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e14070 Background: Selumetinib (AZD6244 hyd sulfate) is an orally bioavailable agent targeting mitogen-activated protein kinase (MEK) 1/2. Selumetinib has shown activity in children with NF1 plexiform neurofibromas and refractory low-grade gliomas. The central nervous system (CNS) penetration of selumetinib in humans is unknown. Methods: We analyzed selumetinib in the cerebrospinal fluid (CSF) of NHP as a surrogate for CNS penetration. Four adult male rhesus macaques were each given a single dose of selumetinib capsules 2.5 mg/kg (human equivalent dose 50 mg/m2) by mouth on an empty stomach. Selumetinib was quantified in serial paired plasma and CSF samples from 0 to 48 hours after administration by Covance Bioanalytical Services (lower limit of quantification 0.5 ng/mL). Free selumetinib was estimated at 2.3% of total based on reported plasma protein binding of 97.7% in NHP. Pharmacokinetic parameters including area under the concentration x time curve (AUC) of selumetinib were calculated using non-compartmental methods and CSF penetration was calculated from the ratio of AUC (CSF): AUC (plasma). Results: Selumetinib was detected in plasma in all four NHPs, but in the CSF of only one NHP (ZH60). The mean (± SD) AUC0-∞ and half-life (t1/2) in plasma were 3350 ± 489 ng•h/mL and 10.8 ± 2.5 hours, respectively. In ZH60, the CSF AUC0-48 hr was 10.6 ng•h/mL. The CSF penetration of total and free selumetinib in this animal was 0.4%, and 14%, respectively. The mean plasma AUC and t1/2 were comparable to that of children (2842 ng•h/mL and 6.9 hours, respectively) at a similar dose in a phase I trial. However, the average peak concentration in children occurred at approximately 1 hour with a Cmax of 841 ng/mL, while the Cmax in NHP plasma ranged from 1 – 4 hours and averaged 198.8 ng/mL. Conclusions: The CSF penetration of oral selumetinib is limited; in one NHP it was 0.4% and 14% when corrected for plasma protein binding of selumetinib. The fact that plasma AUC0-∞ and half-lives in NHP were similar to previously reported human plasma PK indicates that this model may accurately reflect human CSF penetration as well.

The pharmacokinetics of 2',3'-dideoxyadenosine (ddA), didanosine, 2',3'-dideoxyguanosine (ddG), and 6-halogenated prodrugs of ddG, 6-chloro-ddG and 6-iodo-ddG, in plasma and cerebrospinal fluid (CSF) were studied in a non-human primate model. ddA was rapidly and completely deaminated to didanosine, such that didanosine concentration profiles in plasma and CSF were identical following administration of ddA and didanosine. The mean clearance of didanosine was 0.50 liters/h/kg, the terminal half-life was 1.8 h, and the CSF-to-plasma ratio was 4.8%. The disposition of ddG was similar, with a clearance of 0.70 liters/h/kg and a half-life of 1.7 h. The adenosine deaminase-mediated conversion of the 6-halogenated-ddG prodrugs to ddG was rapid but incomplete (48% for 6-chloro-ddG and 29% for 6-iodo-ddG). The CSF-to-plasma ratios of ddG with equimolar doses of ddG, 6-chloro-ddG, and 6-iodo-ddG were 8.5, 24, and 17%, respectively, but the actual ddG exposures in CSF (area under the CSF concentration-time curve) were comparable for ddG (12.1 microM.h) and the 6-halogenated-ddG prodrugs (18.8 microM.h for 6-chloro-ddG, 9.3 microM.h for 6-iodo-ddG).6-Chloro-ddG was not detectable in plasma or CSF, and the CSF-to-plasma ratio of 6-iodo-ddG was 9.4%, so the higher CSF-to-plasma ratios of ddG with the administration of the 6-halogenated-ddG prodrugs does not appear to be the result of enhanced penetration of the prodrug and subsequent dehalogenation to ddG. The penetration of ddG into CSF exceeds that of didanosine and is enhanced by administration of the 6-halogenated prodrugs, although the mechanism of this enhanced penetration is unclear.

Gemcitabine is an excellent candidate for regional therapy. We quantified cerebrospinal fluid (CSF) and plasma concentrations of gemcitabine and its inactive metabolite, 2',2'-difluorodeoxyuridine (dFdU), in nonhuman primates given intrathecal gemcitabine.Three nonhuman primates received 5 mg of gemcitabine via lateral ventricle. CSF was sampled from the fourth ventricle in all of the animals and the lumbar space in one, and one had plasma sampled. One animal had ventricular CSF sampled after receiving 5 mg intralumbar gemcitabine. Gemcitabine and dFdU were measured by high-performance liquid chromatography. Three additional animals had 5 mg intralumbar gemcitabine administered weekly for 4 weeks and were monitored for toxicity.At 37 degrees C in vitro, gemcitabine was stable in CSF. Ventricular delivery of gemcitabine produced peak ventricular CSF gemcitabine concentrations of 297 +/- 105 microg/ml. After 6 h, the concentrations were0.03 microg/ml. Intrathecal gemcitabine was rapidly and extensively converted to dFdU. CSF dFdU concentrations increased to 82 microg/ml at 1 h and then declined to very low values by 24 h. After intraventricular administration, CSF gemcitabine and dFdU area(s) under the curve (AUC) were 251 +/- 85 and 249 +/- 88 microg/ml x h. Intralumbar gemcitabine produced lower ventricular CSF gemcitabine and dFdU concentrations than did intraventricular gemcitabine. The plasma gemcitabine AUC associated with 5 mg of intraventricular gemcitabine was 2 mg/ml x h, which was200-fold lower than the CSF gemcitabine AUC in the same animal. Transient CSF pleocytosis was the only toxicity observed.Our results demonstrate a large pharmacokinetic advantage of intrathecal gemcitabine and support a planned Phase I clinical trial of this dosing strategy.

Purpose: P-glycoprotein (Pgp) is a transmembrane drug efflux pump that is expressed in multidrug-resistant cancer cells and in a variety of normal tissues, including brain capillary endothelial cells which comprise the blood-brain barrier. We studied the effects of the Pgp inhibitor, cyclosporin A (CsA), on the cerebrospinal fluid (CSF) penetration of the Pgp substrate, doxorubicin, in non-human primates. Methods: The animals received doxorubicin alone (2.0 mg/kg i.v. over 60 min) or doxorubicin (1 mg/kg i.v. over 60 min) and CsA (loading dose 4.0 mg/kg i.v. over 2 h, followed by continuous infusion of 12 mg/kg per day over 48 h). Plasma and CSF were collected over 48 h and the doxorubicin concentration was measured by reverse-phase high-pressure liquid chromatography (HPLC) with fluorescence detection (detection limit 5 nM). A two-compartment model was fitted to the plasma concentration-time data. Results: Pgp was demonstrated to be present in the epithelium of the choroid plexus by immunohistochemical methods, indicating that CSF drug penetration could be used as a surrogate for blood-brain barrier penetration. Steady state whole blood CsA concentrations, which were measured with a fluorescence-polarization immunoassay (TDX) that detects both CsA and its metabolites, ranged from 551–1315 μg/l at 24 h. The clearance of doxorubicin in four animals was reduced by 34%, 38%, 45% and 49% when given with CsA. The doxorubicin concentration in the CSF was

Background: We developed a nonhuman primate model using rhesus macaques (Macaca mulatta) to compare drug exposure in cortical brain, pontine tissue, and cerebrospinal fluid (CSF) during systemic administration of chemotherapeutic agents by microdialysis (MD), a continuous in vivo extracellular sampling technique. Pediatric diffuse intrinsic pontine gliomas are aggressive brainstem tumors that respond minimally to chemotherapy. We hypothesize that, because of its role in maintaining basic life-sustaining functions, the protective features of the pons, including the blood brain barrier and the blood-cerebrospinal fluid barrier, may further limit antitumor agents from entering the pons compared to cortical brain tissue. Methods: The coordinates and surgical approach for probe placement were determined using 3T MRI (Philips Healthcare, Best Netherlands) and OsiriX imaging software (v 3.9.4) in 4 adult male rhesus macaques (Macaca mulatta). Custom MD cannulas and probes (CMA, Solna, Sweden) were stereotactically implanted in the brain. The pontine MD cannula (34 mm) was implanted superior to the pons, and the MD probe (34 mm shaft plus 8 mm membrane) was then lowered through the cannula, into the pons. The cortical MD cannula (10 mm) was implanted in frontal cortex and a MD probe (10 mm shaft plus 8 mm membrane) was lowered through the cannula. The MD probe inlet/outlet capillaries were connected to a MD infusion pump (CMA 106, flow rate 0.3 µL/min). Retrodialysis was performed to assess in vivo recovery. A systemic intravenous (IV) drug infusion of temozolomide was then administered over 1hr. Single continuous MD samples were collected from both the pons and cortex over 4 hours with concurrent serial plasma and CSF samples (via a subarachnoid or lumbar catheter). Post-surgical MRI verification of MD probe placement was obtained in 2 of 4 animals. Verification of probe placement was obtained via gross pathology and histology in all 4 animals. Results: MRI-determined coordinates and surgical approach for MD probe placement and sample collection in the pons and cerebral cortex was successful in 3 of 4 animals. Surgical monitoring for heart and respiration rates, ETCO2 and SPO2 remained normal in all animals during probe placement and sample collection. Conclusions: MRI-determined coordinates and surgical methodologies resulted in accurate placement of a MD probe in the pons and cerebral cortex of a rhesus macaque allowing for MD sampling from these sites, in conjunction with CSF and plasma sampling, following systemic drug administration. This new animal model allows for the determination of differences in penetration of chemotherapeutic agents in pons, cerebrum, and CSF following systemic drug administration. Additionally, this model provides the foundation for a subcutaneous, semi-permanent, closed system for CNS MD probe placement and continuous sampling in rhesus macaques. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3789. doi:1538-7445.AM2012-3789

Antifungal prophylaxis in cancer patients who are undergoing chemotherapy is associated with prolonged neutropenia. We measured the effect of fluconazole on doxorubicin pharmacokinetics in nonhuman primates to determine if neutropenia is related to a pharmacokinetic interaction that delays the clearance of the chemotherapeutic agent. Fluconazole pretreatment had no effect on doxorubicin pharmacokinetics.

The pharmacokinetics of subcutaneously administered methotrexate was studied as a parenteral alternative to oral administration. An initial feasibility study was performed in Rhesus monkeys comparing the subcutaneous route to intravenous (IV) injection and oral administration. The subcutaneous dose was completely absorbed and a sustained-release effect was observed when compared with the IV dose. No local or systemic toxicities resulted from subcutaneous methotrexate in the animals. Twelve children with acute lymphoblastic leukemia on maintenance therapy protocols prescribing either 7.5 mg/m2 biweekly or 40 mg/m2 weekly were also monitored after both a subcutaneous and an oral dose of methotrexate. Four children at the higher dosage level were also studied after an equal IV dose. The subcutaneous dose was again completely absorbed in these children at both dose levels, whereas the oral dose, which produced comparable plasma drug concentrations at the lower dosage level, resulted in a total drug exposure (area under the plasma concentration-time curve) that was one third that of the equal subcutaneous dose at the higher dosage level. No local or systemic toxicity was attributed to the subcutaneous methotrexate. Subcutaneous administration of methotrexate is well tolerated and well absorbed and appears to overcome the problems associated with oral administration, including variable absorption and saturation of the absorption mechanism with increasing doses.

The pharmacokinetics of subcutaneous bolus and continuous infusion azidothymidine (AZT) was studied in rhesus monkeys. Three animals received 100 mg/m2 as a bolus injection both intravenously and subcutaneously, with the order of administration randomly determined. Two animals received a continuous subcutaneous infusion of 25 mg/m2 per h for 12 or 24 h. AZT was measured in plasma by a reverse-phase high-pressure liquid chromatographic assay. Following intravenous bolus administration, AZT elimination was rapid, with a mean half-life of 1.2 h and a mean clearance of 318 ml/min per m2 (range, 200 to 441 ml/min per m2). The bolus subcutaneous dose was rapidly (time to peak concentration, 15 to 30 min) and nearly completely (fraction absorbed, 92%) absorbed without evidence of local tissue toxicity. With continuous subcutaneous infusion of AZT, the steady state was attained within 4 h and steady-state concentrations in plasma in the two animals exceeded 3.0 mumol/liter. No local tissue toxicity was observed at the infusion site. The subcutaneous route may be a practical alternative to intravenous administration of AZT and deserves further clinical study.