CA1079736A - Crystalline calcium di-clavulanate dihydrate - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A process is disclosed for the preparation of crystalline calcium di-clavulanate dihydrate by contacting an aqueous solution of a salt of clavulanic acid other than the calcium salt, with a cation exchange resin in the calcium form and eluting the thus formed calcium di-clavulanate from the resin to yield a solution of calcium di-clavulanate substantially free from cations other than calcium and thereafter causing the crystallisation of the calcium di-clavulanate dihydrate from solution and isolating the desired cystalline salt. Pharmaceutical preparations containing the salt are disclosed as well and may contain a penicillin or cephalosporin in addition to the salt.

Description

~1~79736 The present lnvention relates to a new process for the preparation of the crystalllne calcium ~alt of clavular.ic acid, to the salt produced by this process and to pharmaceutical compositions containing it.
Belgian Patent No. 827926 discloses inter alia that the calcium salt of cla~ulanic acid, which is of the formula:

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may be isolated ~rom a fermen~ation broth containing ~alts of clavulanic acld by adsorbing the clavulanic acid resldues onto a weak or strong base ion exchange resin and eluting off with a suitable salt solution ~nd also by hydrogenation of a benzyl or like ester of clavulan~c acid in the presence of a suitable base. We have now found that crystalline calcium di-clavulanate dihydrate may conveniently be prepared from other salts of clavulanic acid in high yield and good purity.

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The present invention provides a process for the preparation of c~ystalline calcium di-clavulanate dihydrate ~Jhich process comprises contacting an aqueous solution of a salt of clavulanic acid other than the calcium salt with a cation exchange rlesin in the calcium form and eluting the thus formed calcium di-clavulanate from the resin to yield a solution of calcium di-clavulanate substantially free from cation~ other than calcium and thereafter causing the crystallisation o~ the calcium di-clavulanate dihydrate from the solution and isolating the desired salt.
The initial salt of clavulanic acid used in this process may be any convenient metal (otner tha~ calcium), ammonium or substi~uted ammonLu~ salt but in ~eneral it is most convenient to use a mono-~alent salt such as ~the lithium, sodium or potassium salt o~ clavulanic acld.
0~ these the lithium and sodium salts are often the most convenient. In general we prefer to use the sodium salt which can lead to a product of particularly acceptable purity.
The salt used to ~orm the solution to be exchanged should be as pure as can be conveniently obtained :Ln order to allow the preparation o~ a pure product.

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~1~797~6 Suitable cation exchange resins for use are cross-lir~ed polystyrene-divinylbenzene co-poly~ers substituted by acid groups ln the form o~ the calcium salt. The preferred acid group is the sulphonic acid group. The resins chosen will generally have 2-20% cross-linking and usually 4 10% cross-linking for example about 8%
cross-linking. The resin 1~ usually in the form of beads, for example spherical beads of 14-52 U.K. mesh size.
B Suitable resins include the calcium ~orm of Amberlite~
resins such as IR-120, IR-l~l, IR-122, 200, 2QOC, 252;
Dowex resins such as 50WX1, 50WX2, 50X4, 50WX8, 5X10, ~10, 50~ 6; Bio Rad resins such as AG 50~X1, 40WX2, AG 50~4, AG 50WX8, AG 50~,~X12; Ionac*resins such as C250, C258 or C255 and Zerollt resins such as 225, ~25, 425, 525, 625; etc.
The resin is normally used in large excess. The quantity of resin used should be suf~icient to provide an exchange capacity at least 10 times, more suitably at least 20 times and preferably at least 30 times of the total cations applied in the solution o~ the salt of clavulanic acid.
In use the resin is in a bed through which the solution is percolated. Gencrally ~hls bad is in the form of a column. .

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1~79736 The concentration of the solution of the salt to be exchanged is not cri-tical but very dilute solutions (for example, less than 1% w/v) should be avoided as low concentrations can lead to inconveniently low loading and incomplete exchange. Similarly very high concentrations (for example those approaching saturation) should be avoided as such solutions can have too high a viscosity for easy use. In general for most salts concentration 2-30~ w/v are acceptable, a range of about 5 to 25% being more suitable and a range of about 8 to 20% being pre~erred, for example about 10%.
The solution to be exchanged is normally applied 510~1y to the top to the column. The band is then allowed to percolate slowly into the top of the res~n a~ter which a little water i8 applied to wash the band a short way into the resin. The remaining necessary water is then ~lowly run through the column so that the band passes through the column in as tight a band as conveniently possible.
The presence of calcium clavulanate in the eluate is usually readily detectable by a change in the refractive inde~ of the eluate. This may be determined using a re~ractometer or ~isually, for example by the presence of striations. Alternati~ely fractions may be taken and tested in convenient manner, ~or example by spotting on a t.l.c. plate and spraying with permanganate which is decoloured by the cla~nLlanate or by methods using the enzyme inhibitory effects of the material.

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The d~sired calcium salt may be obtained from solution in conventional manner, for example ~rom a relatively concentrated solution by the slow addition o~ a water miscible organic solvent such as acetone until crystallisation commences or by concentrating under reduced pressure to a ~yrup followed by adding small quantities of acetone, acetonitrile, acetone/ether or the like to ini-tiate crystallisation.
Most suitably crystallisation is initiated at an ambient or slightly elevated temperature, for example, 12-30C, more suitably 20-25C. Once crystallisation has begun the mixture may be cooled, for example to about -5 to -1~C until no ~urther crystals appear.
Once the desired crystals have formed the~ may be ~llt~red o~f and dried~ ~igorou~ drying c:onditions (such as vacuum drying) are best avoided as they tend to lead to breakdown of the crystal structure and partially dehydrated crystals. Wet air ~hould not be used ~or drying as it can lead to wet crystals. Drying should be effected at atmospheric pressure.
The calcium di-clavulanate dihydrate produced by the process of this invention is also an aspect of this invèntion.
An infra-red spectrum of the salt according to this invention iS,'de~scribed in Table I.

. -- 6 -~07~736 It should be appreciated that the crystalline salt within this invention can contain ~mall amounts of partly dehydrated calcium di-clavulanate or small amounts of water ~thich ~orms no part of the crystal structure and probably simply wets th~ crystal faces.
However, the crystalline salt of this in~ention more suitably contains 6.2 to 7.7% (weight¦~eight) of rater, that is it should not contain water which forms no part o~ the crystal structure although it is acceptable for it to contain small amounts ofpartly dehydrated material. We have found that a salt containing approximately 6.4 to 7.6% (weight/weight) o~ total water to have particularly suitable stability properties. Most suitably the water conten~ of the crystals i8 abou~ 7.5%.
The water contents referred to above are total water, for example as determined by Karl Fisch~r analysis or the equi~alent.
The present i~vention also extends to a pharmaceutical compo3ition ~Ih~ch comprises the said crystalline calcium di-cla~ulanate dihydrate and a pharmaceutically acceptable carrier therefor.
Such ~omposltions are most suitably adapted ~or oral admini~tration.
The compositions of this invention may also comprise a penicillin or cephalosporin. Particularly suitable penicillins include ampicillin (as anhydrate, trihydra~e or salt) and amoxycillin (as trihydrate or salt), `
disodium carbenicillin, disodium ticarcillin, the sodium salts of the phenyl or indanyl ~-esters of carbenicillin ~0 or ticarcillin or the like.

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Su~table forms of the compositions o~ this invention are described in Belgian Patent No. 827926.
TABLE I
Infra Red Spectrum of Calcium Di-Clavulanate Dihydrate ~In Nu~ol Mull):
3630 Shoulder, 3520 Shoulder, 3315 Strong, Broad, 1785 Strong, 1695 Medium, 1660 Shoulder, 1605 Strong, 1575 Shoulder, 1555 Shoulder, 1450 Shoulder, 1418 Weak, 1350 Weak, 1315 Strong, 1200 Medium, 1147 Medium, 1124 Strong, 1096 Medlum, 1072 Medium, 1050 Strong, 1024 Strong, 1004 Strong, 976 We~k, 960 Shoulder, 8~7 Stron~, 850 MedlumJ 803 Medlum, 748 S~rong, 708 We~k, 659 We~k.
(Note No S:lgnlE:Lcnnt P~ak ~t 2330) . ., .~. _ ., .
~ The fvllowing Examples illustrate the inventlon:

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- Sodium clavulanate (290 mg) in water (1 ml) was passed through IR-120 calcium salt (10 ml wet resin).
The eluate (20 ml) ~as concentrated to less than 1 ml volume and acetonitrile (10 ml) added. Fine crystals o~ calcium di-clavulanate dihydrate were obtained which ` had i.r. absorbances thus: 1782 (~-lactam C=0), 1695 (C-C), 1605 cm 1 (C0z ). Water (by Karl Fischer): 7.4%, calcium diclavulanate dihydrate requires 7.6%.

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Exam~le 2 a. Preparation of Resin in Calcium Cycle I~-120 resin (505 ml~ ~as se into a column (15 x 1~ ) and hydrochloric acid (1 M solution) passed through the resin until the pH of the ef~luent was consistently belo~ 0.5. The resin was then washed with distilled wa.ter (initially upflo~ and then downflow) until the pH of the effluent ~as consistently at about 3.5. A solution of calcium chloride (0.25 M) was then passed through the re~in (by downf-o~,~) until the pH
of the percolate dropped to less than 1Ø Further calcium chloride ~olution was passed through the resin until the pH of the percolate was consistently at 3.3-3.5. The resin wa~ then washed (by down~low) with distilled ~ater (about 2 1) until the pH ~f the percolate was conslstently approximately 4.

b. PreParation of solution of Calcium Clavulanate Sodium clavul~nate tetrahydrate (equivalent to 30 g of pure free acid) ~ras made up to 300 ml with distilled water. The solution was stirred with 'Norit~ :
gsx charcoal (3 g) and ~iltered to give a clear pale yellow tinged solution at pH 6.9. The solution was applied to the pre~iously prepared IR-120 calcium cycle column at 200 ml/hour. tnlen loading was complete the column ~as then washed with distilled water at 200 ml/hour.

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~079736 As the calcium salt began to be eluted the p~l of the eluate changed to about 6Ø Fractions (approximately 60 ml) were then collected until a small aliquot of the final fraction produced little or no precipitation withace-tone. This final fraction was colourless and had a pll of about 6.6. The Eirst collected aliquot was discarded and the remainlng aliquots were then combined to yield the desired solution of calcium clavulanate. (If desired the sodium clavulanate tetrahydrate may be replaced by equivalent amounts of potassium clavulanate or lithium clavulanate).

c. Preparation of Crystalline Calcium Clavulanate Dihydrate The solution prepared as described above ~370 ml) was stirred and acetone slowly added. ~Eter about 3.5 volumes of acetonehad be~n added a slightly haæy precLpitate ~ormed and was filtered off via Celite*. ~urther acetonewas added to the solution until a total of 15 volumes had been added.
The resulting solid was filtered off to give a pale buff crystal-line material. This voluminous material collapsed on the filter and was then washed with acetone (3 x 150 ml) and pressed down on the filter to remove residual ace-tone. The solid was then dried under reduced pressure (about 10 torr for 18 hours) to yield the desired crystalline calc:ium clavulanate dihydrate ~28 g, total water 6.4~, purity >90~).

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107g736 The low moisture contents i~ the abo~e sample probably resulted from drying under reduced pressure. Avoidance of reduced pressures leads to the preparation of crystals containing amounts of water closer th~ the theoretical amount needed for the dihydrate. It will be therefore understood that in this example a~r clrying at the flnal step is usually to be preferred.

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9'73~i a. Sodium clavulanate te-trahydrate (150 mg) was dissolved in distilled water (1 ml) l~nd passed do~
B a column of Amberlite IR-120 resin (Ca2+ form, ~7 ml t~et resin). The resin was then washed ~rith distilled water (25 ml). The eluate .tas evaporated under reduced pressure at ambient temperatu:re (about 22C) to a syrup (~0.5 ml) and then triturated with acetonitrile (25 ml). The calcium salt crystallised, and it was cooled to 2-3C for 2 hours, collected by filtration and washed with dry ether (25 ml). The product ~ras a~r-dried to yie].d the de~ired crystalline calcium di-clavulanate dihydrate (75 mg of ~ubstan-tially pure product, water content v7.4%) b. ~mberlite IR-120 in the calcium form was produced as follows:
The resin ~R+ form, 20 ml wet resin exchange capacity 38 m. moles) in water (5~ ml) was treated ~rith calcium hydroxide t0-5 g, excess) with stirring. The exce~s calcium hydroxlde was removed by passing a current of distilled water up through a column containing the resin ('back-washing') until the eluate had a pH
approximately 8-8.5. (This also removes the'~ines'-debris and minor organic impurities).

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Claims (12)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of crystalline calcium di-clavulanate dihydrate which comprises contacting an aqueous solution of a salt of clavulanic acid other than the calcium salt with a cation exchange resin in the calcium form and eluting the thus formed calcium di-clavulanate from the resin to yield a solution of calcium di-clavulanate substantially free from cations other than calcium and thereafter causing the crystallisation of the calcium di-clavulanate dihydrate from solution and isolating the desired crystalline salt.

2. A process as claimed in claim 1 wherein the original salt of clavulanic acid is the lithium, sodium or potassium salt.

3. A process as claimed in claim 1 wherein the original salt of clavulanic acid is the sodium salt.

4. A process as claimed in claim 1 wherein the resin is a cross-linked polystyrene-divinylbenzene co-polymer substituted by sulphonic acid residues in the form of the calcium salt.

5. A process as claimed in claim 1 or 4 wherein the quantity of resin used provides an exchange capacity of at least 10 times the total exchange-able cation capacity in the solution to be applied.

6. A process as claimed in claim 1 or 4 wherein the contacting of the solution with the resin takes the form of percolation through a bed of resin.

7. A process as claimed in claim 1, 2 or 4 wherein the solution of the salt to be exchanged contains 2 - 30% w/v of the salt of clavulanic acid.

8. A process as claimed in claim 1, 2 or 4 wherein the solution of the salt to be exchanged contains 8 - 20% w/v of the salt of clavulanic acid.

9. A process as claimed in claim 1 wherein the crystallisation of the calcium di-clavulanate dihydrate is initiated by the addition of a water miscible organic solvent.

10. A process as claimed in claim 9 wherein the solvent volume is reduced prior to the addition of the organic solvent.

11. A process as claimed in claim 1 wherein the crystals are dried at ambient pressure.

12. Crystalline calcium di-clavulanate dihydrate whenever prepared by a process as claimed in claim 1, 2 or 4.