Abigail D. Davis

Development and validation of LC-MS/MS methods for the measurement of ribociclib, a CDK4/6 inhibitor, in mouse plasma and Ringer's solution and its application to a cerebral microdialysis study.

LC-MS/MS methods to measure ribociclib in mouse plasma and Ringers solution were successfully developed and validated. Reverse phase chromatography was performed with gradient elution using C18 (100A, 50×4.6mm, 3μ) and C8-A (50×2.0mm, 5μ) columns for plasma and Ringers samples, respectively. Mouse plasma samples were extracted using solid phase extraction method, whereas no extraction was required for the Ringers solution samples. Analytes were detected using positive ion MRM mode. The precursor to product ions (Q1→Q3) selected for ribociclib and d6-ribociclib were (m/z) 435.2→252.1 and 441.2→252.1, respectively. The linear range of quantification of ribociclib was 62.5-10,000ng/ml for plasma method and 0.1-100ng/ml for Ringers solution method. The results for the inter-day and intra-day accuracy and precision of quality control samples were within the acceptable range. The lower limit of quantitation (LLOQ) for plasma and Ringers samples were 62.5ng/ml (S/N>30) and 0.1ng/ml (S/N>13), respectively, whereas the limit of detection (LOD) was 6.9ng/ml (S/N>7) and 0.05ng/ml (S/N>3), respectively. The developed methods were successfully applied to the analysis of ribociclib in mouse plasma and dialysate samples collected during a cerebral microdialysis study of ribociclib in a non-tumor bearing mouse.

Development and validation of a sensitive LC MS/MS method for the measurement of the checkpoint kinase 1 inhibitor prexasertib and its application in a cerebral microdialysis study

HighlightsA sensitive LC–MS/MS method for the quantitation of prexasertib in mouse plasma and Ringers solution with 0.5% bovine serum albumin (Ringers/BSA) was developed and validated.Prexasertib was extracted from mouse plasma and Ringers/BSA by Liquid‐liquid extraction.This method demonstrated excellent precision, accuracy, reproducibility for routine analysis, and was successfully applied for a cerebral microdialysis study. ABSTRACT LC MS/MS methods to measure prexasertib in mouse plasma and Ringers solution containing 0.5% BSA (Ringers/BSA) were developed and validated. Liquid‐liquid extraction with tert‐butyl methyl ether was used to extract prexasertib from mouse plasma and Ringers/BSA. Reverse phase chromatography with gradient elution was performed to separate prexasertib from the endogenous interference in the matrix, followed by MS detection using positive ion MRM mode. The initial calibration curve for mouse plasma samples ranged from 1 to 500 ng/ml, and after validation of that curve and use in a preliminary study another calibration curve (0.2–200 ng/ml) was created to enable the quantitation of prexasertib at lower concentrations. The method described was precise and accurate with %CV in precision studies of ≤ 6.7% and accuracies within 95.0–110% of nominal target concentration across all concentrations tested for both matrices. This validated method was successfully applied in the analysis of prexasertib in mouse plasma and dialysate samples collected during a cerebral microdialysis study.

Simvastatin Hydroxy Acid Fails to Attain Sufficient Central Nervous System Tumor Exposure to Achieve a Cytotoxic Effect: Results of a Preclinical Cerebral Microdialysis Study.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors were potent hits against a mouse ependymoma cell line, but their effectiveness against central nervous system tumors will depend on their ability to cross the blood–brain barrier and attain a sufficient exposure at the tumor. Among 3-hydroxy-3-methylglutaryl coenzyme A inhibitors that had activity in vitro, we prioritized simvastatin (SV) as the lead compound for preclinical pharmacokinetic studies based on its potential for central nervous system penetration as determined from in silico models. Furthermore, we performed systemic plasma disposition and cerebral microdialysis studies of SV (100 mg/kg, p.o.) in a murine model of ependymoma to characterize plasma and tumor extracellular fluid (tECF) pharmacokinetic properties. The murine dosage of SV (100 mg/kg, p.o.) was equivalent to the maximum tolerated dose in patients (7.5 mg/kg, p.o.) based on equivalent plasma exposure of simvastatin acid (SVA) between the two species. SV is rapidly metabolized in murine plasma with 15 times lower exposure compared with human plasma. SVA exposure in tECF was <33.8 ± 11.9 µg/l per hour, whereas the tumor to plasma partition coefficient of SVA was <0.084 ± 0.008. Compared with in vitro washout IC50 values, we did not achieve sufficient exposure of SVA in tECF to suggest tumor growth inhibition; therefore, SV was not carried forward in subsequent preclinical efficacy studies.

Abstract 2708: Development of an individualized 3D transport model of topotecan for a patient-derived orthotopic xenograft model of pediatric neuroblastoma

Resistance to chemotherapeutics and targeted therapies in pediatric solid tumors including neuroblastoma is a common cause of poor clinical outcome. These failures in part stem from shortcomings in understanding inter- and intra-tumor heterogeneities of drug penetration due to heterogeneities in blood perfusion. Herein we propose to develop an individualized 3D transport model of topotecan (TPT) for a patient-derived orthotopic xenograft model of pediatric NB5 neuroblastoma to account for inter- and intra-tumor heterogeneities in blood perfusion. The transport model uses a 3D reaction-diffusion equation to simulate diffusion of TPT from blood vessels into the tumor tissue and its flux in and out of intracellular space. Our transport model takes three types of inputs to predict TPT exposure maps defined over the volume of an individual tumor: a) plasma concentration-time profiles from an individualized physiologically-based pharmacokinetic (PBPK) model of TPT (separate cohort), b) 3D blood perfusion map of the individual tumor from contrast enhanced ultrasound (CEUS) using VisualSonics VEVO 2100 imaging system, and c) in vitro TPT cellular uptake and efflux kinetics from two-photon imaging. We use in vitro pharmacodynamics (PD) experiments with NB5 cells exposed to TPT to derive probabilistic PD-rules for drug effects (e.g., γ-H2AX response). Based on these rules and the exposure maps, we then compute probabilities of effects for the entire tumor volume. We will validate the predicted drug effect maps by comparing them to the observed effects measured by immunohistochemistry marker for γ-H2AX from the same tumor (location matched) using spatial correlation techniques. Citation Format: Abbas Shirinifard, Suresh Thiagarajan, Yogesh T. Patel, Abigail D. Davis, Megan O. Jacus, Stacy L. Throm, Jessica Roberts, Vinay Daryani, Clinton F. Stewart, Andras Sablauer. Development of an individualized 3D transport model of topotecan for a patient-derived orthotopic xenograft model of pediatric neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2708.

Abstract 1496: Quantification of tumor blood perfusion of an orthotopic mouse model of neuroblastoma using nonlinear contrast-enhanced ultrasound imaging

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA This study quantifies tumor perfusion in individual tumors to estimate blood flow and blood volume parameters of an individualized tumor compartment of a comprehensive physiologically-based pharmacokinetic model of topotecan using an orthotopic xenograft model of pediatric neuroblastoma. We non-invasively imaged perfusion in orthotopic neuroblastoma (NB5) xenograft tumors (n = 3 CD1 nude mice/time point) using nonlinear contrast enhanced ultrasound technique (CEUS). Tumor tissue and organs from the mice were harvested at predefined time-points. We used a programmable syringe pump to inject MicroMarker® microbubbles via tail vein catheter and acquired images using VisualSonics VEVO 2100 imaging system. We used the burst-replenishment technique to image tumor perfusion, which requires a constant concentration of microbubbles in blood during acquisition. To maintain a steady concentration of microbubbles, we programmed the pump to inject a small bolus followed by constant infusion. Our preliminary analysis showed that healthy kidneys rapidly reach a steady state in less than 1 min, significantly shorter than the commonly used constant infusion without an initial bolus. The nonlinear CEUS signal intensities of kidney cortex showed less than 20% variation between mice. We used a custom program to acquire the CEUS perfusion images over a 3D volume that included the tumor and a kidney. We used the kidney as a reference organ to normalize whole tumor perfusion data. We fitted the log-normal perfusion model to estimate perfusion parameters for individual tumors. Our perfusion quantification over the entire tumor volume represents tumor perfusion more accurately than the commonly used methods based on a single 2D plane without a reference organ. Our approach provides population estimates of blood perfusion based on properly normalized estimates of individual blood perfusion parameters. Citation Format: Suresh Thiagarajan, Abbas Shirinifard, Megan O. Jacus, Abigail D. Davis, Yogesh T. Patel, Stacy L. Throm, Vinay Daryani, Clinton F. Stewart, Andras Sablauer. Quantification of tumor blood perfusion of an orthotopic mouse model of neuroblastoma using nonlinear contrast-enhanced ultrasound imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1496. doi:10.1158/1538-7445.AM2015-1496

Abstract 4519: Development of a whole body physiologically-based pharmacokinetic (PBPK) model with individualized tumor compartment for topotecan (TPT) in mice bearing neuroblastoma (NB)

Intratumoral pharmacokinetic (PK) and pharmacodynamic (PD) heterogeneity contribute to variability in NB tumor response to chemotherapy and can be responsible for tumor relapse. Herein we propose to develop a whole body PBPK model with an individualized tumor compartment to derive individual tumor specific concentration-time profiles for the NB standard of care drug TPT. This model can then relate intratumoral heterogeneity in tumor blood flow to PD response and antitumor effects. PK studies of TPT (0.6, 1.25, 5, and 20 mg/kg, IV bolus) will be performed in CD1 nude mice (n = 3 mice/time point) bearing orthotopic NB (NB5) xenograft. Blood samples will be collected at predetermined time points using cardiac puncture, and plasma separated and stored until analysis. Animals will be perfused using saline solution to remove residual blood, and tissue samples including tumor, muscle, adipose, bone, liver, gallbladder, kidney, spleen, lungs, brain, heart, duodenum, and large intestine collected. TPT concentrations in plasma and tissue homogenate samples will be quantified using a validated HPLC fluorescence spectrophotometry method. Tumor samples will be divided into two sections each, one for TPT quantification and one for immunohistochemistry of PD markers for DNA damage (γ-H2AX) and apoptosis (CASP3). A cohort of mice will be used to quantify tumor blood flow using contrast-enhanced ultrasound (CEUS) using MicroMarker® microbubbles prior to dosing the mice for the PK study. TPT plasma and tissue concentration-time data will be used to develop the whole-body PBPK model with an individualized tumor compartment using NONMEM. Individual tumor perfusion data obtained using CEUS will be combined with the PBPK model to derive tumor specific concentration-time profiles. A preliminary study conducted in non-tumor bearing mice receiving TPT 5 mg/kg showed that TPT plasma and tissue concentration-time data were reasonably described by our PBPK model. As expected from our previous studies, the brain tissue was found to have the lowest exposure to TPT with a brain to plasma partition coefficient (Kp,brain ∼ 8%). We also observed high permeability of TPT (Kp > 1) into the gallbladder, duodenum, large intestine, spleen, liver and kidney. In future we will study the correlations between individual tumor concentrations based on our comprehensive PBPK model and γ-H2AX and CASP3 activity. Citation Format: Yogesh T. Patel, Megan O. Jacus, Abbas Shirinifard, Abigail D. Davis, Suresh Thiagarajan, Stacy L. Throm, Vinay M. Daryani, Andras Sablauer, Clinton F. Stewart. Development of a whole body physiologically-based pharmacokinetic (PBPK) model with individualized tumor compartment for topotecan (TPT) in mice bearing neuroblastoma (NB). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4519. doi:10.1158/1538-7445.AM2015-4519

Abstract 4645: Clofarabine, a potent anticancer compound with limited penetration in an orthotopic murine model of ependymoma

Clofarabine, a deoxyadenosine analog, was a potent hit in our in vitro high-throughput screening against murine ependymoma neurospheres. To prioritize clofarabine for further preclinical efficacy studies, we evaluated the plasma pharmacokinetic (PK) disposition and central nervous system penetration in a murine model of ependymoma. A plasma PK study of clofarabine (45 mg/kg IP) was performed using CD1 nude mice bearing ependymoma cortical allographs (Ink4a/Arf-null + RTBDN) to obtain initial plasma PK parameters. These estimates were used to derive D-optimal plasma sampling time-points (e.g., 0.25, 2.5, and 5 hr) for cerebral microdialysis studies. Comparison of the clofarabine systemic exposure obtained from the plasma PK study and that simulated from pediatric patients using a published population PK model (Bonate, Cancer Chemotherap Pharmacol, 2011) suggested a dosage of 30 mg/kg in mice would be equivalent to a pediatric dosage of 180 mg/m2 given as a 2 hr infusion. Cerebral microdialysis was applied in CD1 nude mice bearing ependymoma cortical allographs (Ink4a/Arf-null + RTBDN), which permitted repeated in situ sampling of clofarabine tumor extracellular fluid (tECF). A microdialysis probe (BASi; 1 mm membrane) was introduced into the tumor through a cannula inserted during tumor cell implantation. After microdialysis probe equilibration, 7 mice were dosed with 30 mg/kg of clofarabine IP. In each mouse, serial plasma samples were collected at 0.25, 2.5, and 5 h post-dose, and tECF dialysate fractions were collected over 60 min intervals for up to 5 h post-dose. To measure clofarabine in both plasma and tECF, a robust, sensitive LC-MS/MS method was developed and validated. Both within-day and between-day precision (%CV) were ≤ 5.1% and accuracy ranged from 86% to 109%. A two-compartment model with absorption and tumor compartments linked to a central compartment was fitted to plasma and tECF concentration-time data using a nonlinear mixed effects modeling approach (NONMEM 7.2.0). For modeling purposes, the volume of the tECF compartment was fixed to published values. Unbound fraction of clofarabine in murine plasma was 0.82 ± 0.14. The model derived area under unbound concentration-time curve (AUCu,0-8) values for 30 and 45 mg/kg dosages were 5185 ± 550 µg/L*hr and 7677 ± 699 µg/L*hr, respectively. Clofarabine was absorbed rapidly from the peritoneal cavity with Tmax (time to reach maximum concentration) value of 0.33 ± 0.17 hr. Tumor to plasma partition coefficient (Kpt,uu: ratio of tumor to plasma AUCu,0-inf) of clofarabine was 0.12 ± 0.05. The model predicted mean tECF clofarabine concentrations were below the in vitro 1-hr IC50 (1.34 µM) for ependymoma neurospheres. In summary, we have shown the tECF clofarabine concentrations were below that required for antitumor effect in our in vitro washout studies, thus we have not pursued clofarabine for detailed efficacy studies in our preclinical pipeline. Citation Format: Yogesh T. Patel, Megan O. Jacus, Abigail D. Davis, Pradeep Vuppala, Jason D. Dapper, Burgess B. Freeman, Nidal Boulos, Stacy L. Throm, Richard J. Gilbertson, Clinton F. Stewart. Clofarabine, a potent anticancer compound with limited penetration in an orthotopic murine model of ependymoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4645. doi:10.1158/1538-7445.AM2014-4645