Hypersensitivity of human tumor xenografts lacking O6-alkylguanine-DNA alkyltransferase to the anti-tumor agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea

Human tumor cell strains having different activities of O⁶-alkylguanine-DNAalkyltransferase (ATR) were transplanted into nude mice andchemotherapeutic responses of tumor xenografts were comparedafter intraperitoneal injection of the anti-tumor drug 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea(ACNU). The tumor strains used were four Mer⁺ strains possessinghigh ATR activity and three Mer^– strains lacking thisactivity. Included in these Mer⁺ strains was a clone 5'dD whichexpresses the Escherichia coli ATR in Mer^– HeLa cellsand thus shows the Mer⁺phenotype. All the Mer^– tumor xenograftswere much more sensitive than tumors of Mer⁺ strains, includingthe clone 5’dD; after the highest ACNU dose (three injectionsof 50 mg/kg), some Mer^– tumors disappeared completelyand the growth of other tumors was severely retarded, whereasall Mer⁺ tumors continued to grow. These results demonstratethat ATR activity in tumor cells is a major determinant of tumorresponse to ACNU, and further suggest that measurement of ATRactivity in biopsy specimens may provide a useful guide to predictthe response to chemotherapy.     

Transfer and expression of human O6-methylguanine-DNA methyltransferase cDNA confers resistance of Mer- HeLa MR cells to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea

Previous studies have demonstrated that O⁶-methylguanine-DNAmethyltransferase (MGMT) is a major contributor to tumor cellularresistance toward chloroethylnitrosoureas. To further clarifythe effect of MGMT gene expression on cellular chemosensitivityto 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea(ACNU) in human cells, a repair-deficient human tumor cell line(HeLa MR) was transfected with a human MGMT expression vector(pSV₂MGMT-neo). Multiple unique transfectants were isolatedwhich exhibited variable levels of MGMT protein by enzymic activityassay and of MGMT mRNA by Northern hybridization analysis. Vector-transfectedcontrols were generated simultaneously. Transfectants expressinghigh levels of MGMT activity showed an increased resistanceto ACNU-induced cytotoxicity. Furthermore, the levels of theprotective effect against ACNU correlated generally with thelevels of introduced MGMT expression. This study further provideddirect evidence of MGMT contribution to ACNU resistance in humantumor cells. Based on the results presented here, we also discussedthe perspective of the clinical utility of MGMT cDNA transferand expression.     

Combination chemotherapy with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride and bleomycin in meningeal carcinomatosis in rats

Combination chemotherapy with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and bleomycin (BLM) was evaluated using an experimental model of meningeal carcinomatosis induced in Sprague-Dawley rats by intracisternal inoculation of 1 X 10(4) Walker 256 tumor cells. Tumor-bearing animals were treated by i.v. administration of cyclophosphamide, BLM, ACNU, or a combination thereof, starting on Day 5 after tumor inoculation. BLM, 5 mg/kg on Day 5 as well as 5 mg/kg/day on Days 5, 7, 9, 11, and 13, was ineffective. Cyclophosphamide, 30 mg/kg, or ACNU, 15 or 30 mg/kg, on Day 5 increased the median survival time by 52, 70, and 82%, respectively. The combination of ACNU, 15 mg/kg, and cyclophosphamide, 15 mg/kg, increased median survival time by 73%, while the combination of ACNU, 15 mg/kg, and BLM, 5 mg/kg, resulted in a maximal increase of median survival time of 200% when the agents were given on Day 5. The combination of ACNU, 15 mg/kg on Day 5, and BLM, 5 mg/kg/day on Days 5, 7, 9, 11, and 13, increased median survival time by over 360% and cured 60% of the animals. These results point to the therapeutic advantage inherent in ACNU and BLM combination therapy.     

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.     

Effects of {alpha}-deuterium substitution on the mutagenicity of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)1

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenictobacco specific nitrosamine, can be converted to electrophilicdiazohydroxide intermediates by metabolic hydroxylalion of eitherthe methylene carbon (carbon 4) or the methyl carbon attachedto the nitrosamine group. To investigate the relative importanceof these two processes in NNK mutagenesis, we synthesized 4,4-dideutero-4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone([4,4,-D₂]NNK)and 4-(trideuteromethylnitrosamino)-1-(3-pyridyl)-1-butanone([CD₃] NNK), and evaluated their mutagenic activities in Salmonellatyphimurium tester strains. In the presence of Aroclor inducedrat liver 9000 g supernatant, NNK and [4,4-D₂]NNK had comparablemutagenic activities towards S. typhimurium TA 1535 and TA 100,but [CD₃]NNK was inactive in both strains. These results suggestthat hydroxylation of the methyl group of NNK is more importantthan hydroxylation of carbon 4 in its activation to a mutagen.To test the inherent mutagenicity of 4-oxo-4-(3-pyridyl)butyldiazohydroxideand methyldiazohydroxide which would be formed by methyl hydroxylationor carbon 4 hydroxylation, respectively, we compared the mutagenicities,without activation, of the corresponding model compounds, 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanoneand carbethoxynitrosaminomethane (methylnitrosourethane). Bothcompounds were highly mutagenic toward S. typhimurium TA 1535and TA 100, but at doses of 4 x 10^–3 to 4 x 10^–4µmol/plate,only 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone wasmutagenic. These results are consistent with those obtainedwith the deuterium substituted compounds and indicate the importanceof 4-oxo-4-(3-pyridyl)butylation of DNA in NNK mutagenesis.     

In vivo Antitumor Activity of Hexamethylmelamine against Human Breast, Stomach and Colon Carcinoma Xenografts

We have evaluated the antitumor activity of Altretamine (hexamethylmelamine, HMM) on human carcinoma xenografts serially transplanted in nude mice. Five human breast carcinoma xenografts, MX-1, T-61, MCF-7, R-27 and Br-10, were inoculated subcutaneously into female nude mice. Two human stomach carcinoma xenografts, SC-1-NU and St-4, and three human colon carcinoma xenografts, Co-3, Co-4 and Co-6, were inoculated subcutaneously into male nude mice. One pellet of 17β-estradiol (0.1 mg/mouse) was inoculated subcutaneously in the mice transplanted with MCF-7 when the tumors were inoculated. HMM was administered per os daily for 4 weeks. MX-1 and T-61 tumors regressed completely after treatment with HMM at a dose of 75 mg/kg (the maximum tolerated dose: MTD) for MX-1 and 25 mg/kg for T-61. Br-10 was sensitive, whereas MCF-7 and R-27 were resistant to HMM at its MTD. HMM exerted the most potent antitumor effect against T-61. Against MX-1, it exerted an antitumor effect equivalent to that of cisplatin or cyclophosphamide. In addition, this agent was effective against all stomach and colon carcinoma xenografts, in particular St-4 (T/C%= 10.7: the mean tumor weight of treated group/the mean tumor weight of control group) and Co-3 (T/C%=31.5%) which are insensitive to presently available agents. HMM seems worthy of further clinical investigation as a candidate agent to treat breast, stomach, colon and other carcinomas.     

Anaerobic metabolism and nuclear binding of the carcinogen 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT)

The anaerobic reductive metabolism of the urinary tract carcinogen2-amino-4-(5-nitro-2-furyl)-[2-¹⁴C]-thiazole ([¹⁴C]ANFT) wasexamined in vitro using rabbit liver and kidney microsomes.The intermediate(s) produced during the reduction binds to tRNA,DNA, and protein. ANFT reduction was inhibited by oxygen, requiredNADPH and was not inhibited by SKF-525A or allopurinol. No bindingto tRNA or DNA was observed if the nudeic acids were added atthe end of the incubation. The covalent binding of an ANFT metabolite(s)to nucleic acids and protein was inhibited by the antioxidantsvitamin E and butylated hydroxytoluene. The stoichiometry ofmicrosomal reduction shows 3 mol of NADPH were used/mol of ANFTreduced. In inner medullary microsomes, the apparent K_m andV_max were 0.05 mM and 0.92 nmol/mg/min, respectively. Two metabolitesfrom the anaerobic incubation of ANFT were isolated. The metaboliteswere tentatively identified as 1-(2-amino-4-thiazolyl)-3-cyano-1-propanoneand 2-amino-4-(5-hydroxylamino-2-furyl)thiazole.     

Potentiation by squalene of antitumor effect of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-nitros ourea in a murine tumor system

The effect of squalene (SQ) on the antitumor activity of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitros our ea (ACNU) was studied in a murine tumor system. SQ at 4.2 g/kg exhibited a significant potentiating effect on the activity of 10 mg/kg of ACNU against lymphocytic leukemia P388 and resulted in some long-term survivors without toxicity to the host. Simultaneous administration of SQ and ACNU was most effective.     

Antitumor Activity and Pharmacokinetics of Estra-1,3,5 (10)-Triene-3,17{beta}-Diol, 3-Benzoate, 17-((4-(4-(Bis (2-Chioroethyl)Amino)Phenyl)-1-Oxobutoxy) Acetate) (Bestrabucil) in Human Tumor Xenografts Serially Transplanted into Nude Mice

Bestrabucil (KM2210), the benzoate of an estradiol-chlorambucilconjugate, was used experimental cancer chemotherapy against13 human tumor xenografts serially transplanted into nude mice,and its pharmacokinetics was studied. The tumors were one esophageal,two gastric, six colon, one cholecystic and three breast carcinomas.Two tumor tissue fragments approximately 3x3x3 mm were inoculatedinto BALB/cA nude mice, which were then treated with KM2210at doses of 100, 200 and 300 mg/Kg/day orally starting 24 hrafter the transplantation or when the tumor reached a weightof 100–300 mg. The concentration of KM2210 and its derivativesin the tumor xenografts, normal muscular tissue and blood wereassayed by high performance liquid chroma-tography. Six out of 13 xenografts were found to be sensitive to KM2210.The concentrations of KM2210 and its derivatives in the tumortissues of the sensitive xenografts were five to 10 times higherthan those in blood and muscular tissue, and the antitumor activitycorrelated well with the area under the curve of active metabolitesof KM2210 in the tumor.     

Antitumor activity and toxicity of ACNU, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride,comparing two divided doses and a single dose

Summary In order to determine whether it is possible to reduce the toxicity of the nitrosoureas, including the delayed type of hematologic toxicity, without diminishing antitumor activity by administering the drug according to a new treatment schedule other than the single high-dose treatment schedule, we examined the antitumor activity against murine tumors and toxicities to host mice of a nitrosourea derivative, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU), which was administered according to two divided doses schedule. Experimental results indicated that the toxicity of ACNU with respect to lethality, as well as weight loss of host mice, was alleviated by administering ACNU (IV) at one-half of LD₁₀ (20 mg/kg) on 2 successive days. However, no impairment of the antitumor activity of ACNU in various murine tumor systems was observed in comparison with that of ACNU administered according to the single high-dose schedule. The delayed type of hematologic toxicity (leukopenia) could not be alleviated by this treatment schedule.     

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.     

Antitumor activity of 7-N-(2-((2-(gamma-L-glutamylamino) ethyl) dithio) ethyl) mitomycin C (KW2149) against human tumor xenografts serially transplanted into nude mice

The antitumor activity and toxicity of 7-N-(2-((2-(gamma-L-glutamylamino) ethyl) dithio) ethyl) mitomycin C (KW2149) were evaluated using a human tumor xenograft--nude mouse system, and compared with those of the maternal compound, mitomycin C. The maximum tolerated dose of KW2149 was estimated to be 15 mg/kg by bolus intraperitoneal or intravenous injection, at which a remarkable reduction of spleen weight was observed, suggesting bone marrow suppression by this agent. A bolus injection of KW2149 seemed to be more effective than a divided injection schedule, when a total of 15 mg KW2149/kg was administered to mice bearing breast (MX-1) and colon (Co-4) carcinomas. The antitumor activity of KW2149 was dose-dependent, and the difference in antitumor effect according to route of administration was minimal. The antitumor spectrum of KW2149 was essentially identical to that of mitomycin C administered intraperiotoneally as a bolus at a dose of 6 mg/kg.     

O6-methylguanine-dna methyltransferase activity and sensitivity of 20 chinese tumor cell strains to l-(4-amino-2-methyl-5-pyrimidinyl) methyl-3- (2-chloroethyl)-3-nitrosourea

O⁶-methylguanine-DNA methyltransferase (MGMT) plays an important role in repairing alkylated DNA. MGMT activity as well as cellular sensitivity to 1- ( 4- amino- 2-methyl - 5 - pyrlmidinyl) methyl - 3 - (2 - chloroethyl) - 3 -nitrosourea (ACNU) of 20 Chinese tumor cell strains were assayed. A linear response between MGMT activity and ACNU sensitivity (D₁₀) was observed. The lower the MGMT activity in the cells, the more the sensitivity to ACNU killing. It suggested that assay of MGMT activity in tumor biopsy could be used as a guide to predict the effectiveness of ACNU treatment in chemotherapy of human cancer.     

Cell kinetics and chemosensitivity of human carcinomas serially transplanted into nude mice

Six human carcinoma xenografts serially transplanted into nude mice were used for the study of chemosensitivity and cell kinetics. Three gastric carcinomas (St-4, St-40 and H-111), two colon carcinomas (Co-3 and Co-4) and one breast carcinoma (MX-1) were inoculated into the subcutaneous tissue of BALB/cA nude mice. The maximum tolerable doses of mitomycin C (MMC), adriamycin (ADM), cyclophosphamide (CPA) and 5-fluorouracil (5-FU) were administered when the tumor weights reached 100-300 mg. The response rates of the tumor to these drugs were found to be 3/6 for MMC, 2/6 for 5-FU and 1/6 for ADM and CPA. Percent labeled mitosis curves obtained from 3H-thymidine pulse labeling were analyzed by the method of Quastler and Sherman. It was found that the antitumor effect of MMC was closely correlated with the growth fractions of the tumors (r = -0.98, P less than 0.001), and it appeared that the tumor cells were more sensitive to MMC in the resting stages during the proliferating phase than in the other cell cycle phases. Cell kinetics is considered to be an important factor in determining chemosensitivity, and the system of human tumor xenografts-nude mice seems to be a suitable experimental model for investigating the correlation between cell kinetics and chemosensitivity in vivo.     

Modulation of O6-methylguanine-DNA methyltransferase-mediated 1-(4-amino-2-methyl-5-Pyrimidinyl)- methyl-3-(2-Chloroethyl)-3-nitrosourea resistance by antisense RNA.

Mer+ HeLa S3 tumor cells with high levels of O6-methylguanine-DNA methyltransferase (MGMT) gene expression were transduced by retroviral-mediated MGMT antisense RNA. The MGMT mRNA, MGMT protein, and MGMT activity in transduced cells were only 28.7%, 32.7%, and 39. 1% of that in HeLa S3 cells, respectively. The transduced cells showed more sensitive to ACNU than HeLa S3 cells both in cell survival and in nude mice experiments. Pathologic examination confirmed that transduced grafts were killed by ACNU. Our results suggested that MGMT gene expression could be modulated by retroviral-mediated antisense RNA and that Mer+ tumor drug resistance to ACNU could be reversed by modulation of MGMT gene expression.     

O~6-METHYLGUANINE-DNA METHYLTRANSFERASE ACTIVITY AND SENSITIVITY OF 20 CHINESE TUMOR CELL STRAINS TO 1-(4-AMINO-2-METHYL-5-PYRIMIDINYL) METHYL-3-(2-CHLOROETHYL)-3-NITROSOUREA

O6-methylguanine-DNA methyltransferase (MGMT) plays an important role in repairing alkylated DNA. MGMT activity as well as cellular sensitivity to 1- ( 4- amino- 2-methyl-5-pyrimidinyl) methyl-3- ( 2-chloroethyl)-3-nitrosourea (ACNU) of 20 Chinese tumor cell strains were assayed. A linear response between MGMT activity and ACNU sensitivity (D10) was observed. The lower the MGMT activity In the cells, the more the sensitivity to ACNU killing. It suggested that assay of MGMT activity in tumor biopsy could be used as a guide to predict the effectiveness of ACNU treatment in chemotherapy of human cancer.     

Carcinogenicity of 1-(4-amino-2-methylpyrimidine-5-yl)-methyl-3-(2-chloroethyl)-3- nitrosoureahydrochloride and three related N-nitroso derivatives following repeated intravenous administration to male Wistar rats

Long-term toxic evaluation of 1-(4-amino-2-methylpyrimidine-5-yl)-methyl-3- (2-chloroethyl)-3-nitrosoureahydrochloride (ACNU), 1-(2-chloroethyl)-1-nitroso-3-(methylene-2-pyridylium)-urea- hydrochloride (CNMPU), 1-(2-chloroethyl)-1-nitroso-3-(4-thiomorpholino)-urea (CNTMU) and 4-[N-(2-chloroethyl)-N-nitrosocarbamoyl]-morpholine (CNCM) which were administered each at five dosages, adapted to the clinical situation, revealed significantly increased tumor risks for the first three agents in the lung and the adrenal gland. Additionally, CNTMU and ACNU induced a significant rise in the tumor load of the neurogenic tissue; the latter compound, being in clinical use, was associated with a significantly increased number of mammary tumors as well. CNCM, however, did not exhibit significantly elevated carcinogenic activity, although the overall tumor load was up to 2.4-fold increased compared to controls. The results indicate no advantage of the newly developed ACNU with respect to its inherent carcinogenic risk as compared to clinically established 2-chloroethyl-N-nitroso derivatives.     

Depletion of O6-methylguanine--DNA methyltransferase and potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea antitumor activity by O6-benzylguanine in vitro

The overcoming effect of O⁶-benzylguanine on O⁶-methylguanine-DNAmethyltransferase (MGMT)-mediated 1,3-bis(2-chloroethyl)-1-nitrosourea(BCNU) resistance in vitro was evaluated. Depletion of MGMTactivity in Mer⁺ HeLa S3 cells by O⁶-benzylguanine was dose-dependentand a complete loss of MGMT activity was achieved at a concentrationof 0.5 µM. The cytotoxic potential of BCNU on MGMT proficientHeLa S3 (1.10 pmol/mg of protein), SMMC-7721 (0.72 pmol/mg ofprotein) and Cc801 (0.39 pmol/mg of protein) was greatly enhancedwhen cells were exposed to 10 µM O⁶-benzylguanine for1 h, but there was a lack of potentiation of BCNU sensitivityin Mer^– HeLa MR cells due to its nearly undetectable levelof MGMT. There existed a correlation between the extent of enhancementand the amount of MGMT activity. The intensity of enhancementexpressed as dose modifying factor = IC₅₀ (BCNU alone)/IC₅₀(10 µM O⁶-benzylguanine + BCNU) was 4.56, 3.89, 3.67 and0.97 in HeLa S3, SMMC-7721, Cc801 and HeLa MR cells respectively.The results further demonstrated that O⁶-benzylguanine may havepotential utility as an adjuvant in combination chemotherapywith chloroethylnitrosourea agents.     

Initiation of hepatocarcinogenesis in infant male B6C3F1 mice by N-hydroxy-2-aminofluorene or N-hydroxy-2-acetylaminofluorene depends primarily on metabolism to N-sulfooxy-2-aminofluorene and formation of DNA-(deoxyguanosin-8-yl)-2-aminofluorene adducts

Previous work from this laboratory provided strong evidencethat N-sulfooxy-2-aminofluorene is the major ultimate electro-philicand carcinogenic metabolite of N-hydroxy-2-acetyl-aminofluorene(N-hydroxy-AAF) in the livers of infant male B6C3F₁ (C57BL/6Jx C3H/HeJ F₁ mice. Over 90% of the hepatic DNA adducts in thesemice consisted of N-(deoxyguan-osin-8-yl)-2-aminofluorene [N-(dGuo-8-yl)]and<10% were deoxyguanosinyl adducts containing 2-acetylaminofluor-ene(AAF) residues. In the present study hepatic DNA adduct formationand tumor initiation by N-hydroxy-2-aminofluor-ene (N-hydroxy-AF)were examined in these mice. N-(dGuo-8-yl)-AF was the only adductdetected in the hepatic DNA; the level at 9 h after a singlei.p. dose of 0.04 or 0.06 µmol/g body wt of [³H]N-hydroxy-AFwas 1.0 or 1.7 pmol/mg DNA. Pre-treatment with a single i.p.dose (0.04 µmol/g body wt) of the sulfotransferase inhibitorpentachlorophenol (PCP) decreased the DNA adduct level by >80%.Similar levels of this adduct were found by ³²P-postlabelinganalysis of DNA from mice treated with unlabeled N-hydroxy-AF.The liver DNA of in-fant male brachyinorphic B6C3F₂ mice [deficientin 3'-phos-phoadenosine-5'-phosphosulfate (PAPS)] containedonly 0.3 pmol/mg DNA of N-(dGuo-8-yl)-AF after an i.p. doseof 0.06 µmol of N-hydroxy-AF/g body wt, while their phenotypi-callynormal (PAPS-sufficient) male littermates had 1.9 pmol/mg DNA.A single i.p. dose of 0, 0.015, 0.03, 0.06 or 0.12 µmol/body wt of N-hydroxy-AF in infant male B6C3F mice induced by10 months an average of 0.2, 2.5, 7, 11 or 14 hepatomas/mouse.Pretreatment with PCP reduced the liver tumor multiplicity ateach dose level by >80%. Essen-tially the same average tumormultiplicities and inhibitions of tumor formation by PCP pretreatmentwere obtained following injections of N-hydroxy-AF or N-hydroxy-AAFat the three lower dose levels. Collectively these data stronglyindicated that N-sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxyAF in the livers of infant male B6C3F₁ mice. Furthermore, sinceonly N-(dGuo-8-yl)-AF adducts were found in the he atic DNAthese lesions appear to be critical in the initiation of hepatocarcinogenesisin these mice by N-hydroxy-AF.     

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.