GB1561395A

GB1561395A - -lactam antibiotic

Info

Publication number: GB1561395A
Application number: GB7544/76A
Authority: GB
Original assignee: Beecham Group PLC
Current assignee: Beecham Group PLC
Priority date: 1976-02-26
Filing date: 1976-02-26
Publication date: 1980-02-20
Also published as: FR2342289B1; BE851872A; AU2264377A; CA1079736A; ZA771089B; DK86177A; IE44704L; DE2708046A1; IE44704B1; AU504721B2; SE7702078L; NL7702036A; JPS52105193A; CH624957A5; FR2342289A1

Abstract

An aqueous solution of a salt of clavulanic acid with the exception of the calcium salt is treated with a cation exchanger resin in the form of the calcium salt. The calcium diclavulanate which is formed is eluted from the exchanger resin, resulting in a solution of calcium diclavulanate which is virtually free of cations other than calcium ions. Calcium diclavulanate dihydrate is allowed to crystallise out of the resulting solution and is isolated in crystalline form. The resulting salt is suitable as active component in pharmaceutical compositions, especially for oral administration.

Description

(54)8 -LACTAM ANTIBIOTIC (71) We, BEECEIAM GROUP LIMI- TED, a British Company, of Beecham House, Great West Road, Brentford, Middlesex, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to a new process for the preparation of the crystalline calcium salt of davulanic acid, to the salt produced by this process and to pharmaceutical compositions containing it.

British Patent Specification No. 1,508,977 discloses inter alia that the calcium salt of davulanic acid, which is of the formula:

may be isolated from a fermentation broth containing salts of clavulanic acid by adsorbing the davulanic acid residues onto a weak or strong base exchange resin and eluting off with a suitable salt solution and also by hydrogenation of a benzyl or like ester of clavulanic acid in the presence of a suitable base. We have now found that crystalline calcium diclavulanate dihydrate may conveniently be prepared from other salts of clavulanic acid in high yield and good purity.

The present invention provides a process for the preparation of crystalline calcium di davulanate dihydrate which process comprises contacting an aqueous solution of a salt of davulanic acid other than the calcium salt with a cation exchange resin in the calcium form and eluting the thus formed calcium diclavulanate 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 diclavulanate dihydrate from the solution and isolating the desired salt The initial salt of clavulanic acid used in this process may be any convenient metal (other than calcium), ammonium or substituted ammonium salt but in general it is most convenient to use a mono-valent salt such as the lithium, sodium or potassium salt of clavulanic acid. Of 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 form the solution to be exchanged should be as pure as can be conveniently obtained in order to allow the preparation of a pure product.

Suitable cation exchange resins for use are cross-linked polystyrene-divinylbenene copolymers substituted by acid groups in the form of the calcium salt. The preferred acid group is the sulphonic acid group. The resins chosen will generally have 2-20% crosslinking and usually 410% cross-linking, for example about 8% cross-linking. The resin is usually in the form of beads, for example spherical beads of 14-52 U.K. mesh size.

Suitable resins include the calcium form of Amberlite resins such as IR--120, IR121, IR-122, 200, 200C, 252; Dowex resins such as 50WX1, 50WX2, 50X4, 50WX8, 5X10, WX10, 50WX16; Bio Rad resins such as AG 50WX1, 40WX2, A < 3 50WX4, AG 50WX8, AG 50WX12; Ionac resins such as C250, C258 or C255; and Zerolit resins such as 225, 325, 425, 525, 625. ("Amberlite", "Dowex" and "Zerolit" are Registered Trade Marks).

The resin is normally used in large excess.

The quantity of resin used should be sufficient to provide an exchange capacity of at least 10 times, more suitably at least 20 times and preferably at least 30 times, the total exchangeable cation capacity in the solution of the salt of clavulanic acid to be applied.

In use the resin is in a bed through which the solution is percolated. Generally this bed is in the form of a column.

The concentration of the solution of the salt to be exchanged is not critical, but very dilute solutions (for example, less than lCgo 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 concentrations of 230% w/v are acceptable, a range of about 5 to 25% being most suitable and a range of about 8 to 20% being preferred, for example about 10%.

The solution to be exchanged is normally applied slowly to the top to the column. The band is then allowed to percolate slowly into the top of the resin after which a little water is applied to wash the band a short way into the resin. The remaining necessary water is then slowly 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 index of the eluate. This may be determined using a refractometer or visually, for example by the presence of striations.

Alternatively fractions may be taken and tested in convenient manner, for example by spotting on a t.l.c. plate and spraying with permanganate which is decolourized by the clavulanate or by methods using the enzyme inhibitory effects of the material.

The desired calcium salt may be obtained from solution in conventional manner, for example from a relatively concentrated solution by the slow addition of a water-miscible organic solvent such as acetone until crystallisation commences or reducing the solvent volume prior to the addition of an organic solvent, for example by concentrating under reduced pressure to a syrup, followed by adding small quantities of acetone, acetonitrile or acetone/ether to initiate cerystallisation.

Most suitably crystallisation is initiated at ambient or slightly elevated temperature, for example, 12--30"C, more suitably 20--25"C.

Once crystallisation has begun the mixture may be cooled, for example to about 5 to C, until no further crystals appear.

Once the desired crystals have formed they may be filtered off and dried. Vigorous drying conditions (such as vacuum drying) are best avoided as they tend to lead to breakdown of the crystal structure and partially dehydrated crystals. Wet air should not be used for drying as it can lead to wet crystals. Drying should preferably be effected at atmospheric pressure.

The calcium di-clavulanate dihydrate pro duced by the process of this invention is also an aspect of this invention.

An infra-red spectrum of the salt according to this invention is shown in Table 1.

It should be appreciated that the crystalline salt within this invention can contain small amounts of partly dehydrated calcium di clavulanate or small amounts of water which forms no part of the crystal structure and probably simply wets the crystal faces. How ever, the crystalline salt of this invention more suitably contains 6:2 to 7.7% (weight/weight) of water, that is it should not contain water which forms no part of the crystal suucture, although it is acceptable for it to contain small amounts of partly dehydrated material. We have found that a salt containing approximately 6.4 to 7.6% (weight/weight) of total water to have particularly suitable stability properties. Most suitably the water content of the crystals is about 7.5%.

The water contents referred to above are total water, for example as determined by Karl Fischer analysis.

The present invention also extends to a pharmaceutical composition which comprises the said crystalline calcium di-clavulanate dihydrate and a pharmaceutically acceptable carrier therefor.

Such compositions are most suitably adapted for oral administration.

The compositions of this invention may also comprise a penicillin or cephalosporin. Particularly suitable penicillins include ampicillin (as the anhydrate or trihydrate or a pharmaceutically acceptable salt) and amoxycillin (as the trihydrate or a pharmaceutically acceptable salt) and also disodium carbenicillin, disodium ticarcillin, and sodium salts of tne phenyl or indanyl a-esters of carbenicillin or ticarcillin.

Suitable forms of the compositions of this invention are described in Belgian Patent Specification No. 827926.

TABLE 1 Infra-red Spectrum of Calcium Di-clavulanate Dihydrate (in Nujol 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 medium, 1072 medium, 1050 strong, 1024 strong, 1004 strong, 976 weak, 9h0 shoulder, 897 strong, 850 medium, 803 medium, 748 strong, 708 weak, 659 weak.

[Note no significant peak at 23301 ["Nujol" is a Registered Trade Mark].

The following Examples illustrate the invention: Example 1.

Sodium clavulanate (290 mg) in water l ml) was passed through Amberlite IR-120 calcium salt (10 ml wet resin). The eluate (20 ml) was concentrated to less than 1 ml volume and acetonitrile (10 ml) added. Fine crystals of calcium di-clavulanate dihydrate were obtained which has i.r. absorbances thus: 1782 (,84actam C=O), 1695 ;C=C), 1605 cm-' (CO2--). Water (by Karl Fischer) 7.4%, calcium diclavulanate dihydrate requires 7.6%.

Example 2.

a. Preparation of Resin in Calcium Cycle.

Amberlite IR-120 resin (500 ml) was set into a column (15" X ") and hydrochloric acid (1 M solution) passed through the resin until the pH of the effluent was consistently below 0.5. The resin was then washed with distilled water (initially upfiow and then down flow) until the pH of the effluent was consistently at about 3.5. A solution of calcium chloride (0.25 M) was then passed through the resin (by downflow) until the pH of the percolate dropped to less than 1.0. Further calcium chloride solution was passed through the resin until the pH of rhe percolate was .onsistentlv at 3.3-3.5 Thk resin was then washed bs downflow with distilled water about 2 1 until the pH of the percolate was onsistently approximately 4 b. Preparation of solution of (:calcium t lavulanate Sodium clavulanate tetrahydrate (equivalent to 30 g of pure free acid) was made up to 300 ml with distilled water. The solution was stirred with 'Norit' (Registered Trade Mark) gsx charcoal (3 g) and filtered to give a clear pale yellow tinged solution at pH 6.9.

The solution was applied to the previously prepared IR-120 calcium cycle column at 200 ml/hr. When loading was complete the column was then washed with distilled water at 200 ml/hr. As the calcium salt began to be eluted the pH of the eluate changed to about 6.0. Fractions (approximately 60 ml; were then collected until a small aliquot of the final fraction produced little or no precipitation with acetone. This final fraction was colourless and had a pH of about 6.6. The first collected aliquot was discarded and the remaining 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. After about 3.5 volumes of acetone had been added a slightly hazv precipitate formed and was filtered .)ff via Elite (Registered rrade ..',Iark F.lrther acetone was added to the solution until a total of 15 volumes had been added. The resulting solid was filtered off to give a pale buff crystalline 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 acetone. The solid was then dried under reduced pressure (about 10 torr for 18 hours) to yield the desired crystalline calcium clavulanate dihydrate (28 g, total water 6A%, purity > 90%).

The low moisture contents in the above sample probably resulted from drying under reduced pressure. Avoidance of reduced pressures leads to the preparation of crystals containing amounts of water closer to the theoretical amount needed for the dihydrate. It will be therefore understood that in this example air-drying at the final step is usually to be preferred.

Example 3.

a. Sodium clavulanate tetrahydrate (150 mg) was dissolved in distilled water (1 ml) and passed down a column of Amberlite IR- 120 resin (Ca2+ form, 27 ml wet resin). The resin was then washed with distilled water (25 ml). The eluate was evaporated under reduced pressure at ambient temperature (about 22 C) to a syrup (zO.S ml) and then triturated with acetonitrile (25 ml). The calcium salt crystal lised, and it was cooled to 2--3"C for 2 hours, collected by filtration and washed with drv ether (25 ml). The product was air-dried .o yield the desired crystalline calcium di-clavulanate dihydrate (75 mg of substantially pure product, water content~7.4%).

b. Amberlite IR-120 in the calcium form was produced as follows: The resin (H+ form, 20 ml wet resin, ex change capacity 38 m.moles) in water (50 ml) was treated with calcium hydroxide (0.5 g, excess) with stirring. The excess calcium hydroxide was removed by passing a current of distilled water up through a column con taining the resin ('back-washing') until eluate had a pH approximately 8-8.5. (This also removes the 'fines'-debris and minor organic impurities).

WHAT WE CLAIM IS: 1. A process for the preparation of crystal line 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 the 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 any of claims 1-3 wherein the resin is a cross-linked polystyrene-divinylbenzene co-polymer substituted by sulphonic acid groups in the form of the calcium salt.

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

6. A process as claimed in any of daims 1-3 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 any of claims 1-6 wherein the solution of the salt to be exchanged contains 230% w/v of the salt of clavulanic acid.

8. A process as claimed in claim 7 wherein the solution contains 8 to 20% w/v of the salt 9. A process as claimed in any of claims 1-8 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 any of claims 1-10 wherein the crystals are dried at atmospheric pressure.

12. A process as claimed in claim 1 substantially as described in any Example herein.

13. Crystalline calcium di-clavulanate dihydrate whenever prepared by a process as claimed in any of claims 1-12.

14. A pharmaceutical composition which comprises the compound claimed in claim 13 and a pharmaceutically acceptable carrier therefor.

15. A composition as claimed in claim 14 adapted for oral administration.

16. A composition as claimed in claim 14 or 15 which also comprises a penicillin or cephalosporin.

17. A composition as claimed in claim 16 which comprises ampicillin anhydrate, ampicillin trihydrate, pharmaceutically acceptable salt of ampicillin, amoxycillin trihydrate or a pharmaceutically acceptable salt of amoxycillin.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **.

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 acetone. The solid was then dried under reduced pressure (about 10 torr for 18 hours) to yield the desired crystalline calcium clavulanate dihydrate (28 g, total water 6A%, purity > 90%).

The low moisture contents in the above sample probably resulted from drying under reduced pressure. Avoidance of reduced pressures leads to the preparation of crystals containing amounts of water closer to the theoretical amount needed for the dihydrate. It will be therefore understood that in this example air-drying at the final step is usually to be preferred.

Example 3.

a. Sodium clavulanate tetrahydrate (150 mg) was dissolved in distilled water (1 ml) and passed down a column of Amberlite IR-

120 resin (Ca2+ form, 27 ml wet resin). The resin was then washed with distilled water (25 ml). The eluate was evaporated under reduced pressure at ambient temperature (about 22 C) to a syrup (zO.S ml) and then triturated with acetonitrile (25 ml). The calcium salt crystal lised, and it was cooled to 2--3"C for 2 hours, collected by filtration and washed with drv ether (25 ml). The product was air-dried .o yield the desired crystalline calcium di-clavulanate dihydrate (75 mg of substantially pure product, water content~7.4%).

b. Amberlite IR-120 in the calcium form was produced as follows: The resin (H+ form, 20 ml wet resin, ex change capacity 38 m.moles) in water (50 ml) was treated with calcium hydroxide (0.5 g, excess) with stirring. The excess calcium hydroxide was removed by passing a current of distilled water up through a column con taining the resin ('back-washing') until eluate had a pH approximately 8-8.5. (This also removes the 'fines'-debris and minor organic impurities).

WHAT WE CLAIM IS: 1. A process for the preparation of crystal line 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 the 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 any of claims 1-3 wherein the resin is a cross-linked polystyrene-divinylbenzene co-polymer substituted by sulphonic acid groups in the form of the calcium salt.
5. A process as claimed in any of claims 1--4 wherein the quantity of resin used provides an exchange capacity of at least 10 times the total exchangeable cation capacity in the solution to be applied.
6. A process as claimed in any of daims 1-3 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 any of claims 1-6 wherein the solution of the salt to be exchanged contains 230% w/v of the salt of clavulanic acid.
8. A process as claimed in claim 7 wherein the solution contains 8 to 20% w/v of the salt
9. A process as claimed in any of claims 1-8 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 any of claims 1-10 wherein the crystals are dried at atmospheric pressure.
12. A process as claimed in claim 1 substantially as described in any Example herein.
13. Crystalline calcium di-clavulanate dihydrate whenever prepared by a process as claimed in any of claims 1-12.
14. A pharmaceutical composition which comprises the compound claimed in claim 13 and a pharmaceutically acceptable carrier therefor.
15. A composition as claimed in claim 14 adapted for oral administration.
16. A composition as claimed in claim 14 or 15 which also comprises a penicillin or cephalosporin.
17. A composition as claimed in claim 16 which comprises ampicillin anhydrate, ampicillin trihydrate, pharmaceutically acceptable salt of ampicillin, amoxycillin trihydrate or a pharmaceutically acceptable salt of amoxycillin.