Visualization of an Endogenous Mitochondrial Azoreductase Activity under Normoxic Conditions Using a Naphthalimide Azo-Based Fluorogenic Probe.

In this paper, we demonstrate the existence of an endogenous mitochondrial azoreductase (AzoR) activity that can induce the cleavage of N═N double bonds of azobenzene compounds under normoxic conditions. To this end, 100% OFF-ON azo-based fluorogenic probes derived from 4-amino-1,8-naphthalimide fluorophores were synthesized and evaluated. The in vitro study conducted with other endogenous reducing agents of the cell, including reductases, demonstrated both the efficacy and the selectivity of the probe for AzoR. Confocal experiments with the probe revealed an AzoR activity in the mitochondria of living cells under normal oxygenation conditions, and we were able to demonstrate that this endogenous AzoR activity appears to be expressed at different levels across different cell lines. This discovery provides crucial information for our understanding of the biochemical processes occurring within the mitochondria. It thus contributes to a better understanding of its function, which is implicated in numerous pathologies.

Efficacy and safety of olmesartan medoxomil-amlodipine besylate tablet in Chinese patients with essential hypertension: A prospective, single-arm, multi-center, real-world study.

There lacks real-world study with a large sample size assessing olmesartan medoxomil-amlodipine besylate (OM-AML) tablet. Therefore, this study aimed to evaluate the efficacy and safety of OM-AML tablet in patients with essential hypertension. Totally, 1341 patients from 36 medical centers with essential hypertension who took OM-AML (20/5 mg) tablet were analyzed in the current prospective, single-arm, multi-center, real-world study (SVK study). Seated systolic blood pressure (SeSBP) and seated diastolic blood pressure (SeDBP) at baseline, week (W)4 and W8 were measured. The mean (±SE) change of SeSBP/SeDBP was -10.8 ± 0.4/-6.6 ± 0.3 mmHg at W4 and -12.7 ± 0.5/-7.6 ± 0.3 mmHg at W8, respectively. At W4, 78.8% and 29.0% patients achieved BP target by China and American Heart Association (AHA) criteria; at W8, 84.7% and 36.5% patients reached blood pressure (BP) target by China and AHA criteria, accordingly. Meanwhile, 80.2% and 86.4% patients achieved BP response at W4 and W8, respectively. Home-measured SeSBP and SeDBP decreased from W1 to W8 (both p < .001). Besides, patients' and physicians' satisfaction were elevated at W8 compared with W0 (both p < .001). The medication possession rate was 94.8% from baseline to W4 and 91.3% from baseline to W8. The most common drug-related adverse events were nervous system disorders (4.6%), vascular disorders (2.6%), and general disorders and administration site conditions (2.3%) by system organ class, which were generally mild and manageable. In conclusion, OM-AML tablet is one of the best antihypertensive agents in patients with essential hypertension.

The enhancement effect of small molecule Lyb24 reveals AzoR as a novel target of polymyxin B.

Given the important role of polymyxin B (PB) in the treatment of drug-resistant Gram-negative bacterial infections, the emergence of PB resistance poses a serious threat to public health. Adjuvant development is a supplementary strategy that can compensate for the lack of novel antibiotics by protecting PB. In this study, we found a small molecule named Lyb24 that showed weak antibacterial activity (minimum inhibitory concentration ≥ 10 µg/ml) but potentiated and revitalized the efficacy of PB against Gram-negative pathogens, including mcr-1- and mgrB-deletion-mediated PB-resistant strains. Our results showed that Lyb24 inhibits the translational levels of genes associated with the modification of lipid A. In addition, Lyb24 increases the permeability, disrupts the integrity and induces the depolarization of the membrane. We further found that both Lyb24 and PB could directly bind to AzoR and inhibit its activity. Structural analysis showed that Lyb24 binds to the isoalloxazine ring of flavin mononucleotide (FMN) through pi-pi stacking and loop η4 of AzoR. A pneumonia model was used to confirm that the activity against clinical PB-resistant Klebsiella pneumoniae was enhanced due to Lyb24 on PB. In conclusion, we provide a potential therapeutic regimen by combining Lyb24 and PB to treat Gram-negative-resistant bacterial infections. Our findings not only explain the synergistic effect of Lyb24, but also expand our knowledge on the mechanism of action of PB.

Azo Reductase Activated Magnetic Resonance Tuning Probe with "Switch-On" Property for Specific and Sensitive Tumor Imaging in Vivo.

Cancer remains a threat to human health. However, if tumors can be detected in the early stage, then the effectiveness of cancer treatment could be significantly improved. Therefore, it is worthwhile to develop more sensitive and accurate cancer diagnostic methods. Herein, we demonstrated an azo reductase (AzoR)-activated magnetic resonance tuning (MRET) probe with a "switch-on" property for specific and sensitive tumor imaging in vivo. Specifically, Gd-labeled DNA1 (DNA1-Gd) and cyclodextrin-coated magnetic nanoparticles (MNP-CD) were employed as enhancer and quencher of MRET, respectively, while DNA2, an azobenzene (Azo) group-modified aptamer (AS1411), served as a linker between enhancer and quencher to construct the MRET probe of MNP@DNA(1-2)-Gd. In tumor tissues with high-level AzoR, the T1-weighted magnetic resonance signal of the MRET probe could be restored by intelligently regulating the switch from "OFF" to "ON" after activation with AzoR, thus accurately indicating the location of the tumor accurately. Moreover, the tumor with a 4 times smaller size than that of the normal tumor model could be imaged based on the proposed MRET probe. The as-proposed MRET-based magnetic resonance imaging strategy not only achieves tumor imaging accurately but also shows promise for early diagnosis of tumors, which might improve patients' survival rates and provide an opportunity for image-guided biomedical applications in the future.

Diverse but desolate landscape of gut microbial azoreductases: A rationale for idiopathic IBD drug response.

Prodrugs reliant on microbial activation are widely used but exhibit a range of efficacies that remain poorly understood. The anti-inflammatory compound 5-aminosalicylic acid (5-ASA), which is packaged in a variety of azo-linked prodrugs provided to most Ulcerative Colitis (UC) patients, shows confounding inter-individual variabilities in response. Such prodrugs must be activated by azo-bond reduction to form 5-ASA, a process that has been attributed to both enzymatic and non-enzymatic catalysis. Gut microbial azoreductases (AzoRs) are the first catalysts shown to activate azo-linked drugs and to metabolize toxic azo-chemicals. Here, we chart the scope of the structural and functional diversity of AzoRs in health and in patients with the inflammatory bowel diseases (IBDs) UC and Crohn's Disease (CD). Using structural metagenomics, we define the landscape of gut microbial AzoRs in 413 healthy donor and 1059 IBD patient fecal samples. Firmicutes encode a significantly higher number of unique AzoRs compared to other phyla. However, structural and biochemical analyses of distinct AzoRs from the human microbiome reveal significant differences between prevalent orthologs in the processing of toxic azo-dyes, and their generally poor activation of IBD prodrugs. Furthermore, while individuals with IBD show higher abundances of AzoR-encoding gut microbial taxa than healthy controls, the overall abundance of AzoR-encoding microbes is markedly low in both disease and health. Together, these results establish that gut microbial AzoRs are functionally diverse but sparse in both health and disease, factors that may contribute to non-optimal processing of azo-linked prodrugs and idiopathic IBD drug responses.

Exploration on the Cr(VI) resistance mechanism of a novel thermophilic Cr(VI)-reducing bacteria Anoxybacillus flavithermus ABF1 isolated from Tengchong geothermal region, China.

Hexavalent chromium resistance and reduction mechanisms of microorganism provide a critical guidance for Cr(VI) bioremediation. However, related researches are limited in mesophiles and deficient for thermophiles. In this work, a novel alkaline Cr(VI)-reducing thermophile Anoxybacillus flavithermus ABF1 was isolated from geothermal region. The mechanisms of Cr(VI) resistance and reduction were investigated. The results demonstrated that A. flavithermus ABF1 could survive in a wide temperature range from 50°C to 70°C and in pH range of 7.0-9.0. Strain ABF1 showed excellent growth activity and Cr(VI) removal performance when initial Cr(VI) concentration was lower than 200 mg L-1 . 93.71% of Cr(VI) was removed at initial concentration of 20 mg L-1 after 72 h. The majority of Cr(VI) was found to be reduced extracellularly by enzymes secreted by cells. XPS and Raman analysis further manifested that Cr2 O3 was the product of Cr(VI) reduction. Moreover, the Cr(VI) transportation-related gene cysP and Cr(VI) reduction-related gene azoR of A. flavithermus ABF1 played key roles in inhibiting Cr(VI) entering cells and promoting extracellular Cr(VI) reduction respectively. This work provides novel insight into the mechanisms of Cr(VI) resistance and detoxication of thermophiles, which leads to a promising alternative strategy for heavy metal bioremediation in areas with elevated temperature.

Sulphonated azo dye decolorization by Alcaligenes faecalis subsp. phenolicus MB207: Insights from laboratory and computational analysis.

Microbial decolorization of azo dyes, mediated by an enzymatic mechanism is an intricate cost-effective, and eco-friendly treatment method of genotoxic azo pollutants. Scientists are on the constant lookout for microbes, enzymes, and mechanisms that could aid remediation of the environment at a fast pace. Alcaligenes faecalis subsp. phenolicus MB207 is one such bacteria, consisting of azoreductase (AzoR) and laccase/multicopper oxidase enzyme responsible for sulphonated mono-azo dye (Methyl orange) and di-azo dye (Congo red) degradation. AzoR degrades dyes by a ping-pong setup while multicopper oxidase achieves this through a non-specified radical approach. We have coupled experimental analysis with bioinformatics for deciphering intricacies of this procedure in tiny scale enzymatic machines of this biotope. The degradation assays were followed by molecular docking of the enzyme-substrate complexes. Key anchoring bonds were detected and mapped H-bonding, electron exchange, ionic interactions, as well as hydrophobic interactions, provided insights into dye-enzyme and NADH-enzyme binding. This study establishes a foundation of the molecular basis of dye interaction with azoR and multicopper oxidase in A. facealis subsp. phenolicus MB207.

Role of AzoR, a LysR-type transcriptional regulator, in SmeVWX pump-mediated antibiotic resistance in Stenotrophomonas maltophilia.

BACKGROUND: The SmeVWX efflux pump of Stenotrophomonas maltophilia contributes to menadione (MD) tolerance and resistance to chloramphenicol, quinolones and tetracycline. The components of the SmeVWX efflux pump are encoded by a five-gene operon, smeU1VWU2X. We have previously demonstrated that the smeU1VWU2X operon is intrinsically unexpressed and inducibly expressed by MD via a SoxR- and SmeRv-involved regulatory circuit in S. maltophilia KJ. We also inferred that there should be other regulator(s) involved in MD-mediated smeU1VWU2X expression in addition to SoxR and SmeRv. OBJECTIVES: To identify novel regulator(s) involved in the regulation of MD-mediated smeU1VWU2X expression and elucidate the regulatory circuit. METHODS: A possible regulator candidate involved in the regulation of MD-mediated smeU1VWU2X expression was identified by a homologue search using the helix-turn-helix domain of SmeRv as a query. Gene expression was assessed using the promoter-xylE transcriptional fusion assay and quantitative RT-PCR. The impact of the regulator on SmeVWX pump-mediated functions was investigated via mutant construction and functional tests (antibiotic susceptibility and MD tolerance). RESULTS: AzoR (Smlt3089), a LysR-type transcriptional regulator, was investigated. In unstressed logarithmically grown cells, AzoR was abundantly expressed and functioned as a repressor, inhibiting the expression of the smeU1VWU2X operon. MD challenge attenuated azoR expression, thus derepressing the expression of the smeU1VWU2X operon in S. maltophilia KJ. AzoR down-regulation-mediated smeU1VWU2X expression was observed in quinolone-resistant and SmeVWX-overexpressing S. maltophilia clinical isolates. CONCLUSIONS: AzoR negatively regulates the expression of the smeU1VWU2X operon and SmeVWX pump-mediated antibiotic resistance in S. maltophilia.

Pancitopenia retardada secundaria a tratamiento con levofloxacino: a propósito de un caso / Delayed pancytopenia due to treatment with levofloxacin: A case report

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Enteropatía asociada a olmesartán: atención a un fenómeno iatrogénico emergente / Olmesartan-associated enteropathy: attention to an emerging iatrogenic phenomenon

El olmesartán es un antagonista del receptor tipo 1 de la angiotensina II utilizado habitualmente en el tratamiento de pacientes con hipertensión arterial. Recientemente se han descrito varios casos de enteropatía sprue-like asociados al uso de este fármaco, con afectación clínica importante y total remisión tras la retirada del mismo. Se presenta el caso de un varón de 64 años, con antecedentes de hipertensión arterial en tratamiento con olmesartan-amlodipino con clínica de diarrea y pérdida de peso severas, atrofia vellositaria en biopsia duodenal sin criterios de enfermedad celiaca y remisión completa del cuadro tras la supresión del olmesartán. Basado en los hallazgos del caso clínico presentado, se revisan las manifestaciones clínicas e histopatológicas así como el curso evolutivo de una posible causa de enteropatía farmacológica recientemente descrita (AU)

Olmesartan is an angiotensin II type 1 receptor blocker commonly used in the treatment of hypertension. Several cases of sprue-like enteropathy associated with the use of this drug have been described which, even with important signs and limitations for the patient, present a full recovery after discontinuing the use of olmesartan. The case of a 64 year-old patient is presented, diagnosed with hypertension, under treatment with olmesartan- amlodipine, with chronic diarrhoea and villous atrophy on intestinal biopsies without diagnostic criteria for celiac disease and with complete remission after suspending discontinuing the use of olmesartan. Based on the clinical features presented by the case reported, the clinical and anatomopathological findings are described as well as the evolution of drug-induced enteropathy (AU)

Tardive akathisia related to the anti-hypertensive agent Sevikar-a case report.

BACKGROUND: Tardive akathisia (TA) is a subtype of tardive syndrome, and its etiology is still uncertain. Sevikar is an anti-hypertensive agent containing both amlodipine and olmesartan, and has never been reported to have an adverse reaction in patients with tardive syndrome. CASE PRESENTATION: A 57-year-old woman who took Sevikar for hypertension for 10 years developed TA one and a half years before receiving any psychiatric treatment. After switching from Sevikar to bisoprolol, she reported obvious improvement in her akathisia. CONCLUSIONS: It is noteworthy that her TA developed before receiving any antidepressant medication, and that her TA improved after discontinuation of Sevikar. In light of these pharmacodynamic properties, it is therefore concluded that use of amlodipine and olmesartan might have caused TA in this patient. We reported this rare case to remind clinicians to be aware of possible akathisia when using amlodipine and olmesartan in combination as anti-hypertensive agents.

Combined treatment with olmesartan medoxomil and amlodipine besylate attenuates atherosclerotic lesion progression in a model of advanced atherosclerosis.

INTRODUCTION: Besides their blood pressure-lowering effects, olmesartan medoxomil and amlodipine besylate exhibit additional anti-inflammatory mechanisms in atherosclerosic disease. Most of the studies investigating the effects of atherosclerosis focused on early atherosclerotic lesions, whereas lesions in human disease, at the time when medical treatment is started, are already well established. Therefore, we set up a model of advanced atherosclerosis and investigated the effects of olmesartan medoxomil, amlodipine besylate, and the combination of both on atherosclerotic lesion size and lesion composition. MATERIALS AND METHODS: Olmesartan medoxomil (1 mg/kg/day), amlodipine besylate (1.5 mg/kg/day), and the combination of both was added to chow and was fed to apolipoprotein E-deficient (ApoE(-/-)) mice at 25 weeks of age. Mice were sacrificed after 25 weeks of drug administration and perfused with formalin. Innominate arteries were dissected out and paraffin embedded. Serial sections were generated, and lesion sizes and their composition - such as minimal thickness of the fibrous cap, size of the necrotic core, and presence of calcification - were analyzed. Electrophoretic mobility shift assays were used to detect DNA-binding activity of the transcription factor nuclear factor-kappa B (NF-κB) in aortic tissue. RESULTS: Treatment with the combination of olmesartan medoxomil and amlodipine besylate led to a significant reduction in atherosclerotic lesion size in ApoE(-/-) mice (olmesartan medoxomil/amlodipine besylate: 122,277±6,795 µm(2), number [n]=14; versus control: 177,502±10,814 µm(2), n=9; P<0.001). Treatment with amlodipine besylate (n=5) alone did not reach significance. However, a trend toward a decrease in lesion size in the amlodipine besylate-treated animals could be observed. In the histological analysis of atherosclerotic lesion composition, significantly thicker fibrous caps were found in treatment with amlodipine besylate (amlodipine: 5.12±0.26 µm, n=6; versus control: 3.98±0.18 µm, n=10; P<0.01). Furthermore, all sections revealed morphological signs of calcification, but no difference could be detected. Treatment with the combination of olmesartan medoxomil and amlodipine besylate showed no effect on lesion composition. Electrophoretic mobility shift assays of nuclear extracts demonstrated reduced activity of the transcription factor NF-κB when treated with olmesartan medoxomil, amlodipine besylate, or their combination, as compared to controls. CONCLUSION: Combined treatment with olmesartan medoxomil and amlodipine besylate attenuated atherosclerotic lesion progression, possibly due to anti-inflammatory mechanisms. Our data support the hypothesis that even in advanced atherosclerosis anti-inflammatory treatment, using angiotensin II type 1 receptor blockers and calcium channel antagonists of the dihydropyridine type can attenuate atherosclerotic lesion progression.

Bioequivalence evaluation of two amlodipine salts, besylate and orotate, each in a fixed-dose combination with olmesartan in healthy subjects.

A fixed-dose combination of amlodipine and olmesartan is used to treat high blood pressure in patients whose hypertension is not sufficiently controlled with either drug alone. The objective of this study was to evaluate the bioequivalence of two fixed-dose combinations, ie, amlodipine orotate/olmesartan medoxomil 10/40 mg and amlodipine besylate/olmesartan medoxomil 10/40 mg, in healthy subjects. A randomized, open-label, single-dose, two-sequence, two-period, crossover study was conducted in 30 healthy adult volunteers. Blood samples were collected for up to 72 hours post-dose in each period. Safety data included the results of physical examinations, clinical laboratory tests, vital signs, an electrocardiogram, and adverse events. For both amlodipine and olmesartan, the 90% confidence intervals for the geometric mean ratios of AUClast and time to peak plasma concentration fell within the bioequivalence acceptance criteria. The two fixed-dose combinations showed similar safety profiles. Amlodipine orotate/olmesartan medoxomil 10/40 mg was bioequivalent to amlodipine besylate/olmesartan medoxomil 10/40 mg.

The effect of dissolution medium, rotation speed and compaction pressure on the intrinsic dissolution rate of amlodipine besylate, using the rotating disk method

The aim of this study was to evaluate the effect of dissolution medium, rotation speed and compaction pressure on the intrinsic dissolution rate (IDR) of the antihypertensive drug amlodipine besylate, using the rotating disk method. Accordingly, a fractional factorial design (33-1) was used, employing dissolution media (water, phosphate buffer pH 6.8 and HCl 0.1 M), rotation speed (50, 75 and 100 rpm), and compaction pressure (1000, 1500 and 2000 psi) as independent variables. The assays were randomized and statistically compared using the Statistica(r) 11 software program. Significance testing (ANOVA) indicated that the dissolution medium had a considerable impact on the IDR of amlodipine besylate. Analysis of the linear and quadratic components of the variables led to the proposition of a mathematical model that describes the IDR as a function of the parameters studied. Conversely, the levels of compaction pressure and rotation speed employed during experimental planning were less relevant, especially when the assay was conducted in the HCl 0.1 M medium.

A finalidade do presente trabalho foi avaliar o efeito do meio de dissolução, velocidade de rotação e pressão de compactação na velocidade de dissolução intrínseca (VDI) do fármaco anti-hipertensivo besilato de anlodipino, usando o método do disco rotativo. Dessa forma, foi utilizado um planejamento experimental do tipo fatorial facionado (33-1) utilizando como variáveis independentes o meio de dissolução (água, HCl 0,1M e tampão fosfato pH 6,8), velocidade de rotação (50, 75 e 100 rpm) e pressão de compactação do fármaco (1000, 1500 e 2000 psi). Os ensaios foram randomizados e comparados estatisticamente pelo software Statistica(r) 11. A análise de variância (ANOVA) indicou que o meio de dissolução exerce considerável impacto na VDI do besilato de anlodipino. A análise das variáveis em seus componentes lineares e quadráticos permitiu a proposição de um modelo matemático que descreve a VDI em função dos parâmetros estudados. Por outro lado, os níveis de pressão de compactação e velocidade de rotação empregados exercem efeito menos relevantes, especialmente quando o ensaio é conduzido em HCl 0,1 M.

Avaliação da segurança e eficácia da combinação ramipril/ anlodipino versus anlodipino em monoterapia no tratamento da hipertensão arterial pela monitorização ambulatorial da pressão arterial: estudo ATAR-MAPA / Evaluation of security and efficiency of the combination ramipril/amlopidine versus amlopidine in monotherapy for the treatment of arterial hypertension by the outpatient monitoring of blood pressure: study ATAR-ABPM

O objetivo do estudo é comparar a eficácia e a tolerabilidade da combinação fixa ramipril/anlodipino e do anlodipino em monoterapia para o tratamento de hipertensão arterial. Após um período de duas semanas de retirada de anti-hipertensivos e uso de placebo (washout), 265 pacientes hipertensos com idades entre 40 e 79 anos foram randomizados para iniciar tratamento com 2,5/2,5 mg de ramipril/anlodipino em combinação fixa ou 2,5 mg de anlodipino, que foram titulados para 5/5 mg e 10/10 mg de ramipril/anlodipino, ou 5 e 10 mg de anlodipino, se necessário. No total, 131 pacientes foram randomizados para terapia combinada e 134 para monoterapia sem diferenças significativas entre os grupos nas características basais e nos níveis de pressão arterial (PA) inicial. A redução média da PA sistólica nos períodos do dia (20,36 ± 13,42 versus 15,86 ± 12,71 mmHg; p = 0,003) e da noite (17,6 ± 17,61 versus 14,09 ± 14,32 mmHg; p = 0,051), avaliada pela monitorização ambulatorial de pressão arterial (MAPA), foi significativamente maior no grupo tratamento com combinação fixa. A redução média da PA diastólica durante o dia à MAPA (11,28 ± 8,29 versus 8,96 ± 8,16 versus mmHg; p = 0,009) foi maior no grupo terapia combinada, mas não durante a noite (8,42 ± 11,16 mmHg versus 7,70 ± 8,63; p = 0,567). A redução média da PA sistólica e diastólica em 24 horas à MAPA também foi maior no grupo tratamento combinado. Ambas as opções terapêuticas promoveram redução significativa da PA sistólica e diastólica; porém, os resultados observados foram melhores no grupo de combinação fixa ramipril/anlodipino

This study aims to compare the efficacy and tolerability of a fixed-dose ramipril/amlodipine combination and amlodipine monotherapy for the treatment of hypertension. After a 2-week placebo washout, 265 hypertensive patients aged 40 to 79 years were randomized for 2.5/2.5 mg ramipril/amlodipine or 2.5 mg amlodipine, titrated to ramipril/amlodipine 5/5 mg and 10/10 mg , or amlodipine 5 and 10 mg, if necessary. A total of 131 patients were assigned to combination therapy, and 134 to monotherapy with no significant differences among them in basal characteristics and blood pressure (BP) levels at the ambulatory blood pressure monitoring (ABPM). Mean reduction in daytime (20.36 ± 13.42 versus 15.86 ± 12.71 mmHg; p = 0.003) and night-time systolic BP on ABPM (17.6 ± 17.61 versus 14.09 ± 14.32 mmHg; p = 0.051) were significantly higher in the combination therapy. Mean daytime diastolic BP reduction on ABPM (11.28 ± 8.29 versus 8.96 ± 8.16 versus mmHg; p = 0.009) was greater in the combination group, but not at night-time (8.42 ± 11.16 mmHg versus 7.70 ± 8.63; p = 0.567). Mean change in 24-h systolic and diastolic BP on ABPM were also greater in the combination treatment. Both treatments promoted a marked reduction in systolic and diastolic BP, and the results observed were better in the ramipril/amlodipine combination group.