Development and Validation of a Reliable UHPLC-MS/MS Method for Simultaneous Quantification of Macrocyclic Lactones in Bovine Plasma.

A fast, accurate and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantification of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) in bovine plasma. A priority for sample preparation was the eradication of possible infectious diseases to avoid travel restrictions. The sample preparation was based on protein precipitation using 1% formic acid in acetonitrile, followed by Ostro® 96-well plate pass-through sample clean-up. The simple and straightforward procedure, along with the short analysis time, makes the current method unique and suitable for a large set of sample analyses per day for PK studies. Chromatographic separation was performed using an Acquity UPLC HSS-T3 column, with 0.01% acetic acid in water and methanol, on an Acquity H-Class ultra-high performance liquid chromatograph (UHPLC) system. The MS/MS instrument was a Xevo TQ-S® mass spectrometer, operating in the positive electrospray ionization mode and two multiple reaction monitoring (MRM) transitions were monitored per component. The MRM transitions of m/z 897.50 > 753.4 for IVER, m/z 921.70 > 777.40 for DORA and m/z 640.40 > 123.10 for MOXI were used for quantification. The method validation was performed using matrix-matched calibration curves in a concentration range of 1 to 500 ng/mL. Calibration curves fitted a quadratic regression model with 1/x2 weighting (r ≥ 0.998 and GoF ≤ 4.85%). Limits of quantification (LOQ) values of 1 ng/mL were obtained for all the analytes, while the limits of detection (LOD) were 0.02 ng/mL for IVER, 0.03 ng/mL for DORA, and 0.58 ng/mL for MOXI. The results of within-day (RSD < 6.50%) and between-day (RSD < 8.10%) precision and accuracies fell within acceptance ranges. No carry-over and no peak were detected in the UHPLC-MS/MS chromatogram of blank samples showing good specificity of the method. The applicability of the developed method was proved by an analysis of the field PK samples.

Macrocyclic Inhibitors of HGF-Activating Serine Proteases Overcome Resistance to Receptor Tyrosine Kinase Inhibitors and Block Lung Cancer Progression.

Hepatocyte growth factor (HGF), the ligand for the MET receptor tyrosine kinase, is a tumor-promoting factor that is abundant in the tumor microenvironment. Proteolytic activation of inactive pro-HGF by one or more of the serine endopeptidases matriptase, hepsin, and HGF activator is the rate-limiting step in HGF/MET signaling. Herein, we have rationally designed a novel class of side chain cyclized macrocyclic peptide inhibitors. The new series of cyclic tripeptides has superior metabolic stability and significantly improved pharmacokinetics in mice relative to the corresponding linear peptides. We identified the lead compound VD2173 that potently inhibits matriptase and hepsin, which was tested in parallel alongside the acyclic inhibitor ZFH7116 using both in vitro and in vivo models of lung cancer. We demonstrated that both compounds block pro-HGF activation, abrogate HGF-mediated wound healing, and overcome resistance to EGFR- and MET-targeted therapy in lung cancer models. Furthermore, VD2173 inhibited HGF-dependent growth of lung cancer tumors in mice.

68Ga-Labeled bismacrocyclic methylene phosphonate as potential bone seeking PET radiopharmaceutical.

Phosphonates-based agents are well-known bone-seeking radiopharmaceuticals with application in detection and therapy. With higher sensitivity and resolution offered by Positron Emission Tomography (PET), tracers based on this technique are gaining huge attention. 68Ga-based generator and radiotracers render independence from the on-site cyclotron. We report the development of 68Ga-labeled DOTA-based bismacrocyclic phosphonate derivative, for bone PET imaging. The synthesis and characterization of 68Ga- DO3P-AME-DO3P was carried out in > 95% purity. The radiotracer displayed high stability and low binding affinity (<3%) to blood serum. High in vitro binding affinity were observed for synthetic hydroxyapatite, SAOS-2, osteoclast and osteoblast cells. In vivo pharmacokinetics revealed fast washout with biphasic release pattern. The deposition of radiotracer in osseous tissues was high (Bone/Muscle ratio:18), as studied from the biodistribution studies. In vivo PET/CT and biodistribution analyses revealed the ability of 68Ga-DO3P-AME-DO3P to target and accumulate in bone, thus displaying its potential as a PET bone imaging agent.

Design, synthesis, and evaluation of a novel macrocyclic anti-EV71 agent.

We describe here the design, synthesis, and evaluation of a macrocyclic peptidomimetic as a potent agent targeting enterovirus A71 (EV71). The compound has a 15-membered macrocyclic ring in a defined conformation. Yamaguchi esterification reaction was used to close the 15-membered macrocycle instead of the typical Ru-catalyzed ring-closing olefin metathesis reaction. The crystallographic characterization of the complex between this compound and its target, 3C protease from EV71, validated the design and paved the way for the generation of a new series of anti-EV71 agents.

Bioanalytical assay for the new-generation ROS1/TRK/ALK inhibitor repotrectinib in mouse plasma and tissue homogenate using liquid chromatography-tandem mass spectrometry.

Repotrectinib, a next-generation ROS1/TRK/ALK tyrosine kinase inhibitor, overcomes resistance due to acquired solvent-front mutations involving ROS1, NTRK1-3, and ALK. A bioanalytical assay for quantification of repotrectinib in mouse plasma and seven tissue-related matrices (brain, liver, spleen, kidney, small intestinal tissue, small intestinal content, and testis homogenates) was developed and validated using liquid chromatography with tandem mass spectrometric detection in a high-throughput 96-well format. Protein precipitation was performed by adding acetonitrile, also containing the internal standard axitinib, to 10-µl samples for all matrices. Chromatographic separation of analytes was done on an ACQUITY UPLC® BEH C18 column by gradient elution using ammonium hydroxide in water and methanol. Compounds were monitored with positive electrospray ionization using a triple quadruple mass spectrometer in selected reaction monitoring mode. The method was successfully validated in the 1-1000 ng/ml calibration range. Precisions (intra- and interday) were in the range of 1.3-8.7% and accuracies were in between 90.5% and 107.3% for all levels in all matrices. The developed method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of repotrectinib in wild-type mice.

Development and Validation of a New LC-MS/MS Analytical Method for Direct-Acting Antivirals and Its Application in End-Stage Renal Disease Patients.

BACKGROUND AND OBJECTIVE: The effectiveness of direct-acting antivirals (DAAs) is not well established in end-stage renal disease (ESRD) patients. Assessment of the plasma concentrations may support understanding of their therapeutic outcomes in this population. The aim of this study is to develop a direct, yet matrix-effect tolerant, analytical method for determining DAAs in the plasma of ESRD patients while maintaining a moderate cost per sample and with an improved analyte extraction recovery. METHODS: In this study, a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the analysis of ombitasvir (OMB), paritaprevir (PRT) and ritonavir (RIT) in plasma. Sample preparation was performed using the liquid-liquid extraction (LLE) method. Isocratic separation was performed using a mixture of methanol and 10 mM ammonium acetate (79:21, v/v) followed by MS/MS detection. The method was validated and applied to determine DAAs in the plasma of ESRD patients (n = 7). RESULTS: The developed method was linear (r2 > 0.995), accurate (89.4 ± 7.8 to 108.3 ± 3.0) and precise (% CV 0.9-15.0) and showed improved recovery (> 80) over previously published ones in the range 5-250, 30-1,500, 20-1,000 ng/mL for OMB, PRT and RIT, respectively. Relative matrix effect was absent, and the method accurately determined the three DAAs in real-life samples (n = 7). CONCLUSIONS: An efficient analytical method for the determination of DAAs is presented. The method overcomes the potential analytical response fluctuation in ESRD. The developed method show improved extraction recoveries and is suitable for routine application in developing economies where hepatitis C virus is most prevalent.

Plasma trough concentrations of antiretrovirals in HIV-infected persons treated with direct-acting antiviral agents for hepatitis C in the real world.

BACKGROUND: Possible drug-drug interactions (DDIs) between antiretrovirals (ARVs) and direct-acting antiviral agents (DAAs) are of some concern. OBJECTIVES: To investigate ARV plasma trough concentrations (Ctrough) before and during DAAs in patients treated in the real world. METHODS: Single-centre, prospective, observational study including HIV/HCV coinfected persons undergoing DAA treatment. Self-reported adherence was assessed and ARVs Ctrough measured by HPLC-UV. Blood samples were collected before and after 2 months of DAA treatment. RESULTS: One-hundred and thirty-seven patients were included: 21.2% treated with ombitasvir/paritaprevir/ritonavir ± dasabuvir (2D/3D) and 78.8% with sofosbuvir-based regimens. Suboptimal Ctrough before and during DAA was found, respectively, in 3 (10.3%) and 3 (10.3%) cases treated with 2D/3D, and 16 (14.8%) and 11 (10.2%) with sofosbuvir-based regimens, even if self-reported ARV adherence was always ≥93%. In 2D/3D-treated patients, median darunavir Ctrough during DAAs was significantly lower than observed before DAAs [1125 ng/mL (IQR, 810-1616) versus 1903 ng/mL (IQR 1387-3983), respectively] (n = 5; P = 0.009), with a 40.9% decrease. In the same group, no differences in atazanavir or raltegravir concentrations were found. In patients treated with sofosbuvir-based regimens, Ctrough of all ARVs were similar before and during DAAs. CONCLUSIONS: In the real world of HIV/HCV coinfected patients, ARV plasma concentrations during DAAs were generally not different from those found before anti-HCV treatment. Although assessed in a small number of patients, darunavir concentrations during 2D/3D showed a significant reduction when compared with those found before DAAs. ARV plasma concentrations measurement during anti-HCV treatment may give useful information for managing HIV/HCV coinfected persons receiving treatment for both infections.

Managing Drug-Drug Interaction Between Ombitasvir, Paritaprevir/Ritonavir, Dasabuvir, and Mycophenolate Mofetil.

No drug-drug interaction study has been conducted to date for the combination of ombitasvir, paritaprevir/ritonavir, dasabuvir (3D), and mycophenolic acid (MPA). We here report the case of a hepatitis C virus-infected patient treated with 3D and MPA for vasculitis. In light of the threat of drug-drug interaction, the concentration of MPA was measured before, during, and 15 days after the end of the 3D treatment. Similar values were found at all 3 time points, thus indicating that there is probably no need to adapt MPA dosage to 3D.

Population pharmacokinetics of paritaprevir, ombitasvir, dasabuvir, ritonavir and ribavirin in hepatitis C virus genotype 1 infection: analysis of six phase III trials.

AIM: The aim of the current study was to characterize the population pharmacokinetics of a triple direct-acting antiviral (DAA) regimen (3D) (ombitasvir, paritaprevir-ritonavir and dasabuvir) and adjunctive ribavirin, and estimate covariate effects in a broad spectrum of subjects with hepatitis C virus (HCV) genotype 1 infection. METHODS: Pharmacokinetic data from six phase III studies and one phase II study in subjects receiving the currently approved doses of the 3D ± ribavirin regimen for treating HCV genotype 1 infection for 12 weeks or 24 weeks were characterized using separate population pharmacokinetic models, built using each component of the regimen from nonlinear mixed-effects methodology in NONMEM 7.3. In the models, demographic and clinical covariates were tested. Models were assessed via goodness-of-fit plots, visual predictive checks and bootstrap evaluations. RESULTS: The population pharmacokinetic models for each component of the 3D ± ribavirin regimen (DAAs and ritonavir, n = 2348) and ribavirin (n = 1841) adequately described their respective plasma concentration-time data. Model parameter estimates were precise and robust, and all models showed good predictive ability. Significant covariate effects associated with apparent clearance and volume of distribution included age, body weight, gender, cirrhosis, HCV subtype, opioid or antidiabetic agent use, and creatinine clearance. CONCLUSION: The population pharmacokinetics of the 3D ± ribavirin regimen components in HCV-infected patients were characterized using phase II and III HCV clinical trial data. Although several statistically significant covariates were identified, their effects were modest and not clinically meaningful to necessitate dose adjustments for any component of the 3D regimen.

Metabolism and Disposition of the Hepatitis C Protease Inhibitor Paritaprevir in Humans.

Paritaprevir (also known as ABT-450), a potent NS3-4A serine protease inhibitor [identified by AbbVie (North Chicago, IL) and Enanta Pharmaceuticals (Watertown, MA)] of the hepatitis C virus (HCV), has been developed in combination with ombitasvir and dasabuvir in a three-direct-acting antiviral agent (DAA) oral regimen for the treatment of patients infected with HCV genotype 1. This article describes the mass balance, metabolism, and disposition of paritaprevir in humans. After the administration of a single 200-mg oral dose of [(14)C]paritaprevir coadministered with 100 mg of ritonavir to four male healthy volunteers, the mean total percentage of the administered radioactive dose recovered was 96.5%, with recovery in individual subjects ranging from 96.0% to 96.9%. Radioactivity derived from [(14)C]paritaprevir was primarily eliminated in feces (87.8% of the dose). Radioactivity recovered in urine accounted for 8.8% of the dose. The biotransformation of paritaprevir in humans involves: 1) P450-mediated oxidation on the olefinic linker, the phenanthridine group, the methylpyrazinyl group, or combinations thereof; and 2) amide hydrolysis at the acyl cyclopropane-sulfonamide moiety and the pyrazine-2-carboxamide moiety. Paritaprevir was the major component in plasma [90.1% of total radioactivity in plasma, AUC from time 0 to 12 hours (AUC0-12hours) pool]. Five minor metabolites were identified in plasma, including the metabolites M2, M29, M3, M13, and M6; none of the metabolites accounted for greater than 10% of the total radioactivity. Paritaprevir was primarily eliminated through the biliary-fecal route followed by microflora-mediated sulfonamide hydrolysis to M29 as a major component in feces (approximately 60% of dose). In summary, the biotransformation and clearance pathways of paritaprevir were characterized, and the structures of metabolites in circulation and excreta were elucidated.

Dose- and Formulation-Dependent Non-Linear Pharmacokinetic Model of Paritaprevir, a Protease Inhibitor for the Treatment of Hepatitis C Virus Infection: Combined Analysis from 12 Phase I Studies.

BACKGROUND AND OBJECTIVES: Paritaprevir is a direct-acting antiviral agent that is a component of approved multidrug regimens used in the treatment of hepatitis C virus (HCV) infection. A population pharmacokinetic model for paritaprevir was developed using data from formulation, bioavailability, and drug-drug interaction studies that evaluated the pharmacokinetics of paritaprevir (coadministered with ritonavir to enhance exposure) with or without ombitasvir and/or dasabuvir at different paritaprevir dose levels. METHODS: A non-linear mixed-effects modeling approach was applied to data from 12 phase I, single- and multiple-dose studies that enrolled a total of 369 healthy volunteers. Age, sex, race, ethnicity, body weight, body surface area, body mass index, and baseline creatinine clearance were evaluated as covariates during model development. In addition, the influences of dose, formulation, and concomitant medications (e.g. ombitasvir and dasabuvir) on paritaprevir bioavailability were included in the model. RESULTS: A two-compartment model with first-order absorption and elimination optimally described paritaprevir plasma concentration-time data. Paritaprevir bioavailability was formulation- and dose-dependent, and increased supraproportionally. The accumulation of paritaprevir was 1.57-fold on repeated dosing compared with the first dose. Coadministration of dasabuvir increased paritaprevir bioavailability by 59 %; however, ombitasvir coadministration did not affect the pharmacokinetic profile of paritaprevir. No subject-specific covariate influenced the paritaprevir pharmacokinetics. The pharmacokinetic model was robust in bootstrap evaluations and was consistent with observed data based on diagnostic goodness-of-fit plots and visual predictive checks. CONCLUSION: The complex pharmacokinetics of paritaprevir were well described by the model, which can be used as a basis for clinical trial dosing and further evaluations in patients with HCV.

Drug Interactions with the Direct-Acting Antiviral Combination of Ombitasvir and Paritaprevir-Ritonavir.

The two direct-acting antiviral (2D) regimen of ombitasvir and paritaprevir (administered with low-dose ritonavir) is being developed for treatment of genotype subtype 1b and genotypes 2 and 4 chronic hepatitis C virus (HCV) infection. Drug-drug interactions were evaluated in healthy volunteers to develop dosing recommendations for HCV-infected subjects. Mechanism-based interactions were evaluated for ketoconazole, pravastatin, rosuvastatin, digoxin, warfarin, and omeprazole. Interactions were also evaluated for duloxetine, escitalopram, methadone, and buprenorphine-naloxone. Ratios of geometric means with 90% confidence intervals for the maximum plasma concentration and the area under the plasma concentration-time curve were estimated to assess the magnitude of the interactions. For most medications, coadministration with the 2D regimen resulted in a <50% change in exposures. Ketoconazole, digoxin, pravastatin, and rosuvastatin exposures increased by up to 105%, 58%, 76%, and 161%, respectively, and omeprazole exposures decreased by approximately 50%. Clinically meaningful changes in ombitasvir, paritaprevir, or ritonavir exposures were not observed. In summary, all 11 medications evaluated can be coadministered with the 2D regimen, with most medications requiring no dose adjustment. Ketoconazole, digoxin, pravastatin, and rosuvastatin require lower doses, and omeprazole may require a higher dose. No dose adjustment is required for the 2D regimen.

Rapid decline of viral RNA in chronic hepatitis C patients treated once daily with IDX320: a novel macrocyclic HCV protease inhibitor.

BACKGROUND: The addition of direct-acting antivirals to pegylated interferon-α plus ribavirin for the treatment of chronic HCV infection can result in an increased sustained viral response rate and may permit reduction in treatment duration. IDX320 is a potent non-covalent macrocyclic inhibitor of the HCV NS3/4A protease. METHODS: This was a randomized double-blind placebo-controlled single- and multiple-dose study to assess the safety, tolerability, antiviral activity and pharmacokinetics of IDX320 in healthy volunteers (HV) and patients with chronic HCV genotype 1 infection. HV (n=48) received single or multiple ascending doses of IDX320. Two HCV-infected patients received a single dose of 200 mg IDX320. Dosages for other HCV-infected patients were as follows: placebo, 50, 100, 200 or 400 mg of IDX320 orally once daily for 3 days (n=30) or placebo/200 mg of IDX320 twice-daily for 3 days (n=8). RESULTS: In total, 48 HV and 40 HCV-infected patients were enrolled and all completed the study. There were no serious adverse events. The majority of adverse events were of mild or moderate intensity. Pharmacokinetics supported a once-daily dosing regimen. A rapid decline in plasma HCV RNA was observed in all patients. In the multiple-dose study, mean HCV RNA reductions were 2.6, 3.1, 3.1, 3.3 and 3.8 log(10) IU/ml after 3 days in the IDX320 50, 100, 200, 400 mg once-daily and 200 mg twice-daily treatment groups, respectively. This compared to a mean HCV RNA reduction of 0.04 log(10) in the placebo group. CONCLUSIONS: Once-daily IDX320 dosing demonstrated potent dose-dependent antiviral activity in treatment-naive HCV genotype-1-infected patients.

Pharmacological demarcation of the growth hormone, gut motility and feeding effects of ghrelin using a novel ghrelin receptor agonist.

The peptide hormone ghrelin exerts a wide spectrum of activities including the stimulation of GH release, feeding, and gastrointestinal motility, purportedly via the activation of a common receptor, GH secretagogue receptor (since renamed the GRLN-R) The aim of the present study was to determine whether these effects can be separated pharmacologically. Tranzyme Pharma (TZP)-101 is a small-molecule agonist with potent binding affinity (inhibitory constant = 16 nm) and full agonist activity (EC50 = 29 nm, maximum response = 111%) at the human recombinant GRLN-R. Pharmacokinetic profiling of TZP-101 in rat determined a plasma elimination half-life of 99 min and low blood-brain barrier permeability (0.09%). The pharmacological response to TZP-101, administered centrally [intracerebroventricular (icv)] or peripherally (iv), was evaluated in comparison with that of acylated ghrelin. Thus, TZP-101 (iv) accelerated gastric emptying of a liquid meal (2% methylcellulose) similarly to ghrelin (iv). IAlso, TZP-101 (icv) stimulated spontaneous, cumulative food intake in a similar manner to ghrelin (icv). However, unlike ghrelin, TZP-101 did not elicit significant GH release on either central or peripheral administration. Moreover, TZP-101 did not alter ghrelin-induced GH release. n total, these data demonstrate that the GH response can be pharmacologically demarcated from the orexigenic and gastrointestinal responses to ghrelin in rats. The observation that the centrally mediated orexigenic response and the peripherally mediated gastric motility response are pharmacologically associated is consistent with the classification of ghrelin as a brain-gut peptide, whereas the additional action of ghrelin to stimulate GH release (possibly via a distinct signaling pathway) may be considered a complementary mechanism to harmonize somatic growth and body composition with the regulation of energy homeostasis.