UV imaging for the rapid at-line content determination of different colourless APIs in their tablets with artificial neural networks.

This paper presents a novel high-resolution and rapid (50 ms) UV imaging system, which was used for at-line, non-destructive API content determination of tablets. For the experiments, amlodipine and valsartan were selected as two colourless APIs with different UV induced fluorescent properties according to the measured solid fluorescent spectra. Images were captured with a LED-based UV illumination (385-395 nm) of tablets containing amlodipine or valsartan and common tableting excipients. Blue or green colour components from the RGB colour space were extracted from the images and used as an input dataset to execute API content prediction with artificial neural networks. The traditional destructive, solution-based transmission UV measurement was applied as reference method. After the optimization of the number of hidden layer neurons it was found that the relative error of the content prediction was 4.41 % and 3.98 % in the case of amlodipine and valsartan containing tablets respectively. The results open the possibility to use the proposed UV imaging-based system as a rapid, in-line tool for 100 % API content screening in order to greatly improve pharmaceutical quality control and process understanding.

Design of Experimental Approach for Development of Rapid High Performance Liquid Chromatographic Process for Simultaneous Estimation of Metoprolol, Telmisartan, and Amlodipine from Formulation: Greenness and Whiteness Evaluation.

The design of an experimental approach, the Box-Behnken design, was implemented to optimize the chromatographic condition to develop a rapid HPLC procedure for quantification of a ternary mixture of metoprolol (MET), telmisartan (TEL), and amlodipine (AML) from the formulation. The perturbation plots, contour, and 3D response surface pictures were developed to study the impact of each variable on the analytes' retention time and the probable interaction between the parameters with fewer chromatographic runs. The optimized HPLC method separated the three analytes within 5 min with excellent selectivity and peak shape on a Zorbax C18 HPLC column using acetonitrile and phosphate buffer (20 mM, pH 5.8) with isocratic elution at a 1.1 mL/min flowrate. A wavelength 230 nm was utilized to monitor the elute. The validation of proposed method demonstrated a wide linearity range of 10-200 µg/mL for MET and TEL and 5-50 µg/mL for AML along with an excellent correlation coefficient. The correctness of the HPLC approach was further confirmed by excellent recovery of the added amount of analytes utilizing the standard addition technique. The recommended HPLC approach was employed safely for quality assurance of the formulation, because the evaluation of the method's greenness and whiteness confirmed the environmentally friendly nature of the approach.

Simultaneous Determination of Amlodipine and Irbesartan in their Pharmaceutical Formulations by Square-Wave Voltammetry.

BACKGROUND: Hypertension is one of the most important health problems in the world and irbesartan and amlodipine are used in combination in various dosages for the treatment of high blood pressure. OBJECTIVE: The aim of this study is to develop a fast, easy, sensitive, accurate, and precise squarewave voltammetry method for simultaneous determination of irbesartan and amlodipine besylate from pharmaceutical formulations at a hanging mercury drop electrode. METHODS: In the applied method, since both active substances gave a peak at different potentials, no interference occurred between them. In optimization studies, Britton-Robinson buffer of pH 8.0 was chosen, in which the most appropriate peak shape and maximum peak current were observed. At the same time, as a result of instrumental parameter optimization to obtain reproducible results, 6 mV for scan increment, 30 mV for pulse amplitude, and 50 Hz for frequency were found suitable. RESULTS: As a result of the calibration studies of the optimized method, linear working ranges were determined as 1.00-13.08 µg mL-1 for irbesartan and 5.83-16.51 µg mL-1 for amlodipine besylate. Limit of detection and limit of quantitation values were respectively calculated as 0.63 and 1.00 µg mL-1 for irbesartan and 0.50 and 1.98 µg mL-1 for amlodipine besylate. The results of precision values (RSD) ranged from 0.67% to 2.31% for irbesartan and 0.65% to 1.49% for amlodipine besylate. Accuracy values were calculated as -0.15% to 1.63% for irbesartan and -0.07% to 3.78% for amlodipine besylate. The results obtained from the recovery studies ranged from 101.05% to 102.78% and from 98.88% to 102.20% for amlodipine besylate and irbesartan, respectively. CONCLUSION: After the validation studies of the developed method were carried out, it was successfully applied to pharmaceutical formulations containing these active substances.

Direct Visualization of Amlodipine Intervention into Living Cells by Means of Fluorescence Microscopy.

Amlodipine, a unique long-lasting calcium channel antagonist and antihypertensive drug, has weak fluorescence in aqueous solutions. In the current paper, we show that direct visualization of amlodipine in live cells is possible due to the enhanced emission in cellular environment. We examined the impact of pH, polarity and viscosity of the environment as well as protein binding on the spectral properties of amlodipine in vitro, and used quantum chemical calculations for assessing the mechanism of fluorescence quenching in aqueous solutions. The confocal fluorescence microscopy shows that the drug readily penetrates the plasma membrane and accumulates in the intracellular vesicles. Visible emission and photostability of amlodipine allow confocal time-lapse imaging and the drug uptake monitoring.

Development and validation of a stability-indicating UPLC method for the determination of olmesartan medoxomil, amlodipine and hydrochlorothiazide degradation impurities in their triple-combination dosage form using factorial design of experiments.

The current work describes the development and validation of a stability-indicating UPLC method for the determination of olmesaratan medoxomil (OLM), amlodipine besylate (AMB), hydrochlorothiazide (HCT) and their degradation products in the triple-combination tablet dosage form. The separation was achieved using a Zorbax Eclipse plus C8 RRHD (100 mm × 3.0 mm), 1.8 µm column with gradient elution of mobile phase A containing 0.02 m of sodium phosphate buffer (pH 3.35) and mobile phase B as acetonitrile and water (90:10, v/v). The detector signal was monitored at UV 250 nm. Analytical performance of the optimized UPLC method was validated as per International Conference on Harmonization guidelines. The linearity ranges for OLM, AMB and HCT were 0.59-240, 0.30-60 and 0.37-150 µg/ml, respectively, with correlation coefficients >0.999. The dosage form was subjected to forced-degradation conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal and photodegradation. The method was proved to be stability indicating by demonstrating the specificity of the drugs from degradation products. The robustness of the method was evaluated through a two-level, three-factorial design with a multivariate approach. Statistical data analysis with best model fit P-value < 0.05 from an ANOVA test indicated that the influence of individual factors is relatively higher than the interaction effects. The method is useful for the analysis of drug products.

Long-acting tunable release of amlodipine loaded PEG-PCL micelles for tailored treatment of chronic hypertension.

Hypertension is a chronic condition that requires lifelong therapeutic management. Strict adherence to drug administration timing improves efficacy, while poor adherence leads to safety concerns. In light of these challenges, we present a nanofluidic technology that enables long-acting drug delivery with tunable timing of drug administration using buried gate electrodes in nanochannels. We developed a poly(ethylene glycol) methyl ether-block-poly(ε-caprolactone) (PEG-PCL)-based micellar formulation of amlodipine besylate, a calcium channel blocker for hypertension treatment. The electrostatically charged PEG-PCL micellar formulation enhanced drug solubility and rendered amlodipine responsive to electrostatic release gating in nanochannels for sustained release at clinically relevant therapeutic dose. Using a low-power (<3 VDC) gating potential, we demonstrated tunable release of amlodipine-loaded micelles. Additionally, we showed that the released drug maintained biological activity via calcium ion blockade in vitro. This study represents a proof of concept for the potential applicability of our strategy for chronotherapeutic management of hypertension.

Fixed Dose Single Tablet Formulation with Differential Release of Amlodipine Besylate and Simvastatin and Its Pharmacokinetic Profile: QbD and Risk Assessment Approach.

PURPOSE: A differential release fixed dose matrix tablet of amlodipine besylate (AML-B) and simvastatin (SIM) was formulated to enhance patient compliance. MATERIAL AND METHOD: In the first phase, release controlling parameters of AML-B and SIM granules were identified and in the second phase a fixed dose AML-B and SIM tablet formulation was prepared and optimized for a differential release of the drugs using a quality by design (QbD) and risk assessment approach. A validated HPLC method was employed for simultaneous determination of AML-B and SIM for FDC formulation. A pharmacokinetics of the above drugs was studied in healthy dogs in the third phase. RESULTS: In QbD-based optimized formulation, Eudragit® RSPO-dicalcium phosphate (DCP) blend controlled the release of AML-B over 8 h, though this diffusion-controlled release assumed first order kinetics. DCP and Eudragit® RS 100 also retarded release of SIM causing SIM release over 8 h after AML-B release from the optimized FDC tablet formulation. The HPLC retention times of AML-B and SIM were 2.10 and 15.52 min, respectively. Linearity for AML-B was 5.0-50 ng/mL and 0.01-2.0 µg/mL for SIM with percent recoveries of 92.85-101.53% and 94.51-117.75% for AML-B and SIM. AUC0-∞ of AML-B was increased 3 fold, while AUC0-∞ of SIM was decreased 2 fold. The tmax values for AML-B and SIM were 12 and 6 h, respectively. AML-B was absorbed without any lag time (tlag) while tlag was 6.33 ± 0.81 h for SIM, thus met the study objective. CONCLUSION: The pharmacokinetic study showed an immediate absorption of AML-B while that of SIM was withheld for 6 h, close to the desired delay time of 8 h.

Structural Basis of the Modulation of the Voltage-Gated Calcium Ion Channel Cav 1.1 by Dihydropyridine Compounds*.

1,4-Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L-type voltage-gated Ca2+ (Cav ) channels. DHP compounds exhibit chirality-specific antagonistic or agonistic effects. The structure of rabbit Cav 1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo-EM structures of nanodisc-embedded Cav 1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)-(+)-Bay K8644, and a titration of its agonistic enantiomer (S)-(-)-Bay K8644 at resolutions of 2.9-3.4 Å. The amlodipine-bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)-(-)-Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration.

Investigation of the enantioselective interaction between selected drug enantiomers and human serum albumin by mobility shift-affinity capillary electrophoresis.

Mobility shift-affinity capillary electrophoresis was employed for enantioseparation and simultaneous binding constant determination. Human serum albumin was used as a chiral selector in the background electrolyte composed of 20 mM phosphate buffer, pH 7.4. The applied setup supports a high mobility shift since albumin and the drug-albumin complex hold negative net charges, while model compounds of amlodipine and verapamil are positively charged. In order to have an accurate effective mobility determination, the Haarhoff-van der Linde function was utilized. Subsequently, the association constant was determined by nonlinear regression analysis of the dependence of effective mobilities on the total protein concentration. Differences in the apparent binding status between the enantiomers lead to mobility shifts of different extends (α). This resulted in enantioresolutions of Rs = 1.05-3.63 for both drug models. R-(+)-Verapamil (KA 1844 M-1 ) proved to bind stronger to human serum albumin compared to S-(-)-verapamil (KA 6.6 M-1 ). The association constant of S-(-)-amlodipine (KA 25 073 M-1 ) was found to be slightly higher compared to its antipode (KA 22 620 M-1 ) when applying the racemic mixture. The low measurement uncertainty of this approach was demonstrated by the close agreement of the association constant of the enantiopure S-(-)-form (KA 25 101 M-1 ).

A Drug Content, Stability Analysis, and Qualitative Assessment of Pharmacists' Opinions of Two Exemplar Extemporaneous Formulations.

Despite a decline in the number of active pharmaceutical ingredients prepared extemporaneously using proprietary products, there remains a need for such products in the community (for example, liquid medicines for paediatrics which may be otherwise commercially unavailable). A lack of experience and quality assurance systems may have diminished pharmacist's confidence in the extemporaneous preparation process; therefore, pharmacists were asked to prepare two proprietary products, omeprazole and amlodipine. The resulting products were characterised in terms of variability in drug quantity, stability, particle size and antimicrobial properties. Furthermore, a self-administered questionnaire was used to assess 10 pharmacists' opinions on the perceived complexity of the extemporaneous compounding process and their overall confidence in the final extemporaneously compounded products. Drug content studies revealed that 88.5% and 98.0% of the desired drug content was obtained for omeprazole and amlodipine, respectively. Antimicrobial properties were maintained for both drugs, however variability in particle size, particularly for amlodipine, was evident between formulations. While pharmacists who partook in the study had some or high confidence in the final products, they reported difficulty formulating the suspensions. Findings from this study provide insight into pharmacists' views on two extemporaneously prepared products and highlight the variability obtained in preparations prepared by different pharmacists.

Desorption of pharmaceuticals from pristine and aged polystyrene microplastics under simulated gastrointestinal conditions.

Microplastics (MPs) in the environment usually undergo extensive weathering and can transport pollutants to organisms once being ingested. However, the transportation mechanism and effect of aging process are poorly understood. This study systematically investigated the desorption mechanisms of pharmaceuticals from pristine and aged polystyrene (PS) MPs under simulated gastric and intestinal conditions of marine organisms. Results showed that the increased desorption in stomach mainly depended on the solubilization of pepsin to pharmaceuticals and the competition for sorption sites on MPs via π-π and hydrophobic interactions. However, high desorption in gut relied on the solubilization of intestinal components (i.e. bovine serum albumin (BSA) and bile salts (NaT)) and the competitive sorption of NaT since the enhanced solubility increased the partition of pharmaceuticals in aqueous phase. Aging process suppressed the desorption of pharmaceuticals because aging decreased hydrophobic and π-π interactions but increased electrostatic interaction between aged MPs and pharmaceuticals, which became less affected by gastrointestinal components. Risk assessment indicated that the MP-associated pharmaceuticals posed low risks to organisms, and warm-blooded organisms suffered relatively higher risks than cold-blooded ones. This study reveals important information to understand the ecological risks of co-existed MPs and pollutants in the environment.

Non-destructive dose verification of two drugs within 3D printed polyprintlets.

Three-dimensional printing (3DP) is a revolutionary technology in pharmaceuticals, enabling the personalisation of flexible-dose drug products and 3D printed polypills (polyprintlets). A major barrier to entry of this technology is the lack of non-destructive quality control methods capable of verifying the dosage of multiple drugs in polyprintlets at the point of dispensing. In the present study, 3D printed films and cylindrical polyprintlets were loaded with flexible, therapeutic dosages of two distinct drugs (amlodipine and lisinopril) across concentration ranges of 1-5% w/w and 2-10% w/w, respectively. The polyprintlets were non-destructively analysed for dose content using a portable near infrared (NIR) spectrometer and validated calibration models were developed using partial least squares (PLS) regression, which showed excellent linearity (R2 Pred = 0.997, 0.991), accuracy (RMSEP = 0.24%, 0.24%) and specificity (LV1 = 82.77%, 79.55%) for amlodipine and lisinopril, respectively. X-ray powder diffraction (XRPD) and thermogravimetric analysis (TGA) showed that sintering partially transformed the phase of both drugs from the crystalline to amorphous forms. For the first time, we report a non-destructive method for quality control of two separate active ingredients in a single 3D printed drug product using NIR spectroscopy, overcoming a major barrier to the integration of 3D printing into clinical practice.

Effect of aging on adsorption behavior of polystyrene microplastics for pharmaceuticals: Adsorption mechanism and role of aging intermediates.

In the environment, aging obviously changes physicochemical properties of microplastics (MPs), but the effects of aging process on adsorption behavior of MPs are not fully understood. In this study, the aging of polystyrene (PS) was accelerated by photo-Fenton reaction. The adsorption mechanism of different aged PS toward atorvastatin (ATV) and amlodipine (AML) and the role of PS-derived intermediates in adsorption process were investigated. Results showed that the adsorption of pristine PS toward pharmaceuticals relied on hydrophobic and π-π interaction, while for aged PS, electrostatic interaction and hydrogen bonding controlled the adsorption. The study revealed that the intermediates released from aging process in high concentration (TOC of 10 mg/L) significantly decreased the adsorption of ATV (10 mg/L) on PS (5.0 g/L) but increased the adsorption of AML (10 mg/L). However, those intermediates at environmental concentration (0.1 mg/L) exhibited low effects on adsorption of pharmaceuticals (1.0 mg/L) on MPs (0.5 g/L of PS). The impact mainly depended on electrostatic interaction between MPs and aging intermediates. Besides, the adsorption of low-degree aged PS was more susceptible to the aging intermediates than that of high-degree aged ones. These findings highlight significant implication of MP-derived intermediates in aquatic environments.

Investigation of complexation of amlodipine with lysozyme and its effect on lysozyme crystal growth.

Lysozyme (LYZ) is a model protein frequently employed to study interaction with drugs and to understand the crystallization process of protein due to its small size and rapid crystallization behavior. Studies related to drug interaction and complexation with proteins will be significantly benefited if a suitable drug-lysozyme crystal is available. This can further aid in the understanding of the mechanism of nucleation, growth and the formation of drug-lysozyme complex. In the present study, amlodipine (AMLD) complexation with LYZ has been monitored, along with its effect on lysozyme crystallization. Different spectroscopic methods have been employed to monitor the nature of complexation, binding mode and changes in helix after interaction with AMLD. The absorbance and fluorescence spectroscopic measurement indicated the probability of a ground state complex between LYZ and AMLD. Further, the temperature dependent fluorescence studies showed an increase in binding constant with temperature, suggesting the static quenching mechanism involved in complex formation due to hydrophobic interactions. CD, FTIR, DLS and DSC techniques confirm the probability of changes in the tertiary structure of protein. Molecular docking was applied to investigate the interaction of amino acid residues of LYZ with AMLD. It was found that the complex formation is spontaneous and the ΔG value obtained (-21. 76 kJ/mol) very well matched with temperature dependent fluorescence study (-24.91 kJ/mol). Crystallization of LYZ was performed with different concentration ranges of AMLD to get a clear picture of its interference on the process. The time required for crystallization of AMLD-LYZ complex and the observed structure of crystal indicates that AMLD influences lysozyme crystallization process by changing the nature of nucleation and rate of crystal growth.

QbD-based development of an extraction procedure for simultaneous quantification of telmisartan, amlodipine besylate and chlorthalidone in combination complex matrix formulation.

The main objective of this study was to establish an efficient extraction procedure for the estimation of telmisartan, amlodipine and chlorthalidone from their combination in sample matrix using an analytical quality by design approach. Initial screening studies were performed for optimization of a suitable diluent to extract active components from sample matrix. Further, the same study was extended for the identification of critical method attributes and the factors affecting the analytical target profile. This study also explains the rugged and robust quantitative determination of combinations drugs with a shorter run time. The design of experimental studies confirms that the current center point parameters are well suited to recoveries. The chromatographic separation was achieved with an X-Terra RP8, 150 × 4.6 mm, 3.5 µm column with an isocratic mobile phase (mixture of 20 mm aqueous ammonium acetate and acetonitrile). To demonstrate the stability-indicating nature of the optimized method, forced degradation studies were conducted and proved. The optimized method was validated according to International Conference on Harmonization guidelines.

Preparation and Evaluation of Poly-γ-glutamic Acid Hydrogel Mixtures with Amlodipine Besylate: Effect on Ease of Swallowing and Taste Masking.

The purpose of the study was to prepare a poly-γ-glutamic acid hydrogel (PGA gel), to evaluate physicochemical properties, its ease of swallowing using texture profile analysis (TPA) and its taste-masking effects on amlodipine besylate (AML) using the artificial taste sensor and human gustatory sensation testing. Using TPA, 0.5 and 1.0% (w/v) PGA gels in the absence of drug were within the range of acceptability for use in people with difficulty swallowing according to permission criteria published by the Japanese Consumers Affairs Agency. The elution of AML from prepared PGA gels was complete within an hour and the gel did not appear to influence the bioavailability of AML. The sensor output of the basic bitterness sensor AN0 in response to AML mixed with 0.5 and 1.0% PGA gels was suppressed to a significantly greater degree than AML mixed with 0.5 and 1.0% agar. In human gustatory sensation testing, 0.5 and 1.0% PGA gels containing AML showed a potent bitterness-suppressing effect. Finally, 1H-NMR spectroscopic analysis was carried out to examine the mechanism of bitterness suppression when AML was mixed with PGA gel. The signals of the proton nearest to the nitrogen atom of AML shifted clearly upfield, suggesting an interaction between the amino group of AML and the carboxyl group of PGA gel. In conclusion, PGA gel is expected to be a useful excipient in formulations of AML, not only increasing ease of swallowing but also masking the bitterness of the basic drug.

Maltodextrin-modified graphene oxide for improved enantiomeric separation of six basic chiral drugs by open-tubular capillary electrochromatography.

An electrochromatographic capillary was modified with graphene oxide (GO), and the coating was characterized by scanning electron microscopy, energy dispersive X-ray spectrometry, and Fourier transform infrared spectra. By utilizing maltodextrin (MD) as the chiral selector, the basic chiral drugs nefopam (NEF), amlodipine (AML), citalopram hydrobromide (CIT), econazole (ECO), ketoconazole (KET) and cetirizine hydrochloride (CET) can be enantiomerically separated on this CEC. Compared with an uncoated silica capillary, the resolutions are markedly improved (AML: 0.32 → 1.45; ECO: 0.55 → 1.89; KET: 0.88 → 4.77; CET: 0.81 → 2.46; NEF: 1.46 → 2.83; CIT: 1.77 → 4.38). Molecular modeling was applied to demonstrate the mechanism of enantioseparation, which showed a good agreement with the experimental results. Graphical abstractSchematic representation of the preparation of graphene oxide-modified capillary (GO@capillary) for enantioseparation of drug enantiomers. The monolayered GO was used as the coating of the GO@capillary. Then the capillary was applied to construct capillary electrochromatography system with maltodextrin for separation of basic chiral drugs.

Physicochemical Stability of Compounded Amlodipine Besylate Suspensions in PCCA Base, SuspendIt.

Amlodipine besylate is an antihypertensive agent recommended for the management of hypertension in children and adolescents. The commercially available 2.5-mg, 5-mg, and 10-mg amlodipine besylate tablets do not provide the necessary flexibility in dosing needed for treating children. This flexibility is readily achieved using an oral, liquid dosage form. However, no commercial liquid dosage form of amlodipine currently exists. An extemporaneously compounded suspension from pure drug powder or commercial tablets would provide a convenient option to meet unique patient needs. The purpose of this study was to determine the physicochemical stability of extemporaneously compounded amlodipine besylate suspensions in the PCCA Base, SuspendIt. This base is a sugar-free, paraben-free, dye-free, and gluten-free thixotropic vehicle containing a natural sweetener obtained from the monk fruit. The study design included two amlodipine besylate concentrations to provide stability documentation over a bracketed concentration range for eventual use by compounding pharmacists. A robust stabilityindicating high-performance liquid chromatographic assay for the determination of the chemical stability of amlodipine besylate in SuspendIt was developed and validated. Suspensions of amlodipine were prepared in SuspendIt at 0.5-mg/mL and 10.0-mg/mL concentrations, selected to represent a range within which the drug is commonly dosed. Samples were stored in plastic amber prescription bottles at two temperature conditions (5°C and 25°C). Samples were assayed initially, and at the following time points: 7 days, 14 days, 29 days, 46 days, 60 days, 90 days, 120 days, and 180 days. Physical data such as pH, viscosity, and appearance were also noted. All measurements were obtained in triplicate. A stable extemporaneous product is defined as one that retains at least 90% of the initial drug concentration throughout the sampling period. This study demonstrates that amlodipine besylate is physically and chemically stable in SuspendIt for 90 days in the refrigerator and 7 days at room temperature, retaining 90% of the label claim (initial drug concentration) at both concentrations. The pH values did not change significantly. The viscosity of the refrigerated samples at both concentrations decreased slightly, while that of the room temperature samples showed a marked increase in viscosity. This study provides a viable, compounded alternative for amlodipine in a liquid dosage form, with an adequate beyond-use-date to meet patient needs. The study further provides stability documentation over a bracketed amlodipine concentration range of 0.5 mg/mL to 10.0 mg/mL, allowing compounding pharmacists more flexibility in customizing their formulations.

Structural Basis for Diltiazem Block of a Voltage-Gated Ca2+ Channel.

Diltiazem is a widely prescribed Ca2+ antagonist drug for cardiac arrhythmia, hypertension, and angina pectoris. Using the ancestral CaV channel construct CaVAb as a molecular model for X-ray crystallographic analysis, we show here that diltiazem targets the central cavity of the voltage-gated Ca2+ channel underneath its selectivity filter and physically blocks ion conduction. The diltiazem-binding site overlaps with the receptor site for phenylalkylamine Ca2+ antagonist drugs such as verapamil. The dihydropyridine Ca2+ channel blocker amlodipine binds at a distinct site and allosterically modulates the binding sites for diltiazem and Ca2+ Our studies resolve two distinct binding poses for diltiazem in the absence and presence of amlodipine. The binding pose in the presence of amlodipine may mimic a high-affinity binding configuration induced by voltage-dependent inactivation, which is favored by dihydropyridine binding. In this binding pose, the tertiary amino group of diltiazem projects upward into the inner end of the ion selectivity filter, interacts with ion coordination Site 3 formed by the backbone carbonyls of T175, and alters binding of Ca2+ to ion coordination Sites 1 and 2. Altogether, our results define the receptor site for diltiazem and elucidate the mechanisms for pore block and allosteric modulation by other Ca2+ channel-blocking drugs at the atomic level. SIGNIFICANCE STATEMENT: Calcium antagonist drugs that block voltage-gated calcium channels in heart and vascular smooth muscle are widely used in the treatment of cardiovascular diseases. Our results reveal the chemical details of diltiazem binding in a blocking position in the pore of a model calcium channel and show that binding of another calcium antagonist drug alters binding of diltiazem and calcium. This structural information defines the mechanism of drug action at the atomic level and provides a molecular template for future drug discovery.

UHPLC-MS/MS assay for simultaneous determination of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin with its active metabolites in human plasma, for population-scale drug-drug interactions studies in people living with HIV.

Thanks to highly active antiretroviral treatments, HIV infection is now considered as a chronic condition. Consequently, people living with HIV (PLWH) live longer and encounter more age-related chronic co-morbidities, notably cardiovascular diseases, leading to polypharmacy. As the management of drug-drug interactions (DDIs) constitutes a key aspect of the care of PLWH, the magnitude of pharmacokinetic DDIs between cardiovascular and anti-HIV drugs needs to be more thoroughly characterized. To that endeavour, an UHPLC-MS/MS bioanalytical method has been developed for the simultaneous determination in human plasma of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin and its active metabolites. Plasma samples were subjected to protein precipitation with methanol, followed by evaporation at room temperature under nitrogen of the supernatant, allowing to attain measurable plasma concentrations down to sub-nanogram per milliliter levels. Stable isotope-labelled analytes were used as internal standards. The five drugs and two metabolites were analyzed using a 6-min liquid chromatographic run coupled to electrospray triple quadrupole mass spectrometry detection. The method was validated over the clinically relevant concentrations ranging from 0.3 to 480 ng/mL for amlodipine, atorvastatin and p-OH-atorvastatin, and 0.4 to 480 ng/mL for pravastatin, 0.5 to 480 ng/mL for rosuvastatin and o-OH-atorvastatin, and 3 to 4800 ng/mL for metoprolol. Validation performances such as trueness (95.4-110.8%), repeatability (1.5-13.4%) and intermediate precision (3.6-14.5%) were in agreement with current international recommendations. Accuracy profiles (total error approach) were lying within the limits of ±30% accepted in bioanalysis. This rapid and robust UHPLC-MS/MS assay allows the simultaneous quantification in plasma of the major currently used cardiovascular drugs and offers an efficient analytical tool for clinical pharmacokinetics as well as DDIs studies.