An efficient method for synthesis, characterization and molecular docking study of new sulfamethoxazole derivatives as antibacterial agents.

A new series of sulfamethoxazole derivatives bearing some biologically active heterocycles such as pyrazole (2), 3,4-dihydropyrimidin (3-7, 11, 12), pyrrole (9) and 1,3-dihydropyrimidin (10) rings were successfully synthesized. Identification of designed compounds was done by physicochemical properties and spectral measurements (1H-NMR, 13C-NMR and FT-IR). New prepared derivatives were assay for their (in vitro) antibacterial efficacy against four types of pathogenic bacterial isolates. Significant of the newly prepared compounds appeared promising activity comparison to the cephalexin standard drug. Most of the active compounds are docked into the effective site of tested bacterial enzymes obtained by crystal structure; results reveal the binding template to enzymes of bacteria, which closely related to the laboratory results.

Occurrence and human health risk assessment of pharmaceuticals and personal care products in real agricultural systems with long-term reclaimed wastewater irrigation in Beijing, China.

Reclaimed wastewater (RW) is increasingly used to irrigate agricultural land and to alleviate agricultural water shortages worldwide. This usage has resulted in concerns about soil contamination by pharmaceuticals and personal care products (PPCPs) and the human health risks associated with dietary crop intake. In this study, we systematically analysed the occurrence and accumulation of 11 PPCPs and one active metabolite in soils and various crops (cucumber, eggplant, long bean and wheat) from realistic RW irrigation fields with different irrigation histories (20, 30 and 40 years) in Beijing and evaluated the human health risks associated with the consumption of these crops. The 11 PPCPs and one active metabolite were detected at concentrations ranging from 0.67 to 22.92 ng L-1 in RW, 0.029-28.13 µg kg-1 in irrigated soil, and <0.01-28.01 µg kg-1 in crops. The concentrations of N4-acetyl-sulfamethoxazole and triclosan were higher than those of other PPCPs, with respective concentrations of 14.39-31.44 ng L-1 and 15.93-26.23 ng L-1 in RW, 10.92-23.29 µg kg-1 and 20.22-28.13 µg kg-1 in irrigated soil and 17.92-28.01 µg kg-1 and 8.92-14.91 µg kg-1 in crops. However, the estimated threshold of toxicological concern (TTC) and hazard quotient (HQ) values revealed that the concentrations of N4-acetyl-sulfamethoxazole and triclosan in crops irrigated with RW should be considered a de minimis risk to human health. The concentrations of 11 PPCPs and one active metabolite in soils and crops and the calculated fruit bioconcentration factors (BCFs) did not display obvious increases associated with the duration of RW irrigation in real agricultural systems (P > 0.05). The concentrations of the studied PPCPs in the RW used for irrigation followed different patterns from the concentrations detected in the irrigated soils and crops. Although the concentrations of sulfamethoxazole, sulfisoxazole, sulfamethazine and trimethoprim in RW were higher than those of many other studied PPCPs, their respective values in the irrigated soils and crops did not display a similar tendency. The uptake and accumulation of PPCPs varied among the crop species (P < 0.05). Although PPCPs were detected in eggplant, long bean and wheat (BCFs: not applicable-1.67, 0.03-1.35 and 0.01-5.01, respectively), PPCPs accumulated at increased levels in cucumber (BCFs 0.03-18.98). The estimated TTC and HQ values showed that the consumption of crops irrigated long-term with RW presents a de minimis risk to human health. However, further studies with more PPCPs and additional crop species need to be conducted, the synergistic effects of chemical mixtures of multiple PPCPs and the toxic effects of PPCP metabolites should be elucidated to obtain more reliable information on the safety of wastewater reuse for irrigation.

The Effect of Inhibitory Signals on the Priming of Drug Hapten-Specific T Cells That Express Distinct Vß Receptors.

Drug hypersensitivity involves the activation of T cells in an HLA allele-restricted manner. Because the majority of individuals who carry HLA risk alleles do not develop hypersensitivity, other parameters must control development of the drug-specific T cell response. Thus, we have used a T cell-priming assay and nitroso sulfamethoxazole (SMX-NO) as a model Ag to investigate the activation of specific TCR Vß subtypes, the impact of programmed death -1 (PD-1), CTL-associated protein 4 (CTLA4), and T cell Ig and mucin domain protein-3 (TIM-3) coinhibitory signaling on activation of naive and memory T cells, and the ability of regulatory T cells (Tregs) to prevent responses. An expansion of the TCR repertoire was observed for nine Vß subtypes, whereas spectratyping revealed that SMX-NO-specific T cell responses are controlled by public TCRs present in all individuals alongside private TCR repertoires specific to each individual. We proceeded to evaluate the extent to which the activation of these TCR Vß-restricted Ag-specific T cell responses is governed by regulatory signals. Blockade of PD-L1/CTLA4 signaling dampened activation of SMX-NO-specific naive and memory T cells, whereas blockade of TIM-3 produced no effect. Programmed death-1, CTLA4, and TIM-3 displayed discrete expression profiles during drug-induced T cell activation, and expression of each receptor was enhanced on dividing T cells. Because these receptors are also expressed on Tregs, Treg-mediated suppression of SMX-NO-induced T cell activation was investigated. Tregs significantly dampened the priming of T cells. In conclusion, our findings demonstrate that distinct TCR Vß subtypes, dysregulation of coinhibitory signaling pathways, and dysfunctional Tregs may influence predisposition to hypersensitivity.

HIV-1 tat expression and sulphamethoxazole hydroxylamine mediated oxidative stress alter the disulfide proteome in Jurkat T cells.

BACKGROUND: Adverse drug reactions (ADRs) are a significant problem for HIV patients, with the risk of developing ADRs increasing as the infection progresses to AIDS. However, the pathophysiology underlying ADRs remains unknown. Sulphamethoxazole (SMX) via its active metabolite SMX-hydroxlyamine, when used prophylactically for pneumocystis pneumonia in HIV-positive individuals, is responsible for a high incidence of ADRs. We previously demonstrated that the HIV infection and, more specifically, that the HIV-1 Tat protein can exacerbate SMX-HA-mediated ADRs. In the current study, Jurkat T cell lines expressing Tat and its deletion mutants were used to determine the effect of Tat on the thiol proteome in the presence and absence of SMX-HA revealing drug-dependent changes in the disulfide proteome in HIV infected cells. Protein lysates from HIV infected Jurkat T cells and Jurkat T cells stably transfected with HIV Tat and Tat deletion mutants were subjected to quantitative slot blot analysis, western blot analysis and redox 2 dimensional (2D) gel electrophoresis to analyze the effects of SMX-HA on the thiol proteome. RESULTS: Redox 2D gel electrophoresis demonstrated that untreated, Tat-expressing cells contain a number of proteins with oxidized thiols. The most prominent of these protein thiols was identified as peroxiredoxin. The untreated, Tat-expressing cell lines had lower levels of peroxiredoxin compared to the parental Jurkat E6.1 T cell line. Conversely, incubation with SMX-HA led to a 2- to 3-fold increase in thiol protein oxidation as well as a significant reduction in the level of peroxiredoxin in all the cell lines, particularly in the Tat-expressing cell lines. CONCLUSION: SMX-HA is an oxidant capable of inducing the oxidation of reactive protein cysteine thiols, the majority of which formed intermolecular protein bonds. The HIV Tat-expressing cell lines showed greater levels of oxidative stress than the Jurkat E6.1 cell line when treated with SMX-HA. Therefore, the combination of HIV Tat and SMX-HA appears to alter the activity of cellular proteins required for redox homeostasis and thereby accentuate the cytopathic effects associated with HIV infection of T cells that sets the stage for the initiation of an ADR.

Sulfamethoxazole derivatives complexed with metals: a new alternative against biofilms of rapidly growing mycobacteria.

Biofilms are considered important sources of infections on biomedical surfaces, and most infections involving biofilm formation are associated with medical device implants. Therefore, there is an urgent need for new antimicrobial compounds that can combat microbial resistance associated with biofilm formation. In this context, this work aimed to evaluate the antibiofilm action of sulfamethoxazole complexed with Au, Cd, Cu, Ni and Hg on rapidly growing mycobacteria (RGM), as well as to evaluate their safety through cytotoxic assays. The results demonstrate potentiation of the novel compounds in antibiofilm activity, mainly in the complex with Au, which was able to completely inhibit biofilm formation and had the capacity to destroy the biofilm at all the concentrations tested. All cytotoxic data suggest that the majority of sulfamethoxazole metallic derivatives are antimicrobial alternatives, as well as safe molecules, which could be used as potential therapeutic agents for bacterial and biofilm elimination.

Improvement of sulfamethoxazole (SMX) elimination and inhibition of formations of hydroxylamine-SMX and N4-acetyl-SMX by membrane bioreactor systems.

Sulfamethoxazole (SMX) has frequently been detected in aquatic environments. In natural environment, not only individual microorganism but also microbial consortia are involved in some biotransformation of pollutants. The competition for space under consortia causing cell-cell contact inhibition changes the cellular behaviors. Herein, the membrane bioreactor system (MBRS) was applied to improve SMX elimination thorough exchanging the cell-free broths (CFB). The removal efficiency of SMX was increased by more than 24% whether under the pure culture of A. faecalis or under the co-culture of A. faecalis and P. denitrificans with MBRS. Meanwhile, MBRS significantly inhibited the formation of HA-SMX, and Ac-SMX from parent compound. Additionally, the cellular growth under MBRS was obviously enhanced, indicating that the increases in the cellular growth under MBRS are possibly related to the decreases in the levels of HA-SMX and Ac-SMX compared to that without MBRS. The intracellular NADH/NAD+ ratios of A. faecalis under MBRS were increased whether thorough itself-recycle of CFB or exchanging CFB between the pure cultures of A. faecalis and P. denitrificans, suggesting that the enhancement in the bioremoval efficiencies of SMX under MBRS by A. faecalis is likely related to the increases in the NADH/NAD+ ratio. Taken together, the regulation of cell-to-cell communication is preferable strategy to improve the bioremoval efficiency of SMX.

Metal-based biologically active azoles and ß-lactams derived from sulfa drugs.

Metal complexes of Schiff bases derived from sulfamethoxazole (SMZ) and sulfathiazole (STZ), converted to their ß-lactam derivatives have been synthesized and experimentally characterized by elemental analysis, spectral (IR, (1)H NMR, (13)C NMR, and EI-mass), molar conductance measurements and thermal analysis techniques. The structural and electronic properties of the studied molecules were investigated theoretically by performing density functional theory (DFT) to access reliable results to the experimental values. The spectral and thermal analysis reveals that the Schiff bases act as bidentate ligands via the coordination of azomethine nitrogen to metal ions as well as the proton displacement from the phenolic group through the metal ions; therefore, Cu complexes can attain the square planner arrangement and Zn complexes have a distorted tetrahedral structure. The thermogravimetric (TG/DTG) analyses confirm high stability for all complexes followed by thermal decomposition in different steps. In addition, the antibacterial activities of synthesized compounds have been screened in vitro against various pathogenic bacterial species. Inspection of the results revealed that all newly synthesized complexes individually exhibit varying degrees of inhibitory effects on the growth of the tested bacterial species, therefore, they may be considered as drug candidates for bacterial pathogens. The free Schiff base ligands (1-2) exhibited a broad spectrum antibacterial activity against Gram negative Escherichia coli, Pseudomonas aeruginosa, and Proteus spp., and Gram positive Staphylococcus aureus bacterial strains. The results also indicated that the ß-lactam derivatives (3-4) have high antibacterial activities on Gram positive bacteria as well as the metal complexes (5-8), particularly Zn complexes, have a significant activity against all Gram negative bacterial strains. It has been shown that the metal complexes have significantly higher activity than corresponding ligands due to chelation process which reduces the polarity of metal ion by coordinating with ligands.

Negative regulation by PD-L1 during drug-specific priming of IL-22-secreting T cells and the influence of PD-1 on effector T cell function.

Activation of PD-1 on T cells is thought to inhibit Ag-specific T cell priming and regulate T cell differentiation. Thus, we sought to measure the drug-specific activation of naive T cells after perturbation of PD-L1/2/PD-1 binding and investigate whether PD-1 signaling influences the differentiation of T cells. Priming of naive CD4(+) and CD8(+) T cells against drug Ags was found to be more effective when PD-L1 signaling was blocked. Upon restimulation, T cells proliferated more vigorously and secreted increased levels of IFN-γ, IL-13, and IL-22 but not IL-17. Naive T cells expressed low levels of PD-1; however, a transient increase in PD-1 expression was observed during drug-specific T cell priming. Next, drug-specific responses from in vitro primed T cell clones and clones from hypersensitive patients were measured and correlated with PD-1 expression. All clones were found to secrete IFN-γ, IL-5, and IL-13. More detailed analysis revealed two different cytokine signatures. Clones secreted either FasL/IL-22 or granzyme B. The FasL/IL-22-secreting clones expressed the skin-homing receptors CCR4, CCR10, and CLA and migrated in response to CCL17/CCL27. PD-1 was stably expressed at different levels on clones; however, PD-1 expression did not correlate with the strength of the Ag-specific proliferative response or the secretion of cytokines/cytolytic molecules. This study shows that PD-L1/PD-1 binding negatively regulates the priming of drug-specific T cells. ELISPOT analysis uncovered an Ag-specific FasL/IL-22-secreting T cell subset with skin-homing properties.

HLA-DQ allele-restricted activation of nitroso sulfamethoxazole-specific CD4-positive T lymphocytes from patients with cystic fibrosis.

BACKGROUND: For certain HLA allele-associated drug hypersensitivity reactions, the parent drug has been shown to associate directly with the risk allele. In other forms of hypersensitivity, HLA risk alleles have not been identified and T cells are activated in an allele unrestricted manner. Chemically reactive drug metabolites bind to multiple proteins; thus, it is assumed that the derived peptide antigens interact with a number of HLA molecules to activate T cells; however, HLA restriction of the drug metabolite-specific T-cell response has not been studied. OBJECTIVE: To utilize T cells from sulfamethoxazole (SMX) hypersensitive patients with cystic fibrosis to examine the HLA molecules that interact with nitroso SMX (SMX-NO)-derived antigens. METHODS: T-cell clones were generated from 4 hypersensitive patients. Drug-specific proliferative responses and cytokine secretion were measured. Anti-human class I and class II antibodies were used to analyse HLA restriction. Antigen-presenting cells expressing different HLA molecules were used to determine the alleles involved in the presentation of SMX-NO-derived antigens to T cells. RESULTS: A total of 976 clones were tested for SMX-NO reactivity. Thirty-nine CD4+ clones were activated with SMX-NO and found to proliferate and secrete cytokines. The SMX-NO-specific response was blocked with an antibody against HLA-DQ. SMX-NO-specific responses were detected with antigen-presenting cells expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles. CONCLUSION AND CLINICAL RELEVANCE: HLA-DQ plays an important role in the activation of SMX-NO-specific CD4+ T cells. Detection of HLA-DQ allele-restricted responses suggests that T cells are activated by a limited repertoire of SMX-NO-modified peptides.

Potential Complementary Therapy for Adverse Drug Reactions to Sulfonamides: Chemoprotection Against Oxidative and Nitrosative Stress by TCM Constituents and Defined Mixtures.

PURPOSE: Our working hypothesis is that bioactive phytochemicals that are important constituents of Traditional Chinese Medicine and their defined mixtures have potential as complementary therapy for chemoprotection against adverse drug reactions whose toxicity is not related to the pharmacological action of the drug but where oxidative and nitrosative stress are causative factors. METHODS: In this investigation we measured cytotoxicity, lipid peroxidation, protein carbonylation and ROS/NOS-mediated changes in the disulfide proteome of Jurkat E6.1 cells resulting from exposure to sulfamethoxazole N-hydroxylamine with or without pre-treatment with low µM concentrations of baicalein, crocetin, resveratrol and schisanhenol alone and in defined mixtures to compare the ability of these treatment regimens to protect against ROS/RNS toxicity to Jurkat E6.1 cells in culture. RESULTS: Each of the Traditional Chinese Medicine constituents and defined mixtures tested had significant chemoprotective effects against the toxicity of ROS/RNS formed by exposure of Jurkat E6.1 cells to reactive metabolites of sulfamethoxazole implicated as the causative factors in adverse drug reactions to sulfa drugs used for therapy. At equimolar concentrations, the defined mixtures tended to be more effective chemoprotectants overall than any of the single constituents against ROS/RNs toxicity in this context. CONCLUSIONS: At low µM concentrations, defined mixtures of TCM constituents that contain ingredients with varied structures and multiple mechanisms for chemoprotection have excellent potential for complementary therapy with sulfa drugs to attenuate adverse effects caused by oxidative/nitrosative stress. Typically, such mixtures will have a combination of immediate activity due to short in vivo half-lives of some ingredients cleared rapidly following metabolism by phase 2 conjugation enzymes; and some ingredients with more prolonged half-lives and activity reliant on phase 1 oxidation enzymes for their metabolic clearance. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Reduction of sulfamethoxazole hydroxylamine (SMX-HA) by the mitochondrial amidoxime reducing component (mARC).

Under high dose treatment with sulfamethoxazole (SMX)/trimethoprim (TMP), hypersensitivity reactions occur with a high incidence. The mechanism of this adverse drug reaction is not fully understood. Several steps in the toxification pathway of SMX were investigated. The aim of our study was to investigate the reduction of sulfamethoxazole hydroxylamine (SMX-HA) in this toxification pathway, which can possibly be catalyzed by the mARC-containing N-reductive enzyme system. Western blot analyses of subcellular fractions of porcine tissue were performed with antibodies against mARC-1, mARC-2, cytochrome b5 type B, and NADH cytochrome b5 reductase. Incubations of porcine and human subcellular tissue fractions and of the heterologously expressed human components of the N-reductive enzyme system were carried out with SMX-HA. mARC-1 and mARC-2 knockdown was performed in HEK-293 cells. Kinetic parameters of the heterologously expressed human protein variants V96L, A165T, M187 K, C246S, D247H, and M268I of mARC-1 and G244S and C245W of mARC-2 and N-reductive activity of 2SF, D14G, K16E, and T22A of cytochrome b5 type B were analyzed. Western blot analyses were consistent with the hypothesis that the mARC-containing N-reductive enzyme system might be involved in the reduction of SMX-HA. In agreement with these results, highest reduction rates were found in mitochondrial subcellular fractions of porcine tissue and in the outer membrane vesicle (OMV) of human liver tissue. Knockdown studies in HEK-293 cells demonstrated that mARC-1 and mARC-2 were capable of reducing SMX-HA in cell metabolism. Investigations with the heterologously expressed human mARC-2 protein showed a higher catalytic efficiency toward SMX-HA than mARC-1, but none of the investigated human protein variants showed statistically significant differences of its N-reductive activity and was therefore likely to participate in the pathogenesis of hypersensitivity reaction under treatment with SMX.

Prevention of virus-induced type 1 diabetes with antibiotic therapy.

Microbes were hypothesized to play a key role in the progression of type 1 diabetes (T1D). We used the LEW1.WR1 rat model of Kilham rat virus (KRV)-induced T1D to test the hypothesis that the intestinal microbiota is involved in the mechanism leading to islet destruction. Treating LEW1.WR1 rats with KRV and a combination of trimethoprim and sulfamethoxazole (Sulfatrim) beginning on the day of infection protected the rats from insulitis and T1D. Pyrosequencing of bacterial 16S rRNA and quantitative RT-PCR indicated that KRV infection resulted in a transient increase in the abundance of Bifidobacterium spp. and Clostridium spp. in fecal samples from day 5- but not day 12-infected versus uninfected animals. Similar alterations in the gut microbiome were observed in the jejunum of infected animals on day 5. Treatment with Sulfatrim restored the level of intestinal Bifidobacterium spp. and Clostridium spp. We also observed that virus infection induced the expression of KRV transcripts and the rapid upregulation of innate immune responses in Peyer's patches and pancreatic lymph nodes. However, antibiotic therapy reduced the virus-induced inflammation as reflected by the presence of lower amounts of proinflammatory molecules in both the Peyer's patches and pancreatic lymph nodes. Finally, Sulfatrim treatment reduced the number of B cells in Peyer's patches and downmodulated adaptive immune responses to KRV, but did not interfere with antiviral Ab responses or viral clearance from the spleen, pancreatic lymph nodes, and serum. The data suggest that gut microbiota may be involved in promoting virus-induced T1D in the LEW1.WR1 rat model.

Direct photolysis of human metabolites of the antibiotic sulfamethoxazole: evidence for abiotic back-transformation.

The presence of potentially persistent and bioactive human metabolites in surface waters gives rise to concern; yet little is known to date about the environmental fate of these compounds. This work investigates the direct photolysis of human metabolites of the antibiotic sulfamethoxazole (SMX). In particular, we determined photolysis kinetics and products, as well as their concentrations in lake water. SMX, N-acetyl sulfamethoxazole, sulfamethoxazole ß-D-glucuronide, 4-nitroso sulfamethoxazole, and 4-nitro sulfamethoxazole were irradiated under various light sources and pH conditions. All investigated metabolites, except sulfamethoxazole ß-D-glucuronide were found to be more photostable than SMX under environmentally relevant conditions. Between two and nine confirmed photoproducts were identified for SMX-metabolites through ultraperformance liquid chromatography/high-resolution mass spectrometry. Interestingly, photolytic back-transformation to SMX was observed for 4-nitroso-SMX, indicating that this metabolite may serve as an environmental source of SMX. Moreover, two human metabolites along with SMX were regularly detected in Lake Geneva. The knowledge that some metabolites retain biological activity, combined with their presence in the environment and their potential to retransform to the parent compound, underlines the importance of including human metabolites when assessing the effects of pharmaceuticals in the environment.

Integrated model for food-energy-water (FEW) nexus to study global sustainability: The main generalized global sustainability model (GGSM).

Over the years, several global models have been proposed to forecast global sustainability, provide a framework for sustainable policy-making, or to study sustainability across the FEW nexus. An integrated model is presented here with components like food-web ecosystem dynamics, microeconomics components, including energy producers and industries, and various socio-techno-economic policy dimensions. The model consists of 15 compartments representing a simplified ecological food-web set in a macroeconomic framework along with a rudimentary legal system. The food-web is modeled by Lotka-Volterra type expressions, whereas the economy is represented by a price-setting model wherein firms and human households attempt to maximize their economic well-being. The model development is done using global-scale data for stocks and flows of food, energy, and water, which were used to parameterize this model. Appropriate proportions for some of the ecological compartments like herbivores and carnivores are used to model those compartments. The modeling of the human compartment was carried out using historical data for the global mortality rate. Historical data were used to parameterize the model. Data for key variables like the human population, GDP growth, greenhouse gases like CO2 and NOX emissions were used to validate the model. The model was then used to make long-term forecasts and to study global sustainability over an extended time. The purpose of this study was to create a global model which can provide techno-socio-economic policy solutions for global sustainability. Further, scenario analysis was conducted for cases where the human population or human consumption increases rapidly to observe the impact on the sustainability of the planet over the next century. The results indicated that the planet can support increased population if the per capita consumption levels do not rise. However, increased consumption resulted in exhaustion of natural resources and increased the CO2 emissions by a multiple of 100.

[Pharmacokinetics of sulfamethoxazole in healthy Han volunteers living at plain and in native Han and Tibetan healthy volunteers living at high altitude].

The paper is to report the pharmacokinetics of sulfamethoxazole in healthy Han volunteers living at plain (PH) and native Han and Tibetan healthy volunteers living at high altitude (HNH and HNT). After healthy volunteers were administrated orally cotrimoxazole tablets, plasma concentration of sulfamethoxazole and metabolite N4-acetylsulfamethoxazole was determined by RP-HPLC, and plasma concentration-time data were analyzed by DAS 2.0 software to get the related pharmacokinetic parameters. The main pharmacokinetic parameters t(1/2) of sulfamethoxazole in PH, HNH and HNT were, respectively, 9.30 +/- 1.11, 10.99 +/- 1.23 and 10.44 +/- 1.05 h; tmax were 1.4 +/- 0.3, 2.0 +/- 1.1 and 1.8 +/- 0.4 h; Cmax were 94.42 +/- 15.26, 89.33 +/- 7.67 and 87.43 +/- 11.61 micro x mL(-1); AUC(0-t) were 1202.5 +/- 238.3, 1 434.7 +/- 193.9 and 1302.8 +/- 103.0 microg x h x mL(-1); AUC(0-infinity) were 1240.7 +/- 255.3, 1511.5 +/- 211.9 and 1363.9 +/- 116.5 microg x h x mL(-1); CL were 1.01 +/- 0.22, 0.81 +/- 0.12 and 0.89 +/- 0.08 L x h(-1) x kg(-1); V were 13.27 +/- 1.73, 12.81 +/- 2.15 and 13.28 +/- 1.20 L x kg(-1). Sulfamethoxazole pharmacokinetic parameters of HNH and HNT were significantly different from that of PH. The t(1/2) was significantly higher and the CL was significantly lower in HNH and HNT than that in PH, and the AUC(0-infinity) was significantly lower in HNT compared with HNH. This study found significant changes in the disposition of sulfamethoxazole under the special environment of high altitude hypoxia. This finding may provide some references for clinical rational application of sulfamethoxazole in HNH and HNT.

Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction.

OBJECTIVES: NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. METHODS: Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. RESULTS: Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (P<0.0001, r=0.42; P=0.01, r=0.23, respectively), and expression of both proteins correlated with one another (P<0.0001; r=0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency. CONCLUSION: These studies indicate that although novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

Metabolic and chemical origins of cross-reactive immunological reactions to arylamine benzenesulfonamides: T-cell responses to hydroxylamine and nitroso derivatives.

Exposure to sulfamethoxazole (SMX) is associated with T-cell-mediated hypersensitivity reactions in human patients. T-cells can be stimulated by the putative metabolite nitroso SMX, which binds irreversibly to protein. The hydroxylamine and nitroso derivatives of three arylamine benzenesulfonamides, namely, sulfamethozaxole, sulfadiazine, and sulfapyridine, were synthesized, and their T-cell stimulatory capacity in the mouse was explored. Nitroso derivatives were synthesized by a three-step procedure involving the formation of nitro and hydroxylamine sulfonamide intermediates. For immune activation, female Balb-c strain mice were administered nitroso sulfonamides four times weekly for 2 weeks. After 14 days, isolated splenocytes were incubated with the parent compounds, hydroxylamine metabolites, and nitroso derivatives to measure antigen-specific proliferation. To explore the requirement of irreversible protein binding for spleen cell activation, splenocytes were incubated with nitroso derivatives in the presence or absence of glutathione. Splenocytes from nitroso sulfonamide-sensitized mice proliferated and secreted interleukin (IL)-2, IL-4, IL-5, and granulocyte monocyte colony-stimulating factor following stimulation with nitroso derivatives but not the parent compounds. Splenocytes from sensitized mice were also stimulated to proliferate with hydroxylamine and nitroso derivatives of the structurally related sulfonamides. The addition of glutathione inhibited the nitroso-specific T-cell response. Hydroxylamine metabolites were unstable in aqueous solution: Spontaneous transformation yielded appreciable amounts of nitroso and azoxy compounds as well as the parent compounds within 0.1 h. T-cell cross-reactivity with nitroso sulfonamides provides a mechanistic explanation as to why structurally related arylamine benzenesulfonamides are contraindicated in hypersensitive patients.

Combined ascorbate and glutathione deficiency leads to decreased cytochrome b5 expression and impaired reduction of sulfamethoxazole hydroxylamine.

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T-cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b (5) (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity toward SMX-NO and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO-related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue-drug adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b (5), as well as significant decreases in SMX-HA reduction in SMX-NO-treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in patients with AIDS with antioxidant depletion.

Comparative study of pharmaceutical properties of some new derivatives of sulfamethoxazole.

This paper reports the changes in various physical properties associated with the derivatization of sulfamethoxazole. The properties studied include moisture content, crystallanity, particle size distribution, porosity, flow, compressibility and compactability. It was found that the derivatives, salicylidene-sulfamethoxazole-Zn(II) • H2O and salicylidene-sulfamethoxazole-Cu(II) • H2O are crystalline substances. The moisture content was found to be highest in salicylidene-sulfamethoxazole-Zn(II) • H2O followed by salicylidene-sulfamethoxazole-Cu(II) • H2O and sulfamethoxazole. The copper complex contained only chemically bonded water, whereas the zinc complex contained both bonded and absorbed water, which affected the strength of the tablets prepared from the three materials accordingly. The particle size decreased on derivatization and complexation with metal ions and the trend was: sulfamethoxazole > salicylidene-sulfamethoxazole-Cu(II) • H2O > salicylidene-sulfamethoxazole-Zn(II) • H2O. This trend was represented by better compactability shown by salicylidene-sulfamethoxazole-Cu(II) • H2O and salicylidene-sulfamethoxazole-Zn(II) • H2O as compared with sulfamethoxazole. Salicylidene-sulfamethoxazole-Zn(II) • H2O had the highest porosity followed by salicylidene-sulfamethoxazole-Cu(II) • H2O, and sulfamethoxazole; this resulted in better compressibility behavior of the complexes. Thus it was observed that salicylidene-sulfamethoxazole-Cu(II) • H2O and salicylidene-sulfamethoxazole-Zn(II) • H2O formed stronger compacts. The values of angle of repose and flow rate show better flow properties for salicylidene-sulfamethoxazole-Cu(II) • H2O as compared with sulfamethoxazole and salicylidene-sulfamethoxazole-Zn(II) • H2O. It was concluded that derivatization substantially changed the pharmaceutical properties, which have important role to play in formulation of solid dosage form.

Role of Oxidative Stress in Hypersensitivity Reactions to Sulfonamides.

Antimicrobial sulfonamides are important medications. However, their use is associated with major immune-mediated drug hypersensitivity reactions with a rate that ranges from 3% to 4% in the general population. The pathophysiology of sulfa-induced drug hypersensitivity reactions is not well understood, but accumulation of reactive metabolites (sulfamethoxazole [SMX] hydroxylamine [SMX-HA] and SMX N-nitrosamine [SMX-NO]) is thought to be a major factor. These reactive metabolites contribute to the formation of reactive oxygen species (ROS) known to cause cellular damage and induce cell death through apoptosis and necroptosis. ROS can also serve as "danger signals," priming immune cells to mount an immunological reaction. We recruited 26 sulfa-hypersensitive (HS) patients, 19 healthy control subjects, and 6 sulfa-tolerant patients to this study. Peripheral blood monocytes and platelets were isolated from blood samples and analyzed for in vitro cytotoxicity, ROS and carbonyl protein formation, lipid peroxidation, and GSH (glutathione) content after challenge with SMX-HA. When challenged with SMX-HA, cells isolated from sulfa-HS patients exhibited significantly (P ≤ .05) higher cell death, ROS and carbonyl protein formation, and lipid peroxidation. In addition, there was a high correlation between cell death in PBMCs and ROS levels. There was also depletion of GSH and lower GSH/GSSG ratios in peripheral blood mononuclear cells from sulfa-HS patients. The amount of ROS formed was negatively correlated with intracellular GSH content. The data demonstrate a major role for oxidative stress in in vitro cytotoxicity of SMX reactive metabolites and indicate increased vulnerability of cells from sulfa-HS patients to the in vitro challenge.