Anti-malarial effect of a combination of risedronate and azithromycin against Plasmodium yoelii nigeriensis infection in Swiss mice.

Combination therapy is used to retard the selection of malaria parasite strains resistant to individual components of a combination of drugs. This approach has proved to be a success in the combination of sulphadoxine and pyrimethamine, which targets two different steps in the folate pathway of malaria parasites. However, after the success of this therapeutic combination, the efficacy of other combinations of drugs that target different enzymes in a particular metabolic pathway has, apparently, not been reported. In the current study, the antimalarial effect of a combination of risedronate (RIS), which is known for its anti-osteoporosis activity, and azithromycin (AZT) was investigated. Peter's suppression test was carried out on mice infected with 1 × 107P. yoelii infected erythrocytes. Drug efficacy was analyzed by comparing the percent reduction in parasitaemia on day 4 post-infection. RIS was observed to be a blood schizonticidal agent against P. yoelii infection which showed ED50 7.0 (4.04-12.13) mg/kg/day x 4. Normalized isobologram showed additive action between RIS 1 mg/kg/day x 4 and AZT 10 mg/kg/day x 4, and antagonistic action for the rest of the combinations (RIS 1 + AZT 20, RIS 1 + AZT 40, RIS 5 + AZT 10, RIS 5 + AZT 20, RIS 5 + AZT 40, RIS 10 + AZT 10, RIS 10 + AZT 20 and RIS 10 + AZT 40 mg/kg/day x 4). Furthermore, a combination of RIS with AZT showed inferior efficacy as compared to AZT treatment alone. This antagonistic interaction may be due to the high accumulation of AZT in WBCs, which will reduce its serum bio-availability, whereas RIS has anti-parasitic activity by increasing WBCs.

Synthesis and Biological Evaluation of 8-Quinolinamines and Their Amino Acid Conjugates as Broad-Spectrum Anti-infectives.

In the search of therapeutic agents for emerging drug-resistant parasites, the synthesis of newer classes of 8-quinolinamines has emerged as a successful chemotherapeutic approach. We report synthesis of 8-quinolinamines bearing 5-alkoxy, 4-methyl, and 2-tert-butyl groups in the quinoline framework and their amino acid conjugates as broad-spectrum anti-infectives. 8-Quinolinamines exhibited potent in vitro antimalarial activity [IC50 = 20-4760 ng/mL (drug-sensitive Plasmodium falciparum D6 strain) and IC50 = 22-4760 ng/mL (drug-resistant P. falciparum W2 strain)]. The most promising analogues have cured all animals at 25 mg/kg/day against drug-sensitive Plasmodium berghei and at 50 mg/kg/day against multidrug-resistant Plasmodium yoelii nigeriensis infections in Swiss mice. The in vitro antileishmanial activities (IC50 = 0.84-5.0 µg/mL and IC90 = 1.95-7.0 µg/mL) comparable to standard drug pentamidine were exhibited by several of the synthesized 8-quinolinamines. At the same time, very promising antifungal activities (Candida albicans-IC50 = 4.93-19.38 µg/mL; Candida glabrata-IC50 = 3.96-19.22 µg/mL; Candida krusei-IC50 = 2.89-18.95 µg/mL; Cryptococcus neoformans-IC50 = 0.67-18.64 µg/mL; and Aspergillus fumigatus-IC50 = 6.0-19.32 µg/mL) and antibacterial activities (Staphylococcus aureus-IC50 = 1.33-18.9 µg/mL; methicillin-resistant S. aureus-IC50 = 1.38-15.34 µg/mL; and Mycobacterium intracellulare-IC50 = 3.12-20 µg/mL) were also observed. None of the 8-quinolinamines exhibited cytotoxicity and therefore are a promising structural class of compounds as antiparasitic and antimicrobials.

Guanylthiourea derivatives as potential antimalarial agents: Synthesis, in vivo and molecular modelling studies.

Guanylthiourea (GTU) derivatives were identified as possible anti-malarial agents, recently, using in vitro studies on Plasmodium falciparum. This article gives an account of the in vivo anti-malarial activity of GTU derivatives against experimental rodent malaria. A total of 20 synthesized GTU derivatives were evaluated for in vivo antimalarial activity, out of which six showed encouraging results; one compound appeared to have curative potential. Molecular docking and molecular dynamics analysis were carried out to understand the molecular level interactions.

Determination of the activity of standard anti-tuberculosis drugs against intramacrophage Mycobacterium tuberculosis, in vitro: MGIT 960 as a viable alternative for BACTEC 460.

BACTEC 460 has now been phased out, so the search for an alternative is imperative. We have determined the activity of standard anti-tuberculosis drugs against intramacrophage Mycobacterium tuberculosis, in vitro, by using BACTEC 460 and MGIT 960 methods. The minimum inhibitory concentrations of isoniazid, rifampicin, ethambutol and streptomycin against intracellular M. tuberculosis H37Rv were found to be 0.2, 0.8, 8.0, and 5.0 µg/mL, respectively, by both methods. These results show a significant (p<0.001) concordance between minimum inhibitory concentrations obtained by these two different methods. MGIT 960 system uses a robust florescence quenching-based oxygen sensor, requires no radioisotope, is safe, and relatively easy to operate. Apparently, this is the first report wherein MGIT 960 has been validated for anti-tubercular susceptibility testing against intracellular M. tuberculosis H37Rv. Our preliminary data thus clearly demonstrate that the MGIT 960 method can be considered as a promising alternative to BACTEC 460 method.

The dichotomy (generation of MAbs with functional heterogeneity) in antimalarial immune response in vaccinated/protected mice: a new concept in our understanding of the protective immune mechanisms in malaria.

Globally, vaccines have emerged as one of the most effective, safe, and cost-effective public health interventions, and are known to save 2-3 million lives, annually. However, despite various commendable efforts, a suitable human malaria vaccine is yet to see the light of the day. The lack of our complete understanding of the molecular mechanisms of pathogenesis and immune protection in malaria appears to be responsible for this state. Earlier, our laboratory has reported that Swiss mice vaccinated with Plasmodium yoelii nigeriensis-total parasite antigens soluble in culture medium and saponin, following a 100% lethal challenge, showed 60% protection. The monoclonal antibodies (MAbs) generated from the splenocytes of these vaccinated/protected mice, following characterization by in vitro merozoite invasion inhibition assay, ex vivo macrophage phagocytosis assay, and in vivo passive transfer of protection test, belonged to 2 distinct groups-a larger group of MAbs inhibited<58% Mz invasion and transferred 30% passive protection, whereas a smaller group of MAbs inhibited 86% Mz invasion and transferred 60% passive protection. Additionally, the MAbs of the smaller group, as compared with the larger one, mediated nearly 2.4-fold enhanced macrophage phagocytosis of infected-erythrocytes, in vitro. These results thus clearly showed a dichotomy among the generated MAbs. An exploration of the phenomenon of dichotomy in protective immunity in malaria by using various hosts and malaria parasite combinations, especially at the level of antibodies, cells, and cytokines, may add new insights to our understanding of the protective immunity, and help in the identification of biomarkers/biosignatures of immune protection and development of future human malaria vaccines.

Interleukin-6: a potent biomarker of mycobacterial infection.

BACKGROUND: Human tuberculosis (TB), a chronic inflammatory disease is caused by Mycobacterium tuberculosis, a facultative intramacrophage pathogen. The highly complex interactions between mycobacteria and macrophages (MΦs), characterized in part by the induction and elaboration of several cytokines including IL-1, IL-6, IL-10, IL-12 p40 and IL-12 p70 are not yet fully understood. The cytokines are known to have important bearing on the pathogenesis and host defense during TB. We thus studied different patterns of cytokines elaborated by mouse peritoneal macrophages (PMs) following their interaction with live and heat-killed, virulent and avirulent, and pathogenic and non-pathogenic mycobacteria, in vitro. MATERIALS AND METHODS: Pathogenic M. tuberculosis H37Rv (virulent) and M. tuberculosis H37Ra (avirulent), and non-pathogenic M. smegmatis were grown in complete Middle Brook 7H9 broth. For some experiments, mycobacteria were heat-killed (80°C; 20 min). The supernatants of cultured PMs, having ingested mycobacteria for 6 h, 24 h, 4 days and 7 days, were harvested for the quantification of IL-1, IL-6, IL-10, IL-12 p40 and IL-12 p70 by using a multiplex suspension cytokine array system. RESULTS: The PMs infected with heat-killed mycobacteria, as compared to their respective live counterparts, invariably elaborated significantly (p < 0.001) increased (approximately 2-3-fold) amounts of IL-6, at all the time-points studied, in vitro. Further, PMs infected with M. tuberculosis H37Ra, as compared to M. tuberculosis H37Rv, elaborated 4-5-fold more (p < 0.001) IL-6. Non-pathogenic M. smegmatis, as compared to pathogenic M. tuberculosis H37Ra and M. tuberculosis H37Rv, following infection, induced the PMs to elaborate highest (p < 0.001) amounts of IL-6 at all the time-points studied. Curiously, none of these mycobacteria-infected PMs elaborated IL-1, IL-10, IL-12 p40 and IL-12 p70, significantly. CONCLUSION: IL-6 appears to be the only major cytokine elaborated by mycobacteria-infected PMs, in vitro, and thus may function as a potent biomarker of mycobacterial infection, either stand-alone or along with other cytokines.

Amino acid, dipeptide and pseudodipeptide conjugates of ring-substituted 8-aminoquinolines: synthesis and evaluation of anti-infective, ß-haematin inhibition and cytotoxic activities.

Three new series of 8-aminoquinolines with modifications in the side-chain by conjugation with amino acids, dipeptides and pseudodipeptides have been synthesized. The synthesized compounds were tested for in vitro antimalarial activity against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains, in vitro cytotoxicity in mammalian kidney cells (Vero), in vitro antileishmanial activity against Leishmania donovani, in vitro antimicrobial activity and in vitro inhibition of ß-haematin formation. The promising compounds were also evaluated for in vivo blood-schizontocidal antimalarial activity against Plasmodium berghei infected mice. The analogues 55 and 101 produced highest antimalarial activities, in vitro. Analogues 52 and 59 exhibited promising antileishmanial and broad spectrum of antifungal activities, respectively.

Comparative drug susceptibility study of five clonal strains of Trichomonas vaginalis in vitro.

OBJECTIVE: To produce comparative data on a group of Trichomonas vaginalis clonal strains with varied drug responses using identical methods and materials. METHODS: Five clonal strains of Trichomonas vaginalis were isolated from reference strain using agar plate technique. The variability of growth kinetic and susceptibility of clonal strain to metronidazole, tinidazole, satranidazole and nitazoxanide were observed in 96 well microtitre plate. RESULTS: Among these clonal strains there was a good correlation between rates of growth with the relative susceptibility of the strains to drugs in vitro. Regarding metronidazole, tinidazole and satranidazole susceptibility, different degrees of susceptibility were determined. However, no difference in nitazoxanide susceptibility was found between the clonal strain tested and a reference strain. CONCLUSIONS: This is the first description of biological variability in clonal stock of Trichomonas vaginalis. Different degrees of drug susceptibility were determined among clonal strains tested. Further studies will be necessary to ascertain the importance of this variability in clinical infection.

Synthesis, antiprotozoal, antimicrobial, ß-hematin inhibition, cytotoxicity and methemoglobin (MetHb) formation activities of bis(8-aminoquinolines).

In continuing our search of potent antimalarials based on 8-aminoquinoline structural framework, three series of novel bis(8-aminoquinolines) using convenient one to four steps synthetic procedures were synthesized. The bisquinolines were evaluated for in vitro antimalarial (Plasmodiumfalciparum), antileishmanial (Leishmaniadonovani), antimicrobial (a panel of pathogenic bacteria and fungi), cytotoxicity, ß-hematin inhibitory and methemoglobin (MetHb) formation activities. Several compounds exhibited superior antimalarial activities compared to parent drug primaquine. Selected compounds (44, 61 and 79) when tested for in vivo blood-schizontocidal antimalarial activity (Plasmodiumberghei) displayed potent blood-schizontocial activities. The bisquinolines showed negligible MetHb formation (0.2-1.2%) underlining their potential in the treatment of glucose-6-phosphate dehydrogenase deficient patients. The bisquinoline analogues (36, 73 and 79) also exhibited promising in vitro antileishmanial activity, and antimicrobial activities (43, 44 and 76) against a panel of pathogenic bacteria and fungi. The results of this study provide evidence that bis(8-aminoquinolines), like their bis(4-aminoquinolines) and artemisinin dimers counterparts, are a promising class of antimalarial agents.

Models of latent tuberculosis: their salient features, limitations, and development.

Latent tuberculosis is a subclinical condition caused by Mycobacterium tuberculosis, which affects about one-third of the population across the world. To abridge the chemotherapy of tuberculosis, it is necessary to have active drugs against latent form of M. tuberculosis. Therefore, it is imperative to devise in vitro and models of latent tuberculosis to explore potential drugs. In vitro models such as hypoxia, nutrient starvation, and multiple stresses are based on adverse conditions encountered by bacilli in granuloma. Bacilli experience oxygen depletion condition in hypoxia model, whereas the nutrient starvation model is based on deprivation of total nutrients from a culture medium. In the multiple stress model dormancy is induced by more than one type of stress. In silico mathematical models have also been developed to predict the interactions of bacilli with the host immune system and to propose structures for potential anti tuberculosis compounds. Besides these in vitro and in silico models, there are a number of in vivo animal models like mouse, guinea pig, rabbit, etc. Although they simulate human latent tuberculosis up to a certain extent but do not truly replicate human infection. All these models have their inherent merits and demerits. However, there is no perfect model for latent tuberculosis. Therefore, it is imperative to upgrade and refine existing models or develop a new model. However, battery of models will always be a better alternative to any single model as they will complement each other by overcoming their limitations.

A short-term model for preliminary screening of potential anti-tubercular compounds.

Screening of new agents for anti-tubercular activity is a challenging task due to the virulent and slow growing nature of mycobacteria. In this study, we explored the use of Mycobacterium smegmatis as an alternate model to virulent strains. We observed that the effect of standard anti-tubercular drugs was similar in mice infected with M. smegmatis and Mycobacterium tuberculosis. The total duration of the experiment, including incubation time, was 10 days in the M. smegmatis-infected mice model compared with 2 months in M. tuberculosis-infected mice. This model of anti-tubercular screening is a simple, easy to carry out, less time-consuming, safer and economical alternative for preliminary in vivo screening of potential anti-tubercular agents.

Evaluation of BACTEC 460 TB system for rapid in vitro screening of drugs against latent state Mycobacterium tuberculosis H37Rv under hypoxia conditions.

Mycobacterium tuberculosis exhibits latent state due to its ability to survive for extended periods under oxygen depletion conditions. The conventional plating method employed for in vitro screening of antitubercular agents against latent state M. tuberculosis is laborious and time consuming exercise. Towards this end, BACTEC 460 TB system of antitubercular screening was evaluated for testing efficacy of antimicrobial agents in hypoxia induced model of latent tuberculosis, in vitro. In this study, drugs like isoniazid, metronidazole and rifampin were tested at concentrations- 2, 10 and 50 microg/ml for their antilatency activity by using BACTEC and conventional methods. Results obtained from both the methods were comparable (P>0.05) and a good correlation was observed between colony forming units and growth index values. Further, time to determine mycobacterial growth was significantly reduced (P

Macromolecular prodrugs. XII. Primaquine conjugates: synthesis and preliminary antimalarial evaluation.

New primaquine conjugates 5-7 with glucosamine and two polymers of polyaspartamide type, poly[alpha,beta-(N-2-hydroxyethyl-DL-aspartamide)] (PHEA) and poly[alpha,beta-(N-3-hydroxypropyl-DL-aspartamide)] (PHPA), were synthesized, characterized and screened for their antimalarial activity. The conjugates differed in the type of covalent bonding, length of the spacer between the polymeric carrier and drug, molecular mass and drug-loading. Blood-schizontocidal activity of the prepared conjugates was tested against Plasmodium berghei infection in Swiss mice. Polymeric conjugates showed better antimalarial activity than the glucosamine conjugate.

Effect of morphine on Mycobacterium smegmatis infection in mice and macrophages.

The immunomodulatory effects of opioids are known in various infections. However, little is known about the effects of opioids in tuberculosis (TB). In the present study, we report the effects of morphine in Mycobacterium smegmatis infection in mice and macrophages. Morphine exerted a dose-dependent suppression of infection in vivo: 50 and 100 mg/kg morphine exerted significant (P<0.05) suppression whereas 5 mg/kg morphine showed no effect. Analogous to the in vivo effects, incubation of M. smegmatis-infected mouse peritoneal macrophages with morphine (100 µM) showed significant reduction in intramacrophage CFU counts. However, morphine did not show any direct antimycobacterial activity in broth dilution assay upto 100 µM concentration. Further, morphine-induced intramacrophage killing of M. smegmatis was abrogated by naloxone and aminoguanidine indicating the involvement of opioid-receptor activation and nitric oxide production in protective effects of morphine. In conclusion, morphine suppressed the progression of experimental TB in both mice and macrophage models.

A comparison of conventional and radiometric methods for the assessment of anti-tubercular activity of drugs against Mycobacterium tuberculosis in mice and macrophage models.

BACKGROUND: Presently, in vitro and in vivo screening of anti-tubercular drugs is a time-consuming exercise. Therefore, it is important to develop faster methods. MATERIAL AND METHODS: Towards this end, conventional plating and radiometric BACTEC methods of anti-tubercular screening were compared to determine the efficacy of anti-tubercular drugs (isoniazid and rifampicin) and morphine in Mycobacterium tuberculosis H37Rv-infected mice and macrophages. RESULTS: A linear correlation (R2 = 0.95) was observed between number of colony forming units (CFUs) and growth index (GI) values. BACTEC method was found to be faster and sensitive as compared to plating method. Further, BACTEC method, being a closed system, appeared to be less susceptible to microbial contamination and poses less biohazard. CONCLUSION: We conclude that BACTEC method can be employed for easy, precise, and rapid screening of anti-tubercular compounds and morphine in mice and macrophage models.