A Thorough QT Study of the Combination Glecaprevir + Pibrentasvir on Cardiac Repolarization in Healthy Subjects.

PURPOSE: Fixed-dose combination glecaprevir (GLE) 300 mg + pibrentasvir (PIB) 120 mg is an orally administered once daily antiviral regimen approved for the treatment of hepatitis C virus (HCV) infection. The objective of this study was to evaluate the potential for cardiac repolarization following GLE + PIB administration in healthy adults. METHODS: This placebo- and active-controlled, randomized, single-dose, 4-period, 4-sequence crossover study enrolled 48 healthy subjects. The doses of GLE 400 mg + PIB 120 mg were selected to provide exposures comparable to those with the doses that are therapeutic in the HCV-infected population, GLE 300 mg + PIB 120 mg. The doses of GLE 600 mg + PIB 240 mg were selected to provide supratherapeutic exposures without exceeding the exposures of the GLE + PIB maximal tolerated doses. Moxifloxacin 400 mg (active control/open label) was used for confirming the sensitivity of the ECG assay in detecting QTc prolongation. Time-matched plasma concentrations and triplicate ECGs were obtained on treatment days -1 and 1. The primary end point was time-matched, placebo-corrected, baseline-adjusted Fridericia-corrected QT interval (ΔΔQTcF). Pharmacokinetic-pharmacodynamic analyses characterized the relationship between GLE and PIB plasma concentrations and ΔΔQTcF using a linear regression model and linear mixed-effects model. Findings from categorical analyses of ECG-interval data were also summarized. Tolerability was evaluated through adverse-events monitoring, physical examination including vital sign measurements, ECGs, and laboratory tests. FINDINGS: A total of 48 subjects (22 women [46%], 26 men [54%]), were enrolled in the study, and 47 subjects completed all 4 periods. None of the subjects had a change from baseline in QTcF interval of >30 msec or an absolute QTcF interval of >450 msec. Peak ΔΔQTcF values observed at 5 h postdose (Tmax) were 2.9 msec (upper 95% confidence limit, 4.9 msec) with the therapeutic dose and 3.1 msec (upper 95% confidence limit, 5.1 msec) with the supratherapeutic dose, with both upper 95% confidence limits well below the 10-msec threshold. Assay sensitivity was confirmed by peak ΔΔQTcF in the positive control (12.8 ms at 2 h postdose). No statistically significant GLE or PIB concentration-dependent effects on ΔΔQTcF were observed. Headache and skin irritation from ECG electrodes were the most commonly reported AEs. No clinically significant vital sign measurements, ECG findings, or laboratory measurements were observed. There were no patterns of T- and U-wave morphologic abnormalities. IMPLICATIONS: The fixed-dose combination regimen of GLE/PIB does not prolong the QTc interval. ClinicalTrials.gov identifier.

Estimating Efflux Transporter-Mediated Disposition of Molecules beyond the Rule of Five (bRo5) Using Transporter Gene Knockout Rats.

Transporter gene knockout models are a practical and widely used tool for pharmacokinetic studies in drug discovery. P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are major efflux transporters that control absorption and bioavailability, and are important when determining oral drug disposition. To the best of our knowledge, beyond the rule of five (bRo5) molecules launched on the market to date tend to be substrates for efflux transporters. The purpose of this study is to evaluate in vivo the impact of efflux transporters on the oral absorption process and systemic clearance using rats which lack P-gp and/or Bcrp expression. We administered five bRo5 substrates (asunaprevir, cyclosporine, danoprevir, ledipasvir, and simeprevir) intravenously or orally to wild-type and Mdr1a, Bcrp, and Mdr1a/Bcrp knockout rats, calculated the clearance, oral bioavailability, and absorption rate profile of each substrate, and compared the results. Systemic clearance of the substrates in knockout rats changed within approximately ±40% compared to wild-types, suggesting the efflux transporters do not have a significant influence on clearance in rats. On the other hand, the oral absorption of substrates in the knockout rats, especially those lacking Mdr1a, increased greatly-between 2- and 5-fold more than in wild-types. This suggests that rat efflux transporters, especially P-gp, greatly reduce the oral exposure of these substrates. Moreover, results on the absorption rate-time profile suggest that efflux transporters are constantly active during the absorption period in rats. Transporter knockout rats are a useful in vivo tool for estimating the transporter-mediated disposition of bRo5 molecules in drug discovery.

Bioanalytical liquid chromatography-tandem mass spectrometric assay for the quantification of the ALK inhibitors alectinib, brigatinib and lorlatinib in plasma and mouse tissue homogenates.

Several second and third generation ALK inhibitors have been introduced in recent years. A bioanalytical assay for simultaneous quantification of alectinib, brigatinib, and lorlatinib was developed and validated for human plasma. The method was also partially validated for diluted mouse plasma and tissue homogenates of brain, liver, kidney, and spleen. Samples (40 µl) were pretreated in a 96-well plate by protein precipitation with acetonitrile containing the internal standard [2H8]-alectinib. After chromatographic separation on an ethylene bridged octadecyl silica column by gradient elution at 600 µl/min using 1% (v/v) formic acid (in water) and acetonitrile, compounds were ionized by a turbo electrospray and monitored by selected reaction monitoring on a triple quadrupole mass spectrometer. Validation was performed in a 2-2000 ng/ml concentration range for alectinib and lorlatinib and a 4-4000 ng/ml range for brigatinib. Precisions (within-day and between-day) were in the range 2.2-15.0% and accuracies were in between 87.2 and 110.2% for all matrices and levels. Compounds were sufficiently stable under most investigated conditions. Results of a pilot pharmacokinetic and tissue distribution study for brigatinib in mice are reported. Finally, successful incurred samples reanalysis of tissue homogenate samples containing brigatinib and lorlatinib is presented. Lorlatinib homogenate samples were also successfully reanalyzed using a second independent assay (cross-validation).

Model System Identifies Kinetic Driver of Hsp90 Inhibitor Activity against African Trypanosomes and Plasmodium falciparum.

Hsp90 inhibitors, well studied in the laboratory and clinic for antitumor indications, have promising activity against protozoan pathogens, including Trypanosoma brucei which causes African sleeping sickness, and the malaria parasite, Plasmodium falciparum To progress these experimental drugs toward clinical use, we adapted an in vitro dynamic hollow-fiber system and deployed artificial pharmacokinetics to discover the driver of their activity: either concentration or time. The activities of compounds from three major classes of Hsp90 inhibitors in development were evaluated against trypanosomes. In all circumstances, the activities of the tested Hsp90 inhibitors were concentration driven. By optimally deploying the drug to match its kinetic driver, the efficacy of a given dose was improved up to 5-fold, and maximal efficacy was achieved with a significantly lower drug exposure. The superiority of concentration-driven regimens was evident in vitro over several logs of drug exposure and was predictive of efficacy in a mouse model of African trypanosomiasis. In studies with P. falciparum, antimalarial activity was similarly concentration driven. This experimental strategy offers an expedient and versatile translational tool to assess the impact of pharmacokinetics on antiprotozoal activity. Knowing kinetic governance early in drug development provides an additional metric for judging lead compounds and allows the incisive design of animal efficacy studies.

Bioanalytical assay for the quantification of the ALK inhibitor lorlatinib in mouse plasma using liquid chromatography-tandem mass spectrometry.

A bio-analytical assay for the first third generation ALK inhibitor lorlatinib in mouse plasma was developed and validated. Ten-µl plasma samples were prepared by adding rucaparib as the internal standard and precipitation of the plasma proteins. For LC-MS/MS analysis, compounds were eluted at 0.5 mL/min and separated on a 3-µm particle-size, polar embedded octadecyl silica column by gradient elution using 0.1% of formic acid (in water) and methanol. Compounds were monitored with positive electrospray ionization using a triple quadrupole mass spectrometer in selected reaction monitoring mode. The assay was fully validated in the 2-2000 ng/mL calibration range. Within-day (8.0-11.6%) and between-day (10.0-15.0%) precisions and accuracies (99.0-113.3%) were within acceptable range. Plasma samples were deemed stable for 6 h at ambient temperature, during three freeze-thaw cycles and for 2 months at -30 °C. Finally, the new assay was applied successfully to pilot pharmacokinetic studies in male and female wild-type mice.

A physiologically based pharmacokinetic model of alvespimycin in mice and extrapolation to rats and humans.

BACKGROUND AND PURPOSE: Alvespimycin, a new generation of heat shock protein 90 (Hsp90) inhibitor in clinical trial, is a promising therapeutic agent for cancer. Pharmacokinetic models of alvespimycin would help in the understanding of drug disposition, predicting drug exposure and interpreting dose-response relationship. In the present study we aimed to develop a physiologically based pharmacokinetic (PBPK) model of alvespimycin in mice and evaluate the utility of the model for predicting alvespimycin disposition in other species. EXPERIMENTAL APPROACH: A literature search was performed to collect pharmacokinetic data for alvespimycin. A PBPK model was initially constructed to demonstrate the disposition of alvespimycin in mice, and then extrapolated to rats and humans by taking into account the interspecies differences in physiological- and chemical-specific parameters. KEY RESULTS: A PBPK model, employing a permeability-limited model structure and saturable tissue binding, was built in mice. It successfully characterized the time course of the disposition of alvespimycin in mice. After extrapolation to rats, the model simulated the alvespimycin concentration-time profiles in rat tissues with acceptable accuracies. Likewise, a reasonable match was found between the observed and simulated human plasma pharmacokinetics of alvespimycin. CONCLUSIONS AND IMPLICATIONS: The PBPK model described here is beneficial to the understanding and prediction of the effects of alvespimycin in different species. It also provides a good basis for further development, which necessitates additional studies, especially those needed to clarify the in-depth mechanism of alvespimycin elimination. A refined PBPK model would benefit the understanding of dose-response relationships and optimization of dosing regimens.

Assessing the impact of HER2 status on the antitumor activity of an HSP90 inhibitor in human tumor xenograft mice using pharmacokinetic-pharmacodynamic modeling.

The purpose of this study is to assess the impact of human epidermal growth factor receptor 2 (HER2) status on the antitumor activity of CH5164840, an orally available heat shock protein 90 (HSP90) inhibitor, using pharmacokinetic-pharmacodynamic modeling. Athymic mice, each implanted with one of eight human tumor xenografts, were treated with CH5164840 once daily at doses of 3.13 to 50 mg/kg. Plasma concentrations of CH5164840 were described by a one-compartment model with first-order absorption rate. Time profiles of tumor growth inhibition in the eight xenograft models were well captured by an indirect response model with a maximum tumor-killing rate constant (Emax model). Threshold plasma concentrations for tumor stasis, which are determined by multiple pharmacodynamic parameters, Emax, EC50 and tumor growth rate constant, were significantly lower in HER2-positive tumors (1.96-3.85 µM) than in HER2-negative tumors (4.48-23.4 µM). The results suggest that CH5164840 was more efficacious in HER2-positive tumors than in HER2-negative tumors in terms of the lower effective concentration of the drug in preclinical animal models.

Tanespimycin and bortezomib combination treatment in patients with relapsed or relapsed and refractory multiple myeloma: results of a phase 1/2 study.

This open-label, dose escalation, multicentre phase 1/2 trial was undertaken to determine the safety and tolerability of the heat shock protein 90 (HSP90) inhibitor tanespimycin (100-340 mg/m(2) )+ bortezomib (0·7-1·3 mg/m(2) ) given on days 1, 4, 8 and 11 in each 21-d cycle. Phase 2 expansion occurred at the highest tested dose of tanespimycin at 340 mg/m(2) and bortezomib at 1·3 mg/m(2) . Seventy-two patients (median age, 60 years) with relapsed or relapsed and refractory multiple myeloma (MM) were enrolled; 63 patients (89%) completed the study. Tanespimycin in combination with bortezomib was well tolerated; few patients experienced significant neutropenia, constipation and anorexia (<10%), and no patients developed severe peripheral neuropathy. Among 67 efficacy-evaluable patients, there were 2 (3%) complete responses and 8 (12%) partial responses, for an objective response rate (ORR) of 27%, including 8 (12%) minimal responses. Response rates were highest among bortezomib-naive patients and proved durable in all patient subgroups, including those with bortezomib-refractory disease. Pharmacodynamic analyses indicated that tanespimycin plus bortezomib effectively inhibited the proteasome, as evidenced by decreased 20S proteasome activity, and inhibited HSP90, as reflected by increased HSP70 expression. The results of this study support additional studies of this combination approach in MM.

Tanespimycin monotherapy in relapsed multiple myeloma: results of a phase 1 dose-escalation study.

Tanespimycin, a heat shock protein 90 (HSP90) inhibitor, induces apoptosis in drug-sensitive and -resistant MM cell lines and in tumour cells from patients with relapsed MM. In this phase 1 dose-escalation study, the safety, plasma pharmacokinetics, and biological/antitumour activity of tanespimycin were evaluated in heavily pretreated patients with relapsed/refractory MM. Tanespimycin (150-525 mg/m(2)) was given on days 1, 4, 8, and 11 of each 3-week cycle for up to 8 cycles. Non-haematological AEs included diarrhoea (59%), back pain (35%), fatigue (38%), and nausea (35%); haematological AEs included anaemia (24%) and thrombocytopenia (21%). One patient (3%) achieved minimal response (MR), with a progression-free survival (PFS) of 3 months, a 41% decrease from baseline in urine M protein, and a 33% decrease from baseline in serum M protein. Fifteen patients (52%) achieved SD with a median PFS of 2.1 months; 5/15 had reductions in serum M protein ranging from 7% to 38% and in urine M protein ranging from 6% to 91%. Mean HSP70 levels increased from day 1 h 0 to day 1 h 4 with further increases on day 11 h 0 and day 11 h 4, consistent with a therapeutic treatment effect. Tanespimycin monotherapy was well tolerated and demonstrated activity across all doses tested.

Development and validation of a rapid and sensitive high-performance liquid chromatography-mass spectroscopy assay for determination of 17-(allylamino)-17-demethoxygeldanamycin and 17-(amino)-17-demethoxygeldanamycin in human plasma.

A sensitive method was developed and validated for the measurement of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) and its active metabolite 17-amino-17-demethoxygeldanamycin (17AG) in human plasma using 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) as an internal standard. After the addition of internal standard, 200 microL of plasma was extracted using ice cold acetonitrile followed by analysis on a Thermo Finnigan triple-quadruple mass spectrometer coupled to an Agilent 1100 HPLC system. Chromatography was carried out on a 50 mm x 2.1 mm Agilent Zorbax SB-phenyl 5 microm column coupled to a 3mm Varian metaguard diphenyl pre-column using glacial acetic acid 0.1% and a gradient of acetonitrile and water at a flow rate of 500 microL/min. Atmospheric pressure chemical ionization and detection of 17AAG, 17AG and 17DMAG were accomplished using selected reaction monitoring of m/z 584.3>541.3, 544.2>501.2, and 615.3>572.3, respectively in negative ion mode. Retention times for 17AAG, 17AG, and 17DMAG were 4.1, 3.5, and 2.9 min, respectively, with a total run time of 7 min. The assay was linear over the range 0.5-3000 ng/mL for 17AAG and 17AG. Replicate sample analysis indicated within- and between-run accuracy and precision within 15%. The recovery of 17AAG and 17AG from 200 microL of plasma containing 1, 25, 300, and 2500 ng/mL was 93% or greater. This high-performance liquid chromatographic tandem mass spectroscopy (HPLC/MS/MS) method is superior to previous methods. It is the first analytical method reported to date for the quantitation of both 17AAG and its metabolite 17AG and can reliably quantitate concentrations of both compounds as low as 0.5 ng/mL.

Predictive physiologically based pharmacokinetic model for antibody-directed enzyme prodrug therapy.

Antibody-directed enzyme prodrug therapy (ADEPT) using anti-TAG-72 antibody and geldanamycin (GA) prodrug were validated in vitro. To understand the complexity and to explore optimal therapeutic regimens for ADEPT in vivo, a physiologically based pharmacokinetic model (PBPK) is applied to analyze each anatomical component/organ. The baseline model predicts that active drug tumor/plasma exposure (AUC) ratio is 2-fold, although antibody-enzyme conjugates (AbE) are distributed into tumors up to 9-fold higher than in plasma. However, the active drug tumor/plasma AUC ratio can be increased up to 100-fold when AbE are depleted from plasma. Similarly, the active drug tumor/plasma AUC ratio can be increased from 2- to 6-fold when the intrinsic clearance of AbE is accelerated by 10-fold. Several sensitive parameters are identified: 1) increasing flow inside tumor (J(iso,tumor)) significantly increases active drug tumor/plasma AUC ratio; 2) increasing permeability of prodrug (from range 1.4 x 10(-6) to 1.4 x 10(-4) cm/s) increases active drug tumor/plasma AUC ratio significantly, whereas active drug permeability enhancement (from range 5 x 10(-4) to 5 x 10(-2) cm/s) has minimal effect; 3) decreasing E(max) and increasing EC(50) for converting prodrug to active drug increase tumor/plasma AUC ratio for active drug. The PBPK model predicts that the optimal dosing interval between AbE and prodrug administration is 5 days, the optimal AbE dose is 0.1 B(max), and the optimal dose for GA prodrug is 60 mg/kg. The current PBPK model successfully identifies sensitive parameters and predicts an optimal dosing regimen for ADEPT.

Synthesis of (131)I-labeled-[(131)I]iodo-17-allylamino-17-demethoxy geldanamycin ([(131)I]iodo-17-AAG) and its biodistribution in mice.

AIM: The aim of this study was to examine the radioiodinating condition of 17-allylamino-17-demethoxy geldanamycin (17-AAG) and observe its biodistribution in the hepatoma cell line HepA tumorearing ICR mice for understanding the possibility of its application in nuclear medicine. METHODS: [(131)I]iodo-17-AAG was prepared by the reaction of 17-AAG with Na[(131)I] in the presence of hydrogen peroxide. [(131)I]iodo-17-AAG was purified by high-performance liquid chromatography (HPLC). The stability of [(131)I]iodo-17-AAG was measured by thin-layer chromatography (TLC). The distributions in HepA tumor-bearing ICR mice at 0.5, 1, 4, 8, 24, and 48 hours after injection of [(131)I]iodo-17-AAG were measured. Tumor uptake studies were performed in HepA tumor-bearing ICR mice. RESULTS: The labeling yield was over 83%. The radiochemical purity of [(131)I]iodo-17-AAG was 99.6% after purification. The specific activity was greater than 4 Ci/micromol. The labeled compound was stable for at least 120 hours in saline at 4 degrees C. It was initially in blood at 5 minutes with 4.79% of injected dose per g of tissue (%ID/g), and then dropped 0.33% ID/g at 24 hours. The uptake in liver, lung, and kidney at 4.44% ID/g, 2.03% ID/g, and 2.17% ID/g decreased with time, and less than 1% ID/g was measured after 24 hours in those organs. There was rapid tumor uptake, which reached 1.26% ID/g at 0.5 hours, the highest uptake at 8 hours. Yet, the [(131)I]iodo-17-AAG in the contralateral muscle was at a low level during the 48 hours. The tumor-contralateral muscle (T/CM) radioactivity ratio for [(131)I]iodo-17-AAG remained constant at all time points. CONCLUSIONS: [(131)I]iodo-17-AAG can be efficiently radiolabeled at high specific activity, purified by HPLC and stored with little radiolysis at this specific activities. [(131)I]iodo-17-AAG is a promising radiopharmaceutical in nuclear medicine, especially for tumor-targeted radionuclide brachytherapy.

Determination of 17-dimethylaminoethylamino-17-demethoxygeldanamycin in human plasma by liquid chromatography with mass-spectrometric detection.

An analytical method was developed and validated for the quantitative determination of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; NSC707545), a novel heat shock 90 inhibitor, in human plasma. Calibration curves were linear in the concentration range of 1-500 ng/mL. Sample pretreatment involved a liquid-liquid extraction of 0.2 mL aliquots of plasma with ethyl acetate. 17-DMAG and the internal standard, beclomethasone, were separated on a Zorbax SB C18 column (75 mm x 2.1 mm, 3.5 microm), using a mobile phase composed of methanol and 0.2% formic acid (55:45, v/v). The column effluent was monitored by mass spectrometry with electrospray ionization. For the quality control samples at four different concentrations that were analyzed in quintuplicate, on four separate occasions, the accuracy and precision ranged from 93.8% to 99.5% and 1.4% to 3.3%, respectively. The assay modifications significantly improve upon our original, validated method. The developed method was subsequently applied to study the pharmacokinetics of 17-DMAG in a group of 23 patients.

Development and validation of a liquid chromatography/tandem mass spectrometry method for the determination of the novel anticancer agent 17-DMAG in human plasma.

An accurate, sensitive, robust and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin hydrochloride (17-DMAG) in human plasma has been developed and validated. Plasma samples were prepared by liquid/liquid extraction with ethyl acetate. The chromatographic separation was achieved within 9 min on a Synergy Polar column with a linear gradient and a mobile phase consisting of methanol and 0.1% formic acid in water. Detection of 17-DMAG and the internal standard (IS), olomoucine, was achieved by MS/MS with electrospray ionisation in positive ion mode. The calibration curve, ranging from 1.89 to 1890 nM, was linear r > 0.994 using a 1/y2 weighted linear regression. The assay showed no significant interferences from endogenous compounds. The lower limit of quantitation (LLOQ) was 1.89 nM, using 250 microL of plasma, with inter-assay precision (%RSD) and accuracy (%RE) values of 11.6% and -5.8%, respectively. Intra-assay precision ranged from 7.8-13.6%. The method described here is being used to evaluate the pharmacokinetic profiles of 17-DMAG given as a once weekly infusion in patients with advanced solid tumours.