Simultaneous determination of dacomitinib and its major metabolite, O-desmethyl dacomitinib in human plasma by LC-MS/MS and its application to clinical testing in patients with non-small cell lung cancer.

Dacomitinib, an irreversible pan-ErbB tyrosine kinase inhibitor targeting the human epidermal growth factor receptor, is used for the treatment of metastatic non-small cell lung cancer. To facilitate the investigations on its metabolism and other relevant studies, based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), a rapid and sensitive bioanalytical technique was established and fully validated for simultaneous quantification of dacomitinib and its main metabolite in human plasma. The plasma samples were treated with acetonitrile containing 0.1% formic acid and the liquid supernatant was collected, dried and dissolved in methanol-water-formic acid (200:800:1, v/v) before injection. The chromatographic separation was performed on an ACE Excel C18 column (2.1 mm × 50.0 mm, i.d., 5 µm) by gradient elution with a mixture of buffer (5 mM ammonium acetate in 0.1% formic acid) and acetonitrile, serving as the mobile phase, with an overall run time of 4 min. Dacomitinib, O-desmethyl dacomitinib and IS were subsequently detected on an AB QTRAP 5500 mass spectrometer in positive ion and multiple reaction monitoring modes at the precursor-to-product transitions of m/z 470.4 â†’ 385.0, m/z 456.0 â†’ 370.9 and m/z 480.2 â†’ 385.1, respectively. The accuracy and precision of determinations were guaranteed within the concentration ranges of 0.25-100 ng/mL for dacomitinib and 0.20-80 ng/mL for O-desmethyl dacomitinib. The intra- and inter-assay accuracy ranged from 92.00% to 104.50% and the intra- and inter-assay precision was less than 8.20% for each analyte. The method was validated and the relevant parameters, including selectivity, interference among analytes and internal standard, carry-over effect, dilution integrity, extraction recovery, matrix effect, and stability, all satisfied the requirements formulated by the US Food and Drug Administration and the European Medicines Agency. The clinical applicability of the fully-validated method was evaluated in medicated samples from patients on dacomitinib.

Validated HPLC-UV method for simultaneous quantification of phosphatidylinositol 3-kinase inhibitors, copanlisib, duvelisib and idelalisib, in rat plasma: Application to a pharmacokinetic study in rats.

Phosphatidylinositol 3-kinase (PI3K) inhibitors are a novel class of anticancer drugs that are approved to treat various malignancies. We report the development and validation of a HPLC method for the simultaneous quantitation of three PI3K inhibitors, namely copanlisib, duvelisib and idelalisib, in rat plasma as per the regulatory guidelines of the United States Food and Drug Administration. The method involves extraction of copanlisib, duvelisib and idelalisib along with an internal standard (IS; filgotinib) from rat plasma (100 µL) using a liquid-liquid extraction process. The chromatographic separation of the analytes was achieved using step-wise gradient elution on a Hypersil Gold C18 column. The UV detection wavelength was set at λmax = 280 nm. Copanlisib, duvelisib, idelalisib and the IS eluted at 7.16, 12.6, 11.9 and 9.86 min, respectively, with a total run time of 15 min. The calibration curve ranged from 50 to 5000 ng/mL for all the analytes. Inter- and intra-day precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated for all three analytes, and the results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in rats.

Validation of a sensitive simultaneous LC-MS/MS method for the quantification of novel anti-cancer thiazolidinedione and quinazolin-4-one derivatives in rat plasma and its application in a rat pharmacokinetic study.

Thiazolidinediones and quinazolin-4-ones compounds, previously known for their activity against Type 2 diabetes and antifungal activity respectively, are currently being investigated for their anti-cancer activity. The determination of pharmacokinetic parameters for these two classes of compounds using a simultaneous chromatographic method with a low detection limit is a challenge. In this study, a highly sensitive and simultaneous LC-MS/MS-based bioanalytical method was developed and validated in rat plasma for the estimation of four novel anti-cancer compounds, BIT-15-67 and BNT-11, belonging to the Thiazolidinedione class, and BNUA-108 and BNUA-48, from the quinazolin-4-one class. The analytes were extracted from plasma samples by protein precipitation and separated on a short reverse phase Hypersil Phenyl BDS, 50 × 4.6 mm, 2.4 µm column at a column oven temperature of 40 °C. An isocratic mobile phase, a 20:80 (v/v) mixture of 5 mM ammonium acetate solution and acetonitrile containing 0.1% formic acid, was used for the elution at a flow rate of 0.4 mL/min. The analytes and internal standard, sulfaphenazole, were quantified in the multiple reaction monitoring mode using positive electrospray ionization with specific pair of mass by charge ratio. All standard validation parameters were assessed as per current bioanalytical method validation guidelines in rat plasma. The area response for the four analytes was found to be linear over the concentration range of 1.00 to 1000 ng/mL in rat plasma. The signal to noise at LLOQ of 1 ng/mL was adequate for application to different pre-clinical studies. The intra- and inter-day precision were <11% and accuracy deviated -1.8 to 9.60% from the nominal. The mean recovery was high (about 90%) and consistent for all the analytes over the linear dynamic range of the method. This simple, robust and validated method can be employed to determine the rat plasma concentrations of the four selected anticancer compounds in preclinical studies such as the pharmacodynamic and the pharmacokinetic studies including tissue distribution and excretion, and the toxicokinetic studies. In this study, pharmacokinetic parameters were determined using this method for all the four compounds individually following intravenous administration in rats.

Quantification of Idelalisib in Human Plasma by Ultra-Performance Liquid Chromatography Coupled to Mass Spectrometry in Negative Ionization Mode.

BACKGROUND: Idelalisib is the first orally active selective phosphatidylinositol 3-kinase delta inhibitor approved by Food and Drug Administration and European Medicines Agency in 2014 for the treatment of several types of blood cancer. Idelalisib is widely used as a monotherapy or in combination with rituximab, bendamustine, or ofatumumab with a significant efficacy. However, idelalisib has shown increased risk of infection and a higher frequency of serious adverse events. It may be useful to determine idelalisib concentration in human plasma to adjust dose and to manage adverse effects in clinical practice. METHODS: After a single-step protein precipitation of plasma samples, the chromatographic separation was performed using an ultra-high performance liquid chromatography system coupled with mass tandem spectrometry in a negative ionization mode using isotope-labeled internal standard. This method was validated by studies of its linearity, accuracy, imprecision, limit of quantification, recovery, matrix effect, selectivity, and stability. RESULTS: The quantification method was linear from 10 to 2500 ng/mL with a 5 ng/mL lower limit of quantification that encompasses the clinical range of drug concentration. The intraday and interday imprecisions were below 8.1% and 11.4%, respectively. The recoveries and matrix effect of idelalisib were 85.6% ± 1.2% and 95.7% ± 3.0%, respectively, which are consistent, precise, and reproducible (coefficient of variation % < 15%). Peak plasma concentration and trough plasma concentration ranges of idelalisib reached 1591-1937 ng/mL and 256.3-303.3 ng/mL, respectively, in 3 follicular lymphoma patients treated with idelalisib 150 mg twice a day. CONCLUSIONS: A robust and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated to quantify idelalisib concentration in human plasma. This method was effectively applied to 3 follicular lymphoma patients.

Impact of a planned dose interruption of dacomitinib in the treatment of advanced non-small-cell lung cancer (ARCHER 1042).

OBJECTIVES: Dacomitinib is a pan-HER inhibitor for advanced non-small-cell lung cancer (NSCLC). We explored the impact of a planned 4-day dacomitinib dose interruption on plasma exposure of dacomitinib and adverse events (AEs) of interest in Cohort III of the ARCHER 1042 study. MATERIALS AND METHODS: Patients, treatment-naïve for advanced NSCLC with EGFR activating mutations, received oral dacomitinib 45mg QD (once daily). A planned dose interruption occurred in Cycle 1 from Days 11 through 14. The primary endpoint was the pharmacokinetic (PK) characteristics of dacomitinib in Cycle 1Day 10 and during dose interruption. Secondary endpoints included safety and concomitant medications used to treat AEs of interest. RESULTS: Cohort III enrolled 25 patients. Median plasma Cmax of dacomitinib in Cycle 1 Day 10 was 83.40ng/mL. Average median plasma dacomitinib concentration during the 4-day dose interruption was 42.63ng/mL. In the first 8 weeks of treatment 1) 80% of patients used concomitant medications for dermatologic AEs, 76% for diarrhea, and 44% for stomatitis, and 2) all patients experienced treatment-emergent AEs and 28% had all-causality Grade 3 AEs. CONCLUSION: At 45mg QD dosing, PK parameters of plasma dacomitinib in Cycle 1 Day 10 were comparable to that obtained in Cycle 1 Day 14 from other dacomitinib studies. Average median plasma dacomitinib concentration during the 4-day dose interruption was approximately half of the median plasma Cmax of dacomitinib observed prior to dose interruption. The toxicity profile was consistent with that from other studies of dacomitinib.

Investigation of the impact of hepatic impairment on the pharmacokinetics of dacomitinib.

Dacomitinib (PF-00299804) is a small-molecule inhibitor of the tyrosine kinases human epidermal growth factor receptor-1 (HER1; epidermal growth factor receptor, EGFR), HER2, and HER4 currently being developed for the treatment of lung cancer with sensitizing mutations in EGFR or refractory to EGFR-directed treatment. Dacomitinib is largely metabolized by the liver through oxidative and conjugative metabolism; therefore, determination of the impact of varying degrees of hepatic impairment on the pharmacokinetics (PK) of dacomitinib was warranted to ensure patient safety. In this phase I, open-label, parallel-group study, a single dose of dacomitinib was administered to healthy volunteers and to subjects with mild or moderate liver dysfunction, as determined by Child-Pugh classification. The primary goal of this study was to evaluate the effects of mild and moderate hepatic impairment on the single-dose PK profile of dacomitinib, as well as to assess the safety and tolerability in these subjects. Plasma protein binding and impact of hepatic function on the PK of the active metabolite PF-05199265 was also investigated. Twenty-five male subjects received dacomitinib 30 mg, with 8 subjects in the healthy- and mild-impairment cohorts and 9 subjects in the moderate-impairment cohort. Compared with healthy volunteers, there was no significant change in dacomitinib exposure in subjects with mild or moderate liver dysfunction and no observed alteration in plasma protein binding. No serious treatment-related adverse events were reported in any group, and dacomitinib was well tolerated. A dose adjustment does not appear necessary when administering dacomitinib to patients with mild or moderate hepatic impairment.

Pharmacokinetic assessment of dacomitinib (pan-HER tyrosine kinase inhibitor) in patients with locally advanced head and neck squamous cell carcinoma (LA SCCHN) following administration through a gastrostomy feeding tube (GT).

BACKGROUND: Dacomitinib is an irreversible oral pan-HER tyrosine kinase inhibitor with antitumor activity demonstrated in patients with recurrent/metastatic (RM) SCCHN. A Phase I trial of dacomitinib with standard therapy in LA SCCHN is ongoing (NCT01737008). As enteral feeding is needed for many SCCHN patients, this study investigated the PK properties of dacomitinib when administered via GT (NCT01484847). Since patients with GT are difficult to recruit, this study also determined the feasibility of PK assessments using a unique design in LA SCCHN patients with GT, by giving a single dose of drug during their radiotherapy (co-administration with chemotherapy avoided). METHODS: Eligible patients were given a single dose of crushed dacomitinib at 45 mg in water suspension via GT. All doses were administered in fasting state and supine position. PK samples were drawn prior to dose (t = 0), 30 min and 1, 2, 3, 4, 6, 12, 24, 48, 72, 96, 144, 168, 192 and 216 hrs post-dose, and analyzed by HPLC-MS/MS. PK parameters (mean [CV%]) of this study were compared with those of dacomitinib given orally using Student t test. RESULTS: Six patients with LA SCCHN patients were enrolled. The median age of patients was 54 years. Two different types of GT were used: 14 F Cope-loop tube (n = 3), 20 F PEG/disc retention tube (n = 3). PK study showed t1/2 of 58 h, Cmax of 17 ng/ml, Tmax of 8 h, AUC0-inf of 1185 ng*hr/ml, Vd/F of 3310 L and CL/F of 41 L/hr. CONCLUSION: Compared with oral dosing of intact immediate release (IR) tablets, GT administration resulted in 34 % reduction in Cmax and 33-44 % decrease in AUC (all p <0.05) (Jänne et al., Clin Cancer Res 2011). Such differences were not detected when compared with the PK properties of dacomitinib administered orally in aqueous suspension (Bello et al., Cancer Chemother Pharm 2013). These differences may be attributed to aqueous suspension of dacomitinib. Caution should be taken with GT administration of orally active small molecule targeted therapy. This study also demonstrated that PK trials in GT patients are feasible using novel designs.

Clinical drug interaction profile of idelalisib in healthy subjects.

Idelalisib, a potent phosphatidylinositol-3-kinase delta (PI3Kδ) inhibitor, is metabolized primarily by aldehyde oxidase to form GS-563117 and to a lesser extent by cytochrome P450 (CYP) 3A and uridine 5'-diphospho-glucuronosyltransferase 1A4. In vitro, idelalisib inhibits P-glycoprotein (P-gp) and organic anion transporting polypeptides 1B1 and 1B3, and GS-563117 is a time-dependent CYP3A inhibitor. This study enrolled 24 healthy subjects and evaluated (1) the effect of idelalisib on the pharmacokinetics (PK) of digoxin, a P-gp probe substrate, rosuvastatin, a breast cancer resistance protein, and OATP1B1/OATP1B3 substrate, and midazolam, a CYP3A substrate; and (2) the effect of a strong inducer, rifampin, on idelalisib PK. On treatment, the most common clinical adverse events (AEs) were headache and pyrexia. Grade 3 transaminase increases were observed in 5 of 24 subjects and were reversible. Two subjects had serious AEs after treatment completion (grade 3 pyrexia and/or drug-induced liver injury). Idelalisib coadministration did not affect digoxin and rosuvastatin PK. Coadministration with idelalisib increased plasma exposures of midazolam (138% and 437% for maximum observed plasma concentration [Cmax ] and area under the plasma concentration-time curve from time 0 extrapolated to infinity [AUCinf ], respectively), consistent with the in vitro finding of CYP3A inhibition by GS-563117. Rifampin caused a substantial decrease in idelalisib (58% and 75%, Cmax and AUCinf , respectively) and GS-563117 exposures, indicating an enhanced contribution of CYP3A to idelalisib metabolism under a strongly induced state.

A phase I open-label study to investigate the potential drug-drug interaction between single-dose dacomitinib and steady-state paroxetine in healthy volunteers.

Dacomitinib is currently in development for the treatment of non-small cell lung cancer. Formation of the major circulating metabolite (PF-05199265) is mediated by cytochrome P450 (CYP) 2D6 and CYP2C9. This phase I, single fixed-sequence, two-period study evaluated the effect of paroxetine, a CYP2D6 inactivator, on dacomitinib pharmacokinetics in healthy volunteers who were extensive CYP2D6 metabolizers. Subjects received a single 45-mg dacomitinib dose alone and in combination with paroxetine (30 mg/day for 10 consecutive days, with dacomitinib administered on day 4) at steady-state levels. Blood samples were collected through 240 hours post-dacomitinib dosing. Dacomitinib exposure (area under the concentration-time curve from 0 to infinity; AUCinf) increased 37%; however a reduction in PF-05199265 AUCinf of approximately 90% was observed during the paroxetine treatment period. The maximum concentration of dacomitinib changed minimally. Adverse events reported with single-dose dacomitinib administered alone or in the presence of steady-state levels of paroxetine were mostly mild, and no serious adverse events were reported. While paroxetine significantly inhibited CYP2D6-mediated metabolism of a single dose of dacomitinib, the modest effect on dacomitinib exposure is unlikely to be clinically relevant when dacomitinib is given daily. Dose adjustment of dacomitinib may therefore not be required upon coadministration with a CYP2D6 inhibitor.

Phase I and pharmacokinetic study of dacomitinib (PF-00299804), an oral irreversible, small molecule inhibitor of human epidermal growth factor receptor-1, -2, and -4 tyrosine kinases, in Japanese patients with advanced solid tumors.

BACKGROUND: Dacomitinib (PF-00299804) is an oral, irreversible, small molecule inhibitor of human epidermal growth factor receptor-1, -2, and -4 tyrosine kinases. METHODS: This phase I, open-label, dose-escalation study (clinicaltrials.gov: NCT00783328) primarily evaluated the safety and tolerability of dacomitinib by dose-limiting toxicity (DLT), and determined the clinically recommended phase II dose (RP2D) in Japanese patients with advanced solid tumors. Dacomitinib was administered orally at three dose levels (15, 30, or 45 mg once daily [QD]). Patients initially received a single dose, and after 9 days of follow-up, continuously QD in 21-day cycles. Endpoints included pharmacokinetics (PK) and antitumor activity. RESULTS: Thirteen patients were assigned to the three dose levels (15 mg cohort: n = 3; 30 mg cohort: n = 3; 45 mg cohort: n = 7) according to a traditional '3 + 3' design. None of the treated patients experienced a DLT. Toxicities were manageable and similar in type to those observed in other studies. PK concentration parameters increased with dose over the range evaluated, with no evidence of accumulation over time. Of 13 evaluable patients, one with NSCLC (adenocarcinoma) had a partial response and nine patients had stable disease. CONCLUSIONS: Dacomitinib 45 mg QD was defined as the RP2D and demonstrated preliminary activity in Japanese patients with advanced solid tumors.