Prediction of quinolone activity against Mycobacterium avium by molecular topology and virtual computational screening.

We conducted a quantitative structure-activity relationship study using a database of 158 quinolones previously tested against Mycobacterium avium-M. intracellulare complex in order to develop a model capable of predicting the activity of new quinolones against the M. avium-M. intracellulare complex in vitro. Topological indices were used as structural descriptors and were related to anti-M. avium-M. intracellulare complex activity by using the linear discriminant analysis (LDA) statistical technique. The discriminant equation thus obtained correctly classified 137 of the 158 quinolones, including 37 of a test group of 44 randomly chosen compounds. This model was then applied to 24 quinolones, including recently developed fluoroquinolones, whose MICs were subsequently determined in vitro by using the Alamar blue microplate assay; the biological results confirmed the model's predictions. The MICs of these 24 quinolones were then treated by multilinear regression (MLR) to establish a model capable of classifying them according to their in vitro activities. Using this model, a good correlation between measured and predicted MICs was found (r(2) = 0.88; r(2)(cv) [cross-validation correlation] = 0.82). Moxifloxacin, sparfloxacin, and gatifloxacin were the most potent against the M. avium- M. intracellulare complex, with MICs of 0.2, 0.4, and 0.9 microg/ml, respectively. Finally, virtual modifications of these three drugs were evaluated in LDA and MLR models in order to determine the importance of different substituents in their activity. We conclude that the combination of molecular-topology methods, LDA, and MLR provides an excellent tool for the design of new quinolone structures with enhanced activity.

Anti-toxoplasma activities of 24 quinolones and fluoroquinolones in vitro: prediction of activity by molecular topology and virtual computational techniques.

The apicoplast, a plastid-like organelle of Toxoplasma gondii, is thought to be a unique drug target for quinolones. In this study, we assessed the in vitro activity of quinolones against T. gondii and developed new quantitative structure-activity relationship models able to predict this activity. The anti-Toxoplasma activities of 24 quinolones were examined by means of linear discriminant analysis (LDA) using topological indices as structural descriptors. In parallel, in vitro 50% inhibitory concentrations (IC(50)s) were determined in tissue culture. A multilinear regression (MLR) analysis was then performed to establish a model capable of classifying quinolones by in vitro activity. LDA and MLR analysis were applied to virtual structures to identify the influence of each atom or substituent of the quinolone ring on anti-Toxoplasma activity. LDA predicted that 20 of the 24 quinolones would be active against T. gondii. This was confirmed in vitro for most of the quinolones. Trovafloxacin, grepafloxacin, gatifloxacin, and moxifloxacin were the quinolones most potent against T. gondii, with IC(50)s of 0.4, 2.4, 4.1, and 5.1 mg/liter, respectively. Using MLR analysis, a good correlation was found between measured and predicted IC(50)s (r(2) = 0.87, cross-validation r(2) = 0.74). MLR analysis showed that the carboxylic group at position C-3 of the quinolone ring was not essential for anti-Toxoplasma activity. In contrast, activity was totally dependent on the presence of a fluorine at position C-6 and was enhanced by the presence of a methyl group at C-5 or an azabicyclohexane at C-7. A nucleophilic substituent at C-8 was essential for the activity of gatifloxacin and moxifloxacin.

Animal models of pneumocystosis.

As in vitro culture systems allowing to isolate Pneumocystis samples from patients or other mammal hosts are still not available, animal models have critical importance in Pneumocystis research. The parasite was reported in numerous mammals but P. carinii pneumonia (PCP) experimental models were essentially developed by using rats, mice, rabbits and ferrets. The rat treated with corticosteroids for 9-12 weeks is a useful PCP model. Like laboratory rats, conventional mice develop PCP after prolonged corticosteroid administration. The ferret (Mustela putorius furo) also develop PCP under corticosteroid regime. Whilst bronchoalveolar lavage (BAL) is really difficult to perform on live laboratory rodents, serial BAL sampling can be performed on live ferrets. Rabbits currently develop spontaneous PCP at weaning without corticosteroid administration. For this reason this model has been used for studying the host immune response as well as Pneumocystis-surfactant interactions. Pigs and horses also develop spontaneous PCP. Treated with corticosteroids, piglets develop extensive PCP and could be used as a non-rodent model. Pneumocystis was detected in many non-human primates. Primates could represent a source of parasites taxonomically related to P. carinii sp. f. hominis. Moreover, primates might be used as experimental hosts to human Pneumocystis. A marked variability of parasite levels among corticosteroid-treated animals and the fact that the origin of the parasite strain remains unknown, are important drawbacks of the corticosteroid-treated models. For these reasons, inoculated animal models of PCP were developed. The intratracheal inoculation of lung homogenates containing viable parasites in corticosteroid-treated non-latently infected rats resulted in extensive, reproducible Pneumocystis infections. Extensive PCP can be obtained within 5-7 weeks, whilst 9-12 weeks are needed in the classical model. The severe combined immunodeficiency (SCID) mouse inoculated by nasal route and the athymic nude rats intratracheally inoculated were used to test the infectivity of Pneumocystis samples coming from cultures or from different hosts. They were also used to test the anti-Pneumocystis activity of antimicrobial molecules.

Evaluation of drug efficacy by using animal models or in vitro systems.

The efficacy of most therapeutic and prophylactic protocols against Pneumocystis carinii pneumonia used in human patients has been tested in animal models, especially in the corticosteroid-treated rat. The advantages and drawbacks of this model have been examined in brief in Chapter 1 of this section. More recently, the nude rat, intratracheally inoculated with Pneumocystis, was used to test new anti-microbian molecules for their anti-Pneumocystis activity. In vitro systems, co-cultures of Pneumocystis with feeder cells as well as axenic cultures, were also used many times for drug screening. In this paper, the most used in vivo or in vitro drug screening systems are described. Moreover, as immunocompromised individuals, AIDS patients, especially, are often infected simultaneously by several infectious agents, a recent co-infection model is described.

Drug evaluation of concurrent Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium complex infections in a rat model.

We present a new experimental model for the simultaneous evaluation of the activities of drugs against Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium complex infections. Rats latently infected with P. carinii were challenged with the MO-1 strain of M. avium and then immunosuppressed with corticosteroids for 7 weeks. At week 5 the RH strain of T. gondii was intraperitoneally injected. Organs were examined for the three pathogens after death or killing of the animals at week 7. Without treatment, rats challenged with T. gondii died with pulmonary P. carinii infection and disseminated T. gondii and M. avium infections. In order to assess the value of the model for evaluation of the activities of drugs, we administered by oral gavage for 7 weeks drugs or combinations of drugs selected for their individual efficacies against at least one pathogen. We found that clarithromycin with sulfamethoxazole, clarithromycin with atovaquone, roxithromycin with sulfamethoxazole or dapsone, and rifabutin with atovaquone were effective against the three infections, whereas PS-15 with dapsone and trimethoprim with sulfamethoxazole were active against Toxoplasma and Pneumocystis infections only. This triple-infection rat model offers a new tool for the simultaneous evaluation of the activities of drugs against three of the major opportunistic infections occurring in immunosuppressed individuals.

Experimental evaluation of roxithromycin combined with dapsone or sulphamethoxazole on Pneumocystis carinii and Toxoplasma gondii dual infections in a rat model.

We have developed a dual infection model in immunosuppressed rats for evaluating drugs against Pneumocystis carinii and Toxoplasma gondii, two important opportunistic pathogens in patients with AIDS. Using this model, we reported that the macrolide roxithromycin was effective at a daily dose of 400 mg/kg in preventing the development of T. gondii infection but did not have a prophylactic effect against P. carinii in the same rats. A lower dose (200 mg/kg/day) had only marginal effects. Extending these experiments, we have now shown that roxithromycin at doses of 400 or 200 mg/kg/day combined with dapsone at doses of 5, 25 or 50 mg/kg/day completely prevented the development of T. gondii infection, with no parasites being detected in any of the tissues sampled. Roxithromycin at either dose combined with dapsone at 25 or 50 mg/kg/day was also effective in preventing the development of P. carinii infection in the lungs. The lowest dose of dapsone (5 mg/kg/day) was not fully effective. Pyrimethamine-dapsone, a combination used clinically, was tested in the same experiment, and gave results comparable to those with roxithromycin-dapsone combinations. In a further experiment combining roxithromycin with sulphamethoxazole, roxithromycin was effective in preventing the T. gondii infection, even when given at only 200 mg/kg/day with 20 mg/kg/day of sulphamethoxazole. When the dose of sulphamethoxazole was reduced to 2 mg/kg/day and given with roxithromycin 200 mg/kg/day, T. gondii infection developed in two of the five rats treated. P. carinii infection was prevented by sulphamethoxazole at 20 mg/kg/day but not completely by 2 mg/kg/day. Roxithromycin also has activity against Mycobacterium avium, another important cause of opportunistic infections in AIDS patients, and the compound penetrates mammalian cells well. Taken together with the favourable pharmacokinetic profile of roxithromycin, these results suggest that it may have a clinical utility, when used with other agents, in controlling the development of opportunistic infections caused by M. avium complex, T. gondii and P. carinii in HIV-infected individuals.

Altered trimethoprim-sulfamethoxazole ratios for prophylaxis and treatment of Toxoplasma gondii and Pneumocystis carinii dual infections in rat model.

Trimethoprim-sulfamethoxazole (TMP-SMX) is the first-line drug for prophylaxis and treatment of Pneumocystis carinii pneumonia (PCP) and for prophylaxis of toxoplasmosis in HIV-infected patients. Evidence indicating intolerance related to the dose of SMX led us to examine the efficacy of altered TMP-SMX ratios in a corticosteroid-treated rat model. Infections were assessed by counting P. carinii cysts in lung and by titration of Toxoplasma gondii burdens in tissue culture. For prophylaxis, the reference regimen of 20 mg/kg TMP plus 100 mg/kg SMX was effective. Reduced doses of SMX (5 and 20 mg/kg) effective against PCP were effective against toxoplasmosis, provided the TMP dose was increased to 100 mg/kg. For curative treatment, the reversed ratio of 100 mg/kg TMP plus 20 mg/kg SMX was not effective. These results may provide a basis for altering the TMP-SMX ratios in setting of prophylaxis for both infections, especially in HIV infected patients who often poorly tolerate sulfonamides.

Combination of PS-15, epiroprim, or pyrimethamine with dapsone in prophylaxis of Toxoplasma gondii and Pneumocystis carinii dual infection in a rat model.

In a rat model of dual infection, we studied such dihydrofolate reductase (DHFR) inhibitors as PS-15 (25 mg/kg of body weight), epiroprim (100 mg/kg), and pyrimethamine (3 mg/kg) alone or in combination with various doses of dapsone (50, 25, or 5 mg/kg) for the prevention of pneumocystosis and toxoplasmosis. Rats latently infected with Pneumocystis carinii were immunosuppressed by corticosteroids for 7 weeks, and the drugs were administered from the initiation of the corticosteroid treatment. At week 5, the rats were inoculated intraperitoneally with the RH strain of Toxoplasma gondii. Infections were monitored by the counting of P. carinii cysts in lung homogenates and the titration of T. gondii in organs by quantitative culture and an indirect immunofluorescence assay. Fourteen of the 15 untreated rats died after T. gondii challenge, with P. carinii infection in the lungs and T. gondii infection in the lungs, liver, spleen, and brain. Of the three tested DHFR inhibitors, only PS-15 exhibited anti-P. carinii activity; none prevented toxoplasmosis in 100% of the rats. After the DHFR inhibitors were combined with dapsone (50 or 25 mg/kg), both pneumocystosis and toxoplasmosis were completely prevented. On the basis of these results, PS-15 and epiroprim combined with dapsone are candidates for use for the prevention of both pneumocystosis and toxoplasmosis.

Influence of Pneumocystis carinii on nitrite production by rat alveolar macrophages.

Nitrite production by rat alveolar macrophages was studied to determine the role of L-arginine oxidation in the interaction between these cells and Pneumocystis carinii. Alveolar macrophages from rats obtained from two different breeders were used: rats from Janvier breeder had latent P. carinii infection, while those from Charles River breeder were bred in a germ-free environment. Pneumocystis carinii increased in vitro nitrite generation by unstimulated alveolar macrophages from Janvier rats only, and this was blocked by NG-monomethyl-L-arginine. Incubation of cells from Janvier and Charles River rats with lipopolysaccharide and/or interferon-gamma increased nitrite production to a similar extent. Pneumocystis carinii partially decreased nitrite release by activated alveolar macrophages, and this was still inhibited by NG-monomethyl-L-arginine. In the presence of P. carinii, superoxide dismutase used as a superoxide anion scavenger had no effect on nitrite production by activated cells. These results show that prior exposure to P. carinii leads to nitric oxide production by rat alveolar macrophages. Although the magnitude of this production seems to be moderate, it is of biological significance since cells of P. carinii-naive rats do not generate nitrite whereas those of latently infected rats do.

Lack of activity of artemether for prophylaxis and treatment of Toxoplasma gondii and Pneumocystis carinii infections in rat.

Artemisinin and its derivatives have been found effective in vivo against Plasmodium and in vitro against Toxoplasma gondii and Pneumocystis carinii. We tested the activity of artemether for prophylaxis and treatment in the rat model of concurrent T. gondii and P. carinii infection. Artemether at doses of 18 and 100 mg/kg administered (s.c.) in prophylaxis did not prevent toxoplasmosis or pneumocystosis, while trimethoprimsulfamethoxazole (reference treatment) was effective for prevention of both infections. Similar results were obtained in curative studies. These results do not support the use of artemether for prevention or treatment of toxoplasmosis or pneumocystosis.

Effects of dietary gamma-linolenic acid on the tissue phospholipid fatty acid composition and the synthesis of eicosanoids in rats.

The purpose of this study was to evaluate the effects of different doses of dietary gamma-linolenic acid (GLA) on the tissue phospholipid fatty acid composition and the synthesis of eicosanoids in growing rats. The supplementation with different oils rich in GLA (borage oil, evening primrose oil, or Spirulina oil) and poor in n-3 polyunsaturated fatty acids or biomass of Spirulina results in a significant dose-related increase of GLA and dihomo-GLA in liver, erythrocyte, and aorta phospholipids in rats fed during 6 weeks different levels of GLA. The arachidonic acid (AA)/dihomo-GLA ratios decreased with increasing intake of dietary GLA, but the AA proportions remained stable. The dietary administration of GLA increased the in vitro production by the aorta of prostaglandin E1 derived from dihomo-GLA, but did not significantly influence the production of prostaglandin E2 derived from AA by the aorta and the thromboxane B2 level in serum.

Polymorphism of the thymidylate synthase gene of Pneumocystis carinii from different host species.

Pneumocystis carinii is an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity of P. carinii. These additions provide new understanding of Pneumocystis transmission and the effect of possible reservoirs of Pneumocystis in the various species. In this study, a 400-bp fragment of the thymidylate synthase (TS) gene of P. carinii has been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat-derived P. carinii was hybridized with the amplified products from rat- and mouse-derived P. carinii, but not with rabbit or human P. carinii DNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplified from rat, mouse, rabbit and human P. carinii were determined. Differences were detected in the gene fragment in P. carinii isolates from the 4 host species; however no difference was revealed in P. carinii isolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of the P. carinii TS gene appeared as host-species specific. A specific probe which recognized all human P. carinii isolates was defined.

Experimental evaluation of combined prophylaxis against murine pneumocystosis and toxoplasmosis.

Prophylactic efficacy of antimicrobial agents against pneumocystosis and toxoplasmosis was examined in a model of concurrent Pneumocystis carinii and Toxoplasma gondii infections in rats. Corticosteroid-treated rats naturally infected by P. carinii were challenged with the RH strain of T. gondii. Infection was assessed by counting P. carinii cysts in lung and by titration of T. gondii in tissues by tissue culture. Untreated rats died after challenge, with P. carinii infection in lungs and T. gondii infection in liver, spleen, lungs, and brain. In rats that received trimethoprim-sulfamethoxazole or pyrimethamine plus dapsone, T. gondii was eradicated and P. carinii pneumonia prevented. Roxithromycin, 200 or 400 mg/kg, provided significant protection against toxoplasmosis but had no efficacy against P. carinii. Atovaquone, 100 or 200 mg/kg, had only partial efficacy against pneumocystosis and toxoplasmosis. These results definitively confirm use of trimethoprim-sulfamethoxazole and pyrimethamine plus dapsone for prophylaxis against combined infection in immunocompromised hosts.

Modulation of rat and human lymphocyte function by n-6 and n-3 polyunsaturated fatty acids and acetylsalicylic acid.

The effects of in vitro additions of between 10 and 100 microM n-6 and n-3 polyunsaturated fatty acids were examined on the proliferation of stimulated T lymphocytes in culture. For both phytohemagglutinin-induced human blood lymphocytes and concanavalin-A-induced rat splenic lymphocytes, the largest inhibitory effects were obtained with 22:4 n-6 and 22:6 n-3, and to a lesser extent with 20:5 n-3. Arachidonic acid 20:4 n-6, the main eicosanoid precursor, was not inhibitory, it even stimulated rat lymphocyte proliferation. Acetylsalicylic acid stimulated both human and rat lymphocyte proliferation. The effects of moderate decreases in the dietary n-6/n-3 ratio by either linseed oil or fish oil maximum eicosapentaenoic acid (MaxEPA) were determined on rat lymphocyte proliferation and natural killer (NK) cell activity in vitro. Dietary changes did not affect mitogen-induced lymphocyte proliferation in vitro, but proliferation of unstimulated lymphocytes was significantly lowered (4-fold) with the n-3-enriched diets. Dietary fish oil but not linseed oil significantly increased the NK cell activity of rat splenic lymphocytes. The n-3-enriched diets, especially the fish oil diets, reduced the stimulatory effect of in vitro added acetylsalicylic acid (aspirin) on lymphocyte proliferation.

Low-dose trimethoprim-sulfamethoxazole alone and in association with zidovudine for prevention and treatment of murine Pneumocystis carinii pneumonia.

Low-dose trimethoprim-sulfamethoxazole (TMP-SMX) alone was found to be as effective as low-dose TMP-SMX plus zidovudine and standard-dose TMP-SMX alone in preventing and treating Pneumocystis carinii pneumonia (PCP) in an immunosuppressed-rat model. Zidovudine alone had no preventive or curative effect on PCP. We conclude that the initially reported reduced incidence of PCP in human immunodeficiency virus-infected patients treated with zidovudine alone is not due to anti-P. carinii activity of zidovudine. Furthermore, the clinical efficacy of low-dose TMP-SMX for the prevention and treatment of PCP should be further investigated.

Use of fluoroquinolones for prophylaxis of murine Pneumocystis carinii pneumonia.

We compared the prophylactic activities of six fluoroquinolones against Pneumocystis carinii pneumonia in immunosuppressed rats. Pefloxacin was the only agent which was as effective as the reference drug trimethoprim-sulfamethoxazole. Clinical trials with pefloxacin in patients at risk for P. carinii pneumonia appear to be justified.

Hydroxymethylthiobutanoic acid spares methionine from catabolism in the chick.

Two groups of chicks were fed, until they were 3 weeks old, diets supplemented with either DL-methionine (MET) or DL-2-hydroxy-4-methylthiobutanoic acid (HMB). Oxidation to CO2 and uric catabolism of intraperitoneally injected L-[1-14C]MET and DL-[1-14C]HMB were studied comparatively for 8 h. HMB supplementation spares available MET from both oxidative and uric catabolism.