A validated HPLC-MS/MS method for estimating the concentration of the ganglioside, GD2, in human plasma or serum.

GD2 is a ganglioside found in the plasma membrane of the neural crest-derived cancer, neuroblastoma. GD2 is shed into the circulation of patients with neuroblastoma and could serve as a tumor biomarker to monitor tumor burden or response to treatment. We developed and validated a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method to quantify the D18:1-18:0 (C18) and the D18:1-20:0 (C20) lipoforms of GD2 in human plasma and serum. Human brain derived GD2 containing a mixture of C18 and C20 was used as the analytical standard. Samples were extracted with methanol containing dueterated-GM1 (internal standard), and analytes were separated on a Phenomenex Kinetex C18 column eluted with a gradient mobile phase composed of ammonium acetate buffer, methanol and isopropanol. An AB Sciex 4500 QTRAP mass spectrometer in negative ion mode was used to quantify the doubly charged GD2 C18 and C20 lipoform precursor ions (m/z 836.8 and m/z 850.8) that both yield a product ion of m/z 290.0. The calibration curves were linear from 4-1000 ng/mL and 6-1500 ng/mL for GD2 C18 and C20 lipoforms respectively. Inter-day and intra-day accuracy were within the acceptable validation range in plasma and serum.

Phase II trial of intravenous lobradimil and carboplatin in childhood brain tumors: a report from the Children's Oncology Group.

BACKGROUND: [corrected] Lobradimil is a synthetic bradykinin analog that rapidly and transiently increases the permeability of the blood-brain barrier (BBB). The combination of lobradimil and carboplatin was studied in pediatric patients with primary brain tumors in a phase II trial, the primary endpoints of which were to estimate the response rate and time to disease progression. PATIENTS AND METHODS: Patients were stratified by histology into five cohorts: brainstem glioma, high-grade glioma, low-grade glioma, medullobastoma/primitive neuroectodermal tumor (PNET), and ependymoma. Patients received carboplatin adaptively dosed to achieve a target AUC of 3.5 mg min/ml per day (7 mg.min/ml/cycle) intravenously over 15 min on 2 consecutive days and lobradimil 600 ng/kg ideal body weight/day on 2 consecutive days each 28 day cycle. RESULTS: Forty-one patients, age 2-19 years, were enrolled; 38 patients, including 1 patient ultimately determined to have atypical neurocytoma, were evaluable for response. No objective responses were observed in the brainstem glioma (n=12) and high-grade glioma (n = 9) cohorts, although two patients with high-grade glioma had prolonged disease stabilization (>6 months). The study was closed for commercial reasons prior to achieving the accrual goals for the ependymoma (n = 8), medulloblastoma/PNET (n = 6) and low-grade glioma (n = 2) cohorts, although responses were observed in 1 patient with PNET and 2 patients with ependymoma. CONCLUSION: The combination of lobradimil and carboplatin was inactive in childhood high-grade gliomas and brainstem gliomas.

Variability in the oral bioavailability of all-trans-retinoic acid.

BACKGROUND: Orally administered all-trans-retinoic acid (all-trans-RA) can induce complete remission in a high proportion of patients with acute promyelocytic leukemia. A previous pharmacokinetic study in patients with acute promyelocytic leukemia raised the possibility that the absorption of orally administered all-trans-RA is a saturable process that would have significant clinical impact on dosing strategies. PURPOSE: This study was specifically designed to examine the saturability of all-trans-RA absorption by measuring the effect of doubling the oral dose of all-trans-RA on plasma drug concentration in patients receiving long-term oral therapy. METHODS: Six patients with solid tumors received oral doses of 10-mg gelatin capsules of all-trans-RA. Patients were studied on 2 consecutive days after they received 28 days of all-trans-RA administered as two daily 78-mg/m2 doses. The study assigned the patients to two groups. Three patients took a 156-mg/m2 dose on day 28 and a 78-mg/m2 dose on day 29; the other three patients took the lower dose on day 28 and the double dose on day 29. Blood samples for the determination of all-trans-RA plasma concentration were obtained at 30-minute intervals starting just prior to drug administration and continuing for a total of 7 hours. The plasma concentration of all-trans-RA was measured by high-performance liquid chromatography. RESULTS: Plasma concentrations following an oral dose of all-trans-RA were highly variable, with peak concentrations ranging from 0.07 to 1.2 microM for the 78-mg/m2 dose level. Doubling the dose from 78 to 156 mg/m2 increased plasma concentration in all six patients, but the increase was unpredictable and not related to dose, ranging from less than a 1.2-fold to more than a 10-fold increase. CONCLUSION: The current study does not support the hypothesis that the gastrointestinal absorption of all-trans-RA involves a saturable process but instead suggests that absorption is highly variable among patients. This wide interpatient variability suggests that pharmacokinetic drug monitoring may have an important role in the management of patients receiving all-trans-RA.

Comparison of one-, two-, and three-dimensional measurements of childhood brain tumors.

BACKGROUND: End points for assessing drug activity in brain tumors are determined by measuring the change in tumor size by magnetic resonance imaging (MRI) relative to a pretreatment or best-response scan. Traditionally, two-dimensional (2D) tumor measurements have been used, but one-dimensional (1D) measurements have recently been proposed as an alternative. Because software to estimate three-dimensional (3D) tumor volume from digitized MRI images is available, we compared all three methods of tumor measurement for childhood brain tumors and clinical outcome. METHODS: Tumor size from 130 MRI scans from 32 patients (32 baseline and 98 follow-up scans, for a total of 130 scans; median, three scans per patient; range, two to 18 scans) was measured by each method. Tumor-response category (partial response, minor response, stable disease, or progressive disease) was determined from the percentage change in tumor size between the baseline or best-response scan and follow-up scans. Time to clinical progression was independently determined by chart review. All statistical tests were two-sided. RESULTS: Concordances between 1D and 2D, 1D and 3D, and 2D and 3D were 83% (95% confidence interval [CI] = 67% to 99%), 61% (95% CI = 47% to 75%), and 66% (95% CI = 52% to 80%), respectively, on follow-up scans. Concordances for 1D and 3D and for 2D and 3D were statistically significantly lower than the concordance for 1D and 2D (P< .001 and P = .003, respectively). Concordance among 1D, 2D, and 3D methods in detecting partial response was high; there was less concordance in classifying tumors in the minor response and progressive-disease categories. Median times to progression measured by the 1D, 2D, and 3D methods were 154, 105, and 112 days, respectively, compared with 114 days based on neurologic symptoms and signs (P = .09 for overall comparison). CONCLUSIONS: Detection of partial responses was not influenced by the measurement method, but estimating time to disease progression may be method dependent for childhood brain tumors.

Dose-dependent pharmacokinetics of all-trans-retinoic acid.

BACKGROUND: Orally administered all-trans-retinoic acid (all-trans-RA) can induce remission in a high proportion of patients with acute promyelocytic leukemia. PURPOSE: To further define the drug's pharmacokinetics, a study of intravenous all-trans-RA was performed in rhesus monkeys. METHODS: A total of nine monkeys received intravenous bolus injections of all-trans-RA. Three different doses (20, 50, and 100 mg/m2) were each tested in three monkeys. Blood samples for determination of all-trans-RA concentration were obtained prior to drug administration and at 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 360, and 480 minutes after drug administration. RESULTS: Plasma disappearance of all-trans-RA was characterized by three distinct phases: a brief, initial exponential decline, followed by a relative plateau in the disappearance curve (the duration of which was dose dependent), and finally a terminal exponential decay. This profile is consistent with a capacity-limited (saturable) elimination process. The first-order (terminal) half-life for all-trans-RA averaged 19 minutes, and the mean clearances were 77, 52, and 59 mL/min for the 20-, 50-, and 100-mg/m2 dose groups, respectively. The mean +/- SD Michaelis constant (Km) for the capacity-limited process was 3.2 +/- 1.9 microM. CONCLUSIONS: Peak plasma concentrations following oral administration of 45 mg/m2 all-trans-RA in humans approach the Km for the capacity-limited process; thus, the dose-dependent pharmacokinetics of all-trans-RA described here may occur within the clinically used dosage range.

Pharmacokinetics of trimetrexate (NSC 352122) in monkeys.

The pharmacokinetics of trimetrexate was studied in Rhesus monkeys following i.v. bolus, continuous i.v. infusion, oral, and subcutaneous administration. Two methods were used to measure drug concentration in plasma, cerebrospinal fluid (CSF), and urine: the dihydrofolate reductase inhibition assay, and a reverse phase high-pressure liquid chromatography assay. The pharmacokinetic behavior of trimetrexate was characterized by triexponential plasma disappearance, elimination primarily by biotransformation, substantial plasma protein binding, poor CSF penetration, and limited oral bioavailability. Methotrexate, administered in an equimolar dose for comparison, was cleared more rapidly from plasma than was trimetrexate. Trimetrexate concentration remained above 0.1 microM 3-fold longer. In contrast to methotrexate, which is cleared almost exclusively by renal excretion, renal clearance of trimetrexate accounted for less than 5% of total clearance. A significant discrepancy was observed in plasma and urine trimetrexate concentrations measured by the two assay methods. The dihydrofolate reductase inhibition assay gave results approximately 2- to 4-fold higher in plasma. Two metabolites of trimetrexate which inhibit dihydrofolate reductase were identified in urine (one was also found in plasma) and appear to account for the different results obtained by the two assays. These metabolites would probably also interfere with the competitive protein binding assay currently being used to measure trimetrexate in ongoing phase I trials.

Pharmacokinetics of oral methotrexate in children.

The absorption and disposition kinetics of p.o. methotrexate were studied in 15 children. Serum levels and urinary excretion of methotrexate, as measured by the dihydrofolate reductase inhibition assay, were monitored following a routine p.o. dose (6.3 to 28.1 mg/sq m) administered after an overnight fast. Significant interindividual variability was noted in peak levels (range, 0.27 to 1.1 microM), time to peak (1 to 5 hr), area under the serum concentration-time curve (1.08 to 5.00 microM . hr), and the fraction of the dose absorbed (23 to 95%). Patients taking doses greater than 12 mg/sq m had a more prolonged absorptive phase and absorbed a smaller fraction of their dose, indicating that the mechanism of absorption may be saturable in some patients within the commonly administered dosage range. Urinary excretion was rapid, and the mean renal clearance of methotrexate was 1.6 times greater than was creatinine clearance, consistent with renal tubular secretion of the drug. While the marked degree of variability observed suggests a potential role for therapeutic drug monitoring in optimizing p.o. methotrexate therapy, the critical time points to monitor, the therapeutic and toxic ranges, and the intrapatient consistency of absorption must be defined before it will be practical and useful.

Preclinical pharmacology of arabinosyl-5-azacytidine in nonhuman primates.

The plasma and cerebrospinal fluid (CSF) pharmacokinetics of arabinosyl-5-azacytidine (AAC) were studied in rhesus monkeys following a 15-min, 1-h, or 12-h i.v. infusion of 200 mg/kg. No clinically significant toxicity was observed with these schedules. The plasma elimination of AAC is rapid and characterized by a triphasic decay with t1/2 alpha = 3.6-5.4 min, t1/2 beta = 18-24 min, and t1/2 gamma = 94-144 min for the above infusion schedules. The CSF penetration of AAC as measured by the CSF:plasma Css ratio for the 12-h infusion was 0.15. The stability of AAC in pooled plasma, phosphate buffered saline, and RPMI 1640 culture media at 37 degrees C was compared with the terminal half-life of AAC observed in vivo. The shorter in vitro AAC half-life in plasma with or without tetrahydrouridine versus that in phosphate buffered saline suggests that the terminal half-life of AAC in vivo is most likely a result of enhanced nucleophilic attack and hydrolytic degradation of the unstable triazine ring in plasma. A triexponential equation modeling the disappearance of AAC was constructed from the in vivo experimental data. Use of this equation in computer-aided simulations of current Phase I doses and schedules of AAC correctly predicts the human plasma concentrations which have been observed. The preclinical pharmacokinetic data provided here may be useful in helping to develop rational human studies with specific concentration x time goals.

Plasma and cerebrospinal fluid pharmacokinetic study of topotecan in nonhuman primates.

Topotecan, a water soluble semisynthetic analogue of camptothecin, is a topoisomerase I inhibitor that has recently entered phase II clinical trials. Topotecan has shown significant preclinical activity in refractory murine tumors and in human tumor xenograft models. In addition, objective antineoplastic activity has been observed in recent adult phase I clinical trials. Topotecan is unstable in solution and is rapidly and spontaneously converted to a less active open ring form which predominates at physiological pH. This study was undertaken to better define the pharmacokinetic behavior of this highly unstable compound in both plasma and cerebrospinal fluid (CSF) and to measure the degree of CSF penetration of this novel antineoplastic agent. Three nonhuman primates with indwelling Ommaya reservoirs received 10 mg/m2 i.v. topotecan administered as a 10-min infusion. Frequent plasma and CSF samples were obtained and immediately extracted and assayed with a reverse phase high performance liquid chromatography assay to quantitate the concentration of topotecan (lactone). Samples were then acidified and reinjected to quantitate total drug (lactone ring plus open ring). Peak plasma concentrations of topotecan ranged from 0.27 to 0.45 microM. Plasma disappearance of the lactone ring was biexponential with a distribution half-life (t1/2 alpha) of 22 +/- 5 min and an elimination half-life (t1/2 beta) of 1.3 +/- 0.1 h. Total body clearance of topotecan was 72.1 +/- 15.8 liters/h/m2. The volume of distribution at steady state was 88.6 +/- 33.2 liters/m2. Peak CSF concentrations of topotecan occurred at 30 min following drug administration and ranged from 0.044 to 0.074 microM. CSF disappearance paralleled that in plasma. The mean ratio of the area under the CSF concentration-time curve to that in plasma was 0.32 (range, 0.29 to 0.37). The mean CSF penetration of topotecan exceeds 30%, which is significantly greater than the penetration of most structurally similar chemotherapeutic agents. The impact of chemotherapy on the survival of patients with primary or metastatic central nervous system malignancies is very limited. Therefore, this novel antineoplastic agent is an excellent candidate for further study in patients with high risk or refractory central nervous system tumors.

Prolongation of drug exposure in cerebrospinal fluid by encapsulation into DepoFoam.

Prolonged maintenance of a therapeutic drug concentration in the cerebrospinal fluid is required for optimal treatment of leptomeningeal leukemia or carcinomatosis with cell cycle-specific antimetabolites. The pharmacokinetics of 1-beta-D-arabinofuranosylcytosine (ara-C) encapsulated into DepoFoam (Depo/Ara-C) was studied in six rhesus monkeys after intrathecal injection into the lumbar sac. Following a single 2-mg dose, the Depo/Ara-C concentration decreased biexponentially with initial and terminal half-lives of 14.6 and 156 h, respectively. The free drug concentration remained above the reported minimal cytotoxic level of 0.1 micrograms/ml (0.4 microM) for more than 672 h (28 days). In contrast, the half-life of ara-C following an intralumbar bolus dose of unencapsulated drug in a single animal was 0.74 h. A single intrathecal injection of Depo/Ara-C can maintain a therapeutic drug concentration in the cerebrospinal fluid for a very prolonged period.

Pharmacokinetics of pyrazoloacridine in the rhesus monkey.

Pyrazoloacridine is a rationally synthesized acridine derivative with in vitro activity against solid tumor cell lines, noncycling and hypoxic cells, and tumor cell lines that exhibit the multidrug resistance phenotype. The pharmacokinetic behavior of pyrazoloacridine after a 1- or 24-h i.v. infusion was studied in 5 rhesus monkeys that received a total of 10 courses of pyrazoloacridine at 300 or 600 mg/m2. Pyrazoloacridine levels in plasma and cerebrospinal fluid were measured by high-pressure liquid chromatography. For 1-h infusions, the plasma disappearance was biexponential with a t 1/2 alpha of 31 min and t 1/2 beta of 11 h. The mean volume of distribution at steady state was 1380 liters/m2. The clearance was 1660 ml/min/m2. For the 300 mg/m2 dose, the mean area under the concentration-time curve was 759 microM.min, and the mean peak concentration was 1.3 microM. For the 600 mg/m2 dose, the area under the concentration-time curve was 1330 microM.min, and the peak concentration was 2.5 microM. The steady-state plasma concentrations during the 24-h continuous infusions were 0.27 microM for the 300 mg/m2 dose and 0.45 microM for the 600 mg/m2 dose. The mean clearance calculated from these steady-state concentrations was 2420 ml/min/m2. Cerebrospinal fluid levels were less than 0.1 microM for all doses and schedules. There was no evidence of toxicity at any dose or schedule. These results contrast strikingly with those obtained in mice and dogs in which, despite a more rapid clearance of pyrazoloacridine, significant toxicities were observed at doses that were nontoxic in the monkey. These interspecies differences in the pharmacokinetic and pharmacodynamic behavior of pyrazoloacridine have important implications for the design of Phase I trials in humans.

A pharmacokinetic model of topotecan clearance from plasma and cerebrospinal fluid.

We present a physiological pharmacokinetic model that describes the plasma and cerebrospinal fluid (CSF) concentrations of topotecan [(S)-9-dimethylaminomethyl-10-hydroxyamptothecin hydrochloride, SK&F 104864-A, NSC 609699] following i.v. and intraventricular administrations in monkeys. The model consists of three physical spaces: the CSF, the plasma, and a body compartment. The model incorporates such processes as reversible conversion of topotecan lactone to an inactive hydroxy acid form, microvascular exchange between CSF and plasma, bulk CSF flow, exchange between plasma and body compartments, and elimination of drug from the plasma compartment. Several parameters in the model were obtained from published literature on the physiology of the monkey. The model was then fit to the plasma and CSF data to deduce the other parameters. Calculated clearances of topotecan lactone and total drug from the CSF after intraventricular injection were 3.9 and 2.2 ml/h, respectively. Clearances of topotecan lactone and total drug from the plasma following a 10-min infusion were 26.3 liters/h/m2 and 17.8 liters/h/m2, respectively. The calculated ratios of the area under the concentration curve in the CSF following i.v. infusion to the area under the concentration curve in plasma were 0.11 and 0.19 for topotecan and total drug, respectively, indicating significant CSF penetration. The volume of distribution was 0.77 liters/kg, which represents distribution in a volume approximating total body water. The forward and reverse rate constants for the lactone-to-hydroxy acid conversion were 1.0 and 0.29 h-1, respectively. Comparison of the clearances (normalized to body surface area) with values reported for mice and humans shows reasonable similarity across species. This pharmacokinetic model may help guide future development and refinement of clinical protocols, especially in the treatment of diseases of the central nervous system.

Plasma and cerebrospinal fluid pharmacokinetics of O6-benzylguanine and time course of peripheral blood mononuclear cell O6-methylguanine-DNA methyltransferase inhibition in the nonhuman primate.

O6-Benzylguanine (O6BG) enhances the cytotoxicity of the nitrosoureas by irreversibly binding and inhibiting the DNA repair enzyme O6-methyl-guanine-DNA methyltransferase (MGMT). The plasma and cerebrospinal fluid (CSF) pharmacokinetics of O6BG and its active metabolite, O6-benzyl-8-oxoguanine, were studied in a nonhuman primate model after 200 mg/m2 had been injected i.v. The parent drug and the metabolite were measured with a reverse-phase HPLC assay. A pharmacokinetic model incorporating separate compartments for O6BG and the O6-benzyl-8-oxoguanine metabolite, first-order conversion of O6BG to the metabolite, and additional first-order elimination rate constants for each compound, was simultaneously fitted to the parent drug and metabolite plasma concentration time data. Elimination of O6BG from plasma was rapid; it had a half-life of 1.6 h and a clearance of 68 ml/min/m2. On the basis of the pharmacokinetic model, essentially all of the O6BG was converted to O6-benzyl-8-oxoguanine. The plasma pharmacokinetic profile of the metabolite differed considerably from that the parent drug. The half-life (14 h) was 10-fold longer and the area under the curve (2420 microM/h) was 11-fold higher than that of O6BG (212 microM/h). The clearance rate of O6-benzyl-8-oxoguanine was 6.4 ml/min/m2. The CSF:plasma ratio was 4.3% for O6BG and 36% for O6-benzyl-8-oxoguanine, and the metabolite area under the curve was 90-fold higher than that of O6BG in CSF. The excellent CSF penetration of the active metabolite provides a rationale for the use of O6BG as a chemosensitizing agent for brain tumors. We also studied the duration of MGMT inhibition in peripheral blood mononuclear cells. By 2 h after a 200 mg/m2 dose of O6BG, > 98% of MGMT activity was suppressed, and > 95% suppression of enzyme activity persisted at 18 and 48 h after the dose. By 2 weeks after the treatment, MGMT levels had returned to baseline. Persistent high concentrations of the active metabolite appear to provide a pharmacological explanation for the prolonged suppression of MGMT activity.

Pharmacokinetics of 9-cis-retinoic acid in the rhesus monkey.

9-cis-Retinoic acid is a naturally occurring biologically active retinoid capable of binding and transactivating both the retinoic acid receptors and the retinoid X receptors. A study was performed to characterize the pharmacokinetics 9-cis-retinoic acid following i.v. bolus administration in the nonhuman primate. Groups of three animals received i.v. bolus doses of 9-cis-retinoic acid of either 50 or 100 mg/m2. Blood and cerebrospinal fluid samples for determination of 9-cis-retinoic acid concentration were obtained prior to and 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 360, and 480 min following drug administration. The plasma drug concentration profile of 9-cis-retinoic acid was consistent with a first-order elimination process, with a harmonic mean half-life of 31 min, and a mean clearance of 97 ml/min/m2. The pharmacokinetics of 9-cis-retinoic acid were linear over the dose range studied. Plasma concentrations of all-trans-retinoic acid following 9-cis-retinoic acid administration were less than the limit of quantitation (0.1 microM), suggesting that isomerization to all-trans-retinoic acid is not a major metabolic pathway. In contrast to all-trans-retinoic acid, the elimination of 9-cis-retinoic acid did not appear to be capacity limited (saturable). Previous studies in the Rhesus monkey have shown that repeated dosing with all-trans-retinoic acid leads to a reduction of this saturable component of elimination and results in reduced exposure to drug. These studies, in an animal model highly predictive of humans, suggest that declines in plasma concentrations of 9-cis-retinoic acid as a result of its repeat administration at doses up to 100 mg/m2 will not occur.

Pharmacokinetics of tiazofurin in the plasma and cerebrospinal fluid of rhesus monkeys.

The pharmacokinetic disposition of tiazofurin in plasma and cerebrospinal fluid was examined in rhesus monkeys. Tiazofurin was readily detectable in both plasma and cerebrospinal fluid within 20 min of commencement and for 24 h after a short i.v. infusion of the drug. The mean clearance of tiazofurin from plasma was 70 +/- 23 (SD) ml/min/sq m after a dose of 100 mg/kg and 106 +/- 38 ml/min/sq m after a dose of 500 mg/kg with no evidence of dose dependency. The data for plasma elimination of tiazofurin were fit to a triexponential equation for comparison with data from other species. The t 1/2 alpha was 0.23 h, t 1/2 beta was 1.9 to 2.0 h, and t 1/2 gamma was 6.8 to 7.1 h. The ratio of area under the cerebrospinal fluid drug concentration-time curve to the area under the plasma drug concentration-time curve was 0.28, which suggests significant penetration of the blood-brain barrier. These results demonstrate the propensity of tiazofurin to enter the cerebrospinal fluid and, probably, the brain, and suggest a potential role for this agent in the treatment of central nervous system cancer.

Intrathecal administration of 4-hydroperoxycyclophosphamide in rhesus monkeys.

The preactivated cyclophosphamide analogue, 4-HC, does not require activation by hepatic microsomal enzymes to express its cytotoxic activity and therefore, unlike cyclophosphamide, may be useful for the regional therapy of cancer. In the present study, the pharmacokinetics and toxicology of 4-HC were studied following intraventricular administration of 0.4 mg to rhesus monkeys with chronic indwelling Ommaya reservoirs. 4-HC was measured in cerebrospinal fluid (CSF) and plasma with a high-performance liquid chromatography assay utilizing a fluorometric detector following derivatization with m-aminophenol. The mean peak level of 4-HC in ventricular CSF was 100 microM 5 min after administration. The drug was cleared rapidly and the elimination was monoexponential with a mean half-life of 22 min. The mean clearance from CSF (0.33 ml/min) was 10-fold higher than CSF bulk flow. The drug was distributed throughout the subarachnoid space with lumbar levels approaching ventricular levels by 60 min. Neither acute nor chronic neurotoxicity or systemic toxicity was observed during the 6-wk observation period. Concentrations of 4-HC demonstrated to be cytocidal in vitro against human breast cancer, lymphoid leukemia, and rhabdomyosarcoma were readily achieved in CSF following intraventricular administration. This study demonstrates that intraventricular therapy with 4-HC is feasible and suggests that further study of this approach in the clinical setting should be considered.

Cerebrospinal fluid penetration of active metabolites of cyclophosphamide and ifosfamide in rhesus monkeys.

The penetration of the active metabolites of cyclophosphamide (CP) and ifosfamide (IF) into cerebrospinal fluid (CSF) was determined in rhesus monkeys following an i.v. infusion of 1 gm/m2 of CP and IF. Active metabolites were measured using a high-performance liquid chromatography assay with fluorometric detection following derivatization with m-aminophenol. CSF to blood ratios of the active metabolites of CP and IF were found to be 0.17 and 0.13 following systemic dosing of CP and IF, respectively. The levels achieved in the CSF, however, were equivalent to levels known to be cytocidal to malignant cell lines derived from tumors which metastasize to the central nervous system. Only one animal demonstrated neurotoxicity with IF. CSF levels of active metabolite in this animal were similar to those observed in the other animals.

Modulation of the cytotoxic effect of cyclopentenylcytosine by its primary metabolite, cyclopentenyluridine.

Cyclopentenylcytosine (CPE-C), a synthetic cytidine analogue with significant preclinical antitumor activity against both solid tumor xenografts and 1-beta-D-arabinofuranosylcytosine resistant murine leukemia cell lines, will soon enter phase I clinical trials. Unlike 1-beta-D-arabinofuranosylcytosine which is activated by deoxycytidine kinase, the enzyme responsible for the phosphorylation of CPE-C is uridine/cytidine kinase. Preclinical pharmacokinetic studies of CPE-C in nonhuman primates revealed that the primary route of elimination in this species was deamination to cyclopentenyluridine (CPE-U), an inhibitor of uridine/cytidine kinase. Since CPE-C is likely to be deaminated in humans, we investigated the modulating effect of CPE-U on the in vitro cytotoxicity of CPE-C in Molt-4 lymphoblasts. Concurrent exposure of cells to cytotoxic concentrations of CPE-C and 50 microM CPE-U resulted in the rescue of 50% of cells and exposure to CPE-U concentrations in excess of 100 microM resulted in the rescue of greater than 90% of cells. Progressive attenuation of the rescue effect was observed with delayed administration of CPE-U and no cells were rescued when addition of CPE-C was delayed for more than 2 h. At the intracellular level it was observed that the formation of the cytotoxic metabolite, cyclopentenylcytosine triphosphate, was blocked by increasing concentrations of CPE-U presumably secondary to inhibition of uridine/cytidine kinase by CPE-U. Although CPE-U can modulate the cytotoxic effects of CPE-C in vitro, the minimum CPE-U levels that are required for modulation coupled with the available preclinical pharmacokinetic data from nonhuman primates suggests that this modulation is not likely to impact on the antitumor effects of CPE-C in humans.

Pediatric phase I trial and pharmacokinetic study of topotecan administered as a 24-hour continuous infusion.

Topotecan, a water-soluble semisynthetic analogue of camptothecin, is the first topoisomerase I inhibitor to undergo evaluation in pediatric patients with refractory malignancies. A phase I and pharmacokinetic study was performed to determine the maximum tolerated dose (MTD) and dose-limiting toxicities, the incidence and severity of other toxicities, and the pharmacokinetics of topotecan in children. Twenty-nine patients received 42 courses of i.v. topotecan administered as a 24-h continuous infusion every 21 days at doses ranging from 2.0 to 7.5 mg/m2. Dose-related hematological toxicity was the dose-limiting toxicity. Leukopenia, neutropenia, and thrombocytopenia occurred sporadically at the 3.0- to 5.5-mg/m2 dose levels, but at 7.5 mg/m2 4 of 5 patients experienced dose-limiting thrombocytopenia (grade 4) and 2 of 5 had dose-limiting neutropenia (grade 4). No other dose-limiting toxicities were observed. Nausea and vomiting were mild and occurred in < 20 and 10% of patients, respectively. Grade 2 hematuria occurred in one patient. No objective responses were observed. Pharmacokinetic studies revealed a linear relationship between the steady-state topotecan concentration and dose. The mean steady-state concentration at the MTD was 18.2 +/- 3.7 nmol/liter and the total body clearance was 28.3 +/- 6.5 liters/h/m2. Elimination was biexponential with a t1/2 alpha of 14.4 +/- 1.8 min and a t1/2 beta of 2.9 +/- 1.1 h. The recommended starting dose for phase II pediatric trials is 5.5 mg/m2. Although this dose exceeds the MTD identified in heavily pretreated adult patients receiving topotecan on the same schedule, it is less than the MTD for minimally pretreated adult patients. Therefore, dose escalation to 7.5 mg/m2 in phase II pediatric trials should be considered for patients who tolerate treatment well at the 5.5-mg/m2 dose.

A phase I study of continuous infusion 5-fluorouracil plus calcium leucovorin in combination with N-(phosphonacetyl)-L-aspartate in metastatic gastrointestinal adenocarcinoma.

Preclinical studies suggest that the biochemical effects of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate carbamoyltransferase (ACTase), may increase the metabolic activation of 5-fluorouracil (5-FU) and enhance its cytotoxicity through both RNA- and DNA-directed mechanisms. In this Phase I trial, 22 evaluable patients with adenocarcinoma of the gastrointestinal tract were entered at escalating doses of 5-FU starting at 1150 mg/m2/day given as a concurrent 72-h i.v. infusion with a fixed dose of leucovorin (LCV), 500 mg/m2/day. The dose of 5-FU was escalated within patients according to individual tolerance, and then PALA at 250 mg/m2 was added 24 h prior to the initiation of the 5-FU/LCV infusion of the subsequent cycle. Dose-limiting mucositis and myelosuppression occurred during the initial cycle in 3 of 5 patients treated with 2300 mg/m2/day 5-FU; therefore, the recommended dose of 5-FU with concurrent LCV is 2000 mg/m2/day. Twenty-seven additional patients were then treated with escalating doses of PALA ranging from 375 to 2848 mg/m2, i.v., followed 24 h later by 2000 mg/m2/day 5-FU with high-dose LCV. Dose-limiting mucositis and myelosuppression occurred during the initial cycle in 2 of 3 patients entered at 2848 mg/m2 PALA. Dose-limiting mucositis and skin rash ultimately required both PALA and 5-FU dose reductions in 4 of 6 patients treated with 1899 mg/m2 PALA. Toxicity was similar, however, in patients receiving PALA at doses ranging from 375 to 1266 mg/m2. The mean steady-state plasma concentration of 5-FU at 2000 mg/m2/day was 6.5 +/- 0.9 microM; patients with 5-FU levels > 9 microM had a significantly higher incidence of serious gastrointestinal and hematological toxicity. Compared to each patient's own baseline, a significant trend for decreasing ACTase activity with increasing PALA dose was evident using cytosol isolated from peripheral blood mononuclear cells 24 h after PALA treatment (P2 = 0.01). PALA < or = 844 mg/m2 failed to appreciably inhibit ACTase activity at 24 h in most patients; furthermore, a decrease in ACTase activity by > 50% from baseline was seen in only 29% of cycles. More consistent inhibition of ACTase activity was seen with PALA > or = 1266 mg/m2. Even with the highest PALA doses, however, ACTase activity returned to baseline by 96 h in most patients.(ABSTRACT TRUNCATED AT 400 WORDS)