In Vitro and In Vivo Trypanosomicidal Action of Novel Arylimidamides against Trypanosoma cruzi.

Arylimidamides (AIAs) have been shown to have considerable biological activity against intracellular pathogens, includingTrypanosoma cruzi, which causes Chagas disease. In the present study, the activities of 12 novel bis-AIAs and 2 mono-AIAs against different strains ofT. cruziin vitroandin vivowere analyzed. The most active wasm-terphenyl bis-AIA (35DAP073), which had a 50% effective concentration (EC50) of 0.5 µM for trypomastigotes (Y strain), which made it 26-fold more effective than benznidazole (Bz; 13 µM). It was also active against the Colombiana strain (EC50= 3.8 µM). Analysis of the activity against intracellular forms of the Tulahuen strain showed that this bis-AIA (EC50= 0.04 µM) was about 100-fold more active than Bz (2 µM). The trypanocidal effect was dissociated from the ability to trigger intracellular lipid bodies within host cells, detected by oil red labeling. Both an active compound (35DAP073) and an inactive compound (26SMB060) displayed similar activation profiles. Due to their high selectivity indexes, two AIAs (35DAP073 and 35DAP081) were moved toin vivostudies, but because of the results of acute toxicity assays, 35DAP081 was excluded from the subsequent tests. The findings obtained with 35DAP073 treatment of infections caused by the Y strain revealed that 2 days of therapy induced a dose-dependent action, leading to 96 to 46% reductions in the level of parasitemia. However, the administration of 10 daily doses in animals infected with the Colombiana strain resulted in toxicity, preventing longer periods of treatment. The activity of the combination of 0.5 mg/kg of body weight/day 35DAP073 with 100 mg/kg/day Bz for 10 consecutive days was then assayed. Treatment with the combination resulted in the suppression of parasitemia, the elimination of neurological toxic effects, and survival of 100% of the animals. Quantitative PCR showed a considerable reduction in the parasite load (60%) compared to that achieved with Bz or the amidine alone. Our results support further investigations of this class with the aim of developing novel alternatives for the treatment of Chagas disease.

Structure-activity relationships of pentamidine-affected ion channel trafficking and dofetilide mediated rescue.

BACKGROUND AND PURPOSE: Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied. EXPERIMENTAL APPROACH: hERG and K(IR)2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit IKr . Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues. KEY RESULTS: Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and K(IR)2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not K(IR)2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective. CONCLUSIONS AND IMPLICATIONS: Drug-induced trafficking defects can be minimized if certain chemical features are avoided or 'synthesized out'; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS.

Surveillance for antibodies to Leishmania spp. in dogs from Sri Lanka.

The global distribution of leishmaniasis is rapidly expanding into new geographic regions. Dogs are the primary reservoir hosts for human visceral leishmaniasis caused by infection with Leishmania infantum. Natural infections with other Leishmania spp. can occur in dogs, but their role as reservoir hosts for other species of Leishmania is uncertain. Leishmania donovani is traditionally considered a visceralizing anthroponotic species; however, cutaneous leishmaniasis caused by L. donovani has been reported in Sri Lanka. In the present study, sera from 114 dogs in Sri Lanka were examined for antibodies to visceralizing Leishmania spp. Sera were tested by the canine immunochromatographic strip assays based on recombinant K39 antigen. Anti-Leishmania spp. antibodies were detectable in 1 of 114 (0.9%) dogs from Sri Lanka. Nonetheless, serological evidence suggests that leishmaniasis may be an emerging zoonosis in Sri Lanka.

Efficacy of the novel diamidine compound 2,5-Bis(4-amidinophenyl)- furan-bis-O-Methlylamidoxime (Pafuramidine, DB289) against Trypanosoma brucei rhodesiense infection in vervet monkeys after oral administration.

Owing to the lack of oral drugs for human African trypanosomiasis, patients have to be hospitalized for 10 to 30 days to facilitate treatment with parenterally administered medicines. The efficacy of a novel orally administered prodrug, 2,5-bis(4-amidinophenyl)-furan-bis-O-methlylamidoxime (pafuramidine, DB289), was tested in the vervet monkey (Chlorocebus [Cercopithecus] aethiops) model of sleeping sickness. Five groups of three animals each were infected intravenously with 10(4) Trypanosoma brucei rhodesiense KETRI 2537 cells. On the seventh day postinfection (p.i.) in an early-stage infection, animals in groups 1, 2, and 3 were treated orally with pafuramidine at dose rates of 1, 3, or 10 mg/kg of body weight, respectively, for five consecutive days. The animals in groups 4 and 5 were treated with 10 mg/kg for 10 consecutive days starting on the 14th day p.i. (group 4) or on the 28th day p.i. (group 5), when these animals were in the late stage of the disease. In the groups treated in the early stage, 10 mg/kg of pafuramidine completely cured all three monkeys, whereas lower doses of 3 mg/kg and 1 mg/kg cured only one of three and zero of three monkeys, respectively. Treatment of late-stage infections resulted in cure rates of one of three (group 4) and zero of three (group 5) monkeys. These studies demonstrated that pafuramidine was orally active in monkeys with early-stage T. brucei rhodesiense infections at dose rates above 3 mg/kg for 5 days. It was also evident that the drug attained only minimal efficacy against late-stage infections, indicating the limited ability of the molecule to cross the blood-brain barrier. This study has shown that oral diamidines have potential for the treatment of early-stage sleeping sickness.

Serological survey of Leishmania infantum and Trypanosoma cruzi in dogs from urban areas of Brazil and Colombia.

Leishmania infantum and Trypanosoma cruzi are zoonotic parasites that are endemic throughout many parts of Latin America. Infected dogs play an important role in transmission of both parasites to humans. A serological survey of Leishmania and Trypanosoma infection was conducted on 365 dogs from São Paulo, Brazil and Bogatá, Colombia, South America. Serum samples were examined by the indirect immunofluorescent antibody test (IFAT). Anti-Leishmania IgG antibodies were detected in 5 of 107 from Brazil (4.7%) and in 4 of 258 dogs (1.6%) from Colombia. Titers ranged from 1:25 to 1:100. Anti-T. cruzi antibodies were not detected in any of the dogs from either Brazil or Colombia. The results show a low prevalence of anti-Leishmania antibodies and no antibodies against T. cruzi in these canine populations. Our study suggests that dogs play a limited role in the spread of L. infantum and T. cruzi in these urban areas of Brazil and Colombia.

Human African trypanosomiasis: pharmacological re-engagement with a neglected disease.

This review discusses the challenges of chemotherapy for human African trypanosomiasis (HAT). The few drugs registered for use against the disease are unsatisfactory for a number of reasons. HAT has two stages. In stage 1 the parasites proliferate in the haemolymphatic system. In stage 2 they invade the central nervous system and brain provoking progressive neurological dysfunction leading to symptoms that include the disrupted sleep wake patterns that give HAT its more common name of sleeping sickness. Targeting drugs to the central nervous system offers many challenges. However, it is the cost of drug development for diseases like HAT, that afflict exclusively people of the world's poorest populations, that has been the principal barrier to new drug development and has led to them becoming neglected. Here we review drugs currently registered for HAT, and also discuss the few compounds progressing through clinical trials. Finally we report on new initiatives that might allow progress to be made in developing new and satisfactory drugs for this terrible disease.

In vitro metabolism of an orally active O-methyl amidoxime prodrug for the treatment of CNS trypanosomiasis.

A new aza-analogue of furamidine, 6-[5-(4-amidinophenyl)-furan-2-yl]nicotinamidine (DB820), has potent in vitro antitrypanosomal activity; however, it suffers from poor oral activity because of its positively charged amidine groups. The dimethoxyamidine prodrug of DB820, N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine (DB844), has potent oral activity in mouse models of both early-stage and CNS African trypanosomiasis. Metabolism of DB844 in human liver microsomes (HLM) was investigated using liquid chromatography-mass spectrometry (LC-MS/MS). The metabolism of DB844 in HLM was NADPH-dependent and resulted in the production of eight metabolites over a 90?min incubation. O-Demethylation and N-dehydroxylation reactions resulted in the metabolic conversion of DB844 to its active DB820 metabolite. Chromatographic conditions used for LC-MS analysis allowed for the separation and identification of all metabolites including positional isomers. Demethylation of either the phenyl or pyridine side of DB844 (DB844 m/z 366.2) resulted in the production of two metabolites (M1A, M1B), each with a molecular ion of m/z of 352.3 and MS(2) fragments of 288.1, 305.2, 321.2 and 335.2. However, the intensities of the MS(2) fragments were different among the two isomeric metabolites, and comparison to an authentic standard allowed for the structural determination of each metabolite. The isomeric metabolites M2A and M2B, resulting from amidoxime reductions of M1A and M1B, were also chromatographically separated and had distinguishable MS(2) profiles that allowed for their structural assignments when compared to an authentic standard. The di-amidoxime product resulting from O-demethylation of either side of DB844 was also identified as an abundant metabolite during microsomal incubations. The active antitrypanosomal metabolite, DB820, was the last metabolite to be formed and thus provides evidence that DB844 may effectively be metabolized to its active metabolite in vivo.

Sequence-dependent binding of bis-amidine carbazole dications to DNA.

The conventional wisdom argues that DNA intercalators possess a condensed polyaromatic ring whereas DNA minor groove binders generally contain unfused aromatic heterocycles, frequently separated by amide bonds. Recently, this view has been challenged with the discovery of powerful intercalating agents formed by unfused aromatic molecules and groove binders containing a polyaromatic nucleus. Bis-amidinocarbazoles belong to this later category of drugs having a planar chromophore and capable of reading the genetic information accessible within the minor groove of AT-rich sequences [Tanious, F.A., Ding, D., Patrick, D.A., Bailly, C., Tidwell, R.R. & Wilson, W.D. (2000) Biochemistry 39, 12091-12101]. But in addition to the tight binding to AT sites, we show here that bis-amidinocarbazoles can also interact with GC sites. The extent and mode of binding of 2,7 and 3,6 substituted amidinocarbazoles to AT and GC sequences were investigated by complementary biochemical and biophysical methods. Absorption, fluorescence, melting temperature and surface plasmon resonance (SPR) measurements indicate that the position of the two amidine groups on the carbazole ring influences significantly the drug-DNA interaction. SPR and DNase I footprinting data confirm the AT-preference of the compounds and provide useful information on their additional interaction with GC sequences. The 3,6-carbazole binds approximately twice as strongly to the GC-containing hairpin oligomer than the 2,7-regioisomer. The high tendency of the 3,6 compound to intercalate into different types of DNA containing G.C base pairs is shown by electric linear dichroism. This work completes our understanding of the sequence-dependent DNA binding properties of carbazole dications.

Separation methods for nucleoside analogues used for treatment of HIV-1 infection.

Clinicians design antiretroviral therapy to prevent HIV-1 replication and resistance, and researchers study antiretroviral concentrations to understand the pharmacokinetics of these drugs. Because drug efficacy and toxicity varies widely between patients receiving the same antiretroviral therapy, there is interest in monitoring individual patient concentrations of antiretroviral drugs. Good science and effective medical care demand inexpensive validated methods with high throughput that are capable of simultaneously analyzing multiple antiretroviral drugs in various matrices. Currently, protease inhibitors, non-nucleoside reverse transcriptase inhibitors, and nucleoside reverse transcriptase inhibitors are used to treat HIV-1 infection. This review summarizes published methods for the quantitation of nucleoside reverse transcriptase inhibitors and their metabolites in different matrices using immunoassays, ultraviolet absorption, and mass spectrometry.

Effects of compound structure on carbazole dication-DNA complexes: tests of the minor-groove complex models.

Carbazole dications have shown excellent activity against opportunistic infections, but they are quite different in structure from previously studied unfused aromatic cations that function by targeting the DNA minor groove. In a previous report [Tanious, F. A., Ding, D., Patrick, D. A., Tidwell, R. R., and Wilson, W. D. (1997) Biochemistry 36, 15315-15325] we showed that, despite their fused ring structure, the carbazoles also bind in A/T sequences of the DNA minor groove and we proposed models for the carbazole-DNA complexes with the carbazole nitrogen facing out of the groove for 3,6 substituted compounds but into the groove in 2,7 carbazoles. To test and refine the models, carbazole-N-methyl substituted derivatives have been synthesized in both the 3,6 and 2,7 series as well as a new 2,6 substituted NH derivative that is intermediate in structure. Footprinting results indicate a broad AT specificity of carbazole binding and a pattern in agreement with a minor groove complex. Surface plasmon resonance biosensor analysis of carbazole binding to an oligomer with an AATT central sequence indicated that the 2,7 NH compound has the largest binding constant. Both the 3,6 NH and NMe compounds bind with similar equilibrium constants that are less than for the 2,7 NH compound. The 2,7 NMe compound has the lowest binding constant of all the carbazoles. Spectroscopic results are also similar for the two 3,6 derivatives but are quite different for the 2,7 NH and NMe carbazole dications. Structural analysis of carbazole complexes with an AATT sequence by 2D NMR methods also supported a minor groove complex of the carbazoles in orientations in agreement with the previously proposed models. From these results, it is clear that the fused ring carbazoles can bind strongly in the DNA minor groove with a broad A/T specificity and that the 2,7 and 3,6 substituted carbazoles bind to the minor groove in opposite orientations.

Simultaneous determination of lamivudine and zidovudine concentrations in human seminal plasma using high-performance liquid chromatography and tandem mass spectrometry.

A HPLC-MS-MS method was developed and validated to measure lamivudine and zidovudine simultaneously in small volumes of human seminal plasma. Sample preparation was simple and rapid, requiring 25 microl of sample, the use of isotopically labeled lamivudine and zidovudine as internal standards and ultrafiltration through a molecular mass cut-off membrane. Lamivudine and its internal standard were separated from zidovudine and its internal standard with isocratic HPLC. Detection was carried out using tandem mass spectrometry. This validated method was used to analyze seminal samples obtained from six HIV-positive patients prescribed lamivudine and zidovudine.

Immunoassays for pentamidine and related compounds: development of a facile inhibitory ELISA suitable for clinical use.

Aromatic dicationic drugs have a broad spectrum of activity against protozoal and fungal pathogens including Pneumocystis carinii, Leishmania mexicana amazonensis, Cryptosporidium parvum and Cryptococcus neoformans. Pentamidine serves as the exemplar for an extensive collection of newly synthesized related compounds, which have reduced toxicity and a wider range of target organisms. Assays of pentamidine and related compounds have depended on HPLC-tandem mass spectrometry (HPLC-TMS) for the quantitation and identification of drug and metabolites. Immunoassays for pentamidine would have many advantages over the HPLC methods including relative simplicity of assay format and required equipment, convenience in sample preparation and reduction in time and cost of assays. In this report we describe a simple ELISA based immunoassay for pentamidine and pentamidine-like drugs with requisite sensitivity and specificity for use as a clinical assay (EC50 value of about 50 nanomolar). Immunogen was synthesized by coupling the hapten aminopentamidine to ovalbumin (chemically modified to provide an optimal number of -SH groups) using sulfo-MBS. Maleic-anhydride activated ELISA plates were covalently sensitized using the aminopentamidine hapten and used in an inhibitory ELISA assay format whereby the ability of analyte to suppress antibody binding to sensitized plate was measured. The assay detects primarily the phenolic amidine of pentamidine when in a para position and hence can also detect structurally related derivatives of pentamidine of potential interest as new therapeutic agents.

Nucleoside analogues achieve high concentrations in seminal plasma: relationship between drug concentration and virus burden.

Human immunodeficiency virus (HIV) can be transmitted in semen from a man to his sexual partners. Antiretroviral drugs are likely to affect the amount of HIV-1 in semen and perhaps transmission of the virus. The concentrations of zidovudine, lamivudine, and HIV-1 RNA in blood and seminal plasma were measured in 9 HIV-positive men over

Prodrugs for amidines: synthesis and anti-Pneumocystis carinii activity of carbamates of 2,5-bis(4-amidinophenyl)furan.

Syntheses of several carbamate analogues of 2, 5-bis(4-amidinophenyl)furan (1) under mild conditions and their evaluation as prodrugs against Pneumocystis carinii pneumonia (PCP) in an immunosuppressed rat model are described. Thus, nine new bis-carbamates: methoxycarbonyl (2), 2,2,2-trichloroethoxycarbonyl (3), ethylthiocarbonyl (4), benzyloxycarbonyl (5), (4-methyl-2-oxo-1, 3-dioxol-4-en-5-yl)methoxycarbonyl (6), phenoxycarbonyl (7), 4-fluorophenoxycarbonyl (8), 4-methoxyphenoxycarbonyl (9), and (1-acetoxy)ethoxycarbonyl (10) and a bis-carbonate ethoxycarbonyloxy (11) of the bis-amidine 1 have been synthesized and evaluated. The in vivo results show that the 4-fluorophenyl carbamate 8 and the 4-methoxyphenyl carbamate 9 in this series had the best anti-PCP activity by both intravenous and oral administration at a dosage level of 22 mol and 33 micromol/kg/day, respectively. Compounds 3-7 were also more active than the parent drug (1) on oral administration. The acute toxicity usually exhibited by the parent amidine 1 at a dosage level of 22 micromol/kg/day on intravenous administration has been significantly reduced by the prodrug modifications, with the exception of compound 10 which exhibited some toxicity. This report also describes the synthesis of several aryl-alkyl and aryl-aryl carbonates (12-14, 16-23) as efficient reagents for the preparation of carbamate derivatives from bis-arylamidines.

In-vitro activity of dicationic aromatic compounds and fluconazole against Cryptococcus neoformans and Candida spp.

We investigated the in-vitro activity of three selected dicationic aromatic compounds for nine clinical isolates of Cryptococcus neoformans and 93 clinical isolates of Candida spp., representing 12 different species, using a broth macrodilution method following NCCLS recommendations. All the clinical isolates were also tested for fluconazole susceptibility. The in-vitro data demonstrate that compounds 39 and 57 have excellent in-vitro activity for all tested strains (MIC 0.19-1.56 mg/L) except Candida pelliculosa. Moreover, compound 39 showed excellent in-vitro fungicidal activity against Candida krusei, Candida glabrata, Candida lusitaniae and Cryptococcus neoformans with MFCs in the range 0.39-6.25 mg/L. Both compounds 39 and 57 showed excellent in-vitro activity against fluconazole-resistant Candida albicans isolates, including a C. albicans strain that contains all known fluconazole-resistant mechanisms. Comparing MIC data from compounds 21, 39 and 57 with fluconazole, we found a statistically significant difference only with compound 39 (P = 0.043). However, comparing MFC data from compounds 21, 39 and 57 with fluconazole, we found statistically significant differences with all three compounds (P < 0.00001). These data indicate the potential antifungal breadth of two bis-benzimidazoles (compounds 39 and 57) as antifungal agents against yeasts. If it can be determined that compounds 39 and 57 are effective and non-toxic in vivo, the prospect of these compounds as clinically useful antifungal agents will be enhanced.

2,4-Diphenyl furan diamidines as novel anti-Pneumocystis carinii pneumonia agents.

Dicationic 2,4-bis(4-amidinophenyl)furans 5-10 and 2, 4-bis(4-amidinophenyl)-3,5-dimethylfurans 14 and 15 have been synthesized. Thermal melting studies revealed high binding affinity of the compounds to poly(dA-dT) and to the duplex oligomer d(CGCGAATTCGCG)2. All of the new compounds were effective against Pneumocystis carinii pneumonia in the immunosuppressed rat model with up to 200-fold increase in activity compared to the control compound pentamidine. No toxicity was noted for 5, 7-10 at the dose of 10 micromol/kg/d; however, the isopropyl analogue 7 showed toxicity comparable to pentamidine at the dosage of 20 micromol/kg/d. Dimethylation of the parent compound on the furan ring resulted in reduced activity and increased toxicity.

Synthesis and anti-Pneumocystis carinii pneumonia activity of novel dicationic dibenzothiophenes and orally active prodrugs.

Dicationic carbazoles have been found to be highly active against a rat model of Pneumocystis carinii pneumonia (PCP). Unfortunately, amidoxime derivatives, designed as prodrugs, were inactive against PCP even though the corresponding amidines were highly active. In the present work, a series of 2,8- and 3,7-bis cationic dibenzothiophenes was synthesized and assayed for anti-PCP activity. Three of the compounds proved to be more potent and less toxic than a standard anti-PCP drug (pentamidine) when given intravenously. Unlike the carbazoles, a dibenzothiophene amidoxime prodrug given orally reduced the parasite load by more than 99%. While no quantitative correlation was seen between anti-PCP activity and DNA binding, a strong level of DNA binding was found to be necessary for antimicrobial activity.

Comparative efficacy evaluation of dicationic carbazole compounds, nitazoxanide, and paromomycin against Cryptosporidium parvum infections in a neonatal mouse model.

The efficacies of dicationic carbazole compounds, nitazoxanide (NTZ), and paromomycin were evaluated against the AUCp1 isolate of Cryptosporidium parvum by using a neonatal mouse model. Compounds were solubilized or suspended in deionized water and administered orally by gavage to neonatal mice at a constant dose rate on days 0 to 5 (treatment started on day 0). Dose rates varied for individual carbazole compounds but ranged from 0.65 to 20 mg/kg of body weight. NTZ was tested at 100 and 150 mg/kg, and paromomycin was tested at 50 mg/kg. Efficacies were determined by comparing numbers of oocysts present in treated versus control mice at necropsy examination on day 6. Demonstrable efficacy was observed for several carbazole compounds, based on significant reductions in the numbers of oocysts recovered from treated mice versus control mice. Compounds 1, 7, and 10 (19.0 mg/kg) reduced oocyst passage in treated mice to less than 5% of that in control mice. Treatment with compounds 6, 8, and 9 (17.0 mg/kg) resulted in reductions of oocyst output to less than 10% of that in controls. Although they were not comparable in efficacy to compounds 1, 6, 7, 8, 9, and 10, treatment with other carbazole compounds resulted in statistically significant reductions in oocyst output in treated versus control mice. Compound 1 retained efficacy resulted in reduction of oocyst output to approximately 6% of that in controls when the dose was reduced to 5 mg/kg. Further reductions in the dose rate resulted in considerable reductions in anticryposporidial activity. Likewise, the efficacies of compounds 9 and 10 were reduced substantially when the doses were lowered to one-half the screening dose. Paromomycin yielded excellent activity (reduction of oocyst output to <2% of that in controls) at a dose of 50 mg/kg. NTZ yielded moderate efficacy as powder and injectable formulations administered at 100 mg/kg orally (reduction of oocyst output to 42 and 26% of that in controls, respectively). Oral administration of the injectable formulation of NTZ at a dose of 150 mg/kg resulted in improved efficacy (oocyst output, <5% of that in controls).

Structure-in vitro activity relationships of pentamidine analogues and dication-substituted bis-benzimidazoles as new antifungal agents.

Twenty analogues of pentamidine, 7 primary metabolites of pentamidine, and 30 dicationic substituted bis-benzimidazoles were screened for their inhibitory and fungicidal activities against Candida albicans and Cryptococcus neoformans. A majority of the compounds had MICs at which 80% of the strains were inhibited (MIC80s) comparable to those of amphotericin B and fluconazole. Unlike fluconazole, many of these compounds were found to have potent fungicidal activity. The most potent compound against C. albicans had an MIC80 of