Talazoparib Exposure-Efficacy Analysis in Patients With Advanced Breast Cancer and Germline BRCA1/2 Mutations in the EMBRACA Trial.

In the phase 3 EMBRACA trial, treatment with the poly(ADP-ribose) polymerase inhibitor, talazoparib, led to significantly improved progression-free survival (PFS) compared with chemotherapy (hazard ratio, 0.54; 95% confidence interval, 0.41-0.71; P < .0001). We conducted an exposure-efficacy analysis using EMBRACA data from 285 patients who were treated with talazoparib and had available pharmacokinetic parameters to evaluate the effect of talazoparib exposure (time-varying average talazoparib concentration [Cavg,t ]) and other baseline variables on PFS. Graphical examination of the relationship between Cavg,t and PFS and a Cox proportional model were used. Exposure-response analyses showed that higher talazoparib exposure, absence of visceral disease, lower baseline lactate dehydrogenase levels, and disease-free interval >12 months were independent covariates associated with longer PFS. The association of talazoparib exposure with PFS (higher exposure, longer PFS) suggests the recommended starting dose of 1 mg once daily (the maximum tolerated dose) is appropriate.

Exposure-Safety Analyses of Talazoparib in Patients With Advanced Breast Cancer and Germline BRCA1/2 Mutations in the EMBRACA and ABRAZO Trials.

Poly(ADP-ribose) polymerase inhibitors, such as talazoparib, may affect hematopoiesis. This analysis characterized the relationship between talazoparib exposure and the most common grade ≥ 3 hematopoietic adverse events (AEs) leading to dose modification in the phase 2 (ABRAZO) and phase 3 (EMBRACA) trials. The relationship between time-varying average talazoparib concentration (Cavg,t ), along with other baseline variables, and grade ≥ 3 anemia, thrombocytopenia, and neutropenia were evaluated both by graphical examination and using univariate and multivariate Cox proportional hazard models. The results indicated that higher Cavg,t was associated with a higher risk of anemia and thrombocytopenia. A trend toward an association between higher Cavg,t and neutropenia was observed, although not statistically significant. Higher risk of all tested safety end points was associated with lower baseline hemoglobin. Higher risk of neutropenia was associated with lower baseline absolute neutrophil count and lower body weight. These findings support the proposed management of AEs through talazoparib dosing modification.

Gender-dependent pharmacokinetics of olaparib in rats determined by ultra-high performance liquid chromatography/electrospray ionization tandem mass spectrometry.

The aim of the present study was to develop a liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of olaparib in rat plasma. The plasma samples were processed using one-step protein precipitation with acetonitrile and then separated on Waters Acquity BEH C18 column (50 × 2.1 mm, particle size 1.7 µm) using water containing 0.1% formic acid and acetonitrile as mobile phase with optimized gradient elution. Mass spectrometric detection was carried out by selective reaction monitoring mode via positive ESI mode with precursor-to-product transitions of m/z 435.3 > 367.1 and m/z 443.1 > 375.2 for olaparib and 2 H8 -olaparib (internal standard). The method was linear over the concentration range 0.1-2000 ng/ml with correlation coefficient >0.9987. The lower limit of quantitation was 0.1 ng/ml. The method showed excellent accuracy and precision, negligible matrix effect and high extraction recovery. The validated method was subsequently utilized to determine the concentration of olaparib in rat plasma and further applied to the pharmacokinetic study of olaparib in rat plasma. Our results demonstrated that olaparib showed gender-dependent pharmacokinetics in rats. Compared with that in males, olaparib showed high plasma exposure, long half-life, low clearance and high bioavailability in females.

Phase I study of intermittent olaparib capsule or tablet dosing in combination with carboplatin and paclitaxel (part 2).

Background In the first part of this extensive phase I study (NCT00516724), continuous olaparib twice daily (bid) with carboplatin and/or paclitaxel resulted in myelosuppression and dose modifications. Here, we report the safety, tolerability, and efficacy of intermittent olaparib dosing combined with carboplatin and paclitaxel. Methods Patients with advanced solid tumors (part D) and enriched for ovarian and breast cancer (part E) received olaparib (capsule and tablet formulations) using intermittent schedules (2 to 10 days of a 21-day cycle) combined with carboplatin/paclitaxel. Safety assessments included evaluation of dose-limiting toxicities (DLTs; cycle 1 only), adverse events (AEs), and physical examinations. Pharmacokinetic assessments of olaparib capsule and tablet combined with carboplatin/paclitaxel were performed. Tumor responses (RECIST) were assessed every 2 cycles. Results In total, 132 heavily pre-treated patients were included. One DLT of grade 3 elevated alanine aminotransferase lasting for 8 days was reported (olaparib tablet 100 mg bid days 3-12, carboplatin area under the curve 4 and paclitaxel 175 mg/m2). The most common hematological AEs were neutropenia (47%) and thrombocytopenia (39%), which frequently led to dose modifications. Non-hematological AEs were predominantly grade 1-2, including alopecia (89%) and fatigue (84%). Overall objective response rate was 46%. Conclusions Discontinuous dosing of olaparib resulted in significant myelosuppression leading to dose interruptions and/or delays. Anti-tumor activity was encouraging in patients enriched with BRCA-mutated breast and ovarian cancer. The most appropriate olaparib tablet dose for use in further studies evaluating olaparib in combination with carboplatin and paclitaxel is 50 mg bid (days 1-5).

Population Pharmacokinetics of Talazoparib in Patients With Advanced Cancer.

Poly(ADP-ribose) polymerase (PARP) inhibitors have been developed to treat cancers associated with somatic BRCA mutations and germline genetic aberrations involved in the DNA damage response. The efficacy, tolerability, and pharmacokinetic/pharmacodynamic (PK/PD) profile of talazoparib, a potent small-molecule PARP inhibitor, was established in 4 clinical studies in cancer patients (2 phase 1 studies PRP-001 and PRP-002, the phase 2 ABRAZO trial, and the phase 3 EMBRACA trial). The current study aimed to describe the population PK of talazoparib and identify covariates that affect talazoparib PK in patients with advanced cancers using pooled data from these 4 studies. Talazoparib PK was well characterized by a 2-compartment model with first-order absorption and absorption lag time. Based on covariate analysis, no dose adjustment for talazoparib is required based on a patient's age, sex, baseline body weight, Asian race, the presence of mild renal or hepatic impairment, or use of acid-reducing agents. A reduced 0.75-mg daily dose is recommended for patients taking a potent P-glycoprotein inhibitor and those with moderate renal impairment. Insufficient data were available to establish dosing recommendations for patients with severe renal and moderate or severe hepatic impairment. The PK of a single 1-mg talazoparib capsule is comparable with 4 0.25-mg capsules. Talazoparib can be taken with or without food. These data provide support for dosing recommendations and labeling information for talazoparib.

UPLC-MS/MS method for the determination of talazoparib in rat plasma and its pharmacokinetic study.

In the present study, an accurate and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of plasma talazoparib concentration in rats was developed and established. The purpose of chromatographic separation of talazoparib and the internal standard (bosutinib) was achieved on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column with a flow rate of 0.40 mL/min, using a gradient elution with acetonitrile and 0.1% formic acid in water as the mobile phase. The detection was performed on a XEVO TQ-S triple quadrupole tandem mass spectrometer coupled with electrospray ionization interface under positive-ion multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions of m/z 381.3 → 285.2 for talazoparib and m/z 530.2 → 141.2 for bosutinib (IS), respectively. The method was linear over the range of 0.5-200 ng/mL for talazoparib. The accuracies and precisions of intra- and inter-day were all within the acceptance limits, and no matrix effect was observed in this method. The validated method was further employed to a pharmacokinetic study of talazoparib after oral treatment with 0.2 mg/kg talazoparib to rats.

Phase I study of continuous olaparib capsule dosing in combination with carboplatin and/or paclitaxel (Part 1).

Background The PARP inhibitor olaparib has shown acceptable toxicity at doses of up to 400 mg twice daily (bid; capsule formulation) with encouraging signs of antitumor activity. Based on its mode of action, olaparib may sensitize tumor cells to DNA-damaging agents. This Phase I trial (NCT00516724) evaluated the safety, pharmacokinetics (PK) and preliminary efficacy of olaparib combined with carboplatin and/or paclitaxel. Methods Patients with advanced solid tumors received olaparib (capsule bid) plus carboplatin (Part A), carboplatin and paclitaxel (Part B), or paclitaxel (Part C). In each part of the study, different drug doses were given to define the most appropriate dose/drug combination to use in further studies. Safety assessments included evaluation of dose-limiting toxicities (DLTs; cycle 1 only), adverse events (AEs) and physical examinations. PK assessments of olaparib, carboplatin and paclitaxel were performed. Tumor responses (RECIST) were assessed every two cycles. Results Fifty-seven patients received treatment. DLTs were reported in two patients (both receiving olaparib 100 mg bid and carboplatin AUC 4; Part A, cohort 2): grade 1 thrombocytopenia with grade 2 neutropenia lasting for 16 days, and grade 2 neutropenia lasting for 7 days. Non-hematologic AEs were predominantly grade 1-2 and included fatigue (70%) and nausea (40%). Bone marrow suppression, mainly neutropenia (51%) and thrombocytopenia (25%), frequently led to dose modifications. Conclusions Olaparib in combination with carboplatin and/or paclitaxel resulted in increased hematologic toxicities, making it challenging to establish a dosing regimen that could be tolerated for multiple cycles without dose modifications.

Bridging Olaparib Capsule and Tablet Formulations Using Population Pharmacokinetic Meta-analysis in Oncology Patients.

BACKGROUND: Olaparib is a first-in-class potent oral poly(ADP-ribose) polymerase inhibitor. OBJECTIVES: The aims of this analysis were to establish an integrated population pharmacokinetic (PK) model of olaparib in patients with solid tumors and to bridge the PK of olaparib between capsule and tablet formulations. METHODS: The population PK model was developed using plasma concentration data from 659 patients in 11 phase I, II, and III studies of olaparib tablets/capsules monotherapy. Relative bioavailability between the tablet and capsule formulations was estimated and the relative exposure between olaparib tablet and capsule therapeutic doses was further assessed. RESULTS: The concentration-time profile was described using a two-compartment model with sequential zero- and first-order absorption and first-order elimination for both capsules and tablets with different absorption parameters. Multiple-dose clearance compared with single-dose clearance was reduced by approximately 15% (auto-inhibition). Disease severity had an impact on olaparib clearance, and tablet strength had an impact on Ka. The olaparib geometric mean area under the curve (AUC) and maximal concentration (Cmax) following a single 300 mg tablet were 42.1 µg h/mL and 5.8 µg/mL, respectively, and the steady-state geometric mean AUC and Cmax following a 300 mg tablet twice daily were 49.0 µg h/mL and 7.7 µg/mL, respectively. The relative exposure (AUC) of the 300 mg tablet formulation is 13% higher than the 400 mg capsule formulation. CONCLUSION: This analysis bridged the olaparib capsule and tablet formulation PK and provided key assessment to support the approval of the olaparib tablet formulation in patients with ovarian cancer, regardless of their BRCA mutation status.

Development and Validation of a Simultaneous Quantification Method of Ruxolitinib, Vismodegib, Olaparib, and Pazopanib in Human Plasma Using Liquid Chromatography Coupled With Tandem Mass Spectrometry.

BACKGROUND: A simple, rapid, and sensitive liquid chromatography coupled with tandem mass spectrometry method has been developed and validated for the quantification of ruxolitinib, olaparib, vismodegib, and pazopanib in human plasma. METHODS: After a simple protein precipitation of plasma samples, the chromatographic separation was performed using an ultraperformance liquid chromatography system coupled with mass tandem spectrometry in a positive ionization mode. The mobile phase consisted of a gradient elution of 10-mmol/L formate ammonium buffer containing 0.1% (vol/vol) formic acid (phase A) and acetonitrile with 0.1% (vol/vol) formic acid (phase B) at a flow rate at 300 µL/min. RESULTS: Analysis time was 5.0 minutes per run, and all analytes and internal standards eluted within 1.5-1.73 minutes. The calibration curves were linear over the range from 10 to 2500 ng/mL for ruxolitinib and from 100 to 100,000 ng/mL for olaparib, vismodegib, and pazopanib with coefficients of correlation above 0.99 for all analytes. The intraday and interday coefficients of variation were below 14.26% and 14.81%, respectively, for lower concentration and below 9.94% and 6.37%, respectively, for higher concentration. CONCLUSIONS: Using liquid chromatography coupled with tandem mass spectrometry, we have developed and validated a simple and rapid assay for the simultaneous quantification of olaparib, vismodegib, pazopanib, and ruxolitinib in human plasma. This method is now part of our therapeutic drug monitoring service provision and is currently used clinically to manage patients prescribed these drugs.

Development and validation of a high-performance liquid chromatography method for the quantification of talazoparib in rat plasma: Application to plasma protein binding studies.

A sensitive and selective RP-HPLC method has been developed and validated for the quantification of a highly potent poly ADP ribose polymerase inhibitor talazoparib (TZP) in rat plasma. Chromatographic separation was performed with isocratic elution method. Absorbance for TZP was measured with a UV detector (SPD-20A UV-vis) at a λmax of 227 nm. Protein precipitation was used to extract the drug from plasma samples using methanol-acetonitrile (65:35) as the precipitating solvent. The method proved to be sensitive and reproducible over a 100-2000 ng/mL linearity range with a lower limit of quantification (LLQC) of 100 ng/mL. TZP recovery was found to be >85%. Following analytical method development and validation, it was successfully employed to determine the plasma protein binding of TZP. TZP has a high level of protein binding in rat plasma (95.76 ± 0.38%) as determined by dialysis method.

AMG 900, a potent inhibitor of aurora kinases causes pharmacodynamic changes in p-Histone H3 immunoreactivity in human tumor xenografts and proliferating mouse tissues.

BACKGROUND: The Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers. In mice, oral administration of AMG 900 blocks the phosphorylation of histone H3 on serine-10 (p-Histone H3), a proximal substrate of aurora-B and inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models. METHODS: In order to establish a preclinical pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 that could be translated to the clinic, we used flow cytometry and laser scanning cytometry detection platforms to assess the effects on p-Histone H3 inhibition in terms of sensitivity, precision, and specificity, in human tumor xenografts in conjunction with mouse skin and bone marrow tissues. Mice with established COLO 205 tumors were administered AMG 900 at 3.75, 7.5, and 15 mg/kg and assessed after 3 hours. RESULTS: Significant suppression of p-Histone H3 in mouse skin was only observed at 15 mg/kg (p <0.0001), whereas in mouse bone marrow and in tumor a dose-dependent inhibition was achieved at all three doses (p ≤ 0.00015). These studies demonstrate that AMG 900 inhibits p-Histone H3 in tumors and surrogate tissues (although tissues such as skin may be less sensitive for assessing PD effects). To further extend our work, we evaluated the feasibility of measuring p-Histone H3 using fine-needle aspirate (FNA) tumor xenograft biopsies. Treatment with AMG 900 significantly inhibited p-Histone H3 (>99% inhibition, p <0.0001) in COLO 205 tumors. Lastly, we illustrate this LSC-based approach can detect p-Histone H3 positive cells using mock FNAs from primary human breast tumor tissues. CONCLUSION: Phosphorylation of histone H3 is a useful biomarker to determine the pharmacodynamics (PD) activity of AMG 900. FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.

Development and validation of a high-performance liquid chromatography-tandem mass spectrometry assay quantifying olaparib in human plasma.

Olaparib is an inhibitor of poly ADP ribose polymerase 1 (PARP-1). Phase I and II trials showed promising results of olaparib against tumours in BRCA mutation carriers. Currently an increasing number of clinical trials with olaparib in combination with other compounds or radiotherapy are conducted. To support these clinical trials an LC-MS/MS method was developed and validated for the quantification of olaparib in human plasma. Human plasma samples were collected in the clinic and stored at nominally -20°C. Olaparib was isolated from plasma by liquid-liquid extraction, separated on a C18 column with gradient elution and analyzed with triple quadrupole mass spectrometry in positive ion mode. A deuterated isotope was used as internal standard for the quantification. The assay, ranging from 10 to 5000ng/mL, was linear with correlation coefficients (r(2)) of 0.9994 or better. The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±7.6% of nominal and inter-assay and intra-assay precision ≤9.3% at the lower limit of quantification and ≤5.7% at the other concentration levels tested. All results were within the acceptance criteria of the US FDA and the latest EMA guidelines for method validation. A quantitative method was developed and validated for the quantification of olaparib in human plasma. The method could successfully be applied for the pharmacokinetic quantification of olaparib in cancer patients treated with olaparib.

Evaluation of the pharmacodynamics and pharmacokinetics of the PARP inhibitor olaparib: a phase I multicentre trial in patients scheduled for elective breast cancer surgery.

Olaparib (AZD2281) is an oral poly(ADP-ribose) polymerase (PARP) inhibitor with antitumour activity in cancer patients with BRCA1/2 germline mutations and in patients with homologous recombination deficiency. In this dose-finding study, patients were randomized to olaparib 10, 30, 100, 200 or 400 mg (capsule formulation) twice daily for the 4-5 days preceding breast cancer surgery. The primary objective was to identify an effective biological dose of olaparib for future trials. Secondary endpoints included evaluation of PARP-1 inhibition dose/exposure-response, and safety. Olaparib plasma pharmacokinetics (PK) and the pharmacodynamics (PD) in tumour and peripheral blood mononuclear cells (PBMCs) were evaluated. Population PK/PD modelling was performed on pooled data from this study and a previously reported study. Sixty patients were randomized (n = 12, each dose). Dose-dependent increases in exposure to olaparib were observed, but at ~50 % lower plasma exposure levels than seen in advanced disease studies. The mean maximal extent of PARP inhibition in PBMCs and tumour tissue was 50.6 % and 70.0 %, respectively, and was similar to inhibitory levels reported previously. No PARP inhibition-dose relationship was observed. Due to the unexpectedly low olaparib exposure, we were unable to determine an effective biological dose. Common adverse events included procedural pain (n = 31 patients), nausea, asthenia, malaise and increased blood creatinine (n = 6, each); these were of mild-to-moderate intensity, and all were manageable. Despite low olaparib exposure, PARP inhibition was consistent with previous reports. Reasons for the inter-study differences in exposure are unclear. The tolerability profile of olaparib was consistent with previous studies.

LC-MS/MS bioanalytical method development for AMG 900: resolution of an isobaric interference in rodent in vivo studies.

AMG 900 is an orally available small molecule that is a highly potent and selective pan-aurora kinase inhibitor currently in development for the treatment of advanced human cancers. A co-eluting, isobaric interference was discovered in preliminary LC-MS/MS analyses of rodent in vivo pharmacokinetic samples during preclinical evaluation of AMG 900. The interference was identified as a major circulating N-oxide metabolite which partially converted to an [M+H-O](+) ion under the conditions of atmospheric pressure chemical ionization. A selective liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of AMG 900 and its N-oxide metabolite in plasma was developed and successfully applied for the bioanalysis of discovery stage preclinical rodent pharmacokinetic studies.

Simultaneous determination of azelastine and its major metabolite desmethylazelastine in human plasma using high performance liquid chromatography-tandem mass spectrometry.

A selective and sensitive high performance liquid chromatography-tandem mass spectrometric method was developed for the analysis of azelastine and its major metabolite, desmethylazelastine, in human plasma. Azelastine-(13)C, d(3) was used as internal standard. Azelastine, desmethylazelastine and the internal standard were extracted by a liquid-liquid extraction method and separation was performed under isocratic chromatographic condition. An abnormal signal loss issue for desmethylazelastine during method development was investigated and resolved. The developed method was precise and reproducible as shown by good intraday assay and interday assay precision (CV%≤ 12.8%). The calibration curve was linear over a range of 10.0/10.0-1000/200 pg/mL for azelastine/desmethylazelastine. The method was successfully applied to a pilot bioequivalence study subsequently.

Gender effects on rat metabolism of AMG 900, an orally available small molecule aurora kinase inhibitor.

AMG 900 is an orally available small molecule that is highly potent and selective as a pan-aurora kinase inhibitor. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. The metabolism of AMG 900 was investigated in both male and female rats. We conducted studies in bile-duct catheterized (BDC) rats where bile, urine and plasma were analyzed to obtain metabolism profiles for each gender. These studies identified gender differences in the metabolism profiles in bile. Bile contained the majority of the drug related material and contained little unchanged AMG 900 which indicated that metabolism was the prominent process in drug elimination. Although bile contained the same metabolites for both genders, the amount of specific metabolites differed. Male rats metabolized AMG 900 primarily through hydroxylation with subsequent sulfate conjugation on the pyrimidinyl-pyridine side-chain whereas female rats favored a different oxidation site on the thiophene ring's methyl group, which is then metabolized to a carboxylic acid with subsequent conjugation to an acyl glucuronide. CYP phenotyping identified the prominent isoforms as being gender specific or biased in the oxidative metabolism of AMG 900. The metabolism in male rats favored both CYP2C11 and CYP2A2 whereas females favored the CYP2C12. The prominent sulfate conjugate identified in the male rat bile could also be due to male biased metabolism since it has been reported that sulfate conjugation is more prevalent in male rats. All the prominent rat metabolism routes for AMG 900 either have male or female bias. These differences in the rat AMG 900 metabolism profiles in bile can be explained by gender specific P450CYP isoforms.

Liquid chromatography-tandem mass spectrometric assay for the PARP-1 inhibitor olaparib in combination with the nitrogen mustard melphalan in human plasma.

A bioanalytical assay for the new poly(ADP-ribose) polymerase-1 inhibitor olaparib in combination with melphalan was developed and validated. For the quantitative assay, human plasma samples were pre-treated on ice using protein precipitation with 2% (v/v) acetic acid in acetonitrile containing erlotinib and melphalan-d8 as internal standards. The extract was diluted with water and injected into the chromatographic system. This system consisted of a sub-2 µm particle, trifunctional bonded octadecyl silica column with an isocratic elution using 0.01% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 10-5000 ng/ml calibration range for both drugs. The lowest level of this range corresponded to the lower limit of quantification. Within day precisions were 3.0-9.3%, between day precisions 6.0-9.8% and accuracies were between 101 and 110% for the whole calibration range. After validation the assay was used to assess the pharmacokinetics of olaparib in a patient with metastatic breast carcinoma. In addition, systemic exposure of melphalan was monitored in patients subjected to isolated hepatic perfusion with this drug. Both applications show that the new assay can be applied for human pharmacokinetic studies for both drugs.

In vitro and in vivo pharmacokinetic characterizations of AMG 900, an orally bioavailable small molecule inhibitor of aurora kinases.

AMG 900 is a small molecule being developed as an orally administered, highly potent, and selective pan-aurora kinase inhibitor. The aim of the investigations was to characterize in vitro and in vivo pharmacokinetic (PK) properties of AMG 900 in preclinical species. AMG 900 was rapidly metabolized in liver microsomes and highly bound to plasma proteins in the species tested. It was a weak Pgp substrate with good passive permeability. AMG 900 exhibited a low-to-moderate clearance and a small volume of distribution. Its terminal elimination half-life ranged from 0.6 to 2.4 h. AMG 900 was well-absorbed in fasted animals with an oral bioavailability of 31% to 107%. Food intake had an effect on rate (rats) or extent (dogs) of AMG 900 oral absorption. The clearance and volume of distribution at steady state in humans were predicted to be 27.3 mL/h/kg and 93.9 mL/kg, respectively. AMG 900 exhibited acceptable PK properties in preclinical species and was predicted to have low clearance in humans. AMG 900 is currently in Phase I clinical testing as a treatment for solid tumours. Preliminary human PK results appear to be consistent with the predictions.

A validated assay for the quantitative analysis of vatalanib in human EDTA plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A sensitive and accurate method for the determination of vatalanib in human EDTA plasma was developed using high-performance liquid chromatography and detection with tandem mass spectrometry. Stable isotopically labeled imatinib was used as internal standard. Plasma proteins were precipitated and an aliquot of the supernatant was directly injected onto a Phenomenex Gemini C18 analytical column (50 mm x 2.0 mm ID, 5.0 microm particle size) and then compounds were eluted with a linear gradient. The outlet of the column was connected to a Sciex API 365 triple quadrupole mass spectrometer and ions were detected in positive multiple reaction monitoring mode. The lower limit of quantification was 10 ng/mL (S/N approximately 10, CV < or = 8.4%). This method was validated over a linear range from 10 to 2500 ng/mL, and results from the validation study demonstrated a good intra- and inter-assay accuracy (inaccuracy < or = 9.57%) and precision (CV < or = 8.81%). This method has been used to determine plasma vatalanib concentrations in patients with advanced solid tumor, enrolled in a phase I pharmacokinetic trial with the drug.

Biomarkers for assessment of pharmacologic activity for a vascular endothelial growth factor (VEGF) receptor inhibitor, PTK787/ZK 222584 (PTK/ZK): translation of biological activity in a mouse melanoma metastasis model to phase I studies in patients with advanced colorectal cancer with liver metastases.

PTK/ZK is a novel, oral angiogenesis inhibitor that specifically targets all 3 vascular endothelial growth factor (VEGF) receptor tyrosine kinases and is currently in phase III clinical trials. In early clinical trials, PTK/ZK demonstrated a dose-dependent reduction in tumor vascular parameters as measured by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and an acute increase in plasma VEGF levels. The reduction in tumor vascularity was significantly correlated with improved clinical outcome in patients with advanced colorectal cancer and liver metastases. To assess the predictive value of a mouse model of tumor metastases, comparisons were performed for the biological activity of PTK/ZK in the mouse model and in patients with liver metastases in the clinical phase I trials. An orthotopic, syngeneic mouse model was used: C57BL/6 mice injected in the ear with murine B16/BL6 melanoma cells which metastases to the cervical lymph-nodes. The primary tumor and spontaneous metastases express VEGF and VEGF receptors and respond to treatment with VEGFR tyrosine kinase inhibitors. PTK/ZK was administered orally, with assessments by DCE-MRI of the metastases and plasma VEGF taken predose and at 3 days posttreatment and efficacy determined at 7 days posttreatment. Dose-ranging studies in naive mice provided preclinical pharmacokinetic data, while two dose-escalation phase I studies provided clinical pharmacokinetic data. An exposure-response relationship was observed both for mouse metastases (measured as % tumor weight treated/control) and for human liver metastases (measured as % regression). In the B16/BL6 model, the active dose of 50 mg/kg PTK/ZK yielded 62.4 (+/- 16.0) h microM plasma exposure, which is comparable to the plasma area under the concentration time curve (AUC) achieved by the 1000 mg dose of PTK/ZK used in clinical trials. At this exposure level in clinical trials, DCE-MRI showed a reduction in the area under the enhancement curve (IAUC) to 47% of baseline. At a similar exposure in the PTK/ZK-treated mice, a reduction in IAUC to 75% of baseline was observed. Furthermore, at doses of 50 mg/kg PTK/ZK and above, an increase in plasma VEGF level 10 h after drug administration was observed in mice which was consistent with findings from the clinical trials. In conclusion, the preclinical pharmacodynamics of PTK/ZK correlate well with clinical activity in phase I trials over comparable exposures to the drug. Thus, data from this preclinical model proved to be consistent with and thus predictive of the biologic effects of PTK/ZK in phase I/II clinical trials.