Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
2001-10-18
2003-11-11
McKane, Joseph K. (Department: 1626)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
active
06645742
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a purifying process for phosphatidylserine, a phospholipid which is useful in particular for the preparation of pharmaceutical compositions used in the treatment of involutional brain syndromes of various nature, but also in the preparation of particular liposome formulations and of dietetic compositions based on natural lecithins.
STATE OF THE ART
Phosphatidylserine is a phospholipid widely present in nature; it is one of the main components of cell membranes in animal organisms and is present in particularly large amounts in mammals' brain tissues. Medical literature mentions interesting properties of phosphatidylserine, among which the most significant relates to its effectiveness in improving mnemonic abilities.
The trans-phosphatidylation of phosphatidylcholine with serine in presence of the enzyme D-phospholipase according to the following scheme is the most convenient reaction for the industrial production of phosphatidylserine.
The reaction can be carried out both in an aqueous ambient, as described in the EP 1 048 738, and in a system containing beyond an aqueous phase also an organic solvent unmixable with water, preferably toluene; this second method is described in U.S. Pat. No. 5,700,668.
Whatever the method of trans-phosphatidylation used, the phosphatidylserine which can be isolated at the end of the process contains in any case large amounts of hydrophilic impurities, such as serine, choline and their salts which are present in the aqueous phase.
Another aspect which should be remarked is that, by measuring the enzymatic activity of the type D-phospholipase of the products according to the method described in related literature (
Biotechn. Techn.,
7, 795 (1993)), every gram of product is found to have about 2 international units of enzymatic activity. In compliance with the general request of strict criteria for purity, the removal of said impurities from the product is highly important.
The methods which are commonly used to remove hydrophilic substances from organic solutions of phospholipids have proved to be ineffective. As a matter of fact, by extracting a toluene solution of the phospholipid mixture obtained as described in U.S. Pat. No. 5,700,668, with the same amount of water, and then by separating the phases, it is found that the content of serine in the organic phase and the activity of D-phospholipase are practically unchanged with respect to the values found before the extraction with water.
Similarly, the elution of the same organic solution on a chromatographic column containing chromatography silica conditioned with toluene, in which the product elution is completed with toluene, results in a very small removal of serine and of the enzymatic activity from the phospholipid.
In another experiment the organic solution of phosphatidylserine to be purified because of serine is added with acetone: the precipitated product shows, after being analyzed, a ratio of serine to phosphatidylserine substantially unchanged with respect to the starting product.
Therefore, there is always the problem related to the availability of an effective purifying process for phosphatidylserines prepared by trans-phosphatidylation of phosphatidylcholine with serine in presence of the enzyme D-phospholipase, and containing as impurities hydrophilic compounds, proteins and inorganic salts.
SUMMARY OF THE INVENTION
The Applicant has now surprisingly found that hydrophilic impurities, proteins and inorganic salts can be successfully removed from a solution of phosphatidylserine in an organic solvent by extraction with water, provided that a polar organic solvent is added to the system.
The object of the present invention is therefore a purifying process for phosphatidylserines having formula (I)
where R
1
e R
2
, identical or different, are a C
10
-C
30
acyl group; X is OH or OM,
where M is chosen from the group of alkali metals, alkaline-earth metals, ammonium and alkyl ammonium,
and where the serine portion is in D, L or racemic form, and preferably in L form, comprising the extraction of said phosphatidylserines from a solution in a hydrocarbon solvent with a mixture of water and a polar organic solvent.
DETAILED DESCRIPTION OF THE INVENTION
In the specific case of phosphatidylserines prepared according to the description contained in U.S. Pat. No. 5,700,668 by trans-phosphatidylation in a two-phase system made of an aqueous phase and a toluene phase, the process according to the invention can be carried out directly on the toluene phase after separating the latter from the aqueous phase at the end of the reaction.
In the case of phosphatidylserines prepared following other methods, the purifying is process can be advantageously carried out by stirring the product in a mixture containing a hydrocarbon solvent, water and a polar organic solvent.
Also in this case the concerned phospholipid is present in the hydrocarbon phase, whereas hydrophilic impurities are to be found in the aqueous phase.
After separating the phases phosphatidylserine can be isolated using known methods such as precipitation with acetone.
According to the present process aromatic or aliphatic solvents can be used as hydrocarbon solvent; among aromatic solvents toluene or xilene are preferred; among aliphatic solvents it is preferable to use n-heptane, n-hexane or cyclohexane.
As polar organic solvent alcoholic solvents can be used, containing for instance 1 to 5 carbon atoms. According to preferred embodiments of the invention, said alcoholic solvent is chosen among secondary and tertiary alcohols; more preferably isopropanol is used.
The extraction according to the invention can be carried out at temperatures between 0 and 70° C., and preferably between 20 and 30° C.
The amount of hydrocarbon solvent is between 4 and 30 liters/kg of phospholipid to be purified, and preferably between 6 and 12 liters.
The volume ratio between water and hydrocarbon solvent is between 0.2 and 5, and preferably between 0.3 and 1. The volume ratio between polar organic solvent and hydrocarbon solvent is between 0.2 and 2, and preferably between 0.3 and 1.2.
The method described can be applied to phosphatidylserines with different acyclic chains and allows to purify both products deriving from the trans-phosphatidylation of phosphatidylcholines of natural origin such as soybean, rape or egg yolk, and phosphatidylcholines synthesized with fat acids, both saturated such as myristic acid, palmitic acid or stearic acid, and unsaturated such as oleic acid or linoleic acid.
The following examples merely aim at disclosing the present invention without limiting its object.
REFERENCES:
patent: 5084215 (1992-01-01), Kearns et al.
patent: 5700668 (1997-12-01), De Ferra et al.
patent: 19917249 (2000-09-01), None
patent: 0922707 (1999-06-01), None
Search Report for EP Application No. 0112 4577-6, dated Mar. 15, 2002.
De Ferra Lorenzo
Massardo Pietro
Chemi S.p.A.
McKane Joseph K.
Morgan & Finnegan , LLP
Shameem Golam M. M.
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