Acarbose purification process

Details Acarbose purification process Acarbose purification process

2002-10-24

2004-05-11

Barts, Samuel (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S018700, C536S017200, C536S055300

Reexamination Certificate

active

06734300

ABSTRACT:

BACKGROUND OF THE INVENTION
Acarbose is a polyglucose-based tetramer oligosaccharide obtained by fermentation of the microorganism Actinoplanes sp. (particularly
A. utahensis
). Its full chemical name is O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-1-yl]amino]-&agr;-D-glucopyranosyl-(1→4)-O-&agr;-D-glucopyranosyl-(1→4)-D-glucose. Acarbose is a white powder having a molecular weight of 645.6. It is freely soluble in water, and its pKa value is 5.1.
Acarbose's empirical formula is C
25
H
43
NO
18
; its structural formula is:
As can be seen from the structural formula, acarbose is a complex oligosaccharide consisting of two glucoses, one deoxyglucose and one dideoxy glucose derivative. The first three monomeric units are bound by O-glycosidic links, while the fourth monomeric unit is bound by an N-glycosidic linkage.
Due to the above structure, the acarbose molecule acts as an antihyperglycemic, which is a consequence of competitive reversible inhibition of the pancreatic &agr;-amylase enzyme and the alimentary tract's &agr;-glucosidohydrolase (Ki≅10
−12
M). This inhibition decreases and postpones the breakdown of ingested carbohydrates, thus decreasing the elevation of blood sugar levels after meals. In type II diabetes, the enzymatic inhibition results in postponed glucose absorption and prevention of hyperglycemia after meals. Acarbose is available on the European market as Glucobay™, and in the United States as Precose™.
Acarbose is obtained by fermentation of a microorganism of the family Acitonoplanaceae, preferably a strain of the genus Actinoplanace, followed by isolation from the fermentation broths by filtration and precipitation. See, for example, U.S. Pat. Nos. 3,876,766; 3,879,546; 4,019,960; and 4,062,950. The purity of the crude acarbose thus produced is inadequate for pharmaceutical use and, therefore, additional purification is necessary to provide the high degree of purity required for clinical application.
As noted above, the fourth glycosidic unit in the acarbose molecule is bound by the N-glycosidic link, imparting weakly basic properties to the molecule. Purification procedures for acarbose previously described in the literature were based on the use of cation-exchangers for adsorbing the acarbose molecule to the binding groups thereon; such groups do not adequately bind the weakly basic acarbose, which accordingly, results in impure acarbose products in the effluent streams therefrom. For example, purification processes have been described (see DE 2,347,782 and U.S. Pat. No. 4,174,439) in which, after the initial treatment of the culture broth with activated carbon, acarbose is bound to strong acid cation-exchangers and eluted with salt solutions or dilute acids. These exchangers are gels of the macro-reticular type based on cross-linked polystyrene, which have poor mass transfer and exhibit broad peaks upon elution, resulting in relatively low product purities. Acarbose products obtained from these processes, after neutralization with basic anion exchangers, are said to have acarbose contents of about 78-88% in the dry matter (by HPLC analysis).
U.S. Pat. Nos. 4,767,850 and 4,666,776 disclose the purification of acarbose solutions by adsorption to strong acid macroporous cation echange resins based on aromatic compounds possessing one or more vinyl groups and at least one hydrophilic monomer.
U.S. Pat. No. 4,904,769, on the other hand, describes an acarbose purification method, using a chromatographic column packed with a weakly acid cation-exchanger having carboxyl binding groups thereon and based on dextran, agarose or cellulose or exchangers which are derived from such exchangers by the addition of polyacrylamide. This patent states that use of the particular purification technique thereof results in acarbose contents of at least 90% and up to as much as 98% by HPLC analysis of the dry matter. It further discloses that the acarbose products thereof contain less than 10%, and preferably 2 to 5% by weight of sugar-like secondary components. During prosecution of the patent it was asserted that strongly acidic cation exchangers could not be utilized to obtain acarbose of such purities.
The method described in U.S. Pat. No. 4,904,769 is, however, difficult to use on a production scale because acarbose is not bound but rather slowed in passing through the column. Moreover, the efficacy of such method depends on a number of factors, including the temperature of the exchanger and the pH range of the acarbose feed, which must be adjusted to optimize purification. In addition, this method involves relatively high cost due to the necessity to use highly pre-purified acarbose crudes and expensive weak acid cation-exchangers.
In International Published Application WO 99/07720 a further acarbose purification method is described, based on the use of non-aromatic strong acid cation-exchanger column chromatography. Exchangers that are hydrophilic and have high mass transfer are disclosed, e.g., a polymer-coated alumina matrix, a sulfoxyethyl cellulose resin or a methacrylate copolymer sulfonate resin. The elution of acarbose products from the column is carried out with ammonia, sodium hydroxide or hydrochloric acid. In the only example of this application, an acarbose product said to possess 98% purity is described. However, it is known that the pH values of such eluates trigger partial acarbose degradation caused by hydrolysis of glycosidic links, and are, therefore, undesirable.
In International Published Application WO 01/30796 an acarbose purification technique is disclosed, utilizing a monodispersed cross-linked polystyrene-based gel strong acid cation exchanger having a hexagonal bed structure and containing a sulfate group. The eluate from such exchanger is obtained with diluted hydrochloric acid (pH 1.0-3.0) to remove remaining salts, sugar-like secondary components and acarbose analogs. The acarbose thus obtained is said to have high purity, not less than 98%.
Yet a further acarbose purification method is disclosed in International Patent Application WO 02/12256. This application describes purifying acarbose employing a strong acid cation-exchanger, e.g., an exchanger incorporating sulfo, sulfomethyl or sulfopropyl groups, in the presence of an anion of a weak acid. The application states that the impurities present in the acarbose fermentation broth cannot adsorb onto the strong acid cation-exchanger when the anion of a weak acid is present in solution. Consequently, only acarbose is said to selectively adsorb onto the strong acid cation-exchanger, resulting in an acarbose product of assertedly high purity containing less than 1% of acarbose-related substances after drying. However, the process described in this application is relatively complex, involving the use of multiple chromatographic columns, using strong cation exchangers in the presence of an anion of a weak acid, and elution with solutions having differing specific pH ranges.
As will be apparent from the preceding background discussion, various of the previously described acarbose purification methods utilize multi-step, complex processes involving the use of both cation exchangers and anion exchangers, and either basic or acidic eluates. The use of such eluates introduces additional ions into the purified acarbose solution and requires subsequent adjustment of pH. The use of multi-step processes additionally imposes substantial additional cost, and may require highly pre-purified acarbose crude feed reactant streams.
Accordingly, it is a primary object of the present invention to provide a relatively simple, direct and economical process for the purification of acarbose, by which that product is produced in purities.
SUMMARY OF THE INVENTION
The present invention relates to a process for the purification of aqueous solutions of crude acarbose, of at least 80% by weight acarbose, which comprises:
(a) passing an aqueous solution of the crude acarbose having a pH value from about 5 to about 7 through a chromatog

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