Evaluation of the role of isocyanates in the action of therapeutic nitrosoureas

The half-lives of chloroethyl and cyclohexyl isocyanate have been determined in tissue culture medium, and the isocyanate concentration produced during the breakdown of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) has been calculated. L1210 or HeLa cells exposed either to the parent nitrosourea or to an equivalent constant isocyanate concentration show no deficiency in the repair of gamma-irradiation damage as measured by DNA strand separation in alkali. Viability studies indicate that the isocyanates play a minor role in the overall cytotoxicity of the nitrosoureas.     

Studies of the mode of action of antitumour triazenes and triazines—II. Investigation of the selective toxicity of 1-aryl-3,3-dimethyltriazenes

The hypothesis that 1-aryl-3-methyltriazenes (monomethyltriazenes) are the active species responsible for the in vivo cytotoxicity of 1-aryl-3,3-dimethyltriazenes (dimethyltriazenes) has been tested in vitro by a comparison of the toxicity of monomethyltriazenes to a tumour which is sensitive to the dimethvltriazenes in vivo (TLX5S lymphoma) and to one which is resistant in vivo (TLX5R lymphoma). The results indicate that monomethyltriazenes are non-selectively cytotoxic. In vitro activation of a dimethyltriazene by a liver homogenate and cofactors was found to produce metabolites which are more toxic to the TLX5S tumour than to the TLX5R whereas when a 1-aryl-3,3-diethyltriazene, which is not an antitumour agent, was activated in the same way, cytotoxic metabolites were generated but the TLX5S tumour showed no preferential sensitivity. This suggests that during metabolic activation of dimethyltriazenes a mixture of selective and non-selective metabolites are generated. In an in vivo test the TLX5R tumour was found to be resistant to a monomethyltriazene, and it is suggested that this may be metabolised to an active species by the host.     

Studies of the mechanism of action of the tumour-inhibitory nitrosoureas.

BCNU [1,3 bis-(2-chloroethyl)-1-nitrosourea] and some related nitrosoureas have been shown to have a wide spectrum of action against a number of transplanted rodent tumours. No correlation was found between the chemical instability of a nitrosourea and its antitumour activity. Unlike difunctional alkylating agents, the nitrosoureas inhibit the incorporation of tritiated precursors in DNA, RNA and protein to equal extents, the inhibition of tritiated thymidine incorporation into DNA occurring within 5 min of incubating cells with BCNU. Although the biological half life of BCNU was found to be very short (15 min by bioassay) a single injection was as effective against the established and widely-disseminated TLX5 lymphoma as against the early transplant. BCNU interfered specifically with the incorporation of labelled thymidine triphosphate into DNA, but no inhibition of DNA polymerase could be demonstrated at physiological dose levels. In their mechanism of action and in their biological properties the tumour inhibitory nitrosoureas are quite distinct from the bifunctional alkylating agents.     

Mechanism of action of the nitrosoureas—IV: Reactions of bis-chloroethyl nitrosourea and chloroethyl cyclohexyl nitrosourea with deoxyribonucleic acid

Calf thymus DNA was reacted with ¹⁴C-labeled bis-chloroethyl nitrosourea (BCNU), and chloroethyl cyclohexyl nitrosourea (CCNU), and the nature of the derivatives investigated in an enzymatic digest. In agreement with earlier studies on polyribonucleotides, evidence was obtained for the formation of 7-hydroxyethyldeoxyguanosine, 3-hydroxyethyldeoxycytidine and 3,N⁴-ethanodeoxycytidine. In addition, significant amounts of 7-aminoethylguanine were identified in the hydrolysate of DNA treated with BCNU, but not in the hydrolysate of DNA treated with CCNU. Aminoethylguanine was also formed when DNA was reacted with chloroethylamine, suggesting that BCNU produced aminoethylguanine via chloroethylamine as an intermediate. Because both BCNU and CCNU are effective antitumor agents, the formation of aminoethylguanine is probably not an important cytotoxic reaction, but it may have significance as far as mutagenic or carcinogenic activities are concerned.     

Studies with a 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP)-resistant L1210 leukemia cell line without cross-resistance to methotrexate

An L1210 leukemia cell line resistant to 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP) (L1210/DDMP) was developed in vivo by treatment of tumor-bearing mice. Resistance to DDMP was confirmed by subsequent in vivo survival experiments and by in vitro dose-response curves. The L1210/DDMP line demonstrated little cross-resistance to another folate analog, methotrexate (MTX). This was confirmed both in vivo, with survival experiments, and in vitro, using dose-response curves. A statistical analysis of the in vivo data confirmed DDMP resistance with lack of MTX cross-resistance. Dihydrofolate reductase (DHFR) activity in the L1210/DDMP/R₅ line was no greater than in the parent cell line (L1210/S). and the K_m of DHFR for dihydrofolate was the same in the L1210/DDMP/R₅ and L1210/S lines. The K_i for DHFR of the L1210/DDMP/R₅ cell line versus the L1210/S cell line was increased 3.0-fold for MTX and 3.5-fold for DDMP. Total accumulation of [¹⁴C]DDMP was identical in the two cell lines. The explanation for the lack of MTX cross-resistance in the L1210/DDMP/R₅ line is unknown.     

Mechanism of the antileukemic effects of 1-p-carboxamidophenyl3,3-dimethyltriazene and its in vitro metabolites

DM-CONH₂, a dimethyltriazene active in prolonging the survival time of mice bearing TLX5 lymphoma, requires metabolic activation by liver homogenate supernatant and cofactors in order to exert in vitro cytotoxic effects on the same tumor cells, as determined by in vivo bioassay of their viability. From the examination of the metabolites produced during these in vitro experiments, it is found that in vitro cytotoxicity is attributable to the generation of MM-CONH₂ by oxidative N-demethylation of DM-CONH₂. Also the generation of DM-COO^? is observed, although this compound is not cytotoxic in vitro. The in vivo effects of DM-CONH₂ and CM-COOK on TLX5 lymphoma are not caused exclusively by cytotoxic effects of the drugs, since they are evident also when no reduction in the number or viability of peritoneal tumor cells is evident, whereas these parameters are significantly reduced by MM-CONH₂. The increase in survival time of mice bearing TLX5 lymphoma caused by the dimethyltriazenes used appears to be caused by the drugs without being subjected to metabolic activation, with a mechanism different from cytotoxicity for tumor cells.     

A chemical basis for the antitumor activity of chloroethylnitrosoureas

A comparison of the aqueous decomposition products of several haloethylnitrosoureas has led to a suggested mode of decomposition and antitumor effect for these compounds. 1,3-Bis-chloroethyl-1-nitrosourea (BCNU), 1-chloroethyl-3-cyclohexyl-1-nitrosourea (CCNU), 1,3-bis-fluoroethyl-1-nitro-sourea (BFNU) and 1-chloroethyl-1-nitrosourea (CNU) decompose in buffered aqueous solution to yield haloethanol, vinyl halide, dihaloethane and acetaldehyde. Evidence is presented that these products are derived from an intermediate haloethyl carbonium ion. On the other hand, 1-chloroethyl-3,3-dimethyl-1-nitrosourea decomposes slowly in aqueous solution, generates acetaldeh?yde, but not the other volatile compounds described above, and is not toxic to murine L1210 leukemia cells in vitro. In contrast to the disubstituted nitrosoureas, chloroethylnitrosourea does not generate an organic isocyanate on aqueous decomposition, but is a very active antitumor agent both in vitro and in vitro. These observations support the hypothesis that the antitumor activity of the chloroethylnitrosoureas is due to the facile decomposition of the parent molecule to form a chloroethyl carbonium ion (or diazonium precursor).     

Mechanism of action of the nitrosoureas -- III. Reactions of bis-chloroethyl nitrosourea and bis-fluoroethyl nitrosourea with adenosine.

Previous papers in this series have provided evidence for the formation of haloethyl nucleoside derivatives from the interaction of the therapeutic nitrosoureas with cytidine and guanosine. Such derivatives could be important in explaining the cytotoxic action of bis-chloroethyl nitrosourea (BCNU), bis-fluoroethyl nitrosourea (BFNU), and related therapeutic agents. We now report the formation of 1-haloethyl adenosines from the reaction of BCNU and BFNU with adenosine. These 1-substituted haloethyl adenosines cyclize to form 1,N⁶-ethanoadenosine: 1-chloroethyladenosine with a half-life of 20 min in neutral aqueous solution at 37°, and 1-fluoroethyladenosine with a half-life of 20 hr under the same conditions. 1-Hydroxyethyladenosine is also a major product of the reaction of either BCNU or BFNU with adenosine, but it is not formed from the hydrolysis of either 1-haloethyladenosine. Accordingly, a reaction mechanism involving a cyclized nitrosourea derivative is proposed to explain the formation of this and other hydroxyethyl nucleosides.     

Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites

All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(β-d-glucopyranosyl)-1-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L121o leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonucleases micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM [¹⁴C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest.64, 1103 (1979)]. In contrast, the ratio of alkylation (L1210: bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210: bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.     

Mechanism of action of 2-haloethylnitrosoureas on deoxyribonucleic acid. Nature of the chemical reactions with deoxyribonucleic acid.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) covalently cross-links DNA under physiological conditions. Methyl substitution at either carbon atom of the 2-chloroethyl portion of the molecule prevents cross-linking. Haloalkyi nitrosoureas including 3-chloropropyl, 4-chlorobutyl and 5-chloropentyl, although they readily alkylate DNA, exhibit no ability to cross-link DNA. 3-(2-Chloroethyl)-1-methylcytosine hydrochloride and N⁴-(2-chloroethyl)- 1-methylcytosine hydrochloride, similar to intermediates suggested in the cross-linking process, alkylate PM2-CCC-DNA readily. These two cytosine derivatives also cyclize readily to give 3,N⁴-ethano-1-methylcytosine closely similar to a species isolated from the treatment of poly-C with BCNU. A number of processes including the extent of DNA alkylation, measured with [¹⁴C]CCNU labeled in the ethylene portion of the molecule, as well as concomitant DNA single strand scission, and intramolecular alkylation and/or hydrolysis of the chloroethyl cytidine intermediate were investigated as to their effects upon the interstrand cross-linking process.     

Studies of the mode of action of antitumour triazenes and triazines—IV. The metabolism of 1-(4-acetylphenyl)-3,3-dimethyltriazene

The metabolism of 1-(4-acetylphenyl)-3,3-dimethyltriazene has been studied in vivo and in vitro in mice. This dimethyltriazene was extensively metabolised in vivo and HPLC analysis of the plasma revealed the presence of two metabolites, the monomethyltriazene, 1-(4-acetylphenyl)-3-methyltriazene, and the arylamine, 4-aminoacetophenone. The dimethyltriazene was also biotransformed in vitro by a 9000 g fraction of mouse liver homogenate to products which were selectively toxic to TLX5 lymphoma cells. HPLC analysis of the products of in vitro metabolism under these conditions showed the presence of the monomethyltriazene but in an amount insufficient to account for the observed cytotoxicity. The monomethyltriazene was itself rapidly biotransformed by a 9000 g fraction of mouse liver homogenate, and by isolated mouse hepatocytes.     

Capillary Gas Chromatography and Thermionic N-P-Specific Detection of 13-Bis(2-chloroethyl)-l-nitrosourea (BCNU) or l-(2-Chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) in Stability and Pharmacokinetic Studies

An expedient, rapid, and sensitive capillary gas chromatographic method for the analysis of l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) or l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) in plasma is described. Separation of the underivatized nitrosourea compounds was performed on a 0.33-mm-i.d., 25-m fused-silica, SE-30 capillary column, and detection was carried out using a thermionic N–P-specific detector. The compounds were extracted from plasma with benzene with a yield of >87%. The assay was linear in the ranges of 0.001 to 0.5 and 0.5 to 25 µg/ml for CCNU or 0.003 to 0.50 and 0.5 to 25 µg/ml for BCNU, with correlation coefficients from 0.9914 to 0.9999 and coefficients of variation (CV) of <3.3%. Other antineoplastic agents did not interfere in the assay. The method was employed to study the pharmacokinetics of BCNU in rabbits. The plasma concentration-time curves were fit to a two-compartment model with a mean (SE) , , and total-body clearance of 2.898 (0.913) hr^–1, 0.1228 (0.0179) hr^–1, and 7.211 (2.862) liters/hr · kg, respectively. Further, the stability of BCNU and CCNU in solution was examined at different temperatures. Both compounds were stable in benzene or acetone (4 to 37°C) but labile in plasma even if refrigerated. The apparent rate constants for degradation of BCNU and CCNU were 0.09921 and 0.02853 hr^–1 at 4°C and 5.998 and 2.553 hr^–1 at 37°C, respectively.     

P-Glycoprotein mediated resistance to 5′-nor-anhydro-vinblastine (Navelbine®)

Summary Navelbine^? (NVB, vinorelbine tartrate) is a semisyntheticVinca alkaloid in which the catharanthine moiety contains an eight-membered ring in place of the nine-membered ring that is present in all naturally occurring members of the vinblastine group. This modification selectively reduces interaction with anoxalvs mititotic microtubules and may account for the lower neurotoxicity with improved antitumor activity that has been observed in clinical trials with breast, lung and ovarian cancer. We were interested in whether the structural modification in NVB would also alter the drug resistance profile. Specifically, our aim was to determine whether NVB, like vinblastine (VBL), participates in P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). NVB-resistant, murine P388 cells (P388/NVB), were derivedin vivo and used in conjunction with a battery of drug-resistant P388 cell linesin vivo and murine and human tumor cell linesin vitro to develop a resistance profile for NVB. P388/NVB bells were cross-resistant to drugs involved in MDR (doxorubicin, etoposide, amsacrine, vinblastine, vincristine and actinomycin D), but not to the alkylating agents, cyclophosphamide, carmustine, and cisplatin, or to the antimetabolites, 5-fluorouracil and methotrexate. P388/NVB cellular resistance to NVB was stable without drug pressure during continuous passagein vivo for more than ten weeks andin vitro for at least five weeks. These cells exhibited increased expression of P-gp, and a 30-fold level of resistance of NVBin vitro, which was completely reversable with verapamil. The MDR phenotype was confirmed in other tumor models. P388 tumors resistant to vinblastine, vincristine, doxorubicin, and etoposide were cross-resistant to NVBin vivo. Likewise, human KB carcinoma cells resistant to colchicine, MDA-A1^R breast carcinoma cells resistant to doxorubicin, and murine B16/F10 melanoma cells transfected with the humanmdr1 gene were cross-resistant to NVBin vitro. Finally, P388 cells resistantin vivo to melphalan, a drug that does not participate in MDR, were cross-resistant to NVB. Therefore, cellular resistance to NVB, like otherVinca alkaloids, may arise by a P-glycoprotein-mediated MDR mechanism, but may also involve non-MDR mechanisms in cells that do not overexpress P-gp.     

In vivo inhibition of adenosine deaminase by 2'-deoxycoformycin in mouse blood and leukemia L1210 cells

Adenosine deaminase (ADA) activities in mouse whole blood, washed erythrocytes and L1210 cells were 0.48, 0.93 and 4.76 units/ml respectively. Methods were developed to determine the second-order association rate constant (k₁) of a tight-binding ADA inhibitor, deoxycoformycin (DCF), and ADA in mouse blood and L1210 cells in vivo. After i.v. injection of DCF, the inhibition of the enzyme was of a monophasic pseudo-first-order nature in blood and biphasic (with an initial lag of 3–5 min) in L1210 cells. In contrast, i.p. injection of DCF produced the opposite pattern, monophasic in L1210 cells and biphasic in blood. The apparent k₁ values determined from the linear portions of these curves were compared with the k₁ values obtained in vitro. The mean k₁ values in vivo were: 4.2 × 10⁴ and 1.4 × 10⁴M^?1 sec^?1 in blood after i.v. and i.p. injections, respectively, and 2.6 × 10³ and 2.2 × 10⁴ M^?1 sec^?1 in L1210 cells after i.v. and i.p. injections respectively. The k₁ values with either whole blood or L1210 in vitro (3.1 × 10⁴ and 5.5 × 10³ M^?1 sec^?1, respectively) were of the same order of magnitude as those obtained with these tissues in vivo. In contrast, the k₁ values were about 150 to 1400-fold higher when either blood hemolysates (4.8 x 10^?6M^?1 sec^?1) or homogenized L1210 cells (7.5 x 10^6?1 sec^?1) were used. The 150 to 1400-fold higher k₁ values for blood hemolysates and homogenized L1210 cells than for intact cell samples (whole blood or whole L1210 ascitic fluid) suggest that the cell membrane plays a role in the interaction of DCF and ADA in these cell lines. The similarity of the rates of association of DCF and ADA in vivo and in vitro for mouse blood and ascites L1210 cells suggests that data obtained in vitro may be used to estimate the k₁ values in in vivo conditions.     

Biochemical studies of a new antitumor agent, O2,2'-cyclocytidine

Cyclocytidine, O²,2′-cyclocytidine (cyclo-C), structurally related to 1-β-d-arabinofuranosyl cytosine (ara-C), inhibits the incorporation of ³H-thymidine into DNA of L1210 leukemia cells both in vivo and in vitro, and human normal marrow cells and leukemic cells in vitro; but it has no effect on uridine or l-valine incorporation. The inhibition is proportional to the dose, and on an equimolar basis in vivo, cyclo-C shows a lesser but longer lasting effect than ara-C. Cyclo-C has no effect on thymidine incorporation into DNA of L1210 leukemic cells resistant to ara-C. Cyclo-C is stable in 0.1 M Tris buffer, pH 7.0, at 37° for the incubated 4-hr period, but at pH 9.0 for 1hr, > 90 per cent is hydrolyzed to ara-C. When cyclo-C was incubated at 37° for 60 min with plasma from various species, the supernatant from boiled human plasma, or Eagle's minimum essential medium, the only product found was ara-C. Ara-C was found in dog's plasma and urine 2 hr after the i.v. injection of ¹⁴C-cyclo-C and in mouse urine 1 hr after the injection (i.p.). The above results suggest that cyclo-C is hydrolyzed to ara-C and may thus serve as a reservoir of ara-C. Intermittent treatment with cyclo-C may, therefore, replace the current clinical practice of 5-day continous intravenous infusion of ara-C.     

Modulation of nitrosourea resistance in human colon cancer by O6-methylguanine.

Human colon cancer is resistant to a variety of alkylating agents including the nitrosoureas. To specifically evaluate nitrosourea resistance, we studied the role of O6-alkylguanine-DNA alkyltransferase (alkyltransferase) which is known to repair nitrosourea-induced cytotoxic DNA damage. Alkyltransferase activity varied over a similar wide range in 25 colon cancer biopsies and 14 colon cancer cell lines but the activity was not correlated with differentiation status, Dukes' classification or in vitro growth characteristics. 1,3-Bis-(2-chloroethyl)-1-nitrosourea (BCNU) resistance and alkyltransferase activity were highly correlated (R2 = 0.929, P less than 0.001) in 7 different colon cancer cell lines, suggesting that the alkyltransferase is an important component of nitrosourea resistance in colon cancer cells. In the BCNU-resistant, high alkyltransferase VACO 6 cell line, inactivation of the alkyltransferase by O6-methylguanine caused a proportional decrease in the BCNU IC50, consistent with that predicted by the regression line. Enzyme inactivation was also associated with a marked increase in DNA cross-link formation. Because alkyltransferase correlates with BCNU resistance in colon cancer, and resistance can be reversed by inactivating the protein, the alkyltransferase may have an important role in nitrosourea resistance in human colon cancer cells. These data provide the rationale for clinical trials in colon cancer with biochemical modulators of the alkyltransferase to increase the therapeutic response to nitrosoureas.     

Nitrosourea-induced DNA single-strand breaks.

Reaction of DNA with nitrosoureas in vitro results in extensive formation of alkali labile sites. Two types of single-strand scission (SSS) processes may be distinguished by their different rates: (1) type I SSS which occurs relatively fast at high pH, and (2) type II SSS which is a much slower process. Neither of these processes is affected by free radical traps. Dimethyl sulfate, which is known to alkylate DNA bases but not phosphate residues, shows no type I SSS but does show extensive type II SSS. That the latter process involves alkylation of bases followed by the formation of apurinic sites was confirmed by using endonuclease VI, an enzyme specific for apurinic positions. Reactions of chloroethylnitrosoureas with DNA produces both type I and type II SSS. Aliphatic amines produced in the decomposition of alkyl nitrosoureas do not contribute significantly to the scission of apurinic sites via Schiff base formation. However, this process may be significant for aryl nitrosoureas. Ethyl nitrosourea (ENU), 1, 3-bis(2-chloroethyl)nitrosourea (BCNU), and 3-cyclohexyl-1-(2-hydroxyethyl)-1-nitrosourea (CHNU) readily degrade poly A by phosphate alkylation, with rates that parallel their relative rates of decomposition. The relative rates of hydrolysis of triethylphosphate and β-hydroxyethyl diethyl phosphate parallel the type I SSS observed for ENU and CHNU with DNA. The type I SSS of DNA by these compounds appears to involve a similar phosphotriester formation and hydrolysis. The type I SSS is in accord with the observed extreme liability of β-hydroxyethyl diethyl phosphate which is attributed to participation of the OH group, and by the fact that methylation of the OH completely inhibits the type I SSS process.     

Pharmacokinetic profile of fotemustine in rat plasma by electrochemical detection

1. A differential pulse polarographic (DDP) assay of diethyl-1-[3-(2-chloroethyl)-3-nitrosoureido] ethylphosphonate (fotemustine) was developed to determine the kinetics of this nitrosourea in plasma, brain, liver, lung and kidney. The optimized polarographic determination, previously applied to BCNU and CCNU, attained a limit of detection of 0.3 micrograms fotemustine/ml plasma and 1 microgram/g in other tissues; the calibration curve in electrolyte or in plasma was linear between 0.5 and 100 micrograms/ml. 2. The choice of electrolyte, the effects of pH, temperature, light, and the stability of fotemustine in samples were investigated. Recovery of fotemustine was 76-90% from lung greater than kidney greater than plasma greater than brain greater than liver; the variability coefficients were low (4.0-7.3%). Tissue samples could be stored for 20 days at -20 degrees C without loss of the compound. 3. Plasma kinetics of fotemustine and BCNU given to male rats at therapeutic doses (20 mg/kg i.v.) fitted a bi-exponential equation. Two minutes after injection plasma, levels of unchanged nitrosoureas were 15 and 11 micrograms/ml respectively. Fotemustine could be measured (0.92 microgram/ml) for 3 h, while BCNU could not be detected after 60 min. Unchanged fotemustine was cleared from the blood stream 3-5 times more slowly than BCNU.     

Antitumor activity of intoplicine (RP 60475, NSC 645008), a new benzo-pyrido-indole: evaluation against solid tumors and leukemias in mice

Summary Intoplicine (RP 60475, NSC 645008) is a new 7H-benzo[e]pyrido[4,3-b] indole derivative which interacts with DNA and inhibits both topoisomerases I and II.In vitro it was found cytotoxic against various cell types with greater cytotoxicity towards solid tumor cells. We report here the anticancer activity of RP 60475 against a variety of transplantable tumors of mice, and also its cross-resistance profile in leukemias. The end points used were % T/C (median tumor weight of the Treated over the Control × 100) and logCK (log₁₀ cell kill total). RP 60475 administered i.v. was found schedule-independent with a peak plasma level problem. It had a good therapeutic index and host recovery usually occurred 7.5 days post last treatment. RP 60475 was found to be highly active against early stage colon 38 (T/C=0%, 2.9 logCK) and could induce 5/5 complete regressions of advanced stage tumor. It was found active against colon adenocarcinoma 51 (T/C=3.6%, 1.9 logCK) and colon carcinoma 26 (T/C=11.7%, 1.2 logCK). Most of the mammary adenocarcinomas were found very responsive, MA16/C (T/C=0%, 2.8 logCK), MA14/A (T/C=0%, 1.4 logCK), MA13/C (T/C=0%, 3.1 log CK) and MA44 (T/C=34%). Excellent activity was also observed against early stage pancreatic ductal adenocarcinoma 03 (T/C=0%) and RP 60475 could achieve 5/5 complete regressions of upstaged tumor. Activity was also obtained on Glasgow osteogenic sarcoma (T/C=0%, 3.3 logCK), on B16 melanoma (T/C=14%, 1.3 logCK) and to a lesser extent on Lewis lung carcinoma (T/C=33.2%). Evaluation of RP 60475 against leukemia sublines with acquired resistance, revealed that L1210/cisplatin and L1210/BCNU were not cross-resistant to RP 60475 whereas P388/vincristine was partially cross-resistant to RP 60475 and P388/doxorubicin was cross-resistant to RP 60475. Based on RP 60475 broad activity against transplantable tumors of mice, its effectiveness against some resistant sublines, its original mechanism of action and its acceptable toxicological profile, this compound was selected for clinical trials.     

Inhibition of intracellular esterases by antitumour chloroethylnitrosoureas. Measurement by flow cytometry and correlation with molecular carbamoylation activity

Antitumour chloroethylnitrosoureas (Cnus) decompose in physiological conditions yielding alkylating species and organic isocyanates. While antitumour activity is mainly attributed to the alkylation of DNA, carbamoylation of intracellular proteins by isocyanates may also have pharmacological and toxicological relevance. We previously reported a novel dynamic flow cytoenzymological assay for esterase inhibition in intact murine cells by BCNU and related isocyanates, and proposed that this might form the basis of an assay for intracellular carbamoylation. We have now examined a wide range of Cnus, isocyanates, and alkylating agents for their ability to inhibit cellular esterases. BCNU, CCNU, their derived isocyanates, and the 4-OH metabolites of CCNU exhibited potent inhibitory activity (I50 values 5.5 x 10(-5)-7.3 x 10(-4) M). Chlorozotocin and GANU were relatively inactive (I50 much greater than 10(-2) M). ACNU, TCNU and the 2-OH metabolites of CCNU exhibited intermediate activity (I50 values 1.1 x 10(-3)-2.3 x 10(-2) M). Compounds not able to form isocyanates were essentially inactive. Poor membrane permeability was also implicated in the weak activity of chlorozotocin and GANU. There was overall a good correlation between esterase inhibition and chemical carbamoylating activity, but some particular differences were identified. We concluded that flow cytoenzymological assay appears to have the potential to provide useful measurement of intracellular protein carbamoylation by existing Cnus and novel derivatives, and also offers the advantage of cell subpopulation identification for in vivo evaluation of these agents.