Losartan Carboxylic Acid Research Articles

Density functional theory and enzyme studies support interactions between angiotensin receptor blockers and angiotensin converting enzyme-2: Relevance to coronavirus 2019

The binding affinities and interactions between eight drug candidates, both commercially available (candesartan; losartan; losartan carboxylic acid; nirmatrelvir; telmisartan) and newly synthesized benzimidazole-N-biphenyltetrazole (ACC519T), benzimidazole bis-N,Nʹ-biphenyltetrazole (ACC519T(2) and 4-butyl-N,N-bis([2-(2H-tetrazol-5-yl)biphenyl-4-yl]) methyl (BV6), and the active site of angiotensin-converting enzyme-2 (ACE2) were evaluated for their potential as inhibitors against SARS-CoV-2 and regulators of ACE2 function through Density Functional Theory methodology and enzyme activity assays, respectively. Notably, telmisartan and ACC519T(2) exhibited pronounced binding affinities, forming strong interactions with ACE2′s active center, favorably accepting proton from the guanidinium group of arginine273. The ordering of candidates by binding affinity and reactivity descriptors, emerged as telmisartan > ACC519T(2) > candesartan > ACC519T > losartan carboxylic acid > BV6 > losartan > nirmatrelvir. Proton transfers among the active center amino acids revealed their interconnectedness, highlighting a chain-like proton transfer involving tyrosine, phenylalanine, and histidine. Furthermore, these candidates revealed their potential antiviral abilities by influencing proton transfer within the ACE2 active site. Furthermore, through an in vitro pharmacological assays we determined that candesartan and the BV6 derivative, 4-butyl-N,N0-bis[20-2Htetrazol-5-yl)bipheyl-4-yl]methyl)imidazolium bromide (BV6(K+)2) also contain the capacity to increase ACE2 functional activity. This comprehensive analysis collectively underscores the promise of these compounds as potential therapeutic agents against SARS-CoV-2 by targeting crucial protein interactions.

Sartan blood pressure regulators in classical and biofilm wastewater treatment – Concentrations and metabolism

Sartans are a group of pharmaceuticals widely used to regulate blood pressure. Their concentration levels were monitored in 80 wastewater treatment plants (WWTP) in the Baltic Sea Region, reached from limit of detection up to 6 µg/L. The concentrations were significantly different in different countries, but consistent within the respective country. The degradation of sartans (losartan, valsartan, irbesartan) in moving bed biofilm reactors (MBBRs) that utilize biofilms grown on mobile carriers to treat wastewater was investigated for the first time, and compared with the degradation in a conventional activated sludge (CAS) treatment plant. The results showed the formation of six microbial transformation products (TPs) of losartan, four of valsartan, and four of irbesartan in biological wastewater treatment. Four of these metabolites have not been described in the literature before. Chemical structures were suggested and selected TPs were verified and quantified depending on availability of true standards. Valsartan acid was a common TP of losartan, valsartan, and irbesartan. Losartan and irbesartan also shared one TP: losartan/irbesartan TP335. Based on the mass balance analysis, losartan carboxylic acid is the main TP of losartan, and valsartan acid is the main TP of valsartan during the biotransformation process. For irbesartan, TP447 is likely to be the main TP, as its peak areas were two orders of magnitude higher than those of all the other detected TPs of this compound. The effects of adapting biofilms to different biological oxygen demand (BOD) loading on the degradation of sartans as well as the formation of their TPs were investigated. Compared to feeding a poor substrate (pure effluent wastewater from a CAS), feeding with richer substrate (1/3 raw and 2/3 effluent wastewater) promoted the metabolism of most compounds (co-metabolization). However, the addition of raw wastewater inhibited some metabolic pathways of other compounds, such as from losartan/irbesartan to TP335 (competitive inhibition). The formation of irbesartan TP447 did not change with or without raw wastewater. Finally, the sartans and their TPs were investigated in a full-scale CAS wastewater treatment plant (WWTP). The removal of losartan, valsartan, and irbesartan ranged from 3.0 % to 72% and some of the transformation products (TPs) from human metabolism were also removed in the WWTP. However, some of the sartan TPs, i.e., valsartan acid, losartan carboxylic acid, irbesartan TP443 and losartan TP453, were formed in the WWTP. Relative high amounts of especially losartan carboxylic acid, which was detected with concentrations up to 2.27 µg/L were found in the effluent.

Hydrochlorothiazide/Losartan Potassium Tablet Prepared by Direct Compression.

Hydrochlorothiazide (HCTZ)/losartan potassium (LOS-K) was used as a model drug to prepare compound tablets through the investigation of the compression and mechanical properties of mixed powders to determine the formulation and preparation factors, followed by D-optimal mixture experimental design to optimize the final parameters. The type and amount of lactose monohydrate (SuperTab®14SD, 19.53–26.91%), microcrystalline cellulose (MCC PH102, 32.86–43.31%), pre-gelatinized starch (Starch-1500, 10.96–15.91%), and magnesium stearate (0.7%) were determined according to the compressive work, stress relaxation curves, and Py value. Then, the compression mechanism of the mixed powder was investigated by the Kawakita equation, Shapiro equation, and Heckel analysis, and the mixed powder was classified as a Class-II powder. The compaction pressure (150–300 MPa) and tableting speed (1200–2400 Tab/h) were recommended. A D-optimal mixture experimental design was utilized to select the optimal formulation (No 1, 26.027% lactose monohydrate, 32.811% MCC PH102, and 15.462% pregelatinized starch) according to the drug dissolution rate, using Hyzaar® tablets as a control. Following oral administration in beagle dogs, there were no significant differences in bioavailability between the No. 1 tablet and the Hyzaar® tablet in HCTZ, losartan carboxylic acid (E-3174), and LOS-K (F < F0.05). Thus, formulation and preparation factors were determined according to the combination of the compression and mechanical properties of the mixed powder and quality of tablets, which was demonstrated to be a feasible method in direct powder compression.

Formulation and Pharmacokinetic Evaluation of Ethyl Cellulose/HPMC-Based Oral Expandable Sustained Release Dosage of Losartan Potassium.

Prolonged retention of losartan potassium in the upper gastrointestinal tract is anticipated to increase its absorption and exposure to CYP450 enzyme subfamilies, undertaking its conversion to more potent (10-40 times) active metabolite, losartan carboxylic acid (LCA). Consistent with this, hydroxypropyl methylcellulose K4M/ethyl cellulose-based novel expandable films (EFs) containing losartan potassium (LP) suitable for prolonged retention in the stomach were developed. The films were prepared by solvent casting method. USP type II dissolution apparatus (0.1N HCl, 37°C, 100rpm) was used to perform the dissolution testing (drug release, unfolding behavior, film integrity, erosion, and water uptake) of the films. In vivo pharmacokinetic studies were carried out in rabbits. An HPLC-UV method was used for the quantification of the drug and its active metabolite in plasma. These folded films placed inside hard gelatin capsule shells unfolded to full dimensions in dissolution medium and provided sustained drug release throughout 12h. The plasma drug concentration-time curves obtained from the in vivo studies were used to determine pharmacokinetic parameters, such as area under the plasma drug concentration-time curve(AUC), area under first moment curve(AUMC), mean residence time (MRT), Cmax, Tmax, t1/2, ke, and Fr in comparison with that of the market formulation, Cozaar®. The novel EFs significantly changed the pharmacokinetic parameters of the drug and its active metabolite. The apparent elimination rate constant (ke) significantly decreased, while MRT and elimination half-life (t1/2) increased in both cases. The relative bioavailabilities (Fr) of both LP and E3174 using the novel formulation were higher than that of Cozaar®.

Evaluation of Potential Herb-Drug Interactions Between Shengmai Injection and Losartan Potassium in Rat and In Vitro

Aim: The present study aimed to explore the potential herb-drug interactions (HDI) between Shengmai injection (SMI) and losartan potassium (LOS) based on the expression profiles of cytochromes P450 (CYP450) and drug transporters in rat and in vitro. Methods: Different concentrations of SMI were used to explore the influence of SMI on the antihypertensive efficacy of LOS in the hypertension rat model established by N (omega)-nitro-L-arginine methyl ester (L-NAME) for 4 weeks. Subsequently, the serum concentration levels of LOS and losartan carboxylic acid (EXP3174) were determined by Liquid Chromatography Mass Spectrometry (LC-MS) and pharmacokinetic analysis. Human liver microsomes, human multidrug resistance protein 1 (MDR1/P-gp), and breast cancer resistance protein (BCRP) vesicles, human embryonic kidney 293 cell line with stable expression of the organic anion transporting polypeptide 1B1 (HEK293-OATP1B1 cells) and mock-transfected HEK293 (HEK293-MOCK) cells were used to verify the effects of SMI on CYP450 enzymes and drug transporters in vitro. Results: Low, medium, and high concentrations of SMI increased the antihypertensive efficacy of LOS to varying degrees. The high dose SMI increased the half-life (t 1/2 ), the maximum plasma concentration (C max), the area under the plasma concentration-time curve (AUC) from time zero to the time of the last measurable plasma concentration (AUC 0-t ), AUC from time zero to infinity (AUC 0-∞ ), and mean residence time (MRT) values of LOS and decreased its apparent volume of distribution (Vd) and clearance (CL) values. The AUC 0-t , AUC 0-∞ , and MRT of LOS were increased, whereas the CL was decreased by the medium concentration of SMI. In addition, the high, medium, and low doses of SMI increased the relative bioavailability (Frel) of LOS. SMI exhibited no significant effects on the pharmacokinetics of EXP3174. In vitro, SMI exhibited different suppressive effects on the enzyme activity levels of CYP1A2 (6.12%), CYP2B6 (2.72%), CYP2C9 (14.31%), CYP2C19 (12.96%), CYP2D6 (12.26%), CYP3A4 (3.72%), CYP2C8 (10.00–30.00%), MDR1 (0.75%), OATP1B1(2.03%), and BCRP (0.15%). Conclusion: In conclusion, SMI improved the antihypertensive efficacy of LOS in the L-NAME-induced hypertension rat model by increasing the concentration of LOS, while leaving the concentration of EXP3174 intact. SMI affected the pharmacokinetic properties of LOS by decreasing the elimination of LOS. These effects might partly be attributed to the inhibition of the activities of CYP3A4, CYP2C9, and of the drug transporters (P-gp, BCRP, and OATP1B1) by SMI, which need further scrutiny.

Evaluation and analytical applicability of a novel volumetric absorptive microsampling strategy for adherence monitoring of antihypertensive drugs by means of LC-HRMS/MS

Volumetric absorptive microsampling (VAMS), an emerging microsampling technique, is expected to overcome some disadvantages of dried blood spots. This study aimed to develop and evaluate a VAMS-based strategy for quantifying ten frequently prescribed antihypertensive drugs (AHD) (amlodipine, bisoprolol, candesartan, carvedilol, lercanidipine, losartan carboxylic acid, metoprolol, nebivolol, telmisartan, valsartan) in finger prick blood (FPB) within the scope of adherence monitoring. The straightforward workflow consisted of VAMS tip hydration and subsequent precipitation. Samples were analyzed by using reversed phase ultra-high performance liquid chromatography coupled to orbitrap mass spectrometry operating in parallel reaction monitoring mode. The analytical procedure was successfully validated based on international recommendations for most of the analytes. Selectivity and within/between-run accuracy and precision were in accordance with the recommendations. Internal standard normalized matrix factor met recommended criteria for all analytes at HT 20%, 40%, and 60% except for amlodipine were the CV exceeded 15% at HT 20% (CV 18%). Dilution integrity was given for all substances, covering the quantification in the upper part of the therapeutic range of selected AHD. Long-term stability in VAMS tips was tested and revealed degradation of lercanidipine after one week of storage at 24 °C. A proof of concept of the analytical applicability was done by quantification of selected AHD in VAMS tips and matched plasma samples. Results revealed that determined concentration in FPB by VAMS and plasma cannot be used interchangeably, and thus that specific reference ranges have to be established. However, a novel VAMS application was implemented in the context of adherence monitoring for at least the investigated AHD.

Amlodipine interfere in serum albumin binding of loasrtan and its active metabolite losartan carboxylic acid (EXP- 1734): Contra-contemporary in vitro approaches

The combination therapy of Losartan (LOS) and amlodipine (AMLO) has proven to be beneficial as compared to either individual drug monotherapy. Nonetheless, the mechanism of a beneficial effect is yet to be established. Conversely, displacement in protein binding may lead to change in free drug concentration. The selected analytes were effectively separated on Thermo β-basic C18 (100×4.6 mm, 5 µm) using mobile phase consisting of 20 mM monobasic potassium phosphate and 0.2 % TEA with acetonitrile and iso-propyl alcohol in gradient mode. The in vitro protein binding experiments were performed under simulated physiological conditions using ultrafiltration as drug-protein binding model and displacement interactions were observed. Amlodipine, losartan and LCA bind more than 95 % to recombinant human serum albumin (rHSA), when studied individually, whereas, protein binding of LCA and Losartan carboxylic acid (LCA) were not affected when spiked concomitantly with AMLO, whereas, binding of Amlodipine to rHSA was decreased by 15-20 % under similar conditions at supra pharmacological concentration level of LOS and LCA. In in-vitro experiments about three to five fold increase in free AMLO concentration was observed in ultrafiltrate in presence of LOS and LCA. The albumin binding capacity of all the analytes was reduced nearly seven to eight-fold in the case of Glycosylated Human serum albumin (Gly-HSA). The in vitro protein binding studies with Gly-HSA have shown alteration in the corresponding albumin binding of AMLO, LOS and LCA.

In Vivo Endothelial NO Activation by Losartan Is Independent of Blood Pressure Lowering and EXP3179/EXP3174 Metabolites Yet Require First Passage Metabolism

Background and Purpose: Losartan is a well-established anti-hypertensive angiotensin II (AngII) receptor type 1 (ATR1) blocker (ARB) with unexpected therapeutic properties in a range of diseases not linked to hypertension, such as chronic lung diseases, Alzheimer's disease, muscular dystrophy and viral infections. This suggests that there is more to losartan than simple AngII-ATR1 inhibition. We have recently shown that losartan can unexpectedly activate endothelial function in vivo via the endothelial release of protective nitric oxide (NO), which is typically associated with aerobic exercise and vascular homeostasis. Hence, losartan activation of endothelial NO release may be behind its protective non-blood pressure-related effects, although how this occurs is unknown. Losartan is a prodrug with many active metabolites that might be behind some of its unexpected therapeutic properties. Losartan first-passage metabolism in the liver converts it to EXP3179 (losartan-carboxaldehyde) believed to be devoid of ATR1 blocking effects followed by further metabolism into EXP3174 (losartan-carboxylic acid), the main ATR1 blocker. Our goal is to determine whether first passage metabolism is required for losartan to activate endothelial NO release, or if bypassing liver metabolism via intraperitoneal (IP) injections of losartan metabolites EXP3179/3174 will result is similar biological activites, an indication that metabolites of losartan are not involved in this biological activity. Experimental Approach: C57B6/J mice were treated with 1) losartan potassium-salt (LOS-PS) for 2 weeks via drinking water (liver first passage-dependent; 0.6 g/L) or 2) daily IP injections (first passage-independent) of LOS-PS, EXP3179 or EXP3174 (10 mg/kg). Systemic BP was noninvasively measured by a tail cuff system in mice anesthetized with isoflurane. NO-dependent vasodilatory properties of losartan, EXP3179 and EXP3174 were evaluated in mouse aortic rings using dual-wire myograph systems. Key Results: When losartan was delivered in drinking water, NO-dependent vasorelaxation was increased by 57.6%. In stark contrast, IP injection of losartan resulted in unexpected increases in contractility by 124.9% compared to vehicle control, whereas IP EXP3179 and EXP3174 also increased vasoconstriction by 69.8% and 72.2% respectively. Vasodilatory effects of losartan were undetected in the presence of L-NAME (NO synthase inhibitor), confirming that losartan exerts its effects in a NO-dependent manner. Moreover, BP was decreased in all drug treated group of mice, confirming that all drugs were biologically active and blocking ATR1. Conclusion and implications: Our data unexpectedly show that losartan increases endothelial function in mice only when given in drinking water, which suggests that non-EXP3179/74 metabolites may play an important role in endothelial NO activation. This also suggests that losartan-induced BP lowering is separate from its endothelial NO activating properties.

CLINICAL IMPLICATION OF ASSESSING QUANTITATIVE PLASMA CONCENTRATIONS OF ANTIHYPERTENSIVE DRUGS

Objective: Dried blood spot (DBS) analysis is a novel analytical method for therapeutic drug monitoring (TDM) to identify non-adherence to antihypertensive drugs. This study was conducted to evaluate the clinical applicability of measuring drug concentrations of eight antihypertensive drugs, using DBS and venipuncture. Furthermore, this study aimed to provide more insight into the between-patient variability in drug concentrations. Design and method: In a previous study a TDM method for eight antihypertensive drugs and four of their metabolites (table 1) was validated in patients assumed to be adherent. The present data was retrieved from this validation study. False negative values from DBS compared to a venipuncture were determined to assess drug adherence. A Generalized Estimating Equation (GEE) was used to assess the influence of covariates including gender, dose, age, weight, and the time interval between drug intake and sampling on the Cplasma (drug concentration in plasma). Results: A total of 135 patients (mean age of 59 ± 12 years, 59% male) were included. The number of samples per drug ranged from 38 (enalaprilate) to 85 (hydrochlorothiazide). When drug concentrations from DBS and plasma were compared, no real false negative values were found. A high variability in Cplasma between patients was observed, especially at peak concentrations with a fold change reaching from 2.3 (canrenone) to 35.2 (losartan-carboxylic acid) (table 1). The time of intake was significantly related to the height of the Cplasma in 7 of the 8 measured drugs with a p-value &lt; 0.05 with the exception of hydrochlorothiazide, but the influence of dose, weight, age and sex on drug levels differed largely between the measured drugs. Conclusions: DBS is a reliable and convenient method to assess non-adherence to antihypertensive drugs in clinical practice. The Cplasma of the eight antihypertensive drugs in this study show a large inter-individual difference and therefore low plasma concentrations do not necessarily mean non-adherence. Non-adherence can only be confirmed if drug levels are undetectable, i.e. values below the lower limit of detection.

Simultaneous Determination of Amlodipine, Losartan, and the Losartan Carboxylic Acid Active Metabolite in Human Plasma by LC-MS/MS

Simultaneous Determination of Amlodipine, Losartan, and the Losartan Carboxylic Acid Active Metabolite in Human Plasma by LC-MS/MS

Target quantification of azole antifungals and retrospective screening of other emerging pollutants in wastewater effluent using UHPLC –QTOF-MS

The information acquired by high resolution quadrupole-time of flight mass spectrometry (QTOF-MS) allows target analysis as well as retrospective screening for the presence of suspect or unknown emerging pollutants which were not included in the target analysis. Targeted quantification of eight azole antifungal drugs in wastewater effluent as well as new and relatively simple retrospective suspect and non-target screening strategy for emerging pollutants using UHPLC-QTOF-MS is described in this work. More than 300 (parent compounds and transformation products) and 150 accurate masses were included in the retrospective suspect and non-target screening, respectively. Tentative identification of suspects and unknowns was based on accurate masses, peak intensity, blank subtraction, isotopic pattern (mSigma value), compound annotation using data bases such as KEGG and CHEBI, and fragmentation pattern interpretation. In the targeted analysis, clotrimazole, fluconazole, itraconazole, ketoconazole and posaconazole were detected in the effluent wastewater sample, fluconazole being with highest average concentration (302.38 ng L−1). The retrospective screening resulted in the detection of 27 compounds that had not been included in the target analysis. The suspect compounds tentatively identified included atazanavir, citalopram, climbazole, bezafibrate estradiol, desmethylvenlafaxine, losartan carboxylic acid and cetirizine, of which citalopram, estradiol and cetirizine were confirmed using a standard. Carbamazepine, atrazine, efavirenz, lopinavir, fexofenadine and 5-methylbenzotriazole were among the compounds detected following the non-targeted screening approach, of which carbamazepine was confirmed using a standard. Given the detection of the target antifungals in the effluent, the findings are a call for a wide assessment of their occurrence in aquatic environments and their role in ecotoxicology as well as in selection of drug resistant fungi. The findings of this work further highlights the practical benefits obtained for the identification of a broader range of emerging pollutants in the environment when retrospective screening is applied to high resolution and high accuracy mass spectrometric data.

Environmental fate of pharmaceutical compounds and antimicrobial-resistant bacteria in hospital effluents, and contributions to pollutant loads in the surface waters in Japan

Environmental fate of 58 pharmaceutical compounds (PhCs) grouped into 11 therapeutic classes in the three different waters, hospital effluent, sewage treatment plant (STP) and river water, was estimated by combination of their quantitative concentration analysis and evaluation of their extent of contribution as loading sources. At the same time, distribution of six classes of antimicrobial-resistant bacteria (AMRB) in the same water samples was estimated by screening of individual PhC-resistant microbes grown on each specific chromogenic medium. The results indicate that 48 PhCs were detected ranged from 1 ng/L (losartan carboxylic acid) to 228 μg/L (acetaminophen sulfate) in hospital effluent, and contribution of the pollution load derived from hospital effluent to STP influent was estimated as 0.1% to 15%. On the other hand, contribution of STP effluent to river water was high, 32% to 60% for antibacterials, antipertensives and X-ray contrast media. In the cases for AMRB, detected numbers of colonies of AMRB in hospital effluent ranged from 29 CFU/mL to 1805 CFU/mL, and the estimated contribution of the AMRB pollution load derived from hospital effluent to STP influent was as low as 0.1% (levofloxacin and olmesartan) to 5.1% (N-desmethyl tamoxifen). Although the contribution of STPs as loading sources of PhCs and AMRB in surface waters was large, ozonation as an advanced water treatment system effectively removed a wide range of both PhCs and AMRB in water samples. These results suggest the importance of reducing environmental pollutant loads (not only at STPs but also at medical facilities) before being discharged into the surface waters, to both conserve water and keep the water environment safe. To our knowledge, this is the first report to show the distribution and contribution of AMRB from hospital effluent to the surface waters.

Conjugation to Ascorbic Acid Enhances Brain Availability of Losartan Carboxylic Acid and Protects Against Parkinsonism in Rats.

Identification of renin-angiotensin system in the interplay of hypertension and neurodegeneration has paved the way for the repurposing of antihypertensive drugs against Parkinsonism. Losartan carboxylic acid (LCA), the potent AT1 blocker metabolite of losartan, suffers from poor bioavailability and brain access. Since ascorbate transporters have earlier shown enough flexibility as carriers, we have conjugated losartan carboxylic acid to ascorbic acid with the aim of achieving higher oral/brain availability. Ester of LCA and ascorbic acid (FED) was developed keeping in view the substrate specificity of ascorbate transporters. Oral/brain bioavailability was assessed using in vivo pharmacokinetic model. Effect on central nervous system (CNS) and protection against Parkinsonism was evaluated using in vivo models. FED enhanced bioavailability of LCA. The higher brain availability of LCA enabled CNS protection as evident from the increase in locomotor activity, improved motor coordination, and protection against drug-induced catatonia. In conclusion, FED offers an approach to repurpose LCA against Parkinsonism. This can encourage further investigation to simultaneously address hypertension and neurodegeneration.

LB.03.10] PHARMACOMETABOLOMIC ANALYZES OF FIVE ANTIHYPERTENSIVE DRUGS FROM HUMAN SERUM SAMPLES OF RESISTANT HYPERTENSIVE PATIENTS

Objective: Our aim was to analyze the serum measurements of five antihypertensive drugs (enalapril and its active metabolite enalaprilat, losartan and its active metabolite losartan carboxylic acid, amlodipine, spironolactone, and clonidine) from 57 resistant hypertensive (RH) patients from a clinical trial, in order to verify if there is a relation between the concentrations of these analytes and the patient's responsiveness. Design and method: From these 57 RH patients, 19 were responsive (ended the clinical trial with controlled blood pressure, less or equal to 140–90 mmHg and/or less or equal to 130–80 mmHg for measurements of ambulatory blood pressure monitoring) and 38 were nonresponsive (ended the study with uncontrolled pressure, greater/equal to the measurements cited above). The drug treatment was the association, when necessary, of until six different antihypertensives: chlortalidone, enalapril, losartan (if enalapril was contraindicated), amlodipine, clonidine, and spironolactone. The last two drugs were indicated in a randomized way. To save time in the analysis and laboratory manual work, we determine and quantify the antihypertensive drugs from lower quantities of sample by a cheaper methodology, compared to other analyzes of this species. We used untreated serum samples in an online preparation, which was performed using a column packed with restricted access carbon nanotubes in a liquid chromatograph coupled to mass spectrometer (LC-MS/MS), employing a column switching mode. Results: We have not found significant differences between the measured concentrations of each analyte in the responsive and nonresponsive patients. Possibly, it is because of our low sample number. The median measurements were: clonidine 0.959 and 1.105micrograms/L; amlodipine 18.706 and 13.939micrograms/L; enalapril 22.972 and 28.112micrograms/L; enalaprilat 19.270 and 16.082micrograms/L; losartan 45.530 and 74.398micrograms/L; losartan carboxylic acid 350.199 and 85.950micrograms/L, for the group that used clonidine. For the group of spironolactone, we had: spironolactone 63.005 and 81.522micrograms/L; amlodipine 14.932 and 14.300micrograms/L; enalapril 107.161 and 33.935micrograms/L; enalaprilat 45.121 and 10.367micrograms/L, losartan 24.207 and 106.996micrograms/L, for responsive and nonresponsive patients, respectively. Conclusions: Although we did not find differences between the measurements, the method can be used as a tool for choosing the best drug to be administered for RH patients, in a fast manner.

High-throughput quantification of 8 antihypertensive drugs and active metabolites in human plasma using UPLC-MS/MS.

To assess drug adherence of patients with hypertension, an analytical method was developed and validated using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The method includes eight frequently prescribed antihypertensive drugs from four classes and their active metabolites: angiotensin converting enzyme inhibitors enalapril and perindopril (active metabolites respectively enalaprilate and perindoprilate), angiotensin II receptor blockers losartan (with the active metabolite losartan carboxylic acid) and valsartan, calcium channel blockers amlodipine and nifedipine and diuretics hydrochlorothiazide and spironolactone (with the active metabolite canrenone). The antihypertensive drugs were analyzed using a simple and fast sample preparation protocol with protein precipitation followed by chromatographic separation using a gradient elution on a reversed phase column. Mass spectrometric detection was conducted by applying both positive and negative electrospray ionization (ESI+/ESI-) and selected reaction monitoring mode (MS/MS). Only 50μl of plasma sample is needed for the simultaneous quantification of all 12 compounds within 6min run-to-run analysis time. Enalapril-d5 was applied as internal standard for all compounds except hydrochlorothiazide (internal standard: Hydrochlorothiazide-13C,d2). The method was validated according to FDA guidelines. Matrix effects were examined using the method of Matuszewski. Correlation coefficients were higher than 0.995 for all compounds. Intra- and inter-day accuracies were <15% for all analytes except spironolactone (-16.8%) in the established linear range. Intra- and inter-day precision were <15% for all analytes. As a result of the lower sensitivity of hydrochlorothiazide, the lowest three calibration levels were excluded. The described method is suitable for the simultaneous quantitative analysis of the most commonly used antihypertensive drugs and their corresponding active metabolites. Major advantages are minimal sample volume and clean up and a short runtime. The method is now available to monitor drug adherence of patients with resistant hypertension in our hospital.

Can the CEIBA Cocktail Designed for Human Cytochrome P450 Enzymes be Used in the Rat for Drug Interaction Studies?

Purpose - The CEIBA cocktail consisting of caffeine (CAF), omeprazole (OZ), dextromethorphan (DM) and losartan (LOS) was previously proposed for the clinical phenotyping of five major human cytochrome P450 (CYP) isoenzymes. This work aimed to assess the usefulness of CEIBA cocktail to study non-clinical drug interactions in the rat. Methods - Wistar rats were divided into five groups to receive a single-oral dose of each probe drug (CAF, OZ, LOS, DM), individually or in combination as a cocktail. Plasma concentrations of the probe drugs and their metabolites [paraxanthine (1,7-X), 5-hydroxyomeprazole (5-OZ), losartan carboxylic acid (E-3174), dextrorphan (DX) and 3-methoxymorphinan (3-MM)] were determined by LC-MS/MS, and the corresponding pharmacokinetic parameters were estimated by non-compartmental analysis. The AUC0-t and Cmax drug/metabolite ratios (phenotypic metrics) were calculated for each probe drug and compared (probe alone versus cocktail). Results - The primary analysis of the pharmacokinetic data suggested the occurrence of pharmacokinetic-based drug interactions when the probe drugs were concurrently administered; such interactions were documented for CAF, 1,7-X, DX and E-3174. Nevertheless, except for the LOS/E-3174 probe drug-metabolite pair (p<0.05), there was little evidence that the probe drugs interacted metabolically as the metabolic ratios calculated were similar in both approaches. Moreover, no evidence was found for relevant pharmacodynamic interactions. Conclusion - CEIBA cocktail seems to be a useful tool to investigate drug interactions involving CYP isoenzymes in the rat, particularly at the level of CYP1A2, CYP2D1/2 and CYP2D2 isoforms using the CAF/1,7-X, OZ/5-OZ and DM/DX metabolic ratios, respectively.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.

Postmortem blood concentrations of sartans measured by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC–MS/MS) method was developed and validated for the analysis of seven common sartans (candesartan, eprosartan, losartan, olmesartan, telmisartan, valsartan, and losartan carboxylic acid (EXP3174), the active metabolite of losartan) in postmortem blood samples with liquid–liquid extraction. Detection was accomplished by triple-quadrupole MS in the selected reaction monitoring mode. The validation procedure showed low limits of quantitation of 5 ng/mL for all compounds with good accuracy and precision. The matrix effect was evaluated from the variation of peak areas and concentrations. The method shows some matrix effect but the effect was efficiently compensated by using an internal standard method. The method was applied to the analysis of sartans in postmortem blood samples, and produced 534 positive findings during 2014–2015. Nine deaths were encountered where the concentration of a sartan was very high, indicating intoxication together with other causes of death or multi-drug intoxication. The quantitative results indicate that sartans do not show significant postmortem redistribution; thus concentrations higher than the therapeutic range can be considered as being toxic or fatal.

Effects of Cytochrome P450 Inhibition and Induction on the Phenotyping Metrics of the Basel Cocktail: A Randomized Crossover Study.

Background and ObjectiveActivity of human cytochrome P450 enzymes (CYPs) shows high inter-and intra-individual variability, which is determined by genetic and non-genetic factors. Using a combination of CYP-specific probe drugs, phenotyping cocktails allow simultaneous assessment of the activity of different CYP isoforms. The objective of this study was to characterize the phenotyping metrics of the Basel cocktail in healthy male subjects with induced and inhibited CYP activity.MethodsIn a randomized crossover study, the probe drugs for simultaneous phenotyping of CYP1A2 (caffeine), CYP2B6 (efavirenz), CYP2C9 (losartan), 2C19 (omeprazole), CYP2D6 (metoprolol), and CYP3A4 (midazolam) were administered to 16 subjects without pretreatment (baseline), after pretreatment with a combination of CYP inhibitors (ciprofloxacin, ketoconazole, and paroxetine), and after CYP induction with rifampicin. All subjects were genotyped. Pharmacokinetic profiles of the probe drugs and their main metabolites and metabolic ratios 2, 4, 6, and 8 h after probe drug application were determined in plasma and compared with the corresponding area under the plasma concentration-time curve (AUC) ratios.ResultsThe Basel phenotyping cocktail was well tolerated by all subjects independent of pretreatment. Good correlations of metabolic ratios with AUC ratios of the corresponding probe drugs and their metabolites for all three conditions (baseline, CYP inhibition, and CYP induction) were found at 2 h after probe drug administration for CYP3A4, at 4 h for CYP1A2 and CYP2C19, and at 6 h for CYP2B6 and CYP2D6. While CYP inhibition significantly changed AUC ratios and metabolic ratios at these time points for all six CYP isoforms, CYP induction did not significantly change AUC ratios for CYP2C9. For CYP3A4, total 1′-hydroxymidazolam concentrations after pretreatment of samples with β-glucuronidase were needed to obtain adequate reflection of CYP induction by the metabolic ratio.ConclusionsInhibition of CYP activity can be detected with the Basel phenotyping cocktail for all six tested CYP isoforms at the proposed time points. The AUC ratio of losartan:losartan carboxylic acid in plasma does not seem suitable to detect induction of CYP2C9. The observed metabolic ratios for inhibited and induced CYP activity need to be confirmed for extensive metabolizers, and typical ratios for subjects with genetically altered CYP activity will need to be established in subsequent studies.ClinicalTrials.gov-ID: NCT01386593.Electronic supplementary materialThe online version of this article (doi:10.1007/s40262-015-0294-y) contains supplementary material, which is available to authorized users.

Simultaneous Determination and Pharmacokinetics of Metolazone, Losartan and Losartan Carboxylic Acid in Rat Plasma by HPLC-ESI-MS-MS.

For the first time, we developed and validated a highly sensitive, selective and rapid HPLC-ESI-MS-MS method for simultaneous quantification of metolazone (MET), losartan (LOS) and its metabolite losartan carboxylic acid (LCA) in rat plasma. After solid-phase extraction, the analytes and internal standard (irbesartan) were extracted from 100 µL plasma sample on an Agilent Poroshell 120, EC-C18 (50 × 4.6 mm, i.d., 2.7 µm) column using 5 µL injection volume with a total run time of 3 min. Acidified methanol/water mixture was used as a mobile phase. The parent → product ion transitions for MET (m/z 366.0 → 258.9), LOS (m/z 423.2 → 207.0), LCA (m/z 437.0 → 235.1) and IS (m/z 429.2 → 207.0) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was found to be linear in the range of 0.05-250 for MET, 2-3,000 for LOS and 4-3,500 ng/mL for LCA. The method was validated with respect to selectivity, linearity, accuracy, precision, recovery and stability according to accepted regulatory guidelines. The described method was successfully applied to preclinical pharmacokinetic studies of analytes after an oral administration of mixture of MET (1 mg/kg) and LOS (10 mg/kg) in rats.

Human bioequivalence evaluation of two losartan potassium tablets under fasting conditions.

The bioequivalence of two different tablet formulations containing losartan potassium 100 mg was determined in healthy volunteers after a single oral dose in a randomized crossover study. Test and reference products were administered to 60 volunteers with 240 ml water after overnight fasting. Plasma concentrations of losartan and its active carboxylic acid metabolite were monitored over a period of 36 h after drug administration by validated LC/MS/MS analytical method. The pharmacokinetic parameters Cmax, AUC0-t, AUC0-∞, AUC0-t/AUC0-∞, tmax, Kel and t½ were determined from plasma concentration time profile of both formulations for losartan and its active metabolite losartan carboxylic acid and were found to be in good agreement. The carboxylic acid metabolite was considered for profiling purpose only. The analysis of variance did not show any significant difference between the two formulations and 90% confidence intervals for the ratio of Cmax (84.89-104.09%), AUC0-t (95.84-102.84%) and AUC0-∞ (96.43-103.25%) values for losartan between the test and reference products were within the 80-125% interval, satisfying the bioequivalence criteria of the US FDA guidelines. These results indicate that the test and the reference products of losartan potassium are bioequivalent and, thus, may be prescribed interchangeably.