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.     

Ventriculolumber perfusion of 3-[(4-amino-2-methyl-5-pyrimidinyl)- methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride for subarachnoid dissemination of gliomas

The toxicity and therapeutic effect of the ventriculolumber perfussion of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl-1-1(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU) against subarachnoid dissemination of gliomas were studied. Twenty-one patients (6 patients with anaplastic glioma, 7 with glioblastoma and 8 with medulloblastoma or PNET) received ventriculolumber perfusion of ACNU when they were diagnosed as having subarachnoid dissemination. The course of perfusion and cumulative dose of ACNU was 10 times and 95 mg on average, respectively. Most of the patients received systemic chemotherapy in combination with perfusion therapy and some patients with radiotherapy. Response rate was 17% and median survival time after the diagnosis of dissemination was 12 months for anaplastic gliomas, 29% and 12 months for glioblastoma, and 88% and over 25 months for medulloblastoma and PNET. The ventriculolumber perfusion of ACNU was performed for prophylactic purpose in 7 patients with high risk at the early postoperative period in combination with conventional adjuvant therapy. The course of perfusion and cumulative dose of ACNU was 2.3 times and 21 mg on average, respectively. One patient developed subarachnoid dissemination and died 22 months after surgery. Other 6 patients survived without dissemination on median over 29 months after surgery. Side effects encountered were headache in 4 patients, nausea and vomiting in 5, a convalsion in 2, right facial weakness in 1, fecal incontinence in 3 and meningitis in 2. They were all temporary except for facial weakness occurred in one patient.These data suggest that the ventriculolumber perfusion of ACNU is a safe and useful in the treatment and prophylaxis against the subarachnoid dissemination of gliomas.     

Central nervous system toxicity and cerebrospinal fluid pharmacokinetics of intraventricular 3-[(4-amino-2-methyl-5-pyrimidinyl)ethyl]-1-(2-chloroethyl)-1-nitro soureas and other nitrosoureas in beagles

The central nervous system toxicity and cerebrospinal fluid (CSF) pharmacokinetics of 3-[(4-amino-2-methyl-5-pyrimidinyl)ethyl]-1-(2-chloroethyl)-1- nitrosoureas, a (ACNU) were determined in beagles and compared to those for three other nitrosoureas, 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, and chlorozotocin. Of the four drugs, ACNU was tolerated best and at doses of 0.2 to 0.8 mg/week for 8 consecutive weeks. We found that the average half-time for CSF elimination of ACNU was 18 min (range, 12 to 38 min). This value exceeded the known rate of ACNU decomposition in aqueous solution (28 to 29 min), implying that the disappearance of ACNU from CSF was due to hydrolytic decomposition and cellular entry and/or transcapillary loss across central nervous system capillaries. The drug exposure integral (C X t) of ACNU in the CSF after a "toxic dose low" of 0.8 mg in the dogs would achieve the equivalent of in vitro cell kills in excess of 3 logs for rat 9L and human glioma 126 cells. As a potential therapeutic agent for meningeal neoplasia, the major limiting factor may be that the CSF elimination of ACNU is rapid compared to its equilibration time from ventricle to spinal- and cerebral convexity-subarachnoid space. Based on these results, we have instituted clinical Phase I trials of intra-CSF ACNU.     

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

Summary 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 using¹⁴C 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 subarach-noid 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.     

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 2mm 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 110min, 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 2mm from the tumor surface.     

MCNU effectiveness on brain tumor. Part I: Antitumor activity in vitro on human glioma and neuroblastoma cell lines

A new water-soluble nitrosourea ( MCNU ) was tested for its antitumor activity against fourteen human glioma cell lines and two neuroblastoma cell lines. Four experiments were performed to determine its antitumor activity: inhibition of cell growth, comparison with ACNU, morphological observation, and analysis of DNA histogram with flowcytometry . Seven out of 14 gliomas (50%) and one neuroblastoma cell lines showed more than 50% inhibition of cell growth in vitro, appearance of giant multinucleated cell morphologically, and DNA accumulation in G2+M and/or S phase of cell cycle in the medium of 10 micrograms/ml MCNU . Antitumor activity and spectrum of MCNU against human brain tumors were almost the same as with ACNU.     

Specific mTOR inhibitor rapamycin enhances cytotoxicity induced by alkylating agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) in human U251 malignant glioma cells

Loss of the PTEN tumor suppressor gene and amplification of the epidermal growth factor receptor (EGFR), which is common in malignant gliomas, result in activation of the mammalian target of rapamycin (mTOR). Rapamycin is a highly specific inhibitor of mTOR and induces a cytostatic effect in various glioma cell lines. DNA-damaging agents such as nitrosourea are widely used in malignant glioma treatment; therefore, we investigated the effect of rapamycin on cell growth and death in combination with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU, nimustine hydrochloride) in human glioma cells. In U251 malignant glioma (U251MG) cells, we confirmed that rapamycin enhanced ACNU-induced apoptosis. We found that rapamysin inhibited ACNU-induced p21 induction, and knocking down of p21 protein by siRNA enhanced ACNU-induced apoptosis in U251MG cells. Furthermore, adenovirus-mediated over-expression of p21 protein rescued U251MG cells from apoptosis induced by ACNU and rapamycin. Finally, treatment of intracerebral U251MG xenografts with a combination of rapamycin and ACNU in vivo resulted in statistically prolonged median survival (P < 0.05). These results suggest that rapamycin in combination with DNA-damaging agents may be efficacious in the treatment of malignant gliomas.     

Interrelationship between O6-alkylguanine-DNA alkyltransferase activity and susceptibility to chloroethylnitrosoureas in several glioma cell lines

Summary In order to study the dynamic relationship in glioma cells between O⁶-alkylguanine-DNA alkyltransferase (AGT) activity and resistance to the cytotoxic effect of chloroethylnitrosoureas (CENUs), we investigated the changes in sensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after modulation of AGT activity. In ACNU-resistant rat glioma cell lines (9LR1, 9LR3, and 9LR12) and a human glioma cell (HNG-1), O⁶-methylguanine enhanced cytotoxicity to ACNU following a depletion of AGT activity. But no enhancement of cytotoxicity was seen in an ACNU-sensitive rat glioma cell line (9L). In the 9L and 9LR12 cells, equivalently sublethal doses of ACNU similarly depleted AGT activity but the regeneration rates of this repair protein were different. In the case of a 7-day pretreatment with human recombinant interferon- (HuIFN-), although it could modulate AGT activity in HNG-1 cells, no definite influence on cellular sensitivity to CENUs was observed. However, a 50-day pretreatment with HuIFN- conferred resistance to CENUs on them despite its effect to reduce AGT activity. Thus, diversity was seen in the relation between AGT activity and resistance to CENUs when AGT activity was modulated by HuIFN-. The results of this study suggest that AGT activity is one of factors affecting cellular sensitivity to CENUs but that alternative mechanisms of tolerance may be induced depending upon some environmental effects.     

Phase I/II study of intraventricular and intrathecal ACNU for leptomeningeal neoplasia

Summary A total of 27 patients with leptomeningeal neoplasia were treated with the water-soluble nitrosourea ACNU given intraventricularly or intrathecally in a phase I/II study. Patients were entered in the study if they showed evidence of either a positive CSF cytology or neurodiagnostic evidence of leptomeningeal disease, or both. Patients were evaluated for toxicity and efficacy; additionally, in 13 patients ACNU pharmacokinetic studies were carried out. A variety of tumor types were represented in the study group, including primary and metastatic CNS tumors. Toxicity was mild and included pain at the injection site (four patients), transient radicular symptoms at a short distance from the injection site (three patients), and nausea and vomiting (one patient). No myelotoxicity was seen. Of 21 patients who presented with positive cytology, 8 (38%) had a conversion from positive to negative cytology, with a range of response durations from 1 to 20+ months. Of the remaining six patients with negative cytology but other neurodiagnostic evidence of leptomeningeal disease, one patient showed an improvement seen on the myelogram and one underwent a brief reduction in CSF protein. ACNU elimination from the ventricular system is rapid, with a beta slope of 0.028 min^-1 and a computed elimination constant, K_o, of 13 min. The mean clearance was 3.8 ml/min (range, 1.0–6.2 ml/min). Peak ACNU levels varied between 108 and 620 g/ml, with the AUC being 1.4–14.7 mg·min/ml. The total dose of ACNU given was between 9 and 104 mg, and the single dose range was 4–16.5 mg. We conclude that ACNU can be given safely with minimal toxicity as intra-CSF therapy, that it demonstrates efficacy in some patients with leptomeningeal disease, and that further studies are warranted to evaluate more fully alternative dosing and drug delivery approaches.     

Antitumor Effect of 3-[(4-Amino-2-Methyl-5-Pyrimidinyl) Methyl]-1-(2-Chloroethyl)-1-Nitrosourea (ACNU) on Human Colon Carcinomas Transplanted into Nude Mice

Two human colon carcinomas serially transplanted into nude micewere used for experimental chemotherapy by 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea(ACNU). Human colon carcinomas Co-3 (well-differentiated adenocarcinoma)and Co-4 (poorly differentiated adenocarcinoma), were transplantedsubcutaneously into the backs of BALB/c male nude mice. Tumorsize was measured three times a week, and treatment was startedwhen the estimated tumor weight reached 100 to 300 mg. Twentyand 40 mg of ACNU per kg was administered intravenously, once,dissolved in 0.2 ml of normal saline. There were marked tumorregression and histological tumor cell destruction in Co-4,whereas no effect was observed in Co-3. Microangiography revealed a similar vascular network in Co-3and Co-4. Whole-body autoradiography was performed 5, 30, 180and 360 minutes after 20 mg (286 µC₁) of [ethylene-2-¹⁴C]-ACNUper kg was injected. ACNU concentration in the Co-3 tumor reacheda peak 30 minutes after injection and diminished promptly withthe decrease of ACNU in the blood, whereas in Co-4 tumors ACNUwas retained in the tumor until 360 minutes after ad ministration.The effect of ACNU was thought to be correlated with the concentrationof the drug in the tumor. Present address: Department of Surgery, Kitasato InstituteHospital, Tokyo, Japan.     

Preliminary Phase II Study of 1-(2-Chloroethyl)-3-(MethylAlpha-D-Glucopyranos-6-Yl)-1-Nitrosourea (MCNU) Against Primary Lung Cancer and Metastatic Pulmonary Tumors

A phase II study of 1-(2-chloroethyl)-3-(methyl--D-glucopyranos-6-yl)-1-nitrosourea(MCNU) was conducted with 16 patients with primary lung canceror metastatic pulmonary tumors who had failed to respond toconventional therapy. MCNU was administered by a single intravenousinjection at a dose of 120 mg/m². There were no patients whoshowed any objective responses. Although stabilization was achievedin 12 patients, four patients with primary lung cancer experiencedprogressive disease. Gastrointestinal toxicities such as anorexia,nausea and vomiting were mild or moderate and readily subsidedwithout any treatment. The major toxic side effects were leukocytopeniaand thrombocytopenia. Five patients (38.4%) had leukocytopeniaof less than 2,000/mm³ and six patients (46.1%) had thrombocytopeniaof less than 5.0x10⁴/mm³.     

Reduction of lethal toxicity of chloroethylnitrosoureas by sugar alcohols without loss of antitumor activity

Summary Sugar alcohols, such as mannitol, sorbitol, galactitol, and inositol, selectively reduced the acute lethal toxicity of 1-(2-chloroethyl)-3-(methyl -d-glucopyranos-6-yl)-1-nitrosourea (MCNU) without reducing its antitumor activity. Fifty mg MCNU/kg killed all CD2F₁ mice within about 10 days, while the administration of 3,000 mg sugar alcohols/kg immediately prior to MCNU protected mice from the lethal toxicity and all survived. The amelioration of MCNU toxicity by sugar alcohols was dose-dependent. Pretreatment with mannitol 1 day before MCNU administration was effective. In addition, a series of five daily treatments with lower doses of mannitol was also effective. This protection was accompanied by the reduction of both body weight loss and myelosuppression. The antitumor effects of MCNU on P388 leukemia and Lewis lung carcinoma were not significantly altered by mannitol treatment. These phenomena were not limited to MCNU, the lethal toxicity of GANU, ACNU, Me-CCNU, and mitomycin C also being reduced by mannitol treatment.     

Effects of Combination Chemotherapy with Biscoclaurine-derived Alkaloid (Cepharanthine) and Nimustine Hydrochloride on Malignant Glioma Cell Lines

In the treatment of malignant glioma, chemotherapy plays a critical role as do surgical resection and irradiation. Cepharanthine (CEP), a biscoclaurine-derived alkaloid, reportedly potentiates the effects of antitumor agents and induces apoptosis in some cancer cells. Here, we examined the effects of CEP, alone and in combination with nimustine hydrochloride (ACNU), on the in vitro proliferation of malignant glioma cells. The cell lines used were U87MG, U251MG, and T98G. At concentrations from 1 to 10g/ml, CEP-promoted cell proliferation somewhat; growth inhibition was noted at concentrations of 15g/ml and higher. Phase-contrast microscopy showed that cells tended to detach from the culture dishes and that cell density became sparse at the higher concentrations. DAPI fluorescence nuclear staining revealed condensation and fragmentation of nuclei, indicating the induction of apoptosis. To examine the cascade of apoptosis, the caspase inhibitors YVAD and DEVD were added. They inhibited CEP-induced apoptosis in U251MG cells (a p53-mutant cell line), but not in U87MG cells (a p53 wild-type cell line), suggesting that in CEP-induced apoptosis two possible cascades are in play. In combination with ACNU, the effects of the higher concentrations of CEP were enhanced.     

Sensitivity of human malignant intracranial tumors against MCNU in vitro in comparison to ACNU and BCNU

Summary An in vitro chemosensitivity test was carried out in 50 specimen of human malignant intracranial tumors. Aim of the study was to evaluate the proportion of sensitivity against MCNU (Ranomustine) in comparison to ACNU and BCNU. 47 tests were evaluable. Mean viability of the specimen was 83.3 ± 18.7%, mean plating efficiency was 0.068 ± 0.051%. 9/47 settings revealed sensitivity against MCNU in vitro (ACNU: 10/47; BCNU: 16/46). There was no advantage of MCNU concerning age or sex of the patients. Brain metastases seemed to be slightly more frequent sensitive against MCNU than primary brain tumors. Cross resistance between ACNU, BCNU and MCNU was rather high. The results of this in vitro series do not encourage a clinical trial of MCNU as an alternative to the commonly used nitrosoureas.Behring Werke, D-3550 Marburg/Lahn, FRG     

The mechanism and overcoming of resistance in ACNU-resistant sublines of C6 and 9L rat glioma

In order to study the mechanism of the resistance to chemotherapeutic agents, especially ACNU [1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride], two variant cell lines (C6/ACNU and 9L/ACNU) resistant to ACNU were selected in vivo from rat C6 and 9L glioma, respectively. Uptake and efflux of ACNU in these resistant cells were studied with Ethylene[¹⁴C]ACNU. The result indicated that the resistance exhibited by both sublines were due to both the reduced uptake of the drug and the increased efflux. The study of the effects of oxidative phosphorylation inhibitor, DNP (2,4-dinitrophenol), on the uptake and retention of ACNU suggested that there is an active outward transport mechanism for ACNU in both glioma sublines and that enhanced activity of this efflux mechanism renders cells highly resistant to the cytotoxic action of ACNU. In an attempt to clarify the more detailed biochemical mechanisms of this active efflux system, we surveyed various membrane-modifying agents which potentiate the sensitivity of these resistant cells to ACNU. Among a number of membrane-modifying agents, reserpine was found to retain ACNU in the resistant cells and to enhance the action of ACNU on these resistant cell lines. It may be concluded that drugs such as reserpine may overcome a mechanism of ACNU resistance.Abbreviations PBS phosphate-buffered saline consisting of 0.02 M sodium phosphate, 0.15 M NaCl pH 7.4 - IC₅₀ concentration of drug required for 50% inhibition of cell growth - C6/ACNU C6 glioma cells resistant to ACNU - 9L/ACNU 9L glioma cells resistant to ACNU - CAP-2 2-(gamma-chloropropyl)2-chloromethylpyrimidine hydrochloride - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea     

MCNU delivery into malignant brain tumor and normal brain tissue by intravenous or intraarterial infusion

In 13 Fischer 344 rats transplanted intracerebrally with 9 L gliosarcoma, 13 normal Fischer 344 rats and 4 clinical cases of malignant glioma, a new water-soluble nitrosourea (MCNU) was given and the concentration was measured in blood, tumor tissues, normal brains around the tumors and normal hemispheres by intravenous or intraarterial infusion of MCNU. At 5 min. after administration of MCNU 20 mg/kg (4-5 mg/body) in 9 L gliosarcoma bearing Fischer rats, mean MCNU concentration in the blood was not different between 20 micrograms/ml intravenous and 23 micrograms/ml intraarterial administrations whereas that in the tumor tissues by intracarotid infusion of MCNU was 40 +/- 14.4 micrograms/g which was about two times as much as 22.9 +/- 8.13 micrograms/g by intravenous infusion of MCNU. Mean MCNU concentration of normal brains around tumor tissues was 2.49 micrograms/g in intravenous and 8.95 micrograms/g in intracarotid infusion. MCNU concentration of tumor tissues in 4 cases of malignant gliomas was higher by intracarotid administration than by intravenous administration compared to that in the blood. Maximum tumor/blood ratio of MCNU was 1.94 in intracarotid administration for the malignant glioma. It is suggested that intraarterial administration was more useful than intravenous infusion as an administration route for malignant brain tumors.     

Comparative effect of administration schedules on the antitumor activities of 3 water-soluble nitrosoureas, ACNU, GANU and MCNU against L1210 leukemia

ACNU, GANU and MCNU, water-soluble nitrosoureas, have been evaluated in terms of influence of treatment schedule on antitumor activity in mice bearing L1210 leukemia. The results obtained were as follows: 1) ACNU produced a significant increase in life span and long-term survivors by administration on day 1 only, once every 8 days for 2 doses or once every 4 days for 3 doses, and the compound was most effective when given on day 1 only. 2) GANU produced a significant increase in life span and long-term survivors by same administration schedules as ACNU, and the compound was most effective when given every 8 days for 2 doses. 3) MCNU produced a significant increase in life span and long-term survivors by each administration including daily treatment, and the compound was most effective when given every 4 days for 3 doses. 4) ACNU and MCNU displayed the same level of activity as CCNU when the drugs were given on day 1 only. Daily treatment with MCNU was as effective as daily treatment with CCNU. Our results suggest that ACNU, GANU and MCNU should be administered by intermittent schedule as lipid-soluble nitrosoureas such as BCNU, CCNU and MeCCNU.     

Acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride in V79 cells through increased removal of O6-alkylguanine

The molecular mechanism of acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU) was investigated using ACNU-resistant clones (ACNUr-1-4) isolated from the V79 cell line. The binding level of alkyl cyanate, a decomposition product of ACNU, to protein in ACNUr-1 cells was not less than that in the parental V79 cells, indicating that the acquired resistance was not due to a reduced intracellular concentration of ACNU. Because O6-chloroethylguanine, an intermediate in cytotoxic interstrand cross-link formation by ACNU, is known to be repaired by the same mechanism as O6-ethyldeoxyguanosine (O6-EtdGuo), we quantitated O6-EtdGuo by radioimmunoassay at various times after exposure of cells to 100 micrograms/ml N-ethyl-N-nitrosourea for 20 min. In V79 cells, elimination of O6-EtdGuo was negligible, but in all four resistant clones, 30 to 59% of the O6-EtdGuo was removed within 24 hr after exposure. This increased removal of O6-EtdGuo among the resistant clones was associated with the activity of O6-alkylguanine DNA alkyltransferase (O6-AGT) determined using cell extracts. The present results indicate that increased removal of O6-chloroethylguanine in ACNU-resistant clones by O6-AGT is mechanistically linked to the acquisition of resistance to ACNU.     

Intrathecal ACNU treatment of B16 melanoma leptomeningeal metastasis in a new athymic rat model

Summary To evaluate new cytotoxic drugs for intrathecal treatment we developed an experimental model of leptomeningeal metastasis by intracisternal injection of 10⁴B16-F10 melanoma cells in nude rats. One hour in vitro incubation with 20 g/ml ACNU (area under the drug concentration-time curve = 1200 gxmin/ml) induced a 4-log kill of B16 melanoma cells. A single or repeated non-toxic dose of 1 mg/kg was injected into the cisterna magna of rats inoculated with tumor (area under the drug concentration-time curve assuming an even cerebrospinal fluid distribution > 7000 gxmin/ml). Median survival free of symptoms was 16 days (range 14–27) for controls (n = 9) and 18 days (range 17–23) for rats treated with ACNU on day 4 (n = 9). Animals treated both on day 2 and 8 (n = 8) developed symptoms on day 21 (range 13–35). Neurological symptoms and neuropathological examination in animals with increased survival indicated local suppression of tumor growth in the cisterna magna but increased spinal seeding and mass growth. From these results and the available pharmacokinetic data on ACNU it is concluded that bolus injection of ACNU — although locally effective — is not a sufficient treatment of widespread leptomeningeal metastasis. An increased therapeutic efficacy might be achieved by ventriculolumbar perfusion.