TECHNICAL FIELD OF THE INVENTION

The present invention relates to antimicrobial pharmaceutical compositions and uses thereof for the treatment or prevention of staphylococcus infections. More particularly the invention relates to anti¬ microbial pharmaceutical compositions containing a bismuth salt and a rifamycin antibiotic agent for the treatment or prevention of staphylococcus infections.

BACKGROUND OF THE INVENTION

Staphylococcus aurβus is encountered as a normal inhabitant of human skin and mucous membranes. However, it is responsible for a variety of clinical disorders in man and in animals ranging from pustules to fulminating septicemia.

The rifamycin antibiotic agents, such as rifabutin, rifamide, rifamycin sodium, rifapentine, rifaximin and rifampicin are complex macrocyclic antibiotics based on natural products of Streptomyces mβditerranei. They inhibit bacterial RNA synthesis by binding to DNA-dependent RNA polymerase. 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.

Rifamycin antibiotics, especially rifampicin, have been used frequently for therapy of selected staphylococcal infections in animals and humans. In vitro rifampicin is extremely active against staphylococci, the growth is usually inhibited by rifampicin concentrations ≤ 0.03 μg/ml. However, mutants resistant to rifampicin can be detected both in vitro and in vivo amongst most of the bacteria usually sensitive to rifampicin. For example S. aurβus has a propensity to rapidly acquire resistance to rifampicin after exposure to the agent as monotherapy. The resistance is usually the result of one-step mutation which alters the β-subunit of RNA-polymerase, reducing its binding affinity for rifampicin.

The rapid development of resistance when rifampicin is used alone is a serious problem and greatly limits the usefulness of rifampicin in the treatment of e.g. 5. aureus infections. The succesful use of rifampicin in e.g. serious staphylococcal infections depends on the elimination of resistant variants present in the bacterial population.

To overcome the problem of rapid emergence of resistant bacteria during a treatment with rifampicin alone in S. aureus infections, combinations with other antibiotics are recommended although they do not necessarily inhibit the development of resistant mutants. Further, of the various antibiotics tested with rifampicin only nafcillin, novobiocin and teicoplanin have been found to suppress the emergence of rifampicin resistance in S. aureus (Eng et al., J. Antimicrob. Chemother.,1985, 15, pp. 201 - 207 and Dixson et al., Eur. J. Clin. Microbiol., 1985; 4/1, 19 - 23).

Colloidal bismuth subcitrate and bismuth subsalicylate have been found to be useful in combination with πitroimidazole by preventing the development of aquired nitroimidazole resistance of Hθlicobactβr pylori (Axon, A. T. R., J. Gastroenterol. Hepatol., 1991, 6/2, 131 - 137).

In vitro synergistic activity of the combination of bismuth subcitrate and rifampicin against Hβlicobactβr pylori has been demonstrated (D.L. Van

Caekenberghe, J. Breyssens; Antimicrobial Agents and Chemotherapy, 1987, 31/9: 1429-1430).

There are no reports concerning the effect of bismuth salts on the development of resistance against antibiotics in staphylococci.

DISCLOSURE OF THE INVENTION

The purpose of this invention is to overcome the problem of rapid development of rifampicin resistance in Staphylococcus by combining a bismuth salt with a rifamycin antibiotic agent. Further the purpose of this invention is to prepare antimicrobial pharmaceutical compositions containing a rifamycin antibiotic and a bismuth salt and thereby to inhibit the development of resistance.

When bismuth subcitrate was combined with rifampicin, it was unexpectedly found that the development of resistance against rifampicin was

suppressed or totally inhibited among S. aureus whereby the effective doses of these substances could be decreased noticeably.

Thus, the present invention provides an antimicrobial pharmaceutical composition comprising a rifamycin antibiotic agent and a bismuth salt in an amount suppressing the development of rifampicin resistance in Staphylococcus.

Staphylococcus infections which may be treated are preferably local rather than systemic infections and include e.g. S. aureus and 5. epidermis.

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

The pharmaceutical compositions according to the invention may be 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 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 (Martindalβ, The Extra

Pharmacopoeia, ed. by James E.F. Reynolds, 30th ed. The Pharmaceutical Press, London, 1993, p. 909).

The concentration of a bismuth salt in a pharmaceutical preparation containing also rifampicin should be in the range of 5 - 3000 μg/ml e.g. 5 to 400 μg/ml. The effect is not concentration dependent and at low bacterial

densities even a relatively low bismuth salt concentration may be sufficient. In topical ointments, powders, gels, rinsing solutions, eye and ear drops and wound dressings etc. the weight ratio of a bismuth salt to rifamycin is preferably between 1 :0.0003 - 1 :50 e.g. 1 :0.0003 to 1 :20, particularly 1 :0.0003 to 1:0.625.

The pharmaceutical compositions containing the active combination may also be used in veterinary medicine.

Further, the compositions according to the present invention are applicable also in the treatment of certain infections caused by either Staphylococcus or Psθudomonas 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 agent is often Ps. aeruginosa or S. aureus. Because of the possibility that these two bacteria are simultaneously present at the infection site, the use of the present combination is especially advantageous for the treatment of this kind of infections / these kinds of infection.

In veterinary clinical praxis a common disease in dogs is otrtis externa 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.

Examples of bismuth salts which may be used in accordance with present invention include bismuth subcitrate, bismuth subnitrate and bismuth subsalicyiate. Bismuth subcitrate is preferred.

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.

EXAMPLE 1 ,

Comparison of the number of Staphylococcus resistant to rifampicin recovered after exposure in MH-broth to rifampicin. rifampicin + bismuth subcitrate. bismuth subcitrate and nil.

Twelve strains of S. aureus were used in the experiment. Strain 1

(FRI-1104) was obtained from Food Research Institute (FRI), Madison, USA and strains 2 - 12 (ATCC-12600, ATCC-8096, ATCC-25923, ATCC-25178, ATCC-8095, ATCC-27664, ATCC-13565, ATCC-19095, ATCC-14458, ATCC- 27154 and ATCC-6538) were obtained from American Type Culture Collection (ATCC), Maryland, USA. All strains were tested by the broth microdilution method for sensitivity of rifampicin. The minimum inhibitory concentration (MIC) was for all strains ≤ 0.008 μg/ml. The strains were similarily tested for their sensitivity of bismuth subcitrate. The MIC for strain 5 was found to be 800 μg/ml and for the others the MIC-values were ≥3200 μg/ml.

The success rate of developing rifampicin resistance after exposure to rifampicin, rifampicin together with bismuth subcitrate, bismuth subcitrate or nil was performed by suspending S. aureus organisms to a density of about 1- 10 ¹⁰ cfu/ml in Mueller-Hinton (MH) broth containing 0.1 μg/ml rifampicin, 0.1 μg/ml rifampicin together with 400 μg/ml bismuth subcitrate, 400 μg/ml bismuth subcitrate or no additions, respectively. After incubating 18 h at 37 °C, an amount of 0,1 ml of each incubation mixture was quantitatively subcultured on plain MH-agar and on MH-agar containing 0.1 or 1 μg/ml rifampicin. The number of viable organisms which appeared on the agars after an overnight incubation at 37 °C was determined.

The results are summarized in tables 1 and 2. The first column (rif) in both tables shows the log cfu/ml of the 12 strains of S. aureus resistant to either 0.1 μg/ml (table 1) or 1 μg/ml (table 2) rifampicin recovered after exposure to 0.1 μg/ml rifampicin for 18 h over the log cfu/ml of total viable organisms. The second column (rif+bic) shows correspondingly the log cfu/ml of the resistant organisms over the log cfu/ml of the total viable organisms after exposure to 0.1 μg/ml rifampicin together with 400 μg/ml bismuth subcitrate. The third column (bic) shows the log cfu/ml of the rifampicin resistant staphylococci over the log cfu/ml of the total viable organisms after exposure to 400 μg/ml bismuth subcitrate only. And the fourth column (nil) shows the log

cfu/ml of the rifampicin resistant organisms over the log cfu/ml of the total viable organisms after exposure only to MH-broth.

The success rate of development of rifampicin resistance after the various exposures (results given in tables 1 and 2) shows that all the 12 S. aureus strains developed resistance against both 0.1 μg/ml and 1 μg/ml rifampicin after 18 h exposure to 0.1 μg/ml rifampicin. The fact that the bacteria did not die during the exposure although their MICs of rifampicin were as low as ≤ 0.008 μg/ml can be partly explained by the well known inoculum effect (Chapman, R. and Steigbiegel, R., 1975, J. Infect .Dis., 147, 156 - 163). The MIC-values were obtained with an inoculum of 10 ⁵ cfu/ml while the initial bacterial density in the exposure studies was about 10 ¹⁰ cfu/ml. When studying the first (rrf) and the second (rif+bic) columns in tables 1 and 2 ^' it can be seen that the emergence of rifampicin resistance was suppressed when the bacteria were exposed to both rifampicin and bismuth subcitrate. The exposure of bismuth subcitrate alone did not significantly affect the total number of vialble organisms or have any clear effect on the number of rifampicin resistant organisms. The comparison of the second (rif+bic) and the fourth (nil) columns in the tables shows that among most of the strains (except perhaps strains 11 and 12) the development of new resistant organisms was possibly totally inhibited by bismuth subcitrate.

The mechanism behind the effect of bismuth subcitrate remain obscure. Rifampicin resistance is primarily caused by alteration in the bacterial RNA- polymerase (Wehrli, W., 1983, Rev. Infect. Dis., 5 (suppl.3), 407 - 411). A change of one amino acid in the subunit causes resistance. This change is apparently inhibited by bismuth salt.

The effects of bismuth subcitrate were measured by using a maximum bismuth subcitrate concentration of 400 μg/ml on a bacterial density of about 10 ¹⁰ cfu/ml. Higher concentrations were avoided because they could have generally suppressed the growth of staphylococci. To have an effect on the development of resistance bismuth salt must apparently react with the individual bacteria. The concentration of bismuth salt that is required for the effect seen thus obviously depends also on the number of bacteria in the study medium. Further, even relatively low bismuth subcitrate concentrations can suppress the emergence of resistance among staphylococci if the density of bacteria in the study system is low enough.

Table 1.

Strain Number of rifampicin resistant organisms / total No: number of organisms after exposure to (log cfu/ml)

1 7.1 / 9.4 4.9 /10.3 2.8 /10.3 5.5/10.6

2 3.6 /10.4 1.8 /9.4 1.2 /9.3 2.9 / 10.4

3 5.2 /10.2 2.4 /10.0 2.9 / 9.9 3.8 / 10.2

4 7.2 /10.5 2.9 /10.3 2.8 /10.3 3.2/10.3

5 5.5 /7.1 1.2 /6.7 1.4 /6.0 2.1 /6.5

6 7.2 /10.6 3.2 /10.4 3.0 /10.3 3.4 / 10.6

7 4.8 /10.3 2.1 /10.0 2.0 / 9.7 2.8/10.2

8 6.9 /10.3 2.4 /10.2 2.1 / 10.1 3.0 / 10.2

9 6.5 /10.0 3.4 /9.8 2.1 /9.5 2.9/9.6

10 3.8 /10.4 2.5 /9.6 1.9 /9.6 2.9 / 10.2

11 7.6 /10.3 5.7 / 9.9 2.4 /10.1 2.9/10.3

12 10.4/10.4 4.4 /10.2 2.5 /10.2 3.1 / 10.5

8

Table 2-

Strain Number of rifampicin resistant organisms / total No: number of organisms after exposure to (log cfuml) if rf+bic bfc ri

1 7.1 /9.4 4.2 /10.3 2.4 /10.3 5.1 /10.6

2 3.5 /10.4 1.4 /9.4 1.3 /9.3 2.8 /10.4

3 3.0 /10.2 1.9 /10.0 2.7 /9.9 3.5 /10.2

4 7.2 /10.5 2.4 /10.3 2.4 /10.3 2.7 / 10.3

5 5.6 /7.1 1.2 /6.7 2.2 /6.0 2.0 /6.5

6 7.0 /10.6 2.8 /10.4 2.4 /10.3 3.1 /10.6

7 4.9 /10.3 1.2 /10.0 1.3 /9.7 2.4 / 10.2

8 6.8/10.3 2.6 /10.2 1.8 / 10.1 2.7 / 10.2

9 6.4 /10.0 3.4 /9.8 1.0 /9.5 2.3 /9.6

10 2.9 /10.4 2.3 / 9.6 1.4 /9.6 2.7 / 10.2

11 7.6 /10.3 5.8 / 9.9 2.3 /10.1 2.7 / 10.3

12 3.1 /10.4 ND/10.2 2.3 /10.2 2.6 / 10.5

EXAMPLE 2

Concentration dependence To study the effect of the concentration of bismuth subcitrate on the development of rifampicin resistance in S. aureus strain 4 was suspended and incubated in MH-broth as discribed in example 1. The broth contained either no addition or 0.1 μg/ml rifampicin or 0.1 μg/ml rifampicin and 50, 100, 200 or 400 μg/ml bismuth subcitrate. An amount of 0.1 ml of each incubation mixture was quantitatively subcultured on MH-agar as described in example 1 except that plates containing 0.1 μgml rifampicin were omitted. The number of viable organisms after an overnight incubation at 37 °C was determined.

The comparative effect of 5 different bismuth subcitrate concentrations (0, 50, 100, 200 and 400 μg/ml ) on the development of rifampicin resistance

in S. aureus strain 4 during an overnight incubation with 0.1 μg/ml rifampicin in MH-broth is presented in table 3.

The results in table 3 do not show any concentration dependent effect of bismuth subcitrate on the development of resistance to rifampicin on S. aureus. These results further show that the number of organisms resistant to rifampicin after overnights exposure to 0.1 μg/ml rifampicin plus 50 - 400 μg/ml bismuth subcitrate do not significantly differ from the number of resistant organisms recovered after overnights incubation in the plain MH-broth. This suggests that bismuth subcitrate totally inhibited the development of rifampicin resistance due to rifampicin exposure on the studied S. aureus strain.

Table 3.

Exposure Number of rifampicin resistant / total number of organisms (log cfu/ml)

Rrf 8.6 / 10.6

Rif + 50 μgAπl BiC 2.7 / 10.6

RH + 100 μg/ml BIC 3.0 / 10.4

Rif + 200 μg/ml BIC 2.6 / 10.3

Rif + 400 μg/ml BIC 2.6 / 10.5 plain MH-broth 2.9 / 10.6

EXAMPLE 3, Ointment or gel formulation for the treatment of skin infections Of

W£U0 £

A topical ointment or gel formulation according to the present invention is prepared by combining rifampicin (0.005 %) and bismuth subcitrate (0.02%) with an appropriate ointment or gel base and with necessary additives.