Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1991-08-15
2001-04-17
Berch, Mark L (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
C540S349000
Reexamination Certificate
active
06218380
ABSTRACT:
BACKGROUND TO THE INVENTION
a.
Streptomyces clavuligerus
has been described in detail by Higgens et al,
Int. J. Systematic Bacteriology,
21, 326 (1971). This streptomycete was of interest because it produced certain &bgr;-lactam antibiotics such as penicillin N, 7-(5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid and 7-(5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl 7-methoxy-3-cephem-4-carboxylic acid. The streptomycete has been deposited in the Agricultural Research Service Collection as NRRL 3585 and in the American Type Culture Collection as ATCC 27064.
Streptomyces clavuligerus
has also been referred to in U.S. Pat. No. 3,770,590 and also by Nagarajan et al,
J. Amer. Chem. Soc.,
93, 2308 (1971), Brannon et al,
Antimicrob. Agents Chemother.,
1, 237 (1972) and
Antimicrob. Agents Chemother,
1, 247 (1972) and Higgens et al,
J. Antibiotics
27, 298 (1974).
b. &bgr;-lactamases are enzymes which open the &bgr;-lactam ring of penicillins and cephalosporins to give products which are devoid of antibacterial activity. These enzymes are produced by many bacteria, notably species or strains of Escherichia, Klebsiella, Proteus, Pseudomonas, Enterobacter and Staphylococcus and are in many instances the explanation for the resistance of certain strains of such organisms to some penicillins and cephalosporins. The importance of &bgr;-lactamase production may be understood when it is realised that a high proportion of clinically isolated organisms produce &bgr;-lactamases (see, for example, M. Wilson and I. A. Freeman,
Bacteriological Proceedings,
30 (1969) where in a paper entitled ‘Penicillin Inactivation by Gram-negative Bacilli’ they showed that 84% of the gram-negative organisms isolated in an American hospital produced &bgr;-lactamase). In many cases, some penicillins or cephalosporins are ineffective in treating diseases ascribed to non &bgr;-lactamase-producing organisms because of the common occurrence of co-infection by a &bgr;-lactamase producer (see, for example, R. May et al;
Brit. J. Dis. Chest.,
66, 185 (1972). Combination of a &bgr;-lactamase inhibiting substance with a penicillin or cephalosporin might be expected to protect the latter from degradation by bacterial &bgr;-lactamase and thereby enhance their antibacterial activity against many infective organisms. This process of enhancement of the antibacterial activity is called synergism when the antibacterial activity of the combination is well in excess of the simple addition of the activities of the two separate substances. The &bgr;-lactamase inhibiting component of the mixture is referred to as a synergist and such substances are valuable for increasing the antibacterial activity of penicillins and cephalosporins against resistant organisms. It is one of the objects of this invention to provide such synergists.
c. Examples of the use of certain &bgr;-lactamase resistant semi-synthetic penicillins and cephalosporins as &bgr;-lactamase inhibitors and synergists for penicillins and cephalosporins have already been described in the literature, see for example, Sutherland et al.,
Nature,
201, 868 (1964); Sabath et al.,
Nature,
204, 1066 (1964); O'Callaghan et al.,
Antimicrob. Agents and Chemotherapy,
1968, 67 (1969). However, none of these known agents have a dramatic effect on the spectrum of the other antibiotic present in the mixture.
d. Certain actinomycete cultures have been described as producing &bgr;-lactamase inhibiting substances which act synergistically with penicillins or cephalosporins, for example, those cultures disclosed in British Patent No. 1,363,075 and those described by Hata et al,
J. Antibiotics,
25, 473 (1972) and Umezawa et al,
J. Antibiotics,
26, 51 (1973). None of these &bgr;-lactamase inhibitors of actinomycetal origin have yet been found to be of use in the clinic. Particularly noteworthy features which distinguish clavulanic acid from other &bgr;-lactamase inhibitors of actinomycetal origin are its extractability into organic solvents from culture filtrate at pH2, its high stability in human blood and its broad spectrum of anti-bacterial and &bgr;-lactamase inhibiting activity, its low molecular weight and its high R
f
values on paper chromatography using a variety of solvent systems.
DESCRIPTION OF THE INVENTION
We have discovered that the aerobic cultivation of
Streptomyces clavuligerus
in conventional nutrient media at about 25-30° C. under roughly neutral conditions produces a &bgr;-lactamase inhibitory substance which also possesses antibacterial activity. We have designated this new material ‘clavulanic acid’.
Clavulanic acid has the following properties:
(a) It is a carboxylic acid.
(b) It forms a sodium salt which has a characteristic infra-red spectrum substantially as shown in the drawing.
(c) It is able to inhibit the growth of strains of
Staphylococcus aureus.
(d) It is able to synergise the antibacterial effect of ampicillin against &bgr;-lactamase producing strains of
Escherichia coli, Klebsiella aerogenes
and
Staphylococcus aureus.
(e) It is able to synergise the antibacterial effect of cephaloridine against the &bgr;-lactamase producing strains of
Proteus mirabilis
and
Staphylococcus aureus.
(f) It forms a methyl ester which has a molecular weight (by mass spectroscopy) of 213.0635 which corresponds to the formula C
9
H
11
NO
5
.
Thus clavulanic acid may be regarded as a monobasic carboxylic acid of the formula C
8
H
9
NO
5
which in the form of its sodium salt has a characteristic infra-red absorption spectrum substantially as shown in the drawing.
The compound produced by
Streptomyces clavuligerus
which has the above properties has the formula (II):
Thus clavulanic acid may be named 3-(&bgr;-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3,2,0] heptane-2-carboxylic acid.
The stereochemistry at C
5
and C
2
of the clavulanic acid is the same as that found in naturally occurring penicillins and cephalosporins so that clavulanic acid may be represented by the structural formula (I):
Thus a fuller chemical name for clavulanic acid is Z-(2R,5R)-3-(&bgr;-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylic acid.
The great usefulness of clavulanic acid may be readily appreciated when it is realised that certain strains of
Klebsiella aerogenes
A, the growth of which is not inhibited by the presence of 125 &mgr;g/ml. of ampicillin, amoxycillin, carbenicillin or benzyl penicillin or by the presence of 10 &mgr;g/ml. of clavulanic acid, are inhibited by the presence of less than 12.5 &mgr;g/ml. of the previously mentioned penicillins when 5 &mgr;g/ml. of clavulanic acid is also present. Similar results have been observed for combinations containing various esters of clavulanic acid. For example, strains of
Klebsiella aerogenes
A, the growth of which is not inhibited by 125 &mgr;g/ml. of ampicillin or by 10 &mgr;g/ml of clavulanic acid methyl ester are inhibited by less than 12.5 &mgr;g/ml. of ampicillin in the presence of 5 &mgr;g/ml. of the clavulanic acid methyl ester. It has also been found that strains of
Staphylococcus aureus
Russell, the growth of which is not inhibited by the presence of 100 &mgr;g/ml. of ampicillin or by 5 &mgr;g/ml of clavulanic acid, are inhibited by the presence of less than 10 &mgr;g/ml. of ampicillin in the presence of 1 &mgr;g/ml. of clavulanic acid. In tests on female mice, it has been found that blood and tissue levels of clavulanic acid considerably in excess of 5 &mgr;g/ml. can readily be achieved by subcutaneous administration of 100 mg/kg of the sodium salt of clavulanic acid and that useful levels of clavulanic acid can be obtained after oral administration of 100 mg/kg of the sodium salt of clavulanic acid.
Accordingly, the present invention provides clavulanic acid as hereinbefore described and its salts and esters.
Most suitably the salts of clavulanic acid will be pharmaceutically acceptable salts such as the sodium, potassium, calcium, magnesium, aluminium, ammonium and substituted ammonium salts such as the trimethylammon
Cole Martin
Howarth Thomas Trevor
Reading Christopher
Berch Mark L
Kinzig Charles M.
SmithKline Beecham p.l.c.
Williams Janice E.
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