Simultaneous quantification of five antiretrovirals in human tissues using ultra-high performance liquid chromatography-tandem mass spectrometry methods for therapeutic drug monitoring at the sites of action.

Although antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection to suppress virus in the blood, HIV persists in tissues. HIV persistence in the tissues is due to numerous factors, and one of those factors are antiretroviral (ARV) concentrations. ARV concentrations in tissues must be adequate to suppress HIV at the sites of action. While therapeutic drug monitoring in the plasma is well-known, drug monitoring in the tissues provides local assessments of adequate ARV exposure to prevent localized HIV resistance formation. Towards these efforts, we validated an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method in human tissues (cervical, rectal, and vaginal tissues) for the simultaneous quantification of five ARVs: bictegravir, cabotegravir, dolutegravir, doravirine, and raltegravir. For this assay, protein precipitation with acetonitrile with stable, isotopically-labeled internal standards followed by supernatant pre-concentration was performed. Analyte separation was accomplished using a multistep UPLC gradient mixture of 0.1 % formic acid in water (A) and acetonitrile (B) with a Waters Cortecs T3 (2.1x100 mm) column. The assay was extensively validated as per the United States Food and Drug Administration Bioanalytical Method Validation Guidance over a clinically observed range (0.05-50 ng/mL) with superb linearity (R2 > 0.99 across all ARVs). The assay run time was 8.5 min. This analytical method achieves appropriate performance of trueness (85.5-107.4 %), repeatability, and precision (CV < 15 %). Our method will be employed for the therapeutic monitoring of guideline-recommended ARVs in human tissues for monitoring therapeutic efficacy in HIV treatment and prevention research efforts.

Population Pharmacokinetic-Pharmacodynamic Modeling and Covariate Analyses of Neutropenia and Thrombocytopenia in Patients With Solid Tumors Treated With Lurbinectedin.

Lurbinectedin is a selective inhibitor of oncogenic transcription. Reversible myelosuppression is its most relevant toxicity. Pharmacokinetic-pharmacodynamic analyses were conducted to characterize the time course of absolute neutrophil count and platelet count recovery and to detect and quantify the effect of relevant covariates in patients with advanced solid tumors treated with lurbinectedin. Absolute neutrophil count, platelet count, and lurbinectedin total plasma concentration were assessed in 244 patients treated with lurbinectedin with varied dosing schedules and doses. A reference extended semimechanistic pharmacokinetic-pharmacodynamic model of myelosuppression was used. Granulocyte colony-stimulating factor (G-CSF) administration was modeled as a dichotomous covariate, and platelet transfusions were included as a bolus dose into the last compartment of the model, representing the central circulation. Final models were suitable to describe the time course of absolute neutrophil count and platelet count recovery. A lurbinectedin dose of 3.2 mg/m2 every 3 weeks can be administered without primary prophylaxis with G-CSF. G-CSF followed by ≤2 dose reductions of 20%, if needed, gradually reduced grade 4 neutropenia from cycle 3 onward. BSA-based dosing reduced the incidence of grade ≥ 3 thrombocytopenia. One-week dose delays because of low absolute neutrophil count occurred in 3.5% of patients, thus supporting every-3-week administration. CYP3A inhibitors produced absolute 11.0% and 6.2% increases in grade ≥ 3 neutropenia and thrombocytopenia, respectively. Neutropenia and thrombocytopenia after lurbinectedin administration to cancer patients are noncumulative, reversible, short lasting, and clinically manageable with secondary prophylaxis of G-CSF or platelet transfusion and, if needed, dose reductions.

Development and validation of a multiplex UHPLC-MS/MS assay with stable isotopic internal standards for the monitoring of the plasma concentrations of the antiretroviral drugs bictegravir, cabotegravir, doravirine, and rilpivirine in people living with HIV.

The widespread use of highly active antiretroviral treatments has dramatically changed the prognosis of people living with HIV (PLWH). However, such treatments have to be taken lifelong raising issues regarding the maintenance of both therapeutic effectiveness and long-term tolerability. Recently approved or investigational antiretroviral drugs present considerable advantages, allowing once daily oral dosage along with activity against resistant variants (eg, bictegravir and doravirine) and also parenteral intramuscular administration that facilitates treatment adherence (eg, long-acting injectable formulations such as cabotegravir and rilpivirine). Still, there remains a risk of insufficient or exaggerated circulating exposure due to absorption issues, abnormal elimination, drug-drug interactions, and others. In this context, a multiplex ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) bioassay has been developed for the monitoring of plasma levels of bictegravir, cabotegravir, doravirine, and rilpivirine in PLWH. A simple and convenient protein precipitation was performed followed by direct injection of the supernatant into the UHPLC-MS/MS system. The four analytes were eluted in less than 3 minutes using a reversed-phase chromatography method coupled with triple quadrupole mass spectrometry detection. This bioassay was fully validated following international guidelines and achieved good performances in terms of trueness (94.7%-107.5%), repeatability (2.6%-11%), and intermediate precision (3.0%-11.2%) over the clinically relevant concentration ranges (from 30 to 9000 ng/mL for bictegravir, cabotegravir, and doravirine and from 10 to 1800 ng/mL for rilpivirine). This sensitive, accurate, and rapid UHPLC-MS/MS assay is currently applied in our laboratory for routine therapeutic drug monitoring of the oral drugs bictegravir and doravirine and is also intended to be applied for the monitoring of cabotegravir/rilpivirine levels in plasma from PLWH receiving once monthly or every 2-month intramuscular injection of these long-acting antiretroviral drugs.

Pharmacokinetics, Safety, and Tolerability of Ledipasvir/Sofosbuvir and Sofosbuvir/Velpatasvir in Healthy Chinese Subjects.

PURPOSE: Ledipasvir/sofosbuvir and sofosbuvir/velpatasvir have been approved worldwide for the treatment of chronic hepatitis C virus (HCV) infection. Although both have been approved in China, there are currently no data on their pharmacokinetic profiles in Chinese individuals. Two studies investigated the pharmacokinetic properties, safety, and tolerability of ledipasvir/sofosbuvir and sofosbuvir/velpatasvir, respectively, in healthy Chinese subjects. METHODS: Two Phase I, open-label, single- and multiple-dose studies were conducted in healthy Chinese subjects. Ledipasvir/sofosbuvir (90/400 mg) or sofosbuvir/velpatasvir (400/100 mg), respectively, was administered orally once daily under fasted conditions. Subjects received a single dose (day 1) and multiple doses (days 8-17 [ledipasvir/sofosbuvir]; days 8-14 [sofosbuvir/velpatasvir]). Plasma pharmacokinetic parameters were estimated by using noncompartmental models, and safety was assessed through clinical evaluation and monitoring of adverse events. FINDINGS: Fourteen subjects were enrolled in each study (7 men, 7 women each; mean age, 30 years [ledipasvir/sofosbuvir] and 29 years [sofosbuvir/velpatasvir]). The pharmacokinetic parameters for sofosbuvir, GS-566500, GS-331007, and ledipasvir or velpatasvir were similar to historical values in non-Chinese subjects. Consistent with the t1/2 of ledipasvir relative to 24-h dosing, accumulation of 177% (AUC) and 107% (Cmax) was observed. There was no significant accumulation of velpatasvir, sofosbuvir, GS-566500, or GS-331007. Both drugs were generally well tolerated; no serious adverse events or discontinuations due to adverse events were reported. IMPLICATIONS: Overall, ledipasvir/sofosbuvir and sofosbuvir/velpatasvir exhibited pharmacokinetic and safety profiles in healthy Chinese subjects similar to those in non-Chinese subjects in historical studies, supporting their use in the Chinese population with HCV infection. ChinaDrugTrials.org.cn identifiers: CTR20160149 (ledipasvir/sofosbuvir); CTR20160602 (sofosbuvir/velpatasvir).

Determination of KD025 (SLx-2119), a Selective ROCK2 Inhibitor, in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry and its Pharmacokinetic Application.

KD025 (SLx-2119), the first specific Rho-associated protein kinase 2 (ROCK2) inhibitor, is a potential new drug candidate currently undergoing several phase 2 clinical trials for psoriasis, idiopathic pulmonary fibrosis, chronic graft-versus-host disease, and systemic sclerosis. In this study, a bio-analytical method was developed and fully validated for the quantification of KD025 in rat plasma and for application in pharmacokinetic studies. KD025 and GSK429286A (the internal standard) in rat plasma samples were analyzed by high-performance liquid chromatography-tandem mass spectrometry with m/z transition values of 453.10 → 366.10 and 433.00 → 178.00, respectively. The method was fully validated according to the United State Food and Drug Administration guidelines in terms of selectivity, linearity, accuracy, precision, sensitivity, matrix effects, extraction recovery, and stability. The method enabled the quantification of KD025 levels in rat plasma following oral administration of 5 mg/kg KD025 and intravenous administration of 2 mg/kg KD025 to rats, respectively. Our findings suggest that the developed method is practical and reliable for pharmacokinetic studies of KD025 in preclinical animals.

UPLC-MS/MS method for the simultaneous quantification of bictegravir and 13 others antiretroviral drugs plus cobicistat and ritonavir boosters in human plasma.

A sensitive and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for 14 antiretroviral drugs and 2 boosters in human plasma. Plasma (100 µL) was precipitated with a solution of acetonitrile containing labelled internal standards. The compounds were separated with a total chromatic run time of 6 min using an Acclaim TM RSLC 120 C18 column (2.1 × 100 mm, 2.2 µm). The method was fully validated according to the European Medecines Agency guidelines. Linearity of all analytes concentrations was validated up to 5000 ng/mL. Lower limits of quantification were ranged from 2.5 ng/mL to 10 ng/mL according to compounds. Intra-day and inter-day precision ranged from 0.2% to 8.9% and accuracies were below 13%. This UPLC-MS/MS method can be applied to clinical pharmacology research and therapeutic drug monitoring in patients living with HIV.

A validated UPLC-MS/MS method for pharmacokinetic study of inflachromene, a novel microglia inhibitor.

Inflachromene (ICM), a novel microglia inhibitor, is under development to treat neuroinflammatory disorders. For the pharmacokinetic evaluation of ICM, we developed quantitative ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method in rat plasma. Protein precipitation method with acetonitrile (ACN) was used for plasma sample preparation. The analyte and carbamazepine (the internal standard) were separated by the chromatography on an ACQUITY UPLC™ BEH C18 column running gradient mobile phase system made up of 0.1% [v/v] formic acid in water and 0.1% [v/v] formic acid in acetonitrile. For the quantification of ICM, the ion transitions, m/z 378.2→187.1 (ICM) and m/z 237.1→194.1 (IS) were monitored in multiple reaction monitoring (MRM) mode. Standard calibration curve showed excellent linearity with the correlation coefficient (R2) of 0.99 within the concentration range of 0.05-10 µg/mL. The precision and accuracy were within acceptable limits (all < 20%). ICM was degraded time and temperature dependently in rat plasma. The analytical method was successfully utilized in a pharmacokinetic study following intravenous and oral administration of ICM in rats.

Development and validation of LC-MS/MS method for quantification of bictegravir in human plasma and its application to an intracellular uptake study.

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantification of bictegravir in human plasma. A small volume of only 50 µL aliquot of plasma was precipitated with acetonitrile containing an internal standard (IS). Chromatographic separation was performed on a Kinetex EVO C18 column, 50 × 3.0 mm, 5 µm using an isocratic mobile phase containing 80:20 acetonitrile-water with 0.1% formic acid. A mass spectrometer was operated in ESI positive multiple reaction monitoring mode using the m/z 450.1/289.1 for bictegravir and 420.1/277.1 for IS. The dynamic range of the method was 1-10,000 ng/mL with a correlation coefficient of ≥0.9991. The precision results of calibration standards were in the range of 0.05-4.57% and accuracies were 95.07-104.70%. The mean extraction recovery was 98.64% with a precision of 2.91%. The method was validated as per US Food and Drug Administration guidelines and was found to be accurate and precise. The method was successfully applied to in vitro cellular uptake study.

Effect of a high-fat meal on the relative bioavailability of H3B-6527, a novel FGFR4 inhibitor, in healthy volunteers.

PURPOSE: This Phase I study estimated the effect of a high-fat meal on the pharmacokinetics (PK) of H3B-6527, a covalent inhibitor of the fibroblast growth factor receptor (FGFR) 4 in clinical development for hepatocellular carcinoma and intrahepatic cholangiocarcinoma. METHODS: In this randomized, single center, single-dose, open-label, 2-period crossover study 12 healthy male volunteers, aged 18-55 years old, received a single 200-mg dose of H3B-6527 (capsule) following an overnight fast or a high-fat breakfast. PK samples were collected serially up to 36 h postdose. H3B-6527 concentrations were measured using a validated high-performance liquid chromatography tandem mass spectrometry method. PK data were analyzed using a noncompartmental approach based on a mixed-effects model. The safety and tolerability of H3B-6527 were also assessed. RESULTS: H3B-6527 plasma exposure increased after a high-fat meal with fed/fasted ratios of the geometric means (90% confidence interval) of 174% (102-298%) for Cmax and 246% (146-415%) for AUC0-t. Food delayed and prolonged absorption of H3B-6527, with a fed/fasted ratio for tmax of 200% (137-263%). PK variability was lower under the fed condition, as illustrated by the CV% for Cmax and AUC0-t of 41.9-54.5% (fed) versus 64.3-70.4% (fasted). CONCLUSIONS: A single 200 mg dose of H3B-6527 was safe and generally well tolerated when administered to healthy adult males. A high-fat meal significantly increased exposure to H3B-6527, from 1.5- to 2.5-fold in the systemic circulation, compared to administration under fasted conditions. Food delayed and prolonged absorption of H3B-6527. In general, lower inter-subject variability was observed in the fed state in healthy volunteers. TRIAL REGISTRATION: ClinicalTrials.gov.: NCT03424577.

Rapid bioanalytical LC-MS/MS method for the simultaneous determination of sofosbuvir and velpatasvir in human plasma-application to a pharmacokinetic study in Egyptian volunteers.

A novel, rapid and validated LC-MS/MS bioanalytical method has been developed for the extraction and determination of sofosbuvir and velpatasvir simultaneously in human plasma using ledipasvir as an internal standard (IS). The simple and reproducible protein precipitation technique with acetonitrile was successfully used for the deproteinization and extraction of the analytes from human plasma matrix. The developed method achieved consistent recoveries over different concentrations with average extraction recoveries of 81.72% and 80.46% for sofosbuvir and velpatasvir, respectively. The chromatographic separation was performed within only 2.80 min as a run time by an isocratic elution through C18 Zorbox eclipse plus (100 × 4.6 mm, 5 µm). Optimum mobile phase consisted of 0.1% formic acid in water: acetonitrile: methanol (30:60:10, v/v/v) pumped at a flow rate of 0.55 mL min-1 and injection volume was 10 µL. LC-MS/MS detection was done by multiple reaction monitoring transitions operating at positive ionization mode for both analytes and IS. Bioanalytical method validation as per EMA guidelines was carried out where the proposed method revealed linearity over the concentration range of 5-5000 and 10-1500 ng mL-1 for sofosbuvir and velpatasvir, respectively. After validation, the method was applied to the analysis of the two drugs after a single oral administration of Epclusa 400/100 mg tablets to Egyptian healthy volunteers.

Study on fluorescence properties of HCV antiviral (velpatasvir) and its fluorimetric determination in presence of sofosbuvir; application to stability study and human plasma.

Velpatasvir (VLP) is a new, oral, direct-acting antiviral with potent inhibitory activity against all hepatitis C virus (HCV) genotypes. A highly sensitive, simple, fast and specific one fluorometric method for determination of VLP in the presence of sofosbuvir was developed and validated. The fluorescence behavior of VLP in different organic solvents was examined and explained. Methanol was concluded to be the best sensitizing reagent. The native fluorescence intensity of VLP was accomplished at 383 nm with 339 nm for excitation wavelength. The impacts of experimental variables included pH, various organized media, and time of stability were examined and optimized. A linear relationship was achieved between the VLP concentration and the fluorescence intensity in a range of 5 to 5 × 103 ng mL-1 with 0.70 and 0.23 ng mL-1 , for quantitation and detection limits respectively. The proposed method was utilized for analyzing of VLP in human plasma and additionally expanded to examine the stability of VLP after its exposure to various stress conditions, like oxidative, alkaline, acidic, UV, daylight and sunlight conditions, according to ICH guidelines. Furthermore, the kinetics of acidic and oxidative degradations of VLP was examined. Moreover, the half-life times of the reaction (t1/2 ) and the first-order reaction rate constants were estimated. Finally, a suggestion for the degradation pathway was presented.

Novel determination of sofosbuvir and velpatasvir in human plasma by UPLC-MS/MS method: Application to a bioequivalence study.

A novel and sensitive LC-MS/MS method was developed, optimized and validated for quantification of sofosbuvir (SOF) and velpatasvir (VEL) in human plasma using ledipasvir as an internal standard (IS). Sample preparation was done using acetonitrile for precipitation of plasma proteins. Chromatographic analysis was done on an Acquity UPLC BEH C18 column using 0.1% formic acid and acetonitrile as a mobile phase. The Xevo TQD LC-MS/MS system was run with electrospray ionization mode. The developed method was optimized and then validated according to the US Food and Drug Administration guidelines. Linearity was found to be in the range of 0.25-3500 ng/mL for SOF and 1-1000 ng/mL for VEL. A short run time of 1.5 min allows swift analysis of many plasma samples per day. The developed method was successfully utilized for estimating both SOF and VEL in the plasma of healthy human volunteers participated in a bioequivalence study.

Determination of corypalmine in mouse blood by UPLC-MS/MS and its application to a pharmacokinetic study.

In this work, a selective and sensitive ultra-performance liquid chromatography tandem mass spectrometry method was established and validated for determination of corypalmine in mouse blood after oral or intravenous administration. A UPLC BEH C18 column was used to separate corypalmine and berberrubine (internal standard) at 40°C. The mobile phase was composed of acetonitrile and 10 mmol/L ammonium acetate (containing 0.1% formic acid) at a flow rate of 0.4 mL/min, and the total run time was 4.0 min. Electrospray ionization in positive ion mode was applied; target fragment ions m/z 342.2 → 178.0 for corypalmine and m/z 322.1 → 307.0 for berberrubine were identified with multiple reaction monitoring mode. The linear range was 1-1000 ng/mL (r > 0.995) and the lower limit of quantification for corypalmine in plasma was 1.0 ng/mL. The intra- and inter-day precisions were both <14%. The range of accuracy in this method was 97.5-109.0%. Mean recovery was >69.6%, and the matrix effect was 96.8-107.6%. Based on its high sensitivity, specificity and reliability, this method was successfully applied to study the pharmacokinetic parameters of corypalmine in mouse by oral and intravenous administration, and finally, the bioavailability of corypalmine was identified at 4.6%.

Pharmacokinetics and Safety of Velpatasvir and Sofosbuvir/Velpatasvir in Subjects with Hepatic Impairment.

BACKGROUND: The pharmacokinetics and safety of velpatasvir, a potent pangenotypic hepatitis C virus NS5A inhibitor, were evaluated in two hepatic impairment studies: a phase I study in hepatitis C virus-uninfected subjects and a phase III study (ASTRAL-4) in hepatitis C virus-infected patients. METHODS: In the phase I study, subjects with moderate or severe hepatic impairment (Child-Pugh-Turcotte Class B or C), and demographically matched subjects with normal hepatic function received a single dose of velpatasvir 100 mg. Pharmacokinetics and safety assessments were performed, and pharmacokinetic parameters were calculated using non-compartmental methods and summarized using descriptive statistics and compared statistically by geometric least-squares mean ratios and 90% confidence intervals. In ASTRAL-4, subjects with decompensated cirrhosis (Child-Pugh-Turcotte Class B) were randomized to receive treatment with either sofosbuvir/velpatasvir ± ribavirin for 12 weeks or sofosbuvir/velpatasvir for 24 weeks. Pharmacokinetic and safety assessments were performed and pharmacokinetic parameters were calculated using a non-compartmental analysis and summarized using descriptive statistics and were compared to pharmacokinetics from ASTRAL-1 [subjects without cirrhosis or with compensated (Child-Pugh-Turcotte Class A) cirrhosis]. RESULTS: In the phase I study, plasma exposures (area under the concentration-time curve) were similar in subjects with Child-Pugh-Turcotte Class B (n = 10) or Child-Pugh-Turcotte Class C hepatic impairment (n = 10) compared with normal hepatic function (n = 13). Percent free velpatasvir was similar in subjects without or with any degree of hepatic impairment. In the phase III study, velpatasvir overall exposure (area under the concentration-time curve over the 24-h dosing interval; AUCtau) was similar and sofosbuvir exposures were higher (~ 100%) for patients with Child-Pugh-Turcotte Class B hepatic impairment compared with the ASTRAL-1 population, which was not considered clinically relevant. CONCLUSIONS: No sofosbuvir/velpatasvir dose modification is warranted for patients with any degree of hepatic impairment.

Describing the holistic toxicokinetics of hepatotoxic Chinese herbal medicines by a novel integrated strategy: Dioscorea bulbifera rhizome as a case study.

It is vital to monitor the holistic toxicokinetics of toxic Chinese herbal medicines (CHMs) for safety. Although an integrated strategy based on the area under the curve (AUC) has been proposed to characterize the pharmacokinetic/toxicokinetic properties of CHMs, improvement is still needed. This study attempted to use 50% inhibitory concentration (IC50) as weighting coefficient to investigate holistic toxicokinetics of the major diosbulbins i.e. diosbulbin A (DA), diosbulbin B (DB), and diosbulbin C (DC) after oral administration of Dioscorea bulbifera rhizome (DBR) extract. Firstly, the cytotoxicities of the three diosbulbins on human hepatic L02 cells were evaluated and the IC50 values were calculated. Then, integrated toxicokinetics of multiple diosbulbins based on AUC and IC50 were determined. Finally, correlations between integrated plasma concentrations and hepatic injury biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bile acid (TBA) were analyzed. As a result, integrated plasma concentrations were correlated well with TBA and the correlation between TBA and IC50-weighting integrated plasma concentrations was better than that of AUC-weighting integrated plasma concentrations. In conclusion, the newly developed IC50-weighting method is expected to generate more reasonable integrated toxicokinetic parameters, which will help to guide the safe usage of DBR in clinical settings.

Development and validation of a LC-MS/MS method for the quantification of the checkpoint kinase 1 inhibitor SRA737 in human plasma.

AIM: SRA737 is an orally active small-molecule inhibitor of checkpoint kinase 1 being investigated in an oncology setting. A HPLC-MS/MS method for quantifying plasma concentrations of SRA737 was validated. METHODS & RESULTS: Sample preparation involved protein precipitation with acetonitrile following addition of 13C15N-deuterated SRA737 as internal standard. A rapid and selective method was fully validated across a range of 5-20,000 ng/ml, exhibiting good sensitivity, overall precision (expressed as coefficient of variation) ≤8.0% and accuracy 96-102%. Consistently high recovery was observed, with no matrix effect and a lower limit of quantitation of 5 ng/ml. CONCLUSION: A novel method for analyzing SRA737 in human plasma has been validated and is now being utilized for quantification of SRA737 in a Phase I trial.

Brain translocator protein occupancy by ONO-2952 in healthy adults: A Phase 1 PET study using [11 C]PBR28.

ONO-2952, a novel antagonist of translocator protein 18 kDa (TSPO), binds with high affinity to TSPO in rat brain and human tumor cell line membrane preparations. This study used the TSPO-specific PET radioligand [11 C]PBR28 to confirm binding of ONO-2952 to brain TSPO in human subjects, and evaluate brain TSPO occupancy and its relationship with ONO-2952 plasma concentration. Sixteen healthy subjects received a single oral dose of 200, 60, 20, or 6 mg ONO-2952 (n = 4 per dose). Two PET scans with [11 C]PBR28 were conducted ≤7 days apart: at baseline and 24 h after ONO-2952 administration. [11 C]PBR28 regional distribution volume (VT ) was derived with kinetic modeling using the arterial input function and a two tissue compartment model. Nonspecific binding (VND ) was obtained on an individual basis for each subject using linear regression as the x-intercept of the Lassen plot. The binding potential relative to VND (BPND ) was derived as the difference between VT in the ROI (VT ROI) and VND , normalized to VND ; BPND = (VT ROI - VND )/VND . TSPO occupancy was calculated as the change in BPND (ΔBPND ) from individual's baseline scan to the on-medication scan to the baseline BPND value. TSPO occupancy by ONO-2952 was dose dependent between 20-200 mg, approaching saturation at 200 mg both in the whole brain and in 15 anatomic regions of interest (ROI). Estimated Ki values ranged from 24.1 to 72.2 nM. This open-label, single-center, single-dose study demonstrated engagement of ONO-2952 to brain TSPO. The relationship between pharmacokinetics and TSPO occupancy observed in this study support the hypothesis that ONO-2952 could potentially modulate neurosteroid production by binding to brain TSPO.

First-in-Human Clinical Trial of Oral ONC201 in Patients with Refractory Solid Tumors.

Purpose: ONC201 is a small-molecule selective antagonist of the G protein-coupled receptor DRD2 that is the founding member of the imipridone class of compounds. A first-in-human phase I study of ONC201 was conducted to determine its recommended phase II dose (RP2D).Experimental Design: This open-label study treated 10 patients during dose escalation with histologically confirmed advanced solid tumors. Patients received ONC201 orally once every 3 weeks, defined as one cycle, at doses from 125 to 625 mg using an accelerated titration design. An additional 18 patients were treated at the RP2D in an expansion phase to collect additional safety, pharmacokinetic, and pharmacodynamic information.Results: No grade >1 drug-related adverse events occurred, and the RP2D was defined as 625 mg. Pharmacokinetic analysis revealed a Cmax of 1.5 to 7.5 µg/mL (∼3.9-19.4 µmol/L), mean half-life of 11.3 hours, and mean AUC of 37.7 h·µg/L. Pharmacodynamic assays demonstrated induction of caspase-cleaved keratin 18 and prolactin as serum biomarkers of apoptosis and DRD2 antagonism, respectively. No objective responses by RECIST were achieved; however, radiographic regression of several individual metastatic lesions was observed along with prolonged stable disease (>9 cycles) in prostate and endometrial cancer patients.Conclusions: ONC201 is a selective DRD2 antagonist that is well tolerated, achieves micromolar plasma concentrations, and is biologically active in advanced cancer patients when orally administered at 625 mg every 3 weeks. Clin Cancer Res; 23(15); 4163-9. ©2017 AACR.

Development and validation of a high performance liquid chromatography quantification method of levo-tetrahydropalmatine and its metabolites in plasma and brain tissues: application to a pharmacokinetic study.

Levo-tetrahydropalmatine (l-THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l-THP and its desmethyl metabolites l-corydalmine (l-CD) and l-corypalmine (l-CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid-liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed-phase Symmetry® C18 column (4.6 × 150 mm, 5 µm) at 25°C. The mobile phase consisted of acetonitrile-methanol-10 mm ammonium phosphate (pH 3) (10:30:60, v/v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1-10,000 ng/mL. The intra- and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l-THP in rats. Taken together, the developed method can be applied for bioanalysis of l-THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples.