Clinical Pharmacokinetic Studies Research Articles

Investigator-Initiated Clinical Pharmacokinetic Studies in Resource-Limited Settings: Minimal Requirements and Practical Guidance.

Clinical pharmacology studies are critical for determining the efficacy and safety of drugs. Due to the resource-intensive nature of these studies, most have been conducted in high-income countries, leading to a significant gap in clinical pharmacology data for patients in low- and middle-income countries. This paper provides an overview of the minimal requirements for performing a clinical pharmacology investigator-initiated trial (IIT), including pharmacokinetic sampling. We identify common challenges in resource-limited settings and propose strategies to overcome them. This guideline covers regulatory approval, participant recruitment, drug storage, sample collection and handling, transport, bioanalytical analysis, and data management tailored to the constraints of resource-limited settings. Strategies are proposed to minimize resource demands, including simplified study designs, the use of technologies like whole blood microsampling, and opportunities for collaboration. The goal is to provide practical guidance for those seeking to perform a clinical pharmacology IIT in resource-limited settings to improve safe and effective drug treatment for patients worldwide. Beyond the scope of this guideline is a detailed step-by-step guide on how to perform clinical pharmacology studies.

Event-driven PrEP beyond cisgender men who have sex with men.

Despite advancements in existing antiretroviral-based prevention strategies, including daily oral, locally acting, and injectable options, there is a pressing need for more inclusive and flexible event-driven pre-exposure prophylaxis (PrEP) strategies for all. Event-driven or intermittent dosing of PrEP in populations beyond cisgender men who have sex with men would offer a promising alternative by fitting prevention into the diverse lifestyles of affected populations and thereby advancing health equity. Evidence from PrEP clinical trials, pharmacokinetic studies, modelling studies, and real-world observational research suggests that event-driven PrEP could be a flexible and inclusive option, yet optimal dosing has not been established across sex and gender spectrums. To advance PrEP equity through inclusivity, studies on event-driven PrEP should include people across the gender spectrum. Real-world demonstration studies and simulation studies of optimal dosing strategies are needed. While awaiting further evidence, clinical providers can offer shared decision making and counselling on available data to include event-driven dosing as an option, especially when daily oral, locally acting, or injectable PrEP are not acceptable or preferred methods. Wider access to diverse PrEP options for all populations fosters a more inclusive and effective global HIV prevention strategy.

Application of Physiologically Based Pharmacokinetic Model to Compare the Biodistribution of Liposomal Amphotericin B With Conventional Amphotericin B Deoxycholate in Humans.

Amphotericin B (AmB) has been a cornerstone in the treatment of invasive fungal infections for over 6decades. Compared with conventional amphotericin B deoxycholate (AmB-DOC), liposomal amphotericin B has comparable efficacy but less nephrotoxicity. The main purpose of this study was to investigate the reason why liposomal amphotericin B has similar therapeutic effects but lower toxicity and the differences of distribution in humans between liposomal amphotericin B and AmB-DOC. To compare the distribution of liposomal amphotericin B and AmB-DOC in humans, the physiologically based pharmacokinetic (PBPK) model was established by bottom-up stepwise method. A rat PBPK model was established firstly, then verified in mouse level in consideration of interspecies differences in physiological- and drug-specific parameters, and finally the PBPK model was extrapolated to humans. Based on preclinical and clinical pharmacokinetic (PK) studies, the AmB-DOC and liposomal amphotericin B PBPK model were established, respectively. The simulated results of human PBPK model showed that the liposomal formulation changed the pharmacokinetic characteristics of AmB. Compared with AmB-DOC, the plasma exposure of liposomal formulation was higher, but the renal exposure was significantly reduced.

Development of a physiologically-based pharmacokinetic model for Ritonavir characterizing exposure and drug interaction potential at both acute and steady-state conditions.

Ritonavir (RTV) is a potent CYP3A inhibitor that is widely used as a pharmacokinetic (PK) enhancer to increase exposure to select protease inhibitors. However, as a strong and complex perpetrator of CYP3A interactions, RTV can also enhance the exposure of other co-administered CYP3A substrates, potentially causing toxicity. Therefore, the prediction of drug-drug interactions (DDIs) and estimation of dosing requirements for concomitantly administered drugs is imperative. In this study, we aimed to develop a physiologically-based PK (PBPK) model for RTV using the PK-sim® software platform. A total of 13 clinical PK studies of RTV covering a wide dose range (100 to 600 mg including both single and multiple dosing), and eight clinical DDI studies with RTV on CYP3A and P-gp substrates, including alprazolam, midazolam, rivaroxaban, clarithromycin, fluconazole, sildenafil, and digoxinwere used for the model development and evaluation. Chronopharmacokinetic differences (between morning vs. evening doses) and limitations in parameter estimation for biochemical processes of RTV from invitro studies were incorporated in the PBPK model. The final developed PBPK model predicted 100% of RTV AUClast and Cmax within a twofold dimension error. The geometric mean fold error (GMFE) from all PK datasets was 1.275 and 1.194, respectively. In addition, 97% of the DDI profiles were predicted with the DDI ratios within a twofold dimension error. The GMFE values from all DDI datasets were 1.297 and 1.212, respectively. Accordingly, this model could be applied to the prediction of DDI profiles of RTV and CYP3A substrates and used to estimate dosing requirements for concomitantly administered drugs.

Quantitative contributions of hepatic and renal organic cation transporters to the clinical pharmacokinetic cimetidine-metformin interaction.

The widely prescribed oral anti-diabetic drug metformin is eliminated unchanged in the urine primarily through active tubular secretion. This process is mediated by organic cation transporter 2 (OCT2), an uptake transporter expressed on the basolateral membrane of renal proximal tubule cells. Metformin uptake into the liver, the site of action, is mediated by OCT1, which is expressed on the sinusoidal membrane of hepatocytes. Sixteen healthy adults participated in a clinical pharmacokinetic drug-drug interaction study in which they were orally administered metformin (50 mg) as a dual OCT1/2 substrate alone (baseline) and with cimetidine (400 mg) as an OCT inhibitor. Relative to baseline, metformin systemic plasma exposure increased by 24% ( p <0.05) in the presence of cimetidine, which was accompanied by a disproportional decrease (8%) in metformin renal clearance ( p =0.005). Genetic variants of OCT1 and OCT2 moderately impacted the significance and magnitude of the interaction. Collectively, we hypothesized that the cimetidine-metformin interaction involves inhibition of hepatic OCT1 as well as renal OCT2. We tested this hypothesis by developing a physiologically based pharmacokinetic (PBPK) model and assessing potential OCT biomarkers in plasma and urine to gain mechanistic insight into the transporters involved in this interaction. The PBPK model predicted that cimetidine primarily inhibits hepatic OCT1 and, to a lesser extent, renal OCT2. The unchanged renal clearance of potential OCT2 biomarkers following cimetidine exposure supports a minimal role for renal OCT2 in this interaction.

Application of liquid chromatography-mass spectrometry for the determination of semaglutide in human serum in clinical pharmacokinetic studies

Introduction. Semaglutide preparations are an important therapeutic option for patients suffering from type 2 diabetes mellitus and obesity due to their high efficacy and the expected increase in the prevalence of these diseases. Consequently, there is a growing need for the development of domestic analogs of semaglutide requiring bioequivalence studies. This study proposes the use of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) as an alternative to the widely used immunoassay for the quantitative determination of semaglutide in human serum.Aim. To develop and validate a method for the quantitative determination of semaglutide in human serum using HPLC-MS/MS.Materials and methods. Serum sample preparation was based on protein precipitation using an acetonitrile-methanol mixture. Liraglutide was selected as the internal standard. The mobile phase consisted of 0.3% formic acid in water and acetonitrile. The stationary phase was represented by a Phenomenex Kinetex C18 chromatographic column 100×3.0 mm, 5 μm, 100 Å. Ionization of semaglutide and liraglutide was performed in positive electrospray mode. Detection was carried out in multiple reaction monitoring mode (MRM).Results. The method demonstrated high accuracy and precision, with relative error and relative standard deviation values of less than 15% across all quality control levels. The confirmed analytical ranges of the method were 0.50–200.00 ng/mL and 1.00–800.00 ng/mL. Over 3.400 volunteer samples were analyzed as part of the studies. Compared to the ELISA method, the proposed method provides higher selectivity and reproducibility of measurements.Conclusions. The method has been developed that provides reproducible quantitative determination of semaglutide in human blood serum. The method was validated in accordance with EAEU requirements and was successfully applied in bioequivalence studies of semaglutide GP40331 (GEROPHARM LLC, Russia). The method is suitable for conducting pharmacokinetic studies of other semaglutide preparations.

Impact of Milk pH and Fat Content on the Prediction of Milk-to-Plasma Ratio: Knowledge Gap and Considerations for Lactation Study Design and Interpretation.

Different empirical lactation models have been published to predict the milk-to-plasma (M/P) ratio of drugs to gain knowledge on the extent of drug distribution to the breastmilk. M/P ratios will likely vary across the lactation period due to differences in physiological milk pH and fat content, which are not routinely reported in clinical lactation pharmacokinetic studies. This work aims to evaluate the sensitivity of two (a theory-based phase distribution and a log-transformed regression) lactation models for M/P prediction at different physiological milk pH and fat content. A literature search was conducted to collate reported M/P ratios for different drugs and their physicochemical parameters required for the prediction of the M/P ratio. Two distribution models were used for M/P ratio predictions. The M/P ratio of drugs was predicted under the physiological milk pHs of 6.8, 7.0, 7.2, and 7.4 and at of 1%, 3%, and 6% fat content. Calculated M/P ratios were compared with the observed M/P ratios. A total of 200 M/P ratios for 130 compounds (40 acids and 90 bases) were collected from clinical studies and included in the analysis. For both model, precision decreases and bias increases outside the milk pH range 7.0-7.2 and fat contents more than 3%. Significant variability exists in the observed M/P ratios. Both milk pH and fat content are important parameters for model prediction. Calculated M/P ratios are influenced by multiple covariates, including milk pH and fat content. The phase distribution model is less sensitive to these covariates than the log-transformed model, especially for acidic compounds. For complex matrices such as breastmilk, the actual physiological parameters of the sampled milk, at least milk fat and pH, and their distributions are required covariates to improve the prediction outcomes, design lactation pharmacokinetic studies, and inform the potential breastfed infant dose.

Role of Physiologically Based Modeling and Simulations in Clinical Pharmacokinetic Study Waivers for Orally Administered Drug Products

Role of Physiologically Based Modeling and Simulations in Clinical Pharmacokinetic Study Waivers for Orally Administered Drug Products

Sample Preparation for Pharmaceuticals using A Bioanalytical Method: A Review

The process of developing a method that will allow a compound of interest to be located and measured in a biological matrix is known as bioanalytical method development. A substance can frequently be measured using a variety of techniques, and selecting an analytical technique requires careful thought. Different extraction techniques, such as liquid-liquid extraction, solid phase extraction (SPE), and protein precipitation, are used to analyse drugs and their metabolites in a biological matrix. Samples from these extraction methods are spiked with calibration (reference) standards and quality control (QC) samples. The process of establishing if a quantitative analytical method is suitable for biomedical applications is known as bioanalytical method. includes all processes that will be presented measuring analytes quantitatively in particular biological samples such blood plasma, serum, or urine. To evaluate a drug's effectiveness and safety, clinical and non-clinical toxicokinetic and pharmacokinetic studies are used. Therefore, in order to produce accurate results, it is crucial that the applied bioanalytical procedures utilised are thoroughly defined, verified, and documented to a suitable quality. An overview of the development and validation of bioanalytical methods is given in this article, along with key considerations for each stage of method validation. The created procedure is then verified. Evaluation and interpretation of bioavailability, bioequivalence, pharmacokinetic, and pharmacodynamic investigations heavily rely on bioanalytical validations. where various parameters are carried out, including accuracy, precision, selectivity, sensitivity, reproducibility, and stability.

Quantitative determination of liposomal irinotecan and SN-38 concentrations in plasma samples from children with solid tumors: Use of a cryoprotectant solution to enhance liposome stability

Preclinical studies have demonstrated that liposomal irinotecan (CPT-11), a topoisomerase I inhibitor, has broad activity against adult cancers, including pancreatic, gastric, colon, lung, glioma, ovarian, and breast cancer. Encapsulation of irinotecan into liposomes can modify its pharmacokinetic properties dramatically. Also, the pharmacokinetic profiles of liposomal drug formulations are not fully understood; thus, bioanalytical methods are needed to separate and quantify nonencapsulated vs. encapsulated concentrations. In this study, two robust, specific, and sensitive LC-MS/MS methods were developed and validated to separate and quantify the nonencapsulated CPT-11 (NE-CPT-11) from the sum-total CPT-11 (T-CPT-11) and its major metabolite, SN-38, in human plasma after intravenous administration of liposomal irinotecan. NE-CPT-11 and SN-38 were separated from plasma samples by using solid-phase extraction, and T-CPT-11 was measured by protein precipitation. The liposomal CPT-11 formulation was unstable during sample storage and handling, resulting in elevated NE-CPT-11 concentration. To improve the stability of liposomal CPT-11, a cryoprotectant solution was added to human plasma samples prior to storage and processing. CPT-11, SN-38, and their respective internal standards, CPT-11-d10 and SN-38-d3, were chromatographically separated on a reversed-phase C18 analytical column. The drugs were detected on a triple quadrupole mass spectrometer in the positive MRM ion mode by monitoring the transitions 587.3 > 124.1 (CPT-11) and 393.0 > 349.1 (SN-38). The calibration curves demonstrated a good fit across the concentration ranges of 10–5000 ng/mL for T-CPT-11, 2.5–250 ng/mL for NE-CPT-11, and 1–500 ng/mL for SN-38. The accuracy and precision were within the acceptable limits, matrix effects were nonsignificant, recoveries were consistent and reproducible, and the analytes were stable under all tested storage conditions. Finally, the LC-MS/MS methods were successfully applied in a phase I clinical pharmacokinetic study of nanoliposomal irinotecan (Onivyde®) in pediatric patients with recurrent solid malignancies or Ewing sarcoma.

Modelling the nicotine pharmacokinetic profile for e-cigarettes using real time monitoring of consumers’ physiological measurements and mouth level exposure

Pharmacokinetic (PK) studies can provide essential information on abuse liability of nicotine and tobacco products but are intrusive and must be conducted in a clinical environment. The objective of the study was to explore whether changes in plasma nicotine levels following use of an e-cigarette can be predicted from real time monitoring of physiological parameters and mouth level exposure (MLE) to nicotine before, during, and after e-cigarette vaping, using wearable devices. Such an approach would allow an -effective pre-screening process, reducing the number of clinical studies, reducing the number of products to be tested and the number of blood draws required in a clinical PK study Establishing such a prediction model might facilitate the longitudinal collection of data on product use and nicotine expression among consumers using nicotine products in their normal environments, thereby reducing the need for intrusive clinical studies while generating PK data related to product use in the real world.An exploratory machine learning model was developed to predict changes in plasma nicotine levels following the use of an e-cigarette; from real time monitoring of physiological parameters and MLE to nicotine before, during, and after e-cigarette vaping. This preliminary study identified key parameters, such as heart rate (HR), heart rate variability (HRV), and physiological stress (PS) that may act as predictors for an individual’s plasma nicotine response (PK curve). Relative to baseline measurements (per participant), HR showed a significant increase for nicotine containing e-liquids and was consistent across sessions (intra-participant). Imputing missing values and training the model on all data resulted in 57% improvement from the original’learning’ data and achieved a median validation R2 of 0.70.The study is in its exploratory phase, with limitations including a small and non-diverse sample size and reliance on data from a single e-cigarette product. These findings necessitate further research for validation and to enhance the model's generalisability and applicability in real-world settings. This study serves as a foundational step towards developing non-intrusive PK models for nicotine product use.

Development of a fluorescent nano-sensing probe for atomoxetine analysis: Additional clinical pharmacokinetic study

Atomoxetine is a psychostimulant drug used for the treatment of attention-deficit/hyperactivity disorder (ADHD) symptoms in people with autism. Herein, eco-friendly fluorescent carbon quantum dots (CQDs) were synthesized using black-eyed pea beans and characterized for the purpose of quantifying atomoxetine in pharmaceutical capsules and human plasma. The selectivity of these CQDs towards atomoxetine was improved by functionalizing their surface with an atomoxetine-tetraphenylborate ion complex. The quantification of atomoxetine is based on measuring the fluorescence quenching of the functionalized CQDs in response to varying concentrations of atomoxetine. The Stern-Volmer plot was employed to investigate the mechanism through which atomoxetine quenches the fluorescence intensity of the CQDs. The outcomes indicated a dynamic quenching mechanism. The applied method was optimized and validated in compliance with ICH requirements, resulting in excellent linearity across the concentration range of 50–800 ng/mL. The developed method was successfully used to quantify atomoxetine in pharmaceutical dosage form and human plasma with acceptable accuracy and precision outcomes. In addition, the method was applied for clinical pharmacokinetic study of atomoxetine in the plasma of children diagnosed with both autism and ADHD. Atomoxetine was rapidly absorbed after a single oral dose of 10 mg, reaching maximum concentration within two hours and having a half-life (t1/2) of 3.11 h. Moreover, the method demonstrates a notable degree of eco-friendliness, as evidenced by two greenness evaluation metrics; Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE).

Development and Validation of a High-Performance Liquid Chromatography Coupled With Ultraviolet Detection Method for Quantification of Bictegravir in Human Plasma.

To establish a method for determining the bictegravir (BIC) concentration in human plasma using high-performance liquid chromatography coupled with ultraviolet detection. The analysis was performed on a CLC-octadecylsilane column (150 × 6.0 mm, 5 μm) using a mixture of phosphate buffer and acetonitrile (62:38, v/v) as the mobile phase at the flow rate of 1.4 mL/min. The column temperature was maintained at 40°C. Using triamcinolone acetonide as the internal standard, 100 μL of plasma sample was extracted by methyl tert-butyl ether, followed by evaporating under nitrogen stream, redissolving with 100 μL mobile phase, and injection of 20-40 μL of supernatant into the chromatographic system. Ultraviolet detection was performed at 260 nm, and the total run time for each sample was 14 minutes. The method exhibited good linearity within the range from 0.10 to 10.0 mcg/mL (r = 0.9995, n = 5). The intraday and interday relative standard deviations for low-, medium-, and high-concentration quality control samples (0.20, 4.00, 8.00 mcg/mL) and the lower limit of quantification (0.10 mcg/mL) were 1.31%-6.20% (n = 10) and 1.18%-2.87% (n = 5), respectively. The intraday and interday accuracies were 100.53%-102.32% and 97.96%-103.84%, respectively. The extraction recovery rates ranged from 80.00% to 88.09% (n = 3). The stability tests showed that the BIC concentration changed by <15%. This study successfully established a high-performance liquid chromatography coupled with ultraviolet detection method for determining plasma BIC concentrations. This method is simple, selective, sensitive, and accurate, making it suitable for clinical monitoring and pharmacokinetic studies of BIC.

Attaining Equity of Access to Research: Perspective on Research in Pregnancy and Breastfeeding Following Dolores Shockley Lecture at ASCPT2024.

Everybody deserves access to evidence-based information to make decisions about their health. However, in many situations, clinical trial eligibility criteria mean that specific data do not exist for certain groups of individuals. These include pregnant and breastfeeding women, children, older people, those with hepatic and renal dysfunction, those with acute severe illness, and those with multiple co-morbidities and interacting medications. Resultantly, there may not be specific drug-dosing information for many patients who are treated in a clinical setting. The ASCPT2024 Dolores Shockley Lecture focused on the equitable access to research with a specific focus on clinical pharmacology studies in pregnancy and breastfeeding. To ensure the safe, effective use of medication in pregnancy and breastfeeding, women should be included in clinical trials and pharmacokinetic studies when a medication is anticipated to be used in women of childbearing potential. Community groups should be involved at all stages of research to maintain transparency and trust. This ensures that local priorities are investigated, that communities understand the findings and are empowered to make evidence-based decisions about their own medication use. Principles informing the design of such studies in pregnancy and lactation are in existence. Mathematical techniques such as physiologically-based pharmacokinetic modeling and stochastic simulation and estimation can enhance study design, and population pharmacokinetic modeling be used to understand variability within and between individuals. Data should be made findable, accessible, interoperable, and reusable (FAIR). Information (and where necessary, training) regarding the use of these approaches should be provided to decision-making stakeholders such as ethics committees and regulatory bodies.

Integrated PBPK-EO modeling of osimertinib to predict plasma concentrations and intracranial EGFR engagement in patients with brain metastases

The purpose of this study was to develop and validate a physiologically based pharmacokinetic (PBPK) model combined with an EGFR occupancy (EO) model for osimertinib (OSI) to predict plasma trough concentration (Ctrough) and the intracranial time-course of EGFR (T790M and L858R mutants) engagement in patient populations. The PBPK model was also used to investigate the key factors affecting OSI pharmacokinetics (PK) and intracranial EGFR engagement, analyze resistance to the target mutation C797S, and determine optimal dosing regimens when used alone and in drug-drug interactions (DDIs). A population PBPK-EO model of OSI was developed using physicochemical, biochemical, binding kinetic, and physiological properties, and then validated using nine clinical PK studies, observed EO study, and two clinical DDI studies. The PBPK-EO model demonstrated good consistency with observed data, with most prediction-to-observation ratios falling within the range of 0.7 to 1.3 for plasma AUC, Cmax, Ctrough and intracranial free concentration. The simulated time-course of C797S occupancy by the PBPK model was much lower than T790M and L858R occupancy, providing an explanation for OSI on-target resistance to the C797S mutation. The PBPK model identified ABCB1 CLint,u, albumin level, and EGFR expression as key factors affecting plasma Ctrough and intracranial EO for OSI. Additionally, PBPK-EO simulations indicated that the optimal dosing regimen for OSI in patients with brain metastases is either 80 mg once daily (OD) or 160 mg OD, or 40 mg or 80 mg twice daily (BID). When used concomitantly with CYP enzyme perpetrators, the PBPK-EO model suggested appropriate dosing regimens of 80 mg OD with fluvoxamine (FLUV) itraconazole (ITR) or fluvoxamine (FLUC) for co-administration and an increase to 160 mg OD with rifampicin (RIF) or efavirenz (EFA). In conclusion, the PBPK-EO model has been shown to be capable of simulating the pharmacokinetic concentration–time profiles and the time-course of EGFR engagement for OSI, as well as determining the optimum dosing in various clinical situations.

Re-discover the value of protein binding assessments in hepatic and renal impairment studies and its contributions in drug labels and dose decisions.

One of the key pharmacokinetic properties of most small molecule drugs is their ability to bind to serum proteins. Unbound or free drug is responsible for pharmacological activity while the balance between free and bound drug can impact drug distribution, elimination, and other safety parameters. In the hepatic impairment (HI) and renal impairment (RI) clinical studies, unbound drug concentration is often assessed; however, the relevance and impact of the protein binding (PB) results is largely limited. We analyzed published clinical safety and pharmacokinetic studies in subjects with HI or RI with PB assessment up to October 2022 and summarized the contribution of PB results on their label dose recommendations. Among drugs with HI publication, 32% (17/53) associated product labels include PB results in HI section. Of these, the majority (9/17, 53%) recommend dose adjustments consistent with observed PB change. Among drugs with RI publication, 27% (12/44) of associated product labels include PB results in RI section with the majority (7/12, 58%) recommending no dose adjustment, consistent with the reported absence of PB change. PB results were found to be consistent with a tailored dose recommendation in 53% and 58% of the approved labels for HI and RI section, respectively. We further discussed the interpretation challenges of PB results, explored treatment decision factors including total drug concentration, exposure-response relationships, and safety considerations in these case examples. Collectively, comprehending the alterations in free drug levels in HI and RI informs treatment decision through a risk-based approach.

Unveiling Pharmacokinetics and Drug Interaction of Linagliptin and Pioglitazone HCl in Rat Plasma Using LC-MS/MS.

The linagliptin (LIN) and pioglitazone HCl (PIO) combination, currently undergoing phase III clinical trials for diabetes mellitus treatment, demonstrated significant improvements in glycemic control. However, the absence of an analytical method for simultaneous determination in biological fluids highlights a crucial gap. This underscores the pressing need for sensitive bioanalytical methods, emphasizing the paramount importance of developing such tools to advance diabetes management strategies and enhance patient care. Herein, a sensitive reverse-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for simultaneous determination of LIN and PIO in rat plasma using alogliptin as an internal standard. Chromatographic separation was performed on an Agilent Eclipse Plus C18 (4.6 × 100 mm, 3.5 μm) using an isocratic mobile phase system consisting of ammonium formate (pH 4.5) and methanol using an acetonitrile-induced protein precipitation technique for sample preparation. Multiple reaction monitoring in positive ion mode was used for quantitation of the precursor to production at m/z 473.2 → 419.9 for LIN, 357.1 → 134.2 for PIO, and 340.3 → 116.1 for ALO. The linearity range was 0.5 to 100 and 1 to 2000 ng/mL for LIN and PIO, respectively. The developed method was validated as per US-FDA guidelines and successfully applied to clinical pharmacokinetic and drug-drug interaction studies with a single oral administration of LIN and PIO in rat plasma. Pharmacokinetic parameters of LIN were significantly influenced by the concomitant administration of PIO and vice versa. Molecular modeling revealed the significant interaction of LIN and PIO with P-glycoprotein. Therefore, the drug-drug interaction between LIN and PIO deserves further study to improve drug therapy and prevent dangerous adverse effects.

Impedimetric sensor based on molecularly imprinted polythionine from deep eutectic solvent for epinephrine determination

Deep eutectic solvents have obtained growing interest in analytical chemistry due to opportunities related the green chemistry paradigm, i.e., substitution of organic solvents, application of nontoxic ingredients and minimal quantities of wastes formed in their synthesis and usage. In this work, the advantages of deep eutectic solvents were shown in the development of electrochemical sensor for epinephrine determination. Polythionine molecularly imprinted with epinephrine (PTNMIP) was electrodeposited from natural deep eutectic solvent (NADES) consisted of citric acid, glucose, and water in 1:1:6 molar ratio and tested with standard solutions of epinephrine, pharmaceutical preparations, and artificial samples of biological liquids. Only one drop (100 µL) of the monomer dissolved in NADES was applied for sensor preparation on the platform of screen-printed carbon electrode. Electropolymerization was performed by multiple scanning of the potential. The formation of molecular imprints was confirmed by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). After the template removal, the charge transfer resistance of the surface layer depended on the epinephrine concentration in the range from 100 nM to 316 µM (limit of detection 50 nM). Selectivity of the signal was confirmed against serotonin and norepinephrine commonly present in biological liquids. Sensor was successfully tested for the determination of epinephrine in spiked solutions mimicking influence of the plasma electrolytes, serum proteins, and in real saliva samples. The recoveries calculated for 10 μM epinephrine ranged from 98.3 to 102.6%. Insignificant influence of the additives and stabilizers present in medications was eliminated by sample dilution. Impedimetric sensor developed showed good opportunities for further application in clinical diagnostics, pharmacokinetics studies and medication manufacture control.

Highly sensitive liquid chromatography-mass spectrometry method for the quantitative analysis of mometasone furoate in human plasma: Method validation and application to clinical pharmacokinetic studies.

We report the development and the validation of a sensitive liquid chromatography-mass spectrometry (LC-MS/MS) method for mometasone furoate (MF) analysis in human plasma. Plasma samples were processed through liquid-liquid extraction and analyzed using LC-MS/MS operating in positive mode using multiple reaction monitoring of transitions m/z 520.9→ 355.0 and m/z 525.8→ 355.0 for MF and the internal standard (IS), respectively. Separation was achieved at 1.0mL/min on a C18 column using a gradient elution of mobile phase of 0.05% ammonia in water (phase A) and acetonitrile (phase B). The assay range was 0.250-100 pg/mL and proved to be accurate and precise MF. Normalized recoveries were consistent and reproducible with a coefficient of variation (CV%) value of 6.0. The CV (%) of the IS normalized matrix factor was not observed in normal, lipemic, and hemolyzed plasmas. Dilutions of 1:10 were accurately quantified. A cycle of three freeze and thaw and stabilities at room temperature and on the autosampler were demonstrated. In addition, MF in the presence of indacaterol and glycopyrronium was proven to be stable at -70°C for at least 157 days. The present method was successfully applied to quantify MF in patients receiving MF, indacaterol, and glycopyrronium as a fixed-dose combination.

498 Bruton’s tyrosine kinase (BTK) inhibitors impede platelet aggregation but not adhesion to collagen.

OBJECTIVES/GOALS: The research objectives of this project are to elucidate the effects of Bruton’s tyrosine kinase inhibitors (BTKi) of varying target specificity on platelet function with regard to platelet aggregation, adhesion, spreading, and intracellular signaling as measured by kinase phosphorylation. METHODS/STUDY POPULATION: Blood from healthy volunteers was obtained and processed to obtain both washed platelets and platelet-rich plasma. The samples were then treated with one of the BTKi drugs or with vehicle (DMSO) at concentrations matching patient blood concentrations derived from clinical trials and pharmacokinetic studies. The incubated samples were then analyzed in an aggregometer using one of several agonists. Aggregation was stopped after five minutes with a perchloric acid-based lysis buffer. The samples were then analyzed by SDS-PAGE and immunoblotting to quantify BTK protein and BTK phosphorylation. Adhesion was assessed by incubating washed platelets treated with BTKi on microtiter wells coated with fibrinogen or collagen and quantifying adherent platelets by their endogenous acid phosphatase activity. RESULTS/ANTICIPATED RESULTS: We found that ibrutinib, zanubrutinib, and pirtobrutinib all completely inhibited collagen-induced platelet aggregation, whereas they did not inhibit aggregation induced by thrombin, ristocetin, arachidonic acid, or the PAR1 activator peptide SFLLRN (T6). Acalabrutinib inhibited collagen-induced platelet aggregation only at high concentrations (1-2 micromolar). At the lower concentration of 200 nanomolar, comparable to the concentration required for the other BTK inhibitors to completely inhibit platelet aggregation, acalabrutinib failed to inhibit aggregation but did inhibit auto-phosphorylation, indicating an impact on signaling. None of the BTKi drugs inhibited adhesion of platelets to collagen-coated surfaces. DISCUSSION/SIGNIFICANCE: Our data show the inhibitory effect of BTKi on collagen-induced platelet aggregation and signaling. However, it remains unclear whether the inhibition is due to an effect on BTK itself or other related kinases. Better insight into the mechanisms of platelet inhibition by BTKi may help guide the development of BTKi with a lower risk of hemorrhage.