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Probability of target attainment (PTA) analyses were conducted to support the recommended ceftolozane/tazobactam dosing regimens, adjusted for renal function, in patients with hospital-acquired/ventilator-associated bacterial pneumonia (HABP/VABP). Previously published population pharmacokinetic models describing the disposition of ceftolozane and tazobactam in plasma and epithelial lining fluid (ELF) in patients with HABP/VABP were used to simulate ceftolozane and tazobactam concentration-time profiles in plasma and ELF over the course of 14 days. The simulations were conducted for patients with normal renal function and for patients receiving adjusted doses for mild, moderate, and severe renal impairment. PTA was calculated using established pharmacokinetic/pharmacodynamic targets for ceftolozane and tazobactam. Across renal function groups, plasma PTA was 100% for ceftolozane and99% for tazobactam; ELF PTA was99% for ceftolozane and87% for tazobactam. These results provided support for the currently recommended ceftolozane/tazobactam dosing regimens for HABP/VABP, which were efficacious and well tolerated in the Ceftolozane-Tazobactam Versus Meropenem for Treatment of Nosocomial Pneumonia (ASPECT-NP) trial.

ASPECT-NP, a phase 3 trial of ceftolozane/tazobactam in hospital-acquired/ventilator-associated bacterial pneumonia (HABP/VABP), excluded patients with end-stage renal disease (ESRD). A modeling/simulation approach was undertaken to inform optimal dosing in this population, using previously developed ceftolozane and tazobactam population pharmacokinetic models informed by data from 16 clinical studies. Stochastic simulations were performed using NONMEM to support dose justification. Probability of target attainment (PTA) simulations in plasma and epithelial lining fluid were conducted using a 14-day treatment, with hemodialysis every other weekday for a high-dose (4X), middle-dose (3X), or low-dose (2X) regimen, where X was the recommended dose in patients with complicated intra-abdominal infection/complicated urinary tract infection and ESRD (500 mg/250 mg ceftolozane/tazobactam loading dose and 100 mg/50 mg ceftolozane/tazobactam maintenance dose administered by 1-hour infusion every 8 hours). PTA was determined using established pharmacokinetic/pharmacodynamic targets: ceftolozane, 30% of the interdose interval (8 hours) in which free ceftolozane concentration exceeded the minimum inhibitory concentration value of 4 µg/mL; tazobactam, 20% of the interdose interval in which free tazobactam concentration exceeded 1 µg/mL. Plasma PTA was90% for both agents for all 3 regimens. Plasma ceftolozane exposures at the high-dose regimen exceeded those from phase 3 study experience. Epithelial lining fluid PTA was90% for high- and middle-dose regimens but was80% for tazobactam on dialysis days at the low-dose regimen. For patients with HABP/VABP and ESRD requiring intermittent hemodialysis, the middle-dose regimen of 1.5 g/0.75 g ceftolozane/tazobactam loading + 300 mg/150 mg maintenance every 8 hours by 1-hour infusion is recommended.

Supplementary Methods

Tumor proliferation measured as Ki67 index in (A) mSEP-CR(-)RTBDNb and (B) mCPC treated with the indicated dose and route of administration of gemcitabine.

information on drugs used in preclinical studies

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.

Supplementary Material and Methods : Plasma pharmacokinetics of topotecan with and without the ABCG2 inhibitor Ko143. Supplementary Figure S1: RNA expression of ABC transporters in human and mouse Group3 medulloblastoma. Supplementary Figure S2: Abcg2 protein expression in mouse bone marrow from Abcg2-null mice and Group3 medulloblastoma tumor cells. Supplementary Figure S3: Dose-response of chemotherapeutic agents alone or with two Abcg2 inhibitors, Ko143 or Tariquidar. Supplementary Figure S4: Weight measurement and Abcg2 protein expression in mouse Group3 medulloblastoma during treatment. Supplementary Table 1: Affymetrix gene array analysis of genes in the porphyrin pathway in granule neuron progenitors (GNPs) and Mouse Group3 medulloblastoma tumor cells. Supplementary Table 2: Chemotherapeutic drugs tested on mouse Group3 medulloblastoma tumor cells. Supplementary Table 3: Plasma PK paramaters of topotecan after administration as single agent or in combination with the Abcg2 inhibitor Ko143.

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. Cancer Res; 75(18); 3879–89. ©2015 AACR.

An exposure–efficacy analysis of the phase 3 ASPECT-NP trial was performed to evaluate the relationship between plasma exposure of ceftolozane and tazobactam and efficacy endpoints (primary: 28-day all-cause mortality; key secondary: clinical cure at test-of-cure visit) in adult participants with hospital-acquired or ventilator-associated bacterial pneumonia (HABP/VABP). Participants ( N = 231) from the ceftolozane/tazobactam treatment group in the intention-to-treat population who had pharmacokinetic data available and relevant baseline lower respiratory tract (LRT) pathogen(s) susceptibility data were included.

Ceftolozane/tazobactam (C/T) is a combination of a novel cephalosporin with tazobactam, recently approved for the treatment of hospital‐acquired and ventilator‐associated pneumonia. The plasma pharmacokinetics (PK) of a 3‐g dose of C/T (2 g ceftolozane and 1 g tazobactam) administered via a 1‐hour infusion every 8 hours in adult patients with nosocomial pneumonia (NP) were evaluated in a phase 3 study (ASPECT‐NP; NCT02070757). The present work describes the development of population PK models for ceftolozane and tazobactam in plasma and pulmonary epithelial lining fluid (ELF). The concentration‐time profiles of both agents were well characterized by 2‐compartment models with zero‐order input and first‐order elimination. Consistent with the elimination pathway, renal function estimated by creatinine clearance significantly affected the clearance of ceftolozane and tazobactam. The central volumes of distribution for both agents and the peripheral volume of distribution for tazobactam were approximately 2‐fold higher in patients with pneumonia compared with healthy participants. A hypothetical link model was developed to describe ceftolozane and tazobactam disposition in ELF in healthy participants and patients with pneumonia. Influx (from plasma to the ELF compartment) and elimination (from the ELF compartment) rate constants were approximately 97% lower for ceftolozane and 52% lower for tazobactam in patients with pneumonia versus healthy participants. These population PK models adequately described the plasma and ELF concentrations of ceftolozane and tazobactam, thus providing a foundation for further modeling and simulation, including the probability of target attainment assessments to support dose recommendations of C/T in adult patients with NP.

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.

Selumetinib (AZD6244, ARRY-142886), a mitogen activated protein kinases (MEK1 and 2) inhibitor, has been granted orphan drug designation for differentiated thyroid cancer. The primary aim of this analysis was to characterize the population pharmacokinetics of selumetinib and its active metabolite N-desmethyl-selumetinib in patients with cancer. Concentration-time data from adult and pediatric clinical trials were pooled to develop a population pharmacokinetic model using a sequential approach where selumetinib and N-desmethyl-selumetinib data were modeled separately. A sequential zero- and first-order absorption with lag time with a two-compartment model for selumetinib and a two-compartment model for N-desmethyl-selumetinib best described the concentration-time data. Intrapatient variability in absorption was higher than interpatient variability. The apparent drug clearance (CL/F) from the central compartment was 13.5 L/hr (RSE 4.9%). Significant covariates for CL/F were age, alanine aminotransferase, and body surface area. This study confirms that flat dosing is appropriate in adults, whereas body-surface area based dosing should be used in pediatric patients.

AIMS: No population pharmacokinetic studies of high‐dose methotrexate (HDMTX) have been conducted in infants with brain tumours, which are a vulnerable population. The aim of this study was to evaluate HDMTX disposition in these children to provide a rational basis for MTX dosing. METHODS: Patients received 4 monthly courses of HDMTX (5 g/m(2) or 2.5 g/m(2) for infants aged ≤31 days) as a 24‐h infusion. Serial samples were analysed for MTX by an enzyme immunoassay method. Pharmacokinetic parameters were estimated using nonlinear mixed effects population modelling. Demographics, concomitant medications and genetic polymorphisms were considered as pharmacokinetic covariates while MTX exposure and patient age were considered as covariates for Grade 3 and 4 toxicities. RESULTS: The population pharmacokinetics of HDMTX were estimated in 178 patients (age range 0.02–4.7 years) in 648 courses. The population clearance and volume were 90 mL/min/m(2) and 14.4 L/m(2), respectively. Significant covariates on body surface area adjusted MTX clearance included estimated glomerular filtration rate and co‐treatment with dexamethasone or vancomycin. No significant association was observed between MTX toxicity and MTX exposure, patient age, leucovorin dosage or duration. MTX clearance in infants ≤31 days at enrolment was 44% lower than in older infants, but their incidence of toxicity was not higher since they also received a lower MTX dosage. CONCLUSIONS: By aggressively following institutional clinical guidelines, HDMTX‐related toxicities were low, and using covariates from the population pharmacokinetic model enabled the calculation of a rational dosage for this patient population for future clinical trials.

PURPOSE: Cyclophosphamide is widely used to treat children with medulloblastoma; however, little is known about its brain penetration. We performed cerebral microdialysis to characterize the brain penetration of cyclophosphamide (130 mg/kg, IP) and its metabolites [4-hydroxy-cyclophosphamide (4OH-CTX) and carboxyethylphosphoramide mustard (CEPM)] in non-tumor bearing mice and mice bearing orthotopic Group 3 medulloblastoma. METHODS: A plasma pharmacokinetic study was performed in non-tumor-bearing CD1-nude mice, and four cerebral microdialysis studies were performed in non-tumor-bearing (M1 and M3) and tumor-bearing mice (M2 and M4). Plasma samples were collected up to 6-hours post-dose, and extracellular fluid (ECF) samples were collected over 60-minute intervals for 24-hours post-dose. To stabilize and quantify 4OH-CTX, a derivatizing solution was added in blood after collection, and either directly in the microdialysis perfusate (M1 and M2) or in ECF collection tubes (M3 and M4). Plasma/ECF cyclophosphamide and CEPM, and 4OH-CTX concentrations were separately measured using different LC-MS/MS methods. RESULTS: All plasma/ECF concentrations were described using a population-based pharmacokinetic model. Plasma exposures of cyclophosphamide, 4OH-CTX, and CEPM were similar across studies (mean AUC=112.6, 45.6, and 80.8 µmol∙hr/L). Hemorrhage was observed in brain tissue when the derivatizing solution was in perfusate compared with none when in collection tubes, which suggested potential sample contamination in studies M1 and M2. Model-derived unbound ECF to plasma partition coefficients (Kp,uu) were calculated to reflect CNS penetration of the compounds. Lower cyclophosphamide Kp,uu was obtained in tumor-bearing mice versus non-tumor bearing mice (mean 0.15 versus 0.22, p=0.019). No differences in Kp,uu were observed between these groups for 4OH-CTX and CEPM (overall mean 0.10 and 0.07). CONCLUSIONS: Future studies will explore potential mechanisms at the brain-tumor barrier to explain lower cyclophosphamide brain penetration in tumor-bearing mice. These results will be used to further investigate exposure-response relationships in medulloblastoma xenograft models.

Ceftolozane-tazobactam, a combination of the novel antipseudomonal cephalosporin ceftolozane and the well-established extended-spectrum β-lactamase inhibitor tazobactam, is approved for treating complicated urinary tract infections (cUTI) and complicated intra-abdominal infections (cIAI) in adults. To determine doses likely to be safe and efficacious in phase 2 pediatric trials for the same indications, single-dose ceftolozane-tazobactam plasma pharmacokinetic data from a recently completed phase 1 trial in pediatric patients (birth to

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

Despite significant improvement in outcomes for patients with hematologic malignancies and solid tumors over the past 10 years, patients with primary or metastatic brain tumors continue to have a poor prognosis. A primary reason for this is the inability of many chemotherapeutic drugs to penetrate into the brain and brain tumors at concentrations high enough to exert an antitumor effect because of unique barriers and efflux transporters. Several studies have been published recently examining the central nervous system pharmacokinetics of various anticancer drugs in patients with primary and metastatic brain tumors. To summarize recent advances in the field, this review critically presents studies published within the last 9 years examining brain and cerebrospinal fluid penetration of clinically available anticancer agents for patients with central nervous system tumors.

An LC-MS/MS method to measure ribociclib in mouse plasma and Ringer’s solution was successfully developed and validated. Reverse phase chromatography was performed with gradient elution using C18 (100A, 50x4.6 mm, 3μ) and C8-A (50x 2.0 mm, 5 μ) columns for plasma and Ringer’s samples, respectively. Mouse plasma samples were extracted using solid phase extraction method, whereas no extraction was required for the Ringer’s 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 62.5–10000 ng/ml for plasma method and 0.1–100 ng/ml for Ringer’s 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 Ringer’s samples were 62.5 ng/ml (S/N > 30) and 0.1 ng/ml (S/N > 13), respectively, whereas the limit of detection (LOD) was 6.9 ng/ml (S/N > 7) and 0.05 ng/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.

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.

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.

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. Cancer Res; 75(18); 3879–89. ©2015 AACR.

Clofarabine, a deoxyadenosine analog, was an active anticancer drug in our in vitro high-throughput screening against mouse ependymoma neurospheres. To characterize the clofarabine disposition in mice for further preclinical efficacy studies, we evaluated the plasma and central nervous system disposition in a mouse model of ependymoma. A plasma pharmacokinetic study of clofarabine (45 mg/kg, IP) was performed in CD1 nude mice bearing ependymoma to obtain initial plasma pharmacokinetic parameters. These estimates were used to derive D-optimal plasma sampling time points for cerebral microdialysis studies. A simulation of clofarabine pharmacokinetics in mice and pediatric patients suggested that a dosage of 30 mg/kg IP in mice would give exposures comparable to that in children at a dosage of 148 mg/m2. Cerebral microdialysis was performed to study the tumor extracellular fluid (ECF) disposition of clofarabine (30 mg/kg, IP) in the ependymoma cortical allografts. Plasma and tumor ECF concentration–time data were analyzed using a nonlinear mixed effects modeling approach. The median unbound fraction of clofarabine in mouse plasma was 0.79. The unbound tumor to plasma partition coefficient (K pt,uu: ratio of tumor to plasma AUCu,0–inf) of clofarabine was 0.12 ± 0.05. The model-predicted mean tumor ECF clofarabine concentrations were below the in vitro 1-h IC50 (407 ng/mL) for ependymoma neurospheres. Thus, our results show the clofarabine exposure reached in the tumor ECF was below that associated with an antitumor effect in our in vitro washout study. Therefore, clofarabine was de-prioritized as an agent to treat ependymoma, and further preclinical studies were not pursued.

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 present an individualized predictive three-dimensional (3D) model of tumor response to topotecan (TPT) for a patient-derived orthotopic xenograft model of pediatric NB5 neuroblastoma. This model is integrated with an individualized physiologically-based pharmacokinetic (PBPK) model of TPT and accounts for the effects of inter- and intra-tumor heterogeneities in blood perfusion on tumor response. This model uses a set of 3D reaction-diffusion equations to simulate transport of TPT from blood vessels into the tumor tissue and its flux in and out of intracellular space. The transport model takes inputs from three spatial scales (systemic, tissue and cells) 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, b) 3D blood perfusion map of the individual tumor from contrast enhanced ultrasound (CEUS) using VEVO 2100 imaging system, and c) in vitro TPT cellular uptake and efflux kinetics from two-photon imaging. The transport model also gives feedback to the PBPK model. We use in vitro pharmacodynamics (PD) experiments with NB5 cells exposed to TPT to derive probabilistic PD-rules for drug effects (γ-H2AX response). Based on these rules and exposure maps predicted by the transport model, we then compute probabilities of effects for the entire tumor volume. We validate the predicted drug effect maps by comparing them to observed effect data (from three dosage levels) measured by immunohistochemistry marker for γ-H2AX from the same tumor (location matched serial sections) using spatial correlation techniques. Citation Format: Abbas Shirinifard, Suresh Thiagarajan, Yogesh T. Patel, Abigail D. Davis, Megan O. Jacus, Jessica Roberts, Clinton F. Stewart, András Sablauer. An individualized predictive 3D model of tumor response to topotecan for a patient-derived orthotopic xenograft model of pediatric neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A33.

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, András 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.

This work was supported by grants from the NIH (R.J.G., P01CA96832 and P30CA021765), the American Lebanese Syrian Associated Charities and Cancer Research UK.

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.

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, András 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

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

An ongoing trial of risk-adapted therapy for infants and young children < 3 yrs old diagnosed with brain tumors includes IV CTX (NCT00602667). One aim is to describe the pharmacokinetics (PK) of CTX, and 2 of its metabolites, the inactive carboxyethylphosphoramide mustard (CEPM) and the active 4-hydroxycyclophosphamide (4-OH CTX) in children < 3 yrs old. This report describes the disposition of CTX and metabolites in infants ≤ 1 yr old. Induction therapy includes 1.5 gm/m2 CTX IV over 1 hr on day 9 of a 28 day course. PK studies for CTX were performed in consenting patients and samples were collected prior to drug infusion, at the end of the infusion (EOI), and 3, 6, and 24 h after EOI. Blood was processed to plasma for determination of CTX and CEPM using a LC-MS/MS method. For 4-OH CTX, samples were immediately derivatized using phenylhydrazine to ensure 4-OH CTX stability until analysis using a LC-MS/MS method. A model with one compartment for CTX and each metabolite was fit to the data using nonlinear mixed effects modeling. In 27 infants ≤ 1 yr, 133 samples were obtained and analyzed for CTX, CEPM, and 4-OH CTX. The median (range) age, BSA, and weight were 6.9 months (0.2 - 12 months), 0.4 m2 (0.2 - 0.6 m2), and 8.1 kg (3.6 - 14.1 kg), respectively. As depicted in the Table below, CTX and 4-OH CTX AUC were 50% and 30% higher in the youngest age group, respectively. Our preliminary analysis showed inclusion of age as a covariate on CTX clearance to 4-OH CTX improved model fits, decreasing the variability observed in 4-OH CTX AUC. Since high 4-OH CTX exposure is associated with increased toxicities, our results indicate the need for better models to accurately simulate 4-OH CTX exposures in infants with the goal of developing rational dosing approaches for CTX in this population. This is the first analysis of CTX and metabolite PK in infants ≤ 1 yr old with brain tumors. Future studies will include a full covariate analysis and an evaluation of exposure-toxicity relationships associated with CTX and metabolites. Age group (months)# ptsCTX AUC0-∞ (μM*hr)4-OH CTX AUC0-∞ (μM*hr)0-363556 (2423-6170)†227 (158-263)†3-652854 (2174-3312)174 (170-237)6-972454 (2356-3281)179 (138-220)9-1292461 (1793-2735)175 (136-210)†Kruskal-Wallis, p Citation Format: Vinay M. Daryani, Thandranese S. Owens, K. Elaine Harstead, Yogesh T. Patel, David C. Turner, Stacy L. Throm, John C. Panetta, Amar Gajjar, Clinton F. Stewart. Age dependent disposition of cyclophosphamide (CTX) and metabolites in infants ≤ 1 year old with brain tumors. [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 4526. doi:10.1158/1538-7445.AM2015-4526

Medulloblastoma is the most common malignant childhood brain tumor, consisting of four molecularly and clinically distinct subtypes. Recent work has advanced our understanding about the basic pathological processes, identifying unique mutations and anatomical locations that contribute to medulloblastoma subtype heterogeneity. Recognizing how subtype-specific genetic alterations dictate treatment response is critical to advancing treatments for all patients. The central nervous system (CNS) contains a unique vascular system that tightly regulates exchange between the blood and brain, termed the blood brain barrier (BBB). How brain tumors specify BBB properties in newly formed blood vessels, and if this dictates tumor behavior remains unanswered. Here we show that WNT-subtype medulloblastomas create a microenvironment that inhibits BBB formation, producing tumors with a highly permeable aberrant vascular network. We found human and mouse WNT-subtype tumors display increased vascularization and hemorrhaging, along with decreased expression of endothelial BBB associated genes. Fenestrated blood vessels, normally associated with peripheral organs, are highly enriched in WNT-subtype tumors, allowing unrestricted exchange of substances between blood and tumor. Canonical Wnt signaling is essential for normal CNS BBB formation and maintenance. WNT-subtype tumors express high levels of secreted Wnt antagonists, creating a microenvironment that is inhibitory to canonical Wnt signaling and BBB formation in neighboring endothelial cells. Differences in BBB status between WNT and SHH-subtype tumors result in differential in vivo treatment response and drug penetration, suggesting the abnormal vasculature might contribute to the excellent clinical prognosis associated with WNT-subtype patients. Our data offer insight into brain tumor-vascular BBB specification, and should benefit ongoing efforts to improve response, survival, and quality of life for all medulloblastoma patients.

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

Chemotherapies active in preclinical studies frequently fail in the clinic due to lack of efficacy, which limits progress for rare cancers since only small numbers of patients are available for clinical trials. Thus, a preclinical drug development pipeline was developed to prioritize potentially active regimens for pediatric brain tumors spanning from in vitro drug screening, through intracranial and intra-tumoral pharmacokinetics to in vivo efficacy studies. Here, as an example of the pipeline, data are presented for the combination of 5-fluoro-2′-deoxycytidine and tetrahydrouridine in three pediatric brain tumor models. The in vitro activity of nine novel therapies was tested against tumor spheres derived from faithful mouse models of Group 3 medulloblastoma, ependymoma, and choroid plexus carcinoma. Agents with the greatest in vitro potency were then subjected to a comprehensive series of in vivo pharmacokinetic (PK) and pharmacodynamic (PD) studies culminating in preclinical efficacy trials in mice harboring brain tumors. The nucleoside analog 5-fluoro-2′-deoxycytidine (FdCyd) markedly reduced the proliferation in vitro of all three brain tumor cell types at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in brain tumors necessary to inhibit tumor cell proliferation, but no tumor displayed a significant in vivo therapeutic response. Despite promising in vitro activity and in vivo PK properties, FdCyd is unlikely to be an effective treatment of pediatric brain tumors, and therefore was deprioritized for the clinic. Our comprehensive and integrated preclinical drug development pipeline should reduce the attrition of drugs in clinical trials. Electronic supplementary material The online version of this article (doi:10.1007/s11060-015-1965-0) contains supplementary material, which is available to authorized users.