Quantitative determination of liposomal irinotecan and SN-38 concentrations in plasma samples from children with solid tumors: Use of a cryoprotectant solution to enhance liposome stability.

Preclinical studies have demonstrated that liposomal irinotecan (CPT-11), a topoisomerase I inhibitor, has broad activity against adult cancers, including pancreatic, gastric, colon, lung, glioma, ovarian, and breast cancer. Encapsulation of irinotecan into liposomes can modify its pharmacokinetic properties dramatically. Also, the pharmacokinetic profiles of liposomal drug formulations are not fully understood; thus, bioanalytical methods are needed to separate and quantify nonencapsulated vs. encapsulated concentrations. In this study, two robust, specific, and sensitive LC-MS/MS methods were developed and validated to separate and quantify the nonencapsulated CPT-11 (NE-CPT-11) from the sum-total CPT-11 (T-CPT-11) and its major metabolite, SN-38, in human plasma after intravenous administration of liposomal irinotecan. NE-CPT-11 and SN-38 were separated from plasma samples by using solid-phase extraction, and T-CPT-11 was measured by protein precipitation. The liposomal CPT-11 formulation was unstable during sample storage and handling, resulting in elevated NE-CPT-11 concentration. To improve the stability of liposomal CPT-11, a cryoprotectant solution was added to human plasma samples prior to storage and processing. CPT-11, SN-38, and their respective internal standards, CPT-11-d10 and SN-38-d3, were chromatographically separated on a reversed-phase C18 analytical column. The drugs were detected on a triple quadrupole mass spectrometer in the positive MRM ion mode by monitoring the transitions 587.3 > 124.1 (CPT-11) and 393.0 > 349.1 (SN-38). The calibration curves demonstrated a good fit across the concentration ranges of 10-5000 ng/mL for T-CPT-11, 2.5-250 ng/mL for NE-CPT-11, and 1-500 ng/mL for SN-38. The accuracy and precision were within the acceptable limits, matrix effects were nonsignificant, recoveries were consistent and reproducible, and the analytes were stable under all tested storage conditions. Finally, the LC-MS/MS methods were successfully applied in a phase I clinical pharmacokinetic study of nanoliposomal irinotecan (Onivyde®) in pediatric patients with recurrent solid malignancies or Ewing sarcoma.

Entinostat as a combinatorial therapeutic for rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. For the alveolar subtype (ARMS), the presence of the PAX3::FOXO1 fusion gene and/or metastases are strong predictors of poor outcome. Metastatic PAX3::FOXO1+ ARMS often responds to chemotherapies initially, only to subsequently relapse and become resistant with most patients failing to survive beyond 8 years post-diagnosis. No curative intent phase II or phase III clinical trial has been available for patients in the past 10 years (ARST0921). Thus, metastatic ARMS represents a significantly unmet clinical need. Chemotherapy resistance in ARMS has previously been attributed to PAX3::FOXO1-mediated cell cycle checkpoint adaptation, which is mediated by an HDAC3-SMARCA4-miR-27a-PAX3::FOXO1 circuit that can be disrupted by HDAC3 inhibition. In this study, we investigated the therapeutic efficacy of combining the epigenetic regulator entinostat, a Class I Histone Deacetylase (HDAC1-3) inhibitor, with RMS-specific chemotherapies in patient derived xenograft (PDX) models of RMS. We identified single agent, additive or synergistic relationships between relapse-specific chemotherapies and clinically relevant drug exposures of entinostat in three PAX3::FOXO1+ ARMS mouse models. This preclinical data provides further rationale for clinical investigation of entinostat, already known to be well tolerated in a pediatric phase I clinical trial (ADVL1513).

Population pharmacokinetics of methotrexate and 7-hydroxymethotrexate and delayed excretion in infants and young children with brain tumors.

PURPOSE: The objectives of this study were to develop a population pharmacokinetic model of methotrexate (MTX) and its primary metabolite 7-hydroxymethotrexate (7OHMTX) in children with brain tumors, to identify the sources of pharmacokinetic variability, and to assess whether MTX and 7OHMTX systemic exposures were related to toxicity. METHODS: Patients received 2.5 or 5 g/m2 MTX as a 24-hour infusion and serial samples were analyzed for MTX and 7OHMTX by an LC-MS/MS method. Pharmacokinetic parameters were estimated using nonlinear mixed-effects modeling. Demographics, laboratory values, and genetic polymorphisms were considered as potential covariates to explain the pharmacokinetic variability. Association between MTX and 7OHMTX systemic exposures and MTX-related toxicities were explored using random intercept logistic regression models. RESULTS: The population pharmacokinetics of MTX and 7OHMTX were adequately characterized using two-compartment models in 142 patients (median 1.91 y; age range 0.09 to 4.94 y) in 513 courses. The MTX and 7OHMTX population clearance values were 4.6 and 3.0 l/h/m2, respectively. Baseline body surface area and estimated glomerular filtration rate were significant covariates on both MTX and 7OHMTX plasma disposition. Pharmacogenetic genotypes were associated with MTX pharmacokinetic parameters but had only modest influence. No significant association was observed between MTX or 7OHMTX exposure and MTX-related toxicity. CONCLUSIONS: MTX and 7OHMTX plasma disposition were characterized for the first time in young children with brain tumors. No exposure-toxicity relationship was identified in this study, presumably due to aggressive clinical management which led to a low MTX-related toxicity rate.

Effect of Obesity on the Pharmacokinetics and Pharmacodynamics of Anticancer Agents.

An objective of the Precision Medicine Initiative, launched in 2015 by the US Food and Drug Administration and National Institutes of Health, is to optimize and individualize dosing of drugs, especially anticancer agents, with high pharmacokinetic and pharmacodynamic variability. The American Society of Clinical Oncology recently reported that 40% of obese patients receive insufficient chemotherapy doses and exposures, which may lead to reduced efficacy, and recommended pharmacokinetic studies to guide appropriate dosing in these patients. These issues will only increase in importance as the incidence of obesity in the population increases. This publication reviews the effects of obesity on (1) tumor biology, development of cancer, and antitumor response; (2) pharmacokinetics and pharmacodynamics of small-molecule anticancer drugs; and (3) pharmacokinetics and pharmacodynamics of complex anticancer drugs, such as carrier-mediated agents and biologics. These topics are not only important from a scientific research perspective but also from a drug development and regulator perspective. Thus, it is important to evaluate the effects of obesity on the pharmacokinetics and pharmacodynamics of anticancer agents in all categories of body habitus and especially in patients who are obese and morbidly obese. As the effects of obesity on the pharmacokinetics and pharmacodynamics of anticancer agents may be highly variable across drug types, the optimal dosing metric and algorithm for difference classes of drugs may be widely different. Thus, studies are needed to evaluate current and novel metrics and methods for measuring body habitus as related to optimizing the dose and reducing pharmacokinetic and pharmacodynamic variability of anticancer agents in patients who are obese and morbidly obese.

Topotecan clearance based on a single sample and a population pharmacokinetic model: Application to a pediatric high-risk neuroblastoma clinical trial.

BACKGROUND: Topotecan, an antitumor drug with systemic exposure (SE)-dependent activity against many pediatric tumors has wide interpatient pharmacokinetic variability, making it challenging to attain the desired topotecan SE. The study objectives were to update our topotecan population pharmacokinetic model, to evaluate the feasibility of determining individual topotecan clearance using a single blood sample, and to apply this approach to topotecan data from a neuroblastoma trial to explore exposure-response relationships. PROCEDURE: Our previous population pharmacokinetic and covariate model was updated using data from 13 clinical pediatric studies. A simulation-based Bayesian analysis was performed to determine if a single blood sample could be sufficient to estimate individual topotecan clearance. Following the Bayesian approach, single pharmacokinetic samples collected from a Children's Oncology Group Phase III clinical trial (ANBL0532; NCT0056767) were analyzed to estimate individual topotecan SE. Associations between topotecan SE and toxicity or early response were then evaluated. RESULTS: The updated population model included the impact of patient body surface area (BSA), age, and renal function on topotecan clearance. The Bayesian analysis with the updated model and single plasma samples showed that individual topotecan clearance values were estimated with good precision (mean absolute prediction error ≤16.2%) and low bias (mean prediction error ≤7.2%). Using the same approach, topotecan SE was derived in patients from ANBL0532. The exposure-response analysis showed an increased early response after concomitant cyclophosphamide and topotecan up to a topotecan SE of 45 h ng/mL. CONCLUSIONS: A simple single-sample approach during topotecan therapy could guide dosing for patients, resulting in more patients reaching target attainment.

Non-chemotherapy adjuvant agents in TP53 mutant Ewing sarcoma.

Ewing sarcoma (EWS) is a malignant tumor arising in bone or soft tissue that occurs in adolescent and young adult patients as well as adults later in life. Although non-metastatic EWS is typically responsive to treatment when newly diagnosed, relapsed cases have an unmet need for which no standard treatment approach exists. Recent phase III clinical trials for EWS comparing 7 vs 5 chemotherapy drugs have failed to improve survival. To extend the durability of remission for EWS, we investigated 3 non-chemotherapy adjuvant therapy drug candidates to be combined with chemotherapy. The efficacy of these adjuvant drugs was investigated via anchorage-dependent growth assays, anchorage-independent soft-agar colony formation assays and EWS xenograft mouse models. Enoxacin and entinostat were the most effective adjuvant drug in both long-term in vitro and in vivo adjuvant studies. In the context that enoxacin is an FDA-approved antibiotic, and that entinostat is an investigational agent not yet FDA-approved, we propose enoxacin as an adjuvant drug for further preclinical and clinical investigation in EWS patients.

Using the LeiCNS-PK3.0 Physiologically-Based Pharmacokinetic Model to Predict Brain Extracellular Fluid Pharmacokinetics in Mice.

INTRODUCTION: The unbound brain extracelullar fluid (brainECF) to plasma steady state partition coefficient, Kp,uu,BBB, values provide steady-state information on the extent of blood-brain barrier (BBB) transport equilibration, but not on pharmacokinetic (PK) profiles seen by the brain targets. Mouse models are frequently used to study brain PK, but this information cannot directly be used to inform on human brain PK, given the different CNS physiology of mouse and human. Physiologically based PK (PBPK) models are useful to translate PK information across species. AIM: Use the LeiCNS-PK3.0 PBPK model, to predict brain extracellular fluid PK in mice. METHODS: Information on mouse brain physiology was collected from literature. All available connected data on unbound plasma, brainECF PK of 10 drugs (cyclophosphamide, quinidine, erlotonib, phenobarbital, colchicine, ribociclib, topotecan, cefradroxil, prexasertib, and methotrexate) from different mouse strains were used. Dosing regimen dependent plasma PK was modelled, and Kpuu,BBB values were estimated, and provided as input into the LeiCNS-PK3.0 model to result in prediction of PK profiles in brainECF. RESULTS: Overall, the model gave an adequate prediction of the brainECF PK profile for 7 out of the 10 drugs. For 7 drugs, the predicted versus observed brainECF data was within two-fold error limit and the other 2 drugs were within five-fold error limit. CONCLUSION: The current version of the mouse LeiCNS-PK3.0 model seems to reasonably predict available information on brainECF from healthy mice for most drugs. This brings the translation between mouse and human brain PK one step further.

Genetic susceptibility to cognitive decline following craniospinal irradiation for pediatric central nervous system tumors.

BACKGROUND: Survivors of pediatric central nervous system (CNS) tumors treated with craniospinal irradiation (CSI) exhibit long-term cognitive difficulties. Goals of this study were to evaluate longitudinal effects of candidate and novel genetic variants on cognitive decline following CSI. METHODS: Intelligence quotient (IQ), working memory (WM), and processing speed (PS) were longitudinally collected from patients treated with CSI (n = 241). Genotype-by-time interactions were evaluated using mixed-effects linear regression to identify common variants (minor allele frequency > 1%) associated with cognitive performance change. Novel variants associated with cognitive decline (P < 5 × 10-5) in individuals of European ancestry (n = 163) were considered replicated if they demonstrated consistent genotype-by-time interactions (P < .05) in individuals of non-European ancestries (n = 78) and achieved genome-wide statistical significance (P < 5 × 10-8) in a meta-analysis across ancestry groups. RESULTS: Participants were mostly males (65%) diagnosed with embryonal tumors (98%) at a median age of 8.3 years. Overall, 1150 neurocognitive evaluations were obtained (median = 5, range: 2-10 per participant). One of the five loci previously associated with cognitive outcomes in pediatric CNS tumors survivors demonstrated significant time-dependent IQ declines (PPARA rs6008197, P = .004). Two variants associated with IQ in the general population were associated with declines in IQ after Bonferroni correction (rs9348721, P = 1.7 × 10-5; rs31771, P = 7.8 × 10-4). In genome-wide analyses, we identified novel loci associated with accelerated declines in IQ (rs116595313, meta-P = 9.4 × 10-9), WM (rs17774009, meta-P = 4.2 × 10-9), and PS (rs77467524, meta-P = 1.5 × 10-8; rs17630683, meta-P = 2.0 × 10-8; rs73249323, meta-P = 3.1 × 10-8). CONCLUSIONS: Inherited genetic variants involved in baseline cognitive functioning and novel susceptibility loci jointly influence the degree of treatment-associated cognitive decline in pediatric CNS tumor survivors.

CNS penetration of methotrexate and its metabolite 7-hydroxymethotrexate in mice bearing orthotopic Group 3 medulloblastoma tumors and model-based simulations for children.

The brain penetration of methotrexate (MTX) and its metabolite 7-hydroxymethotrexate (7OHMTX) was characterized in non-tumor bearing mice and mice bearing orthotopic Group 3 medulloblastoma. Plasma pharmacokinetic studies and cerebral and ventricular microdialysis studies were performed in animals dosed with 200 or 1000 mg/kg MTX by IV bolus. Plasma, brain/tumor extracellular fluid (ECF) and lateral ventricle cerebrospinal fluid (CSF) MTX and 7OHMTX concentration-time data were analyzed by validated LC-MS/MS methods and modeled using a population-based pharmacokinetic approach and a hybrid physiologically-based model structure for the brain compartments. Brain penetration was similar for MTX and 7OHMTX and was not significantly different between non-tumor and tumor bearing mice. Overall, mean (±SD) model-derived unbound plasma to ECF partition coefficient Kp,uu were 0.17 (0.09) and 0.17 (0.12) for MTX and 7OHMTX, respectively. Unbound plasma to CSF Kp,uu were 0.11 (0.06) and 0.18 (0.09) for MTX and 7OHMTX, respectively. The plasma and brain model were scaled to children using allometric principles and pediatric physiological parameters. Model-based simulations were adequately overlaid with digitized plasma and CSF lumbar data collected in children receiving different MTX systemic infusions. This model can be used to further explore and optimize methotrexate dosing regimens in children with brain tumors.

Phase II study of alisertib as a single agent for treating recurrent or progressive atypical teratoid/rhabdoid tumor.

BACKGROUND: Recurrent atypical teratoid/rhabdoid tumor (AT/RT) is, most often, a fatal pediatric malignancy with limited curative options. METHODS: We conducted a phase II study of Aurora kinase A inhibitor alisertib in patients aged <22 years with recurrent AT/RT. Patients received alisertib once daily (80 mg/m2 as enteric-coated tablets or 60 mg/m2 as liquid formulation) on Days 1-7 of a 21-day cycle until progressive disease (PD) occurred. Alisertib plasma concentrations were measured in cycle 1 on Days 1 (single dose) and 7 (steady state) and analyzed with noncompartmental pharmacokinetics. Trial efficacy end point was ≥10 participants with stable disease (SD) or better at 12 weeks. RESULTS: SD (n = 8) and partial response (PR) (n = 1) were observed among 30 evaluable patients. Progression-free survival (PFS) was 30.0% ± 7.9% at 6 months and 13.3% ± 5.6% at 1 year. One-year overall survival (OS) was 36.7% ± 8.4%. Two patients continued treatment for >12 months. PFS did not differ by AT/RT molecular groups. Neutropenia was the most common adverse effect (n = 23/30, 77%). The 22 patients who received liquid formulation had a higher mean maximum concentration (Cmax) of 10.1 ± 3.0 µM and faster time to Cmax (Tmax = 1.2 ± 0.7 h) than those who received tablets (Cmax = 5.7 ± 2.4 µM, Tmax = 3.4 ± 1.4 h). CONCLUSIONS: Although the study did not meet predetermined efficacy end point, single-agent alisertib was well tolerated by children with recurrent AT/RT, and SD or PR was observed in approximately a third of the patients.

Development and Validation of a Sensitive and Specific LC-MS/MS Method for IWR-1-Endo, a Wnt Signaling Inhibitor: Application to a Cerebral Microdialysis Study.

IWR-1-endo, a small molecule that potently inhibits the Wnt/ß-catenin signaling pathway by stabilizing the AXIN2 destruction complex, can inhibit drug efflux at the blood−brain barrier. To conduct murine cerebral microdialysis research, validated, sensitive, and reliable liquid chromatography−tandem mass spectrometry (LC-MS/MS) methods were used to determine IWR-1-endo concentration in the murine plasma and brain microdialysate. IWR-1-endo and the internal standard (ISTD) dabrafenib were extracted from murine plasma and microdialysate samples by a simple solid-phase extraction protocol performed on an Oasis HLB µElution plate. Chromatographic separation was executed on a Kinetex C18 (100A, 50 × 2.1 mm, 4 µm particle size) column with a binary gradient of water and acetonitrile, each having 0.1% formic acid, pumped at a flow rate of 0.6 mL/min. Detection by mass spectrometry was conducted in the positive selected reaction monitoring ion mode by monitoring mass transitions 410.40 > 344.10 (IWR-1-endo) and 520.40 > 307.20 (ISTD). The validated curve range of IWR-1-endo was 5−1000 ng/mL for the murine plasma method (r2 ≥ 0.99) and 0.5−500 ng/mL for the microdialysate method (r2 ≥ 0.99). The lower limit of quantification (LLOQ) was 5 ng/mL and 0.5 ng/mL for the murine plasma and microdialysate sample analysis method, respectively. Negligible matrix effects were observed in murine plasma and microdialysate samples. IWR-1-endo was extremely unstable in murine plasma. To improve the stability of IWR-1-endo, pH adjustments of 1.5 were introduced to murine plasma and microdialysate samples before sample storage and processing. With pH adjustment of 1.5 to the murine plasma and microdialysate samples, IWR-1-endo was stable across several tested conditions such as benchtop, autosampler, freeze−thaw, and long term at −80 °C. The LC-MS/MS methods were successfully applied to a murine pharmacokinetic and cerebral microdialysis study to characterize the unbound IWR-1-endo exposure in brain extracellular fluid and plasma.

Combination of Ribociclib and Gemcitabine for the Treatment of Medulloblastoma.

Group3 (G3) medulloblastoma (MB) is one of the deadliest forms of the disease for which novel treatment is desperately needed. Here we evaluate ribociclib, a highly selective CDK4/6 inhibitor, with gemcitabine in mouse and human G3MBs. Ribociclib central nervous system (CNS) penetration was assessed by in vivo microdialysis and by IHC and gene expression studies and found to be CNS-penetrant. Tumors from mice treated with short term oral ribociclib displayed inhibited RB phosphorylation, downregulated E2F target genes, and decreased proliferation. Survival studies to determine the efficacy of ribociclib and gemcitabine combination were performed on mice intracranially implanted with luciferase-labeled mouse and human G3MBs. Treatment of mice with the combination of ribociclib and gemcitabine was well tolerated, slowed tumor progression and metastatic spread, and increased survival. Expression-based gene activity and cell state analysis investigated the effects of the combination after short- and long-term treatments. Molecular analysis of treated versus untreated tumors showed a significant decrease in the activity and expression of genes involved in cell-cycle progression and DNA damage response, and an increase in the activity and expression of genes implicated in neuronal identity and neuronal differentiation. Our findings in both mouse and human patient-derived orthotopic xenograft models suggest that ribociclib and gemcitabine combination therapy warrants further investigation as a treatment strategy for children with G3MB.

Phase I study of ribociclib and everolimus in children with newly diagnosed DIPG and high-grade glioma: A CONNECT pediatric neuro-oncology consortium report.

Background: Genomic aberrations in the cell cycle and PI3K/Akt/mTOR pathways have been reported in diffuse intrinsic pontine glioma (DIPG) and high-grade glioma (HGG). Dual inhibition of CDK4/6 and mTOR has biologic rationale and minimal overlapping toxicities. This study determined the recommended phase 2 dose (RP2D) of ribociclib and everolimus following radiotherapy in children with DIPG and HGG. Methods: Patients were enrolled according to a Rolling-6 design and received ribociclib and everolimus once daily for 21 and 28 days, respectively. All patients with HGG and biopsied DIPG were screened for retinoblastoma protein presence by immunohistochemistry. Pharmacokinetics were analyzed. Results: Nineteen patients enrolled (median age: 8 years [range: 2-18]). Three patients enrolled at each dose level 1 and 2 without dose-limiting toxicities (DLT). Thirteen patients were enrolled at dose level 3, with one patient experiencing a DLT (grade 3 infection). One patient came off therapy before cycle 9 due to cardiac toxicity. The most common grade 3/4 toxicities were neutropenia (33%), leucopenia (17%), and lymphopenia (11%). Steady-state everolimus exposures in combination were 1.9 ± 0.9-fold higher than single-agent administration. Median overall survival for 15 patients with DIPG was 13.9 months; median event-free survival for four patients with HGG was 10.5 months. Two longer survivors had tumor molecular profiling identifying CDKN2A/B deletion and CDK4 overexpression. Conclusion: The combination of ribociclib and everolimus following radiotherapy in children with newly diagnosed DIPG and HGG was well tolerated, with a RP2D of ribociclib 170 mg/m2 and everolimus 1.5 mg/m2. Results will inform a molecularly guided phase II study underway to evaluate efficacy.

Population pharmacokinetics of crenolanib in children and young adults with brain tumors.

PURPOSE: Crenolanib, an oral inhibitor of platelet-derived growth factor receptor, was evaluated to treat children and young adults with brain tumors. Crenolanib population pharmacokinetics and covariate influence were characterized in this patient population. METHODS: Patients enrolled on this phase I study (NCT01393912) received oral crenolanib once daily. Serial single-dose and steady-state serum pharmacokinetic samples were collected and analyzed using a validated LC-ESI-MS/MS method. Population modeling and covariate analysis evaluating demographics, laboratory values, and comedications were performed. The impact of significant covariates on crenolanib exposure was further explored using model simulations. RESULTS: Crenolanib serum concentrations were analyzed for 55 patients (2.1-19.2 years-old) and best fitted with a linear two-compartment model, with delayed absorption modeled with a lag time. A typical patient [8-year-old, body surface area (BSA) 1 m2] had an apparent central clearance, volume, and absorption rate of 41 L/h, 54.3 L, and 0.19 /h, respectively. Patients taking acid reducers (histamine H2 antagonists or proton pump inhibitors) concomitantly exhibited about 2- and 1.7-fold lower clearance and volume (p < 0.0001 and p = 0.018, respectively). Crenolanib clearance increased with BSA (p < 0.0001), and absorption rate decreased with age (p < 0.0001). Model simulations showed cotreatment with an acid reducer was the only covariate significantly altering crenolanib exposure and supported the use of BSA-based crenolanib dosages vs flat-dosages for this population. CONCLUSIONS: Crenolanib pharmacokinetics were adequately characterized in children and young adults with brain tumors. Despite marked increased drug exposure with acid reducer cotreatment, crenolanib therapy was well tolerated. No dosing adjustments are recommended for this population.

Serial assessment of measurable residual disease in medulloblastoma liquid biopsies.

Nearly one-third of children with medulloblastoma, a malignant embryonal tumor of the cerebellum, succumb to their disease. Conventional response monitoring by imaging and cerebrospinal fluid (CSF) cytology remains challenging, and a marker for measurable residual disease (MRD) is lacking. Here, we show the clinical utility of CSF-derived cell-free DNA (cfDNA) as a biomarker of MRD in serial samples collected from children with medulloblastoma (123 patients, 476 samples) enrolled on a prospective trial. Using low-coverage whole-genome sequencing, tumor-associated copy-number variations in CSF-derived cfDNA are investigated as an MRD surrogate. MRD is detected at baseline in 85% and 54% of patients with metastatic and localized disease, respectively. The number of MRD-positive patients declines with therapy, yet those with persistent MRD have significantly higher risk of progression. Importantly, MRD detection precedes radiographic progression in half who relapse. Our findings advocate for the prospective assessment of CSF-derived liquid biopsies in future trials for medulloblastoma.

Population pharmacokinetic analysis of crizotinib in children with progressive/recurrent high-grade and diffuse intrinsic pontine gliomas.

PURPOSE: Crizotinib, a potent oral tyrosine kinase inhibitor, was evaluated in combination with dasatinib in a phase 1 trial (NCT01644773) in children with progressive or recurrent high-grade and diffuse intrinsic pontine gliomas (HGG and DIPG). This study aimed to characterize the pharmacokinetics of crizotinib in this population and identify significant covariates. METHODS: Patients (N = 36, age range 2.9-21.3 years) were treated orally once or twice-daily with 100-215 mg/m2 crizotinib and 50-65 mg/m2 dasatinib. Pharmacokinetic studies were performed for crizotinib alone after the first dose and at steady state, and for the drug combination at steady state. Crizotinib plasma concentrations were measured using a validated LC-MS/MS method. Population modeling was performed (Monolix) and the impact of factors including patient demographics and co-medications were investigated on crizotinib pharmacokinetics. RESULTS: Crizotinib concentrations were described with a linear two-compartment model and absorption lag time. Concomitant dasatinib and overweight/obese status significantly influenced crizotinib pharmacokinetics, resulting in clinically relevant impact (> 20%) on drug exposure. Crizotinib mean apparent clearance (CL/F) was 66.7 L/h/m2 after single-dose and decreased to 26.5 L/h/m2 at steady state when given alone, but not when combined with dasatinib (mean 60.8 L/h/m2). Overweight/obese patients exhibited lower crizotinib CL/F and apparent volume V1/F (mean 46.2 L/h/m2 and 73.3 L/m2) compared to other patients (mean 75.5 L/h/m2 and 119.3 L/m2, p < 0.001). CONCLUSION: A potential pharmacokinetic interaction was observed between crizotinib and dasatinib in children with HGG and DIPG. Further, crizotinib exposure was significantly higher in overweight/obese patients, who may require a dosing adjustment.

Fusion Oncoproteins in Childhood Cancers: Potential Role in Targeted Therapy.

Cancer remains the leading cause of death from disease in children. Historically, in contrast to their adult counterparts, the causes of pediatric malignancies have remained largely unknown, with most pediatric cancers displaying low mutational burdens. Research related to molecular genetics in pediatric cancers is advancing our understanding of potential drivers of tumorigenesis and opening new opportunities for targeted therapies. One such area is fusion oncoproteins, which are a product of chromosomal rearrangements resulting in the fusion of different genes. They have been identified as oncogenic drivers in several sarcomas and leukemias. Continued advancement in the understanding of the biology of fusion oncoproteins will contribute to the discovery and development of new therapies for childhood cancers. Here we review the current scientific knowledge on fusion oncoproteins, focusing on pediatric sarcomas and hematologic cancers, and highlight the challenges and current efforts in developing drugs to target fusion oncoproteins.

Development and validation of an LC-MS/MS method to quantify the bromodomain and extra-terminal (BET) inhibitor JQ1 in mouse plasma and brain microdialysate: Application to cerebral microdialysis study.

JQ1, is a cell-permeable small-molecule inhibitor of bromodomain and extra-terminal protein (BET) function with reportedly good CNS penetration, however, unbound and pharmacologically active CNS JQ1 exposures have not been characterized. Additionally, no quantitative bioanalytical methods for JQ1 have been described in the literature to support the CNS penetration studies. In the present article, we discuss the development and validation of a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitative methods to determine JQ1 in mouse plasma and brain microdialysate. JQ1 and the internal standard, dabrafenib (ISTD), were extracted from plasma and microdialysate samples using a simple solid phase extraction protocol performed on an Oasis HLB µElution plate. Chromatographic separation of JQ1 and ISTD was achieved on a reversed phase C12 analytical column with gradient elution profile of mobile phases (MP A: water containing 0.1 % formic acid and MP B: acetonitrile containing 0.1 % formic acid) at a flow rate of 0.6 mL/min. The mass spectrometric detection was performed in the positive MRM ion mode by monitoring the transitions 457.40 > 341.30 (JQ1) and 520.40 > 307.20 (ISTD). The calibration curves demonstrated good linearities over the concentration range of 5-1000 ng/mL for the mouse plasma method (r2 ≥ 0.99) and 0.5-500 ng/mL for the microdialysate method (r2 ≥ 0.99). The experimental limit of quantification obtained was 5 and 0.5 ng/mL for the mouse plasma and microdialysate method, respectively, with the coefficient of variation less than 10 % for the analyte peak area. All the other validation parameters, including intra-and inter-day accuracy and precision, matrix effect, selectivity, carryover effect, and stability, were within the USFDA bioanalytical guidelines acceptance limits. The LC-MS/MS method was successfully applied to a mouse pharmacokinetic and cerebral microdialysis study to characterize the unbound JQ1 exposure in brain extracellular fluid and plasma.

Pharmacokinetically guided dosing of oral sorafenib in pediatric hepatocellular carcinoma: A simulation study.

Sorafenib improves outcomes in adult hepatocellular carcinoma; however, hand foot skin reaction (HFSR) is a dose limiting toxicity of sorafenib that limits its use. HFSR has been associated with sorafenib systemic exposure. The objective of this study was to use modeling and simulation to determine whether using pharmacokinetically guided dosing to achieve a predefined sorafenib target range could reduce the rate of HFSR. Sorafenib steady-state exposures (area under the concentration curve from 0 to 12-h [AUC0->12 h ]) were simulated using published sorafenib pharmacokinetics at either a fixed dosage (90 mg/m2 /dose) or a pharmacokinetically guided dose targeting an AUC0->12 h between 20 and 55 h µg/ml. Dosages were either rounded to the nearest quarter of a tablet (50 mg) or capsule (10 mg). A Cox proportional hazard model from a previously published study was used to quantify HFSR toxicity. Simulations showed that in-target studies increased from 50% using fixed doses with tablets to 74% using pharmacokinetically guided dosing with capsules. The power to observe at least 4 of 6 patients in the target range increased from 33% using fixed dosing with tablets to 80% using pharmacokinetically guided with capsules. The expected HFSR toxicity rate decreased from 22% using fixed doses with tablets to 16% using pharmacokinetically guided dosing with capsules. The power to observe less than 6 of 24 studies with HFSR toxicity increased from 51% using fixed dosing with tablets to 88% using pharmacokinetically guided with capsules. Our simulations provide the rationale to use pharmacokinetically guided sorafenib dosing to maintain effective exposures that potentially improve tolerability in pediatric clinical trials.