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Furins are serine endoproteases that are involved in many biological processes, where they play important roles in normal metabolism, in the activation of various pathogens, while they are a target for therapeutic intervention. Dichlorophenyl-pyridine "BOS" compounds are well known drugs that are used as inhibitors of human furin by an induced-fit mechanism, in which tryptophan W254 in the furin catalytic cleft acts as a molecular transition energy gate. The binding of "BOS" drug into the active center of furin has been computationally studied using the density functional theory (DFT) and ONIOM multiscaling methodologies. The binding enthalpies of the W254 with the furin-BOS is -32.8 kcal/mol ("open") and -18.8 kcal/mol ("closed"), while the calculated torsion barrier was found at 30 kcal/mol. It is significantly smaller than the value of previous MD calculations due to the relaxation of the environment, i.e., nearby groups of the W254, leading to the reduction of the energy demands. The significant lower barrier explains the experimental finding that the dihedral barrier of W254 is overcome. Furthermore, sartans were studied to evaluate their potential as furin inhibitors. Sartans are AT1 antagonists, and they effectively inhibit the hypertensive effects induced by the peptide hormone Angiotensin II. Here, they have been docked into the cavity to evaluate their effect on the BOS ligand via docking and molecular dynamics simulations. A consistent binding of sartans within the cavity during the simulation was found, suggesting that they could act as furin inhibitors. Finally, sartans interact with the same amino acids as W254, leading to a competitive binding that may influence the pharmacological efficacy and potential drug interactions of sartans.

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N-nitrosamines (NAs) are prevalent mutagenic impurities in various consumer products. Their discovery in valsartan-containing medicines in 2018 prompted global regulatory agencies to set guidelines on their presence and permissible levels in pharmaceuticals. In order to determine the NAs content in medicines, efficient and sensitive analytical methods have been developed based on mass spectrometry techniques. Direct analysis in real time-mass spectrometry (DART-MS) has emerged as a prominent ambient ionization technique for pharmaceutical analysis due to its high-throughput capability, simplicity, and minimal sample preparation requirements. Thus, in this study DART-MS was evaluated for the screening and quantification of NAs in medicines. DART-MS analyses were conducted in positive ion mode, for both direct tablet analysis and solution analysis. The analytical performance was evaluated regarding linearity, precision, accuracy, limits of detection, and quantification. The DART-MS proved to be suitable for the determination of NAs in medicines, whether through direct tablet analysis or solution analysis. The analytical performance demonstrated linearity in the range from 1.00 to 200.00 ng mL-1, limits of quantification about 1.00 ng mL-1, precision and accuracy lower than 15%, and no significant matrix effect for six drug-related NAs. In conclusion, the DART-MS technique demonstrated to be an alternative method to determine NAs in medicines, aligning with the principles of green chemistry.

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Introduction: Transforming growth factor ß (TGFß) metabolism plays an important role in the pathogenesis of Marfan syndrome (MFS). Accordingly, drug therapy uses TGFß receptor blockade to slow down the cardiovascular manifestations, above all aortic root dilatation. Angiotensin II type 1 receptor blockers (ARBs) have been shown to reduce TGFß levels in adults. Data on childhood are lacking and are now being investigated in the TiGer For Kids study presented here. Methods: We examined 125 children without chronic disease and 31 pediatric Marfan patients with a proven FBN1 variant with regard to TGFß levels. In addition, we measured TGFß levels during the initiation of ARB therapy in pediatric Marfan patients. Results: In children without chronic disease, TGFß levels were found to decrease from childhood to adolescence (p < 0.0125). We could not measure a relevantly increased TGFß level in pediatric Marfan patients. However, we showed a significant suppression of the TGFß level after treatment with ARBs (p < 0.0125) and a renewed increase shortly before the next dose. Discussion: The TGFß level in childhood changes in an age-dependent manner and decreases with age. The TGFß level drops significantly after taking ARBs. Based on our experience and data, a TGFß receptor blockade in childhood seems reasonable. So far, TGFß level cannot be used as an MFS screening biomarker.

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Nitrosamines (NAs) are molecules that include the nitroso functional group. In 2018, the US Food and Drug Administration (FDA) received its first report of NAs in pharmaceuticals. The fact that NA impurities are likely human carcinogens is relevant to these compounds. Furthermore, prolonged exposure to NA contaminants above safe limits may raise the risk of cancer. The goal of this article was to assess the amounts of six different NAs in Sartan group medicines purchased from formal pharmacies in Istanbul, Türkiye, using a validated LC-MS/MS assay. An LC-MS/MS-based analytical assay was undertaken. The separation was performed with a HR ODS 150mm×3.0mm and 5-analytical columns, providing effective separation of major peaks from NA impurities. In mobile phase A, formic acid was 0.10% in water, while in mobile phase B, formic acid was 0.10% in methanol. The flow rate was 0.4mL/minute, and the total runtime was 18minutes with the gradient elution mode. The validation was conducted in line with ISO/IEC 17025 requirements. Up to 100µg/L, linearity was determined using correlation coefficients (r2>0.995) for all NAs. The limit of quantification values for all NAs analyses were below 1.0µg/L. The mean recovery value obtained during the spike experiment was 95.18%, demonstrating the accuracy of the procedure. In addition, the accuracy was shown by a certified reference analysis, which yielded relative standard deviation and relative error values of 1.82% and 3.34%, respectively. During the intermediate precision testing, bias and relative standard deviation were 0.96 and 2.87%, respectively. Of the 75 study samples involving Sartan group medical products, no nitrosamine impurities were detected, demonstrating that pharmaceutical companies have adequate medication safety precautions in place in accordance with FDA and European Medicines Agency (EMA) regulations published to prevent NA contaminants in human medicinal products.

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Background: Lung toxicity of angiotensin receptor blockers (sartans) have very seldom been reported in the literature despite their wide use. We here report a case of interstitial lung disease elicited by sartans, with two episodes induced by two different sartans at 10 years of interval. Case presentation: In 2012, eprosartan was the very likely cause of a drug induced interstitial lung disease in a 60 year old man. Indeed, his symptoms, consisting in a MMRC2 dyspnea and recurrent hemoptysis, completely disappeared after the removal of this drug. When the circumstances rendered it necessary to start another angiotensin receptor blocker (namely valsartan) ten years later we did not expect the same reaction to occur given among other things a very poor literature on the topic. After a few months with this medication, he however developed similar symptoms and a Chest CT imaging that was comparable to what he had in 2012.This time also the clinical picture resolved completely when the sartan was stopped. Conclusion: We report this first case of a drug induced interstitial lung disease induced by two different angiotensin receptor blockers (sartans) with a new drug challenge ten years after the first one.

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AIMS: In genetic aortopathies (GA) particular attention is paid to aortic root dilatation which has an impact on morbidity and mortality. This study focuses on the effects of therapy with angiotensin-II-receptor-blockers (ARB) or beta-blockers (BB) on aortic root growth and the question which therapy should be initiated at which dosage and at what age. METHODS: Since 1998 we diagnosed 208 patients with GA (170 FBN-1). 81 patients between 5 months and 18 years receiving either ARB or BB therapy were included. We retrospectively analyzed the progression of the dilatation of Sinus Valsalva aortae (SV) using calculated z-scores before and after therapy initiation and compared BB and ARB treatment. RESULTS: Both ARB and BB (p < 0.05) therapy showed significant improvement in aortic root growth, while the effect is significantly more pronounced in ARB (p < 0.01) independent of age and genetic cause. A detailed comparison of the two drug groups showed a more sustained effect in limiting the progression of the dilatation of the aortic root in patients treated with ARB. Progression of dilatation of the SV was significantly lower in children treated with ARBs compared to BB (delta z-score, p < 0.05). In addition, ARBs were better tolerated and had a significantly lower discontinuation rate (3%) compared to BB (50%) (p < 0.01). Independently of age at initiation all children and adolescents were able to reach the target dose under ARB. CONCLUSION: We demonstrated a significant change in both treatment options, with the effect of ARB being more pronounced while being better tolerated throughout the treatment period.

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N-nitrosamines (NA) impurities have unexpectedly been found in sartan products, angiotensin II receptor antagonists that are used to control hypertension, representing an urgent concern for industry, global regulators and for the patients. In this study, an HPLC-MS/MS method was developed and validated for the quantification of six NA (N-nitrosodimethylamine, N-Nitroso-N-methyl-4-aminobutyric acid, N-Nitrosodiethylamine, N-ethyl-N-nitroso-2-propanamine, N-nitroso-diisopropylamine and N-nitroso-di-n-butylamine) in losartan, valsartan, olmesartan, irbesartan, candesartan and telmisartan products. The method was validated in terms of sensitivity, linearity, accuracy, precision, robustness and stability. The limits of quantification were 100, 31.25, 250, 33, 312.5 and 125 µg kg-1 in losartan, valsartan, olmesartan, irbesartan, candesartan and telmisartan samples, respectively, which met the sensitivity requirements for the limits set by Food and Drug Administration of the United States. The standard curves showed good linearity. The recoveries ranged from 93.06 to 102.23% in losartan matrix, 83 to 85.9% in valsartan, 96.1 to 101.2% in olmesartan, 89.2 to 97.5% in irbesartan, 93.4 to 132.0% in candesartan and 62.3 to 106.2% in telmisartan matrix. The other parameters met the validation criteria, the good sensitivity and precision, high accuracy and simple and fast analysis provides a reliable method for quality control of NA in sartan pharmaceutical products. The developed method was successfully applied for the determination of N-nitrosamines in 71 sartan products marketed in Brazil.

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N-nitroso-N-methyl-4-aminobutyric acid (NMBA) is the third N-nitrosamine impurity found in sartans. Herein, a sensitive and stable LC-MS/MS method with multiple reactions monitoring mode has been developed for the quantitative determination of NMBA in four sartan substances. The effective separation of NMBA and sartan substances was achieved on a C18 column under gradient elution conditions. The mass spectrometry method of the atmospheric pressure chemical ionization source and internal standard method was selected as the quantitative analysis method of NMBA. Then, this proposed LC-MS/MS analysis method was validated in terms of specificity, sensitivity, linearity, accuracy, precision and stability. Good linearity with correlation coefficient over 0.99 was obtained at the NMBA concentration of 3-45 ng/mL, and the limit of quantification was 3 ng/mL. Additionally, the recoveries of NMBA in four sartan substances ranged from 89.9% to 115.7%. The intra-day and inter-day relative standard deviation values were less than 5.0%. In conclusion, this developed determination method for NMBA through liquid chromatography-tandem mass spectrometry showed the characteristics of good sensitivity, high accuracy and precision, which will be of great help for the quantitative analysis of NMBA in sartan products.

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Two physical and climatic factors that were distant and recent prerequisites and a transformation trigger for a clone of the ancestral pseudotuberculous microbe Yersinia pseudotuberculosis O:1b (the causative agent of the Far East scarlet-like fever (FESLF)) into a population of the plague microbe derivative Y. pestis are considered. One remote prerequisite was the aridification of the Central Asian landscapes in the second half of the Cenozoic period and the formation of the Gobi Arid Zone. The arid conditions of Central Asia determined the formation of adaptive species-specific protective behavior in the Tarbagan marmot (Marmota sibirica) when installing the plug of a wintering hole, which later contributed to the massive infection of the animals with FESLF by the aberrant (traumatic, not alimentary) method during hibernation. A recent prerequisite and a real trigger of Y. pestis speciation was the onset of the last maximum (Sartan) ice age in Central Asia at the turn of the Pleistocene and Holocene, 22 000-15 000 years ago. Freezing of the cooling burrows of the Tarbagan marmot caused a behavioral shift in the larval population of the marmot flea Oropsylla silantiewi and the transition to the cold winter-spring months of the year from saprophagy in the nesting litter to hematophagy on the bodies of sleeping animals. Larval scarifications in the oral cavity of sleeping marmots have become the entrance gate for a unique traumatic FESLF infection. The constellation of climatic changes, the heterothermal (and, accordingly, heteroimmune) condition of the family groups of sleeping marmots, and the year-round propagation of marmot fleas in wintering burrows, combined with behavioral shifts in marmots and fleas caused by climatic changes, led to the formation of conditions in the parasitic system M. sibirica-O. silantiewi in which the transformation of the FESLF microbe into the causative agent of the plague occurred according to peripatric speciation. Thus, the climatic changes that happened at different times in the Cenozoic initially led to a shift in behavior of the Tarbagan marmot and, subsequently, to a shift in the behavior of the fleas parasitizing it. Ultimately, the change in the behavior of marmots and fleas caused the transition of the clone(s) of the FESLF causative agent into a new ecological niche and adaptive zone, as well as the transformation into a population(s) of the plague microbe.

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Background and aim: Tuberculosis (TBC) is a deadly disease and major health issue in the world. Emergence of drug resistant strains further worsens the efficiency of available anti-TBC drugs. Natural compounds and particularly traditional medicines such as Unani drugs are one of the promising alternatives that have been widely used nowadays. This study aims to evaluate the efficacy of unani drug Qurs-e-Sartan Kafoori (QSK) on Mycobacterium tuberculosis (MTB). Experimental procedures: Drug susceptibilities were estimated by broth microdilution assay. Cell surface integrity was assessed by ZN staining, colony morphology and nitrocefin hydrolysis. Biofilms were visualized by crystal violet staining and measurement of metabolic activity and biomass. Lipidomics analysis was performed using mass spectrometry. Host pathogen interaction studies were accomplished using THP-1 cell lines to estimate cytokines by ELISA kit, apoptosis and ROS by flow cytometry. Results: QSK enhanced the susceptibilities of isoniazid and rifampicin and impaired membrane homeostasis as depicted by altered cell surface properties and enhanced membrane permeability. In addition, virulence factor, biofilm formation was considerably reduced in presence of QSK. Lipidomic analysis revealed extensive lipid remodeling. Furthermore, we used a THP-1 cell line model, and investigated the immunomodulatory effect by estimating cytokine profile and found change in expressions of TNF-α, IL-6 and IL-10. Additionally, we uncover reduced THP-1 apoptosis and enhanced ROS production in presence of QSK. Conclusion: Together, this study validates the potential of unani formulation (QSK) with its mechanism of action and attempts to highlight its significance in MDR reversal.

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A genotoxic carcinogen, N-nitrosodimethylamine (NDMA), was detected as a synthesis impurity in some valsartan drugs in 2018, and other N-nitrosamines, such as N-nitrosodiethylamine (NDEA), were later detected in other sartan products. N-nitrosamines are pro-mutagens that can react with DNA following metabolism to produce DNA adducts, such as O6 -alkyl-guanine. The adducts can result in DNA replication miscoding errors leading to GC>AT mutations and increased risk of genomic instability and carcinogenesis. Both NDMA and NDEA are known rodent carcinogens in male and female rats. The DNA repair enzyme, methylguanine DNA-methyltransferase can restore DNA integrity via the removal of alkyl groups from guanine in an error-free fashion and this can result in nonlinear dose responses and a point of departure or "practical threshold" for mutation at low doses of exposure. Following International recommendations (ICHM7; ICHQ3C and ICHQ3D), we calculated permissible daily exposures (PDE) for NDMA and NDEA using published rodent cancer bioassay and in vivo mutagenicity data to determine benchmark dose values and define points of departure and adjusted with appropriate uncertainty factors (UFs). PDEs for NDMA were 6.2 and 0.6 µg/person/day for cancer and mutation, respectively, and for NDEA, 2.2 and 0.04 µg/person/day. Both PDEs are higher than the acceptable daily intake values (96 ng for NDMA and 26.5 ng for NDEA) calculated by regulatory authorities using simple linear extrapolation from carcinogenicity data. These PDE calculations using a bench-mark approach provide a more robust assessment of exposure limits compared with simple linear extrapolations and can better inform risk to patients exposed to the contaminated sartans.

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INTRODUCTION: Based on the observation of beneficial effects on cancer metabolism, microenvironment, or VEGF-signaling, several non-anticancer drugs have been discussed as useful in renal cell carcinoma (RCC). In the present study, we investigated the prognostic impact of concomitant medication in RCC and correlated comedication with cell-cycle and proliferation activity in corresponding surgical specimen. METHODS: A total of 388 patients who underwent surgery for localized RCC were included. The individual medication was evaluated according to substance classes. Tissue microarrays from corresponding tumor specimen were immunohistochemically (IHC) stained for Cyclin D1 and Ki67 and semi-quantitatively evaluated. Uni- and multivariate analyses were used to compare survival outcomes. For the comparison of IHC expression according to medication subgroups, Kruskal-Wallis analysis was performed. RESULTS: Median follow-up was 57.93 months (95% CI 53.27-69.43) and median OS accounted for 181.12 months (129.72-237.17). Univariate analysis identified pathological standard variables (T-stage > T2, Grading > G2, L1, N1, M1, sarcomatoid subtype, necrosis) as significant determinants of OS. Moreover, statin use (p = 0.009) and sartan use (p = 0.032) were significantly associated with improved OS. Multivariate analysis identified M1-stage (p < 0.001), statin and sartan use (p = 0.003 and p = 0.033, respectively) as independent prognosticators of survival. Expression of Ki67 was significantly reduced in patients with statin use (p = 0.013), while Cyclin D1 expression showed no correlation with comedication. CONCLUSIONS: Concomitant intake of statins and sartans identifies as an independent predictor of OS in RCC, and reduced Ki67 expression was significantly associated with statin use. Further evaluation of drug repurposing approaches with these substances in RCC appear warranted.

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Quaternary amine functionalized metal-organic framework MIL-101(Cr) (MIL-101(Cr)-NMe3) was prepared as the sorbent for the magnetic solid-phase extraction (MSPE) of azide from sartan drugs before ion chromatography determination. Magnetization of MIL-101-NMe3 were achieved concurrently by adding MIL-101-NMe3 and Fe3O4@SiO2 to the sample solution under ultrasonication. The prepared Fe3O4@SiO2/MIL-101-NMe3 gave the adsorption capacity of 37.5mgg-1. The developed method had a detection limit of 0.24µgL-1 and quantitation limit of 0.79µgL-1 for azide. The relative standard deviations for the intra-day retention time and peak area were 0.52% and 0.36% (n=5), respectively. The developed method was successfully applied for the determination of azide in sartan drugs with the recoveries from 96.5% to 100.5%.

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OBJECTIVE:To establish the method for simultaneous determination of related substances in Amlodipine hydrochlo-rothiazide and valsartan tablets. METHODS:HPLC method was used. The determination was performed on Symmetry C18 column with mobile phase consisted of water-acetonitrile-trifluoroacetic acid(90:10:0.1,V/V/V)and water-acetonitrile-trifluoroace-tic(10:90:0.1,V/V/V)(gradient elution)at the flow rate of 0.6 mL/min. The detection wavelength was set at 237 nm(impurity A) and 225 nm(impurity B,C,D). The column temperature was 30 ℃. RESULTS:The linear range of impurity A,B,C,D were 93.43-987.34 ng/mL(r=0.9994),12.27-255.92 ng/mL(r=0.9996),78.96-657.17 ng/mL(r=0.9999),28.39-218.16 ng/mL(r=0.9997),respectively. The limits of quantitation were 91.27,11.35,78.31,26.56 ng;the limits of stability and detection were 22.98,3.13,19.17,8.16 ng. RSDs of precision tests were lower than 2.0%. Only impurity A was found in repeatability test,RSD=0.79%. Recoveries were 99.4%-100.6%(RSD=0.41%,n=9),98.9%-102.0%(RSD=1.04%,n=9),99.4%-100.9%(RSD=0.56%,n=9),98.6%-101.2%(RSD=0.92%,n=9),respectively. CONCLUSIONS:The method is simple,rapid,sensitive and repeatable,and can be used for the determination of related substance in Amlodipine hydrochlorothiazide and valsartan tablets.

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AIMS: To evaluate the benefit of adding Losartan to baseline therapy in patients with Marfan syndrome (MFS). METHODS AND RESULTS: A double-blind, randomized, multi-centre, placebo-controlled, add on trial comparing Losartan (50 mg when <50 kg, 100 mg otherwise) vs. placebo in patients with MFS according to Ghent criteria, age >10 years old, and receiving standard therapy. 303 patients, mean age 29.9 years old, were randomized. The two groups were similar at baseline, 86% receiving ß-blocker therapy. The median follow-up was 3.5 years. The evolution of aortic diameter at the level of the sinuses of Valsalva was not modified by the adjunction of Losartan, with a mean increase in aortic diameter at the level of the sinuses of Valsalva of 0.44 mm/year (s.e. = 0.07) (-0.043 z/year, s.e. = 0.04) in patients receiving Losartan and 0.51 mm/year (s.e. = 0.06) (-0.01 z/year, s.e. = 0.03) in those receiving placebo (P = 0.36 for the comparison on slopes in millimeter per year and P = 0.69 for the comparison on slopes on z-scores). Patients receiving Losartan had a slight but significant decrease in systolic and diastolic blood pressure throughout the study (5 mmHg). During the study period, aortic surgery was performed in 28 patients (15 Losartan, 13 placebo), death occurred in 3 patients [0 Losartan, 3 placebo, sudden death (1) suicide (1) oesophagus cancer (1)]. CONCLUSION: Losartan was able to decrease blood pressure in patients with MFS but not to limit aortic dilatation during a 3-year period in patients >10 years old. ß-Blocker therapy alone should therefore remain the standard first line therapy in these patients.

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New preventive strategies for atherosclerosis are needed. In this study, we tested whether a new therapeutic approach consisting of low-dose treatment with a statin and sartan combination could prevent atherogenic diet-induced impairment of the arterial wall in guinea pigs. Twenty-five Dunkin-Hartley guinea pigs were randomly assigned to five experimental groups: 1) normal diet; 2) atherogenic diet (AD); 3) AD + a low-dose atorvastatin and valsartan combination (5mg/kg/day and 2.4mg/kg/day, respectively); 4) AD + low-dose atorvastatin (5mg/kg/day); 5) AD + low-dose valsartan (2.4mg/kg/day). After 8 weeks of treatment, the animals were killed, blood samples collected and thoracic and abdominal aortas isolated. The atherogenic diet significantly impaired maximal thoracic aorta endothelium-dependent relaxation by 40.1% relative to the normal diet. The low-dose combination, compared to the separate drugs, completely preserved thoracic aorta endothelium-dependent relaxation at the level of the group receiving normal diet. This substantial effect was associated with a significant change in the expression of NOS3 (R=0.93; P=0.0002) and IL1b (R=-0.79; P=0.003) genes. In addition, treatment with the low-dose combination or the separate drugs also prevented atherosclerotic plaque formation. We found that treatment with the low-dose atorvastatin and valsartan combination has the capability to completely protect the arterial wall from atherogenic diet-induced damage in the guinea pig model. Further studies evaluating this new therapeutic approach are desirable.

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Renin-angiotensin-system (RAS) activation plays a key role in the development of hypertension and cardiovascular disease. Drugs that antagonize the RAS (angiotensin-converting enzyme [ACE] inhibitors and angiotensin receptor blockers [ARBs]) have proven clinical efficacy in reducing blood pressure values and cardiovascular morbidity and mortality. ACE inhibitors partially inhibit plasma ACE, and angiotensin II generation. Thus, ARBs, which block selectively type 1 angiotensin II receptor (AT(1)R), have been developed and used in the clinical management of hypertension and cardiovascular disease. Experimental and clinical trials with ARBs indicate that this class of drug represents an effective, safe and well tolerated therapeutic option for the prevention and care of hypertension, even though there is no proven superiority as compared to ACE inhibitors except for the better tolerability. Most ARBs may not completely inhibit the AT(1)R at the approved clinical doses. Azilsartan medoxomil is a newly approved ARB for the management of hypertension. This ARB induces a potent and long-lasting antihypertensive effect and may have cardioprotective properties. This article reviews the current evidence on the clinical effectiveness of azilsartan in hypertension.