Yogesh T. Patel

Medulloblastoma Genotype Dictates Blood Brain Barrier Phenotype.

The childhood brain tumor, medulloblastoma, includes four subtypes with very different prognoses. Here, we show that paracrine signals driven by mutant β-catenin in WNT-medulloblastoma, an essentially curable form of the disease, induce an aberrant fenestrated vasculature that permits the accumulation of high levels of intra-tumoral chemotherapy and a robust therapeutic response. In contrast, SHH-medulloblastoma, a less curable disease subtype, contains an intact blood brain barrier, rendering this tumor impermeable and resistant to chemotherapy. The medulloblastoma-endothelial cell paracrine axis can be manipulated in vivo, altering chemotherapy permeability and clinical response. Thus, medulloblastoma genotype dictates tumor vessel phenotype, explaining in part the disparate prognoses among medulloblastoma subtypes and suggesting an approach to enhance the chemoresponsiveness of other brain tumors.

Deriving therapies for children with primary CNS tumors using pharmacokinetic modeling and simulation of cerebral microdialysis data.

The treatment of children with primary central nervous system (CNS) tumors continues to be a challenge despite recent advances in technology and diagnostics. In this overview, we describe our approach for identifying and evaluating active anticancer drugs through a process that enables rational translation from the lab to the clinic. The preclinical approach we discuss uses tumor subgroup-specific models of pediatric CNS tumors, cerebral microdialysis sampling of tumor extracellular fluid (tECF), and pharmacokinetic modeling and simulation to overcome challenges that currently hinder researchers in this field. This approach involves performing extensive systemic (plasma) and target site (CNS tumor) pharmacokinetic studies. Pharmacokinetic modeling and simulation of the data derived from these studies are then used to inform future decisions regarding drug administration, including dosage and schedule. Here, we also present how our approach was used to examine two FDA approved drugs, simvastatin and pemetrexed, as candidates for new therapies for pediatric CNS tumors. We determined that due to unfavorable pharmacokinetic characteristics and insufficient concentrations in tumor tissue in a mouse model of ependymoma, simvastatin would not be efficacious in further preclinical trials. In contrast to simvastatin, pemetrexed was advanced to preclinical efficacy studies after our studies determined that plasma exposures were similar to those in humans treated at similar tolerable dosages and adequate unbound concentrations were found in tumor tissue of medulloblastoma-bearing mice. Generally speaking, the high clinical failure rates for CNS drug candidates can be partially explained by the fact that therapies are often moved into clinical trials without extensive and rational preclinical studies to optimize the transition. Our approach addresses this limitation by using pharmacokinetic and pharmacodynamic modeling of data generated from appropriate in vivo models to support the rational testing and usage of innovative therapies in children with CNS tumors.

ABCG2 Transporter Expression Impacts Group 3 Medulloblastoma Response to Chemotherapy

While a small number of plasma membrane ABC transporters can export chemotherapeutic drugs and confer drug resistance, it is unknown whether these transporters are expressed or functional in less therapeutically tractable cancers such as Group 3 (G3) medulloblastoma. Herein we show that among this class of drug transporters, only ABCG2 was expressed at highly increased levels in human G3 medulloblastoma and a mouse model of this disease. In the mouse model, Abcg2 protein was expressed at the plasma membrane where it functioned as expected on the basis of export of prototypical substrates. By screening ABC substrates against mouse G3 medulloblastoma tumorspheres in vitro, we found that Abcg2 inhibition could potentiate responses to the clinically used drug topotecan, producing a more than 9-fold suppression of cell proliferation. Extended studies in vivo in this model confirmed that Abcg2 inhibition was sufficient to enhance antiproliferative responses to topotecan, producing a significant survival advantage compared with subjects treated with topotecan alone. Our findings offer a preclinical proof of concept for blockade of ABCG2 transporter activity as a strategy to empower chemotherapeutic responses in G3 medulloblastoma.

Translational Pharmacokinetic-Pharmacodynamic Modeling and Simulation: Optimizing 5-Fluorouracil Dosing in Children With Pediatric Ependymoma

We previously investigated novel therapies for pediatric ependymoma and found 5‐fluorouracil (5‐FU) i.v. bolus increased survival in a representative mouse model. However, without a quantitative framework to derive clinical dosing recommendations, we devised a translational pharmacokinetic‐pharmacodynamic (PK‐PD) modeling and simulation approach. Results from our preclinical PK‐PD model suggested tumor concentrations exceeded the 1‐hour target exposure (in vitro IC90), leading to tumor growth delay and increased survival. Using an adult population PK model, we scaled our preclinical PK‐PD model to children. To select a 5‐FU dosage for our clinical trial in children with ependymoma, we simulated various 5‐FU dosages for tumor exposures and tumor growth inhibition, as well as considering tolerability to bolus 5‐FU administration. We developed a pediatric population PK model of bolus 5‐FU and simulated tumor exposures for our patients. Simulations for tumor concentrations indicated that all patients would be above the 1‐hour target exposure for antitumor effect.

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.

Establishing a Preclinical Multidisciplinary Board for Brain Tumors

Purpose: Curing all children with brain tumors will require an understanding of how each subtype responds to conventional treatments and how best to combine existing and novel therapies. It is extremely challenging to acquire this knowledge in the clinic alone, especially among patients with rare tumors. Therefore, we developed a preclinical brain tumor platform to test combinations of conventional and novel therapies in a manner that closely recapitulates clinic trials. Experimental Design: A multidisciplinary team was established to design and conduct neurosurgical, fractionated radiotherapy and chemotherapy studies, alone or in combination, in accurate mouse models of supratentorial ependymoma (SEP) subtypes and choroid plexus carcinoma (CPC). Extensive drug repurposing screens, pharmacokinetic, pharmacodynamic, and efficacy studies were used to triage active compounds for combination preclinical trials with “standard-of-care” surgery and radiotherapy. Results: Mouse models displayed distinct patterns of response to surgery, irradiation, and chemotherapy that varied with tumor subtype. Repurposing screens identified 3-hour infusions of gemcitabine as a relatively nontoxic and efficacious treatment of SEP and CPC. Combination neurosurgery, fractionated irradiation, and gemcitabine proved significantly more effective than surgery and irradiation alone, curing one half of all animals with aggressive forms of SEP. Conclusions: We report a comprehensive preclinical trial platform to assess the therapeutic activity of conventional and novel treatments among rare brain tumor subtypes. It also enables the development of complex, combination treatment regimens that should deliver optimal trial designs for clinical testing. Postirradiation gemcitabine infusion should be tested as new treatments of SEP and CPC. Clin Cancer Res; 24(7); 1654–66. ©2018 AACR.

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 IA21: Pediatric medulloblastoma: Drug screens and preclinical studies

Medulloblastoma (MB) is the most common malignant brain tumor that develops in the cerebellum. MB occurs mostly in children between the ages of 3-7 years. Human MBs are classified into four subgroups: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 (G3) and G4, each of which with distinct molecular signatures. SHH MBs with MYCN amplification and TP53 mutations and G3 MBs represent the most aggressive subgroups and the least curable with current therapeutic regimen. Whole genome sequencing and gene expression analysis of human G3 MBs demonstrated C-MYC (MYC) overexpression, from gene amplification in ~17% of cases, stem like properties and high levels of EZH2 and histone 3 lysine 27 trimethylation (H3K27me3) marks. G3 MBs have few, recurrent oncogenic point mutations that might be targeted therapeutically but contain large chromosomal copy number changes and an aberrant epigenome. We developed a mouse model of G3 MB using orthotopic transplantation as well as in utero electroporation approaches by enforcing the expression of Myc with loss of p53 function that shares many characteristics of its human counterpart including high levels of Ezh2 and H3K27me3 marks. A high throughput screen of FDA-approved drugs on mouse G3 MB neuropsheres identified Pemetrexed and Gemcitabine that, in combination with standard of care chemotherapy, greatly increased the survival of mice bearing mouse or human G3 MBs. Because genetic alterations found in human G3 MBs occur in epigenetic regulators, we tested several drugs that target the epigenome to re-establish normal gene expression profiles, including those of tumor suppressor genes. Using a preclinical drug development pipeline, we evaluated nine compounds that are believed to modulate the epigenome. Of the nine compounds tested we found that the nucleoside analog 5-fluoro-2′-deoxycytidine (FdCyd) markedly reduced the proliferation of G3 MBs in vitro at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in the tECF (tumor extra cellular fluid) necessary to inhibit tumor cell proliferation in vivo. However, despite promising in vitro activity and in vivo PK properties, FdCyd was completely ineffective in treating mouse and human G3 MBs in vivo. Our studies highlight the requirement for a comprehensive and integrated preclinical drug development pipeline with mouse and human medulloblastoma to identify the best candidate drug to be translated into the clinic and to avoid taking a drug forward that looks feasible in in vitro screens but which efficacy does not translate into an in vivo setting. Citation Format: Marie Morfouace, Yogesh Patel, Anang Shelat, Daisuke Kawauchi, Giles W. Robinson, Kip Guy, Richard J. Gilbertson, Clinton Stewart, Amar Gajjar, Martine F. Roussel. Pediatric medulloblastoma: Drug screens and preclinical studies. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA21.