by complex binding of the substance to the pnstein and hence steric fixing of the 35 protein molecule; b) binding of the free bulk water by polar substances and hence stabilising of the protein by influencing its hydrate coat. The hydrophobic t 1 - 5 - 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 5 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. 10 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 15 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 20 substances are preferably contained in the preparation in an amount of from 0.1 to 3.0%.

In order to ensure that the stabiliser is finely divided and-the distribution is stable, emulsifiers may be 25 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, in particular, non-ionic emulsifiers such as 30 polysorbates (polyoxyethylene (n) sorbitanmonolaurate, e.g. TweenR 20), nonoxynol (polyoxyethylene (n) -nonylphenylether, e.g. TritonR N101, TritonR Nlll), and poloxamer (polyethylenepolypropyleneglycol, PluronicR F68) . If the pharmaceutical preparation is in the form 35 of a hydrogel, the emulsifiers will not only bring about a fine distribution of the stabiliser but will also improve the spreading of the gels. - 6 - The pharmaceutical preparations according to the invention are suitable for the administration of human and animal proteins such as those listed as follows, including their structurally similar bioactive 5 equivalents (by equivalents is meant those proteins which have substantially the same biological activity with a different amino acid -sequence): cytokines, e.g. interferons such as huIFNalpha, hulFNbeta, huIFNgamma, huIFNomega, hybrid interferons, animal interferons such 10 as EqIFNbeta, EqIFNgamma, or lymphokines such as interleukin-2, TNFbeta, or monokines such as interleukin-1, TNFalpha,- growth factors, e.g. epidermal growth factor (E6F); anticoagulants, e.g. vascular anticoagulant proteins (e.g. VAC alpha, VAC beta), 15 antithrombins; fibrinolytics, e.g. tPA, urokinase; proteins with an anti-allergic activity, e.g. IgE binding factor; therapeutically active enzymes, (A;kA e.g. lysozyme, superoxide dismutases. 20 The proteins used may either be of natural origin or produced by the recombinant method. The range of indications depends on the biological activity of the protein which is to be applied; within the specific spectrum for each protein any application is possible 25 which requires topical administration of the active substance. The content of therapeutically active protein in the pharmaceutical preparation will naturally depend on the activity of the protein, the needs of the particular indication and the type of preparation used. 30 It may span a wide range of quantities.

Suitable forms for administration include, in particular, hydrogels, suppositories and forms for vaginal use. 35 The use of excipients, carriers and additives will depend on the particular application selected, whilst - 7 - care should be taken to ensure that they do not affect the stability of the protein by the type and quantity used. 10 15 35 The pharmaceutical preparations according to the invention may contain, as additives, preservatives such as p-hydrobenzoates (nipa esters, methylparaben) 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 tauroglycolic acid may be added to the pharmaceutical preparation.

Hydrogel forming agents which may be used include gelatine and cellulose derivatives such as methyl-cellulose, hydroxypropylcellulose and, in a particularly preferred embodiment, hydroxyethylcellulose, as well as 20 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 distribution of the stabiliser it should be borne in mind, in this 25 connection, 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. 30 The buffer systems used are selected according to the optimum 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 preparation - 8 - takes the form of a hydrogel the carrier is water.

The additives which may be present also include moisture-retaining substances such as glycerol, 5 sorbitol, 1, 2-propyleneglycol, butyleneglycol and polyols.

The preparations in the form of hydrogels according to the invention are so-called "low-filled" emulsions, 10 because of their low oil content, which tend to break down easily, as is well known. The preparation of these emulsions is therefore of particular importance with regard to their stability. 15 To ensure the gentlest possible production of a stable emulsion, with relatively little technical intervention, in which the stability of the fine distribution of the stabiliser and hence its activity over a long period of time is ensured, a two-step process is preferably used 20 in the manufacture of the preparations according to the invention, particularly hydrogels.

In the first step, in a system of water/stabiliser/optionally emulsifier, a phase inversion from a W/0 emulsion to an 0/W emulsion is 25 brought about and the fine pre-emulsion thus obtained is combined with the majority of the aqueous phase.

The following procedure is particularly preferred: first of all, a pre-emulsion is produced by the so-called 30 "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 35 broken off 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 - 9 - inverted to form a fine 0/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. 5 The time at which the protein solution is added is not critical; this is preferably the final step of the process. Using the process preferred according to the invention, extremely stable emulsions are obtained which show no tendency to separate after half a year's storage 10 at room temperature.

In the case of smaller quantities or when technically more complicated homogenisers such as nozzle homogenisers are available, an 0/W emulsion may also be 15 produced in a single step without the preparation of a preemulsion; 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 20 technology and is at the same time gentle.

The Examples which follow are intended to illustrate the invention with reference to hydrogel formulations containing IFN alpha, IFN gamma, TNF alpha, TNF beta, 25 lysozyme or VAC alpha, as the therapeutically active protein: Example l 30 100 grams of gel contains: IFN gamma 0 .1 9 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 - 10 - Deionised water ad 100 g 96.76 g The hydrogel was produced by the preferred two step method: 5 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. The 10 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/0 emulsion. This emulsion was briefly left to stand, whereupon it 15 separated. After the stirrer was switched on again the emulsion was brought to the point of phase inversion, to produce a very finely divided 0/W emulsion. b) Preparation of the hydrogel 20 The paraffin oil emulsion was stirred into the sterile-filtered buffer solution and finely divided therein.

Then microbiologically pure hydroxyethylcellulose was sprinkled into the emulsion and distributed therein with 25 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. 30 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 I FN-gamma, measured by the ELISA test (the antibodies used bind -35 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 - 11 - distribution.

Pig. 2 shows a comparison test with gelatine as a constituent of a hydrogel formulation without the 5 separate addition of a stabiliser, showing the clearly 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. 10 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). 15 Example 2 100 g of gel contains: 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 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 30 emulsifier Pluronic F68 were dissolved in hot water at 80°C with stirring and the solution 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 35 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 l. - 12 - Example 3 100 g of gel contains: TNF alpha 0.1 g Methylparaben 0.213 g Sodium dihydrogen phosphate monohydrate 0.053 g Dipotassium hydrogen phosphate trihydrate 0.0427 g Natrosol 250 HX 00 « H g Polysorbate 20 0 .107 g Thin liquid paraffin oil 1.07 g Deionised water ad 100 g 96.5443 g The hydrogel was prepared as described in Example 1.

Example 4 100 g of gel contains: IFN alpha 0 .0005 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 96 .8595 g The hydrogel was prepared as described in Example 1.

Example 5 100 g of gel contains: IFN gamma 0.100 g- Methylparaben 0.2 g Sodium dihydrogen phosphate monohydrate 0.05 g Dipotassium hydrogen phosphate trihydrate 0.04 g - 13 - 5 10 15 20 25 30 Tauroglycolic acid 0.01 g Natrosol 250 HX 1.75 g Polysorbate 20 0.1 g Thin liquid paraffin oil 1.0 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.

Example 6 100 g of gel contains: IFN gamma 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 .1 g Thin liquid paraffin oil 0 .6 g Thick liquid paraffin oil 0 .4 g Deionised water ad 100 g 96 .81 g The hydrogel was prepared as described in Example 1.

Example 7 100 g of gel contains: 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 - 14 - The hydrogel was prepared as described in Example 2. The tnyristyl alcohol was distributed in the sterile-filtered buffer solution which had been heated to about 60°C.

After the buffer solution had cooled, the procedure was 5 continued as described in Example 2.

Example 8 100 g of gel substance contains: 10 IFN gamma 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 20 0 .0952 g Thin liquid paraffin oil 0 .952 g Deionised water ad 100 g 20 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 25 after manufacture.

Example 9 100 g of gel substance contains: 30 IFN gamma 0.025 g Methylparaben 0.20 g Succinate (buffer pH 6.2) 0.2362 g Sodium chloride 0.8766 g • 35 Natrosol 250 HX 1.75 g Polysorbate 20 0.1 g LABRAFIL 1944 CS 1.0 g - 15 - Deionised water ad 100 g The hydrogel was prepared as described in Example 1. 5 Eyaiflplft 10 100 g of gel substance contains: IFN gamma 0.025 g 10 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 15 LABRAFIL 2735 CS 1.0 g Deionised water ad 100 g The hydrogel was prepared as described in Example 1.

Example 11 20 100 g of gel substance contains: IFN-gamma 0.025 g Methylparaben 0.20 g Succinate (buffer pH 6.2) 0.2362 g Sodium chloride 0.90 g 25 Natrosol 250 HX 1.75 g Polysorbate 20 0.1 g Myristyl alcohol 1.0 g Deionised water ad 100 g 30 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 l but at 50-60°C. The rest of the method was as in Example 1. The stability curve is shown in Fig. 6. - 16 - 0.05 9 0.2 g 0.05 g ! 0.04 g 1.75 g 0.2 g 2.0 g ad 100 g Example 12 100 g of gel substance contains: TNF beta Methylparaben Sodium dihydrogen phosphate monohydrate Dipotassium hydrogen phosphate trihydrate Natrosol 250 HX Polysorbate 20 Thin liquid paraffin oil Deionised water The hydrogel was prepared as described in Example 1. Example 13 100 g of gel substance contains: Lysozyme 2.4 million units 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 in Example 1. - 17 - Example 14 100 g of gel substance contains: 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 10 Deionised water ad 100 g The hydrogel was prepared as in Example l. <4 - 18 -

Claims (17)

1. A pharmaceutical preparation for topical 5 application containing one or more stabilised, therapeutically active proteins and conventional excipients, carriers and additives, characterised in that it contains as stabiliser one or more physiologically acceptable hydrophobic substances, 10 particularly paraffin oil or oils, in finely divided form, in an amount which stabilises the protein and is less than the amount in which the substance has a carrier function. 15

2. A pharmaceutical preparation according to claim 1, characterised in that it contains a natural or recombinant, human or animal protein, selected from the group comprising the interferons, TNFof, TNFJ3, t-PA, including the structurally similar bioactive equivalents 20 thereof, individually or in a therapeutically useful mixture, in a therapeutically active quantity.

3. A pharmaceutical preparation according to one of the preceding claims, characterised in that it contains 25 as hydrophobic substance paraffin oil or mixtures of paraffin oils in a quantity of from 0.1 to 3%, based on the total mass.

4. A pharmaceutical preparation according to one of 30 the preceding claims, characterised in that, in addition to the finely distributed hydrophobic substance, it contains an emulsifier, more particularly a non-ionic emulsifier. 35

5. A pharmaceutical preparation according to one of the preceding claims, characterised in that it is in the form of a hydrogel. - 19 -

6. A pharmaceutical preparation according to claim 5, characterised in that it contains a hydrogel forming agent from the group of gelatines, cellulose derivatives such as hydroxyethylcellulose, or synthetic polymers 5 such as polyvinyl alcohol, either individually or in admixture.

7. A pharmaceutical preparation according to claim 6, characterised in that it contains hydroxyethyl-cellulose 10 as hydrogel forming agent.

8. A pharmaceutical preparation according to one of the preceding claims, characterised in that it contains a physiologically acceptable preservative such as p- 15 hydroxybenzoate, sorbic acid, chlorhexidine digluconate, benzalkonium chloride and hexadecyltrimethyl ammonium bromide, either individually or in admixture.

9. A pharmaceutical preparation according to one of 20 the preceding claims, characterised in that it contains, as further additives, permeation accelerators and/or moisture retaining agents and/or a buffer system.

10. • A process for preparing a pharmaceutical 25 preparation according to one of claims 1-9, characterised in that, in a first step of the process, in the system comprising the stabiliser/water/optionally emulsifier, phase inversion is effected from a W/0 emulsion to an 0/W emulsion and the fine pre-emulsion 30 thus obtained is combined with the majority of the aqueous phase.

11. A process according to claim 10, characterised in that, first of all, a pre-emulsion is prepared by adding 35 water to the stabiliser and the finely divided emulsifier optionally contained therein, with stirring, until a coarse W/0 emulsion is obtained, the stirring - 20 - process is stopped until the emulsion has sedimented, then by the resumption of stirring and the addition of more water to give a content of about 50%, with phase inversion, a fine 0/W emulsion is produced, this pre-5 emulsion is finely divided in the buffer solution, which optionally contains preservatives and other additives, the hydrogel forming agent is introduced and allowed to swell and finally the protein solution is added. 10

12. Use of physiologically acceptable hydrophobic substances for stabilising therapeutically active proteins in pharmaceutical preparations for topical application, with the proviso that the hydrophobic substance is contained in the preparation in an amount 15 which is less than the amount in which the svibstance has a carrier function.

13. Use according to claim 12, with the proviso that the hydrophobic substance is paraffin oil. .20

14. Use according to one of claims 12 and 13, with the proviso that the pharmaceutical preparation is in the form of a hydrogel.

15. A pharmaceutical composition as claimed in claim 1 substantially as described herein with reference to the Examples and/or the accompanying drawings.

16. A process as claimed in claim 10 substantially as described herein with reference to the Examples and/or the accompanying drawings.

17. A pharmaceutical composition whenever prepared by a process as claimed in any of claims 10, 11 or 16. T0MKINS & CO.