Karen Godwin

Plasma and cerebrospinal fluid pharmacokinetics of intravenous temozolomide in non-human primates

Temozolomide is a prodrug that undergoes spontaneous chemical degradation at physiologic pH to form the highly reactive alkylating agent, methyl-triazenyl imidazole carboxamide (MTIC). In clinical trials, temozolomide has activity in gliomas and is approved for recurrent anaplastic astrocytoma. We, therefore, studied the penetration of temozolomide into the cerebrospinal fluid (CSF) as a surrogate for blood–brain barrier penetration in a non-human primate model. Three Rhesus monkeys with indwelling Ommaya reservoirs received 7.5mg/kg (150mg/m2) of temozolomide as a 1h intravenous infusion. Frequent blood and CSF samples were obtained over 24h, plasma was immediately separated by centrifugation at 4°C, and plasma and CSF samples were acidified with HCl. Temozolomide concentration in plasma and CSF was measured by reverse-phase high-pressure liquid chromatography. Plasma temozolomide concentration peaked 0.5h after the end of the infusion and was 104±3μM. The mean peak CSF temozolomide concentration was 26±4μM at 2.5h. The mean areas under the temozolomide concentration–time curves in plasma and CSF were 392±18 and 126±18μMh, respectively, and the CSF:plasma ratio was 0.33±0.06. Clearance of temozolomide was 0.116±0.004l/kg/h, and the volume of distribution at steady state was 0.254±0.033l/kg. In this non-human primate model, temozolomide penetrated readily across the blood–brain barrier. These findings are consistent with the activity of temozolomide in brain tumors.

Plasma and cerebrospinal fluid pharmacokinetics of 9-aminocamptothecin (9-AC), irinotecan (CPT-11), and SN-38 in nonhuman primates

Purpose: The plasma and cerebrospinal fluid (CSF) pharmacokinetics of the camptothecin analogs, 9-aminocamptothecin (9-AC) and irinotecan, were studied in a nonhuman primate model to determine their CSF penetration. Methods: 9-AC, 0.2 mg/kg (4 mg/m2) or 0.5 mg/kg (10 mg/m2), was infused intravenously over 15 min and irinotecan, 4.8 mg/kg (96 mg/m2) or 11.6 mg/kg (225 mg/m2), was infused over 30 min. Plasma and CSF samples were obtained at frequent intervals over 24 h. Lactone and total drug forms of 9-AC, irinotecan, and the active metabolite of irinotecan, SN-38, were quantified by reverse-phase HPLC. Results: 9-AC lactone had a clearance (CL) of 2.1 ± 0.9 l/kg per h, a volume of distribution at steady state (Vdss) of 1.6 ± 0.7 l/kg and a half-life (t1/2) of 3.2 ± 0.8 h. The lactone form of 9-AC accounted for 26 ± 7% of the total drug in plasma. The CSF penetration of 9-AC lactone was limited. CSF 9-AC lactone concentration peaked 30 to 45 min after the dose at 11 to 21 nM (0.5 mg/kg dose), and the ratio of the areas under the CSF and plasma concentration-time curves (AUCCSF: AUCP) was only 3.5 ± 2.1%. For irinotecan, the CL was 3.4 ± 0.4 l/kg per h, the Vdss was 7.1 ± 1.3 l/kg, and the t1/2 was 4.9 ± 2.2 h. Plasma AUCs of the lactone form of SN-38 were only 2.0% to 2.4% of the AUCs of irinotecan lactone. The lactone form of irinotecan accounted for 26 ± 5% of the total drug in plasma, and the lactone form of SN-38 accounted for 55 ± 6% of the total SN-38 in plasma. The AUCCSF: AUCP ratio for irinotecan lactone was 14 ± 3%. SN-38 lactone and carboxylate could not be measured (<1.0 nM ) in CSF. The AUCCSF: AUCP ratio for SN-38 lactone was estimated to be ≤ 8%. Conclusion: Despite their structural similarity, the CSF penetration of 9-AC and SN-38 is substantially less than that of topotecan which we previously found to have an AUCCSF: AUCP ratio of 32%.

Rescue of experimental intrathecal methotrexate overdose with carboxypeptidase-G2.

The carboxypeptidase G class of enzymes rapidly hydrolyze methotrexate (MTX) into the inactive metabolites 4-deoxy-4-amino-N10-methylpteroic acid (DAMPA) and glutamate. This study evaluated the use of carboxypeptidase-G2 (CPDG2) as a potential intrathecal (IT) rescue agent for massive IT MTX overdose. The CSF pharmacokinetics of MTX with and without CPDG2 rescue was studied in adult rhesus monkeys (Macaca mulatta) using a nontoxic IT 5 mg dose (equivalent to 50 mg in humans). Without CPDG2 rescue, peak CSF MTX concentration was 2,904 +/- 340 mumol/L. Within 5 minutes of administration of 30 U IT CPDG2, CSF MTX concentrations decreased greater than 400-fold to 6.55 +/- 6.7 microM. Subsequently, groups of three monkeys received either 25 mg IT MTX (equivalent to 250 mg in humans) followed by 150 U IT CPDG2 or 50 mg IT MTX (equivalent to 500 mg in humans) followed by 300 U IT CPDG2. All animals survived without neurotoxicity. Our studies suggest that CPDG2 may prove to be an important addition to the currently recommended strategy for the management of IT MTX overdose.

Intrathecal administration of topotecan in nonhuman primates

The cerebrospinal fluid (CSF) pharmacokinetics of topotecan were studied in a nonhuman primate model following intraventricular administration of 0.1 mg. Lactone and total drug concentrations were measured using a reverse-phase HPLC method with fluorescence detection. The mean peak concentrations of lactone and total drug in ventricular CSF were 83±18 μM and 88±25 μM, respectively. CSF drug elimination of the lactone was bi-exponential with a terminal half-life of 1.3 h. The mean clearance from ventricular CSF was 0.075 ml/min for the lactone and 0.043 ml/min for total drug. The ventricular CSF drug exposure (AUC) to lactone was 450-fold greater following intraventricular administration of 0.1 mg topotecan than after systemic intravenous administration of a 40-fold higher dose (10 mg/m2). Peak lumbar concentrations (n=1), which occurred 2 h after intraventricular drug administration, were 0.98 μM and 2.95 μM for the lactone and total drug, respectively. A transient CSF pleocytosis was observed in one animal following intraventricular topotecan administration and in one animal following intralumbar topotecan administration. No other acute or chronic neurologic or systemic toxicities were observed following a single intraventricular dose or weekly (×4) intralumbar topotecan. Compared with systemic topotecan, intrathecal administration provided a significant pharmacokinetic advantage in terms of CSF drug exposure and did not produce any significant neurotoxicity in a nonhuman primate model. Intrathecal topotecan should be evaluated clinically as a potential alternative therapy for refractory meningeal tumors.

Methotrexate pharmacokinetics following administration of recombinant carboxypeptidase-G2 in rhesus monkeys.

PURPOSE Carboxypeptidase-G2 (CPDG2) is a bacterial enzyme that rapidly hydrolyzes methotrexate (MTX) into inactive metabolites. As an alternative form of rescue after high-dose MTX (HDMTX), CPDG2 has more potential advantages than standard leucovorin (LV) rescue. In this study, the plasma pharmacokinetics of MTX with and without CPDG2 were evaluated in adult rhesus monkeys. MATERIALS AND METHODS The plasma pharmacokinetics of MTX were determined in groups of animals that had received a 300-mg/m2 loading dose of MTX followed by a 60-mg/m2/h infusion during an 18-hour period. One group received CPDG2 at the end of the infusion, and the other group served as a control. Two additional animals with high titers of anti-CPDG2 antibody also were studied. RESULTS During infusion, the steady-state MTX plasma concentration was 11.3 +/- 4.8 mumol/L. Without CPDG2, the postinfusion plasma MTX concentration remained above 0.1 mumol/L for more than 6 hours. After the administration of 50 U/kg of CPDG2, plasma MTX concentrations decreased to nontoxic levels (less than 0.05 mumol/L) within 30 minutes. The initial half-life (t1/2 alpha) of MTX decreased from 5.8 +/- 2.1 minutes to 0.7 +/- 0.02 minutes after enzyme administration. The postinfusion area under the plasma concentration time curve of MTX was 301 +/- 171 mumol/L/min without CPDG2 compared with 19.6 +/- 6.1 mumol/L/min with CPDG2. The immunogenicity studies performed indicated that although animals developed anti-CPDG2 antibodies, none of them manifested allergic symptoms. The effectiveness of CPDG2 was diminished but not eliminated in animals with high titers of anti-CPDG2 antibody. CONCLUSIONS CPDG2 is capable of rapidly decreasing plasma MTX concentrations to nontoxic levels. The administration of CPDG2 seems safe, well tolerated, and it may be useful as an alternative to LV rescue.

Pharmacokinetics of PEG-l-asparaginase and plasma and cerebrospinal fluidl-asparagine concentrations in the rhesus monkey

The pharmacokinetics of the polyethylene glycol-conjugated form of the enzymel-asparaginase and the depletion ofl-asparagine from the plasma and cerebrospinal fluid (CSF) following an i.m. dose of 2500 IU/m2 PEG-l-asparaginase was studied in rhesus monkeys. PEG-l-asparaginase activity in plasma was detectable by 1 h after injection and maintained a plateau of approximately 4 IU/ml for more than 5 days. Subsequent elimination from plasma was monoexponential with a half-life of 6±1 days. Plasmal-asparagine concentrations fell from pretreatment levels of 14–47 μM to <2 μM by 24 h after injection in all animals and remained undetectable for the duration of the 25-day observation period in four of six animals. In two animals, plasmal-asparagine became detectable when the PEG-l-asparaginase plasma concentration dropped below 0.1 IU/ml. Pretreatment CSFl-asparagine levels ranged from 4.7 to 13.6 μM and fell to <0.25 μM by 48 h in five of six animals. CSFl-asparagine concentrations remained below 0.25 μM for 10–14 days in four animals. One animal had detectable CSFl-asparagine concentrations within 24 h and another had detectable concentrations within 1 week of drug administration despite a plasma PEG-l-asparaginase activity profile that did not differ from that of the other animals. These observations may be useful in the design of clinical trials with PEG-l-asparaginase in which correlations among PEG-l-asparaginase pharmacokinetics, depletion ofl-asparagine, and clinical outcome should be sought.

The plasma pharmacokinetics and cerebrospinal fluid penetration of the thymidylate synthase inhibitor raltitrexed (Tomudex) in a nonhuman primate model

Purpose: Raltitrexed (Tomudex™), ZD1694) is a novel quinazoline folate analog that selectively inhibits thymidylate synthase. Intracellularly, raltitrexed is polyglutamated to its active form which can be retained in cells for prolonged periods. The pharmacokinetics of raltitrexed in plasma and cerebrospinal fluid (CSF) were studied in a nonhuman primate model. Methods: Animals received 3 mg/m2 (n= 1), 6 mg/m2 (n= 3), or 10 mg/m2 (n= 3) i.v. over 15 min, and frequent plasma samples were obtained over 48 h. CSF samples were drawn from an indwelling 4th ventricular Ommaya reservoir over 48 h. Plasma and CSF raltitrexed concentrations were measured with a novel, sensitive enzyme inhibition assay with a lower limit of quantification of 0.005 μM. A three-compartment pharmacokinetic model was fitted to the raltitrexed plasma concentration-time data. Results: The plasma concentration-time profile of raltitrexed was triexponential with a rapid initial decline and a prolonged terminal elimination phase (t1/2 > 24 h), which was related to retention of raltitrexed in a deep tissue compartment. At the peak approximately 30% of the administered dose was in the deep tissue compartment, and 24 h after the dosing >20% of the administered dose remained in the body with >99% in the deep tissue compartment. The mean peak (end of infusion) plasma concentrations after the 3, 6, and 10 mg/m2 doses were 1.5, 2.4 and 4.8 μM, respectively. The clearance of raltitrexed ranged from 110 to 165 ml/min per m2, and the steady-state volume of distribution exceeded 200 l/m2. The CSF penetration of raltitrexed was limited (0.6 to 2.0%) and drug could only be detected in the CSF following a 10 mg/m2 dose. Conclusions: The elimination of raltitrexed is triexponential with a prolonged terminal elimination phase. The pharmacokinetic profile is consistent with extensive polyglutamation and intracellular retention of ralitrexed. The three-compartment model presented here may be useful for the analysis of the pharmacokinetics of raltitrexed in humans.

Pharmacokinetics and cerebrospinal fluid penetration of daunorubicin, idarubicin, and their metabolites in the nonhuman primate model

PURPOSE Idarubicin (4-demethoxy-daunorubicin) is more potent and less cardiotoxic than the commonly used anthracyclines, doxorubicin and daunorubicin. In addition, idarubicin is metabolized to an active metabolite, idarubicinol, in contrast to other anthracyclines whose alcohol metabolites are much less active than the parent drug. The current study was performed in nonhuman primates to determine the plasma and cerebrospinal fluid (CSF) pharmacokinetics of idarubicin and idarubicinol and to compare them to the pharmacokinetics of daunorubicin and daunorubicinol. METHODS A dose of 30 mg/m2 of daunorubicin or 8 mg/m2 of idarubicin was administered intravenously over 15 minutes. Plasma and CSF were sampled frequently from the end of the infusion to 72 to 96 hours after infusion. Drug and metabolite concentrations were measured using high-pressure liquid chromatography (HPLC). RESULTS Daunorubicin elimination from plasma was triphasic with a terminal half-life of 5.9 +/- 1.8 hours, area under the concentration-time curve (AUC) 22.5 +/- 9.2 mumol/L.min, and clearance 2790 +/- 960 mL/min/m2. Daunorubicinol elimination was biphasic with a terminal half-life 10.2 +/- 2.3 hours and an AUC 74.5 +/- 5.3 mumol/L.min. Idarubicin elimination was triphasic with terminal half-life of 12.3 +/- 11.4 hours, a AUC 10.8 +/- 3.7 mumol/L.min, and clearance 1650 +/- 610 mL/min/m2. Idarubicinol elimination was biphasic with a terminal half-life 28.7 +/- 4.2 hours and AUC 67 +/- 9.8 mumol/L.min. CSF penetration was low for both parent drugs and their metabolites. CSF idarubicin was measurable at a single time point (1 hour after administration) for 2 animals, and was not measurable for the third. The CSF to plasma concentration ratio at that time point was 8% in 1 animal and 15% in the other. Idarubicinol was detected in 2 to 4 samples at various times, appearing as early as 1 hour in 1 animal and persisting as late as 48 hours in another. The CSF to plasma concentration ratio at corresponding time points was 1.9 +/- 0.6%. Daunorubicin was measurable for < 6 hours after intravenous administration. For individual animals, the mean CSF to plasma concentration ranged from 4% to 12%. Daunorubicinol was detectable by 1 hour in 2 of 3 animals and by 3 hours in the other, and remained detectable at 24 hours in 2 of 3. The terminal half-life of daunorubicinol in CSF was 8.8 +/- 1.3 hours, the AUC was 1.8 +/- 1.5 mumol/L.min, and the AUCCSF to AUCplasma ratio was 2.4 +/- 1.9%. CONCLUSION Idarubicin, idarubicinol, daunorubicin, and daunorubicinol penetrate poorly into the CSF after intravenous administration.

Pharmacokinetics, cerebrospinal fluid penetration, and metabolism of piroxantrone in the Rhesus monkey

SummaryPiroxantrone is an anthrapyrazole derivative with broad anti-tumor activityin vitro and less cardiac toxicity than the anthracyclines. The metabolic pathways and central nervous system penetration of piroxantrone have not been determined. In this study we examined the pharmacokinetic behavior of piroxantrone in plasma and cerebrospinal fluid in a non-human primate model. In addition, a urinary metabolite of piroxantrone was isolated and its cytotoxicity evaluatedin vitro. The disappearance of piroxantrone from plasma after an intravenous dose of 150 mg/m2 given over 60 minutes was biexponential with mean t1/2 alpha of 1.0 minutes and a mean t1/2 beta of 180 minutes. The mean area under the curve was 220 ΜM·min and the clearance was 1420 ml/min/m2. Piroxantrone was not detectable in the cerebrospinal fluid.Piroxantrone and three other compounds not present in pre-treatment samples were detected in urine. The major urinary metabolite was isolated. Its cytotoxicity against MOLT-4 cellsin vitro was at least one log less than that of piroxantrone. In addition, one of the other compounds detected in urine was determined to be a glucuronide conjugation product of the major metabolite.The results of this study may be useful in the interpretation of the activity and toxicity of piroxantrone in clinical trials.