Abstract:
The anthocyanidins pelargonidin and delphidin, have been found to possess novel and unexpected activity in the ophthalmic field, specifically that they are active in reducing the permeability of ciliary body vessels. According to one aspect of the present invention there is provided a method of reducing the hyperpermeability of the ciliary body ocular vessels in a subject which comprises administering to the subject an effective dose of pelargonidin or delphidin. The invention further provides the use of pelargonidin or delphidin in the manufacture of pharmaceutical compositions for carrying out the method of treatment referred to above.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to methods and pharmaceutical compositions for the treatment of ophthalmic diseases, and specifically to the use of selected anthocyanidins to elicit a specific ophthalmic pharmacological effect. 
     2. Description of Related Art 
     Anthocyanidins are known to have valuable pharamacological properties in treating peripheral vascular diseases. Thus our British Patent Nos. 1 589 294 and 1 595 531 describe and claim anthocyanidins which have cicatrizing, epithelium-regenerating, anti-inflammatory, vaso-protective, hypolipaemic, hypocholesterolaemic and hypoglycaemic activity. Owing to their low toxicity, these compounds are particularly useful for the prolonged treatment of patients with impaired peripheral circulation. 
     The glycosides of anthocyanidins (anthocyanosides) which are present as mixtures in extracts of certain fruits, have been described as being active in improving the visual function, particularly night vision (French Patent No. 1369 M, Jun. 25, 1962, Chibret). These compounds apparently accelerate the regeneration of rhodopsin, and thus facilitate adaptation to darkness. 
     SUMMARY OF THE INVENTION 
     We have now found that two of the anthocyanidins described in GB 1 589 294, namely pelargonidin and delphidin, have a novel and unexpected activity in the ophthalmic field, specifically that they are active in reducing the permeability of ciliary body vessels. 
     These blood vessels differ in their anatomic structure from the other ocular blood vessels and from the blood vessels of the general circulation and are involved in the regulation of endo-ocular pressure and of the blood-water barrier, and in the production of aqueous humor. 
     This newly-discovered activity of reducing the permeability of ciliary body vessels could not have been forecast from the known properties of anthocyanins or anthocyanosides. 
     The structure of pelargonidin and delphidin is as follows: ##STR1## wherein in pelargonidin (Compound I) each R represents hydrogen, 
     in delphinidin (Compound II) each R represents hydroxyl, and 
     X -  is a pharmaceutically acceptable anion, e.g. Cl - . 
     The novel ophthalmic activity of these compounds has been established by various in vivo experiments in animal models. 
     Thus according to one aspect of the present invention there is provided a method of reducing the hyperpermeability of the ciliary body ocular vessels in a subject which comprises administering to the subject an effective dose of an anthocyanidin of formula ##STR2## wherein (I) each R represents hydrogen, or 
     (II) each R represents hydroxy, and 
     X -  is a pharmaceutically acceptable anion, e.g. Cl - . 
     The method of the invention is of particular utility in reducing the hyperpermeability of the ciliary body ocular vessels induced by injury or by harmful agents. 
     The invention further provides the use of an anthocyanidin of formula (I) and (II) above in the manufacture of pharmaceutical compositions for carrying out the methods of treatment referred to. 
     Various routes of administration may be used in carrying out the method of the invention. For example, the anthocyanidins may be administered orally or they may be administered locally to the eye. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The specific pharmacological effects obtained according to the invention will now be described in more detail. 
     A. EFFECT ON THE PERMEABILITY OF THE HAEMATO-OPHTHALMIC BARRIER 
     The effect on the permeability of the haemato-ophthalmic barrier was measured by inducing an increase in the permeability of the vessels of the ciliary body, either by paracentesis or by instillation of sodium hydroxide into the conjunctival sac. The reduction in permeability induced by oral administration of the products under examination was then measured. 
     1. The Paracentesis Method 
     (i) Acute Administration 
     The experiment was carried out on 80 New Zealand albino rabbits, average body weight 2.8 kg, divided into eight groups each of ten animals. 
     The vasal permeability was measured by the dye method: 1 ml of Evans Blue in a concentration of 0.5% was injected into the marginal vein of the auricle. 
     After 10 minutes the left eye was subjected to paracentesis and after 12 minutes a sample of aqueous humour was taken. The quantity of Evans Blue in the sampled aqueous humour was used to assess the permeability of the haemato-ophthalmic barrier. Immediately afterwards, compounds I and II were administered in doses of 100 or 200 mg/kg by stomach probe. 
     Two or four hours after administration, paracentesis was carried out on the right eye and after 12 minutes a sample of aqueous humour was taken. 
     The quantity of Evans Blue in the aqueous humour was evaluated by spectrophotometer reading at 623 nm. 
     (ii) Repeated Administration 
     The experiment was carried out on 20 New Zealand rabbits, average body weight 2.5 kg, using the same experimental technique as for acute administration. 
     The substances were administered in doses of 100 mg/kg os/day for seven days, and the effect on the permeability of the barrier was measured on the eighth day, 24 hours after the last administration. 
     Table 1 gives the results obtained with compounds I and II administered in a single dose of 100 or 200 mg/kg. The amount of dye coming out of the vessels of the ciliary body, two hours or four hours after administration, was reduced in a statistically significant manner. When the substances are repeatedly administered their effectiveness increases. The inhibition of the amount of dye emerging rose from 15% to 21-22% and was still statistically significant 24 hours after the last administration. 
     
                                           TABLE 1__________________________________________________________________________Effect On The Permeability Of The Haemato-Ophthalmic Barrier Induced ByParacentesis In The Rabbit                       Evans Blue (mcg/ml of aqueous humour)          No. of  No. of                       m ± s.d.Substance  Dose mg/kg os          Administrations                  Animals                       Time 0                             2 hours                                    4 hours                                           24 hours__________________________________________________________________________Compound I  100     1       10   41.2 ± 10.6                             34.9 ± 8.5**                             (-15.4%)  100     1       10   55.5 ± 20.6                                    46.9 ± 17.5**                                    (-15.5%)  200     1       10   50.5 ± 17.9                             42.2 ± 15.0**                             (-16.4%)  200     1       10   53.1 ± 23.7                                    44.8 ± 20.2**                                    (-15.6%)  100     7       10   56.9 ± 14.8      44.2 ± 11.7**                                           (-22.4%)Compound II  100     1       10   48.3 ± 7.2                             41.5 ± 7.9**                             (-14.1%)  100     1       10   53.6 ± 12.3                                    45.6 ± 9.7**                                    (-14.9%)  200     1       10   53.1 ± 12.2                             45.1 ± 10.3**                             (-15.1%)  200     1       10   54.1 ± 11.5                                    45.9 ± 12.2**                                    (-15.1%)  100     7       10   53.7 ± 12.8      42.5 ± 13.3**                                           (-20.8%)__________________________________________________________________________ The brackets give the percentage variations relative to time 0 **p &lt; 0.01 Student&#39;s &#34;t&#34; for paired data 
    
     2. Sodium Hydroxide Method 
     The experiment was carried out on 50 New Zealand rabbits, average body weight 2.2 kg. The substance was administered once a day for seven days in doses of 50 or 100 mg/kg. A control group was treated with tap water. 
     Thirty minutes after the last administration, 1 ml of a 0.5% solution of Evans Blue was administered in the marginal vein of the ear. After 30 minutes, inflammation of the eye tissue was produced by instilling two drops of 0.25N sodium hydroxide in the conjunctival sac. Two hours later the animal was anesthesized and a sample of aqueous humour was taken in order to measure the proportion of dye. 
     As Table 2 shows, compounds II and I both induced a dose-dependent reduction in the permeability of the vessels of the ciliary body caused by the irritating agent. The reduction following administration of 100 mg/kg was statistically significant compared with the controls. 
     
                       TABLE 2______________________________________Effects Of Compounds I And II On The Permeability Of TheHaemato-Ophthalmic Barrier Induced By NaOH In the Rabbit              Dose      Evans Blue    No. of    mg/kg/day mcg/ml  PercentageSubstance    Animals   os        m ± s.e.                                Variation______________________________________Controls 10        --        5.9 ± 0.6Compound II    10         50       5.1 ± 0.7                                -14    10        100        3.2 ± 0.4*                                -45Compound I    10         50       5.8 ± 0.8                                 -2    10        100        3.5 ± 0.6*                                -40______________________________________ *p &lt; 0.05 according to Student&#39;s &#34;t&#34; test 
    
     3. Corneal Lens Method 
     The experiment was carried out on 40 New Zealand rabbits, average body weight 2.5 kg, divided into three groups of ten animals each. 
     The vasal permeability was measured by the dye method: 1 ml of Evans Blue in a concentration of 0.5% was injected into the marginal vein of the ear. After 10 min a soft corneal lens embedded in a 2% solution of histamine hydrochloride was applied for 30 min to the left eye. 
     The left eye was subjected to paracentesis and a sample of aqueous humour was taken. 
     Immediately after, the substances were given by gavage at the dose of 100 mg/kg. Two or four hours after administration a lens embedded with histamine as described before was applied for 30 min on the right eye. Paracentesis was carried out and aqueous humor was taken. 
     The quantity of Evans Blue in the aqueous humor was evaluated as described in 1(i). 
     As Table 3 shows, compounds II and I both reduced permeability of the vessels of the ciliary body caused by histamine. The effects after 2 and 4 hours were statistically significant compared with the basal values. 
     
                                           TABLE 3__________________________________________________________________________Effect on the Permeability of the Haemato-OphthalmicBarrier Induced by Histamine             Evans Blue (mcg/ml of aqueous humour)  Dose  No. of             m ± sdSubstance  mg/kg/os        animals             Time 0 2 hours                           4 hours__________________________________________________________________________Compound II  100   10   110.9 ± 32.7                    82.0 ± 7.0***                    (-25.3)  100   10   100.6 ± 38.9                           68.8 ± 8.7***                           (-31.7)Compound I  100   10   106.4 ± 29.5                    86.2 ± 6.1**                    (-19.0)  100   10   108.2 ± 16.4                           75.9 ± 10.3***                           (-29.9)__________________________________________________________________________ ***P &lt; 0.001 **P &lt; 0.01 Student&#39;s &#34;t&#34; for paired data In brackets the percentage variations relative to time 0. 
    
     B. ACUTE TOXICITY 
     This was tested on mice and rats after administration by stomach probe. Doses up to 6 g/kg of compounds I and II did not cause toxic symptoms or death. The LD 50 must therefore be above 6 g/kg. 
     C. OCULAR TOLERABILITY 
     The ocular tolerability of compounds I and II was measured in New Zealand rabbits on the anterior segment (conjunctiva, cornea, iris and crystalline lens) and on the posterior segment (vitreous humour and retina) after oral administration, local application and intra-ocular injection. 
     1. Oral Administration 
     Both products were administered by stomach probe, either in a single dose of 200 mg/kg or in repeated doses of 100 mg/kg for 7 days. Surveys were made before and 1, 2 and 4 hours after acute administration, on the third and seventh days after repeated administration and 5 days after suspension of treatment. 
     2. Local Application 
     The substances were instilled into the conjunctival sac in a quantity of 0.5 ml day of 1% aqueous suspensions for 30 days. 
     3. Intra-Ocular Injection 
     The substances were injected under anaesthesia into the front chamber at a concentration of 0.1%, after evacuation of the chamber. The ocular structures were examined by slit lamp, 1, 24 and 48 hours after injection. 
     Single or repeated oral administration of compounds I and II did not induce modifications in the conjunctive, cornea, iris, crystalline lens, vitrous humour or retina. 
     Local application was also perfectly tolerated. 
     After intracamerular injection, a blue coloration of the intracamerular structures was observed, together with hyperaemia of the conjunctiva and an inflammatory reaction which, in most cases, disappeared in 48 hours. 
     The intensity of the reactions was similar for both products. 
     On the basis of the test results for pharmacological and clinical activity, compounds I and II can be used in the treatment of conditions of increased capillary permeability following a noxious stimulus resulting in opening of intercellular junctions, break of blood aqueous barrier and leakage of seric proteins in the anterior chamber. 
     Such conditions of capillary fragility are frequent complications of diabetes. Therefore a direct action of compounds I and II on vasal permeability can improve ophthalmic affections in diabetic patients. 
     Various routes of administration may be used in carrying out the method of the invention. For example, the anthocyanidins may be administered orally or they may be administered locally to the eye. 
     The products according to the invention can be used in oral pharmaceutical forms such as tablets, capsules and sachets of granulate. 
    
    
     The following examples illustrate the invention without being limitative. 
     
         ______________________________________EXAMPLE 1 (Compound I, 100 mg, capsules)Each capsule, size 2, contains:Compound I                  100    mgLactose                     138    mgMethylcellulose             4      mgSodium carboxymethylcellulose                       4      mgMagnesium stearate          2      mgTalc                        2      mgEXAMPLE 2 (Compound II, 300 mg, capsules)Each capsule, size 1, contains:Compound II                 300    mgMethylcellulose             6      mgSodium carboxymethylcellulose                       6      mgTalc                        3      mgMagnesium stearate          3      mgEXAMPLE 3) (Compound II, 100 mg, tablets)Each 160 mg tablet contains:Compound II                 100    mgMethylcellulose             2      mgSodium carboxymethylcellulose                       10     mgMicrogranular cellulose     30     mgLactose                     12     mgTalc                        3      mgMagnesium stearate          3      mgEXAMPLE 4) (Compound I, 300 mg, tablets)Each 500 mg tablet contains:Compound I                  300    mgMethylcellulose             6      mgMicrogranular cellulose     100    mgLactose                     70     mgSodium carboxymethylcellulose                       18     mgMagnesium stearate          3      mgTalc                        3      mgEXAMPLE 5 (Compound I, 100 mg, granulate sachets)Each 1 g sachet contains:Compound I                  100    mgMannitol                    400    mgAmmonium glycyrrhizinate    10     mgFlavouring powder           10     mgMethylcellulose             5      mgLactose                     475    mgEXAMPLE 6 (Compound II, 300 mg, granulate sachets)Each 1 g sachet contains:Compound II                 300    mgMannitol                    300    mgAmmonium glycyrrhizinate    10     mgFlavouring powder           10     mgMethylcellulose             5      mgLactose                     375    mgExample 7 (Compound II, 100 mg, lyophilized collyrium)Each &#34;lyophilized&#34; ampoule containsCompound II                 100    mgMannitol                    300    mgEach &#34;Solvent&#34; ampoule contains:Hydroxypropylmethylcellulose                       40     mgBenzalkonium chloride       1      mgMonobasic sodium phosphate  82.8   mgDibasic sodium phosphate    23.9   mgWater for injection q.s. to 10     ml______________________________________