Metabolism and toxicity of two new benzodiazepine-type antileishmanial agents in hepatocytes and macrophages

With increasing reports of resistance of Leishmania to antimonials (Thakur et al., 2004) and other traditional antileishmanial drugs, the need for the discovery of new antileishmanial agents is rising. In an attempt to find new antileishmanial agents, two new benzodiazepine (BNZ) analogues (7-chloro-4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benodiazepine-2,5-dione (BNZ-1) and 4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-2)) were synthesised, and found to be effective against leishmaniasis in mice. This study investigates the metabolism of these two drugs together with the prototype BNZ, flurazepam (FZP), using rat hepatocytes, and investigates their interaction with glutathione in macrophages. Hepatocytes (>80% viability by Trypan Blue exclusion isolated by liver perfusion with collagenase) were prepared from male Sprague-Dawley rats (200-250 g). Drugs (100 μM) were incubated with 2 × 106 viable cells/ml in Krebs-Hepes buffer, pH 7.4, in rotating round bottomed flasks under an atmosphere of 95% O2/5% CO2, at 37 °C for 3 h, and timed samples taken for metabolite measurement. Samples were extracted twice with ethyl acetate at pH 10, the combined organic phases evaporated to dryness and stored at −20 °C until analysis. Metabolites were separated by HPLC using an ACE C18 column (50 mm × 3.0 mm i.d., 5 μm packing), and a solvent gradient consisting of 0.1% formic acid: acetonitrile (starting composition 95:5%, and composition after 25 min 65:35% for FZP and 70:30% for both BNZ 1 and 2). Flow rate was 0.5 ml/min, and detection was at 230 nm. Identification of the metabolites was by mass spectrometry with both positive and negative ion electronspray ionization. The effects of 24 h exposure to the compounds (100 μM) was investigated in the macrophage cell line J774.1 in terms of reduced glutathione content (GSH) and the activity of glutathione reductase (GR). There was no evidence of significant cytotoxicity with any of the compounds at the concentration used. FZP (m/z 388) was metabolised by dealkylation of the two N-1 ethyl substituents (m/z 360 and m/z 332), followed by hydroxylation on the BNZ ring. There was no evidence for either N- or O-glucuronidation of the resulting metabolites. BNZ-1 (m/z 321) was metabolised by N-demethylation (m/z 307) followed by hydroxylation (m/z 323), mono- and di-hydroxylation of the parent (m/z 337 and m/z 353) and by glucuronidation of the mono-hydroxylated metabolite (m/z 513). BNZ-2 (m/z 287) was transformed by N-demethylation (m/z 273) and hydroxylation of the parent (m/z 303), with the latter further metabolised by O-glucuronidation (m/z 479). In addition, the hydroxylated N-demethylated product (m/z 289) was also formed. Macrophages did not produce detectable metabolism of any of the drugs. All the drugs depleted macrophage GSH significantly (p < 0.05 by ANOVA followed by Dunnett's test) with BNZ-1 and BNZ-2 causing greater depletion than FZP (40.6 ± 4.0 and 45.8 ± 8.4, respectively, compared with 55.5 ± 4.9 nmol/mg protein with FZP, n = 3). Control macrophage GSH was 74.1 ± 6.6 nmol/mg protein. The depletion in GSH was not due to inhibition of GR: only FZP caused a significant decrease in macrophage GR activity (28.0 ± 5.9 compared with 42.1 ± 8.0 nmol/ml/min in control cells, p < 0.05 by ANOVA followed by Dunnett's test, n = 3). The marked depletion of macrophage GSH indicates a potential toxic interaction in mammalian cells. The new BNZ analogues were rapidly metabolised by hepatocytes, producing N-dealkylated and multiple hydroxylated phase I metabolites, followed by glucuronidation. This rapid metabolism may limit the therapeutic effect of BNZ 1 and 2 if their metabolites are inactive against leishmaniasis and suggest the need to optimise these lead structures further to obtain compounds with reduced rates and extent of metabolism

First Foods Nutrition Curriculum for New Immigrant Families: A Pilot Study

Background: Immigrant families arrive in the US from a variety of nutritional landscapes and educational experiences. Early childhood is a key time to intervene to set children on a healthy path. Creating nutritional education programs tailored for immigrant families may improve nutrition and health outcomes. Objective: To evaluate the First Foods curriculum as a tool for knowledge and behavior change for new immigrant families of young children. Methods: Immigrant caregivers of children less than 2 years old were invited to attend First Foods, a 4-class series. Each series was offered in 1 of 5 different languages (Arabic, Dari, Somali, Burmese, and Nepali). Recruitment occurred through community organizations, primary care clinics and Women, Infants, and Children (WIC), and classes were held in King County, Washington. The curriculum was developed and taught by a registered pediatric dietitian with input from general pediatricians, all experienced in the care of immigrant families. Classes were interpreted in the relevant language and course materials were translated. The classes were based on 4 themes -- 1) Child Eating and Development, 2) Eating Together, 3) Food Safety, and 4) Health Living -- and incorporated positive parenting and child development. Attendees completed pre- and post-surveys in their respective languages or in English. Descriptive statistics, chi-squared analyses, t-tests, and a multi-level linear regression model were conducted in Stata v14.0. Results: Participants in the classes included 47 caregivers (91% mothers). Nearly one-third had previously lived in a refugee camp. They had lived in the US a mean 5.5 years (95% CI: 3.8-7.2 years), attended a mean 8.6 years of school (95% CI: 7.1-10.1 years), and had a mean of 2.8 children (95% CI: 2.3-3.3 children). Classes ranged in size from 5 to 14 caregivers. Caregivers reported an improved understanding of 2 out of 4 methods to decrease risk of dental caries (drinking tap water, p = \u3c0.001; going to the dentist, p=0.02). They reported a decreased use of food as a reward from the pre- to the post-survey (p=0.027). Additionally, the caregivers reported increased frequency of considering sugar content in family foods (p=0.033), and decreased frequency of purchasing food at a convenience store, after participating in the curriculum (p=0.001). Conversely, there were several domains where caregivers did not show a change in their response. Conclusion: First Foods, a community-tailored, early childhood feeding curriculum for immigrant parents of young children, improved knowledge and behavior among caregivers from a variety of immigrant communities in some domains. In the other domains, there may be opportunities to further optimize the educational messages and approach

Prevention of Group B Streptococcal Disease in the Newborn.

Group B streptococcus (GBS) is a leading cause of morbidity and mortality among newborns. Universal screening for GBS among women at 35 to 37 weeks of gestation is more effective than administration of intrapartum antibiotics based on risk factors. Lower vaginal and rectal cultures for GBS are collected at 35 to 37 weeks of gestation, and routine dindamycin and erythromycin susceptibility testing is performed in women allergic to penicillin. Women with GBS bacteriuria in the current pregnancy and those who previously delivered a GBS-septic newborn are not screened but automatically receive intrapartum antibiotics. Intrapartum chemoprophylaxis is selected based on maternal allergy history and susceptibility of GBS isolates. Intravenous penicillin G is the preferred antibiotic, with ampicillin as an alternative. Penicillin G should be administered at least four hours before delivery for maximum effectiveness. Cefazolin is recommended in women allergic to penicillin who are at low risk of anaphylaxis. Clindamycin and erythromycin are options for women at high risk for anaphylaxis, and vancomycin should be used in women allergic to penicillin and whose cultures indicate resistance to clindamycin and erytbromycin or when susceptibility is unknown. Asymptomatic neonates born to GBS-colonized mothers should be observed for at least 24 hours for signs of sepsis. Newborns who appear septic should have diagnostic work-up including blood culture followed by initiation of ampicillin and gentamicin. Studies indicate that intrapartum prophylaxis of GBS carriers and selective administration of antibiotics to newborns reduce neonatal GBS sepsis by as much as 80 to 95 percent

Tailoring amide N-substitution to direct liquid crystallinity in benzanilide-based dimers

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