WO1994017800A1

WO1994017800A1 - AN IMPROVED β-LACTAM ANTIBIOTIC COMPOSITION

Info

Publication number: WO1994017800A1
Application number: PCT/DK1994/000064
Authority: WO
Inventors: Poul Børge POULSEN
Original assignee: Gist-Brocades B.V.
Priority date: 1993-02-15
Filing date: 1994-02-15
Publication date: 1994-08-18
Also published as: AU6106794A

Abstract

An improved pharmaceutical composition comprising a very pure semisynthetic β-lactam antibiotic synthesized by enzymatic synthesis is provided. The composition is completely or substantially devoid of the disagreeable smell and/or taste which many β-lactam antibiotics impart to pharmaceutical compositions containing them. The invention also relates to the use of a very pure semisynthetic β-lactam antibiotic produced by enzymatic synthesis for providing such an improved pharmaceutical composition.

Description

AN IMPROVED 3-LACTAM ANTIBIOTIC COMPOSITION

FIELD OF THE INVENTION

The present invention relates to an improved pharmaceutical composition comprising a very pure semisyntetic /3-lactam antibiotic synthesized by enzymatic synthesis which composition is completely or substantially devoid of the disagreeable smell and/or taste which many /3-lactam antibiotics impart to pharmaceutical compositions containing them and to a method of producing it. The invention also relates to the use of a very pure semisyntetic /3-lactam antibiotic produced by enzymatic synthesis for providing such an improved pharmaceutical com¬ position.

BACKGROUND OF THE INVENTION

It has long been realized by persons of the medical profession that an important factor in achieving a good compliance with a prescribed regimen which comprises ingestion of a medicine is the smell and/or the taste of the particular composition (Ruff et al. Antimicrobial drug suspensions: a blind comparison of taste of fourteen common paediatric drugs. Pediatr.Infect.Dis.J. 10 (1991) , 30 - 33) . If a medicine has an offensive smell and/or taste, this significantly reduces the patients' compliance with the prescribed regimen. This is particularly pronounced with young children who furthermore often find it difficult to swallow a tablet or a capsule.

Various attempts have been made with a view to providing pharmaceutical compositions having improved properties as regards their smell and/or their taste.

When a medicine has an offensive smell and/or taste, this can be due to a property inherent in the pharmacologically active substance itself. Thus, many pharmacologically active substances have an inherent bitter taste. In such cases, more acceptable compositions as regards their smell and/or taste can only be achieved by masking the smell and/or the taste of the active substance.

However, an offensive smell and/or taste may also be imparted to a pharmaceutical composition because the pharmacologically active substance contained therein is accompanied by evil- smelling and/or bad-tasting impurities which originate from its synthesis or which are formed by degradation after the synthesis. Depending on their nature, even minute traces of such impurities can cause troubles. Also in such cases, it can of course be attempted to provide acceptable pharmaceutical compositions by masking the unacceptable smell and/or taste.

Examples of pharmaceutical compositions widely recognized to be rather troublesome as regards their smell and/or their taste are those containing /3-lactam antibiotics. Accordingly, attempts have been made to produce dosage forms accepted by young children of various penicillins, e.g. formulated as syrups or as oil suspensions with a variety of flavouring agents added to impart a flavour of chocolate, vanilla or various fruits. However, the flavouring agents added do not always effectively mask the offensive flavour originating from the antibiotic. Also, many patients dislike the oily feel in the mouth caused by the oil suspensions and some doctors and parents are reluctant to let children have the syrups because of their high sugar contents.

Some /3-lactam antibiotics have been formulated as effervescent tablets containing in addition to the 3-lactam antibiotic various flavouring agents and a system which releases carbon dioxide on contact with water so that the tablets on dissolution in water produce a lemonade-like drink. However, also in these cases the attempt to mask the flavour of the antibiotic is not always successful.

Dry powder compositions of penicillins and cephalosporins containing a buffer salt and coated with insoluble material to mask the taste are disclosed in German Offenlegungsschrift No. 25 09 572 (to Beecham Group Ltd.) Masking of the flavour of i.a. the potassium salt of phenoxymethylpenicillin by using an ice-cream based composition is described in Danish Patent Application No. 3113/89 (to Novo Nordisk A/S) .

According to the United States Pharmacopeia (U.S.P.) the purity of bulk amoxicillin must be in the range of from 90.0% to 105.0% when compared to a U.S.P. standard which is defined to be 99.5% pure. This range has been established on the basis of what was a realistic demand for bulk amoxicillin produced by non-enzymatic methods. The extraordinary broadness of this range compared to the range allowed for most other drug compounds reflects the difficulties involved in providing pure and reproducible bulk amoxicillin by non-enzymatic methods.

When a pharmaceutical composition has an offensive smell and/or taste originating from impurities which accompany the pharmacologically active substance contained therein, an obvious solution to the problem would be to purify the phar¬ macologically active substance so as to remove the impurities. However, so far it has not been possible to solve the problems related to the offensive smell and/or taste encountered with pharmaceutical compositions containing semisyntetic /3-lactam antibiotics in this way and thus there still is a need for improved pharmaceutical compositions containing semisyntetic 3-lactam antibiotics and for a method of providing them.

SUMMARY OF THE INVENTION

Surprisingly, it has now turned out that when a pharmaceutical composition containing a semisyntetic /3-lactam antibiotic is manufactured from a semisyntetic /3-lactam antibiotic produced by enzymatic synthesis, e.g. as described in WO 92/01061 or in WO 93/12250 (both to Novo Nordisk A/S) followed by extensive purification by a method known per se the product is completely or substantially devoid of the offensive smell and/or taste which a semisyntetic /3-lactam antibiotic produced by non- enzymatic synthesis imparts to a pharmaceutical composition containing it.

Thus, in its broadest aspect the present invention relates to a pharmaceutical composition comprising a semisyntetic /3-lactam antibiotic produced by enzymatic synthesis, to a method of producing a pharmaceutical composition comprising a semisyntetic /3-lactam antibiotic, which composition is completely or substantially devoid of disagreeable flavour, the composition being produced using a semisyntetic /3-lactam antibiotic produced by enzymatic synthesis, and to the use of an enzymatically produced semisynthetic /3-lactam antibiotic for providing a pharmaceutical composition comprising it which composition is completely or substantially devoid of disagreeable flavour.

In a first preferred embodiment the invention relates to a pharmaceutical composition comprising an enzymatically produced penicillin.

In a further preferred embodiment the invention relates to a pharmaceutical composition comprising enzymatically produced amoxicillin.

In a further preferred embodiment the invention relates to a pharmaceutical composition comprising an enzymatically produced cephalosporin.

In a further preferred embodiment the invention relates to a pharmaceutical composition comprising enzymatically produced cephalexin.

In a further preferred embodiment the invention relates to a pharmaceutical composition comprising an enzymatically produced /3-lactam antibiotic wherein the bulk /3-lactam antibiotic used for preparing the composition contains less than 2%, more preferred less than 1,5%, still more preferred less than 1%, most preferred less than 0.5% of impurities.

In a further preferred embodiment the invention relates to a pharmaceutical composition comprising enzymatically^' produced amoxicillin wherein the bulk amoxicillin used for preparing the composition contains less than 2%, more preferred less than 1,5%, still more preferred less than 1%, most preferred less than 0.5% of impurities.

In a further preferred embodiment the invention relates to a method of producing a pharmaceutical composition comprising a semisyntetic /3-lactam antibiotic, which composition is completely or substantially devoid of disagreeable flavour, the composition being produced using a semisyntetic /3-lactam antibiotic produced by enzymatic synthesis.

In a further preferred embodiment the invention relates to a method of producing a pharmaceutical composition comprising a semisyntetic penicillin, which composition is completely or substantially devoid of disagreeable flavour, the composition being produced using a semisyntetic penicillin produced by enzymatic synthesis.

In a further preferred embodiment the invention relates to a method of producing a pharmaceutical composition comprising semisyntetic amoxicillin, which composition is completely or substantially devoid of disagreeable flavour, the composition being produced using semisyntetic amoxicillin produced by enzymatic synthesis.

In a further preferred embodiment the invention relates to a method of producing a pharmaceutical composition comprising a semisyntetic cephalosporin which composition is completely or substantially devoid of disagreeable flavour, the composition being produced using a semisyntetic cephalosporin produced by enzymatic synthesis.

In a further preferred embodiment the invention relates to a method of producing a pharmaceutical composition comprising semisyntetic cephalexin which composition is completely or substantially devoid of disagreeable flavour, the composition being produced using semisyntetic cephalexin produced by enzymatic synthesis.

In a further preferred embodiment the invention relates to a method of producing a pharmaceutical composition comprising a semisyntetic /3-lactam antibiotic, which composition is completely or substantially devoid of disagreeable flavour, the composition being produced using a semisyntetic /3-lactam antibiotic produced by enzymatic synthesis and containing less than 2%, more preferred less than 1,5%, still more preferred less than 1%, most preferred less than 0.5% of impurities.

In a further preferred embodiment the invention relates to the use of an enzymatically produced semi-synthetic /3-lactam antibiotic containing less than 2%, more preferred less than 1,5%, still more preferred less than 1%, most preferred less than 0.5% of impurities for providing a pharmaceutical composition which is completely or substantially devoid of disagreeable flavour.

In a further preferred embodiment the invention relates to the use of an enzymatically produced semi-synthetic penicillin for providing a pharmaceutical composition which is completely or substantially devoid of disagreeable flavour.

In a further preferred embodiment the invention relates to the use of enzymatically produced semi-synthetic amoxicillin for providing a pharmaceutical composition which is completely or substantially devoid of disagreeable flavour.

In a further preferred embodiment the invention relates to the use of an enzymatically produced semi-synthetic cephalosporin for providing a pharmaceutical composition which is completely or substantially devoid of disagreeable flavour.

In a further preferred embodiment the invention relates to the use of enzymatically produced semi-synthetic cephalexin for providing a pharmaceutical composition which is completely or substantially devoid of disagreeable flavour.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated with reference to the appended drawings wherein

Fig. 1 shows the HPLC chromatogram of a sample of amoxicillin prepared by non-enzymatic synthesis; and

Fig. 2 shows the HPLC chromatogram of a sample of purified amoxicillin prepared by enzymatic synthesis.

DETAILED DESCRIPTION OF THE INVENTION

As used in the present specification the expression "enzymatic synthesis" designates that the introduction of the acyl group in the 6-amino group of a penicillin nucleus, e.g. 6-APA, or in the 7-amino group of a cephalosporin nucleus, e.g. 7-ACA or 7-ADCA, is performed under the influence of an enzyme. Similarly, the expression "non-enzymatic synthesis" indicates that the side chain is introduced without using an enzyme.

Wherever "amoxicillin" is mentioned in the present specification, what is meant is "amoxicillin trihydrate". Wherever in the present specification mention is made of the maximum amount of impurities contained in the /3-lactam antibiotics of this invention it is to be understood as determined by HPLC and calculated on basis of % area of the peaks, the preferred HPLC method being the one described in Example 3.

A preferred method of preparing the 3-lactam antibiotic described herein is to treat the parent a ino /3-lactam, e.g. 6-APA, with an acylating agent in the presence of an enzyme in a manner known per se. for example as described in WO 92/01061. The enzyme to be used in the process of this invention may be any enzyme catalyzing the reaction in question. Such enzymes have been known since around 1966. Enzymes to be used are, for example, termed penicillin amidase or penicillin acylase and classified as E.C. 3.5.1.11. A number of microbial enzymes are known to have this activity, derived from for example Aceto- bacter, Xanthomonas, Mvcoplana. Protaminobacter. Aeromonas (West German patent application having publication No. 2,163,792) Pseudo onas (Austrian Patent No. 243986), Flavobacterium (Dutch patent application No. 70-09138) , Aphano- cladium. Cephalosporium (West German patent application having publication No. 2,621,618), Acetobacter pasteurianum. Bacillus megaterium, Xanthomonas citrii (European patent application having publication No. 339,751), Kluyvera citrophila (Aqr.Biol.Chem. 37 (1973), 2797 - 2804) and Escherichia coli (West German patent application having publication No. 2,930,794). The Escherichia coli enzyme is commercially available. The enzyme also may be a so-called ampicillin hydrolase, acylase or amidase. In this connection, reference is, inter alia, made to Hakko to Kogyo 38 (1980) , 216 et seq. , the content of which is incorporated by reference.

Fig. 1 shows a HPLC chromatogram of an ordinary good quality commercial sample of amoxicillin prepared by non-enzymatic synthesis. As it appears from the chromatogram, the sample contains a vast number of impurities. To the best of the inventor's knowledge, the identity of most of the impurities is not known.

Fig. 2 shows a HPLC chromatogram of purified amoxicillin, prepared by enzymatic synthesis. The chromatograms of Figs. 1 and 2 which are recorded under similar conditions very clearly illustrate the improved quality of the enzymatically prepared amoxicillin relative to the amoxicillin prepared by non- enzymatic synthesis.

The use of very pure 3-lactam antibiotics in pharmaceutical compositions has several advantages. Evidently, as already mentioned, the production of compositions with an acceptable smell and/or taste is greatly facilitated when the drug compound is completely or substantially devoid of evil smelling and/or evil tasting impurities and it does not have an inherent bad smell and/or taste. When there is no bad smell and/or taste which has to be masked, it may even be appropriate to omit flavouring agents from the composition. This is in good agreement with the present widespread endeavours in society to use as few additives as possible in food and drink with a view to avoiding allergic reactions. In keeping with this, ingestion of as many as possible of the impurities of unknown identity - even those which do not contribute to the bad smell and/or taste of a drug compound - should also be avoided. For this reason it is of course an advantage to use an enzymatically produced /3-lactam antibiotic which contains a significantly reduced amount of impurities relative to the same 3-lactam antibiotic produced by a non-enzymatic process.

Some of the impurities found in non-enzymatically produced β- lactam antibiotics are known to be partly ring opened dimers and trimers. These molecules have reactive sites and a molecular weight of the order of magnitude 1000 Daltons which implicates that they may cause allergy. Accordingly, it can be expected that there will be fewer cases of allergic reactions following the use of /3-lactam compositions based on enzymatically produced 3-lactam antibiotics than after the use of 3-lactam compositions based on non-enzymatically produced /3-lactam antibiotics.

A further advantage of the very pure bulk /3-lactam antibiotics which can be obtained by enzymatic synthesis is their improved stability. This is illustrated in Example 4 which describes an accelerated stability testing of a sample of amoxicillin prepared by enzymatic synthesis and a sample of amoxicillin prepared by non-enzymatic synthesis. It is reasonable to anticipate that the improved stability of the bulk product produced by enzymatic synthesis will also be reflected in the stability of the pharmaceutical compositions containing it.

Examples of /3-lactam antibiotics which are suited for incorporation in the pharmaceutical compositions according to the present invention are ampicillin, amoxicillin, ticarcillin, cefaclor, cefatrizine, cefaparol, cephradine, cephalexin, cefadroxil, cephaloglycin and cephalothin.

The pharmaceutical compositions according to the present invention can be intended for any route of administration commonly used for the administration of /3-lactam antibiotics, the oral route being preferred.

The daily dosage of the /3-lactam compositions to be given which i.a. depends on the particular compound and on the condition of the patient is determined by those skilled in the art. Generally, an amount of about 0.1 to 200 g/kg body weight is administered. Dosage unit compositions may contain such amounts or submultiples thereof to make up the daily dose.

Pharmaceutical compositions according to the invention for oral administration may be solid or liquid. Solid dosage forms for oral administration may comprise tablets, pills, capsules, powders and granules. In such solid dosage forms, the _/3-lactam antibiotic may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, according to the known art, additional substances other than inert diluents, e.g. lubricating agents, such as magnesium stearate. In the case of capsules, tablets and pills the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with coatings of various kinds, e.g. enteric coatings.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavouring, and perfuming agents.

The expression "flavour" as used in the present description is meant to signify any sense impression of smell and taste and any combination thereof.

The features disclosed in the present description, examples and claims may, both separately and in any combination thereof, be material for realising this invention in diverse forms thereof. The invention is further illustrated in the following examples which are not to be construed as limiting but merely as an illustration some preferred features of the invention for which protection is sought.

EXAMPLE 1

Tablets according to the invention each containing 250 mg or 500 mg of amoxicillin trihvdrate.

A suitable formulation for tablets containing 250 mg or 500 mg of amoxicillin trihydrate is, by weight:

Amoxicillin trihydrate 62.5% Microcrystalline cellulose 30.60% Aerosil 200 0.80

Polyvidon VA 64 0.50%

Primojel C 5.00% Magnesium stearate 0.60%

Amoxicillin prepared as described in WO 92/01061 is used and the tablets are manufactured in the following manner:

Amoxicillin, Aerosil and Avicel are mixed in a blender and the resulting mixture is sprayed with an aqueous solution of the polyvidon. The damp mass is passed through a 12 mesh screen and the granules are dried. The dried granules are again passed through a 12 mesh screen and the Primogel and the magnesium stearate are added little by little to the screened product. After a final blending the dry granules are compressed into tablets. Compression of 400 mg of the granules into a tablet provides a tablet containing 250 mg of amoxicillin trihydrate while compression of 800 mg of the granules into a tablet provides a tablet containing 500 mg of amoxicillin trihydrate. These tablets exhibit considerably improved properties as regards their flavour compared to tablets manufactured from amoxicillin trihydrate produced by non-enzymatic synthesis.

EXAMPLE 2

Triangle Tests.

A triangle test was carried out in which ten persons tasted 250 mg samples in random order of amoxicillin trihydrate manufactured by non-enzymatic synthesis (two samples for each person) or by the enzymatic method described in WO 92/01061 (one sample for each person) .

The result of the test was that 10 out of 10 persons could clearly distinguish between the two products tested. The enzymatically produced amoxicillin did not develop an off-taste on the tongue, even after 15 seconds, whereas the amoxicillin produced by non-enzymatic synthesis clearly did.

On a different day the smell of the same products was evaluated. Also in this case it was found that 10 out of 10 persons could clearly distinguish the enzymatically produced amoxicillin which was found to have virtually no recognisable smell from the amoxicillin produced by non-enzymatic synthesis since this product was found to have a characteristic /3-lactam smell.

EXAMPLE 3

Comparison by HPLC of samples of amoxicillin produced bv enzymatic and bv non-enzymatic synthesis.

HPLC analysis was performed as follows:

Column: RP C-18, 5 μm, YMC 120 A, OdMeSi (25 x 0.4 cm) Guard column: Supelguard LC-18, 2 cm.

The following solvents were used for gradient elution:

A: 25mM phosphate buffer, pH 6.5

B: acetonitrile

Gradient:

Time (min) %A %B

0 100 0

40 70 30

45 70 30

46 100 0

60 100 0

Flow: 1 ml/min UV-detection at 215 nm Attenuation: 16 Chart speed: 0.5 cm/min

Sample: 0.2 mg/ml, 20 μl injected

The chromatogram of a sample of amoxicillin prepared by enzymatic synthesis is shown in Fig. 1 and the chromatogram of a sample of amoxicillin prepared by non-enzymatic synthesis is shown in Fig. 2. In Fig. 1 the retention time of the amoxicillin peak is 16.39 min and in Fig. 2 it is 16.45 min. The figures, in particular Fig. 2, also show the retention times of several other peaks caused by impurities. The identity of most of the impurities is unknown. However, it is quite evident from an inspection of the chromatograms that the number and the height of peaks caused by impurities is considerably lower in Fig. 1 than in Fig. 2 which means that the amoxicillin prepared by enzymatic synthesis is more pure than the amoxicillin prepared by non-enzymatic synthesis.

EXAMPLE 4

Accelerated stability testing of samples of amoxicillin produced bv enzymatic and bv non-enzvmatic synthesis.

40 mg samples of amoxicillin produced by enzymatic synthesis (A) were weighed into each of four 50 ml vials which were then sealed. Simililarly, 40 mg samples of amoxicillin produced by non-enzymatic synthesis (B) were weighed into each of four 50 ml vials which were sealed. One vial from each lot was placed in a refrigerator while the remaining three vials from each lot were placed in a thermostat at 100°C. After 24, 48, and 72 hours one vial from each lot was transferred from the thermostat to the refrigerator and subsequently analysed by HPLC for the amount of amoxicillin remaining. As it appears from Table 1, which shows the amount of amoxicillin remaining as percentage of the starting value, the amoxicillin prepared by enzymatic synthesis, A, is considerably more stable in the accelerated stability test than the amoxicillin prepared by non-enzymatic synthesis. Table l

Hours at 100°C

0 24 48 72

A 98.69 79.11 71.70 71.23

B 96.15 52.62 44.68 39.25

EXAMPLE 5 Preparation of very pure amoxicillin trihydrate.

The method of Example 9 in WO 92/01061 was modified as follows: In a 10 litre reactor 200 mM 6-APA and 650 inM D-p_- hydroxyphenylglycine was allowed to react under vigorous stirring at 25°C and pH 6.0 in the presence of 120,000 Units of immobilized penicillin G acylase from E. coli. After 3 hours the crystals formed were separated from the reaction liquid and the immobilized enzyme. The crystals were dissolved in a continuous mode in water by addition of acid so that a pH value of 1.0 was reached. Holding time: 0.5 min. After crystallisation at pH 4.1, the product was isolated and dried. The purity (HPLC) of the resultant amoxicillin trihydrate was 99.3%.

Claims

1. A pharmaceutical composition comprising a semi¬ syntetic /3-lactam antibiotic produced by enzymatic synthesis and a suitable carrier.

2. A pharmaceutical composition according to claim 1, wherein the /3-lactam antibiotic is a penicillin.

3. A pharmaceutical composition according to claim 1, wherein the /3-lactam antibiotic is amoxicillin.

4. A pharmaceutical composition according to claim 1, wherein the /3-lactam antibiotic is a cephalosporin.

5. A pharmaceutical composition according to claim 1, wherein the /3-lactam antibiotic is cephalexin.

6. A pharmaceutical composition according to any one of the preceding claims, wherein the bulk /3-lactam antibiotic used for preparing the composition contains less than 2% of impurities.

7. A method of producing a pharmaceutical composition comprising a semisyntetic /3-lactam antibiotic, which composition is completely or substantially devoid of dis- agreeable flavour, wherein the composition is produced using a semisyntetic /3-lactam antibiotic produced by enzymatic synthesis.

8. A method according to claim 7 wherein the _/3-lactam antibiotic used contains less than 2% of impurities.

9. A method according to claim 7 wherein the /3-lactam antibiotic used is amoxicillin.

10. Use of a semisynthetic /3-lactam antibiotic produced by enzymatic synthesis for providing a pharmaceutical composition comprising it which composition is completely or substantially devoid of disagreeable flavour.

11. Use according to claim 10 of a semisynthetic β- lactam antibiotic produced by enzymatic synthesis and containing less than 2%, more preferred less than 1,5%, still more preferred less than 1%, most preferred less than 0.5% of impurities.

12. Use according to claim 10 - 11 of a penicillin pro- duced by enzymatic synthesis.

13. Use according to claim 10 - 12 of amoxicillin pro¬ duced by enzymatic synthesis.

14. Use according to claim 10 - 12 of a cephalosporin produced by enzymatic synthesis.

15. Amoxicillin, completely or substantially devoid of disagreeable flavour.

16. Amoxicillin containing less than 2%, more preferred less than 1,5%, still more preferred less than 1%, most preferred less than 0.5% of impurities and produced by enzymatic synthesis.