WO1996008259A1

WO1996008259A1 - Synergistic antimicrobial composition

Info

Publication number: WO1996008259A1
Application number: PCT/FI1995/000494
Authority: WO
Inventors: Timo Juhani Pekkanen
Original assignee: Orion-Yhtymä Oy
Priority date: 1994-09-12
Filing date: 1995-09-12
Publication date: 1996-03-21
Also published as: AU3388595A; GB9418346D0; GB2293323A

Abstract

A synergistic, antimicrobial pharmaceutical composition for the treatment or prevention of Pseudomonas infections contains a bismuth salt and as an antibiotic agent either a rifamycin antibiotic or fusidic acid or its pharmaceutically or veterinarily acceptable salt.

Description

SYNERGISTIC ANTIMICROBIAL COMPOSITION

TECHNICAL FIELD OF THE INVENTION

The present invention relates to synergistic, antimicrobial pharmaceutical compositions and uses thereof for the treatment or prevention of Pseudomonas infections. More particularly the invention relates to synergistic, antimicrobial pharmaceutical compositions for the treatment or prevention of Pseudomonas infections containing a bismuth salt and as an antibiotic agent either a rifamycin antibiotic, a sulfonamide, vancomycin or fusidic acid or pharmaceutically or veterinariiy acceptable salt thereof.

BACKGROUND OF THE INVENTION

The use of antimicrobial agents is a subject of continuing research, much of which, in addition to the discovery of new agents, is directed to the discovery of means for the enhancement of the activity of known active agents.

Although Pseudomonas spp. are quite apathogenic to humans, they are very important in causing opportunistic infections particularly in hospitalized patients with severe underlaying illnesses. Ps. aeruginosa, the most pathogenic member of this group, is often associated with infections related to injured skin of burned patients, wounds on legs and bedsores. It may also occur in otitis media and in infections of the eye, the joints and the urinary, genitourinary and respiratory tracts. Other infectious strains are Ps. fluorescβns, Ps. putida and Ps. cepacia.

In domestic animals similar infections caused by Pseudomonas spp, mainly Ps. aeruginosa are relatively common. Ps. aeruginosa is an important θtiologic factor with Staphylococcus aureus in otitis externa in dogs. Mastitis of dairy cows caused by Ps. aeruginosa is often a serious life threatening disease where treatment alternatives are very few.

Because Pseudomonas are naturally very resistant to antibiotics and further, mutations and R-factors make them even more resistant, only few antibiotic agents are effective in the treatment of Pseudomonas infections.

Some new semisynthetic penicillins, cefalosporins and aminoglygosides are ₂

currently used in the therapy of Pseudomonas infections and combined use of two anti-microbial agents is preferred in severe infections.

Rifamycin antibiotics, such as rifabutin, rifamide, rifamycin sodium, rifapentine, rifaximin and rifampicin are complex macrocyclic antibiotics based on natural products of Streptomyces mediterranei. They inhibit bacterial RNA synthesis by binding to DNA-dependent RNA poiymerase. Rifampicin (chemically 3-4(4-methylpiperazinylimino-methylidene)-rifamycin SV) is an extremely efficient inhibitor of the bacterial enzyme. It inhibits the growth of most gram-positive bacteria, as well as many gram-negative microorganisms and mycobacteria. Although rifamycin antibiotic agents have quite broad spectrum anti-microbial activity, they lack the desired activity against Pseudomonas.

The activity of rifampicin against some common pathogenic bacteria (MlC:mg/l) is presented below (Antibiotic and Chemotherapy, 1992, 6th edition, edited by Lambert, H.P. and O'Grady F.W., Churchill Livingstone, Great Britain).

Staph. aureus 0.008-0.06

Strep, pyogenes 0.03-0.1

Strep, pneumoniae 0.06-4

M. tuberculosis 0.1-1

H. influenzae 0.5-1

E. coli 8-16

Salmonella 8-16

Ps. aeruginosa 32-64

Rifampicin is readily absorbed from the gastrointestinal tract. After a single oral dose of 600 mg plasma peak concentrations of 7 to 9 μg/ml have been reported (Martindale, The Extra Pharmacopoeia, ed. by James E.F. Reynolds, 30th ed., The Pharmaceutical Press, London, 1993, p.199). On the other hand, the ratio of rifampicin concentration at the infection site e.g. in skin blisters to its concentration in serum is 1 :5 (Gerding, D.N. et al., 1991 , in

Antibiotics in Laboratory Medicine, edited by V. Lorian, Williams & Wilkins. Baltimore, Maryland, USA). So, by administering rifampicin doses sufficiently high to give a microbiologically active (> MIC) concentration against Ps. aeruginosa e.g. in skin blisters serious adverse effects of overdosage, for example hepatic and hematological abnormalities, are likely to occur.

Vancomycin is an amphoteric glycopeptide produced by Streptomyces orientalis. It inhibits the synthesis of bacterial cell wall by inhibiting the peptidoglycan synthesis inside the bacterial cell. Vancomycin is active only against Gram-positive organisms, notably staphylococci, including β-lactamase-producing and methicillin-resistant strains, and streptococci.

The susceptibility of some pathogenic bacteria to vancomycin (MIC:mg/l) is presented below (Antibiotic and Chemotherapy, 1992, 6th edition, edited by Lambert, H.P. and O'Grady F.W., Churchill Livingstone, Great Britain).

Staph. aureus 0.25-4

Staph. epidermis 0.25-8

Strep, pneumoniae 0.25-1

E. faecalis 0.25-4

C. difficile <0.1-0.5

Enterobacteria R

Ps. aeruginosa R

Sulphonamides such as sulphadiazine and sulphafurazole, act as inhibitors of folic acid synthesis. They are active against a broad spectrum of bacteria. For example group A streptococci, pneumococci and Neisseria are highly susceptible and staphylococci moderately so. Pseudomonas aeruginosa, however, is usually resistant.

Fusidic acid is a steroidal antibiotic produced by the growth of certain strains of Fusidium coccineum. It inhibits bacterial protein synthesis by binding to elongation factor G which is necessary for translocation. Fusidic acid and its salts have been used succesively in the treatment of staphylococcal infections in human and veterinary medicine especially topically in eye infections and infections of the skin. Fusidic acid and its salts however lack the desired activity against Pseudomonas.

The activity of sodium fusidate against some common pathogenic bacteria (MIC:mg/l) is presented below (Antibiotic and Chemotherapy, 1992, 6th edition, edited by Lambert, H.P. and O'Grady F.W., Churchill Livingstone, Great Britain).

Staph. aureus 0.03 - 0.1

Strep, pyogenes 4 - 16

Strep, pneumoniae 2 - 16

M. meningitidis 0.06 - 0.25

Enterobacteria R

Ps. aeruginosa R Bismuth salts such as subsalicylate and subcitrate have been demonstrated to have antibacterial action against some microorganisms, for example Clostridium difficile and Helicobacter pylori (Cornick, N. A. et al.; Reviews of Infectious Diseases, 1990, 12 (suppl. 1), s9 - s10 and Lee S.P.; Scand J. Gastroenterol., 1991 , 26 (suppl. 185), 1 - 6). The mechanism of this antibacterial activity is not known. It has been suggested that bismuth disrupts bacterial cell integrity causing leakage of intracellular components or displaces magnesium ions, that are important to the integrity of the gram- negative outer membrane, thereby destabilizing the permeability barrier, and enhancing antibiotic permeation (Domenico et al.; Jounal of Antimicrobial Chemotherapy, 1991 , 28, 801-810).

In vitro synergistic activity of the combination of bismuth subcitrate and rifampicin against Helicobacter pylon has been demostrated. (D.L. Van Caekenberghe, J. Breyssens; Antimicrobial Agents and Chemotherapy, 1987, 31/9: 1429-1430). Further, the influence of bismuth subsalicylate and bismuth subnitrate on the antibiotic action of aminogiycosides and some beta-lactam antibiotics against Pseudomonas aeruginosa have been studied (Domenico et al., 1992, Eur. J. Clin. Microbiol. Infect. Dis., 11 : 170-174). However, beta- lactam antibiotics (the bacterial cell wall), aminogiycosides (protein synthesis by binding to the bacterial ribosome), rifamycin antibiotics (nucleic acid synthesis), vancomycin (peptidoglycan synthesis), sulfoanamides (folic acid synthesis) and fusidic acid and its salts (protein synthesis by inhibiting transiocation) differ functionally and structurally from each other. Further, it has also been shown that the antibacterial activity of some antibiotics (e.g. cefoperazone, imipenem and mezlocillin) against Ps. aeruginosa is decreased by bismuth salts (Domenico et al., 1992, Eur. J. Clin. Microbiol. Infect. Dis., 1 : 170-174). Thus, results with one antibiotic cannot be applied to another antibiotic belonging to structurally and/or functionally different group.

DISCLOSURE OF THE INVENTION

In accordance with the present invention the activity of rifamycin antibiotics, vancomycin, sulfonamides and/or fusidic acid and its pharmacologically or veterinarily acceptaple salts against Pseudomonas are enhanced by combining a bismuth salt with a rifamycin antibiotic, vancomycin, a sulfonamide or fusidic acid or its pharmacologically or veterinarily acceptaple salt. The present invention also provides a synergistic antimicrobial pharmaceutical composition containing a bismuth salt and a rifamycin antibiotic or vancomycin or a suifonamide or fusidic acid or pharmacologically or veterinarily acceptable salt thereof.

When bismuth subcitrate was combined with rifampicin or with vancomycin or with sulphadiazine or with sodium fusidate, it was found that the presence of the bismuth salt enhanced the antimicrobial activity of the antibiotic agent against Pseudomonas to an unexpected extent and because of this high-level synergistic action, the effective doses of these substances could be decreased noticeably.

Thus, the present invention provides an antimicrobial pharmaceutical o composition comprising a bismuth salt and a rifamycin antibiotic or a bismuth salt and vancomycin or a bismuth salt and a suifonamide or a bismuth salt and fusidic acid or its pharmacologically or veterinarily acceptable salt, in an amount having a synergistic effect against Pseudomonas spp.

The pharmaceutical compositions according to the invention include for 5 example granulates, tablets, capsules, dragees, powders, sprays, ointments, gels, emulsions, suspensions and infusions (solutions) and can be administered for example orally, rectally or topically. Both agents are preferably administered concurrently, but the pharmaceutical effect of the present composition will be present if both agents exist concurrently for a 0 certain duration in the body, particularly at the infection site. So, the bismuth salt can be administered separately from or simultaneously with the antibiotic agent. These two agents can be administered via different routes, for example antibiotic agent orally and bismuth salt topically.

The pharmaceutical compositions according to the invention may be 5 formulated and employed in the usual manner.

These compositions are particularly well-suited for ophthalmic, otic or topical uses (gels, ointments, powders, sprays and aqueous or oily emulsions and suspensions) but could also find other applications. However, topical use of bismuth salts is preferred. Systemic treatment of infections with bismuth o salts is not recommended because of the possibility of serious side effects.

Nausea and vomiting as well as darkening of the tongue have been reported. Further, the possibility of bismuth encephalopathy exists after prolonged use. (Martindale, The Extra Pharmacopoeia, ed. by James E.F. Reynolds, 30th ed. The Pharmaceutical Press, London, 1993, p. 909). The pharmaceutical compositions containing the synergistic active combination may also be used in veterinary medicine.

Enhancement of the antibacterial activity of rifamycin antibiotics, vancomycin, sulfonamides and fusidic acid and its pharmaceutically or veterinarily acceptable salt in the treatment of Pseudomonas infections is one of the advantages achieved with the combined use of a rifamycin antibiotic or vancomycin or a suifonamide or fusidic acid or its pharmaceutically or veterinarily acceptable salt and a bismuth salt. When a rifamycin antibiotic or vancomycin or a suifonamide or fusidic acid or its pharmaceutically or o veterinarily acceptable salt and a bismuth salt are used together, they produce a synergistic effect which permits a reduction in the dosage of one or both drugs with achievement of a similar therapeutic effect and the combination produces a more rapid or complete bactericidal effect than could be achieved with either drug alone.

5 Further, the compositions according to the present invention are applicable also in the treatment of certain infections caused by either Staphylococcus or Pseudomonas such as hospital infections which in many instances are extremely resistant to antibiotics. The most common hospital infections are the urinary tract and wound infections where the causative 0 agent is often Ps. aeruginosa or S. aureus. Because of the possibility that these two bacteria are simultaneously present in the infection site, the use of the present combination is especially advantageous for the treatment of this kind of infections.

In veterinary clinical praxis a common disease in dogs is otitis externa 5 which is often caused by Ps. aeruginosa or S. aureus. Because of the difficulty of reliable laboratory diagnosis it is advantageous to treat the infection with the present combination which has a good effect on both types of bacteria.

The pharmaceutical composition of the present invention exhibit o synergistic antimicrobial activity against Pseudomonas. It has been found that in order to obtain the desired synergistic antimicrobial activity in accordance with the present invention the ratio (by weight) of rifampicin to bismuth subcitrate should be in the range of 1 :1.6 to 1 : 13333, preferably 1 :25 to 1 :200, the ratio (by weight) of vancomycin to bismuth subcitrate should be in 5 the range of 1 :444 to 10:1 , preferably 1 :6 to 1 :2, the ratio (by weight) of sulphadiazine to bismuth subcitrate should be in the range of 1 :2162 to 1:2, preferably 1 :34 to 1 :42 and the ratio (by weight) of sodium fusidate to bismuth subcitrate should be in the range of 1 :512 to 64:1 , preferably 1 :2 to 4:1. (1g BiC contains about 185 mg Bi).

The preferred bismuth salt used in accordance with the present 5 invention is bismuth subcitrate. Other bismuth salts which may be used include bismuth subnitrate, bismuth subsalicylate.

The following examples are presented to further illustrate the present invention. They should not be interpreted as limiting the scope of the invention in any way.

ι o EXA PLE 1

Svnerqv study and the enhancement of the antibacterial activity of rifampicin with bismuth subcitrate

Eight Pseudomonas aeruginosa strains were used in the experiment. Five were ATCC (American Type Culture Collection) strains isolated from 1 5 human infections and three were EK (College of Veterinary Medicine,

Helsinki, Finland) strains originated from animal infections. These EK-strains were cheked by morphological characteristics and conventional biochemical methods to be Pseudomonas aeruginosa.

Rifampicin (R^'rf) stock solution was prepared daily in absolute methanol 20 and appropriately diluted with Mueller-Hinton broth. Bismuth subcitrate (BiC) powder was brought into solution with 1 N NaOH and further diluted as above. The poor solubility of other bismuth salts prevented their testing.

After initial cultivation on blood agar at 37 °C typical colonies of the strain to be studied were transferred to Mueller-Hinton broth and incubated

25 20 h at 37 °C. A 10 -³ dilution of the culture with Mueller-Hinton broth was used for the determination of the minimum inhibitory concentration (MIC) by the standard microdilution broth procedure (Amterdam, D.; 1991, Susceptibility Testing of Antimicrobials in Liquid Media. In Antibiotics in Laboratory Medicine, 3th edition, ed. by V. Lorian. Williams & Wilkins,

30 Baltimore, Maryland, USA, pp. 53-105). A 10 "³ dilution of the culture with Mueller-Hinton broth was used also for the synergism studies by the checker board microtiter tray method (G.M. Eliopoulos and R. C. Moellering Jr., 1991. Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 3th edition, ed. by V. Lorian. Williams & Wilkins, Baltimore, Maryland, USA, pp. 432-492). Synergy was quaπtitatθd by calculating fractional inhibitory concentration index (FIC index) as decribed by Eliopoulos and Moellering. With this method synergy is defined as a FIC index of ≤ 0.5.

Because of the complexity of the synergy assay, only rifampicin was chosen for testing.

The MICs (μg/ml) of bismuth subcitrate and rifampicin and FIC-indexes at the range of synergism in addition to the lowest MlC-values of rifampicin when used with bismuth subcitrate and the corresponding concentration of bismuth subcitrate together with the ratios of rifampicin/bismuth subcitrate at o the observed range of synergism are presented in table 1.

The results in table 1 show that the MlC-values of rifampicin for the tested eight Pseudomonas strains which varied from 8 μg/ml to 32 μg/ml decreased with the presence of bismuth subcitrate to 0.06 - 1 μg/ml. The FIC- indexes were in the range of 0.13 - 0.37 indicating synergism. Further, the 5 obtained FIC-indexes did not indicate indifference or antagonism between rifampicin and bismuth subcitrate. The lowest MIC of rifampicin when combined with bismuth subcitrate was 0.06 μg/ml. This was obtained when bismuth subcitrate concentration was 800 μg/ml. When the MlC-values of rifampicin were compared with the respective values of rifampicin with 0 bismuth subcitrate, 16 to 266 fold increase in the sensitivity of Ps. aeruginosa strains was observed. The ratio of rifampicin to bismuth subcitrate varied between 1 : 13333 - 1 :1.6 at the observed range of synergism. The concentrations of bismuth subcitrate necessary for synergism were far below the corresponding MICs of bismuth subcitrate for the microbes.

5 The present study shows that rifampicin is useful antibiotic in the treatment of Pseudomonas infections if it is combined with proper amount of bismuth subcitrate. Also in infections where the simultaneous presence of Pseudomonas and Staphylococci is suspected or when it is not known which of the bacteria are involved the combined use of rifampicin and bismuth o subcitrate is advantageous. The results further suggest that in order to achieve synergism against Pseudomonas with rifampicin/bismuth subcitrate mixtures, the concentration of bismuth subcitrate should be higher than the corresponding concentration of rifampicin. Table t

Strain MIC of BiC MIC of Rif FIC-index at The lowest MIC of Rif Rif /BiC ratios at μg/ml μg/ml the range of with BiC and the the observed synergism corresponding BiC range of concentration μg/ml synergism

ATCC-10145 800 16 0.25 - 0.31 1/200 1 :200 - 1:3

ATCC-14210 6400 16 0.25 - 0.31 0.25/3200 1 :1600 - 1 :25

ATCC- 15692 3200 16 0.16 - 0.27 0.125/1600 1 :3200 - 1 :50

ATCC- 19660 1600 16 0.28 - 0.37 0.125/800 1 :800 - 1:12

ATCC-43390 1600 32 0.25 - 0.37 0.125/800 1 :400 - 1 :1.6

EK-81 > 3200 16 0.25 - 0.37 0.06/800 1 :13333 - 1:3

EK-163 6400 8 0.13 - 0.34 0.06/800 1 :13333- 1 :25

EK-168 > 3200 16 0.25 - 0.37 0.06/800 1 :13333- 1 :6

EXAMPLE 2

Dose response study with rifampicin and bismuth subcitrate

In the study on the possible dose response relationship of the bismuth subcitrate with the sensitivity of Pseudomonas aeruginosa to rifampicin, the MIC of rifampicin for Pseudomonas strain EK 81 was determined as above in example 1 but having in the wells of each tray 50, 100, 200, 400 or 800 μg/ml bismuth subcitrate.

The concentration dependent effect of bismuth subcitrate on the sensitivity of Ps. aeruginosa strain EK-81 to rifampicin is presented in table 2. The MlC-values of rifampicin were determined by the standard microdilution broth procedure.

The results in table 2 show that bismuth subcitrate increased in dose dependent manner the sensitivity of Ps. aueruginosa EK-81 to rifampicin. Without the presence of bismuth subcitrate the MIC-value of rifampicin for strain EK-81 was 16 μg/ml but with bismuth subcitrate concentration of 800 μg/ml the MIC-value was found to be 0.06 μg/ml. Further, even a relatively low concentration of bismuth subcitrate enhanced considerably the activity of rifampicin against Ps. aeruginosa. The MIC of bismuth subcitrate for Ps. aeruginosa EK-81 was >3200 μg/ml.

Table 2.

BiC μg/ml MIC Rif μg/ml

0 16

50 2

100 1

200 1

400 0.125

800 0.062

EXAMPLE 3

Svnergv study and the enhancement of the antibacterial activity of vancomycin with bismuth subcitrate

Eight Pseudomonas aeruginosa strains were used in the experiment.

Seven were ATCC (American Type Culture Collection) strains isolated from human infections and one was EK (College of Veterinary Medicine, Helsinki, Finland) strain originated from animal infection. This strain was choked by morphological characteristics and conventional biochemical methods to be Pseudomonas aeruginosa.

Vancomycin (Va) stock solution was prepared and appropriately diluted with Mueller-Hinton broth daily. Bismuth subcitrate (BiC) power was brought into solution with 1 N NaOH and further diluted as above. The poor solubility of other bismuth salts prevented their testing.

Pseudomonas aeruginosa strains were cultivated and the MIC- and

FlC-values were determined as described in example 1. The MICs (μg/ml) of bismuth subcitrate and vancomycin and FIC- indexes at the range of synergism in addition to the lowest MlC-values of vancomycin when used with bismuth subcitrate and the corresponding concentration of bismuth subcitrate together with the ratios of vancomycin/ bismuth subcitrate at the observed range of synergism are presented in table 3.

The results in table 3 show that the tested Pseudomonas aeruginosa strains were quite resistant to vancomycin as well as to bismuth subcitrate. The MIC-value of vancomycin for the tested strains which varied from o 500 μg/ml to 1000 μg/ml decreased with the presence of bismuth subcitrate to 1.8 - 62.5 μg/ml. The FIC-indexes were in the range of 0.06 - 0.5 indicating synergism. Further, the obtained FIC-indexes did not indicate antagonism or indifference between vancomycin and bismuth subcitrate. The lowest MIC of vancomycin when combined with bismuth subcitrate was 1.8 μg/ml. This was 5 obtained when the concentration of bismuth subcitrate was 800 μg/ml. When the MlC-values of vancomycin were compared with the respective values of vancomycin with bismuth subcitrate, 8 to 278 fold increase in the sensitivity of Ps. aeruginosa strains was observed. The ratio of vancomycin to bismuth subcitrate varied between 1:444 to 10:1 at the observed range of 0 synergism.The concentrations of bismuth subcitrate necessary for synergism were below the corresponding MICs of bismuth subcitrate for these microbes.

The present study shows that vancomycin is useful antibiotic in the treatment of Pseudomonas infections if it is combined with proper amount of bismuth subcitrate. Also in infections where the simultaneous presence of 5 Pseudomonas and Staphylococci is suspected or when it is not known which of the bacteria are involved the combined use of vancomycin and bismuth subcitrate is advantageous.

Table 3.

Strain MIC of BiC MICofVa FIC-index at The lowest MIC of Va Va/BiC ratios at μg/ml μg/ml the range of with BiC and the the observed synergism corresponding BiC range of concentration μg/ml synergism

ATCC-27853 >6400 >500 0.27 - 0.46 31.25/400 1:12.5-10:1

ATCC- 14204 6400 1000 0.11 -0.14 12.5/800 1:64-8:1

ATCC- 14210 6400 >500 0.07 - 0.25 31.25/1600 1:51 -10:1

ATCC- 15692 3200 >500 0.125-0.16 31.25/400 1:13-5:1

ATCC- 19660 1600 500 0.28 - 0.5 62.5/400 1:6-1:1.6

ATCC-10145 800 500 0.15-0.375 62.5/400 1:6-5:1

ATCC-9027 6400 500 0.13-0.19 1.8/800 1 :444 - 12

EK-81 >3200 1000 0.06-0.13 7.8/800 1:102- 1:1

EXAMPLE 4

Svnerφv study and the enhancement of the antibacterial activity of sulDhadiazine with bismuth subcitrate

Eight Pseudomonas aeruginosa strains were used in the experiment. Seven were ATCC (American Type Culture Collection) strains isolated from human infections and one was EK (College of Veterinary Medicine, Helsinki, Finland) strain originated from animal infection. This strain was cheked by morphological characteristics and conventional biochemical methods to be Pseudomonas aeruginosa.

Sulphadiazine (SDZ) stock solution was prepared and appropriately diluted with Mueller-Hinton broth daily. Bismuth subcitrate (BiC) power was brought into solution with 1 N NaOH and further diluted as above. The poor solubility of other bismuth salts prevented their testing. Further, because of the complexity of the synergy assay, only sulphadiazine was chosen for testing.

Pseudomonas aeruginosa strains were cultivated and the MIC- and FlC-vaiues determined as decribed in example 1. The MICs (μg/ml) of bismuth subcitrate and sulphadiazine and FIC- indexes at the range of synergism in addition tϋ the lowest MlC-values of sulphadiazine when used with bismuth subcitrate and the corresponding concentration of bismuth subcitrate together with the ratios of sulphadiazine/ bismuth subcitrate at the observed range of synergism are presented in table 4.

The results in table 4 show that the tested Pseudomonas aeruginosa strains were quite resistant to sulphadiazine as well as to bismuth subcitrate. The MlC-values of sulphadiazine for the tested eight strains which varied from 95 μg/ml to 400 μg/ml decreased with the presence of bismuth subcitrate to 0.74 - 5.9 μg/ml. The FIC-indexes were in the range of 0.01 - 0.28 indicating synergism. Further, the obtained FIC-indexes did not indicate antagonism or indifference between sulphadiazine and bismuth subcitrate. The lowest MIC of sulphadiazine when combined with bismuth subcitrate was 0.74 μg/ml. This was obtained when the concentration of bismuth subcitrate was 1600 μg/mi. When the MlC-values of sulphadiazine were compared with the respective values of sulphadiazine with bismuth subcitrate, 32 to 270 fold increase in the sensitivity of Ps. aeruginosa strains was observed. The ratio of sulphadiazine to bismuth subcitrate varied between 1 :2162 to 1 :2 at the observed range of synergism.The concentrations of bismuth subcitrate necessary for synergism were below the corresponding MICs of bismuth subcitrate for these microbes.

The present study shows that sulphadiazine is useful antibiotic in the treatment of Pseudomonas infections if it is combined with proper amount of bismuth subcitrate. Also in infections where the simultaneous presence of

Pseudomonas and Staphylococci is suspected or when it is not known which of the bacteria are involved the combined use of sulphadiazine and bismuth subcitrate is advantageous. Table 4.

Strain MIC of BiC MIC of FIC-index at The lowest MIC of SDZ/BiC ratios at μg nl SDZ the range of SDZ with BiC and the the observed μg/ml synergism corresponding BiC range of concentration μg/ml synergism

ATCC-27853 > 6400 380 0.25 - 0.28 1.48/25 1 :18 - 1 :1081

ATCC- 14204 6400 400 0.02 • 0.25 1.48/1600 1 :42 - 1 :1081

ATCC- 14210 6400 190 0.04 - 0.25 0.74/1600 1:2 - 1:2162

ATCC- 15692 3200 190 0.04 • 0.26 2.97/800 1 :4 - 1269

ATCC- 19660 1600 190 0.05 - 0.28 5.9/25 1 :4 - 1 :136

ATCC- 10145 800 400 0.06 - 026 5.9/200 1 -2 - 1 :34

ATCC-9027 6400 95 0.01 - 0.13 1.48/25 1 :18 - 1 :1081

EK-81 > 6400 200 0.03 - 0.26 1.48/1600 1 :42 - 1 :1081

EXAMPLE 5

Svnerαv study and the enhancement of the antibacterial activity of sodium fusidate with bismuth subcitrate

Sodium fusidate (SF) stock solution was prepared daily in distilled water and appropriately diluted with Mueller-Hinton broth. Bismuth subcitrate (BiC) power was brought into solution with 1 N NaOH and further diluted as above. The poor solubility of other bismuth salts prevented their testing.

Because of the complexity of the synergy assay, only sodium fusidate was chosen for testing. Pseudomonas aeruginosa strains were cultivated and the MIC- and FlC-values were determined as described in example 1.

The MICs (μg/ml) of bismuth subcitrate and sodium fusidate and FIC- indexes at the range of synergism in addition to the lowest MlC-values of 5 sodium fusidate when used with bismuth subcitrate and the corresponding concentration of bismuth subcitrate together with the ratios of sodium fusidate/ bismuth subcitrate at the observed range of synergism are presented in table 5.

The results in table 5 show that the tested Pseudomonas aeruginosa o strains were quite resistant to sodium fusidate as well as to bismuth subcitrate. The MlC-values of sodium fusidate for the tested eight strains which varied from 1600 μg/ml to 6400 μg/ml decreased with the presence of bismuth subcitrate to 3.6 - 200 μg/ml. The FIC-indexes were in the range of 0.02 - 0.31 indicating synergism. Further, the obtained FIC-indexes did not indicate 5 antagonism or indifference between sodium fusidate and bismuth subcitrate.

The lowest MIC of sodium fusidate when combined with bismuth subcitrate was 3.6 μg/m. This was obtained when the concentration of bismuth subcitrate was 800 μg/ml. When the MlC-values of sodium fusidate were compared with the respective values of sodium fusidate with bismuth 0 subcitrate, 8 to 444 fold increase in the sensitivity of Ps. aeruginosa strains was observed. The ratio of sodium fusidate to bismuth subcitrate varied between 1 :512 to 64:1 at the observed range of synergism.The concentrations of bismuth subcitrate necessary for synergism were below the corresponding MICs of bismuth subcitrate for these microbes.

5 The present study shows that sodium fusidate is useful antibiotic in the treatment of Pseudomonas infections if it is combined with proper amount of bismuth subcitrate. Also in infections where the simultaneous presence of Pseudomonas and Staphylococci is suspected or when it is not known which of the bacteria are involved the combined use of fusidic acid or its o pharmaceutically or veterinarily acceptable salt and bismuth subcitrate is advantageous. Table 5.

Strain MIC of BiC MICof SF FIC-index at The lowest MIC of SF SF/BiC ratios at μg/ml μg/ml the range of with BiC and the the observed synergism corresponding BiC range of concentration μg/ml synergism

ATCC-27853 > 6400 1600 0.05 - 0.26 3.6/800 1 :512 - 4:1

ATCC- 14204 6400 1600 0.09 - 0.26 6.25/1600 1256 - 4:1

ATCC-14210 6400 >3200 0.14 - 0.19 50/1600 1256 - 4:1

ATCC-15692 3200 >3200 0.02 - 0.27 6.25/1600 1:16 - 8:1

ATCC- 19660 1600 1600 0.03 - 0.31 100/400 1:4 - 16:1

ATCC-10145 800 1600 0.14 - 0.31 100/200 12 - 64:1

ATCC-9027 6400 6400 0.03 - 0.26 50/1600 1 :32 - 8:1

EK-81 > 3200 1600 0.09 - 0.13 6.25/1600 1:512 - 4:1

EXAMPLE 6

Dose response study with sodium fusidate and bismuth subcitrate

In the study on the possible dose response relationship of the bismuth subcitrate with the sensitivity of Pseudomonas aeruginosa to sodium fusidate, the MlC-values of sodium fusidate for three Pseudomonas aeruginosa strains were determined as above in example 5 but having in the wells of each tray 50, 100, 200, 400 or 800 μg/ml bismuth subcitrate.

The effect of the increasing concentration of bismuth subcitrate on the MICs of sodium fusidate for strains ATCC 25004, ATCC 10145 and ATCC 14204 determined by the standard microdilution broth procedure are presented in table 6.

The results in table 6 show that bismuth subcitrate increased in dose dependent manner the sensitivity of the three Ps. aeruginosa strains to sodium fusidate. Further, even a relatively low concentration of BiC enhanced considerably the activity of sodium fusidate against Ps. aeruginosa and increase in the sensitivity of Pseudomonas to sodium fusidate by bismuth subcitrate increases with increasing concentration of bismuth subcitrate.

The MlC-values of sodium fusidate and bismuth subcitrate for the strains used in this experiment are: Strain MIC SF μg/ml MIC BiC μg/ml

ATCC-25004 3200 6400

ATCC-10145 1600 800

ATCC-14204 1600 6400

Table 6.

BiC MIC of SF μg/ml μg/ml ATCC-25004 ATCC-10145 ATCC-14204

50 200 100 100

100 100 -

200 100 50 100

400 50 625 50

800 25 625

EXAMPLE 7,

A αel containing rifampicin and bismuth subcitrate for the treatment of wounds

A gel formulation according to the present invention is prepared by combining rifampicin (0.001 - 0.1 %) and bismuth subcitrate (0.1 -0.5 %) with a suitable gel base containing for example a carboxyvinyl polymer as a gel former.

EXAMPLE 8.

Eve drops containing sodium fusiriatβ and bismuth subcitrate

An ophtalmic formulation according to the present invention is prepared by combining sodium fusidate (0.01 - 0.1 %), bismuth subcitrate (0.1 - 0.5 %) and other necessary ingredients with an aqueous phosphate buffer solution.

Claims

1. A product which is an antimicrobial pharmaceutical composition 5 comprising (i) an antibiotic agent which is a rifamycin antibiotic or vancomycin or a suifonamide or fusidic acid or a pharmaceutically or veterinarily acceptable salt thereof and (ii) a bismuth salt for use in the treatment of a Pseudomonas infection.

2. A product comprising (i) an antibiotic agent which is a rifamycin o antibiotic or vancomycin or a suifonamide or fusidic acid or a pharmaceutically or veterinarily acceptable salt thereof and (ii) a bismuth salt as a combined preparation for simultaneous, separate or sequential use in the treatment of a Pseudomonas infection.

3. A product according to claim 1 or 2 wherein the bismuth salt is 5 bismuth subcitrate, bismuth subsalicylate or bismuth nitrate.

4. A product according to claim 3 wherein the bismuth salt is bismuth subcitrate.

0 5. A product according to any one of claims 1 to 4 wherein the antibiotic agent is rifampicin.

6. A product according to any one of claims 1 to 4 wherein the antibiotic agent is sodium fusidate. 5

7. A product according to any one of claims 1 to 4 wherein the antibiotic agent is sulphadiazine.

8. A product according to any one of claims 1 to 4 wherein the antibiotic o agent is vancomycin.

9. A product according to any one of claims 1 to 5 comprising a rifamycin antibiotic wherein the weight ratio of a rifamycin antibiotic to bismuth salt is in the range of from 1 :13333 to 1 :1.6 5

10. A product according to claim 6 wherein the weight ratio of sodium fusidate to bismuth salt is in the range of from 1 :512 to 64:1

11. A product according to claim 7 wherein the weight ratio of sulphadiazine to bismuth salt is in the range of from 1 :2162 to 1 :2.

5 12. A product according to claim 8 wherein the weight ratio of vancomycin to bismuth salt is in the range of from 1 :444 to 10:1.

13. A product according to any one of claims 1 to 12 wherein the bismuth salt is in a form suitable for administration simultaneously with the antibiotic o agent.

14. A product according to any one of claims 1 to 12 wherein the bismuth salt is in a form suitable for administration separately from the antibiotic agent.

5 15. Use of a bismuth salt and an antibiotic agent which is a rifamycin antibiotic or vancomycin or a suifonamide or fusidic acid or a pharmaceutically or veterinarily acceptable salt thereof in the manufacture of a medicament for use in the treatment of a Pseudomonas infection.

0 16. A method of treatment or prevention of a Pseudomonas infection comprising administering to a subject an effective amount of an antibiotic agent which is a rifamycin antibiotic or vancomycin or a suifonamide or fusidic acid or a pharmaceutically or veterinarily acceptable salt thereof and an effective amount of a bismuth salt. 5

17. A pharmaceutical or veterinary formulation comprising as active ingredients an antibiotic agent which is a rifamycin antibiotic or vancomycin or a suifonamide or fusidic acid or a pharmaceutically or veterinarily acceptable salt thereof, and a bismuth salt formulated for pharmaceutical or veterinary o use respectively.

18. Use of a bismuth salt to enhance the antimicrobial activity of an antibiotic agent which is a rifamycin antibiotic or vancomycin or a suifonamide or fusidic acid or a pharmaceutically or veterinarily acceptable salt thereof 5 against Pseudomonas.