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1.  Artemisinins, New Miconazole Potentiators Resulting in Increased Activity against Candida albicans Biofilms 
Mucosal biofilm-related fungal infections are very common, and the incidence of recurrent oral and vulvovaginal candidiasis is significant. As resistance to azoles (the preferred treatment) is occurring, we aimed at identifying compounds that increase the activity of miconazole against Candida albicans biofilms. We screened 1,600 compounds of a drug-repositioning library in combination with a subinhibitory concentration of miconazole. Synergy between the best identified potentiators and miconazole was characterized by checkerboard analyses and fractional inhibitory concentration indices. Hexachlorophene, pyrvinium pamoate, and artesunate act synergistically with miconazole in affecting C. albicans biofilms. Synergy was most pronounced for artesunate and structural homologues thereof. No synergistic effect could be observed between artesunate and fluconazole, caspofungin, or amphotericin B. Our data reveal enhancement of the antibiofilm activity of miconazole by artesunate, pointing to potential combination therapy consisting of miconazole and artesunate to treat C. albicans biofilm-related infections.
PMCID: PMC4291436  PMID: 25367916
2.  Plant-Derived Decapeptide OSIP108 Interferes with Candida albicans Biofilm Formation without Affecting Cell Viability 
We previously identified a decapeptide from the model plant Arabidopsis thaliana, OSIP108, which is induced upon fungal pathogen infection. In this study, we demonstrated that OSIP108 interferes with biofilm formation of the fungal pathogen Candida albicans without affecting the viability or growth of C. albicans cells. OSIP108 displayed no cytotoxicity against various human cell lines. Furthermore, OSIP108 enhanced the activity of the antifungal agents amphotericin B and caspofungin in vitro and in vivo in a Caenorhabditis elegans-C. albicans biofilm infection model. These data point to the potential use of OSIP108 in combination therapy with conventional antifungal agents. In a first attempt to unravel its mode of action, we screened a library of 137 homozygous C. albicans mutants, affected in genes encoding cell wall proteins or transcription factors important for biofilm formation, for altered OSIP108 sensitivity. We identified 9 OSIP108-tolerant C. albicans mutants that were defective in either components important for cell wall integrity or the yeast-to-hypha transition. In line with these findings, we demonstrated that OSIP108 activates the C. albicans cell wall integrity pathway and that its antibiofilm activity can be blocked by compounds inhibiting the yeast-to-hypha transition. Furthermore, we found that OSIP108 is predominantly localized at the C. albicans cell surface. These data point to interference of OSIP108 with cell wall-related processes of C. albicans, resulting in impaired biofilm formation.
PMCID: PMC3993227  PMID: 24566179
3.  Evaluation of the In Vitro Antiplasmodial, Antileishmanial, and Antitrypanosomal Activity of Medicinal Plants Used in Saudi and Yemeni Traditional Medicine 
The antiplasmodial, antileishmanial, and antitrypanosomal activity of twenty-five medicinal plants distributed in Saudi Arabia and Yemen was evaluated. The plants were extracted with methanol and screened in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi, and free trypomastigotes of T. brucei. To assess selectivity, cytotoxicity was determined on MRC-5 cells. Criteria for activity were an IC50 < 10 μg/mL and high selectivity (SI). Seven plants showed interesting antiprotozoal activity in one or more models. Extracts of Caralluma penicillata and Acalypha ciliata showed fairly good activity against P. falciparum with IC50 of 6.7 and 10.8 μg/mL and adequate selectivity (SI > 9.6 and >5.9). Interesting activity against L. infantum was obtained with Verbascum bottae (IC50 of 3.2 μg/mL, SI 10.2) and Solanum glabratum (IC50 8.1 μg/mL, SI 3.4). The extracts of C. penicillata, Leucas virgata, Loranthus regularis, and V. bottae exhibited moderate activity against T. brucei (IC50 8.5, 8.1, 8.3, and 2.3 μg/mL; SI > 7.6, 7.7, 4.3, and >14.1). These results partly support the traditional use of some of the selected medicinal plants and warrant further investigations into the putative active constituents.
PMCID: PMC4055400  PMID: 24963330
4.  Selective Inhibitors of Fibroblast Activation Protein (FAP) with a (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine Scaffold 
ACS Medicinal Chemistry Letters  2013;4(5):491-496.
Fibroblast activation protein (FAP) is a serine protease that is generally accepted to play an important role in tumor growth and other diseases involving tissue remodeling. Currently there are no FAP inhibitors with reported selectivity toward both the closely related dipeptidyl peptidases (DPPs) and prolyl oligopeptidase (PREP). We present the discovery of a new class of FAP inhibitors with a N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold. We have explored the effects of substituting the quinoline ring and varying the position of its sp2 hybridized nitrogen atom. The most promising inhibitors combined low nanomolar FAP inhibition and high selectivity indices (>103) with respect to both the DPPs and PREP. Preliminary experiments on a representative inhibitor demonstrate that plasma stability, kinetic solubility, and log D of this class of compounds can be expected to be satisfactory.
PMCID: PMC4027141  PMID: 24900696
Fibroblast activation protein (FAP); dipeptidyl peptidase IV (DPPIV); prolyl oligopeptidase (PREP); seprase
5.  Transcript and Protein Analysis Reveals Better Survival Skills of Monocyte-Derived Dendritic Cells Compared to Monocytes during Oxidative Stress 
PLoS ONE  2012;7(8):e43357.
Dendritic cells (DCs), professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS). A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (mo)DCs and monocytes in response to oxidative stress.
Oxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min). Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H2DCFDA). Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels.
Tert-BHP increased CM-H2DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress–related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2), an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation.
Our results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.
PMCID: PMC3419731  PMID: 22916248
6.  Experimental Induction of Paromomycin Resistance in Antimony-Resistant Strains of L. donovani: Outcome Dependent on In Vitro Selection Protocol 
Paromomycin (PMM) has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 µM), either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 µM inclusion level that did not affect normal growth. Comparison of the IC50 values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC50 = 199 µM) compared to the parent strain (IC50 = 45 µM). In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field.
Author Summary
Leishmaniasis is caused by protozoan parasites of the genus Leishmania and is transmitted by inoculation of infective promastigotes by the female sand fly. In the mammalian host, amastigotes live inside macrophage cells which may lead to various clinical symptoms. First-line treatment relies mainly on antimonials and miltefosine; however, drug resistance is a growing problem. The antibiotic paromomycin (PMM) has recently been added as treatment option, but it is now essential to proactively assess the likelihood of resistance development to safeguard its long term effectiveness. Since ‘resistant’ patient isolates are not yet available, we artificially selected for PMM resistance using two different in vitro protocols with drug pressure on either the extracellular promastigote or on the intracellular amastigote stage. Resistance in promastigotes was obtained after about 25 weeks and persisted in the intracellular amastigote. High levels of resistance were obtained within two selection cycles on amastigotes, but with the unexpected observation that the promastigotes remained fully susceptible. In addition, the resistance proved to be stable. We could clearly demonstrate that a different PMM-resistance is obtained dependent on the ‘stage-selection’ protocol. These findings have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field.
PMCID: PMC3362622  PMID: 22666513
7.  In vitro antiplasmodial, antileishmanial and antitrypanosomal activities of selected medicinal plants used in the traditional Arabian Peninsular region 
Worldwide particularly in developing countries, a large proportion of the population is at risk for tropical parasitic diseases. Several medicinal plants are still used traditionally against protozoal infections in Yemen and Saudi Arabia. Thus the present study investigated the in vitro antiprotozoal activity of twenty-five plants collected from the Arabian Peninsula.
Plant materials were extracted with methanol and screened in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxic activity was determined against MRC-5 cells to assess selectivity. The criterion for activity was an IC50 < 10 μg/ml (<5 μg/ml for T. brucei) and selectivity index of >4.
Antiplasmodial activity was found in the extracts of Chrozophora oblongifolia, Ficus ingens, Lavandula dentata and Plectranthus barbatus. Amastigotes of T. cruzi were affected by Grewia erythraea, L. dentata, Tagetes minuta and Vernonia leopoldii. Activity against T. brucei was obtained in G. erythraea, L. dentata, P. barbatus and T. minuta. No relevant activity was found against L. infantum. High levels of cytotoxicity (MRC-5 IC50 < 10 μg/ml) and hence non-specific activities were noted in Cupressus sempervirens, Kanahia laniflora and Kniphofia sumarae.
The results endorse that medicinal plants can be promising sources of natural products with antiprotozoal activity potential. The results support to some extent the traditional uses of some plants for the treatment of parasitic protozoal diseases.
PMCID: PMC3493369  PMID: 22520595
8.  α-Ketoheterocycles as inhibitors of Leishmania mexicana cysteine protease CPB 
ChemMedChem  2010;5(10):1734-1748.
Cysteine proteases of the papain superfamily are present in nearly all eukaryotes and also play pivotal roles in the biology of parasites. Inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Inspired by the in vivo antiparasitic activity of the vinyl sulfone based cysteine protease inhibitors (CPIs), a series of α-ketoheterocycles 1-15 has been developed as reversible inhibitors of a recombinant L. mexicana cysteine protease CPB2.8. The isoxazoles 1-3 and especially the oxadiazole 15 are potent reversible inhibitors of CPB2.8, however, in vitro whole-organism screening against a panel of protozoan parasites did not fully correlate with the observed inhibition of the cysteine protease.
PMCID: PMC3245848  PMID: 20799311
cysteine proteases; inhibitors; ketoheterocycle; parasite CPB; Trypanosoma
9.  Quorum Sensing Inhibitors Increase the Susceptibility of Bacterial Biofilms to Antibiotics In Vitro and In Vivo▿† 
Although the exact role of quorum sensing (QS) in various stages of biofilm formation, maturation, and dispersal and in biofilm resistance is not entirely clear, the use of QS inhibitors (QSI) has been proposed as a potential antibiofilm strategy. We have investigated whether QSI enhance the susceptibility of bacterial biofilms to treatment with conventional antimicrobial agents. The QSI used in our study target the acyl-homoserine lactone-based QS system present in Pseudomonas aeruginosa and Burkholderia cepacia complex organisms (baicalin hydrate, cinnamaldehyde) or the peptide-based system present in Staphylococcus aureus (hamamelitannin). The effect of tobramycin (P. aeruginosa, B. cepacia complex) and clindamycin or vancomycin (S. aureus), alone or in combination with QSI, was evaluated in various in vitro and in vivo biofilm model systems, including two invertebrate models and one mouse pulmonary infection model. In vitro the combined use of an antibiotic and a QSI generally resulted in increased killing compared to killing by an antibiotic alone, although reductions were strain and model dependent. A significantly higher fraction of infected Galleria mellonella larvae and Caenorhabditis elegans survived infection following combined treatment, compared to treatment with an antibiotic alone. Finally, the combined use of tobramycin and baicalin hydrate reduced the microbial load in the lungs of BALB/c mice infected with Burkholderia cenocepacia more than tobramycin treatment alone. Our data suggest that QSI may increase the success of antibiotic treatment by increasing the susceptibility of bacterial biofilms and/or by increasing host survival following infection.
PMCID: PMC3101409  PMID: 21422204
10.  In Vitro Profiling of Pramiconazole and In Vivo Evaluation in Microsporum canis Dermatitis and Candida albicans Vaginitis Laboratory Models▿  
Antimicrobial Agents and Chemotherapy  2010;54(11):4927-4929.
The triazole antifungal pramiconazole (Stiefel, a GSK company) was compared with itraconazole, miconazole, and terbinafine in vitro and in vivo. Potent in vitro activities against Candida spp. (50% inhibitory concentration [IC50], 0.04 to 1.83 μM) and Microsporum and Trichophyton spp. (IC50, 0.15 to 1.34 μM) were obtained but not, however, against other filamentous molds and zygomycetes. In the M. canis guinea pig model and C. albicans vulvovaginitis rat model, pramiconazole was superior to the reference compounds after oral and topical administration.
PMCID: PMC2976107  PMID: 20805398
11.  Structure-Activity Relationship of Cinnamaldehyde Analogs as Inhibitors of AI-2 Based Quorum Sensing and Their Effect on Virulence of Vibrio spp 
PLoS ONE  2011;6(1):e16084.
Many bacteria, including Vibrio spp., regulate virulence gene expression in a cell-density dependent way through a communication process termed quorum sensing (QS). Hence, interfering with QS could be a valuable novel antipathogenic strategy. Cinnamaldehyde has previously been shown to inhibit QS-regulated virulence by decreasing the DNA-binding ability of the QS response regulator LuxR. However, little is known about the structure-activity relationship of cinnamaldehyde analogs.
Methodology/Principal Findings
By evaluating the QS inhibitory activity of a series of cinnamaldehyde analogs, structural elements critical for autoinducer-2 QS inhibition were identified. These include an α,β unsaturated acyl group capable of reacting as Michael acceptor connected to a hydrophobic moiety and a partially negative charge. The most active cinnamaldehyde analogs were found to affect the starvation response, biofilm formation, pigment production and protease production in Vibrio spp in vitro, while exhibiting low cytotoxicity. In addition, these compounds significantly increased the survival of the nematode Caenorhabditis elegans infected with Vibrio anguillarum, Vibrio harveyi and Vibrio vulnificus.
Several new and more active cinnamaldehyde analogs were discovered and they were shown to affect Vibrio spp. virulence factor production in vitro and in vivo. Although ligands for LuxR have not been identified so far, the nature of different cinnamaldehyde analogs and their effect on the DNA binding ability of LuxR suggest that these compounds act as LuxR-ligands.
PMCID: PMC3020944  PMID: 21249192
12.  Evaluation of Nucleoside Hydrolase Inhibitors for Treatment of African Trypanosomiasis ▿ †  
In this paper, we present the biochemical and biological evaluation of N-arylmethyl-substituted iminoribitol derivatives as potential chemotherapeutic agents against trypanosomiasis. Previously, a library of 52 compounds was designed and synthesized as potent and selective inhibitors of Trypanosoma vivax inosine-adenosine-guanosine nucleoside hydrolase (IAG-NH). However, when the compounds were tested against bloodstream-form Trypanosoma brucei brucei, only one inhibitor, N-(9-deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (UAMC-00363), displayed significant activity (mean 50% inhibitory concentration [IC50] ± standard error, 0.49 ± 0.31 μM). Validation in an in vivo model of African trypanosomiasis showed promising results for this compound. Several experiments were performed to investigate why only UAMC-00363 showed antiparasitic activity. First, the compound library was screened against T. b. brucei IAG-NH and inosine-guanosine nucleoside hydrolase (IG-NH) to confirm the previously demonstrated inhibitory effects of the compounds on T. vivax IAG-NH. Second, to verify the uptake of these compounds by T. b. brucei, their affinities for the nucleoside P1 and nucleoside/nucleobase P2 transporters of T. b. brucei were tested. Only UAMC-00363 displayed significant affinity for the P2 transporter. It was also shown that UAMC-00363 is concentrated in the cell via at least one additional transporter, since P2 knockout mutants of T. b. brucei displayed no resistance to the compound. Consequently, no cross-resistance to the diamidine or the melaminophenyl arsenical classes of trypanocides is expected. Third, three enzymes of the purine salvage pathway of procyclic T. b. brucei (IAG-NH, IG-NH, and methylthioadenosine phosphorylase [MTAP]) were investigated using RNA interference. The findings from all these studies showed that it is probably not sufficient to target only the nucleoside hydrolase activity to block the purine salvage pathway of T. b. brucei and that, therefore, it is possible that UAMC-00363 acts on an additional target.
PMCID: PMC2863631  PMID: 20194690
13.  In Vitro Sensitivity Testing of Leishmania Clinical Field Isolates: Preconditioning of Promastigotes Enhances Infectivity for Macrophage Host Cells▿  
Antimicrobial Agents and Chemotherapy  2009;53(12):5197-5203.
Diagnostic material from patients with leishmaniasis is generally available as promastigotes, and proper testing for susceptibility to first-line drugs by the intracellular amastigote assay is frequently hampered by the poor infectivity of the promastigotes for the macrophage host cell. Several conditions for optimization of the in vitro metacyclogenesis and cell infectivity of Leishmania donovani, L. guyanensis, and L. braziliensis field strains obtained from patients receiving standard antimony medication were investigated. Triggering log-phase promastigotes to become amastigote-like by increasing the temperature or acidifying the culture medium was not successful. Adequate metacyclogenesis and the highest levels of macrophage infection were obtained after 5-day-old late-log-phase promastigote cultures were preconditioned at 25°C to pH 5.4 for 24 h in Schneider's medium prior to infection. The susceptibility assay with primary peritoneal mouse macrophages included pentavalent antimony (SbV; sodium stibogluconate), trivalent antimony (SbIII; potassium antimonyl tartrate), miltefosine, and the experimental drug PX-6518. All strains were sensitive to miltefosine (50% inhibitory concentration [IC50] < 10 μM) and PX-6518 (IC50 < 2 μg/ml) but showed distinct susceptibility to SbV and/or SbIII, depending on whether they were derived from cured, relapse, or nonresponder patients. Within the available set of Leishmania species and strains, simultaneous SbV-SbIII resistance was clearly associated with treatment failure; however, a larger set of isolates is still needed to judge the predictive value of SbV-SbIII susceptibility profiling on treatment outcome. In conclusion, the proposed conditioning protocol further contributes toward a more standardized laboratory model for evaluation of the drug sensitivities of field isolates.
PMCID: PMC2786351  PMID: 19752271
14.  Design and evaluation of Trypanosoma brucei metacaspase inhibitors 
Metacaspase (MCA) is an important enzyme in Trypanosoma brucei, absent from humans and differing significantly from the orthologous human caspases. Therefore MCA constitutes a new attractive drug target for antiparasitic chemotherapeutics, which needs further characterization to support the discovery of innovative drug candidates. A first series of inhibitors has been prepared on the basis of known substrate specificity and the predicted catalytic mechanism of the enzyme. In this Letter we present the first inhibitors of TbMCA2 with low micromolar enzymatic and antiparasitic activity in vitro combined with low cytotoxicity.
PMCID: PMC2845880  PMID: 20167486
15.  In Vitro Susceptibilities of Leishmania donovani Promastigote and Amastigote Stages to Antileishmanial Reference Drugs: Practical Relevance of Stage-Specific Differences▿  
The in vitro susceptibilities of the reference strain Leishmania donovani MHOM/ET/67/L82 to sodium stibogluconate, amphotericin B, miltefosine, and the experimental compound PX-6518 were determined for extracellular log-phase promastigotes, established axenic amastigotes, fresh spleen-derived amastigotes, and intracellular amastigotes in primary mouse peritoneal macrophages. Susceptibility to amphotericin B did not differ across the various axenic models (50% inhibitory concentrations [IC50], 0.6 to 0.7 μM), and amphotericin B showed slightly higher potency against intracellular amastigotes (IC50, 0.1 to 0.4 μM). A similar trend was observed for miltefosine, with comparable efficacies against the extracellular (IC50, 0.4 to 3.8 μM) and intracellular (IC50, 0.9 to 4.3 μM) stages. Sodium stibogluconate, used either as Pentostam or as a crystalline substance, was inactive against all axenic stages (IC50, >64 μg SbV/ml) but showed good efficacy against intracellular amastigotes (IC50, 22 to 28 μg SbV/ml); the crystalline substance was about two to three times more potent (IC50, 9 to 11 μg SbV/ml). The activity profile of PX-6518 was comparable to that of sodium stibogluconate, but at a much higher potency (IC50, 0.1 μg/ml). In conclusion, the differential susceptibility determines which in vitro models are appropriate for either drug screening or resistance monitoring of clinical field isolates. Despite the more complex and labor-intensive protocol, the current results support the intracellular amastigote model as the gold standard for in vitro Leishmania drug discovery research and for evaluation of the resistance of field strains, since it also includes host cell-mediated effects. Axenic systems can be recommended only for compounds for which no cellular mechanisms are involved, for example, amphotericin B and miltefosine.
PMCID: PMC2737839  PMID: 19546361
16.  High dose folic acid pre-treatment blunts cardiac dysfunction during ischemia coupled to maintenance of high energy phosphates and reduces post-reperfusion injury 
Circulation  2008;117(14):1810-1819.
The B-vitamin folic acid (FA) is important to mitochondrial protein and nucleic acid synthesis, is an anti-oxidant, and enhances nitric oxide synthase activity. Here, we tested whether FA reduces myocardial ischemic dysfunction and post-reperfusion injury.
Wistar rats were pretreated with either FA (10mg/d) or placebo for 1-wk, and then underwent in vivo transient left coronary artery occlusion for 30min with or without 90min reperfusion (total:n=131; sub-groups used for various analyses). FA (4.5•10-6M i.c) pretreatment and global ischemia/reperfusion (30 min/30min), was also performed in vitro (n=28).
After 30min ischemia, global function declined more in controls than FA-pretreated rats (ΔdP/dtmax -878±586 mmHg/s vs. placebo -1956±351 mmHg/s, p=0.03), and regional thickening was better preserved (37.3±5.3% vs. 5.1±0.6%-placebo, p=0.004). Anterior-wall perfusion fell similarly (-78.4±9.3% vs. -71.2±13.8%-placebo at 30 min); yet myocardial high energy phosphates ATP and ADP reduced by ischemia in controls were better preserved by FA-pretreatment (e.g. ATP: control: 2740±58; ischemia: 947±55; ischemia+FA: 1332±101 nmol/g, p=0.02). Basal oxypurines (xanthine, hypoxanthine, and urate) rose with FA-pretreatment, but increased less during ischemia than in controls. Ischemic superoxide generation declined (3124±280 FA vs. 5898±474 placebo, cpm/mg, p=0.001). After reperfusion, FA-treated hearts had smaller infarcts (3.8±1.2% vs 60.3±4.1%-placebo area at risk, p<0.002), less contraction band necrosis, TUNEL-positivity, superoxide, and nitric oxide synthase uncoupling. Infarct size declined similarly with 1 mg/d FA as well.
FA-pretreatment blunts myocardial dysfunction during ischemia, and ameliorates post-reperfusion injury. This is coupled to preservation of high energy phosphates, reducing subsequent ROS-generation, eNOS-uncoupling and post-reperfusion cell death.
PMCID: PMC2474781  PMID: 18362233
Ischemia; Folic acid; High Energy Phosphates; Superoxide; eNOS-uncoupling
17.  Comparative Activities of the Triterpene Saponin Maesabalide III and Liposomal Amphotericin B (AmBisome) against Leishmania donovani in Hamsters 
Maesabalide III (MB-III), an oleane triterpene saponin isolated from the Vietnamese plant Maesa balansae, is a new antileishmanial lead compound whose activity against Leishmania donovani (MHOM/ET/67/L82) in groups of five golden hamsters was evaluated after administration of a single subcutaneous dose on either day 1 (prophylactic treatment) or day 28 (curative treatment) after infection. Liposomal amphotericin B (AmBisome), administered intravenously at 5 mg/kg of body weight, was used as the reference drug. Amastigote burdens in liver, spleen, and bone marrow were determined either 7 days (early effects) or 56 days (late effects) after treatment. Prophylactic administration of MB-III at 0.2 mg/kg reduced liver amastigote burdens by 99.8 and 83% within 7 and 56 days after treatment, respectively. In the latter group, however, all animals became ill and some died. Both MB-III at 0.8 mg/kg and liposomal amphotericin B were 100% effective against liver stages, but clearance from the spleen and bone marrow was not achieved. Curative administration of MB-III at 0.2 and 0.4 mg/kg was not protective, as no survivors were left at the termination of the experiment on day 84. Despite the high level of reduction of the liver amastigote burden after treatment with MB-III at 0.8 mg/kg (94.2%) or liposomal amphotericin B (99.4%), clinical protection could not be obtained in either group, with two deaths occurring and the residual liver burdens persisting. It is concluded that administration of a single dose of MB-III at 0.8 mg/kg has efficacy potential comparable to that of a single dose of liposomal amphotericin B at 5 mg/kg and is therefore considered a promising new antileishmanial lead compound. However, multiple-dose pharmacological, toxicological, and pharmacokinetic studies are still needed before it can become a valid drug candidate for development.
PMCID: PMC415622  PMID: 15155199
18.  In Vitro and In Vivo Activities of a Triterpenoid Saponin Extract (PX-6518) from the Plant Maesa balansae against Visceral Leishmania Species 
The in vitro and in vivo activities of a mixture of six oleane triterpene saponins, recovered from the methanolic extract of the leaves of the Vietnamese plant Maesa balansae (PX-6518), were evaluated against drug-sensitive visceral Leishmania strains. The in vitro 50% inhibitory concentration (IC50) against intracellular Leishmania infantum amastigotes was 0.04 μg/ml. The cytotoxic concentrations causing 50% cell death (CC50s) were about 1 μg/ml in murine macrophage host cells and >32 μg/ml in human fibroblasts (MRC-5 cell line). Evaluation in the Leishmania donovani BALB/c mouse model indicated that a single subcutaneous administration of 0.4 mg/kg at 1 day after infection reduced liver amastigote burdens by about 95% in all treated animals. If treatment was delayed until 14 days after infection, a dose of 1.6 mg/kg of body weight was required to maintain the same level of activity. Single 250-mg/kg doses of sodium stibogluconate (Pentostam) 1 and 14 days after infection produced comparable efficacies. A single dose of PX-6518 at 2.5 mg/kg administered 5 days before infection was still 100% effective in preventing liver infection, suggesting a particularly long residual action. Spleen and bone marrow could not be cleared by PX-6518 nor sodium stibogluconate. PX-6518 did not show activity after oral dosing at up to 200 mg/kg for 5 days. This study concludes that triterpenoid saponins from M. balansae show promising in vitro and in vivo antileishmanial potential and can be considered as new lead structures in the search for novel antileishmanial drugs.
PMCID: PMC310194  PMID: 14693530

Results 1-18 (18)