Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~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:
CH2H
~~
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` C02H
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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3736
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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~C)7973~;
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.
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~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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10'~973~
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)
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~ The fvllowing Examples illustrate the inventlon:
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~ Exam~le 1
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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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~07973~
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~).
* Trade Mark for diatomaceous earth
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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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