WO2000071680A1

WO2000071680A1 - A process for producing prostaglandin synthetase and prostaglandins

Info

Publication number: WO2000071680A1
Application number: PCT/AU2000/000485
Authority: WO
Inventors: Andrew Bending
Original assignee: Sphinx Millenium Pty Ltd
Priority date: 1999-05-24
Filing date: 2000-05-19
Publication date: 2000-11-30
Also published as: CA2374345A1; AUPQ053999A0

Abstract

The specification describes a method of preparing prostaglandin synthetase from a natural source, incubating the synthetase with diperlinolenic acid and purifying the prostaglandin E1 produced. The prostaglandin synthetase is prepared by homogenising a natural source of the synthetase in a halide of an alkali metal such as potassium chloride and separating the homogenised mixture into a liquid phase and a solid phase. The pH of the liquid phase is adjusted to a level in a range from 5 to 5.5 to cause the prostaglandin synthetase to form as a solid phase. The prostaglandin synthetase is then separated from the liquid phase and used to synthesise prostaglandin E1.

Description

A PROCESS FOR PRODUCING PROSTAGLANDIN SYNTHETASE AND PROSTAGLANDINS

The present invention relates to a process for the production of Prostaglandin Ei (PGEi) . The specification of US Patent No. 3,069,322 contains claims to prostaglandin and describes a process for producing it from natural sources. The specification of US Patent No. 3,069,322 states that crude prostaglandin that had been produced previously was not sufficiently pure for parenteral applications. In particular, the crude prostaglandin available up until that time contained antigens, pyrogens, tissue fragments, lipids, cellular debris, foreign proteins and other similar pollutants.

The process described in the specification of US Patent No. 3,069,322 comprised the following steps:

(1) Mincing freeze-dried sheep prostate glands in a meat grinder.

(2) Suspending the minced glands in distilled water using 4 litres of water per kilogram of dried glands. (3) Allowing the suspension to stand for 15 minutes.

(4) Adding 12 litres of 95% ethanol per kilogram of dried glands.

(5) Stirring the resultant mixture for about 1 hour.

(6) Allowing the mixture to stand overnight to form a sediment and a supernatant ethanol solution.

(7) Decanting the supernatant ethanol solution and straining the insoluble residue through cheesecloth adding the filtrate to the supernatant ethanol solution and filtering the resultant solution. (8) Evaporating the ethanol solution in vacuo to about 1/20 its original volume.

(9) Adding 3 litres of ether per kilogram of dried glands to form a water phase and an ether phase.

(10) Acidifying the water phase to increase the pH of the water phase to 3.5.

(11) Adding and mixing a further 3 litres of ether per kilogram of original dried glands and again with two further lots of ether.

(12) Combining the ether extracts and extracting the ether extracts 6 times with a 1/4 of the volume of 0.2 molar phosphate buffer having a pH of 8. (13) Acidifying the combined buffer phases to reduce the pH to 3 with 6 normal hydrochloric acid.

(14) Extracting with one volume of ether.

(15) Extracting a further 3 times with 1/2 the previous volume of ether. (16) Combining the ether extracts and washing until free of chloride ions with small portions of water, each portion of water being passed through a second ether phase.

(17) Evaporating the ether in vacuo leaving a solid residue, and subjecting the residue to a 5 stage countercurrent distribution between equal volumes of ether and 0.5 molar phosphate buffer at pH 6.4, 200 millilitres being used per 5 grams of extract.

(18) Acidifying the buffer phases.

(19) Extracting 3 times with ether and evaporating all phases to dryness to produce crude PGE from the ether phase, the crude PGE thus produced was then purified by a process comprising the following steps:

(i) Dissolving the crude PGE in an isooctanol chloroform ethanol water mixture having the ratio of 1:1:10:10 at the rate of 100 milligrams of extract to 5 millilitres of mixture, (ii) Passing the solution through a column as a mobile phase in which 4 millilitres of static phase comprising an isooctanol chloroform methanol water mixture in the ratio of 1:1:10:10 was supported on

4.5 grams of hydrophobic diatomite. (iii) Discarding the first 50 millilitres of eluates and collecting the next 65 millilitres of eluates. The PGE concentrate thus obtained was further purified and separated by reverse phase chromatography using a methanol water isoamyl acetate chloroform mixed solvent system in the ratio of 35:65:4:6 respectively.

According to Volume 8 of a book entitled "Progress in Organic Chemistry" published by Wiley, New York in 1973, prostaglandin Ej_. and prostaglandin E₂ were biosynthesised until recently from the essential fatty acids bishomo-γ-linolenic acid and arachidonic acid using an enzyme preparation obtained from ram seminal vesicles. According to the book, the limitations of such a biosynthesis are evident from the fact that the seminal vesicles of one ram weigh approximately 20 grams, and it takes 1 kilogram of seminal vesical tissue to produce sufficient enzyme to transform 1 gram of essential fatty acid to 250-300 milligrams of prostaglandin.

A paper entitled "Biosynthesis of Prostaglandins" by B. Samuelsson, presented at a symposium in Milan in September 1968, provides more information about the biosynthesis of prostaglandin from essential fatty acids. The paper was published in 1969 in Volume 5 of Progress in Biochemical Pharmacology at pages 109-128. On page 110, the article states as follows, "fractionation of homogenates from vesicular glands of sheep shows that the conversion of eicosatrienoic acid into prostaglandins requires the microsomal fraction and boiled supernatant. The supernatant can be replaced by glutathione or tetrahydrofolate" . On page 114, the paper states as follows, "The enzymatic activity of the material purified by DEAE cellulose was stimulated by hydroquinone in the presence of glutathione or tetrahydrofolate whereas the antioxidant had no effect when supernatant was used as cofactor or when the crude microsomal fraction was incubated. "

The present invention provides a method of extracting prostaglandin synthetase from natural sources. Accordingly, the present invention provides a method of preparing prostaglandin synthetase which method includes homogenising a natural source of prostaglandin synthetase in an aqueous solution of a halide of an alkali metal to form a first solid phase homogenised within a first liquid phase, separating the first solid phase from the first liquid phase, adjusting the pH of the first liquid phase to a pH in a range from 5 to 5.5 and separating a second solid phase from the first liquid phase, the second solid phase being prostaglandin synthetase. Preferably the alkali metal halide employed in producing the synthetase is potassium chloride.

Prostaglandin Ex may then be produced by incubating a mixture of diperlinolenic acid (8,11,14- eicosatrienoic acid) , -glutathione, hydroquinone and the prostaglandin synthetase .

PGEi may be extracted from the incubated mixture with acetone followed by filtration, concentration of the filtrate to 1/3 volume at a reduced pressure, eluting with petroleum benzene and then dichloro methane. Semi-finished PGEi may then be recovered from the filtrate by cooling to 4°C and leaving until the PGEi crystallises.

The semi-finished PGEi may be further purified by dissolving it in a small volume of chloroform, mounting the solution on a chromatographic column and eluting with a mixture of ethyl acetate, glacial acetic acid, petroleum benzene and water in the ratio of 200:22.5:12.5:5. Pure PGEi crystals may be obtained by cooling the eluate to 4°C until crystallisation has stopped.

A preferred embodiment of the invention is described below by way of example.

EXAMPLE

Adipose and connective tissue are dissociated from the seminal vesicles of goats. The seminal vesicles are homogenised in a 0.154 molar potassium chloride solution to form a homogenised suspension. The homogenised suspension is centrifuged at 7,900 rpm for 20 minutes. Additional potassium chloride solution is added to the precipitate and the precipitate and potassium chloride solution again homogenised before again centrifuging the homogenised mixture. The supernatant liquids resulting from centrifuging the homogenised mixtures are combined and filtered, the pH adjusted to a pH in the range from 5 to 5.5 and again centrifuged at 7,900 rpm for 20 minutes. The resultant supernatant is discarded and the precipitate eluted with a 0.125 M solution of the disodium salt of EDTA. Following elution, the pH of the precipitate is adjusted to a pH in the range from 7.5 to 8. The precipitate is then mixed with 0.045 gram of hydroquinone, 0.5 gram of L-glutathione and 1 gram of diperlenolenic acid per kilogram of seminal vesical tissue. The suspension is then incubated for 1 hour whilst ventilating with air by vortexing.

Three volumes of acetone are added to the suspension which is stored overnight. The suspension is filtered and the filtrate concentrated to 1/3 of its volume by reducing the pressure. The filtrate is then washed 3 times with petroleum benzene and after adjusting the pH to a pH in the range from 2 to 2.5, is washed 3 times with dichloromethane. The concentrated washed filtrate is then left in a refrigerator at 4°C until semi-finished PGEi precipitates. The semi-finished PGE is dissolved in a small volume of chloroform and mounted on a silver nitrate silica gel column. The column is eluted with a mixture of ethyl acetate, glacial acetic acid, petroleum benzene and water in the ratio of 200:22.5:12.5:5. The eluate is stored at 4°C to enable pure crystals of PGEi to form. The crystals have a purity in excess of 95% PGEi.

Claims

1. A method of preparing prostaglandin synthetase which method includes homogenising a natural source of prostaglandin synthetase in an aqueous solution of a halide of an alkali metal to form a first solid phase homogenised within a first liquid phase, separating the first solid phase from the first liquid phase, adjusting the pH of the first liquid phase to a pH in a range from 5 to 5.5 and separating a second solid phase from the first liquid phase, the second solid phase being prostaglandin synthetase.

2. A method according to claim 1, wherein the alkali metal halide is potassium chloride.

3. A method according to claim 1 or claim 2, including the step of incubating a mixture which includes the prostaglandin synthetase, diperlinolenic acid (8,11,14- eicosatrienoic acid) , L-glutathione and hydroquinone to produce prostaglandin Ei (PGEi) •

4. A method according to claim 3, including the steps of mixing the PGEi with acetone, filtering to produce a filtrate, concentrating the filtrate to about a third of its volume by application of a reduced pressure, eluting with petroleum benzene and dichloro methane and cooling the filtrate to about 4°C and allowing crystals of PGEi to form.

5. A method according to claim 4, including the steps of dissolving the crystals of PGEi in a small volume of chloroform, mounting the solution on a chromatographic column and eluting with a mixture of ethyl acetate, glacial acetic acid, petroleum benzene and water in a ratio of 200:22.5:12.5:5 by volume and cooling the eluate to 4°C until crystallisation has stopped to form crystals of PGEi having a purity in excess of 95% by weight.