AU4080193A

AU4080193A - Anti-inflammatory agent

Info

Publication number: AU4080193A
Application number: AU40801/93A
Authority: AU
Inventors: David Russell Blake; Christopher John Morris; Richard Bartholomew Williams; Paul Graham Winyard
Original assignee: British Technology Group Ltd
Current assignee: BTG International Ltd
Priority date: 1992-06-01
Filing date: 1993-05-14
Publication date: 1993-12-30
Also published as: WO1993024137A1; EP0648125A1; JPH07507289A; CA2136646A1; GB9211538D0

Description

ANTI-INFLAMMATORY AGENT This Invention relates to antl-Inflammatory agents. Inflammation Is associated with a wide range of disorders resulting from both external and internal factors. For example, skin inflammation occurs in many conditions as a result of disease or injury. Other disorders result in internal inflammation, for example in joints affected by arthritis and in intestinal Inflammatory conditions such as colitis. Inflammation is also postulated as participatory in atherosclerosis, diabetes and other endocrine disorders.

One serious disorder which gives rise to secondary disabilities which are associated with secondary inflammation is haemophilia. Two types of haemophilia are recognised, both being X-linked, recessive bleeding disorders with indistinguishable clinical manifestations. The more common type, haemophilia A, is attributable to decreased blood levels of properly functioning clotting factor VIII, whilst in haemophilia B, the defect is a decreased level of functional blood clotting factor IX. Haemophilia patients commonly suffer from progressive arthritis and musculoskeletal disability as a consequence of bleeding into joints and muscles. Much effort is put into management routines for haemophilia by lifestyle adjustments to protect affected joints. In the acute phase of a joint bleeding episode, which is the primary manifestation of the disorder, management comprises intravenous administration of Factor VIII concentrate, together with rest and simple or sometimes opiate analgesia. Simple analgesics are used in the subacute arthropathy that may persist following an acute bleeding episode. Non-steroidal anti- inflammatory drugs are only seldom used, and then only for pain control in the chronic arthropathy - when advanced joint damage has occurred. They are not generally used in the acute and subacute stages. It is against this background that it has surprisingly been found that factor VIII has an anti-inflammatory utility independent of its recognised blood clotting function.

Accordingly, the present invention relates to the use of factor VIII in the preparation of a pharmaceutical composition intended for introduction to the non-haemophiliac human body for the alleviation of inflammation.

While it is recognised that in some circumstances it may be necessary to introduce the factor VIII by intravenous injection, it has further been found that local administration of factor

VIII, for example by a topical or intradermal route, is also effective in alleviating inflammation.

Therefore, according to a further aspect of the invention, there is provided a pharamaceutical composition, for use in local administration for the alleviation of Inflammation, comprising factor VIII and a suitable carrier therefor.

Preferably, the local administration is carried out topically or intradermally at or near the site of inflammation.

The dosage employed will of course vary widely with the severity of the condition being treated and also with the method of introduction. For most applications it is envisaged that a unit dose will range from 1 to 4000 international units of factor VIII, preferably from 100 to 1500. Of course the amount actually employed depends on the weight of the patient and is preferably from 1 to 50 per kilogram of body weight.

The carrier will depend on the method of introduction of the factor VIII to the patient. Thus, if intravenous injection is employed, the carrier may conveniently be saline. For intradermal application, a saline carrier may again prove to be most suitable. However, for topical administration, it is envisaged that a percutaneous penetration enhancer may be included in the carrier. Drug transport through human skin has been reviewed by Barry, Molec. Aspects Med., 12, 1991, 195-241. Suitable enhancers cover a wide diversity of chemical types - solvents (such as water, alcohols, dimethylsulphoxide, dimethyl- formamide and dimethylacetamide, pyrrolidones, propylene glycol), Azone and its derivatives, surfactants (anionic, cationic and non-ion1c), fatty acids and alcohols, terpenes and their derivatives, alkyl sulphoxides, phosphine oxides and sugar esters and miscellaneous materials such as urea and its long chain analogues, N, N-diethyl-m-toluamide, calcium thioglycolate and anticholinergic agents. Various transdermal drug delivery systems have been developed and are envisaged as applicable to the present invention - see for example the systems described by Ranade, J. Clin. Pharmacol., 21, 1991, 401-418.

Other pharmaceutically acceptable additives may be included in the preparations dependent on the intended condition to be treated and intended mode of application, for example human serum albumin, mannitol and histidine. The factor VIII employed in the composition is preferably human factor VIII and may be that presently available commercially with varying degrees of purity. Different commercial examples of factor VIII have been found to exhibit anti- inflammatory activity as described in the examples which follow. While presently available factor VIII 1s in general derived from blood, it 1s envisaged that factor VIII or a constituent peptide derived from factor VIII manufactured by recombinant DNA technology will be equally suitable.

The pharmaceutical compositions of the invention may be employed to treat a wide variety of inflammatory conditions. Such conditions include early acute inflammation, as often found in skin and mucosal inflammation, or more persistent inflammation as often affects the joint and periodontium, (especially in rheumatoid arthritis and periodontitis) . Life threatening inflammatory conditions both acute and chronic including burns and septic shock would also be included. With a suitable delivery vehicle, it is envisaged that the compositions may also find use in the treatment of specific mucosal inflammatory disorders, such as colitis, for example by pessary or suppository, or even in the treatment of conditions such as atherosclerosis which are associated with inflammation. In fact it is envisaged that factor VIII may be employed for both primary and secondary inflammatory conditions, either external, internal or mucosal . The invention will now be further described by way of example with reference to Figures 1 to 10. In Figure 1 to 4, the symbol

I 1 represents the largest standard deviation observed in the experiment. The symbol 1 in Figs. 5 to 10 represents the standard deviation for each group. Example 1.

Anti-inflammatory effect of i.v. introduced Factor VIII on carraαeenan induced paw Oedema

The factor VIII evaluated in this example was freeze-dried factor VIII (heat treated : 8Y 250 - obtained from Bio Products Laboratory, Herts, U.K.). Group A (Control) and Groups B, each of six male rats (Wistar, Albino, weight 180-200g) were employed in the following procedure:

Group A. Rats were anaesthetised by 1ntraper1toneal (i.p.) injection with 0.1 ml of 6% w/v pentobarbitone sodium (Sagatal - available from May & Baker, Essex, U.K.) using a sterile syringe and dosing needle (size 25G). After achievement of anaesthesia, the rat penile vein was exposed and injected with 0.1ml sterile saline. Immediately after i.v. injection of saline the rats were injected subcutaneously in the subplantar region of the left paw with 0.1 ml (1.5% w/v) carageenan solution in sterile saline. (Before use, and after making up as a solution of 0.15g carrageenan in 10 ml sterile saline, the carrageenan solution was allowed to stand in an incubator (60°C) for 4 - 5 hours followed by sterilisation at 100°C for 35 minutes and autoclaving) . Groups B. As for group A, the rats were anaesthetised with Sagatal and then received 0.1 ml of the appropriate factor VIII solution in sterile saline to deliver doses of 15 U/Kg, 7.5U/kg, or 3.75U/kg, where U represents one international unit of anti- hae ophiHc activity which is equivalent to that quantity of anti-haemophilic factor present in 1ml of normal human serum, via the penile vein. Immediately after factor VIII injection, the rats were injected subcutaneously in the subplantar region of the left paw with 0.1 ml carrageenan solution.

The time was noted at the beginning of i.v. saline and factor

VIII injections. After carrageenan induction the paw circumference (mm) was assessed at a point proximal to the hindmost spurs on the subplantar surface at hourly intervals for

5 to 6 hours.

At each time point the mean ± standard deviation of each group was calculated and the data subjected to a twin tailed students t-test and the degree of statistical significance calculated.

The results are shown in Fig. 1.

It will be seen from Fig. 1 that administration of 15U/kg of factor VIII Into the penile vein immediately prior to induction of carrageenan paw oedema produced a significant (P< 0.05 to P< 0.01) suppression of paw swelling compared to saline controls at 2 - 3 hours. The suppression effects declined after 4 hours. Lower doses of factor VIII showed less significant activity. Example 2. Ant1-inflammatory effect i.v. introduced Factor VIII on carrageenan induced paw oedema

Example 1 was repeated employing an alternate source of factor VIII (essentially free of Von Willebrand's Factor), i.e. Factor VIII (Monoclate - P available from Armour Pharmaceuticals Co. Ltd, U.K. The Monoclate-P was employed in heparized sterile saline. As the commercial preparation also contains a mixture of 2% albumin, 0.8% mannltol and 1.2 mM histidine, that mixture was also tested in the model. The procedure outlined Example 1 was employed except that factor VIII was used at a dose of 5.5 U/rat. The results are shown in Fig.2 for factor VIII and in Fig.3 for injection of 0.1 ml. of the albumin/mannitol/histidine mixture.

It will be seen from Figs. 2 and 3 that administration of factor VIII into the penile vein immediately prior to induction of carrageenan paw oedema produced a significant ( P< 0.05 to P< 0.01) suppression of the paw swelling compared to saline controls between 2 and 4 hours. The suppressive effects began to decline after 4 hours. The albumin/mannitol/histidine mixture produced a suppression in the paw swelling, but this was not significant. Example 3

Anti-inflammatory effect of Factor VIII on carraoenan induced paw oedema-dose response.

Using the source of factor VIII described in Example 1 and the procedure described in Example 1, groups of rats were injected i.v. with doses of 100 UKg ^_1 , 50 Ukg ^_1 and 10 Ukg ^{~ ]} of factor VIII. Control groups received either sterile water or heparin (0.2U/0.1ml) in sterile water, again injected i.v. The results are shown in Fig.4 It will be seen that administration of factor VIII to rats through the penile vein immediately prior to the carrageenan induction produced marked suppression in the degree of inflammation. Initial signs of factor VIII activity were observed at 2 hours with 100 Ukg ^-1 producing the largest suppression (P< 0.01). In contrast, at 3 hours 50 UKg^-1 factor VIII showed greater anti-inflammatory (P<0.001) activity compared to 100 and lOUKg^-1 (P<0.01). At 4 hours factor VIII effects were similar to those observed at 3 hours with the exception of lOUkg^-1 factor VIII which showed a diminished response. The effects of factor VIII at 50 and lOOUkg^-1 <P 0.05 were reduced at 5 hours whilst 10 UKg^-1 dose showed no activity.

In this model of acute inflammation, administration of heparin failed to show any significant effects. Example 4 Anti-inflammatorv effect of topically and intradermallv administered Factor VIII

An alternative model system to the carragenan induced paw oedema model used in Examples 1 to 3 was employed. This alternative model, the glucose-oxidase-polyethylene glycol (GO -PEG) model shows an acute inflammatory response (initiated by H₂0₂) caused by the enzyme glucose oxidase and, possibly, 0₂ ^~, as shown by the protective effects of the oxygen radical scavengers catalase and superoxide dis utase (SOD). The response is exacerbated by i.v. iron dextran at 24 and 48 hours post GO injection. The model consists of a vascular component assessed clinically by measuring oedema and a cellular component consisting of an inflammatory cell infiltrate.

The source of factor VIII used in Example 1 was employed. The rats employed (6 per group A,B,C and D) were male Wistar, Albino, weight 200 - 250 g. The procedure was as follows: Group A. The rats anaesthetised by intraperitoneal injection with 0.1ml Sagatal ⁽30 mg kg^-1 using sterile syringe and dosing needle (size 25G) . After achievement of anaesthesia the rats were shaved and injected intradermally (i.d.) at two sites with 0.1ml sterile saline containing 64μg GO-PEG (The glucose oxidase PE6 was obtained from Sigma Chemical Co., Dorset, U.K.). One site was left constituting the positive control as Group A.

Group B. The other site was injected i.d. with 2.47 Ukg^-1 factor VIII in saline with the GO-PEG.

Group C. This group of 6 rats was anaesthetised with Sagatal and received 64μg GO-PEG in 0.1ml. saline at two sites. One site received 4.93U factor VIII topically in 0.1ml oil vehicle to constitute Group C.

Group D. The other site received topically 4.93U factor VIII in 0.1ml saline vehicle. The time was noted at the beginning of injections. After inflammation induction the inflammatory index (mm) was assessed using calipers measuring the diameter of the oedematous response at time periods of 2,4,6,24 and 48 hours. Standard deviations were calculated as described in Example 1. The results are shown in Fig. 5.

It will be noted that GO-PEG- induced inflammation increasing to a maximum 6 hours post administration but persisted for 48 hours at least. As shown in Fig. 5, i.d. administration of Factor VIII showed significant inhibition of inflammation at 1 (P>0.01), 2 (P>0.0001), 4 (P>0.01) and 6 hours (P> 0.025) post administration. Topical application of Factor VIII in a saline solution showed a slight but significant inhibition at 2 (P> 0.001⁾ and 4 hours (P>0.05) post administration whereas in an oil based solution , significantly decreased inflammation at 2 hours (P>0.001) post administration. Example 5.

Anti-inflammatorv effect of intradermal 1v and penetration-enhanced topically administered factor VIII

The procedure described in Example 4 was repeated but employing the following dosed groups of rats. As penetration enhancers there were employed dimethyl sulphoxide (DMSO, supplied by Sigma Chemical Co.) and propylene glycol (PG, supplied by

Sigma Chemical Co.).

Group A: Intradermal injection at three shaved sites on the lumbrosacral area with 64μg GO-PEG in 0.1ml sterile saline. Each site received either 24.7U (approx lOOUKg^-1), 12.35U (approx. δOUKg-¹) or 2.47U (approx. lOUKg^"1) Factor VIII injected intradermal ly with GO-PEG.

Group B: Intradermal injection at three shaved sites with 64μg GO-PEG. These sites received either 36.05U (approx. IδOUKg-¹) or 24.7U (approx. 100 UKg^"1) Factor VIII topically in 0.1ml 75% DMSO. The remaining site received topical DMSO alone.

Group C: Intradermal injection at two shaved sites with 64μg GO-PEG. These sites received either 12.35U (approx. 50UKG^"1) or 2.47 U (approx. lOUKg^"1) Factor VIII topically in 0.1ml 75% DMSO. One other site received topical DMSO alone.

Group D: Intradermal injection at three shaved sites with 64μg GO-PEG. These sites received either 36.05U (approx. lSOUKg^"1) or 24.7U (approx. lOOUKg^"1) Factor VIII topically in 0.1ml PG. The remaining site received topical PG alone.

Group E: Intradermal injection at two shaved sites with 64μg GO-PEG. These sites received either 12.35U (approx. 50UKg^_1) or 2.47U (approx. lOUKg^"1) Factor VIII topically in 0.1ml PG. One other site received topical PG alone. All Factor VIII solutions were prepared immediately before application.

The results are shown for Group A in Fig.6, for Groups B and

C in Fig. 7 and Group D and E in Fig. 8 It is to be noted that GO-PEG- induced inflammation increasing to a maximum at 6 hours post administration but persisted for 48 hours at least. As shown in Figure 6, i.d. administration of Factor VIII showed significant inhibition of inflammation at 2, 4, 6 hours and 48 hours post administration. Topical application of Factor VIII 1n a DMSO solution showed a significant Inhibition at 2 (150, 50 and 10 UKg^-1),4 (all doses), 6 (100 and 50 UKg^-1) and 48 (150,100 and lOUKg ^_1) hours post administration (Fig. 7). Topical application of Factor VIII in a PG solution showed a significant inhibition at 2 (50UKg^-1 only), 4 (150, 100 and lOUKg^"1), 6 (150, 100 and 50UKg^_1) and 48

(100 and lOUKg^-1) hours post administration (Figure 8). However, the topical vehicle DMSO also suppressed Inflammation at 4 and 6 hours post induction and the other vehicle PG at 6 hours (Figures

6 and 7). Topical application of DMSO, PG, Factor VIII and intradermal injection of Factor VIII, showed no adverse effects on normal skin.

Surprisingly, both topical and intradermal administration showed a late suppression at 48 hours given that only one application was given.

Example 6

Comparison of anti-inflammatory effect of different sources of

Factor VIII on carrageenan induced paw Oedema

The samples of Factor VIII used in this Example were as follows:

Monoclate P - obtainable from Armour

Pharmaceuticals Co. Ltd. 8-SM-1000 - obtained from Bio Products

Laboratory. This commercial preparation has been affinity purified with monoclonal antibody followed by addition of L-histidine, polyethylene glycol, calcium chloride, sodium chloride and human albumin.

Highly purified Factor obtained by further purifying VIII 8-SM-1000 by anion exchange chromatography. The procedure outlined in Example 1 was followed using Wistar, Albino rats (190-200g) randomised into the following eight groups (6 per group), Groups B to H which were injected intravenously (lOOμl via penile dorsal vein)

Group A Absolute control (carrageenan injected, non treated)

Group B Sterile saline Group C Buffer A ⁽50mM tris, 0.64M NaCl , 5mM CaCl ₂, 0.1% Tween 20, pH 7.0)

Group D Monocl te - P (50 Ukg^"1) Group E Monoclate - P (25 Ukg^"1) Group F Highly purified Factor VIII (50 Ukg^-1) Group G Highly purified Factor VIII (25 Ukg^-1) Group H 8-SM-1000 (25 Ukg^"1) The results are shown in Figures 9 and 10. It can be seen that Factor VIII in both the bulked out forms and the highly purified form exert anti-inflammatory effects at the P<0.05 and P<0.01 levels. (P values are as compared to group A) For 8-SM-1000 (25 Ukg^-1) and highly purified Factor VIII (50 Ukg^-1) inhibition was from 1 to 4 hours.

Monoclate P at 25 and 50 Ukg^-1 was anti-inflammatory for a 2 to 4 hour period. Low dose highly purified Factor VIII (25 Ukg^-1) exerted inhibition at 3 and 4 hours post challenge.

No significant differences were found between the Factor VIII preparations at any time point.

Sterile saline showed no anti-inflammatory effect at any time point, but Buffer A had some inhibitory effect (not significant) at 1 and 2 hours but not at the later time points.

Claims

1. The use of Factor VIII in the preparation of a pharmaceutical composition intended for introduction to the non-haemophiliac human body for the alleviation of inflammation.

2. A pharmaceutical composition, for use in local administration for the alleviation of inflammation, comprising factor VIII and a suitable carrier therefor.

3. A composition according to claim 2 for use in intradermal administration.

4. A composition according to claim 2 for use in topical administration.

5. A composition according to any one of claims 2 to 4 wherein the carrier comprises saline and optionally one or more bulking agents and/or stabilizers.

6. A composition according to claim 4 wherein the carrier comprises a percutaneous penetration enhancer.

7. A composition according to any one of claims 2 to 6 in unit dosage form, each dosage comprising from 1 to 4000 international units of factor VIII.