Phenylbenzothiazole derivatives: effects against a Trypanosoma cruzi infection and toxicological profiles.

Current treatments for Chagas disease have a limited impact during the chronic stage and trigger severe side effects. Treatments target Trypanosoma cruzi, the etiological agent of the disease. The aims of this study were to evaluate the trypanocidal activity of four 2-phenylbenzothiazole derivatives (BZT1-4) in vitro by using the infectious and non-infectious forms of T. cruzi (trypomastigotes and epimastigotes, respectively) and to test the most promising compound (BZT4) in vivo in mice. Additionally, the toxicological profile and possible neuronal damage were examined. In relation to trypomastigotes, BZT4 was more selective and effective than the reference drug (benznidazole) during this infective stage, apparently due to the synergistic action of the CF3 and COOH substituents in the molecule. During the first few hours post-administration of BZT4, parasitemia decreased by 40% in an in vivo model of short-term treatment, but parasite levels later returned to the basal state. In the long-term assessment, the compound did not produce a significant antiparasitic effect, only attaining a 30% reduction in parasitemia by day 20 with the dose of 16 mg/kg. The toxicity test was based on repeated dosing of BZT4 (administered orally) during 21 days, which did not cause liver damage. However, the compound altered the concentration of proteins and the proteinic profile of neuronal cells in vitro, perhaps leading to an effect on the central nervous system. Further research on the low trypanocidal activity in vivo compared to the better in vitro effect could possibly facilitate molecular redesign to improve trypanocidal activity.

Mapping benznidazole resistance in trypanosomatids and exploring evolutionary histories of nitroreductases and ABCG transporter protein sequences.

The nitro-heterocyclic compound benznidazole (BZ) is the first-line drug for the treatment of Chagas disease, caused by the protozoan Trypanosoma cruzi. However, therapeutic failures are common for reasons that include the influences of parasite and host genetics, the effects of toxicity on adherence to treatment, and difficulties in demonstrating parasitological cure. To obtain information on the origin of the resistance to BZ and eliminate from the scenery the participation of the host, initially we mapped the susceptibility to the drug in thirteen species of seven genera of the family Trypanosomatidae. We verified that all Trypanosoma species are sensitive to low concentrations of the drug (IC50 2.7 to 25 µM) while Non-Trypanosoma species are highly resistant to these concentrations. The two groups of parasites correspond to the major phylogenetic lineages of trypanosomatids. Next, we searched in the trypanosomatid genome databases homologs of two type-I nitroreductases (NTR-1 and OYE) and an ABC transporter (ABCG1) that have been associated with BZ resistance in T. cruzi. The predicted proteins were characterized regarding domains and used for phylogenetic analyses. Homologous NTR-1 genes were found in all trypanosomatids investigated and the structural characteristics of the enzyme suggest that it may be functional. OYE genes were absent in BZ-sensitive African trypanosomes, which excludes the participation of this enzyme in BZ bio-activation. Two copies of ABCG1 genes were observed in most BZ resistant species, while Trypanosoma species exhibit only one copy per haploid genome. Functional studies are required to verify the involvement of these genes in BZ resistance. In addition, since multiple mechanisms can contribute to BZ susceptibility, our study poses a range of organisms highly resistant to BZ in which these aspects can be investigated. Preliminary studies on BZ uptake indicate marked differences between BZ-sensitive and BZ-resistant species.

In vitro genotoxicity of nitroimidazoles as a tool in the search of new trypanocidal agents.

BACKGROUND: Only benznidazole (Bnz) (1) and nifurtimox (Nfx) (2) are licensed for the treatment of Chagas disease although their safety and efficacy profile are far from ideal. Farmanguinhos from Fiocruz has developed seven nitroimidazole compounds (4-10) analogs of megazol (3). OBJECTIVES: To evaluate whether the genotoxic effect of 3 was abolished in the seven nitroimidazoles (4-10) analogs using the in vitro alkaline comet assay (CA) and the in vitro cytokinesis-block micronucleus assay (CBMN) in whole human blood cells (WHBC) and correlate this effect with their trypanocidal activity using bloodstream trypomastigote forms of Trypanosoma cruzi. METHODS: The toxicity of 3-10 to WHBC in the in vitro CA was determined using the fluorescein diacetate/ethidium bromide assay. DNA damage in the in vitro CA was evaluated according to tail size in four classes (0-3) and methyl methane-sulfonate (MMS) was used as a positive control. The cytotoxicity of 3-10 to WHBC in the CBMN was measured using the cytokinesis-block proliferation index and the replication index. The number of the micronucleate cells in 2,000 binucleate cells by experimental group was determined. Mitomycin C and N-deacetyl-N-methylcolchicine were used as positive controls. FINDINGS: Compound 3 showed a significant DNA strand break effect through the in vitro CA and highly significant clastogenic and/or aneugenic effect in the CBMN. Compounds 5, 6, 8, 9 and 10 showed negative results in the CBMN and positive results in the in vitro CA, while the inverse effect was observed for 4 and 7. MAIN CONCLUSIONS: Compound 10 was the most promising to proceed with the development as a drug candidate in the treatment of Chagas disease showing absence of chromosomal cytogenetic damage and high activity against T. cruzi, about two times higher than 3 and the clinical drug 1.

Alkylated/aminated nitroimidazoles and nitroimidazole-7-chloroquinoline conjugates: Synthesis and anti-mycobacterial evaluation.

The success in exploring anti-tubercular potency of nitroimidazole and quinoline, the core moieties of recently approved anti-tubercular drugs instigated us to synthesize a series of alkylated/aminated 2-methyl-5-nitroimidazoles and nitroimidazole-7-chloroquinoline conjugates and to evaluate them for their activities against Mycobacterium tuberculosis as well as for their cytotoxicity towards the J774 murine macrophage cell line. Although the synthesized compounds did not surpass the activity of the standard drug Isoniazid, they have appreciable activities with minimal cytotoxicity. The synthesized nitroimidazole-7-chloroquinoline conjugate, 11c, having butyl chain as linker, proved to be the most potent among the series with an MIC50 value of 2.2 µg/mL.

Next-Generation Hypoxic Cell Radiosensitizers: Nitroimidazole Alkylsulfonamides.

Innovations in the field of radiotherapy such as stereotactic body radiotherapy, along with the advent of radio-immuno-oncology, herald new opportunities for classical oxygen-mimetic radiosensitizers. The role of hypoxic tumor cells in resistance to radiotherapy and in suppression of immune response continues to endorse tumor hypoxia as a bona fide, yet largely untapped, drug target. Only nimorazole is used clinically as a radiosensitizer, and there is a dearth of new radiosensitizers in development. Here we present a survey of novel nitroimidazole alkylsulfonamides and document their cytotoxicity and ability to radiosensitize anoxic tumor cells in vitro. We use a phosphate prodrug approach to increase aqueous solubility and to improve tumor drug delivery. A 2-nitroimidazole and a 5-nitroimidazole analogue demonstrated marked tumor radiosensitization in either ex vivo assays of surviving clonogens or tumor regrowth delay.

Delamanid is not metabolized by Salmonella or human nitroreductases: A possible mechanism for the lack of mutagenicity.

Nitro-containing compounds such as nitrofuran and nitroimidazole are drugs used for the treatment of infectious diseases. However, many of these nitro-containing drugs are positive in the bacterial reverse mutation assay (Ames test). The recently approved anti-multidrug-resistant tuberculosis (MDR-TB) drug, delamanid (Deltyba™; OPC-67683), a derivative of 4-nitroimidazole, was negative for mutagenicity in the Ames assay. In Salmonella typhimurium, mutagenicity of nitro compounds has been closely associated with the ability of nitroreductase to metabolize (degradation)these compounds. To explore the lack of mutagenicity for delamanid, we examined the initial metabolic rate and mutagenic-specific activity of a series of nitro compounds in S. typhimurium TA100. The order of maximum mutagenic-activity was nitrofuran > 2-nitroimidazole > 5-nitroimidazole ≥ 4-nitroimidazole, which is very similar to the order of initial metabolic rate, i.e., the Pearson's correlation coefficient (r = 0.85) showed a correlation between metabolic rate and mutagenic-activity. No metabolism of delamanid was detected even after 60 h of treatment. In addition, delamanid was not reduced by two human nitroreductases. These facts may explain the absence of genotoxicity of delamanid in both in vitro and in vivo tests.

Evaluation of Cytotoxicity and Hypoxic Effect of Nitroimidazole Embedded Nanoparticles.

Adenylate cyclase is a key intracellular enzyme involved in energy imbalance leading to tumor hypoxia and cytotoxicity. In this study, adenylate cyclase activities in isolated hepatocytes and Kupffer cells were compared in the presence of several metabolic stimulators. In cultured hepatocyte cells, adenylate cyclase was stimulated by guanylyl imidotriphosphate (GITP), guanosine triphosphate (GTP), progesterone and nitroimidazole embedded nanoparticle (NNP) effectors, while prostaglandin E2 and F2α were used as effectors in cultured Kupffer cells. The results showed that NNPs decreased adenylate cyclase specific activity in a dose-dependent manner after preincubation of hepatocytes with NNPs. The NNPs stimulated adenylate cyclase activities in hepatocytes were evaluated based on measurement of cyclic adenosine monophosphate (cAMP). The stimulatory effects of NNPs on adenylate cyclase were independent of the presence of GTP and may have been due to a direct effect on the catalytic subunit of adenylate cyclase. In addition, basal cAMP generation in hepatocyte cells was efficiently suppressed by the NNPs. In conclusion, NNPs exerted direct effects on the catalytic subunit of the adenylate cyclase system, and adenylate cyclase was hormone sensitive in liver cells.

Phase Transitions of Isotropic to Anisotropic Biocompatible Lipid-Based Drug Delivery Systems Overcoming Insoluble Benznidazole Loading.

Previous studies reported low benznidazole (BNZ) loading in conventional emulsions due to the weak interaction of the drug with the most common oils used to produce foods or pharmaceuticals. In this study, we focused on how the type of surfactant, surfactant-to-oil ratio w/w (SOR) and oil-to-water ratio w/w (OWR) change the phase behavior of different lipid-based drug delivery systems (LBDDS) produced by emulsion phase inversion. The surfactant mixture composed of soy phosphatidylcholine and sodium oleate (1:7, w/w, hydrophilic lipophilic balance = 16) stabilized medium chain triglyceride in water. Ten formulations with the clear aspect or less turbid dispersions (five with the SOR ranging from 0.5 to 2.5 and five with the OWR from 0.06 to 0.4) were selected from the phase behavior diagram to assess structural features and drug-loading capacity. The rise in the SOR induced the formation of distinct lipid-based drug delivery systems (nanoemulsions and liquid crystal lamellar type) that were identified using rheological measurements and cross-polarized light microscopy images. Clear dispersions of small and narrow droplet-sized liquid-like nanoemulsions, Newtonian flow-type, were produced at SOR from 0.5 to 1.5 and OWR from 0.12 to 0.4, while clear liquid or gel-like liquid crystals were produced at SOR from 1.5 to 2.5. The BNZ loading was improved according to the composition and type of LBDDS produced, suggesting possible drug location among surfactant layers. The cell viability assays proved the biocompatibility for all of the prepared nanoemulsions at SOR less than 1.5 and liquid crystals at SOR less than 2.5, demonstrating their promising features for the oral or parenteral colloidal delivery systems containing benznidazole for Chagas disease treatment.

Norisoboldine, an alkaloid from Radix linderae, inhibits NFAT activation and attenuates 2,4-dinitrofluorobenzene-induced dermatitis in mice.

CONTEXT: The nuclear factor of activated T-cells (NFAT) is a family of transcription factors, essential for T-cell activation. Norisoboldine (NOR), an isoquinoline alkaloid from Radix linderae, has been demonstrated to possess anti-inflammatory activity. OBJECTIVE: This study examines NOR's effect on NFAT activation and its therapeutic potential for atopic dermatitis (AD). MATERIALS AND METHODS: The transcriptional activity of NFAT was examined with luciferase reporter assay, using K562-luc cells, stimulated with 20 ng/mL PMA plus 1 µM ionomycin. NFAT dephosphorylation was examined by immuno-blotting in K562-luc cells and Jurkat cells. Interleukin-2 (IL-2) expression in Jurkat cells was examined by real-time PCR. A mouse model of dermatitis, induced by 2,4-dinitrochlorobenzene (DNCB), was used to test NOR's therapeutic potential for AD. RESULTS: NOR, dose-dependently, inhibited PMA and ionomycin-induced NFAT reporter gene expression in K562-luc cells in the range of 2-50 µM. NOR also inhibited PMA and ionomycin-induced NFAT dephosphorylation in K562-luc cells and Jurkat cells. Consequently, NOR suppressed PMA plus ionomycin-induced IL-2 expression in Jurkat cells. The administration of NOR (10 mg/kg, i.p.), alleviated DNCB-induced dermatitis in mice, by the reduction of ear swelling and attenuation of inflammatory infiltration into ear tissue. Moreover, mRNA levels of INF-γ, TNF-α, IL-4 and IL-6 in ears of NOR-treated mice were reduced by 78.4, 77.8, 72.3 and 73.9%, respectively, compared with untreated controls. DISCUSSION AND CONCLUSION: This study demonstrates that NOR inhibits NFAT activation in T-cells and alleviates AD-like inflammatory reaction in a DNCB-induced dermatitis model, highlighting NOR as a potential therapeutic agent for AD.

Evaluation of cytokine profile and HLA association in benznidazole related cutaneous reactions in patients with Chagas disease.

BACKGROUND: Benznidazole is the drug of choice for Chagas disease. The major drawback of this drug is the high adverse events rate, being cutaneous reactions the most frequent one, leading to definitive withdrawal of treatment in 15%-30% of patients. METHODS: Prospective observational study where adult Chagas disease patients accepting to receive benznidazole (100 mg/8 hours for 60 days) were included. The objective was to characterize the skin toxicity of benznidazole in patients with Chagas disease, determine the serum cytokine profile, and evaluate the potential association with specific HLA alleles and benznidazole concentration. Serum cytokine levels were measured at day 0, 15, and 60 of treatment. Class I and II HLA alleles were determined. When cutaneous reaction was detected, a skin biopsy was performed. Serum benznidazole concentration was determined at the time of cutaneous reaction, or at day 15 of treatment. RESULTS: Fifty-two patients were included, 20(38.5%) had cutaneous reaction, and median time of appearance was 9 days. Skin biopsies showed histopathological findings consistent with drug eruption. Patients with cutaneous drug-reaction had higher proportion of eosinophilia during treatment, and higher interleukin (IL)-5 and IL-10 serum concentrations at day 15 of treatment than those without cutaneous reaction. Treatment interruption (that included moderate-severe cutaneous reactions) was more frequent in patients carrying HLA-B*3505 allele (45.5% vs 15.4%, P = .033). No differences in benznidazole serum concentration were found. CONCLUSIONS: Benznidazole related cutaneous reaction rate is high, and it was produced by a delayed hypersensitivity reaction with a Th2 response. Carrying HLA-B*3505 allele could be associated with moderate-severe cutaneous reaction.

Studies of genotoxicity and mutagenicity of nitroimidazoles: demystifying this critical relationship with the nitro group

Nitroimidazoles exhibit high microbicidal activity, but mutagenic, genotoxic and cytotoxic properties have been attributed to the presence of the nitro group. However, we synthesised nitroimidazoles with activity against the trypomastigotes of Trypanosoma cruzi, but that were not genotoxic. Herein, nitroimidazoles (11-19) bearing different substituent groups were investigated for their potential induction of genotoxicity (comet assay) and mutagenicity (Salmonella/Microsome assay) and the correlations of these effects with their trypanocidal effect and with megazol were investigated. The compounds were designed to analyse the role played by the position of the nitro group in the imidazole nucleus (C-4 or C-5) and the presence of oxidisable groups at N-1 as an anion receptor group and the role of a methyl group at C-2. Nitroimidazoles bearing NO2 at C-4 and CH3 at C-2 were not genotoxic compared to those bearing NO 2 at C-5. However, when there was a CH3 at C-2, the position of the NO2 group had no influence on the genotoxic activity. Fluorinated compounds exhibited higher genotoxicity regardless of the presence of CH3 at C-2 or NO2 at C-4 or C-5. However, in compounds 11 (2-CH3; 4-NO2; N-CH2OHCH2Cl) and 12 (2-CH3; 4-NO2; N-CH2OHCH2F), the fluorine atom had no influence on genotoxicity. This study contributes to the future search for new and safer prototypes and provide.

Studies of genotoxicity and mutagenicity of nitroimidazoles: demystifying this critical relationship with the nitro group.

Nitroimidazoles exhibit high microbicidal activity, but mutagenic, genotoxic and cytotoxic properties have been attributed to the presence of the nitro group. However, we synthesised nitroimidazoles with activity against the trypomastigotes of Trypanosoma cruzi, but that were not genotoxic. Herein, nitroimidazoles (11-19) bearing different substituent groups were investigated for their potential induction of genotoxicity (comet assay) and mutagenicity (Salmonella/Microsome assay) and the correlations of these effects with their trypanocidal effect and with megazol were investigated. The compounds were designed to analyse the role played by the position of the nitro group in the imidazole nucleus (C-4 or C-5) and the presence of oxidisable groups at N-1 as an anion receptor group and the role of a methyl group at C-2. Nitroimidazoles bearing NO2 at C-4 and CH3 at C-2 were not genotoxic compared to those bearing NO 2 at C-5. However, when there was a CH3 at C-2, the position of the NO2 group had no influence on the genotoxic activity. Fluorinated compounds exhibited higher genotoxicity regardless of the presence of CH3 at C-2 or NO2 at C-4 or C-5. However, in compounds 11 (2-CH3; 4-NO2; N-CH2OHCH2Cl) and 12 (2-CH3; 4-NO2; N-CH2OHCH2F), the fluorine atom had no influence on genotoxicity. This study contributes to the future search for new and safer prototypes and provide.

Novel solid dispersions of benznidazole: preparation, dissolution profile and biological evaluation as alternative antichagasic drug delivery system.

Solid dispersions (SD) of benznidazole (BNZ) in sodium deoxycholate (NaDC) or low-substituted hydroxypropylcellulose (L-HPC) were developed by freeze-drying process to improve the solubility of this low water-soluble drug and consequently, its trypanocidal activity. Although the dissolution studies showed a progressive decrease in the release rate of BNZ when formulated in the presence of NaDC, the increase in the surfactant concentration resulted in a better trypanocidal profile on epimastigotes, as well as in an enhancement of the unspecific cytotoxicity. However, such an effect was not so evident on amastigotes and in vivo (blood-trypomastigotes), where high concentrations of surfactant (BNZ:NaDC ≥ 1:6) experimented a loss of activity, correlating this fact with the minor cession of BNZ these formulations accomplished in acidic locations (i.e., dissolution test medium). According to the in vitro results, we reformulated the promising SD-1:3 (IC50 epimastigotes = 33.92 ± 6.41 µM, IC50 amastigotes = 0.40 ± 0.05 µM and LC50 = 183.87 ± 12.30 µM) replacing NaDC by L-HPC, which achieved the fastest dissolution profile. This fact, together with the safety this carrier ensures (LC50 > 256 µM), prompted us to evaluate the cellulose SD in vivo, improving the effectiveness of its NaDC equivalent (%AUPC = 96.65% and 91.93%, respectively). The results compiled in the present work suggest these solid dispersions as alternative drug delivery systems to improve the limited chemotherapy of Chagas disease.

Alterations of mitochondria in liver but not in heart homogenates after treatment of rats with benznidazole.

The mitochondrial oxidative phosphorylation system was studied in liver and heart homogenates after treatment of rats with benznidazole. The drug was given by oral gavage to adult female Wistar rats for 9 consecutive days (100 mg benznidazole/kg body weight as a daily dose). The mitochondrial state 4 and state 3 respiration rates, respiratory control, efficiency of oxidative phosphorylation (ADP/O), and ATPsynthase activity were assayed. The results showed that according to all these parameters, the mitochondria in cardiac homogenates were not affected in the rats treated with benznidazole. By contrast, mitochondria in the liver homogenates of drug-treated rats were altered, showing decreased respiratory control and a lower coefficient of ADP/O as a result of an increase in the state 4 respiration rate. These data indicate the possibility of production of an uncoupling factor leading to increased proton leakage through the inner mitochondrial membrane as a result of a 9-day treatment of rats with benzonidazole. The obtained experimental data might at least partly explain the nature of benznidazole toxicity in the liver treated with benznidazole.

Selective activity of 2,4-diaryl-1,2,3,4-tetrahydroquinolines on Trypanosoma cruzi epimastigotes and amastigotes expressing ß-galactosidase.

The growth inhibitory effect on Trypanosoma cruzi epimastigotes and the unspecific cytotoxicity over NCTC-929 fibroblasts of two series of previously synthesized 2,4-diaryl-1,2,3,4-tetrahydroquinolines (THQ), have been studied in vitro and compared with those of benznidazole (BZ). Derivatives AR39, AR40, AR41, AR91 and DM15 achieved outstanding selectivity indexes (SI) on the extracellular form (SITHQ>SIBZ>9.44) and thus, were tested in a more specific in vitro assay against amastigotes, showing less effectiveness than the reference drug (SIBZ>320) but also accomplishing great selectivity on the intracellular stage (SITHQ>25). These promising results, supported by the in silico prediction of high bioavailability and less potential risk than benznidazole, reveal several tetrahydroquinolines as prototypes of potential antichagasic drugs.

Effect of chronic alcohol drinking on rat liver microsomal nitroreductive metabolism of nifurtimox and benznidazole.

Nifurtimox (Nfx) and benznidazole (Bz) have serious toxic side effects. Manufacturers warn about significant adverse effects when simultaneous alcohol consumption is being made, but its mechanism is not known. The levels and toxicity of these drugs are linked to their liver microsomal nitroreduction to reactive metabolites. In this study, we analyzed whether alcohol drinking enhanced those nitroreductive processes. Male and female Sprague-Dawley rats, 5-6 weeks old (125-150 g body weight) were used. They were fed ad libitum for 28 days with Lieber and De Carli control or alcohol regular liquid diets. The rats were separated into two dietary groups: ethanol and control group. Both were pair fed with the respective diet. Their liver microsomes were isolated and the nicotinamide adenine dinucleotide phosphate-dependent nitroreduction of Nfx and Bz were determined. Alcohol drinking significantly induced microsomal nitroreduction of these drugs in male rats (11% for Nfx and 41% for Bz) but not in females. The activity observed in the alcohol-induced male rats was 100% inhibited by diphenyleneiodonium and attributable to P450 reductase. Inductive effects of alcohol drinking on nitroreductive activation of both drugs might be only partially involved in the harmful interactions described.

Genotoxicity profile of fexinidazole--a drug candidate in clinical development for human African trypanomiasis (sleeping sickness).

The parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness, is a highly neglected fatal condition endemic in sub-Saharan Africa, which is poorly treated with medicines that are toxic, no longer effective or very difficult to administer. New, safe, effective and easy-to-use treatments are urgently needed. Many nitroimidazoles possess antibacterial and antiprotozoal activity and examples such as tinidazole are used to treat trichomoniasis and guardiasis, but concerns about toxicity including genotoxicity limit their usefulness. Fexinidazole, a 2-substituted 5-nitroimidazole rediscovered by the Drugs for Neglected Diseases initiative (DNDi) after extensive compound mining of public and pharmaceutical company databases, has the potential to become a short-course, safe and effective oral treatment, curing both acute and chronic HAT. This paper describes the genotoxicity profile of fexinidazole and its two active metabolites, the sulfoxide and sulfone derivatives. All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative. Thus, fexinidazole does not pose a genotoxic hazard to patients and represents a promising drug candidate for HAT. Fexinidazole is expected to enter Phase II clinical trials in 2012.

In vitro activity of N-benzenesulfonylbenzotriazole on Trypanosoma cruzi epimastigote and trypomastigote forms.

Chagas disease is still an important health problem in Central and South America. However, the only drugs currently available for specific treatment of this disease may induce toxic side effects in the host. The aim of this work was to determine the activity of N-benzenesulfonylbenzotriazole (BSBZT) against the protozoan parasite Trypanosoma cruzi. The effects of BSBZT and benzotriazole (BZT) were compared to those of benznidazole (BZL) on epimastigote and trypomastigote forms. BSBZT was found to have an in vitro growth inhibitory dose-dependent activity against epimastigotes, with flow cytometry analysis confirming that the treated parasites presented size reduction. BSBZT showed an IC(50) of 21.56 µg/mL (81.07 µM) against epimastigotes at 72 h of incubation, whereas BZT did not affect the growth of this parasite form. Furthermore, the toxic effect of BSBZT, was stronger and appeared earlier (at 24h) in trypomastigotes than in epimastigotes, with the LC(50) of this compound being 28.40 µg/mL (106.79 µM) against trypomastigotes. The concentrations of BSBZT used in this study presented low hemolytic activity and cytotoxicity. Consequently, at concentrations near IC(50) and LC(50) (25µg/mL), BSBZT caused only 2.4% hemolysis and 15% of RAW 264.7 cell cytotoxicity. These results reveal the potential of BSBZT as a prototype in drug design for developing new anti-T. cruzi compounds.

Pharmacokinetics of TH-302: a hypoxically activated prodrug of bromo-isophosphoramide mustard in mice, rats, dogs and monkeys.

PURPOSE: To characterize the pharmacokinetics of the prodrug, TH-302, and its active metabolite, bromo-IPM (Br-IPM), in nonclinical species. METHODS: TH-302 was administered in single oral, intraperitoneal and intravenous bolus doses to mice, rats, dogs and monkeys as well as in acute and chronic safety studies in rats and dogs as a 30-min intravenous infusion given once a week for 3 weeks. Assessments were made using liquid chromatography-tandem mass spectrometry. RESULTS: TH-302 was extensively distributed with high systemic clearance exceeding hepatic plasma flow in all species studied, resulting in half-lives ranging between 8 min (mice) and over 4 h (rats). In rats, TH-302 exhibited linear kinetics following intravenous administration and good oral bioavailability. In acute and chronic safety studies, there was no accumulation of TH-302 following once weekly dosing for 3 weeks in the rat and dog. Br-IPM plasma concentrations were a small fraction of the TH-302 plasma concentrations with significantly smaller percentages present in dogs than in rats. Allometric scaling predicted that the systemic clearance and steady-state volume of distribution in humans would be 38.8 l/h/m(2) and 34.3 l/m(2), respectively, resulting in a terminal elimination half-life of about 36 min. These values were similar to those observed in patients with solid tumors (27.1 l/h/m(2), 23.5 l/m(2) and 47 min). CONCLUSIONS: TH-302 exhibited good safety, efficacy and pharmacokinetic properties in nonclinical species, translating into favorable properties in humans.

Metabolism, pharmacokinetics and excretion of a novel hypoxia activated cytotoxic prodrug, TH-302, in rats.

The metabolism, pharmacokinetics and excretion of a hypoxically activating prodrug developed for the treatment of cancer, TH-302, were studied in rats following intravenous administration of 50 mg/kg [(14)C]-TH-302. The pharmacokinetics of TH-302 was characterized by a short half-life of 12.3 min, a high clearance of 2.29 L/h/kg and a volume of distribution of 0.627 L/kg. In intact and bile duct-cannulated rats, TH-302 was extensively metabolized with total recovery in excreta of 68.1% and 85.8%, respectively, with equal amounts excreted through urine and bile. Quantitative whole body autoradiography showed rapid distribution of [(14)C]-TH-302 associated radioactivity with the highest concentrations in the kidney and small intestinal content, suggesting significant biliary excretion and/or gut secretion. TH-302 was metabolized via (i) hydrolysis to form 2-bromoethyl amine RM3 (7.5%); (ii) monoglutathione conjugation and subsequently to the mercapturic acid RM13 (7.5%); and (iii) diglutathione conjugation followed by hydrolysis to form the dicysteine conjugate RM5 (6.5%). A large percentage (19.7%) of the dose in the excreta was associated with unidentified polar metabolites RM1 and RM2. TH-302 was the predominant circulating component in plasma and the two major metabolites in plasma were the cysteine conjugate RM8 and mercapturic acid RM13.