In-vitro activity of rifabutin and albendazole singly and in combination with other clinically used antimicrobial agents against Pneumocystis carinii

Oscar Cirioni*, Andrea Giacometti, Francesco Barchiesi, Moira Fortuna and Giorgio Scalise

Institute of Infectious Diseases and Public Health, University of Ancona, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in-vitro activity of rifabutin and albendazole alone and in combination with clarithromycin, etoposide, minocycline and pyrimethamine was investigated against four clinical isolates of Pneumocystis carinii. The susceptibility tests were performed by inoculation of the isolates on to cell monolayers and by determining the parasite count after 72 h incubation at 37°C. The culture medium was supplemented with serial dilutions of each agent. Albendazole tested alone was more active than rifabutin. Albendazole suppressed the growth of cysts and trophozoites by >50% at 4 mg/L. Rifabutin, at the same concentration, produced about 40% reduction in the mean cyst and trophozoite counts. Albendazole (4 mg/L) combined with etoposide 4 mg/L showed the highest anti-P. carinii activity, with a decrease of 86.3% and 90.1% in cyst and trophozoite counts, respectively. The greatest synergic interaction was detected when rifabutin (4 mg/L) was combined with clarithromycin (4 mg/L). Our study suggests that clinically used antimicrobial agents may be effective in inhibiting P. carinii growth in vitro and that, above all, some of these agents possess a positive interaction upon combination with other clinically used compounds. These findings may be useful in the establishment of a prophylaxis regimen for multiple opportunistic pathogens.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pneumocystis carinii is one of the leading causes of opportunistic infection in immunocompromised patients. Several new compounds are being evaluated extensively in vitro and in vivo and some are now available to treat the infection clinically. The anti-P. carinii agents in current clinical use have important limitations, and there is a need for more potent and less toxic molecules or new drug combinations.1,2,3

Chemoprophylaxis is being used increasingly to prevent a growing number of opportunistic infections that occur in immunocompromised patients. Multiple opportunistic pathogen prophylaxis (MOPP) may result in reduced toxicity, fewer drug interactions and better patient compliance. Consequently, several studies have been focused on the antimicrobial spectrum of clinically used drugs.1,4

Co-trimoxazole is the most widely used prophylactic and therapeutic combination against P. carinii pneumonia. Drugs which inhibit folic acid synthesis have long been a mainstay in the treatment of P. carinii related infections. The dihydrofolate reductase inhibitors (DHFRs) such as pyrimethamine and trimethoprim, only have a static effect on parasite growth and frequently cause adverse effects in AIDS patients. The antifolate combination of DHFR and sulphonamide or sulphone acts synergically against P. carinii. Recent investigations have demonstrated that sulphamethoxazole accounts almost entirely for the anti-Pneumocystis activity of co-trimoxazole.5,6

A number of other single drugs and combinations of drugs are being tested in vitro and in vivo. Rifabutin has been shown to be clinically or experimentally effective against Mycobacterium spp. and Toxoplasma gondii, and it is being used extensively for prophylaxis against Mycobacterium avium complex (MAC) infections in immunocompromised HIV-positive patients.3,7 Albendazole, an agent with an important role in the treatment of cestode infections such as echinococcosis and cysticercosis, is now being used in human AIDS patients for treatment of intestinal microsporidiosis with varying results.8,9 The possible use of rifabutin and albendazole for other opportunistic infections deserves further study, although recent investigations provide evidence that both rifabutin and albendazole exhibited in-vivo activity against P. carinii in several rat models.3,10

Several studies have been performed to investigate synergy between various drugs. Recent reports provide evidence that other combinations besides co-trimoxazole have increased activity against P. carinii in vitro and in the rat model: macrolides and sulphamethoxazole, clindamycin and primaquine, atovaquone and DHFRs or rifabutin.11,12,13

In the present study we investigated the in-vitro activity of rifabutin and albendazole singly and in combination with other clinically used antimicrobial agents against P. carinii and compared their activity with that of co-trimoxazole.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Parasite preparation and cell culture

These procedures have been described in detail previously.6Briefly four clinical isolates of P. carinii were obtained from bronchoalveolar lavages of four distinct immunocompromised patients who had not received prior anti-P. carinii therapy. Specimens were homogenized in an equal volume of phosphate-buffered saline. The homogenate was centrifuged (1800g for 15 min) and the pellet was resuspended in physiological saline. Contaminating bacteria were partially eliminated by four washes in 10 mL of physiological saline followed by incubation in phosphate-buffered saline containing ampicillin (2000 mg/L) and streptomycin (2000 mg/L), for 4 h at 37°C. Organisms were pelleted by centrifugation (1800g for 20 min), and resuspended in Dulbecco's modified Eagle's medium (DMEM) (Bio-Whittaker, Walkersville, MD, USA), counted by methenamine silver stain to detect cysts and with Giemsa stain for counting of P. carinii nuclei prior to use for culture. The final inoculum was 103–104 P. carinii organisms/mL.

The human lung epithelial cell line A-549 was maintained in 96-well tissue culture plates. Culture was initiated by adding 0.1 mL of inoculum to an adherent layer of 50–70% confluent A-549 cells. After incubation for 4 h at 37°C in 5% CO2 to allow attachment and penetration of sporozoites, the monolayers were washed with DMEM to remove non-invasive sporozoites, residual cysts and nonadherent epithelial cells, and 0.2 mL of new growth medium with or without antimicrobial agents was added. Infected cell cultures were kept at 37°C in 5% CO2 throughout the study.

Drugs

All compounds were purchased commercially (Sigma-Aldrich, Milan, Italy), with the exception of clarithromycin (Abbott, Rome, Italy) and rifabutin (Pharmacia & Upjohn, Milan, Italy). Trimethoprim, pyrimethamine, clarithromycin and rifabutin were dissolved in methanol/acetone (1/1) at a concentration of 1 mg/mL. Sulphamethoxazole, albendazole and etoposide were dissolved in dimethylsulphoxide at 1 mg/mL. Minocycline was dissolved in distilled water at a concentration of 1 mg/mL. Solutions of drugs were made fresh on the day of assay or stored at –80°C in the dark for short periods.

Susceptibility testing

Serial dilutions of each drug were prepared in DMEM. All drugs were tested at concentrations close to those that could be achieved clinically. Albendazole and rifabutin were tested at concentrations of 0.5, 1, 2 and 4 mg/L. Clarithromycin, minocycline and etoposide were each tested at concentrations of 1, 2 and 4 mg/L. Pyrimethamine was tested at concentrations of 0.1, 0.2 and 0.4 mg/L. Co-trimoxazole used as a reference drug combination was tested at concentrations of 0.8, 1.6 and 3.2 mg/L, and 4, 8 and 16 mg/L of the two components, respectively. Preliminary experiments indicated that the final concentrations of methanol, acetone and dimethylsulphoxide used in the dilution of drugs did not inhibit the growth of P. carinii. In experiments to test drug interactions, the four above-mentioned concentrations of albendazole and rifabutin were added to the three concentrations of each other agent. Antibiotic-free plates were used as controls in the study. Experiments were performed in triplicate.

P. carinii were added at a concentration of 102–103 organisms/well. The monolayers were incubated at 37°C in 5% CO2 atmosphere. After 72 h the supernatant was withdrawn from each triplicate well and P. carinii trophozoites and cysts counted following Giemsa and methenamine silver staining. Viability assays were performed in conjunction with microscopic enumeration of the organisms, by using the dual-fluorescence staining method.14

Analysis of results

The activity of each agent and combination was evaluated by parasite count from plates with antimicrobial-supplemented medium compared with parasite count from control plates without drugs. The average number of P. carinii parasites/mL was calculated by counting 50 oil immersion fields (x1000 magnification) of each of three slides.

The 50% and 90% inhibitory concentrations (IC50 and IC90, respectively) of each drug were defined as the concentrations required to produce a 50% and 90% reduction in the mean cyst or trophozoite counts, respectively, compared with controls without drug after 72 h incubation in the presence of drugs.

The activity of each compound was also expressed by calculating the ratio of the cyst and trophozoite numbers in cultures containing albendazole and rifabutin at a concentration of 4 mg/L, singly or in combination with other agents, to the cyst and trophozoite number in control cultures after 72 h incubation.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In control plates without drugs, at least a three-fold increase in the number of P. carinii nuclei occurred, with peak numbers reached on day 3: for this reason, the drug activities in plates with antibiotic-supplemented medium were evaluated on the basis of the parasite counts performed after 72 h incubation. Our data showed the presence of a slight reduction in the number of trophozoites and cysts after day 5 and a rapid decrease after day 10. The average number of parasites grown in the absence of antibiotic was 44.7 (range 19–61) when calculated by counting 50 oil immersion fields.

Albendazole and rifabutin had different effects (Table I). Albendazole tested alone was shown to be more effective than rifabutin, with a higher activity against trophozoites than cysts. Albendazole suppressed the growth of cysts and trophozoites by >50% at 4 mg/L (IC50 4 mg/L). Rifabutin, at the same concentration, showed less potency against either cysts or trophozoites producing about 40% reduction in the mean cyst and nucleus counts (IC50 >4 mg/L). Neither albendazole nor rifabutin achieved a 90% inhibitory effect (IC90 >4 mg/L).


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Table I. Anti-Pneumocystis spp. activity of albendazole and rifabutin alone or in combination with other antimicrobial agents
 
The results of combination studies are shown in Tables II and III. It is important to note that the agents used in combination were superior to single-agent activity in reducing the cyst and trophozoite counts. Albendazole (4 mg/L) combined with etoposide (4 mg/L) showed the highest anti-P. carinii activity, with a decrease of 86.3% and 90.1% in cyst and trophozoite counts, respectively. When rifabutin (4 mg/L) was combined with etoposide and clarithromycin, it produced a decrease of 72.2% and 75.6% in cyst counts and a decrease of 76.9% and 79.9% in trophozoite counts, respectively. Of particular interest was the combination of rifabutin with clarithromycin. The most clearly positive synergic response was detected when rifabutin (4 mg/L) was combined with clarithromycin (4 mg/L).


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Table II. Inhibitory effects of albendazole in combination with other agents: per cent reduction of no. of parasites versus control plates without drugs
 

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Table III. Inhibitory effects of rifabutin in combination with other agents: per cent reduction of no. of parasites versus control plates without drugs
 
Co-trimoxazole suppressed the growth of cysts and trophozoites by >50% at concentrations of 0.8/4 mg/L and 1.6/8 mg/L, respectively, and by >90% at concentrations of 3.2/16 mg/L and 1.6/8 mg/L, respectively (Table I).

The evaluation of the ratios of the peak organism counts in drug-treated cultures to the peak organism counts in control cultures, showed that rifabutin at a concentration of 4 mg/L showed the lowest activity against either cysts or trophozoites (peak ratio, 0.60 and 0.58, respectively). Rifabutin (4 mg/L) combined with clarithromycin (4 mg/L) and etoposide (4 mg/L) showed an in-vitro anti-P.carinii activity (cyst peak ratio, 0.24 and 0.27, and trophozoite peak ratio, 0.20 and 0.23, respectively) slightly lower than co-trimoxazole (cyst peak ratio, 0.08; trophozoite peak ratio, 0.06). Albendazole tested alone at a concentration of 4 mg/L also showed weak activity (cyst peak ratio, 0.43; trophozoite peak ratio, 0.40). Albendazole (4 mg/L) upon combination with etoposide (4 mg/L) exhibited an in-vitro anti-P. carinii activity (cyst peak ratio, 0.14; trophozoite peak ratio, 0.10), similar to co-trimoxazole. These results are summarized in Table I.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In immunocompromised individuals MOPP is based on the concept of the advantages of the use of agents with activity against a variety of opportunistic pathogens. Despite advances in chemoprophylaxis, no comprehensive strategy for the prevention of opportunistic infections in immunocompromised subjects, such as HIV-infected patients, has been developed.15 In this study, we have explored the feasibility of combining clinically used antimicrobial agents at concentrations achieved in serum during therapy, for the control of the in-vitro replication of P. carinii. The parasite growth was assessed by quantifying cysts and trophozoites in the supernatant after 72 h incubation at 37°C in 5% CO2. Preliminary experiments indicated that P. carinii grows in clusters, which can be seen both in the supernatant and attached to the cell monolayer.6 Currently, a continuous in-vitro culture system for P. carinii remains elusive and a generally accepted method for in-vitro screening has not been established. Nevertheless, in the present study, control plates and tests were included in each run of the experiments to evaluate the presence of toxic effect on the cell monolayer and the viability of the organism. As a control drug we used the combination co-trimoxazole, which has proved to be extremely effective in treating P. carinii pneumonia. Currently, resistance of P. carinii to co-trimoxazole does not represent a clinical problem. However, the finding of isolates with recently acquired point mutations in the gene that codes for the enzyme dihydropteroate synthetase, a key enzyme target of co-trimoxazole, may indicate that susceptibility to this drug will not continue indefinitely.4,16

Albendazole, a benzimidazole developed more than 20 years ago, is a compound that disrupts microtubule function by blocking polymerization of the {alpha}- and ß-tubulin subunits.10 It is thought to act by binding to parasite tubulins, inhibiting their polymerization and impairing glucose uptake.17 It has proved useful in treating echinococcosis, cysticercosis and strongyloidosis. Albendazole has also been shown to be effective in the treatment of infections caused by the protozoans Enterocytozoon bieneusi, Septata intestinalis and Giardia intestinalis. In addition, single doses of albendazole have been shown to achieve cure rates of >90% against Enterobius vermicularis, Ascaris lumbricoides, hookworm and cutaneous larva migrans.17 Recently, it was evaluated for its effect on P. carinii in vitro and in vivo and was found to be inhibitory.10,18 In our study, criteria for synergy were not defined and we did not calculate values such as the fractional inhibitory concentration index; thus, our findings indicate only the presence of additive anti-P. carinii activity when albendazole was combined with etoposide, clarithromycin, minocycline or pyrimethamine. The different biochemical pathways can explain the lack of synergy between albendazole and the other antimicrobial agents.

Rifabutin is a spiro-piperidyl-rifamycin derived from rifamycin-S. It is structurally related to and shares many properties of rifampin.19 Rifabutin has a potential role to play in a MOPP regimen because it is active against mycobacteria, Gram-positive cocci, Haemophilus influenzae, Legionella pneumophila, Helicobacter pylori, Campylobacter jejuni, Chlamydia trachomatis and Toxoplasma gondii.7 The antimicrobial activity of the rifamycins is produced by inhibition of DNA-dependent RNA polymerase and subsequent initiation of transcription. This results in inhibition of protein synthesis.19 It is currently approved in the United States, Canada and many other countries for use as prophylaxis for Mycobacterium avium- complex infection in HIV-infected patients with low CD4 cell counts. Rifabutin was shown to be highly protective against development of cryptosporidiosis in immunosuppressed HIV-infected patients.20 Recently, it was found to be highly effective in a mouse model of toxoplasmosis.21 When administered alone, rifabutin showed weak efficacy in protecting immunosuppressed rats and mice against P. carinii related pneumonia.3,13,22 In our experiments rifabutin was found to be moderately active against P. carinii, but its activity was highly enhanced upon combination with clarithromycin. Taken together, these data are in agreement with previous in-vivo investigations, in which rifabutin showed synergic activity with other clinically used agents, although these combinations were not as effective as co-trimoxazole.3 The mechanism of this synergic effect between rifabutin and clarithromycin appears to be complex. Several anti-mycobacterial in-vitro studies have shown that the effects of this combination are highly variable and dependent on the strain tested.19,23 Interestingly, studies of pharmacokinetic interaction of clarithromycin and rifabutin showed that the macrolide, by inhibiting the cytochrome P-450 pathway, reduces the metabolism of rifabutin, thus resulting in increased serum and tissue concentrations.24

Finally, in our study only the combination of co-trimoxazole suppressed the growth of cysts and trophozoites by >90%. Neither albendazole nor rifabutin, singly or in combination with other agents, achieved a 90% inhibitory effect, with the exception of albendazole (4 mg/L) associated with etoposide (4 mg/L) which produced a 90.1% reduction in trophozoite counts.

Patients with advanced HIV infection have overlapping, superimposed risks for developing protozoal, fungal, viral, bacterial and mycobacterial infections.7 The concept of MOPP is based on the premise that the parsimonious use of agents that protect against several classes of opportunistic pathogens that cause infections, will result in better efficacy, enhanced patient compliance, reduced toxicity, fewer drug interactions and lower cost. Our data showed a good anti-Pneumocystis activity of albendazole and rifabutin, used alone or in combination. However, despite this fact and the attractiveness of the concept of MOPP, there are still some significant obstacles to its implementation. The spectrum of opportunistic pathogens in immunocompromised patients is wide, and the variety of mechanisms responsible for their inhibition may necessitate the use of an array of antimicrobial agents with different modes of antimicrobial activity. In addition multidrug prophylaxis may be associated with risks of synergic or complementary toxicity and drug interaction. Clinical trials have been planned but have proved to be complex. The results of such trials would probably not be available for many years. Nevertheless albendazole and rifabutin deserve serious consideration, since these drugs, used alone or in combination, are active against many pathogens that infect individuals with HIV disease.


    Notes
 
* Correspondence address. Clinica Malattie Infettive, c/o Azienda Ospedaliera Umberto I, Piazza Cappelli, 1, I-60121 Ancona, Italy. Tel: +39-71-596-3467; Fax: +39-71-596-3468; E-mail: cmalinf{at}popcsi.unian.it Back


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 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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2 . Cushion, M. T., Chen, F. & Kloepfer, N. (1997). A cytotoxicity assay for evaluation of candidate anti-Pneumocystis carinii agents. Antimicrobial Agents and Chemotherapy 41, 379–84.[Abstract]

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9 . Dieterich, D. T., Lew, E. A., Kotler, D. P., Poles, M. A. & Orenstein, J. M. (1994). Treatment with albendazole for intestinal disease due to Enterocytozoon bieneusi in patients with AIDS. Journal of Infectious Diseases 169, 178–83.[ISI][Medline]

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Received 21 October 1998; returned 13 April 1999; revised 28 April 1999; accepted 29 July 1999





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