IE47223B1 - Anti-tussic or anti-thrombotic pharmaceutical compositions and their preparation - Google Patents

Description

This invention relates to pharmaceutical compositions having antithrombotic (thrombozyte aggregation inhibiting) or anti-tussic effects, and to methods of preparing such compositions.

It is known that papaverine has an anti-thrombotic effect, but in 5 effective concentration has such strong side effects that it is in practice not suitable for this purpose.

A medical suppression of the cough reflex occurring in bronchial and pulmonary illnesses for freeing the breathing tract from mechanical or pathological irritations is desirable when a dry, irritated cough occurs, as it does for example in asthma, whooping cough, tuberculosis and lung cancer.

. The best known cough-inhibiting substance is codeine which acts on the cough centre, but when given orally has a slight addictive effect because demethylation in the organism produces morphine. This effect is even more pronounced when codeine is injected. The most important side effects in codeine therapy are constipation and depression of the breathing action, and for this reason the use of codeine is in many cases contra-indicated or at least entails disadvantages. A further objection to codeine is that its manufacture requires the cultivation of opium poppies.

It is also known that the alkaloid d-glaucine which is isolated from the flowers of the yellow poppy, can be used in cough therapy.

The poppy does not contain 1-glaucine, but at least four other alkaloids, - 2 47223 whose separation from d-glaucine is extremely difficult. The d,l-glaucine synthesized, starting from papaverine, by oxidative ring-closing reaction of laudanosoline (Angewandte Chemie 1967, pages 815 and 816) contains considerable quantities of an hitherto not recognised by-product which crystallizes together with the glaucine salt. This by-product has been found to be 1-(N,N-dimethylaminoethyl)-3,4,6,7-tetramethoxyphenanthrene, which is apparently produced by Hofmann degradation. In view of the phenanthrene structure and the often sympathomimetically active diemthylami noethyl group of this compound, glaucine contaminated with such a by-product cannot be used in pharmaceutical products.

The pharmaceutical compositions of the present invention have antithrombotic (thrombozyte aggregation inhibiting) or anti-tussic effects which are therapeutically active at a low concentration of the active compound and are largely free from undesired side effects.

The anti-tussic compositions when given orally, have an improved longlasting central cough-inhibiting effect, are not addictive and largely free from the undesired gastrointestinal side effects of the known products.

The invention provides a pharmaceutical composition comprising a physiologically acceptable solid or aqueous liquid carrier and at least one therapeutically active agent which is an aporphine derivative selected from 1,2,9,10-tetrahydroxyaporphine (hereinafter referred to as tetrahydroxyaporphine), and glaucine containing 1-glaucine: or physiologically acceptable salts or quaternary N-alkyl-ammonium derivatives thereof.

Examples of salts of such active agents are a halogen acid salt, a tartrate, an N-methoxyhalogenide, and embonate.

Even at active substance concentrations far below what is necessary for papaverine the pharmaceutical compositions according to the invention have pronounced anti-thrombotic effect and are largely free from the side - 3 47323 effects which occur when papaverine is used.

Preferably the said active agent is glaucine of which 50% to 100% is 1-glaucine, or its physiologically acceptable salt or li-methohalogenide.

A further preferred composition according to the invention is one in which the glaucine is d,1-glaucine, or a derivative thereof as hereinbefore set forth.

Thus when the said active agent is glaucine, the glaucine preferably consists of from 50% to 100% by weight 1-glaucine, and may be d,1-glaucine.

To achieve an anti-thrombotic (thrombozyte aggregation inhibiting) effect, the compositions of the present invention may be in dosage unit form comprising an aqueous liquid or solid carrier and from 100 to 800 mg, preferably from 100 to 500 mg of active agent per dosage unit. Thus the said composition may be in the form of a pill, dragee, tablet or capsule. Alternatively such compositions may be adapted for injection, and comprise a solution of the active agent in a physiologically acceptable aqueous liquid carrier.

To achieve an anti-tussic effect, the compositions of the present invention may be in dosage form adapted for oral administration, comprising aqueous liquid or solid carrier and from 1 to 1000 mg of active agent per dosage unit. Thus the composition may be in the form of a pill, dragee, tablet or capsule, containing for example from 5 to 50 mg of active agent per dosage unit, or may be in the form of a syrup containing for example from 1 to 10 mg of the active agent per ml.

Such compositions provide improved effects. Thus when compared with previously used anti-tussic compositions containing codeine they are substantially free of spasm-inducing side effects in the intestinal region, and when compared with compositions containing d-glaucine as the active agent, 1-glaucine, or combinations of d-glaucine and 1-glaucine, e.g. d,l- 4 47223 glaucine, or derivatives thereof as hereinbefore referred to, provide an improved effect.

Pharmaceutical compositions according to the invention preferably contain glaucine embonate or glaucine tartrate. Glaucine embonate provides a considerably prolonged anti-cough effect, without delaying the onset of said effect.

The invention also provides compositions comprising glaucine, glaucine salts or N-methohalogenides, free from l-(N,N-dimethylaminoethyl)3,4,6,7-tetramethoxyphenanthrene.

The invention also provides a method of preparing a pharmaceutical composition for administration to humans to obtain an anti-tussic or a thrombozyte aggregation inhibiting effect, comprising formulating a mixture or solution of a physiologically acceptable solid or aqueous liquid carrier with at least one active agent selected from glaucine containing 1-glaucine, or its physiologically acceptable salt or quaternary N-alkyl ammonium derivative.

The invention is hereinafter particularly described and illustrated by the following Examples and Comparative Tests.

Preparation of Tetrahydroxyaporphine Hydrochloride.

For making laudanosine there was added to 225 g (0.6 Mol) of papaverine hydrochloride in 2 litres of 50% aqueous methyl alcohol a solution of 30 g sodium hydroxide in 300 ml water, the addition being made slowly. The resulting precipitate was filtered off and dried for 20 hours at 60°C.

The product was 201 g of papaverine base with a melting point of 144 to 146°C (yield 98%). 200 g (0.6 Mol) of the papaverine base was dissolved in 200 ml of methyl alcohol. To the solution there was added 120 ml of methyl iodide and the whole heated to boiling during 6 hours. After cooling, the crystalline - 5 product was filtered off and dried. The product was 280 g of papaverine methiodide with a melting point of 127 to 129°C (yield 97%). 200 g (0.4 Mol) of the papaverine methiodide was suspended in 2 litres of a 10% aqueous methyl alcohol and to this mixture there was added sodium borohydride until complete solution was obtained and the yellow-orange colouration has disappeared. The resulting solution was poured into 12 litres of water and the resulting white precipitate was filtered off and dried, The product was 230 g of laudanosine with a melting point of 113 to 115°C'-(yield 78%).

To make laudanosoline hydrobromide 121 g (0.34 Mol) of laudanosine was heated in 600 ml of 40% hydrobromic acid for about 10 hours, until all the methyl hromide was removed. After cooling the solution, the crystalline product was separated. The product was 100 g of laudanosoline hydrobromide with a melting point of 230°C (yield 77%).

To make tetrahydroxyaporphine hydrochloride, 98 g (0.26 Mol) of laudanosoline hydrobromide was dissolved in 1.2 litres of a 50% aqueous methyl alcohol at 80°C. After complete solution, the solution was cooled down to 6°C by adding ice, after which there was added a filtered solution, which had also been cooled to 6°C, of 100 g(0.62 Mol) of iron (III) chloride in 500 ml of 50% aqueous methyl alcohol. After one minute there was added 1,5 litres of concentrated hydrochloric acid and the solution was allowed to stand at room temperature. The precipitated grey-brown cyrstals were filtered off, washed with acetone and dried. The product was 43 g of tetrahydroxyaporphine hydrochloride showing a melting point of 242 to 244°C (yield 50%).

Preparation of d^l-Glauaine Hydrobromide.

For making d,l-glaucine hydrobromide, 6 g (0.018 Mol) of tetrahydroxyaporphine hydrochloride was dissolved with warming in 850 ml of methyl alcohol. In a separate operation, 21.6 g (0.126 Mol) of tri methyl phenylammonium chloride in methyl alcohol was reacted with a solution of 8.5 g - 6 47223 (0.15 Mol) of KOH in methyl alcohol. After filtering off the precipitated potassium chloride, the filtrate was made up to 840 ml with methyl alcohol. The two solutions were then mixed together slowly under a protective gas and added slowly over 6 hours to anisol which had been heated to 110°C, the methyl alcohol distilling off. After completion of the addition the solution was cooled and a black, amorphous residue was removed by filtering.

The resulting dark green filtrate was evaporated under vacuum until dry.

To the residue there was added 5 ml of ethanol, 15 mis of hydrobromic acid and 20 mis of ethyl acetate. From the resulting mixture a product crystallized out and was filtered off and dried. The product was 5.5 g of d,l-glaucine hydrobromide with a melting point of 235°C (with decomposition) (yield 67%).

Purification and Resolution of d,l-glauaine.

A thin-layer chromatograph showed that the d,l-glancine hydrobromide made by the above process contained about 10% of l-(N,N-dimethylaminoethyl)3,4,6,7- tetramethoxyphenanthrene. 60 g of this d,l-glaucine was dissolved in 200 ml of 50% aqeuous ethanol and reacted with an excess of 8.5 g of potassium hydroxide in 50 ml of water.

The resulting mixture was shaken with about 500 ml of chloroform to extract the free d,l-glaucine base. The separated organic phase was dried over anhydrous sodium sulphate and filtered. After evaporating off the solvent 50 g of an oily residue was obtained and this was recrystallized from 75 ml of ethyl acetate. After filtering and drying there was obtained .4 g of d.l-glaucine base showing a melting point of 128 to 130°C. After repeated recrystallization from further 100 ml portions of ethyl acetate, there was obtained 25.15 g of d,l-glaucine base with a melting point of 138 to 140°C. By making a thin-layer chromatograph it was determined that this contained less than 0.1% of impurities. - 7 47 283 After recrystallizing the combined residues from ethyl alcohol there was obtained about 5 g of l-(N,N-dimethylaminoethyl)-3,4,6,7-tetramethoxyphenanthrene with a melting point of 248 to 250°C and a molecular weight, determined by mass spectrometry, of 369.

NMR spectrum: 9.13(s,l), 7.88(d,l), 7.66(d,l), 7.46(s,2), 3.97(s,3), 3.93(s,6), 3.86(s,3), 3.44-3.14(m,4), 2.73(s,3), 2.52(s,3).

To separate the isomers, 5.09 g (0.014 Mol) of d,l-glaucine was dissolved in 70 ml of ethyl alcohol and the solution reacted with a solution of 2.2 g (0.014 Mol) of d-tartaric acid in 70 ml of ethyl alcohol (50°C).

Slow cooling resulted in a fine crystalline precipitate which was filtered off, washed with ether and dried. The product was 3.6 g of 1-glaucine-dbitartrate with a melting point of 210 to 212°C and a specific rotation in water of -26° (yield 93%).

The sill impure 1-glaucine-d-bitartrate was reacted with an aqueous solution of sodium hydroxide and extracted with ether. After evaporating the solvent, the residue was dissolved in 50 ml of ethyl alcohol and reacted with a solution of 1.15 g of d-tartaric acid in 50 ml of ethyl alcohol. After separation there was obtained 3.39 g of 1-glaucine-dbitartrate showing a melting point of 212 to 215°C and a specific rotation in water of -32°. The specific rotation of the 1-glaucine base in ethyl alcohol was -101° (94% optical purity). 2.48 g of 1-glaucine was reacted in 15 ml of ethyl alcohol with a small excess of 48% hydrobromic acid. After separation there was obtained 2.78 g of 1-glaucine hydrobromide with a melting point of 235°C (with decomposition) (yield 98.5%).

From the mother liquors of the first and second d-bitartrate crystallization there was obtained, after evaporation, a greenish residue, - 8 47223 which was dissolved in 20 ml of water, treated with aqueous sodium hydroxide and then extracted with 250 ml of ether. After drying and filtering, the solvent was evaporated, giving 1.57 g of d-glaucine with a melting point of 120°C. The specific rotation in alcohol was 104.6°. By reacting this product with hydrobromic acid there was obtained a d-glaucine hydrobromide with a melting point of 235°C (with decomposition). The d-glaucine obtained by methylation from d-boldine showed a specific rotation in ethyl alcohol of +115°.

Preparation of 1-Glaucine Hydrochloride.

To make 1-glaucine hydrochloride, a quantity of 1-glaucine was dissolved in a little methyl alcohol and to the solution there was added a small excess of concentrated hydrochloric acid. To the resulting voluminous precipitate there was added ethyl acetate, giving whitish to pink crystals. After filtering, washing with acetone and drying there were obtained white to pink fine crystals of 1-glaucine hydrochloride with a melting point of 232 to 233°C.

Preparation of 1-Glaucine Hydroiodide For making 1-glaucine hydroiodide, a quantity of 1-glaucine was dissolved in 2 n hydrochloric acid and the solution reacted with saturated potassium iodide solution. The resulting crystalline precipitate was recrystallized from a mixture of methyl alcohol and ether. This gave a crystalline, yellowish glaucine hydroiodide with a melting point of 238°C. Preparation of d-Glaucine Methiodide A mixture of 3.92 g (0.01 Mol) of glaucine, 1.5 g (0.01 Mol) of potassium carbonate and 5 ml of methyl iodide was heated for 6 hours in methanol. The still hot solution was filtered and the solvent removed under vacuum, giving 4.2 g of d-glaucine methiodide with a melting point of 218 to 220°C (Yield 85%). - 9 47223 Preparation of Tetrahydroxyaporphine Melkoohloride. g (0.126 Mol) of laudanosine was dissolved in methanol and heated with 15 ml of methyl iodide for 2 hours under reflux. The resulting solid residue was filtered, washed and dried, giving 56 g of laudanosine meth5 iodide monohydrate with a melting point of 238 to 240°C. g of laudanosine methiodide hydrate was dissolved in 150 ml of 48% hydrobromic acid and heated under reflux for 15 hours. The resulting, yellow precipitate was filtered and dried, giving 22.1 g of laudanosoline methobromide with a melting point of 237 to 239°C. 50 g (0.0125 Mol) of laudanosoline methobromide was dissolved in 500 ml of water at 20°C and reacted with a filtered aqueous solution containing 40.5 g of iron (III) chloride in 500 ml of water. After 24 hours there was added to the dark violet solution 500 ml of concentrated HCl. The mixture was evaporated down to about half its original volume. After adding methanol a yellow precipitate crystallized out. The product was 27.6 g of tetrahydroxyaporphine methochloride with a melting point of 236 to 239°C (yield 63%).

The resulting product, and also the previously obtained products, were identified by ultra-violet, infra-red and NMR spectra and by their melting points.

Preparation of 1-Glaueine Embonate For making 1-glaucine embonate, 841 mg of 1-glaucine was reacted in 10 ml of dimethyl formamide with 459 mg of embonic acid. After heating to complete solution, the mixture was poured into 100 ml of water and the precipitated product filtered and dried. The product was 1.32 g of powdery, slightly brownish 1-glaucine embonate with a melting point of 189 to 192°C (yield 100%). - 10 47223 Preparation of d, l-Glauaine Elaborate Far making d,l-glaucine embonate, a mixture of 1.42 g of d,1-glaucine and 0.78 g of embonic acid was dissolved in 20 ml of dimethyl formamide and the resulting brown solution was introduced into 400 ml of water. The resulting slightly brownish precipitate was filtered off, washed with water and dried. The product was 1.75 g of d,1-glaucine embonate with a melting point of 188 to 190°C (yield 80%). found: calculated: C71.O258 H6.05% N2.55% 069.36% HS.98% N2.52% Preparation of d,l-Glauoine, d, l-Tartrate For making k,1-glaucine tartrate, 355 mg (1 Mol) of d,l-glancine in 10 ml of ethyl alcohol was reacted with 150 mg of d,l-tartaric acid in 10 ml of ethyl alcohol at 50°C. After cooling the solution there was obtained 220 mg of optically inactive d,l-glaucine-d,l-tartrate with a melting point of 215°C (yield 97%).

Example 1.

Coated pills with an anti-cough effect were made of the following components; Core: 1-glaucine hydrobromide lactose starch talcum Coating: gum arabic talcum crystallized sugar white wax red dye - 11 20 mg mg 40 mg 10 mg 130 mg 4.5 mg 120 mg mg mg 0.1 mg 0.4 mg Example 2 For making capsules with long-period anti-cough effect, stretchcapsules of hard gelatin were each filled with 160 mg of a mixture of the following components: d,1-glaucine hydrochloride 10 mg d,1-glaucine embonate 25 mg lactose 60 mg starch 60 mg magnesium stearate 5 mg 160 mg Example 3 For making a syrup with an anti-cough effect the following components were mixed together: 1,glaucine hydrobromide 133 mg saccharose 20 g sorbitol 45 mg citric acid 125 mg p-hydroxybenzoic methyl ester 100 mg aroma (essence of sweet oranges) l,5g distilled water, up to lOOg Example 4 For making a medicine intended for injection, ampoules were filled with the following solution: tetrahydroxyaporphine hydrochloride 11.22 mg (corresponding to 10 mg of tetrahydroxyaporphine base) sodium bisulphite 0.7 mg - 12 47223 tartaric acid 5.01 mg sodium hydrogen tartrate 10.2 mg propylene glycol 300 mg distilled water to 2 ml Example 5.

Although the active compounds used according to the invention have an anti-thrombotic effect already in small dosages, the exact amount of active agent to be used in an anit-thrombotic tablet, dragee or capsule will vary with the severity of the thrombogenic risk of the patient, his weight and his response to the active compound. The active compound shall preferably be compounded with non-toxic edible excipient chemically inert to the active compound. The amount of excipient should preferably be sufficient to separate the particles of the active agent from each other and to cause quick solution or dispersion in the gastric juices in the stomach. To this purpose the composition may comprise 10 to 99%, preferably 20 to 75% of active agent, the rest being carrier material and conventional adjuvants. Suitable excipients are lactose, sucrose, starch, talcum, stearic acid and its salts, and other commonly used excipients for tabletting and granulation and mixtures thereof.

For preparing anti-thrombotic tablets there were used the following ingredients: d,1-glaucine hydrobromide 100,0 mg sucrose 25,9 mg starch 22,1 mg acacia 7,8 mg talc 3,1 mg magnesium stearate 1,5 mg stearic acid 1,6 mg 162,0 mg - 13 * 47 223 The active compound was mixed with the sucrose and the gum acacia, and then with the starch made previously into a paste with a small amount of distilled water. This mixture was dried, converted into a granular powder and then blended with the talc, magnesium stearate and the stearic acid which act as mold lubricants. After mixing in a pony mixer the mixture was tabletted on a conventional tabletting machine.

Example 6.

There were prepared anti-thrombotic capsules each containing: Tetrahydroxyaporphine hydrochloride 400,0 mg magnesium stearate 4,0 mg 404,0 mg Example 7.

There were prepared anti-thrombotic tablets each containing: 1-glaucine hydrohromide 200,0 mg magnesium stearate 2,0 mg 202,0 mg Example 8.

In a conventional tabletting machine there were prepared anti- thrombotic tablets each containing: d,1-glaucine hydrobromide 200,0 mg polyvi nyl pyrrolidone 15,0 mg corn starch 20,0 mg 235 ,0 mg 25 Alternatively there can be prepared tablets adapted to be subsequently cut, each containing 400 mg active compound.

Example 9.

For making anti-thrombotic capsules hard gelatine capsules were each - 14 4 7223 filled with a mixture of: d,1-glaucine hydrobromide silicagel magnesium stearate 200,0 mg ,0 mg 2,0 mg 212,0 mg Comparative test 1.

In order to compare the anti-cough effectiveness of codeine phosphate with the effectivenesses of the different isomers of glaucine hydrobromide, the testing method of Friebel and Reichle was used. 60 guinea pigs with weights between 200 and 300 g were divided up into ten groups of different sizes,. Before administering the active substance, each animal was subjected, on the same day as the day of test with the active substance, for a period of 8 minutes to an aerosol of 20% citric acid, the animal being confined in a chamber at constant air pressure. During this control test the number of cough pulses was counted with the help of a pressure transmitter. From these measurements an average control value for each group was calculated. After this preliminary control text there was administered subcutaneously to each animal of each group a suspension of the active substance in 1% by weight sodium carboxymethyl cellulose. After 30 minutes each animal was again exposed in the test chamber for 8 minutes to the aerosol of 20% by weight citric acid and the number of cough pressure pulses recorded. The different active substances were adninistered in increasing doses ranging from 3 mg/kg to 100 mg/kg of body weight. Altogether eight different doses were administered. The results were expressed as percent deviation from the average control value for the group on the same day, the deviation being entered in a diagram against the logarithm of the dose administered. From the resulting effectiveness curve, based on the logarithm - 15 47223 of the dose, the coefficient of correlation and the ED5Q were calculated for each active substance, by the regression analysis method of Downie and Heath (1965). The values obtained for codeine phosphate, for d-glaucine hydrobromide, for d,1-glaucine hydrobromide and for 1-glaucine hydrobromide thirty minutes after administering the active substance are summarized in Table 1.

TABLE 1.

Active substance Number of animals Correlation coeff.* Rising slope of curveED50 mg/kg codeine phosphate** 41 -0.3956 -67.4 20.6 d-glaucine hydrobromide** 59 -0.5340 -60.8 54.0 1-glaucine hydrobromide 38 -0.6867 -70.96 31.2 d,1-glaucine hydrobromide 25 -0.4874 -57.8 27.2 * statistically significant (p<0.01) for each active substance. ** not according to invention.

These results show that 1-glaucine hydrobromide has a considerably lower effective dose Εϋ^θ value compared to d-glaucine hydrobromide, and that surprisingly the d,1-glaucine hydrobromide has an even lower EDgQ value than 1-glaucine hydrobromide.

Comparative test 2.

One of the most unpleasant side effects of anti-cough medicine containing codeine is constipation and the occurrence of intestinal spasms.

A model test for determining the effects of anti-cough active substances on intestinal motility and on the evacuation action of the stomach-intestinal tract of mice was therefore made. After subcutaneous injection of the active substance, each animal was given, through a throat probe, a 10% by weight suspension of carbon in a 5% by weight aqueous slurry of gum arabic. - 16 47223 Two hours later the intestine was sectioned and the length of stomach coecum measured, and also the distance travelled by the carbon expressed as a percent of intestine length. The results are set forth in Table II.

TABLE II. 5 Active substance ! ι Dost mg/kg Number of animals Distance travelled, average % Standard deviation % CONTROL 40 74.3 3.6 codeine phosphate** 3 7 70.4 3.5 10 IB 74.3 2.4 30 17 34.8* 9.5 100 18 20,6* 4.5 d-glaucine hydrobromide** 10 18 74.6 2.5 30 18 80.1 2.6 100 18 36.4* 4.9 1-glaucine hydrobromide 3 8 74.3 6.3 10 8 94.3* 2.0 30 8 85.4 4.3 100 8 51.8* 10.6 * significantly different from the control value (p<0.05). ** not according to invention.

The effects of codeine phosphate, of d-glaucine hydrobromide and of 1-glaucine hydrobromide on intestinal motility were determined at different 25 doses. For each dose the average value was calculated for a sufficient number of animals.

These results show that 1-glaucine hydrobromide, in contrast to d-glaucine hydrobromide and codeine phosphate, administered in a dose of - 17 47223 mg/kg resulted in a significant increase in intestinal activity. And even when the dose of 1-glaucine hydrobromide is increased to 30 mg/kg and 100 mg/kg, the intestinal motility is considerably more than the corresponding values for d-glaucine hydrobromide and codeine phosphate.

Comparative test 3.

Experiments were conducted in vitro to determine the effects of different active substances on the smooth muscles of isolated guinea pig intestine samples suspended in a bath. After adding to the bath different doses of the active substances, the contractive force of the muscle material was recorded. It was found that codeine at molar concentrations between 1 χ ΙΟ-® and 32 χ 105 produces contractions which depend on the dose, d-glaucine showed a dose-dependent increase of spontaneous activity and tone at molar concentrations between 1 x 10-3 arid 8 x 10 . On the other hand, -5 -5 using 1-glaucine at molar concentrations between 1 X 10 and 8 x 10 , a reduction in spontaneous activity and no increase in tone were observed. This shows that 1-glaucine, in contrast to d-glaucine and codeine, has no spasmogenic activity.

In further tests it was investigated in what concentrations the active substances being compared suppress spasms induced in isolated guinea pig intestine by carbachol or histamine. It was found that both codeine and d-glaucine are effective against carbachol or histamine at a molar con-3 -5 centration of 1 x 10 or 4 x 10 . On the other hand d,1-glaucine was -8 effective against histamine at molar concentrations of only 4 x 10 to β 7 x 10 and against carbachol at molar concentrations of only 4 x 10 .

Comparative test 4.

In a further series of tests the effects of different doses of codeine phosphate in suppressing contractions induced in isolated guinea pig intestines by electric voltages were tested. For this purpose one electrode was immersed in the solution surrounding the intestinal preparation. - 18 47223 The second electrode was connected to the preparation at the attachment point. Alternating current pulses were applied for 5 seconds and it was found that the resulting contracting reflex is reduced by adding active substance. The results of the tests are shown in Table III.

TABLE III.

Acti ve Substance Molar concentration 4x IO’6 5x IO-6 8x IO'6 lx 10-5 2x 10-5 4x IO’5 6x 10’5 8x IO’5 codeine* - 85 - 49 36 - - - phosphate - 51 - 49 52 58 - - d-glaucine* - - - 77 74 44 - - hydrobromide - - - 95 79 36 - - 1-glaucine - - - 71 64 24 - 7 hydrobromide - - - 74 32 23 - - d,1-glaucine - - - 63 48 8 - - hydrobromi de 81 - 63 - - - - - * not according to invention.

These results show that codeine phosphate at a molar concentration of about 10 suppresses by about 50% the electrically induced contraction reflexes. But increased codeine doses do not result in increased suppression because the spontaneous activity induced by the codeine phosphate itself increases the contractions. It was found that d-glaucine hydrobromide at a dose of 4 x 10 suppresses electrically induced contraction reflexes. But at higher concentrations the increasing spontaneous activity prevents any distinguishing between direct production and electric production of the - 19 47223 activity. Using 1-glaucine hydrobromide, it was found that at a concentration of 4 x 10”® the electrically induced spasms are more effectively suppressed, compared to what is obtained using d-glaucine hydrobromide.

At a concentration of 8 x 10” of 1-glaucine hydrobromide the electrically induced contractions are suppressed even more effectively. Using d,l-5 glaucine hydrobromide it was found possible at concentrations of 4 x 10 to 6 x 10'5 to suppress the electrically induced spasms practically completely, without at the same time inducing any spontaneous activity. In comparing the values shown in the table it should be observed that when the d,l-5 glaucine hydrobromide is at the concentration of 4 x 10 , the d-glaucine hydrobromide and the 1-glaucine hydrobromide are each at a molar concen-5 tration of 2 x 10 .

Comparative text 5.

In a series of tests conducted by the method of Domenjoz, the effects of the different active substances on the coughing centre was examined by electrical stimulation of the upper laryngeal nerves of anaesthetized cats. Electrical stimulation of the nerve produces a coughing reflex and the intention is to reduce this by intravenous injection of the active substance. The minimal effective dose MED is determined by increasing the dose from 0.1 mg/kg of body weight up to 0.3 mg/kg, 10. mg/kg, 3.0 mg/kg and 6.0 mg/kg, until the dose is sufficient for completely suppressing the coughing reflex.

In these tests each animal was given, in the one hand, codein and, on the other hand, at sufficient time intervals also glaucine, so that the ratio of the minimal effective doses of glaucine and codein can be deduced. Table IV shows the minimal effective doses MED and also the observed duration of cough suppression.

TABLE IV Active substance MED mg/kg Durations minutes codeine phosphate* 6 18 - 54 d-glaucine hydrobromide* 1 5-39 d,1-glaucine hydrobromide 1 44 - 62 * not according to invention.

These results show that both the d-glaucine hydrobromide and the d,lglaucine hydrobromide completely suppress the coughing reflex already at a dose of 1 mg/kg of body weight. The dose of codeine phosphate required for this is 6 mg/kg. And it will be observed that the duration of the effect, at this minimal dose, is considerably greater for the d,1-glaucine hydrobromide than for the d-glaucine hydrobromide.

Comparative test 6.

The equivalent effective doses for codeine phosphate and d,1-glaucine 20 hydrobromide with oral administration were determined on 20 female guinea pigs with body weights between 230 and 600 g. The guinea pigs were divided into four groups. Each animal was then tested as described further above by subjecting the animal for 8 minutes to a 20% citric acid aerosol, the number of cough pulses produced during the 8 minutes being recorded. The control average/value obtained in this way for each group of guinea pigs was calculated. After this control test each animal was given, by means of a throat probe in a volume of lOml/kg either doses of 50 or 100 mg/kg of body weight of codeine phosphate, or doses of 100 or 200 mg/kg of d,1-glaucine hydrobromide. - 21 4 7 323 One hour after administering the active substance, each animal was once more subjected for 8 minutes to the citric acid aerosol and the number of cough pulses recorded. The results were compared with the control values and expressed as percentages of the control values. The results are shown in Table V.

TABLE V Active substance Dose mg/kg Cough pulses, percent of control value codeine phosphate* 50 37 100 50 d,1-glaucine hydrobromide 100 30 200 40 * not according to invention.

The results show that a dose of 75 mg of codeine phosphate per kg of body weight has about the same effect as a dose of 150 mg of d,120 glaucine hydrobromide per kg of body weight.

In a further comparative test, 40 female guinea pigs with body weights between 230 and 600 g were divided into 4 groups and each animal was again subjected to the action of a 20% citric acid aerosol for 8 minutes. From the control values thus obtained the average control value for each group was calculated. Each animal of each group was then given, by means of a throat probe, either 75 mg/kg of codeine phosphate, or 135 mg/kg of d,1-glaucine hydrochloride, or 150 mg/kg of d,1-glaucine hydrobromide, or 378 mg/kg of d,1-glaucine embonate. These doses are the equimolar quantities, based on glaucin. After a period of 1, or 3, or 5, or 24 hours each animal was again subjected for 8 minutes to the - 22 47223 action of the citric acid aerosol, and the number of cough pulses during this period was recorded. The values thus obtained were compared with the control values and the percent deviation of the average measured value for each group, from the average control value, was calculated.

The results are shown in Table VI.

TABLE VI.

Active substance Cough pulses during 8 minutes, percent of control value, after: 1 hour 3 hours 5 hours 24 hours codeine phosphate* 84 70 84 95 d,1-glaucine hydrobromide 90 47 59 113 d,1-glaucine hydrochloride 80 72 69 102 d,1-glaucine embonate 80 51 57 81 * not according to invention.

These results show that in all the tests the d,1-glaucine salts have a greater effect than codeine phosphate in reducing the number of cough pulses. In particular it will be observed that d,1-glaucine embonate has a considerably longer-lasting effect without any delay in the beginning of the effect.

Comparative test 7.

The toxicity thresholds of codeine phosphate, of d-glaucine hydrobromide and of d,1-glaucine hydrobromide were determined in mice by the Wilcox method. It emerged, as shown in Table VII, that d,1-glaucine is considerably less toxic than 1-glaucine. The toxicities of codeine and d,1-glaucine hydrobromide are about equal. - 23 47223 TABLE VII Active substanceLD50 peroral mg/kgLD50 subcutaneous mg/kg codeine phosphate* 640 230 d-glaucine hydrobromide* 345 125 d,1-glaucine hydrobromide 686 320 *not according to invention.

Comparative test 8.

The effectiveness of several compounds corresponding to Formula (1) in influencing the aggregation of blood platelets in platelet-rich human blood plasma was compared with the effectiveness of papaverine hydrochloride.

For this purpose highly diluted solutions were prepared of the active substance in physiological NaCl solution, the solutions showing definite molar concentrations. 10 microlitres of each solution was tested, after 5 minutes incubation at 37°C, for its effect on aggregation of the blood platelets under the influence of adenosine diphosphate added to give the critical concentration. The measurements were made by the turpi dity method described by Born in Nature (1962) on page 927. The critical concentration is the least concentration of adenoside diphosphate which results in primary aggregation of the blood platelets. This is followed by an irreversible secondary aggretation. Table VIII shows the results when compositions according to the invention gave superior results to the known papaverine hydrochloride.

TABLE VIII Compound Aggregation inhibition at 5 x 10'® molar concentration papaverine hydrochloride* - tetrahydroxyaporphorine hydrochlori de + tetrahydroxyaporphori ne methochlori de + d,1-glaucine hydrobromide + 1-glaucine hydrobromide + * not according to invention.

Comparative test 9.

The active compounds d,1-glaucine hydrobromide and tetrahydroxyaporphine hydrochloride were selected for in vivo assessment of the anti-thrombotic activity using a modified hamster cheek pouch technique described by Duling, Berne and Born (1968) and Begent and Born (1970).

Male golden hamsters, weighing 80 - 120g, were anaesthetised with intraperitoneal pentobarbitone. The cheek pouch was everted using a cotton bud and spread out over a special Perspex stage. The top layer and connective tissue were removed leaving a thin vascular membrane which was transilluminated from below. The preparation was observed using a Leitz Dialux microscope and long working range objectives at a magnification of x 250. The cheek pouch remained in good condition for the duration of the experiment by continually bathing with Tyrode solution at a temperature of 37°C.

A micropipette of tip diameter 1 - 2pm was filled with a 0,01 M solution of the sodium salt of adenosine diphosphate in distilled water.

The micropipette was manipulated close to a venule of diameter 16 - 40um.

The reference electrode was placed in contact with the animal. When the negative potential was applied from the external circuit the resultant current of approximately 300 nA ejected adenosine diphosphate (of the Order of 2 x 10-^ Itioles/sec) from the pipette. This caused the formation of a white body (platelet thrombus) at the tip of the pipette or slightly downstream. The growth rate of the thrombus was quantified by noting the time taken for 30%, 50% and 90% of the white body to form. When the current was switched off the white body rapidly embolised and no new white bodies could be formed until the current was reapplied.

In this study the effect of the drug v/as assessed over a period between 30 and 90 minutes after oral administration of each drug at the dose levels of 2, 5, 10 and 20 mg/kg.

The compound was tested at four dose levels in 5 animals each with one group of 5 animals serving as a control. Preparation of the cheek pouch commences at 15 minutes after dosing and thrombus stimulation commenced at 30 minutes after dosing each animal.

The growth rates were calculated as gradients of the regression lines by a standard programme in a 9100B Hewlett Packard calculator. Dose response curves are obtained by plotting maximum inhibition expressed in percent of control group on thrombus induction in the micro-circulation of the hamster cheek pouch against the dose level. The compounds were shown to inhibit thrombus formation when administered orally. The results are . shown in Table IX: Product Oral dose for maximal inhibition mg/kg Maximal inhibition % Time of maximal inhibition min (after dosing) glaucine hydrobromide 10 41 60 - 70 tetrahydroxyaporphine hydrochloride 20 29 50 - 60 - 25 47223 The dose response curves indicated that the compounds were active at relatively low dose levels and the inhibitory capacity of each compound tended to plateau at approximately 10 mg/kg.

Comparative test 10.

In order to investigate the anti-cough effectiveness of orally given codein phosphate, d-glaucine hydrobromide, d,1-glaucine hydrobromide , 1-glaucine hydrobromide and 1-glaucine-d-tartrate, respectively, guinea pigs were orally dosed with one of the active agents mentioned one hour before being exposed for ten minutes at a 5% citric acid aerosol. In each case the last 5 minutes of the exposure were used for the measurements.

The effective doses ED^„ are shown in Table X: TABLE X Compound Oral ED50 mg/kg Codeine phosphate* 94.9 d-glaucine hydrobromide* 198.96 d, 1-glaucine hydrobromide 16.0 20 1-glaucine hydrobromide 7.21 1-glauci ne-d-tartrate 6.8 * not according to invention.

Claims (19)

1. A pharmaceutical composition comprising a physiologically acceptable solid or aqueous liquid carrier and at least one therapeutically active agent which is an aporphine derivative selected from tetrahydroxyaporphine, and glaucine containing 1-glaucine; or physiologically acceptable salts or quaternary N-alkyl-ammonium derivatives thereof.

2. A composition according to Claim 1, wherein the said active agent is glaucine of which 50% to 100% by weight is 1-glaucine, or its physiologically acceptable salt or N-methohalogenide.

3. A composition according to Claim 2. in which the glaucine is d,lglaucine or its physiologically acceptable salt or N-methohalogenide.

4. A composition according to any of Claims 1 to 3, in dosage unit form for administration to humans to obtain a thrombozyte aggregation inhibiting effect comprising a liquid or solid carrier and from 100 to 800 mg of the said active agent per dosage unit.

5. A composition according to Claim 4, containing from 100 to 500 mg of the said active agent per dosage unit.

6. A composition according to Claim 4 in the form of a pill, dragee, tablet or capsule.

7. A composition according to Claim 4, for injection and comprising a solution of the said active agent in a physiologically acceptable aqueous liquid.

8. A composition according to any of Claims 1 to 3 in dosage unit form for oral administration to obtain an anti-tussic effect comprising a liquid or solid carrier and from 1 to 1000 mg of said active agent per dosage unit. - 28 47223

9. A composition according to Claim 8 in the form of a pill, dragee, tablet of capsule.

10. A composition according to Claim 9, which contains from 5 to 50 mg of said active agent per dosage unit.

11. A composition according to Claim 8 in the form of a syrup.

12. A composition according to Claim 11, wherein the said syrup contains from 1 to 10 mg of the said active agent per ml of syrup.

13. A composition according to any of Claims 1 to 10, comprising glaucine, or glaucine salts of N-methohalogenides, free from 1-(N,N-Dimethylaminoethyl)3,4,6,7-tetramethoxyphenanth rene.

14. A composition according to any of Claims 1 to 13, comprising glaucine embonate of d,l-glaucine-d,l-tartrate.

15. A composition according to Claim 1, substantially as hereinbefore described, with particular reference to the Examples.

16. A method of preparing a pharmaceutical composition for administration to humans to obtain an anti-tussic or a thrombozyte aggregation inhibiting effect, comprising formulating a mixture or solution of a physiologically acceptable solid or aqueous liquid carrier with at least one active agent selected from glaucine containing 1-glaucine, or its physiologically acceptable salt or quaternary N-alkyl ammonium derivative.

17. A method according to Claim 16, wherein the said active agent consists of from 50% to 100% by weight of 1-glaucine.

18. A composition according to Claim 1 which is an anti-thrombotic compositi on. - 29 47223

19. A composition according to Claim 1 which is an anti-tussic composition.