Pharmacokinetic Properties of Anticancer Agents for the Treatment of Central Nervous System Tumors: Update of the Literature.

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.

PU.1 regulates glutathione peroxidase expression in neutrophils.

Based on knockout models, the transcription factor PU.1 has been shown to be important for the maturation of neutrophils. As the list of genes PU.1 directly regulates in neutrophils is still quite limited, defining PU.1 target genes for this lineage will provide valuable insight into how this factor regulates neutrophil development and terminal function. Using the combined techniques of representational difference analysis and a cDNA library screen, we identified four genes that were differentially expressed in the PU.1-expressing 503PU myeloid cell line but not the PU.1 null parent cell line 503. Two of these genes, glutathione peroxidase (GPx) and serine leukoprotease inhibitor, are involved in protecting neutrophils from the products they make to destroy pathogens and were analyzed further to determine if PU.1 directly regulates their expression. These studies showed that PU.1 directly regulated the expression of only the GPx gene through binding sites in the promoter and a 3' regulatory region. Thus, PU.1 not only regulates the expression of molecules involved in the production of reactive oxygen species but also a gene that protects the neutrophils from these same destructive enzymes.

Low-level GATA2 overexpression promotes myeloid progenitor self-renewal and blocks lymphoid differentiation in mice.

The transcription factor GATA2 is highly expressed in hematopoietic stem cells and is downregulated during lineage maturation. Gain of function mutations, loss of function mutations, and overexpression of GATA2 have been reported in acute myeloid leukemia. In previous studies, we and others showed that GATA2 overexpression at high levels, similar to that seen in hematopoietic stem cells, blocked differentiation of hematopoietic stem cells and progenitors. To better understand the effects of GATA2, we designed a Tamoxifen-inducible GATA2-estrogen receptor (ERT) vector. In the absence of Tamoxifen, small amounts of GATA2-ERT were still able to enter the nucleus in mouse bone marrow (BM) cells, providing us with a tool to test the effects of low-level GATA2 overexpression. We observed that this low-level GATA2 overexpression enhanced self-renewal of myeloid progenitors in vitro and resulted in immortalization of BM cells to myeloid cell lines. Continuous GATA2-ERT expression was required for the proliferation of these immortalized lines. Myeloid expansion and a block in T and B lineage differentiation were observed in mice transplanted with GATA2-ERT-expressing BM cells. Myeloid expansion occurred after the granulocyte monocyte progenitor stage, and lymphoid block was distal to the common lymphoid progenitor in transgenic mice. GATA2 appeared to induce growth via downstream activation of Nmyc and Hoxa9. Our results demonstrate that GATA2 overexpression at low level confers self-renewal capacity to myeloid progenitors and is relevant to myeloid leukemia development.

Observational Evaluations of Mice during Cerebral Microdialysis for Pediatric Brain Tumor Research.

In vivo animal experiments are critical in the process of finding and developing new treatments for children with CNS tumors. Cerebral microdialysis, which enables researchers to measure drug concentrations in the brain or tumor tissue of unanesthetized mice, is a highly specialized procedure that provides valuable information that cannot be gained by using an in vitro system. When designing any in vivo animal study, 3 Rs principles (replacement, reduction, and refinement) must be considered to ensure that the highest standards of care are followed. As part of the refinement process, the objectives of this study were to collect behavioral monitoring data from mice undergoing cerebral microdialysis, to identify any behaviors predictive of significant pain or distress that could affect the animal's welfare, and to use these data to refine the existing monitoring checklist and schedule for its use by others performing this procedure. We developed a monitoring checklist for assessing wellbeing and distress of mice during cerebral microdialysis experiments. Comparison of 79 mice that underwent cerebral microdialysis experiments with a control group of 20 mice revealed that cerebral microdialysis and tethering of mice are well tolerated for as long as 24 h with only minor evidence of stress.

Preclinical examination of clofarabine in pediatric ependymoma: intratumoral concentrations insufficient to warrant further study.

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/m(2). 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.

Population pharmacokinetics of bevacizumab in children with osteosarcoma: implications for dosing.

PURPOSE: To describe sources of interindividual variability in bevacizumab disposition in pediatric patients and explore associations among bevacizumab pharmacokinetics and clinical wound healing outcomes. EXPERIMENTAL DESIGN: Before tumor resection, three doses of bevacizumab (15 mg/kg) were administered to patients (median age, 12.2 years) enrolled in a multi-institutional osteosarcoma trial. Serial sampling for bevacizumab pharmacokinetics was obtained from 27 patients. A population pharmacokinetic model was fit to the data, and patient demographics and clinical chemistry values were systematically tested as predictive covariates on model parameters. Associations between bevacizumab exposure and wound healing status were evaluated by logistic regression. RESULTS: Bevacizumab concentration-time data were adequately described by a two-compartment model. Pharmacokinetic parameter estimates were similar to those previously reported in adults, with a long median (range) terminal half-life of 12.2 days (8.6 to 32.4 days) and a volume of distribution indicating confinement primarily to the vascular space, 49.1 mL/kg (27.1 to 68.3 mL/kg). Body composition was a key determinant of bevacizumab exposure, as body mass index percentile was significantly (P < 0.05) correlated to body-weight normalized clearance and volume of distribution. Furthermore, bevacizumab exposure before primary tumor resection was associated with increased risk of major wound healing complications after surgery (P < 0.05). CONCLUSION: A population pharmacokinetic model for bevacizumab was developed, which demonstrated that variability in bevacizumab exposure using weight-based dosing is related to body composition. Bevacizumab dosage scaling using ideal body weight would provide an improved dosing approach in children by minimizing pharmacokinetic variability and reducing likelihood of major wound healing complications.

Combination metronomic oral topotecan and pazopanib: a pharmacokinetic study in patients with gynecological cancer.

BACKGROUND: Combination metronomic topotecan plus pazopanib is active against preclinical models of gynecological cancer. Both agents are substrates for ATP-binding cassette family transporters so there is an increased likelihood for pharmacokinetic (PK) drug-drug interactions. PATIENTS AND METHODS: PK analyses of topotecan were performed during three cycles of a phase I dose-escalation study of metronomic topotecan and pazopanib in consenting adult patients with gynecological cancer. Concentration time data were analyzed using a population PK approach. RESULTS: Twenty-one patients were evaluable for serial PK studies. Considerable inter- and intra-patient variability was observed in the PK parameters, attributable primarily to highly variable oral bioavailability. No difference in topotecan disposition was detected between administration cycles, nor between the off- versus on-pazopanib studies. CONCLUSION: The lack of a statistically significant drug-drug interaction agrees with preclinical findings suggesting that pazopanib does not influence the PK of metronomic topotecan. No adjustment of low dose metronomic topotecan dosing is merited when used in conjunction with pazopanib.

Determination of crenolanib in human serum and cerebrospinal fluid by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

A LC-ESI-MS/MS method for the determination of crenolanib (CP-868,596) in human serum was developed and validated employing d4-CP-868,596 as an internal standard (ISTD). In addition to human serum, the method was also partially validated for crenolanib determination in human cerebrospinal fluid (CSF) samples. Sample aliquots (50µl of serum or CSF) were prepared for analysis using liquid-liquid extraction (LLE) with tert-butyl methyl ether. Chromatography was performed using a phenomenex Gemini C18 column (3µm, 100mm×4.6mm I.D.) in a column heater set at 50°C and an isocratic mobile phase (methanol/water/formic acid at a volume ratio of 25/25/0.15, v/v/v). The flow rate was 0.45mL/min, and the retention time for both analyte and ISTD was less than 3.5min. Samples were analyzed with an API-5500 LC-MS/MS system (ESI) in positive ionization mode coupled to a Shimadzu HPLC system. The ion transitions monitored were m/z 444.4→373.1 and m/z 448.2→374.2 for crenolanib and ISTD, respectively. The method was linear over the range of 5-1000ng/mL for serum and 0.5-1000ng/mL for CSF. For human serum, both intra-day and inter-day precision were <4%, while intra-day and inter-day accuracy were within 8% of nominal values. Recovery was greater than 50% for both the analyte and ISTD. For CSF samples, both intra-day and inter-day precision were <9% except at the lower limit of quantification (LLOQ) which was <17%. The intra-day and inter-day accuracy were within 11% of the nominal CSF concentrations. After validation, this method was successfully applied to the analysis of serial pharmacokinetic samples obtained from a child treated with oral crenolanib.

Magnetic resonance imaging-guided microdialysis cannula implantation in a spontaneous high-grade glioma murine model.

Cerebral microdialysis is used to study anticancer drug penetration in the central nervous system (CNS) and brain tumors in animal models. Genetically engineered murine models (GEMMs) have been recently used to study many aspects of CNS tumors since they represent a more relevant model than orthotopic brain tumor xenograft models. However, it is challenging to implant microdialysis cannula in these animals because T2-weighted magnetic resonance imaging (MRI) does not show the reference point (bregma) traditionally used to obtain stereotactic coordinates. Thus, an alternative reference point that can be visualized on MRI images is needed. In this study, a novel reference point, identified as the intersection between the olfactory bulb/frontal lobe border and the midline between cerebral hemispheres on T2-weighted MRI images, was used to calculate anterior-posterior and medial-lateral coordinates of brain tumors in a GEMM. This point overlies a visible crossover between the rostral rhinal vein and the midline suture on the mouse skull, allowing for the conversion of the MRI coordinates into surgical stereotactic coordinates. Postmortem MRI and histological examination confirmed accurate probe placement. This procedure will facilitate the accurate and precise implantation of microdialysis probes for the study of anticancer drug penetration in brain tumors of GEMMs.

MDM2 antagonist nutlin-3a reverses mitoxantrone resistance by inhibiting breast cancer resistance protein mediated drug transport.

Breast cancer resistance protein (BCRP; ABCG2), a clinical marker for identifying the side population (SP) cancer stem cell subgroup, affects intestinal absorption, brain penetration, hepatobiliary excretion, and multidrug resistance of many anti-cancer drugs. Nutlin-3a is currently under pre-clinical investigation in a variety of solid tumor and leukemia models as a p53 reactivation agent, and has been recently demonstrated to also have p53 independent actions in cancer cells. In the present study, we first report that nutlin-3a can inhibit the efflux function of BCRP. We observed that although the nutlin-3a IC(50) did not differ between BCRP over-expressing and vector control cells, nutlin-3a treatment significantly potentiated the cells to treatment with the BCRP substrate mitoxantrone. Combination index calculations suggested synergism between nutlin-3a and mitoxantrone in cell lines over-expressing BCRP. Upon further investigation, it was confirmed that nutlin-3a increased the intracellular accumulation of BCRP substrates such as mitoxantrone and Hoechst 33342 in cells expressing functional BCRP without altering the expression level or localization of BCRP. Interestingly, nutlin-3b, considered virtually "inactive" in disrupting the MDM2/p53 interaction, reversed Hoechst 33342 efflux with the same potency as nutlin-3a. Intracellular accumulation and bi-directional transport studies using MDCKII cells suggested that nutlin-3a is not a substrate of BCRP. Additionally, an ATPase assay using Sf9 insect cell membranes over-expressing wild-type BCRP indicated that nutlin-3a inhibits BCRP ATPase activity in a dose-dependent fashion. In conclusion, our studies demonstrate that nutlin-3a inhibits BCRP efflux function, which consequently reverses BCRP-related drug resistance.

UGT1A1 promoter genotype correlates with SN-38 pharmacokinetics, but not severe toxicity in patients receiving low-dose irinotecan.

PURPOSE: To study the association between UDP-glucuronosyltransferase 1A1 (UGT1A1) genotypes and severe toxicity as well as irinotecan disposition in pediatric patients with solid tumors receiving low-dose, protracted irinotecan (15 to 75 mg/m2 daily for 5 days for 2 consecutive weeks). PATIENTS AND METHODS: Seventy-four patients on five institutional clinical trials received irinotecan (15 to 75 mg/m2) daily intravenously or orally for 5 days for 2 consecutive weeks. Genomic DNA was genotyped for UGT1A1*28, and patients were designated as 6/6, 6/7, or 7/7 depending on the number of TA repeats in the UGT1A1 promoter region. Patients were evaluated for gastrointestinal and hematologic toxicity, as well as baseline and maximal serum bilirubin levels. Toxicity and pharmacokinetic results were evaluated during courses 1 and 2 of irinotecan therapy. RESULTS: The frequencies of 6/6, 6/7, and 7/7 genotypes were 27 (36.5%), 36 (48.6%), and 9 (12.2%) of 74 patients, respectively. Patients with 7/7 genotype had a statistically greater baseline total bilirubin than patients with 6/6 or 6/7 genotype (P = .005). UGT1A1*28 genotype was not associated with grade 3 and 4 neutropenia (P = .21 for course 1; P = .23 for course 2) or diarrhea (P = .176 for course 1; P = .87 for course 2). However, patients with the 7/7 genotype tended to have higher SN-38 area under the plasma time-concentration curve (AUC) values and lower SN-38G/SN-38 AUC ratios. CONCLUSION: Severe toxicity was not increased in pediatric patients with the 7/7 genotype when treated with a low-dose protracted schedule of irinotecan. Therefore, UGT1A1 genotyping is not a useful prognostic indicator of severe toxicity for patients treated with this irinotecan dosage and schedule.