Concentration rather than dose defines the local brain toxicity of agents that are effectively distributed by convection-enhanced delivery.

BACKGROUND: Convection-enhanced delivery (CED) has been developed as a potentially effective drug-delivery strategy into the central nervous system. In contrast to systemic intravenous administration, local delivery achieves high concentration and prolonged retention in the local tissue, with increased chance of local toxicity, especially with toxic agents such as chemotherapeutic agents. Therefore, the factors that affect local toxicity should be extensively studied. NEW METHOD: With the assumption that concentration-oriented evaluation of toxicity is important for local CED, we evaluated the appearance of local toxicity among different agents after delivery with CED and studied if it is dose dependent or concentration dependent. RESULTS: Local toxicity profile of chemotherapeutic agents delivered via CED indicates BCNU was dose-dependent, whereas that of ACNU was concentration-dependent. On the other hand, local toxicity for doxorubicin, which is not distributed effectively by CED, was dose-dependent. Local toxicity for PLD, which is extensively distributed by CED, was concentration-dependent. COMPARISON WITH EXISTING METHOD: Traditional evaluation of drug induced toxicity was dose-oriented. This is true for systemic intravascular delivery. However, with local CED, toxicity of several drugs exacerbated in concentration-dependent manner. From our study, local toxicity of drugs that are likely to distribute effectively tended to be concentration-dependent. CONCLUSION: Concentration rather than dose may be more important for the toxicity of agents that are effectively distributed by CED. Concentration-oriented evaluation of toxicity is more important for CED.

Safety and feasibility of convection-enhanced delivery of nimustine hydrochloride co-infused with free gadolinium for real-time monitoring in the primate brain.

OBJECTIVES: Convection-enhanced delivery (CED) has been developed as an effective drug-delivery strategy for brain tumors. Ideally, direct visualization of the tissue distribution of drugs infused by CED would assure successful delivery of therapeutic agents to the brain tumor while minimizing exposure of the normal brain tissue. We previously showed the anti-tumor efficacy of nimustine hydrochloride (ACNU) delivered via CED against a rodent intracranial xenografted tumor model. Here, we developed a method to monitor the drug distribution using a non-human primate brain. METHODS: CED of a mixture of ACNU with gadodiamide was performed using three non-human primates under real-time magnetic resonance imaging monitoring. Animals were clinically observed for any toxicity after infusion. Two months later, their brains were subjected to histological examination for the evaluation of local toxicity. Another one animal was euthanized immediately after CED of a mixture of ACNU, gadodiamide, and Evans blue dye to evaluate the concordance between ACNU and gadodiamide distributions. The harvested brain was cut into blocks and the ACNU content was measured. RESULTS AND DISCUSSION: Real-time magnetic resonance imaging monitoring of co-infused gadodiamide confirmed the success of the infusion maneuver. In the monkey that also received Evans blue, the distribution of Evans blue was similar to that of gadodiamide and paralleled the measured ACNU content, suggesting concordance between ACNU and gadodiamide distributions. Histological examination revealed minimum tissue damage with the infusion of ACNU at 1 mg/ml, determined as a safe dose in our previous rodent study. CED of ACNU can be co-administered with gadodiamide to ensure successful infusion and monitor the distribution volume.

Safety and efficacy of convection-enhanced delivery of ACNU, a hydrophilic nitrosourea, in intracranial brain tumor models.

Convection-enhanced delivery (CED) is a local infusion technique, which delivers chemotherapeutic agents directly to the central nervous system, circumventing the blood-brain barrier and reducing systemic side effects. CED distribution is significantly increased if the infusate is hydrophilic. This study evaluated the safety and efficacy of CED of nimustine hydrochloride: 3-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU), a hydrophilic nitrosourea, in rat 9 L: brain tumor models. The local neurotoxicity of ACNU delivered via CED was examined in normal rat brains, and the maximum tolerated dose (MTD) was estimated at 0.02 mg/rat. CED of ACNU at the MTD produced significantly longer survival time than systemic administration (P < 0.05, log-rank test). Long-term survival (80 days) and eradication of the tumor occurred only in the CED-treated rats. The tissue concentration of ACNU was measured by high-performance liquid chromatography, which revealed that CED of ACNU at the dose of 100-fold less total drug than intravenous injection carried almost equivalent concentrations of ACNU into rat brain tissue. CED of hydrophilic ACNU is a promising strategy for treating brain tumors.

Pharmacokinetics of nimustine, cytosine arabinoside, and methotrexate in cerebrospinal fluid during cerebrospinal fluid perfusion chemotherapy.

This report investigates the pharmacokinetics of nimustine (ACNU), cytosine arabinoside (Ara-C), and methotrexate (MTX) in cerebrospinal fluid (CSF) during CSF perfusion chemotherapy. A 47-year-old Japanese man with spinal cord, cerebellum and brain stem dissemination of oligo-astrocytoma received nine courses of CSF perfusion chemotherapy with ACNU, Ara-C, and MTX. A CSF perfusion chemotherapy solution was perfused via an Ommaya reservoir in the ventricle, and was discharged by drainage though another Ommaya reservoir in the lumbar spinal canal. CSF samples via Ommaya reservoirs in the lumbar spinal canal were obtained during the fifth and eighth courses of treatment. The concentrations of ACNU and Ara-C in CSF were measured by HPLC, and the MTX concentrations by fluorescence polarization immunoassay. In the fifth course of treatment, a CSF injection chemotherapy solution, consisting of 5 mg of ACNU dissolved in 20 ml of artificial CSF, was injected over a few minutes using the Ommaya reservoir. Next, a CSF perfusion chemotherapy solution, consisting of 10 mg of Ara-C and 5 mg of MTX dissolved in 100 ml of artificial CSF, was perfused over 2 h. In the eighth course of treatment, a CSF perfusion chemotherapy solution, consisting of 5 mg of ACNU, 10 mg of Ara-C and 5 mg of MTX dissolved in 100 ml of artificial CSF, was perfused over 2 h. In both treatments, the highest concentrations of Ara-C and MTX in CSF were observed 1 or 2 h after the end of perfusion, with the values of each drug being similar. The CSF AUCs of Ara-C and MTX in each treatment were of similar values. Although the highest concentration of ACNU in CSF was observed in the fifth treatment 1 h after injection (an injection chemotherapy of ACNU plus a perfusion chemotherapy of Ara-C and MTX), the concentration of ACNU in CSF was undetectable in the eighth treatment (a perfusion chemotherapy of ACNU, Ara-C and MTX). We were successful in administering all anticancer drugs, and reaching a level of over 1.0 microg/ml concentration in CSF of the lumbar spinal canal, using an injection chemotherapy of ACNU plus a perfusion chemotherapy of Ara-C and MTX; this was done even though the drugs, in particular ACNU, underwent some perfusion-period dependent decomposition.

Pharmacokinetics of cytosine arabinoside, methotrexate, nimustine and valproic acid in cerebrospinal fluid during cerebrospinal fluid perfusion chemotherapy.

This report investigates the pharmacokinetics of cytosine arabinoside (Ara-C), methotrexate (MTX), nimustine (ACNU) and valproic acid (VPA) in cerebrospinal fluid (CSF) during CSF perfusion chemotherapy. A 28-year-old Japanese woman with disseminated glioblastoma was, on admission, on a stable oral regimen of prolonged-release VPA tablets (Depakene-R), 400 mg twice a day, for seizure control. Twelve courses of CSF perfusion chemotherapy with Ara-C, MTX, and ACNU were administered. Plasma samples and CSF samples via Ommaya reservoirs were obtained during the eleventh course of treatment. The Ara-C and ACNU concentrations were measured by HPLC. The MTX and VPA concentrations were measured by fluorescence polarization immunoassay. During CSF perfusion chemotherapy, the highest CSF concentrations of Ara-C, MTX, and ACNU were observed at the end of the perfusion and decreased in a monoexponential pattern. The half-lives of Ara-C, MTX, and ACNU were 2.65, 3.52, and 0.71 h, respectively. No anticancer drugs were detectable in plasma during CSF perfusion chemotherapy. Before CSF perfusion chemotherapy, the free VPA concentration in plasma was 14.4% of the total VPA concentration. The mean total and free VPA concentrations in plasma were 78.0+/-0.8 and 10.9-0.3 microg/ml, respectively. The free VPA concentrations in plasma and in CSF were of similar values. At the end of perfusion, the lowest free VPA concentration in CSF was 30.3% of that at the initiation of perfusion. The free VPA concentrations in CSF at 3, 7, 23, and 47 h after the end of perfusion were 79.8, 94.5, 100.9, and 100.9% respectively of that at the initiation of perfusion. During CSF perfusion chemotherapy, the ratio of free VPA concentrations to the total VPA in CSF was 86.3+/-6.9%. The VPA concentrations in CSF rapidly decreased during the CSF perfusion but recovered to pre-treatment levels within 7 h.

ACNU, MTX and 5-FU penetration of rat brain tissue and tumors.

The distribution of radio-labeled ACNU, MTX and 5-FU in brain and tumor tissue was studied in female Wistar rats by macroautoradiography after intrathecal administration. In normal rats, ACNU and 5-FU, administered intracisternally, distributed rapidly in the subarachnoid space, ventricular system and cerebrospinal fluid (CSF). 5-FU and MTX penetrated the brain deeply; the diffusional transport of ACNU was limited to a depth of 1 or 2 mm from the CSF surface of the brain. MTX and 5-FU clearance into the blood circulation was rather slow while ACNU cleared relatively quickly. The half time of ACNU, 5-FU and MTX radioactivity at the ventricular surface was 10, 21, and 110 min, respectively, at their maximal concentration after intracisternal administration. In rats with leptomeningeal tumor induced by intracisternal inoculation of Walker 256 cells, the distribution patterns of ACNU, 5-FU, and MTX were essentially the same as in normal rats despite 10-20 cell layers of tumor growing in the subarachnoid space. 5-FU and MTX were able to penetrate tumor masses in the subarachnoid space; MTX penetration was slower than that of 5-FU and ACNU failed to penetrate to more than a depth of 1 or 2 mm from the tumor surface.

Pharmacokinetics of anticancer drugs in cerebrospinal fluid.

OBJECTIVE: To examine the pharmacokinetics of anticancer drugs in the cerebrospinal fluid (CSF) during chemotherapy by the lumbar-ventricular (LV) and ventricular-lumbar (VL) routes. CASE SUMMARY: A 69-year-old Japanese woman with disseminated glioblastoma received two LV and four VL courses of CSF perfusion chemotherapy with methotrexate, nimustine, and cytarabine hydrochloride. Samples of CSF from the ventricles and lumbar spinal canal were obtained via Ommaya reservoirs during one LV and one VL course. Drug concentrations in the CSF were measured by fluorescence polarization immunoassay or HPLC. RESULTS: During LV CSF perfusion, the highest CSF drug concentrations in both the ventricles and the lumbar spinal canal were observed at the end of perfusion. During treatment, the concentrations of all three drugs in the lumbar spinal canal were higher than those in the ventricles. The CSF AUC of methotrexate in the ventricles was 16.1% of that in the lumbar spinal canal. During VL CSF perfusion, the highest drug concentrations were also observed at the end of perfusion. The drug concentrations in the lumbar spinal canal were initially lower than those in the ventricles. However, the concentrations of methotrexate and cytarabine in the lumbar spinal canal exceeded those in the ventricles 3 hours after perfusion. The AUC of methotrexate in the lumbar spinal canal was 174.9% of that in the ventricles. CONCLUSIONS: The pharmacokinetics of anticancer drugs in ventricular CSF differ from those in lumbar CSF during LV and VL perfusion chemotherapy.

A strategy for selective anti-cancer drug concentration increase in rat glioma tissue with Ca(2+)-channel blocker co-administration: calcium kinetics in intra-glioma arteriolar smooth muscle cells.

A rat glioma model was employed to estimate the Ca2+ kinetics in the tumor arteriolar smooth muscle cells. Electron microcytochemistry revealed that the density of intracellular Ca2+ deposits in the intra-tumor arteriolar smooth muscle cells was significantly greater, with slightly higher membrane Ca(2+)-adenosine triphosphatase (ATPase) activity, compared to the contralateral cerebral arterioles. Furthermore, the administration of tyrphostin, a tyrosine kinase inhibitor, specifically increased only the intra-tumor blood flow. These findings suggest that the condition of the intra-tumor arteriole alters the susceptibility to contraction by the accelerated Ca2+ influx into the cytoplasm mediated through the tyrosine kinase pathway. After the administration of diltiazem, which also has a blocking effect on the Ca(2+)-channel mediated through this pathway, the local intra-tumor blood flow showed an increase of 39% with a marked decrease of intracellular Ca2+ concentration of the arteriolar smooth muscle cells in the tumor, while the blood flow in the basal ganglia increased by only 8%. The intra-tumor concentration of Nimustine-HCl (ACNU) with co-administration of diltiazem was significantly increased compared to that without the co-administration. Co-administration of diltiazem may be a valuable strategy in chemotherapy for glioma in affording the selective increase of intra-tumor concentration of the anti-cancer drug.

Timing of hypertonic glucose and thermochemotherapy with 1-(4-amino-2-methylpyrimidine-5-yl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) in the BT4An rat glioma: relation to intratumoral pH reduction and circulatory changes after glucose supply.

PURPOSE: Intraperitoneal hypertonic glucose has previously been shown to induce hyperglycemia, hemo-concentration, and to influence systemic and tumor circulation, and, thus, enhance the effect of thermochemotherapy with 1-(4-amino-2-methylpyrimidine-5-yl)methyl-3-(2-chloroethyl)-3-nitrosoure a (ACNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). However, the optimal timing and the precise mechanisms responsible are not known. The effect of different time intervals between glucose load and thermochemotherapy with ACNU in the treatment of BT4An tumors, therefore, was investigated. Changes of serum glucose (Se-glucose), hemoglobin, systemic circulation parameters, tumor pH, and tumor temperature, induced by intraperitoneal glucose and/or hyperthermia, were measured to assess their effect on tumor growth. METHODS AND MATERIALS: (a): Inbred BD IX rats with BT4An tumors on the hind leg were treated with ACNU 7 mg/kg intravenously just before waterbath hyperthermia, and intraperitoneal hypertonic glucose (6 g/kg) at different time intervals before (240-0 min) or immediately after thermochemotherapy. (b): Intratumoral pH and temperature were measured at different intervals after intraperitoneal glucose, and during hyperthermia with or without previous glucose. (c): Hemoglobin, hematocrit, and Se-glucose were measured at different times after intraperitoneal glucose. (d): Mean arterial pressure, pulse pressure, and heart rate were measured for 120 min after intraperitoneal glucose. RESULTS: (a): The number of tumor controls and the growth delay was greatest with glucose 45 min before thermochemotherapy, and least with a time interval of 240 min. Glucose after thermochemotherapy delayed tumor growth. (b): After intraperitoneal glucose alone, intratumoral pH decreased gradually from 6.76 to 5.86 after 240 min. Hyperthermia 120 min after glucose induced a rapid further pH drop, while hyperthermia alone had no significant influence on pH. Intratumoral temperature was higher during hyperthermia in animals given glucose. (c): A substantial rise of Se-glucose and hemoglobin developed. The hemoconcentration was maintained also after reduction of Se-glucose towards normal values. (d): An initial tachycardia, and a reduction of the mean arterial pressure of about 10% 5-45 min after was measured. CONCLUSION: The data indicate that a complex interaction between gradually reduced tumor pH, hyperglycemia, hemoconcentration, and reduced tumor blood flow, and not a breakdown of systemic circulation, is responsible for the effect of intraperitoneal glucose on thermochemotherapy with ACNU. Interestingly, enhancement of thermochemotherapy effect was also seen when intraperitoneal glucose was given after heat and ACNU.

Neurotoxicity and pharmacokinetics of ventriculolumbar perfusion of methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-glucopyranoside (MCNU) in dogs.

Ventriculolumbar perfusion of methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-glucopyranoside (MCNU), a water soluble nitrosourea with log P -0.71, may be efficacious in the treatment of subarachnoid dissemination of malignant glioma. We used 2 dogs to study the neurotoxicity and pharmacokinetics of MCNU. MCNU (1 mg), dissolved in 10 ml of artificial CSF, was administered via the right lateral ventricle during a period of 18 to 42 min and the CSF was drained by lumbar puncture. The perfusion was repeated once a week for 10 consecutive weeks. No neurological and systemic symptoms were noted after perfusion. Histological examination of the brain and spinal cord showed local denudation of the ependyma and local subependymal spongy degeneration and gliosis in the lateral ventricle into which MCNU was administered in one dog and local denudation of the ependyma in the other. When administration was over a period of 21 to 38 min, the MCNU concentration in the lumbar CSF peaked at 11.11 to 50.67 micrograms/ml, in 28 to 78 min. The area under the drug concentration-time curve (AUC) was 1152 micrograms x min/ml on average, significantly larger than that of ACNU. The elimination phase followed linear kinetics and the half-time was 41.1 min on average, significantly longer than that of ACNU. These findings suggest that ventriculolumbar perfusion of MCNU may be effective in the treatment of subarachnoid dissemination of malignant glioma notwithstanding some local histological changes.

Pharmacokinetics of intrathecal 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride in rats.

The pharmacokinetics of intrathecal 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) were studied in female Wistar rats by macroscopical autoradiography using 14C labeled ACNU. In normal rats, ACNU rapidly distributed in the subarachnoid space and ventricles after intracisternal administration. Diffusional transport into the brain tissue was limited to a depth of 1 or 2 mm from the cerebrospinal fluid (CSF) surface of the brain. Clearance of ACNU from the CSF space and brain was relatively fast and the half time of ACNU concentration at the cortical or ventricular surface was 10 min. In rats with leptomeningeal tumor induced by intracisternal inoculation of Walker 256 carcinosarcoma cells, the distribution pattern of ACNU after intracisternal administration was essentially the same as in normal rats until the tumor had grown in the subarachnoid space to form more than 10 or 20 layers of tumor cells. ACNU was distributed in the tumor as well. When the tumor had grown to form masses in the subarachnoid space, ACNU failed to penetrate to more than a depth of 1 or 2 mm from the tumor surface. Our results suggest that intrathecal ACNU administration may have no, or minor side effects on the brain and that it can eliminate floating or thin layered tumor cells in the subarachnoid space but not bulky tumors.

Ventriculolumbar perfusion of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosou rea hydrochloride.

We report on the toxicity, intrathecal pharmacokinetics, and therapeutic effect of the ventriculolumbar perfusion of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitros our ea hydrochloride (ACNU) against the subarachnoid dissemination of primary central nervous system tumors. Fifteen patients received ventriculolumbar perfusion of ACNU. One was treated with ventriculolumbar perfusion of ACNU alone, and the others underwent concomitant systemic chemotherapy; three of these patients received irradiation as well. ACNU was administered at an initial dose of 0.5 and was increased to 1.5 to 10.0 mg in six patients. Because of a lack of Level 2 or greater toxicity, the subsequent seven patients received 8.7 to 10.0 mg of ACNU dissolved in artificial cerebrospinal fluid (CSF) at a concentration of 0.1 mg/ml, from the start of the treatment. During ACNU administration, the lumbar CSF was drained at approximately the same rate as that of the infusion. Twelve patients received from 3 to 42 courses (average, 14 courses). The cumulative dose of ACNU ranged from 5 to 330.4 mg (average, 82.9 mg). One patient had a convulsion; two patients experienced transient headache, nausea, and vomiting; two others reported transient headache, nausea, vomiting, and fecal incontinence; and one experienced transient nausea, vomiting, and fecal incontinence. No side effects were noted in the other nine patients. When 9.0 to 9.5 mg of ACNU, dissolved in 90 to 95 ml of artificial CSF, was administered for 37 to 52 min, the maximum concentration of ACNU in the lumbar CSF was 9.86 to 12.79 micrograms/ml and the area under the drug concentration-time curve was 260.8 to 502.5 micrograms.min/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of intrathecally administered ACNU in mongrel dogs: pharmacokinetics and quantitative autoradiographic study.

The pharmacokinetics of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) in the cerebrospinal fluid (CSF), were determined in dogs after ventriculolumbar perfusion (VLP, n = 6), and bolus injection into the ventricle (VB, n = 2), cisterna magna (MB, n = 5), and lumbar cistern (LB, n = 3), by high-performance liquid chromatography. The VLP method introduced effective amounts of ACNU into the lumbar cistern for cell kill in vitro. That is, the areas under the time concentration curve (AUC) of ACNU in the lumbar CSF for those receiving a 1.5 mg perfusion of ACNU were 481, 791, and 520 micrograms.min/ml and those receiving a 5 mg perfusion were 1,081, 2,048, and 1,215 micrograms.min/ml, respectively. These values were superior to 3-log cell kill condition of 9L gliosarcoma and 1.5-log cell kill of HU-126 human glioma cell line. Among the groups to which 5 mg of ACNU was administered, the VLP method attained significantly higher AUC values in the lumbar CSF than MB method. Quantitative autoradiography using an imaging plate system was performed in the VLP group (n = 2), VB group (n = 1), MB group (n = 2), and LB group (n = 2) using a 10 microCi/kg [ethylene-14C] ACNU dose which is thought to be related to the alkylating activity of ACNU. The VLP method attained a stable and abundant distribution of ACNU in the neural axis from the ventricular cavity to the lumbar cistern, but the cerebral convexity surface was devoid of a significant level of ACNU. When the MB method was used, the pharmacokinetic data varied in the cisterna magna and lumbar region, and again no significant level of ACNU was detected in the ventricular cavity. With the LB method, although a rich distribution was detected in the spinal cord, the concentration decreased abruptly at the upper cervical level. The VB method was unsatisfactory for obtaining an effective amount of ACNU in the lumbar region. The research and testing to date indicate that the VLP method is the procedure of choice in the treatment of meningeal dissemination.

[Intrathecal distribution of ACNU by various modes of its administration analyzed by HPLC and autoradiography].

Various modes of administration of ACNU (nimustine hydrochloride) were tried to make clear which mode is the best method to obtain intrathecal diffuse distribution of ACNU to match the condition of killing of glioma cells (10 micrograms/ml; greater than 30 min.). Tried modes of administration included 1)bolus injection into ventricular cavity, 2)bolus injection into cisterna magna, 3)bolus injection into lumbar subarachnoid space, 4)ventriculo-lumbar perfusion, 5)chiasmatic cistern-lumbar perfusion. Used dose of ACNU was 5 mg/body for all modes of administration. ACNU level in CSF was measured by HPLC method specially developed by authors. To make clear intrathecal distribution of ACNU, autoradiography using 14C-ethylene-ACNU was studied after administration of 10 muCi/Kg of radioactive ACNU. The images were studied by image analyzer system (BAS-2,000 system developed by Fuji Film Co. Ltd). Among the modes of administration tried, ventriculo-lumbar perfusion method gave the best results in terms of lumbar, ventricular, cisterna magna, and basal cistern distribution of ACNU to match the cell kill condition experimentally ascertained. Although, bolus injection of ACNU into cisterna magna gave sufficient amount of ACNU in lumbar region, the initial level of ACNU was too high in cisterna magna, and administration of ACNU once a week for three times in a canine cisterna magna resulted in considerable deterioration of brain stem and basal structure. In addition to it, the level of ACNU in ventricular cavity was not detectable. Lumbar bolus injection resulted in also too much ACNU accumulation at the injected lumbar area, and at the cisterna magna region, ACNU was not detectable.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-resistance patterns in ACNU-resistant glioma sublines in culture.

Three ACNU-resistant clones (R1, R3, and R12) were isolated from 9L rat glioma cells under selection pressure of ACNU in vitro. The authors have investigated the mechanisms of resistance and characteristics of these clones at the cellular level by studying cross-resistance patterns to chemical and physical agents. Although these resistant sublines showed complete cross-resistance to methyl-chloroethylnitrosourea (MCNU), no cross-resistance was observed for other alkylating agents, while each of the resistant sublines showed partial cross-resistance to structurally dissimilar toxic agents (vinblastine, Adriamycin, and VP-16). No difference in ACNU uptake was observed between 9L and R3 cells, and resistance patterns among alkylating agents suggested that the mechanism of ACNU resistance was specific to bifunctional nitrosoureas. Based on a transport study, this multidrug resistance could be explained by reduced intracellular uptake of these drugs, but there seemed little possibility that membrane P-glycoprotein, which usually is observed in typical multidrug-resistant cells, was expressed in these ACNU-resistant cells because enhanced drug efflux was not found in ACNU-resistant sublines. Significant collateral sensitivity to L-asparaginase indicated that ACNU might disturb the asparagine synthetic pathways by its mutagenic action. The increased level of total glutathione in the resistant sublines may be one mechanism of radiation or ACNU resistance.

Neurotoxicity and pharmacokinetics of intrathecal perfusion of ACNU in dogs.

To test the feasibility of intrathecal perfusion of ACNU (3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitro sou rea hydrochloride) in the treatment of subarachnoid dissemination of malignant glioma, the neurotoxicity and pharmacokinetics of ACNU were studied in dogs. ACNU [1-2 mg dissolved in 10-20 ml of lactated Ringer's solution or artificial cerebrospinal fluid (CSF)] was administered via the right lateral ventricle by constant drip infusion and CSF was drained by lumbar puncture. The infusion time was from 15 to 71 min. For the control, a bolus injection was given. No neurological and systemic symptoms were noted after perfusion. Histological examination of the brain and spinal cord revealed only mild denudation of ependyma in the wall of the ventricles in a dog treated three times with 2 mg ACNU (perfusion twice, bolus injection once) and in 2 dogs perfused with 1 mg ACNU once a week for 10 weeks. ACNU was not detected in lumbar CSF after bolus injection into the lateral ventricle. When 1 mg of ACNU, dissolved in 10 ml of artificial CSF, was perfused for a duration of 22 to 31 min, it started to appear in the lumbar CSF 10 to 15 min after the start of perfusion, reaching a maximum concentration of 13.88 to 22.31 micrograms/ml. The area under the drug concentration-time curve was 344 to 706 micrograms x min/ml; the half-time was 15.5 to 19.5 min. The distribution volume was 30.6 to 54.1 ml. These findings suggest the feasibility of intrathecal perfusion of ACNU in the treatment of patients with subarachnoid dissemination of glioma.

Rapid and simple method for the determination of nimustine hydrochloride in human blood and brain by high-performance liquid chromatography.

A simple method for the determination of nimustine hydrochloride in blood and brain by high-performance liquid chromatography was developed. A pH 4.52 buffer was used in the extraction from blood and a pH 5.0 buffer was used for brain. A pre-packed Extrelut column was used to make the extraction procedure uncomplicated. At room temperature light-resistant test-tubes were unnecessary. The lower limit of detection was 50 ng/ml for blood and 100 ng/g for brain. This method may be useful for the determination of nimustine hydrochloride in blood and brain samples from patients.

Prediction of ACNU plasma concentration-time profiles in humans by animal scale-up.

Plasma concentration-time profiles of nimustine hydrochloride, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride (ACNU), in the mouse, rat, rabbit, and dog were determined by high-performance liquid chromatographic analysis. The pharmacokinetic parameters for these four animal species and previously reported clinical data were analyzed for investigation of interspecies correlation. Log-log plots of body weight (W; kg) vs total plasma clearance (CLtot,p; ml/min) and steady-state distribution volume (Vd,ss; l) for the four animal species were linear, with high correlation coefficients (r 0.996 for both parameters), despite the fact that the nonrenal clearance was greater than 97% in these species. Linear regression on the plots excluding human data yielded allometric equations (CLtot,p = 50.6 W0.957; Vd, ss = 1.29 W1.03) that were extrapolated to predict ACNU pharmacokinetic parameters in humans. For both parameters, however, there were 3-fold differences between the predicted and observed parametric values. To investigate these discrepancies, we measured serum protein binding of ACNU in these animal species and in humans. The values of CLtot,p and Vd,ss were converted into those of CLutot,p and Vd,uss, which correspond to the parameters for unbound ACNU. In this case, correlation coefficients of the log-log plots excluding human data (CLutot,p = 71.7 W0.891; Vd,uss = 1.82 W0.966) were also high (r greater than or equal to 0.991). The extrapolated values vs those observed in a 70-kg human were the following: CLutot,p, 3,160 vs 2,290 ml/min; Vd,uss, 110 vs 106 l. Thus, the animal data were successfully extrapolated to yield better predictions of human pharmacokinetic parameters if the analysis was based on the unbound plasma concentration of ACNU. In addition, the predicted plasma concentration-time profile for humans also showed good agreement with the observed ones. These results suggest the importance of measuring unbound fractions of drugs for more accurate prediction of human pharmacokinetic parameters by extrapolation of animal data to the human situation.

Evaluation of antitumor activity in a human breast tumor/nude mouse model with a special emphasis on treatment dose.

Eight lines of human breast tumors implanted in nude mice were treated with various antitumor agents at two different doses, maximum tolerated doses (MTD) and rational doses (RD) that were pharmacokinetically equivalent to the clinical doses; the response rates to both doses were compared. With MTD, the response rates to mitomycin C and vinblastine were 100%, and those to other agents including cyclophosphamide, nimustine (a water-soluble nitrosourea), vincristine, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), 5-fluorouracil (5-FU), and methotrexate were 30%-50%, indicating high responsiveness to the former two agents. In contrast, when the RD were used, the response rates to the majority of these agents were 25%-40%, and those to vincristine and nimustine were 13% and 0%, respectively. These results agree with the reported clinical results compared with those with MTD, suggesting the importance of the use of clinically equivalent doses in the evaluation of antitumor efficacy in a human tumor/nude mouse system.