CA1098035A - Anti-tussive and anti-thrombotic compositions containing tetrahydroxyaporphine or l-glaucine - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
An anti-tussive or anti-thrombotic composition comprising a pharmaceutical composition comprising a physiologically acceptable carrier and an effective amount of at least one therapeutically active compound se-lected from 1, 2, 9, 10-tetrahydroxyaporphine and glaucine containing 1-glaucine, and the physiologically acceptable salts and quaternaty N-alkyl-ammonium derivatives of such compounds.

Description

9~;35i This invention relates to pharmaceutical compositions having anti-thrombotic or anti-tussive effects, to the use of specified active compo~mds in such compositions, and methods for preparing purified glaucine.
It is known that papaverine has an anti-thrombotic effect, but in effective concentration has such strong side effects on the vessels that it is useless in practice for this purpose.
A medical suppression of the cough refle~ 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, lung cancer and tl~e like. The best known cough-inhibiting substance is codeine, which acts on the cough centre, but when given orally has a slight addictive effect, because de~methylation 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 application of codeine is in many cases contra-indicated or at least entails disadvantagesO A further objection to codeine is that its manufacture requires a planlationwise cultivation of opium poppies, It is known that the alkaloid d-glaucine, which is isolated from the flowers of the yellow poppy, can be applied in cough therapy. The chemi-cal structure of glaucine corresponds to general formula (I) 3 ~
~ R40 - ~ ~ " ~ ~ - R7 ~ ~/

5 ~
o - 1 - ~c wherein Rl and R2 represent a carbon-carbon bond, and R3 to R7 all are methyl groups The poppy does not contain l-glaucine, but at least four other alkaloids, whose separation from d-glaucine is extremely difficult. The d,l-glaucine synthesi~ed, starting from papaverin, by oxidative ring-closing reaction of laudanosoline ("Angewandte Chemie", 1967, pages 815 and 816) contains consider- -able quantities of an hitherto not recognised by-product, which crystallizes together with the glaucine salt. Our research has re-vealed this by-product to be l-(N,N-dimethylaminoethyl)-3,4,6,7-tetra- -~
methoxyphenanthrene, which is apparently produced by Hofmann degrada-tion. In view of the phenanthrene structure and the often sympatho-mimetically active dimethylaminoethyl group of this compound, glaucine contaminated with such a by-product cannot be used in pharmaceutical products.
This invention seeks to provide pharmaceutical compositions having anti-thrombotic or anti-tussive effect which are therapeuti-cally active at a low concentration of the active compound and are largely free from undesired side effects.
This invention further seeks to provide anti-tussive com-positions and anti-tussive agents which, even when given orally, have an improved long-lasting central cough-inhibitive effect) are not addictive and are largely free from the undesired gastrointestinal side effects of the known products.
According to the invention there is provided a pharmaceutical composition comprising a physiologically acceptable carrier and an effective amount of at least one therapeutically active compound selected from 1, 2, 9, 10-tetrahydroxyaporphine and glaucine contain-. ~.

~ - 2 -~ ~7~ 5 ing l-glaucine, and the physiologically acceptable salts and quater-nary N-alkyl-ammonium derivatives of such compounds.
Even at active substance concentrations far below what is necessary for papaverine, the pharmaceutical compositions according to the invention have a pronounced anti-thrombotic effect and are largely free from the side effects which occur when papaverine is used. The anti-thrombotic compositions, which may be adapted for oral administration or for injection, contain either 1,2,9,10-tetra-hydroxyaporphine or glaucine containing l-glaucine, or a physiolog-ically acceptable salt or quaternary N-alkyl-ammonium derivatives thereof, in an amount of about 5 to 30, preferably 8 to 20, mg/kg ¦ ~ body weight~ ~t~ s~g~7 Preferred compositions include pills, dragees, tablets and capsules each containing 100 to 800, preferably 200 to 500, mg of active compound. Other preferred compositions include such dosage unit forms containing 100 to 500, preferably 200 to 400, mg glaucine containing l-glaucine, or salt thereof, per dosage unit.
The anti-tussive compositions are characterized by a thera-:, peutically effective content of at least one of glaucine containing at least some l-glaùcine, and its physiologically acceptable salts.
For thls purpose, the therapeutically effective amounts of the glaucine or salt thereof are from 0.01 to 40, preferably 0.3 to ?, mg/kg body weight~ particularly when the glaucine contains 50 to 100%
l-glaucine. Preferred compositions include syrups containing 1 to 10 mg of active compound per ml and pills, dragees and tablets each containing 3 to 100, preferably 5 to 40, mg of the active compound.

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3~i Such solid dosage units generally contain 4 tQ 99%, preferably 10 to 19%, by weight of the active compound.
The compositions according to the invention are advantage-ous compared to the known anti-tussive medicines containing codeine or d-glaucine and are not only free from spasm including side effects in the intestinal region, but also have an unexpectedly increased anti-tussive effect compared to preparations containing d-glaucine.
The preferred anti-tussive compositions may contain, based on the total content of l-glaucine and l-glaucine salts, an at most equimolar quantity of d-glaucine and its unobjectionable salts with inorganic or organic acids. This combined application of l-glaucine and d-glaucine and their salts, within the molar ratios given above, unexpectedly provides a further improvement of the anti-tussive effect compared to that obtained by the same quantity of l-glaucine or the even less effective d-glaucine.
The pharmaceutical compositions may preferably contaln glaucine embonate, which shows a considerably prolonged anti-cough ~-effect, without delaying the beginning of said effect.
In the following there will be described in greater detail the L .~i;
~ - 4 -pharmaceutical compositions, the preparation of the active compounds and their therapeutical effects on the basis of examples and comparative tests.
Example 1.
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 60Co The product was 201 g of papaverine base with a melting point of 1~4 to 146C
~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 refluxed for 6 hours. After cooling, the crystalline product was filtered of and dried. The product was 280 g of papaverine methiodide with a melting point of 127 to 129C ~yield 97%)O
200 g C0~4 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 had disappeared. The resulting solution was poured into 12 litres o$ ~ter. T~e resulting white precipitate was ~iltered off and dried. The product was 230 g o laudanosine with a melting point of 113 to 115C (yield 78%~.
Example 2.
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 bromide was removed. After cooling the solution, the crystalline product was separated. me produet was 100 g of laudanosoline hyd~obromide with a melting point of 230C (yield 77%)~
Example 3.
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 80Co After complete solution, the solution was cooled down to 6C by adding ice, after which there was added a filtered solution, which had also been cooled to 6C, 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 crystals were filtered off, washed with acetone and dried. The product was 43 g of tetrahydroxy-aporphine hydrochloride showing a melting point of 242 to 244C (yield 50%).
Example 4.
For making d,l-glaucine hydrobromide, 6 g (0.018 Mol) of tetra-hydroxyaporphine hydrochloride was dissolved with warming in 840 ml of methyl alcoholO In a separate operation, 21.6 g (0.126 Mol) of trimethylphenyl~
ammonium chloride in methyl alcohol was reacted with a solution of 8.5 g (0.15 Mol) of ~OH in methyl alcoholO AEter filtering off the precipitated potassium chloride, the filtrate was made up to 840 ml with methyl alcoholO
The two solutions were then mixed together s]owly under a protective gas, and added slowly over 6 hours to anisol which had been heated to 110C, the methyl alcohol distilling ofE. After complet:ion of the addition the solution was cooled and a black, amorphous residue was removed by iltering. The resulting dark green filtrate was evaporated under vacuum until dry. To the residue there was added 5 ml of ethanol, 15 ml of hydrobromic acid and 20 ml of ethyl acetate. Prom the resulting mixture a product crystallized out and was filtered off and driedO The product was 5.5 g of d~l-glaucine hydro-bromide with a melting point of 235~ (with decomposition) (yield 67%).
Example 5.
A thin-layer chromatograph showed that the d~l~glaucine hydro-bromide made by the process oE Example 4 contained about 10% of l-(NjN-dimethyl-aminoethyl)-3,4,6,7-tetrametho,Yyphenanthrene. 60 g of this d,l-glaucine hydrobromide was dissolved in 200 ml of 50% aqueous ethanol and reacted with an excess of 8.5 g of potassium hydroxide in 50 ml of water.

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:~9~35 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 30.4 g of d,l-glaucine base showing a melting point of 128 to 130C. After repeated recrystallization ~rom further 100 ml portions of ethyl aceta~e, there was obtained 25.15 g of d,l-glaucine base with a melting point o-f 13~ to 140C.
By making a thin-layer chromatograph it was determined that this contained less than 0.1% of impurities.
After recrystallizing the combined residues from ethyl alcohol there was obtained about 5 g of 1-~N,N-dimethylaminoethyl)-3,4,6,7-tetra-methoxyphenanthrene with a melting point of 248 to 250C and a molecular weight, determined by mass spectrometry, of 369.
NMR spectrum: 9.13~s,1), 7.88(d,1)~ 7.66(d,1), 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 ~ol) of d,l-glaucine was dissolYed ~l 70 ml of ethyl alcohol and the solution reacted with a solution of 2.2 g Co.014 Mol) of d-tartaric acid in 70 ml of ethyl alcohol ~50C).
Slow cooling resulted in a fine crystalline precipitate which was filtered of~ washed with ether and dried. The product was 3.6 g of l-glaucine-d-bi~artrate with a melting point of 210 tc 212C and a specific rotation in water of -26 ~yield 93%).
The still impure l-glaucine-d-bitartra~e was reached with an aqueous solution of sodium hydroxide and extracted with ether. After eva-porating the solvent~ the residue was dissolved in 50 ml of ethyl alcohol and reacted with a solution of 1.15 g of d-tarkaric acid in 50 ml of ethyl alcohol. After separation there was obtained 3.39 g of l-glaucine-d-bitar-trate showing a melting point of 212 to 215G and a specific r~tation in water

3~5 of -32. The specific rotation of the l-glaucine base in ethyl alcohol was ~101 (94%)optical purity.
2.~8 g of l-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 l-glaucine hydrobromide with a melting point of 235C ~with decom-position) (yield 98.5%).
From the mother liquors of the first and second d-bitartrate crystallization there was obtained, after evaporation, a greenish residue, wilich was dissolved in 20 ml of wa*er, treated with aqueous sodium hydroxide and then extracted with 250 ml of ether. After drying and filtering, the solvent was evaporated, giving 1.67 g of d-glaucine with a melting point of 120C. The speciEic rotation in alcohol was 104.6. By reacting this product with hydrobromic acid there was obtained a d-glaucine hydrobromide with a melting point o~ 235C (with decomposition). The d-glaucine obtained by methylation from d-boldine showed a specific rotation in ethyl alcohol of ~115, ~ .
To make l-glaucine hydrochloride, a quantity of l-glaucine was dissolved in a little methyl alcohol and ~o the solution there was added a small excess of concentrated hydrochloric acid. To the resulting volu~ninous precipitate there was added ethyl acetate, giving whitish to pink crystals.
After ~iltering, washing with acetone and drying there were obtained white to pink fine crystals of l-glaucine hydrochloride with a melting point of 232 to 233C.
; Example_ .
For making l~glaucine hydToiodide, a quantity of l-glaucine was dissolved in 2 n hydrochloric acid and the solution reacted wi-th saturated potassium iodide solution. The resulting crystalline precipitate was re-crystallized from a mixture of methyl alcohol and ether. This gave a crystal-line, yellowisll glaucine hydroiodide with a melting point of 238Co Example 8.
A mixture of 3.92 g (0.01 Mol) of glaucine, 1.5 g (0.011 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%).
Example 9.
45 g (0.126 Mol) of laudanosine was dissolved in methanol and heated ~Yith 15 ml of methyl iodide for 2 hours under reflux. ~le resulting solid residue was filtered, washed and dried, giving 56 g of laudanosine methiodide monohydrate with a melting point of 238 to 2~0C.
Example 10.
30 g o laudanosine methiodide hydrate was dissolved in 150 ml o$ 48~ hydrobromic acid and heated under reflux for 15 hours. The resul~ing, yellow precipitate was filtered and dried, giving 22.1 g of laudanosoline methobromide with a melting point of 237 to :739C.
Example 11.
50 g ~0~0125 Mol) of laudanosoline methobromide was dissolved in 5~Q ml of water at 20C and reacted with a filtered aqueous solution contain-in~ 40.5 g of iron ~III) chloride in 500 ml of water. AEter 24 hours there was added to the dark violet solution 500 ml of concentrated HCl. The mi~ture was evaporated down to about half its original volume. After adding methanol a yellow precipitate crystallized out. The product was 27.6 g of tetra- -hydroxyaporphine methochloride with a melting point of 236 to 239C (yield 63%).
The resulting product, and also the previously obtained products, were identified b~ ultra-violet, infra-red and NMR spectra and by their melting points.
Example 12.
For making l-glaucine embonate, 841 mg of l-glaucine was reacted _ g~

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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 l-glaucine embonate with a melting point of 189 to 192C
(yield 100%).
Example 13.
For making d,l-glaucine embonate, a mixture of 1.42 g of d,l-glaucine and Q.78 g of embonic acid was dissolved in 20 ml of dimethyl form-amide and the resulting brown solution was introduced into 400 ml of water.
The resulting slig~tly brownis~ precipitate was filtered off, washed with wate~ and dried. T~e product was 1.75 g of d,l-glaucine embonate with a melting point of 188 to 190C (yield 80%).
AnalysiS: C65H66N2 14 calculated: C 71.02% H 6.05% N 2.55%
found: C 69.36% H 5.98% N 2.52%
Example 14.
For making d,l-glaucine tartrate, 355 mg Cl Mol) of d~l-glaucine in 10 ml of ethyl alcol~ol was reacted with 150 mg of d,l~tartaric acid in 10 ml of ethyl alcohol at 50C. After cool mg the solution there was obtained 220 mg of optically inactive d,l~glaucine-d,l-tartrate with a melting point of 215C ~yield 97%).
E ample_15.
Coated pills with an anti-cough effect were made of the following ~ components:
; Core: 1 glaucine hydrobromide 20 mg lactose 60 mg starch 40 mg talcum 10 mg 130 mg -` 1 0 - .,:, : .

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Coating: gum arabic 4.5 mg ~alcum 35 mg crystallized sugar 80 mg ::
white wax 0.1 mg red d~e 0.4 mg 120 mg Example 16.
For making capsules with long-period anti-cough effect~ stretch-capsules of hard gelatin were each filled with 160 mg of a mixture of the ; 10 following components:
d~l-glaucine hydrochloride 10 mg d,l-glaucine embonate 25 mg lactose 60 mg : starch 60 mg magnesium stearate 5 mg 160 mg Example 17.
~cr making a syrup w~th an anti~cough effect the ollo~ing com-; ~ ponents~were mixed together~
;20~ : l-glaucine~hydrobromide 133 mg: :
saccharose~
sorbitol citric acid 125 mg ~ p-h~droxybenzolc~meth~l~ester lOQ mg : ~ aroma ~essence of:sweet oranges) 1.5 g : ~ :
~ ~ distilled wa~er, up to : 100 g : For:making a medlcine~intended:for m~ection, ampoules were ~ fllled wlth a solution~having the following solution: :

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~9~3~3~i tetrahydroxyaporphine hydrochloride 11.22 mg (corresponding to 10 mg of tetrahydroxyaporphine base) sodium bisulphate 0.7 mg tartaric acid 5.01 mg sodium hydrogen tartrate 10.2 mg propylene glycol 300 mg distilled water to 1 ml.
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Although the active compounds used according to the invention have an anti-thrombotic affect already in small dosages~ the exact amount of active agent to be used in an anti-thrombotic tablet, dragee or capsule will ~ary with the severity of the thrombogenic risk of the patient, his weight and his response to the active compowld. The active compound should prefer-ably be compounded witll 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 solu~ion or dispersion in the gastric ~uices in the stomach. To this purpose the composition may comprise about 10 to 99%, preferably 20 to 75% of active ; 20 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 follow-ing ingredients:
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d, l-glaucine 100.0 mg sucrose 25.9 mg starch 22.1 mg acacia 7.8 mg talc 3.1 mg magnesiwn stearate 1.5 mg stearic acid 1.6 mg 162.0 mg The active compound was mixed wi~h the sucrose and the gum 10acacia, 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.

Fxample 20 There were prepared anti-thrombotic capsules each containing:
tetrahydroxyaporph~ne hydrochloride 400.0 mg ;; magnesium stearate 4.0 mg 404.0 mg ~:

Example 21 ~ :
There were prepared an~ thrombotic tablets each containing:
1-glau~cine hydrobromide 200.0nlmg : magnesiwn stearate 2~0 mg 202.0 mg :

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In a conventional tabletting machine there were prepared anti-thrombotic tablets each containing: .
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d,l-glaucine h~drobromide 200.0 mg polyvinylpyrrolidonc 15.0 mg corn starch 20.0 mg 23500 mg Alternatively there can be prepared tablets adapted to be subse-quently cut, each containing 400 mg active compound.
Example 23.
For making anti~thrombotic capsules hard gelatine capsules were each filled with a mixture of:
d,l-glaucine hydrobromide 200.0 mg silicagel lO.0 mg magnesium stearate 2~0 mg 212.0 mg Comparative test l.
In order to compare the anti-cougrh effectiveness of codeine phos-phat0 with the effectivenesses of the different isomers of glaucine hydro-bromide, the testing method of Friebel and Reichle was used~ 60 guinea pigs with weights between 200 and 300 g ~ere divided up into ten groups of different ~izes. Before administering the ac~ive substance, each animal was ~Z0 ~subjected, on the same day as the day of test with the active substance, for a period of~8 minutes to an aerosol o 20% citric acid, the animal being con- ~ - ;
fmed 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 test, there was given subcutaneously to each~
animal of each group a suspenslon of the active substance in 1% sodium carboxy-methyl ce~llulose. After 30 minutes each animal was again exposed in the test :
chamber for 8 minutes to the aerosol of 20% citric acid and the number of cough pressure pulses recorded. The different active substances were administered in increasing~doses ranging from 3 mg/kg to 100 mg/kg of body .~ .

i35i weight. Altogether eight different doses were given. 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 tne resulting effectiveness curve, based on the logarithm of the dose, the coefficient of correlation and the ED50 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,l-glaucine hydrobromide and for l-glaucine hydrobromide thirt~ minutes after administering the active substance are summarized in Table I.
Table I

active substance number correl- rising ED50 of ation slope mg'k _ _ ani als coeff. of curve _ _ _ codeine 41 -0.3956 -~7.~ 2006 d-glaucine hydrobromide 59 -0.53~0 -60.8 54.0 l-glaucine hydrobromide 3~ -0.6~67 -70.96 31.2 d,l-glaucine hydrobromide 25 -0.487~ -57.8 27.2 *statis-ticall~ signiEicant ~p <0.01) for each active substance.
These results show that l-glaucine hydrobromide has a considerably lo~er effective dose ED50 value compared to d-glaucine hydrobromide, and that surprisingly the dll-glaucine hydrobromide has an even lower ED50 value than l-glaucme hydrobromide, Comparative test 2, One of the most unpleasant side effects of anti-cough medicine containing codeine is cons~ipation 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 o$ mice was therefore made, AEter subcutaneous injection of the ac~ive substance, each animal was given, through a throat probe, a 10% suspension of carbon in a 5% aqueous slurry of gum arabic. Two hours later the intestine ~L~9~35 was sectloned and the distance of stomach coecum measured, and also the distance travelled by the carbon, which was expressed as percent of intestine length.
Table II

active substance dose number of distancestandard mg/kg animals travelled, deviation avera~e % %
CONTROL 40 74.3 3.6 codeine phosphate 3 7 70.4 3.5 18 74.3 2.4 17 ~4.8* 9.5 100 18 20.6* 4.5 d-glaucine hydrobromide 10 18 74.6 2.5 18 80.1 2.6 100 18 36.4* 4.9 l-glaucine hydrobromide 3 8 74.3 6.3 8 94.3* 2.0 8 85.4 4.3 100 8 51.8* 10.6 *slgnlficantiy different from the control value ~p ~0.05)0 The efects of codeine phosphate, of d-glaucine hydrobromide and of l-glaucine hydrobromide on intestinal motility were determined at different doses. For~each do~se the average value was calculated for a sufficient number of animals. The results of these tests are shown in Table II above.
These results show that l-glaucine hydrobromide~ in contrast to ~: :
d-glaucine hydrobromide and codeine phosphate, administered in a dose of 10 mgjkg~resulted in a significant increase in intestinal activity. And even~
when the dose of l-glaucine hydrobromide is increased to 30 mg/kg and 100 mg/kg, the intestinal motility is considerably more ~han the corresponding values -~or d~glaucine h~drobromide and codeine phosphate.

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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 x 10 5 and 32 x 10 5 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 5 and 8 x 10 5. On the other hand, using l-glaucine at molar concentrations between 1 x 10 5 and 8 x 10 5, a reduction in spontaneous activity and no increase in tone were observed. This sho~s that l glaucine, in contrast to d-glaucine and codeine, has no spasmo-genic activityO
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 fo~md that both codeine and d glaucine are effective against carbachol or histamine at a molar concen-tration of 1 x 10 3 or 4 x 10 5. On the other hand, d,l-glaucine was effective against histamine at molar concentrations of only 4 x 10 8 to 4 x 10 6 and - .
against carbachol at molar concentrations of only 4 x 10 7.
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C~omparative test 4.
In a forther serles 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 m the solution surrounding the intestinal preparation. 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.
active substallce molar concentration ~x 5x 8x lx 2x 4x 6x 8x lO 10-5 10-5lO l0_ 10 codeine - 85 - 49 36 - - -phosphate - 51 - 49 52 58 d-glaucine - - - 77 7~ 4~1 - -hydrobromide - - - 95 79 36 l~glaucine - - - 71 64 24 - 7 hydrob:romide ~ 74 32 23 - -d,l-glaucine - - - 63 48 8 hydrobromide 81 - 63 These results show that codeine phosphate at a molar concentration of about lO 5 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 5 suppresses electrically induced contraction reflexes. But at higher concentrations the increasing spontaneous activity prevents any distinguishing between direct production and electric production of the activity. Using l-glaucine hydrobromide, it was found that at a concentration ;~ of 4 x 10 5 the electrically induced spasms are more effect~vely suppressed, compared to what is obtained using d-glaucine hydrobromide. At a concentration o~ 8 x lO 5 of l-glaucine hydrobromide the elec~rically induced contractions .
are suppressed even more ~effectively~ Using d,l-glaucine hydrobromide it was found possible at concentrations of 4 x lO 5 to 6 x lO 5 to suppress the electrically induced spasms practically completely) without at the same time ; inducing any spontaneous actlvity. In comparing the values shown in the table it should be observed that when the d,l-glaucine hydrobromide is at the concentration o 4 x lO 5, the d-glaucine hydrobromide and the l-glaucine 2Q hydrobromide are each at a molar concentration of 2 x lO 5.

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.... . : . , Comparative test 5.
In a series of tests conducted by the method of Domenjo~, the effects of the different active substances on the coughing centre was examined by electrical stimulation of the upper laryngeal nerves of anaesthetized catsO 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, 1.0 mg/kgJ 3O0 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, codeine 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 ME4D duration _ m~kg minutes codeine phosphate 6 18 - 54 d-glaucine hydrobromide 1 5 - 39 d,l-glaucine hydrobromide 1 44 - 62 These results show that ~oth the d-glaucine hydrobromide and the d,l~glaucine h~droh~omide completely suppress the coughing reflex already at a dose of 1 mg/kg of body weightO The dose of codeine phosphate required for this is 6 mg~kg. A~d it will be observed that the duration of the effect, at this minimal dose, is considerably greater for the d,l-glaucine hydro-bromide than for the d-glaucine hydrobromide.
Comparative test_6.
The equivalent effective doses for codeine phosphate and d~l-glaucine 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 :

83~i 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 average control value obtained in this way for each group of guinea pigs was calculated. Af~er this control test each animal was given, by means of a throat probe in a volume of 10 ml/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,l-glaucine hydrobromide~ One hour after administering the active substance, each animal was once more subjected for 8 minutes to the citric acid aerosol and the num~er 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

active substance dose cough pulses, percent m~/k~ of control value ,, , ~
codeine phosphate 50 37 d,1-glaucine hydrobromide 100 30 The results show that a dose of 75 mg of codeine phosphate per o~ ~ody weigh~ has about the same effect as a dose of 150 mg of d,l-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,l-glaucine hydrochloride, or 150 mg/kg of d,l-glaucine hydrobromide, or 378 mg/kg of d,l-glaucine embonate. These doses are the equimolar quantities~

based on glaucine. After a period of 1, or 3, or 5, or 24 hours each animal was again subjected for 8 minutes to the action of the citric acid aerosol, and ~he 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 h 3 h 5 h 24 h . _ . . _ . . . .
codeine phosphate 84 70 84 95 d,l-glaucine hydrobromide 90 47 59 113 d,l-glaucine hydrochloride 80 72 69 102 d,l-glaucine embonate 80 51 57 81 These results show that in all the tests the d,l-glaucine salts have a greater effect than codeine phosphate in reducing the number of cough pulses. In particular it will be observed that d,l-glaucine embonate has a considerably longer-lasting effect without any delay in the beginning of the effect.
Com~arative test 7.
The toxicity thresholds of codeine phosphate, of d-glaucine hydro~romide and of d,l-glaucine hydrobromide were determined in mice by the ~lcox method. It emerged, as shown in Table VII, that d,l-glaucine is considerably less toxic than d glaucine. The toxicities of codeine phosphate and d,l-glaucine hydrobromide are about equal.
Table VII.

active substance LD50 peroral LD50 subcutaneous - - mg/kg mg/kg codeine phosphate 640 230 d~glaucine hydrobromide 345 125 d,l-glaucine hydrobromide 686 320 Com~_~ative test 8.
~le effectiveness of several compounds corresponding to Formula (I) 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 sub-stances in physiological NaCl solutions, the solutions showing def~nite molar concentrations 10 microlitres of each solution was tested, after 5 minutes incubation at 37C, for its effect on aggregation of the blood platelets under the influence of adenosine diphosphate added to give the critical con-centration. The measurements were made by the turbidity method described by Born in "Nature" (1962) on page 927. The critical concentration is the least concentration of adenosine diphosphate which results in primary ~-aggregation of the blood platelets. This is followed by an irreversible secondary aggregation. The following table shows the results:

aggregation inhibition, at 5 x 10-6 molar Compound concentration _ papaverine hydrochloride tetrahydroxyaporphine hydrochloride +
tetrahydroxyaporphine methochloride +
. .
d~ l-glaucine hydrobromide l-glaucine hydrobrvmide ; ~0 Comparative test 9 The active compounds, namely d, l-glaucine hydrobromide and tetrahydroxyaporphine hydrochloride were selected for in vivo assessment of the anti-thrombic activity using a modified hamster cheek pouch tech-nique described by Duling, Berne and Born (196&) and Begent and Born ~1970).
Male golden hamsters, weighing 80-120 g, were anaesthetised with intraperitoneal pentobarbitone. The cheek pouch was everted using a cotton .~ ',.

'~i . .

~0~ 3~i bud and spread out over a special Perspex stage. The top layer and connective t:issue were removed leaving a thin vascular membrane which was trans-illuminated 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 37C.
A micropipette of tip diameter 1-2 ~m 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 16-40 ~m. The re~erence electrode was placed in contact with the animal. When the negative potential was applied from the ex~ernal circuit the resultant current of approximately 300 nA ejected adenosine diphosphate ~of the order of 2 x 10 14 moles/sec) from the pipette. This caused the formation of a white body (platelet thrombus) at the tip of the pipette or slightly downstream. The ~ro~th 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 each drug was assessed over a period be~cween 30 and 90 minutes after oral administration of each drug at the dose levels o Z~ 5J 10 aDd 20 mg/kg.
Each compound was tested at four dose levels in 5 animals each with one group of 5 animals serving as a control. Preparation o the cheek pouch commenced at lS minutes after dosing and thrombus stimulation commenced at 30 minut~s 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 o~ control group on thrombus induction in the micro-circulation of the ~ ' ..
3Q ~ hamster cheek pouch against ~he dose level. All ~h~ compounds were shown ' . ~

3~

to inhibit thrombus ormation when administered orally. The results are shown in the following table IX:
Table IX

~roduct oral dose for maximal time of maximal maximal inhibition inhibition inhibition _ _ mg/kg % _min (after dosing) glaucine hydrobromide 10 41 ~}~ 3-¢~
tetrahydroxyaporphine ~ ~
hydrochloride 20 29 ~ t~
The dose response curves indicated that all compounds were ac*ive at relatively low dose levels and the inhibi*ory 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 codeine phosphate, d-glaucine hydrobromide, d,l-glaucine hydrobromide, l-glaucine hydrobromide and l-glaucine-d-tartrate, respectively, guinea pigs were orally dosed with one of the active agents mentioned one hour before being exposed for ten minutes to a 5% citric acid aerosol. In each case the last 5 minutes of the exposure were used for the measurements. The effective doses ED50 are shown in Table IX:
Table IX
Oral ED50 mg/kg Codeine phosphate 94-9 .
d~glaucine hydrobromide 198096 d,l-glaucine hydrobromide 16.0 1 glaucine hydrobromide 7.21 l-glauc~ne-d-tartra~e 608

Claims (17)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A pharmaceutical composition comprising a physiologically accept-able carrier and an effective amount of at least one therapeutically active compound selected from 1, 2, 9, 10-tetrahydroxyaporphine and glaucine contain-ing 1-glaucine, and the physiologically acceptable salts and quaternary N-alkyl-ammonium derivatives of such compounds.

2. A composition according to claim 1, wherein said active compound is selected from glaucine containing 50 to 100% 1-glaucine, and the physiologi-cally acceptable salts and quaternary N-methohalogenides thereof.

3. A composition according to claim 2, wherein said active compound is selected from d,1-glaucine and the physiologically acceptable salts thereof.

4. A composition according to any of claims 1 to 3, in dosage unit form for administration to humans to obtain a thrombocyte aggregation inhibit-ing effect, comprising a solid or aqueous liquid carrier and from 100 to 800 mg of the active compound per dosage unit.

5. A composition according to any of claims 1 to 3, in dosage unit form for administration to humans to obtain a thrombocyte aggregation inhibiting effect, comprising a solid or aqueous liquid carrier and from 200 to 500 mg of the active compound per dosage unit.

6. A composition according to any of claims 1 to 3 in the form of dosage units selected from pills, dragees, tablets and capsules, suitable for oral administration to humans to obtain a thrombocyte aggregation inhibiting effect, containing from 100 to 800 mg of the active compound per dosage unit.

7. A composition according to any of claims 1 to 3 in the form of a solution of the active compound in a physiologically acceptable aqueous liquid, suitable for administration to humans by injection to obtain a thrombocyte aggregation inhibiting effect, containing from 100 to 800 mg of the active compound per dosage unit.

8. A composition according to claim 2 in dosage unit form for oral administration to humans to obtain an anti-tussive effect, comprising a solid or aqueous liquid carrier and a unit dosage of about 0.01 to 40 mg/kg.

9. A composition according to claim 2 in dosage unit form for oral administration to humans to obtain an anti-tussive effect, comprising a solid or aqueous liquid carrier and a unit dosage of about 0.1 to 3 mg/kg.

10. A composition according to claim 8 or 9 in the form of a syrup con-taining 1 to 10 mg of the active compound per ml.

11. A composition according to claim 8 or 9 in the form of a pill, dragee, capsules or tablet each containing 3 to 100 mg of the active compound.

12. A composition according to claim 8 or 9 in the form of a pill, dragee, capsules or tablet each containing 5 to 40 mg of the active compound.

13. A composition according to claim 8 or 9 in the form of a pill, dragee, capsules or tablet each containing 3 to 100 mg of the active compound, and containing 4 to 99% by weight of the active compound.

14. A composition according to claim 8 or 9 in the form of a pill, dragee, capsules or tablet each containing 5 to 40 mg of the active compound, and containing 10 to 19% by weight of the active compound.

15. A pharmaceutical composition according to any one of claims 1 to 3 comprising glaucine embonate as the active compound.

16. A pharmaceutical composition according to any one of claims 8 and 9 comprising glaucine embonate as the active compound.

17. A composition according to claim 8 or 9 in the form of a pill, dragee, capsules or tablet each containing 5 to 40 mg of the active compound, and containing 10 to 19% by weight of glaucine embonate.