NZ243365A - Use of 9(z),12(z),15(z)-octadecatrien-6-ynoic acid (dicranine) in pharmaceutical and cosmetic compositions - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £43365 24 3 3 65 Priority "■■ — "■';•■• "25 NoV 1993 Pub'ic-':-n Cr.r.-.

P.O. _ R | p?s» jr\ », p i»sf p ^ ^ 1« %J? CsJ1' i' b jp*& \j 'a s a Xi '<$ Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION USE OF 9, 12, 15-OCTADECATRIENE 6-YNOIC ACID WE, SOCIETE DES PRODUITS NESTLE S.A., a Swiss company of Entre-deux-Villes, Vevey, SWITZERLAND hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page la) la This invention relates to the use of 9(Z),12(Z),15 (Z)-octadecatriene-6-ynoic acid which will be referred to hereinafter as "dicranine".

Dicranine is a polyunsaturated fatty acid of which the methyl ester can be extracted from the moss Ceratodon purpureus, as shown in the Article by J. Gellerman et al. published in Biochemistry (1977), Vol. 16, 7, 1258-1262.

It has surprisingly been found that dicranine has therapeutic properties, more particularly anti-aggregating and anti-inflammatory properties and also anti-bacterial properties.

Accordingly, the present invention relates to the use of dicranine as a therapeutically active substance. More particularly, the present invention relates to the use of dicranine as an anti-aggregating, anti-inflammatory and anti-bacterial agent.

The present invention also relates to a pharmaceutical composition containing dicranine as its therapeutically active substance in conjunction with a pharmaceutically inert excipient.

The invention provides a chemical compound 9(Z),12(Z),15(Z)-octadecatriene-6-ynoic acid as a therapeutically active substance.

Specifically the invention provides use of a chemical compound 9(Z),12(Z),15(Z)-octadecatriene-6-ynoic acid together with a pharmaceutically acceptable excipient to produce a pharmaceutical composition.

The invention also provides a pharmaceutical composition containing 9(Z),12(Z),15(Z)-octadecatriene-6-ynoic acid as its therapeutically active substance in conjunction with a pharmaceutically inert excipient.

The invention further provides a cosmetic composition containing 9(Z),12(Z),15(Z)-octadecatriene-6-ynoic acid as the therapeutically active ingredient, together with an acceptable carrier or diluent.

The pharmaceutical composition according to the invention may be formulati as an injectable solution or as tablets (followed by page lb) lb 24 The present invention also relates to a cosmetic composition containing dicranine.

The cosmetic composition according to the invention may be formulated, for example, as a cream, ointment or lotion.

The present invention is illustrated by the following Examples in which the following abbreviations are used: - 13-HODE 13-hydroxyoctadecadienoic acid - 12-HETE 13-hydroxyeicosatetraenoic acid -HETE 5-hydroxyeicosatetraenoic acid - HHT 12-hydroxyheptadecatrienoic acid - 12-HPETE 12-hydroperoxyeicosatetraenoic acid •X £ n r ■<z - - - rn r 16 JUL1993 (followed by page 2) 24 3 3 6 5 - LTB4 (5S,12R)-5,12-dihydroxy-6,14-cis—8,10-trans- eicosatetraenoic acid - PGG2 15-hydroperoxy-9a,lla-peroxidoprosta-5,13- dienoic acid - PGH2 15-hydroxy-9a,lla-peroxidoprosta-5,13 - dienoic acid.

Example 1 describes a process for the synthesis of dicranine.

Examples 2 to 6 demonstrate the anti-inflammatory, 10 anti-aggregating and anti-bacterial properties of dicranine.

Example 1 a) 50 mg Fe(N03)3 and 10.9 g metallic sodium are added to 15 200 ml liquid NH3.

After stirring for 1 hour at ambient temperature, 50 ml propargyloxytetrahydropyran are added dropwise. After stirring for another hour, 30 ml ethyl iodide are added. Stirring is continued for 12 hours to allow the ammonia to 20 escape. Sulfuric acid is then added to a pH value of approximately 4, after which the mixture is extracted with ether.

The ether phase is recovered and the solvent is evaporated. The residue is dissolved in 50 ml methanol, 25 10 ml 25% sulfuric acid is added to the resulting solution and, after stirring for 1 hour, the mixture is diluted with water. The diluted mixture is then extracted with ether. The ether phase is recovered and dried over magnesium sulfate, after which the solvent is evaporated under 30 reduced pressure. The residue is distilled and 22 g 2-pentine-l-ol are obtained. b) 0.5 ml pyridine is added to a solution of 10.5 g 2-pentine-l-ol in 150 ml ether, after which 4.2 ml phosphorus tribromide are added dropwise. 24 3 3 6 5 3 The mixture is heated under reflux for 3 hours, after which ice is added.

The mixture is then extracted with ether. The ether phase is washed with a potassium carbonate solution and 5 dried over magnesium sulfate and the solvent is evaporated under reduced pressure. The residue is distilled and 9.75 g l-bromo-2-pentyne are obtained. c) 25.5 g ethyl bromide are added with stirring to a 10 solution of 100 ml THF containing 5.0 g magnesium.

The mixture is cooled to 0°C and a solution of 6.0 g propargyl alcohol in 10 ml tetrahydrofuran (THF) is gently added.

The mixture is stirred for 2 hours at 20°C and then 15 cooled to 0°C. 0.5 g cuprous chloride and a solution of 9.7 g l-bromo-2-pentyne in 15 ml THF are then added in that order. The mixture is heated under reflux for 19 hours, after which an excess of cuprous chloride is added and heating is continued for 5 hours. A mixture of ice and 20 sulfuric acid is then added and the resulting mixture is extracted with ether. The ether phase is washed with a potassium carbonate solution and dried over magnesium sulfate and the solvent is removed under reduced pressure. The residue is distilled and 6.8 g 2,5-octadiyne-l-ol are 25 obtained. d) 6.5 g 2,5-octadiyne-l-ol and 2 ml phosphorus tribro-mide in 75 ml ether are reacted in the same way as described in b) to obtain 6.3 g l-bromo-2,5-octadiyne. e) 3.6 ml propargyl alcohol and 6.0 g l-bromo-2,5-octadiyne are added to 9.6 ml ethyl bromide and 2.8 g magnesium in 50 ml THF in the same way as described in c) . 4.5 g 2,5,8-undecatriyne-l—ol are obtained in the form 24 3 3 6 5 4 of a white solid. f) 0.5 ml pyridine and 1.0 g Lindlar catalyst are added to a solution of 2.0 g 2,5,8-undecatriyne-l-ol in 30 5 ml ethyl acetate. The mixture is placed under hydro gen at 1 bar for 24 hours.

The catalyst is recovered by filtration and the solvent is evaporated under reduced pressure.

Distillation of the residue gives 0.8 g 2,5,8-undeca-10 triene-l-ol. g) A mixture of 0.8 g 2,5,8-undecatriene-l-ol and 0.3 ml phosphorus tribromide in 20 ml ether is heated under reflux for 30 minutes in the same way as described in 15 b) . Distillation gives 0.52 g l-bromo-2,5,8-undeca- triene. h) Bromine is added a solution of 5.0 g 6-heptenoic acid in 70 ml ether to obtain the dibrominated compound (verified by NMR).

A solution of 5 g sodium in 150 ml liquid NH3 is added to this mixture which is then stirred for 12 hours to allow the ammonia to escape. 10.0 g solid ammonium chloride and 200 ml water are then added. The mixture is acidified by addition of 6 N HCl and is then extracted with ether. The ether phase is washed with water and dried over magnesium sulfate and the solvent is evaporated under reduced pressure. Distillation of the residue gives 3.5 g heptyne-6-oic acid. i) 550 mg heptyne-6-oic acid in 5 ml THF are added dropwise to a solution of ethyl magnesium bromide prepared from 0.21 g magnesium and 0.97 g ethyl bromide in 50 ml THF.

The mixture is stirred for 1 hour at 20°C, after which 24 3 3 65 mg CuCN and 500 mg l-bromo-2,5,8-undecatriene are added in that order. The mixture is heated under reflux for 6 hours, cooled and, after addition of ice, is acidified with dilute HCl.

The mixture is extracted with ether, the ether phase is recovered and dried over magnesium sulfate, the residual solvents are evaporated under pressure and a yellow oil is obtained.

Column chromatography and distillation give 260 mg of 10 ' a colourless oil which is indentified by NMR of the carbon and proton and by mass spectrometry as being 9(Z),12(Z),15-(Z)-octadecatriene-6-ynoic acid.

Example 2 This Example tests the effect of dicranine on the lipoxygenase and the cyclooxygenase of isolated human blood platelets.

Platelets are isolated from human blood on an anticoagulant of the ACD type (Lagarde et al., Thrombos. Res. 20 (1979) 17, 581-588). Dicranine solubilized in ethanol is added to the platelets in suspension in a physiological buffer (Hepes) in a quantity of approx. 3.105 cells/^l to obtain a dicranine concentration of 10~* or 10"6 M.

The platelets are incubated for 10 minutes at 37 °C and 25 are then stimulated by addition of an arachidonic acid solution to obtain an arachidonic acid concentration of 10~5 M, followed by incubation for another 10 minutes at 37"C. The whole is then acidified to pH 3 by addition of 3N HCl. The total lipids are extracted with ethyl ether and the mono-30 hydroxylated fatty acids are separated therefrom by thin-layer chromatography using the method described by Guichar-dant et al. in Biochem. Biophys. Acta, (1985) , 836, 210-214.

The monohydroxylated fatty acids are measured by high-35 performance liquid chromatography (HPLC) and are quantified 24 3 3 6 5 by comparison with the standard 13-HODE under the assumption that they all have the same molar extinction coefficient at 3.10* M"1 cm"1.

During activation of the platelets, two metabolic pathways of the arachidonic acid are activated, namely: the lipoxygenase pathway which leads to the formation of 12-HPETE which is reduced to 12-HETE, the cyclooxygenase pathway which forms the metabolites responsible for platelet aggregation, namely:: the endoperoxides PGG2, PGH2 and the thromboxan A2 (TXA2) which stabilizes into TXB2 the prostaglandins of series 2 and - a monohydroxylated fatty acid, HHT.

Determination of the 12-HETE enables the lipoxygenase activity to be estimated. Determination of HHT enables the cyclooxygenase activity to be estimated.

The following results are obtained: Dicranine concentra t i on Visually observed effect Increase in 12-HETE relative to the control Reduction in HHT relative to the control Control 10"6 M "* M Aggregation — Slight inhibition 40% Inhibition of platelet aggregation 654% 18% 31% Very strong activation of the lipoxygenase pathway and a reduction in the cyclooxygenase pathway are observed for a dicranine cocnentration of 10~4 M. This result shows that 35 the increase in 12-HETE is not associated with greater availability of the arachidonic acid which is less well utilized by the cyclooxygenase. Accordingly, dicranine is 2 4 3 3 6 5 7 effective in increasing the 12-HETE. Now, it is known that 12-HETE is involved in the regulation of immunological processes. Accordingly, dicranine may be assumed to have an immunological effect.

The same effect is observed, albeit less strongly, for a dicranine concentration of 10"6 M.

Example 3 This Example tests the anti-agglomerating properties 10 of dicranine on isolated human blood platelets. The test used is based on Born's turbidimetric method described in Nature (1962), 194, 927-929.

Several inductors each acting specifically at various levels of the action of arachidonic acid were used, namely: 15 - thrombin which takes into account the entire enzymatic mechanism resulting in aggregation, - arachidonic acid, a direct substrate of cyclooxygenase which forms the mediators responsible for aggregation, namely the endoperoxides PGG2, PGH2 and thromboxan A2 20 (TXA2) , 9-methano-PGH2 (structural analog of PGH2, reference U46619) which causes aggregation on its own. The following results were obtained for a dicranine concentration of 10"* M: 1. Without preliminary incubation of the dicranine: When thrombin (0.1 unit/ml) or arachidonic acid (5.10~6 M) is used as inductor, normal aggregation of the platelets is observed (no inhibition). When 9-methano-PCH2 30 (1 jug/ml) is used as inductor, aggregation is completely inhibited. Accordingly, it may be concluded that dicranine does not act at the level of the enzymes phospholipase or cyclooxygenase; on the contrary, it seems to act directly on the receptor site of PGH2/TXA2 to inhibit aggregation. 35 If a second dose of 9-methano-PGH2 (1 ng/ml) is added, no 24 3 3 6 8 aggregation is observed. Accordingly, the inhibition process appears to be irreversible. 2. With incubation of dicranine: After incubation for 10 minutes at 37"C, aggregation is slightly inhibited when thrombin is used as inductor. After incubation for 20 minutes at 37°C, aggregation is inhibited to a far greater extent with thrombin. One explanation may be that the inhibition of aggregation necessitates the formation of a metabolite of dicranine which takes place by slow kinetics.

The results are set out in the following Table: Inductors Thrombin . Without incubation . 10 Mins. . 2 0 Mins.

Arachidonic acid . Without incubation 9-Methano-PGH, . Without incubation Level of aggregation (%) Control Dicranine (10"* M) 65 70 68 70 66 70 69 44 70 This effect may also arise out of the fact that the synthesis of 12-HETE, which is known as an inhibitor of platelet aggregation, is considerably increased in the presence of dicranine. The same applies to the synthesis of 12-HPETE, its precursor.

It may be concluded from the present Example that, in a concentration of at least 10"*M and after incubation, dicranine has anti-aggregating properties towards human blood platelets. 24 3 3 6 5 9 In addition, the dicranine appears to act through its metabolites (monohydroxylated fatty acids) rather than directly.

Example 4 One of the principal metabolites of dicranine was isolated by HPLC, namely 13-hydroxy-9(Z),12(Z),15 (Z) -octadecatriene-6-ynoic acid.

The anti-aggregating properties of this metabolite 10 were studied in accordance with Example 3 using thrombin as inductor in a quantity of 0.1 unit per ml.

Slow inhibition of platelet aggregation induced by thrombin is observed at a metabolite concentration of 10-6M both without incubation and after incubation for 10 15 minutes.

Example 5 The object of this Example is to test the effects of dicranine on isolated human leucocytes.

Human leucocytes are isolated by the method described by Guichardant et al. in Biochem. J. (1988), 256, 79-883. The leucocytes are then optionally incubated for 30 minutes at 37°C in the presence of dicranine in a concentration of 10"* or 10_6M in ethanol, after which the optionally incu-25 bated leucocytes are stimulated by addition of arachidonic acid in a concentration of 10_5M and ionophoric calcium (Oven A23187-Sigma) in a concentration of 2.10_6M and are then left for 10 minutes at 37°C. The whole is then acidified to pH 3 by addition of 3N hydrochloric acid. The 30 total lipids are extracted with a mixture of ethanol and chloroform (1:2), after which the lipids extracted are separated by two-dimensional thin-layer chromatography.

The monohydroxylated fatty acids are developed in the first dimension with a solvent mixture of hexane, ethyl 35 ether and glacial acetic acid in a ratio by volume of 24 3 3 6 5 59:40:1, the dihydroxylated fatty acids remaining at the beginning of the plate. The monohydroxylated fatty acids are collected from the plate together with the 13-HODE and are extracted with ethyl ether. After evaporation of the 5 solvent under nitrogen, they are separated by inverse-phase HPLC with an elution solvent mixture of methanol and aqueous acetic acid, pH 3, in a ratio by volume of 73:27. They are detected by UV spectroscopy at a wavelength of 234 nm and are quantified by comparison with the standard 13-10 HODE on the assumption that they all have the same molar extinction coefficient at 3 x 10* M"1cm"1.

The dihydroxylated fatty acids remaining at the beginning of the plate are then developed in the second dimension perpendicularly to the first using a solvent 15 mixture of hexane, ethyl ether and glacial acetic acid in a ratio by volume of 25:74:1. They are collected, extracted and separated as described above except that the elution solvent mixture used for the HPLC is methanol/aqueous acetic acid, pH 3, in a ratio by volume of 66:34. PGB2 is 20 used as the quantitative evaluation standard for the dihydroxylated fatty acids according to their respective maximum UV absorptions (wavelength 280 nm and molar extinction coefficient 2.8 x 10* M^cnf1 for PGB2; wavelength 270 nm and molar extinction coefficient 5 x 10* M"1cm"1 for the 25 dihydroxylated derivatives).

It is known that stimulation of the leucocytes by ionophoric calcium in the presence of arachidonic acid activates 5-lipoxygenase and that this activation translates into a significant synthesis of 5-HETE and leucotri-30 enes, such as LTB4.

This is observed for the control leucocytes which were not incubated in the presence of dicranine.

In the case of the leucocytes incubated in the presence of 10"* M dicranine, the activation of 5-lipoxygenase 35 is inhibited. This inhibition causes a significant reduc- 24 3 3 n tion in the synthesis of 5-HETE and LTB*. This reduction is of the order of 50% for their synthesis by comparison with the control which is significant at 5% for LTB<, according to the Student-Fischer paired test.

In the case of the leucocytes incubated in the pres ence of 10"6 M dicranine, the activation of 5-lipoxygenase is not inhibited. In this concentration, dicranine is not active.

In a concentration of 10"* M, dicranine inhibits the 10 synthesis of leucotrienes which are the mediators of inflammation. Accordingly, dicranine has anti-inflammatory properties.

Example 6 These Examples describe the anti-bacterial properties of dicranine towards the following bacteria: - Bacillus stearothermophilus - Bacillus cereus - Bacillus subtilis - Staphylococcus aureus - Streptococcus faecalis Example a 3 ml nutrient gelose (SNA) inoculated with 1% of a 25 culture of the bacterium in question previously incubated for 16 to 18 hours are applied to a Petri dish of 15 ml gelose (PCA). A certain quantity of dicranine in the form of a solution in ethanol is applied to a 6.5 mm or 13 mm diameter disc of sterile filter paper. After evaporation 30 of the solvent, the disc is placed at the centre of the Petri dish.

The whole is then incubated for 24 hours at a temperature of 30 to 37 °C for the mesophilic bacteria and at a temperature of 55°C for the thermophilic bacteria. Visual-35 ly, it can be seen that, in a zone surrounding the paper 24 3 3 65 12 disc, the microorganism has not developed. The diameter in mm of this inhibition zone is measured, the following results being obtained: Dicranine concentration 50 (Mg/disc) 75 100 125 250 500 1000 B. stearoth. 0 9 11 14 16 17 26 32 B. cereus 0 9 11 16 19 22 B. subtilis 0 8 11 13 13 14 16 19 St. aureus 0 11 12 16 17 21 22 23 Str. faecalis 6 12 13 Not determined Accordingly, inhibition of the development of the bacteria is observed from 25 /xg dicranine per disc or even - for Str. faecalis - from 10 jug dicranine/disc.

Example b 200 jul of an infusion based on brain and heart con taining 0 to 200 /ug/ml dicranine in the form of its sodium salt are introduced into each cup of a 96 cup plate and 50 Hi of an aqueous suspension containing 103 to 105 germs per ml are added. The whole is then incubated at the tempera-25 tures mentioned in Example a.

Samples (100 nl) are taken after 0, 2, 4, 6, 8 and 24 hours and are diluted with 900 /nl physiological salt solution containing 0.85% sodium chloride and 0.1% tryptone to obtain approximately 10 to 300 bacteria per gelose 30 plate. After incubation for 24 hours, the colonies formed are counted. The following results are obtained: 24 3 3 6 5 13 a) number of B. Stearothermophilus still viable per ml solution (logarithmic value): Dicranine concentration (Mg/ml) Control 50 100 150 200 0 Hour 3.699 3.699 3.724 3.699 3.711 3.699 2 Hours 4.009 3.845 3.806 3.176 2.736 2.477 4 Hours .826 3.146 2.816 1.937 6 Hours 6.975 6.628 6.439 3.263 2.766 1.544 8 Hours 7.975 8.312 7.161 3.845 2.602 1.454 24 Hours 7.484 7.628 7.366 .866 2.093 0.004 b) Number of B.cereus till viable per ml solution (logarithmic value): Dicranine concentration (jug/ml) Control 50 100 150 200 0 Hour 3.699 3.628 3.628 3.415 3.146 2.826 2 Hours 3.900 3.699 3.602 3.149 2.107 0.875 4 Hours 2.924 2.049 0.041 6 Hours 6.591 6.337 .734 2.778 1.648 0.176 8 Hours 8.238 7.945 6.301 2.767 1.439 0.301 24 Hours 7.544 7.544 7.0 6.071 1.124 0.041 24 3 3 6 5 14 c) Number of B. subtilis still viable per ml solution (logarithmic value): Dicranine concentration Control (/ig/ml) 50 100 150 200 0 Hour 4.301 4.301 4.301 4.000 3.954 2.204 2 Hours 4.439 4.204 3.903 3.748 3.716 1.342 4 Hours 3.740 3.556 3.699 1.079 6 Hours 6.954 6.851 3.732 3.665 3.618 1.021 8 Hours 8.423 7 .756 3.991 3.782 3.679 0.301 24 Hours 7.724 7.484 4.114 3.342 3.277 0.001 Accordingly, it can be seen that, in concentration of 150 /xg/ml, dicranine is bactericidal towards the mesophilic bacteria B. cereus and B. subtilis. In a concentration of 100 /ig/ml, a bacteriostatic effect is observed for 4 hours.

Dicranine is bactericidal towards the thermophilic 20 bacterium B. stearothermophilus from 200 /ig/ml. In concentration of 50 to 150 /ig/ml, a bacteriostatic effect is observed for 4 hours.

Claims (9)

WHAT WE CLAIM IS:

1. Use of a chemical compound 9 (Z),12(Z),15(Z)-octadecatriene-6-ynoic acid together with a pharmaceutically acceptable excipient to produce a pharmaceutical composition.

2. Use of the chemical compound claimed in claim 1 wherein the pharmaceutical composition is suitable for use as an anti-aggregating agent.

3. Use of the chemical compound claimed in claim 1 wherein the pharmaceutical composition is suitable for use as an antiinflammatory agent.

4. Use of the chemical compound claimed in claim 1 wherein the pharmaceutical composition is suitable for use as an antibacterial agent.

5. A pharmaceutical composition containing 9(Z),12(Z),15(Z)-octadecatriene-6-ynoic acid as its therapeutically active substance in conjunction with a pharmaceutically inert excipient.

6. A cosmetic composition containing 9 (Z) , 12 (Z), 15 (Z) -octadecatriene-6-ynoic acid as the therapeutically active ingredient, together with an acceptable carrier or diluent.

7. Use of a compound as claimed in claim 1, substantially as herein described with reference to any one of the Examples.

8. A composition as claimed in claim 5, substantially as herein described with reference to any one of the Examples.

9. A cosmetic composition as claimed in claim 6, substantially as herein described with reference to any one of the Examples. SOCIETE PES PRODUITS NESTLE S A By their attorneys BALDWIN, SON & CAREY