Synthesis and anti-HIV activity of prodrugs derived from saquinavir and indinavir

With a view to improving the pharmacological properties, safety and pharmacokinetic profiles of current protease inhibitors, the synthesis of various acyl-substituted saquinavir and indinavir prodrugs, their in vitro stability with respect to hydrolysis and their anti-HIV (LAI and HTLV IIIB) activity and cytotoxicity in CEM-SS and MT4 cells have been investigated. Hydrolysis of the ester bond and liberation of the active free drug was found to be crucial for HIV inhibition: the faster the hydrolysis, the closer the anti-HIV activity was to that of the respective parent drug. This is the case for most of the C-14-substituted indinavir and saquinavir derivatives (IC50 from 10 to 360 nM for ester half-lives of 90 min to 40 h). Concomitantly, the level of HIV inhibition is very low for the prodrugs for which hydrolysis is very slow. This is the case with the myristoyl or oleyl saquinavir esters, owing to the stable masking of the hydroxyl that is part of the peptidomimetic non-cleavable transition state isostere responsible for the inhibitory potency of saquinavir (and indinavir). In contrast, the anti-HIV activity of the monosubstituted C-8 indinavir prodrugs seems not to be correlated with their resistance to hydrolysis, as expected (the C-8 hydroxyl of indinavir is not involved in the transition state isostere). No cytotoxicity was detected for the indinavir and saquinavir prodrugs for concentrations as high as 10 or even 100 microM, thus indicating promising therapeutic potential.     

Prevalence of methylenetetrahydrofolate reductase 677T and 1298C alleles and folate status: a comparative study in Mexican, West African, and European populations

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism is heterogeneously distributed worldwide, with the highest and lowest frequencies of the T allele in Mexico and Africa, respectively, and a south-to-north gradient in Europe. Distribution of MTHFR 1298A-->C is less well known. It has been hypothesized that 677T frequency could result in part from gene-nutrient interactions. OBJECTIVE: The objective was to compare the association of 677T and 1298C alleles with plasma concentrations of homocysteine, folate, and vitamin B-12 in geographical areas with contrasting 677T allele frequencies. DESIGN: Healthy young adults (n = 1277) were recruited in Mexico City, the West African countries of Bénin and Togo, France, and Sicily (Italy). Homocysteine, folate, and vitamin B-12 were measured in plasma, and MTHFR polymorphisms were measured in genomic DNA. RESULTS: Mexico City and Sicily reported the highest and Bénin and Togo reported the lowest plasma concentrations of folate. Mexico City had the highest 677T allele prevalence and the lowest influence of 677TT genotype on homocysteine, whereas the opposite was observed in Africa. The prevalence of the 1298C allele was lowest in the Mexicans and Africans and highest in the French. The percentage of the 677T genotype was significantly associated with the folate concentrations in 677CC carriers in a univariate analysis (R = 0.976; 95% CI: 0.797, 0.996; P < 0.0002) and in a multiple regression model that included homocysteine, vitamin B-12, and age (P = 0.0002). CONCLUSION: Our data agree with the hypothesis of a gene-nutrient interaction between MTHFR 677C-->T polymorphism and folate status that may confer a selective advantage of TT-homozygous genotype when dietary intake of folate is adequate, at least in the areas studied.     

The hamster cheek pouch as a model of oral cancer for boron neutron capture therapy studies: selective delivery of boron by boronophenylalanine.

Herein we propose and validate the hamster cheek pouch model of oral cancer for boron neutron capture therapy (BNCT) studies. This model serves to explore new applications of the technique, study the biology and radiobiology of BNCT, and assess the uptake of boron compounds and response of tumor, precancerous tissue, and clinically relevant normal tissues. These issues are central to evaluating and improving the therapeutic gain of BNCT. The success of BNCT is dependent on the absolute amount of boron in the tumor, and the tumor:blood and tumor:normal tissue boron concentration ratios. Within this context, biodistribution studies are pivotal. Tumors were induced in the hamsters with a carcinogenesis protocol that uses dimethyl-1,2-benzanthracene and mimics spontaneous tumor development in human oral mucosa. The animals were then used for biodistribution and pharmacokinetic studies of boronophenylalanine (BPA). Blood, tumor, precancerous pouch tissue surrounding tumor, normal pouch tissue, tongue, skin, cheek mucosa, palate mucosa, liver, and spleen, were sampled at 0-12 h after administration of 300 mg BPA/kg. The data reveal selective uptake of BPA by tumor tissue and, to a lesser degree, by precancerous tissue. Mean tumor boron concentration was 36.9 +/- 17.5 ppm at 3.5 h and the mean boron ratios were 2.4:1 for tumor:normal pouch tissue and 3.2:1 for tumor:blood. Higher doses of BPA (600 and 1200 mg BPA/kg) increased tumor uptake. Potentially therapeutic absolute boron concentrations, and tumor:normal tissue and tumor:blood ratios can be achieved in the hamster oral cancer model using BPA as the delivery agent.     

Reduced l-Arginine Transport and Nitric Oxide Synthesis in Human Umbilical Vein Endothelial Cells from Intrauterine Growth Restriction Pregnancies is Not Further Altered by Hypoxia

Intrauterine growth restriction (IUGR) is associated with chronic fetal hypoxia, altered placental vasodilatation and reduced endothelial nitric oxide synthase (eNOS) activity. In human umbilical vein endothelial cells (HUVEC) from pregnancies complicated with IUGR (IUGR cells) and in HUVEC from normal pregnancies (normal cells) cultured under hypoxia l-arginine transport is reduced; however, the mechanisms leading to this dysfunction are unknown. We studied hypoxia effect on l-arginine transport and human cationic amino acid transporters 1 (hCAT-1) expression, and the potential NO and protein kinase C α (PKCα) involvement. Normal or IUGR HUVEC monolayers were exposed (0–24 h) to 5% O₂ (normoxia), and 1 or 2% O₂ (hypoxia). l-Arginine transport and hCAT-1 expression, phosphorylated and total PKCα or eNOS protein and mRNA expression were quantified. eNOS involvement was tested using a siRNA against eNOS (eNOS-siRNA) adenovirus. IUGR cells in normoxia or hypoxia, and normal cells in hypoxia exhibited reduced l-arginine transport, hCAT-1 expression, NO synthesis and eNOS phosphorylation at Serine¹¹⁷⁷, effects reversed by calphostin C (PKC inhibitor) and S-nitroso-N-acetyl-l,d-penicillamine (SNAP, NO donor). However, N^G-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor) reduced hCAT-1 expression only in normal cells in normoxia. Increased Thr⁶³⁸-phosphorylated PKCα was exhibited by IUGR cells in normoxia or hypoxia and normal cells in hypoxia. The effects of hypoxia in normal cells were mimicked in eNOS-siRNA transduced cells; however, IUGR phenotype was unaltered by eNOS knockdown. Thus, IUGR- and hypoxia-reduced l-arginine transport could result from increased PKCα, but reduced eNOS activity leading to a lower hCAT-1 expression in HUVEC. In addition, IUGR endothelial cells are either not responsive or maximally affected by hypoxia. These mechanisms could be responsible for placental dysfunction in diseases where fetal endothelium is chronically exposed to hypoxia, such as IUGR.     

The relationship between urban airborne pollution and short-term mortality: Quantitative and qualitative aspects

The influence of airborne pollution on mortality rates has been examined since the well known severe pollution episodes of Pennsylvania (1948) and London (1952). Three main epidemiological approaches are available: transversal studies, time series studies, prospective studies. The most frequently used method is the time series analysis, which retrospectively determines daily mortality rates as compared to daily pollution levels in a defined period. The vast majority of studies applying this approach confirm the existence of a positive correlation between mortality and airborne pollution. The relative risk of death during the most severely polluted days as compared to the least polluted days ranges between 1.02 and 1.13. Airborne pollution affects mortality as related to cardiovascular and chronic obstructive pulmonary diseases mainly in subjects previously affected by these pathologies. The dose–response mortality–pollution curve shows linear increments of mortality at low pollution levels, weak increments at high pollution levels. This phenomenon is attributed to the existence of subjects highly susceptible to pollution induced adverse health effects. These subjects, dying at low pollution levels, are not available to increase mortality rates at high pollution levels. Mortality is mainly related to the concentration of single pollutants, such as suspended particle matter, sulfur oxides, nitric oxides and ozone. Elderly are highly susceptible to pollution-induced mortality increases. Therefore, the study of mortality–pollution correlation is of particular interest in cities characterized by a relative prevalence of the elderly in the population. The latency period between pollution increases and related mortality increases falls in the 1–3 days interval range. The pollution–mortality relationship is influenced by many factors, such as the occurrence of exceedingly high or low environmental temperatures, influenza epidemics, etc. Many hypotheses has been raised to support the causality of the mortality–pollution association, including a direct effect of pollutants on cardiovascular and respiratory apparatus, and the release of inflammatory mediators affecting blood viscosity and pneumocytes homeostasis. On the whole, available data suggest that pollution is able to increase mortality only if associated to other risk factors determining an increased susceptibility in the exposed population.