Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2000-01-12
2001-07-31
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C514S451000, C514S460000, C549S271000, C549S292000
Reexamination Certificate
active
06268186
ABSTRACT:
The present invention relates to a process of preparing, purifying and/or isolating compounds which are hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors (or their precursors) such as lovastatin. The invention in particular relates to purifying such a compound from a composition comprising microorganisms that have produced it (such as by using an adsorbent resin), performing an extraction using toluene, performing a lactonization reaction, if necessary (e.g. in the toluene), and washing the toluene with water before isolating the final compound.
It is known that certain mevalonate derivatives are active as hypercholesterolemic agents, and these function by limiting cholesterol biosynthesis by inhibiting the enzyme HMG-CoA reductase. Mevalonate derivatives include the naturally occurring fungal metabolites lovastatin and compactin.
Lovastatin can be produced by fermentation of various microorganisms including
Aspergillus terreus,
during which it is produced in a free hydroxy acid form along with a number of by-products. Consequently, isolation usually involves removal of the microorganisms (usually referred to as the biomass) and by-products, followed by lactonization (to the closed ring or lactone form, called lovastatin) and removal of by-products. Several methods of isolation have been developed, but they are usually multi-step processes involving extraction from a fermentation broth (or a filtrate thereof) using organic solvents. Due to the number of steps involved the yield of lovastatin is generally low. In addition, large quantities of potentially hazardous solvents are usually required which not only entails the requisite safety measures but additionally increases costs dealing with solvent disposal.
The present invention seeks to provide a process of purifying or isolating an (3-hydroxy-3-methyl glutaryl-coenzyme A) HMG-CoA reductase inhibitor, such as lovastatin, or a precursor thereof, starting from a composition comprising cells (e.g. microorganisms) which have produced the precursor or inhibitor, in good yield and/or using reduced quantities of solvent.
In a first aspect the invention provides a process of purifying a compound, which is preferably an HMG-CoA reductase inhibitor, or a precursor thereof, of the general formula I or II:
wherein:
each of R
1
and R
2
independently represents a hydrogen atom, a methyl group or a hydroxyl group; and
R
3
represents a (straight chain or branched) C
2-6
alkyl group;
or a salt or isomer thereof;
and there is one double bond present in the first ring, between either carbon atoms 3 and 4 or atoms 4 and 4a, and no or one double bond in the second ring, which if present is between either carbon atoms 4a and 5 or atoms 5 and 6;
from an (e.g. aqueous) composition comprising cells, such as microorganisms, that have produced the compound, the process comprising:
(a) adjusting, if necessary, the pH of the composition to be at least 7.5;
(b) removing (e.g. by filtering) from the composition the cells to obtain a solution of the compound (e.g. a filtrate);
(c) contacting the solution with a resin so that the compound is adsorbed onto the resin; and
(d) removing the compound from the resin.
By this process the compound can be absorbed onto a resin and so purified. The cells can be removed by filtration (in which case one is left with a filtrate), centrifugation (so one obtains a supernatant) or even by decantation. Whichever method is employed, the remaining cells (or biomass), or the waste liquid resulting from passage over the resin, can then be discarded, without further processing. This can be achieved because no (non-aqueous) solvent need be added to either the composition or the solution prior to contact with the resin, unlike prior art processes. The compound is suitably removed from the resin by elution.
The purification process can thus result in a concentration of the compound, and when the resin is a hydrophobic one, it is possible to remove polar components present in the original composition.
The adjustment to a pH of at least 7.5 has been found to improve purification because this assists in the dissolution of the compound. Several prior art processes, such as those described in U.S. Pat. Nos. 4,231,938 and 4,294,926 (Merck) purify the precursor (to lovastatin) from an essentially neutral fermentation-derived medium, but that is not basified, unlike the process of the present invention, and as a result not all the precursor has dissolved. Therefore, some of the precursor can remain either with the microorganisms when they are removed (by centrifugation).
A precursor is a compound that can be converted into the reductase inhibitor by a relatively simple or straightforward chemical conversion, such reactions being known in the art. This may involve several steps, although sometimes only one reaction is required, and that is lactonization (described later in relation to the second aspect of the invention). This is the closing of the “open” ring and results in a compound of the formula II. However, many compounds of formula I are inhibitors although they are sold (and often administered) in the lactone form of formula II. These are converted to the open ring form of formula I in vivo.
Alternatively, a precursor can be converted into a reductase inhibitor by a hydroxylation reaction, such as at the 6-position, and this can be achieved by using various microorganisms, for example those described in GB-A-2,077,264 (Sankyo).
Preferred reductase inhibitors are able to inhibit the biosynthesis of cholesterol, and so can be useful as hypercholesterolemic agents. The test for HMG-CoA reductase inhibition is well known in the art, but for example one can use the methodology of Beg et al, FEBS Letters 80:123-129 (1977) or described in J. Biol. Chem. 234:2835 (1959). Suitable enzymes can be prepared as described by Kleinsek et al, PNAS 74:1431-5 (1977).
In formulae I and II, suitably R
2
represents a hydrogen atom. Preferably R
3
represents a 1-methyl propyl group (as is the case for lovastatin, pravastatin and compactin) or a 1,1-dimethyl propyl group (as is the case with simvastatin).
Preferably the compound will have one double bond in each of the first and second rings, suitably located between carbon atoms 3 and 4 and carbon atoms 4a and 5 (as is the case with lovastatin, pravastain, compactin and simvastatin, although compounds which have only one double bond, located in the first ring, are contemplated (such as between carbon atoms 3 and 4, in the case of dihydrolovastatin). Other compounds that are contemplated have a double bond in the first ring between carbon atoms 4 and 4a and a double bond in the second ring between carbon atoms 5 and 6 and are described in GB-A-2,077,264 (Sankyo)
The (usually aqueous) composition comprising the cells (usually microorganisms although plant or animal cells modified to produce the precursor can be used) can be any composition which comprises the compound of general formula I or II. It will often contain water and microorganisms, particularly when the composition comprises a fermentation broth, a sample removed from such a broth (e.g. before fermentation is complete) or a sample from the broth which has been stored, for example at a low temperature.
The microorganism can be any microorganism that is capable of producing the compound and this includes bacteria, yeasts and (preferably) fungi. Preferred microorganisms are fungal, for example of the genus Aspergillus, Monascus, Penicillium , Paecilomyces, Hypomyces, Phoma, Pleurotus, Doratmyces, Eupenicillium, Gymnoaxus and Trichoderma.
Of these, one can optimally use a fungus of the species
Penicillium citrinum
or
Aspergillus terreus,
for example strain AD43.
Preferably, in (a) the composition is adjusted to have a pH of from 9 to 13, such as from 10 to 11. The composition may be diluted with water. The pH can be adjusted by any suitable alkali. Preferably, it is an alkali metal hydroxide, for example sodium hydroxide, for example 1-3N, such as about 2N, NaOH.
At this stage in the process the composition is usually a fe
Bouman Aad Johannes
De Pater Robertus Mattheus
Purmer Cornelis Frederik
Sibeijn Mieke
DSM N.V.
Lilling Herbert J.
Morrison & Foerster / LLP
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