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.     

Stereoselective monooxygenation of carcinostatic 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by purified cytochrome P-450 isozymes

Three highly purified forms of liver microsomal cytochrome P-450 (P-450a, P-450b and P-450c) from Aroclor 1254-treated rats catalyzed 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) monooxygenation in the presence of purified NADPH-cytochrome P-450 reductase, NADPH, and lipid. Differences in the regioselectivity of CCNU and MeCCNU monohydroxylation reactions by the cytochrome P-450 isozymes were observed. Cytochrome P-450-dependent monooxygenation of CCNU gave only alicyclic hydroxylation products, but monooxygenation of MeCCNU gave alicyclic hydroxylation products, an αhydroxylation product on the 2-chloroethyl moiety, and a trans-4-hydroxymethyl product. A high degree of stereoselectivity for hydroxylation of CCNU and MeCCNU at the cis-4 position of the cyclohexyl ring was demonstrated. All three cytochrome P-450 isozymes were stereoselective in primarily forming the metabolite cis-4-hydroxy-trans-4-Methyl-CCNU from MeCCNU. The principal metabolite of CCNU which resulted from cytochromes P-450a and P-450b catalysis was cis-4-hydroxy CCNU, whereas the principal metabolites from cytochrome P-450c catalysis were the trans-3-hydroxy and the cis-4-hydroxy isomers. Total amounts of CCNU and MeCCNU hydroxylation with cytochrome P-450b were twice that with hepatic microsomes from Aroclor 1254-treated rats. Catalysis with cytochromes P-450a and P-450c was substantially less effective than that observed with either cytochrome P-450b or hepatic microsomes from Aroclor 1254-treated rats.     

Morphological transformation of hamster embryo cells by cancer chemotherapeutic agents

Studies were made on induction of morphological transformation of hamster embryo cells by various types of cancer chemotherapeutic agents, including alkylating agents, antimetabolites, antibiotics and natural products. Cyclophosphamide, actinomycin D, mitomycin C and adriamycin hydrochloride, which are widely used as cancer chemotherapeutic agents and are also known to be carcinogenic in animals, produced significant morphological transformation. Two other compounds that are known to be carcinogenic in vivo, thio-TEPA and daunomycin hydrochloride, did not induce transformation. The anti-metabolites 6-mercaptopurine, 5-fluorouracil, cytosine arabinoside and methotrexate also did not produce morphological transformation of hamster embryo cells.     

Mechanism of action of 2-haloethylnitrosoureas on deoxyribonucleic acid. Nature of the intermediates from nitrosourea decomposition.

Direct current (DC) and differential pulse polarographic analyses were used to measure the rates of decomposition of a series of 2-haloethylnitrosoureas in aqueous solution. Measured by these methods, the rates of the first and rate-determining step which show a marked pH and solvent dependence agree with the overall rate of decomposition measured by gas evolution. In the 1,3-bis(haloethyl)-1-nitrosourea series, changing the nature of the halogen X has a small effect on the rate of decomposition. In the 3-cyclohexyl-1-(2-haloethyl)-1-nitrosourea series, changing X for OH or OCH₃ results in the rate of hydrolysis being reduced considerably. A free—NH₂ group in the nitrosourea structure as in CNU, MNU, ENU, CPNU, 4-CBNU and 5-CPNU accelerates considerably the rate of decomposition relative to the BCNU and CCNU series. Arrhenius parameters for the decomposition in aqueous pH 7.1 solution in the temperature range 28–47° were obtained for BFNU, BCNU and BBNU: log A, ?20.1± 1.4,?21.6± 0.7 and ?22.3±1.6; E_a, 24.4 ± 2.0, 26.5± 1.0 and 27.2 m 2.3 kcal/mole. The corresponding values for BINU were estimated as log A,?23.3± 3.0; E_a, 28.0± 3.0 kcal/mole. Examination of the decomposition products of 1,3-bis(2-chloropropyl)-1-nitrosourea (BCNU-β-Me) and 1,3-bisl 1-(chloromethyl)ethyl]-1-nitrosourea (BCNU-α-Me) favors decomposition pathway B via the diazohydroxide and cyclic chloronium ion for BCNU-β-Me and via the diazohydroxide and/or 2-chloro-1-methylethyl carbonium ion for BCNU-α-Me. While there is no evidence for the contribution of pathway A via a 2-imino-N-nitrosooxazolidinone for these compounds, consideration of product type and yields implicates a third decomposition pathway, via a 1,2,3-oxadiazoline intermediate. Additional evidence for an oxadiazoline intermediate is obtained by the isolation of 2-bromoethanol when BCNU is decomposed in the presence of a high concentration of sodium bromide.     

Influx and efflux transport as determinants of melphalan cytotoxicity: Resistance to melphalan in MDR1 overexpressing tumor cell lines

There is a considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) and other hematopoietic tumors. We hypothesized that this may be due to variations in the expression of influx and efflux transporters of melphalan. We measured the expression of the influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines. Variation in the intracellular accumulation accounted for nearly half of the variation in the cytotoxicity of melphalan in MM cell lines (r² = 0.47, P = 0.04). High expression of the efflux transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of melphalan (r² = 0.56, P = 0.03 and r² = 0.62, P = 0.02, respectively). The effect was reversed by the MDR1 inhibitor cyclosporine. In addition, the MDR1 overexpressing HL-60 cell line showed 10-fold higher resistance to melphalan than the non-MDR1 expressing one. Again, the resistance was reversed by cyclosporine and by MDR1-specific shRNA.LAT1 was the major influx transporter in tumor cell lines with 4000-fold higher expression than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity.     

Novel amidine analogue of melphalan as a specific multifunctional inhibitor of growth and metabolism of human breast cancer cells

A novel amidine analogue of melphalan (AB4) was compared to its parent drug, melphalan in respect to cytotoxicity, DNA and collagen biosynthesis in MDA-MB-231 and MCF-7 human breast cancer cells. It was found that AB4 was more active inhibitor of DNA and collagen synthesis as well more cytotoxic agent than melphalan. The topoisomerase I/II inhibition assay indicated that AB4 is a potent catalytic inhibitor of topoisomerase II. Data from the ethidium displacement assay showed that AB4 intercalated into the minor-groove at AT sequences of DNA. The greater potency of AB4 to suppress collagen synthesis was found to be accompanied by a stronger inhibition of prolidase activity and expression compared to melphalan. The phenomenon was related to the inhibition of beta(1)-integrin and IGF-I receptor mediated signaling caused by AB4. The expression of beta(1)-integrin receptor, as well as Sos-1 and phosphorylated MAPK, ERK(1) and ERK(2) but not FAK, Shc, and Grb-2 was significantly decreased in cells incubated for 24h with 20 microM AB4 compared to the control, not treated cells, whereas in the same conditions melphalan did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. These results indicate the amidine analogue of melphalan, AB4 represent multifunctional inhibitor of breast cancer cells growth and metabolism.     

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.     

In vitro pharmacokinetics and pharmacodynamics of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

The relationship between treatment efficacy and the pharmacokinetics (PK) and pharmacodynamics (PD) of anticancer drugs is poorly defined. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an alkylating agent used in the treatment of brain and other forms of cancer. It is postulated that BCNU kills cells by forming DNA interstrand cross-links. The present study was undertaken to characterize the PK and PD of BCNU in mouse L1210 cells. L1210 cells were exposed to BCNU (0-160 microM) and analyzed for intracellular BCNU concentrations, DNA interstrand cross-links, cell cycle phase, and cytotoxicity. The half-life of BCNU in cells was approximately 40 min. The maximum reduction of mitochondrial enzyme activity (maximum cell death) achieved within 24 hr after exposure to BCNU was concentration-dependent and could be described by a Hill equation. At lower concentrations, the area under the DNA interstrand cross-link-time curve linearly correlated with the maximum cell death and the area under the BCNU concentration-time curve. BCNU induced cell accumulation in the G(2)/M phase of the cell cycle, which continued even after apparent completion of cross-link repair. Loss of membrane permeability was minimal (approximately 2%) during the first 24 hr. Thereafter, cells died exponentially over the next 9 days, primarily by necrosis. In conclusion, while cytotoxicity was concentration-dependent, an indirect relationship was found among the time-course of BCNU concentrations, DNA interstrand cross-links, and cell death. Because of the disparity between the time-scale of PK and PD, focusing only on the early events may provide limited information about the process of anticancer drug-induced cell death.     

Brain uptake and CNS levels of 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)urea (HECNU)

The uptake of ¹⁴C-labeled 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl) urea (HECNU) into the brain was investigated in the rat after intracarotid injection according to the method of OLDENDORF, as well as in cisternal cerebrospinal fluid obtained by suboccipital puncture after i.v. injection of the drug.The brain uptake index was 31.9 ± 2.9%. Cerebrospinal fluid/blood quotients after i.v. injection were 0.82 at 10 min and 1.10 at 60 min. The results of both methods clearly show that HECNU, in spite of its hydrophilic property, easily penetrates the blood-brain barrier.     

A structure--activity study of seven new water soluble nitrosoureas.

The in vitro alkylating activity, carbamoylating activity, decomposition rates and octanol-water partition coefficients (Log P) of seven water soluble chloroethylnitrosourea antitumor agents and a reference lipid soluble analog were correlated with their biological activities in mice. The alkylating activity of each compound demonstrated a significant inverse linear correlation with both the decomposition rate in 0.1 M sodium phosphate buffer. pH7.4 (r = -0.92,P< 0.01), and the molar ld₁₀ dose (r = 0.87, P< 0.01). A direct relationship was found between the Log P values and both the alkylating activity (r = -0.86. P< 0.01) and the molar ld₁₀ dose (r = 0.77, P< 0.025). However, the addition of the variable. Log P, in multiple regression analysis did not contribute significantly to any of the direct correlations of chemical parameters with biological variables. In comparison, carbamoylating activity did not function as an independent variable for the relative myelotoxicity or lethality of each compound. All water soluble drugs except for chlorozotocin and 1-(2 chloroethyl)-3-(β-d-glucopyranosyl)-1-nitrosourea, the two analogs with glucose carriers, produced a significant reduction in circulating neutrophils at their respective ld₁₀ doses. There was no correlation between relative myelotoxicity and alkylating activity, carbamoylating activity or Log P. The glucose moiety appears to function as an independent variable for reducing nitrosourea cytotoxicity to bone marrow cells without significantly altering antitumor activity.     

KS900: A hypoxia-directed, reductively activated methylating antitumor prodrug that selectively ablates O-alkylguanine-DNA alkyltransferase in neoplastic cells

To most effectively treat cancer it may be necessary to preferentially destroy tumor tissue while sparing normal tissues. One strategy to accomplish this is to selectively cripple the involved tumor resistance mechanisms, thereby allowing the affected anticancer drugs to gain therapeutic efficacy. Such an approach is exemplified by our design and synthesis of the intracellular hypoxic cell activated methylating agent, 1,2-bis(methylsulfonyl)-1-methyl-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS900) that targets the O-6 position of guanine in DNA. KS900 is markedly more cytotoxic in clonogenic experiments under conditions of oxygen deficiency than the non-intracellularly activated agents KS90, and 90M, when tested in O⁶-alkylguanine-DNA alkyltransferase (AGT) non-expressing cells (EMT6 mouse mammary carcinoma, CHO/AA8 hamster ovary, and U251 human glioma), and than temozolomide when tested in AGT expressing cells (DU145 human prostate carcinoma). Furthermore, KS900 more efficiently ablates AGT in HL-60 human leukemia and DU145 cells than the spontaneous globally activated methylating agent KS90, with an IC₅₀ value over 9-fold lower than KS90. Finally, KS900 under oxygen-deficient conditions selectively sensitizes DU145 cells to the chloroethylating agent, onrigin, through the ablation of the resistance protein AGT. Thus, under hypoxia, KS900 is more cytotoxic at substantially lower concentrations than methylating agents such as temozolomide that are not preferentially activated in neoplastic cells by intracellular reductase catalysts. The necessity for intracellular activation of KS900 permits substantially greater cytotoxic activity against cells containing the resistance protein O⁶-alkylguanine-DNA alkyltransferase (AGT) than agents such as temozolomide. Furthermore, the hypoxia-directed intracellular activation of KS900 allows it to preferentially ablate AGT pools under the oxygen-deficient conditions that are present in malignant tissue.     

Inhibition of calcium uptake and catecholamine release by 8-(n,n-diethylamino)- octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) In cultured bovine adrenal chromaffin cells

Effects of intracellular calcium antagonists, 8-(f,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) and 1-(5-(p-nitrophenyl)-furfurylidene-amino) hydantoin sodium hydrate (dantrolene sodium), on catecholamine release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. TMB-8 inhibited carbamylcholine-evoked catecholamine release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner with a similar potency. On the contrary, dantrolene sodium did not show obvious inhibitory effects of catecholamine release and ⁴⁵Ca²⁺ uptake. Although TMB-8 inhibited the high K⁺-evoked catecholamine release and ⁴⁵Ca²⁺ uptake, the potency of the drug was approximately 100-fold less than when used to inhibit the carbamylcholine-evoked catecholamine release and ⁴⁵Ca²⁺ uptake. The inhibitory effect of TMB-8 on the carbamylcholine-evoked catecholamine release was not overcome by an increase in an extracellular calcium concentration, and was not due to competitive antagonism at the nicotinic receptor site. Moreover, TMB-8 inhibited the carbamylcholine-stimulated ⁴⁵Ca²⁺ efflux, but dantrolene sodium failed to affect it. These results suggest that TMB-8, a well-known intracellular calcium antagonist, prevents the cellular calcium uptake in cultured adrenal chromaffin cells, and thus prevents catecholamine release.     

Reductive activation of the prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively occurs in oxygen-deficient cells and overcomes O-alkylguanine-DNA alkyltransferase mediated KS119 tumor cell resistance

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) is a prodrug of the 1,2-bis(sulfonyl)hydrazine class of antineoplastic agents designed to exploit the oxygen-deficient regions of cancerous tissue. Thus, under reductive conditions in hypoxic cells this agent decomposes to produce the reactive intermediate 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which in turn generates products that alkylate the O⁶-position of guanine in DNA. Comparison of the cytotoxicity of KS119 in cultured cells lacking O⁶-alkylguanine-DNA alkyltransferase (AGT) to an agent such as Onrigin™, which through base catalyzed activation produces the same critical DNA G-C cross-link lesions by the generation of 90CE, indicates that KS119 is substantially more potent than Onrigin™ under conditions of oxygen deficiency, despite being incompletely activated. In cell lines expressing relatively large amounts of AGT, the design of the prodrug KS119, which requires intracellular activation by reductase enzymes to produce a cytotoxic effect, results in an ability to overcome resistance derived from the expression of AGT. This appears to derive from the ability of a small portion of the chloroethylating species produced by the activation of KS119 to slip through the cellular protection afforded by AGT to generate the few DNA G-C cross-links that are required for tumor cell lethality. The findings also demonstrate that activation of KS119 under oxygen-deficient conditions is ubiquitous, occurring in all of the cell lines tested thus far, suggesting that the enzymes required for reductive activation of this agent are widely distributed in many different tumor types.     

CCNU-Adriamycin association induces earlier and more severe nephropathy in rats

Adriamycin (ADR) has a broad spectrum of antitumoral activity but is ineffective against human brain tumors. However, such tumors can be sensitive to a combination of adriamycin and lipophilic antineoplastic agents such as the nitrosoureas. CCNU, a nitrosourea, induces cholestasis in the rat and ADR is predominantly excreted via the biliary route. We decided to investigate the effect of CCNU on the nephrotic syndrome induced by ADR. Female Wistar rats were injected with a single dose of 10 mg/kg ADR and 24 h later were force fed 20 mg/kg CCNU in a single dose. Animals were sacrificed 4, 8, 15, 21, 28 or 60 days after the injection of ADR. A high rate of fatality (60%) occurred after the 21st day of treatment. Biological changes (alkaline phosphatase, SGPT, bilirubin) and ultrastructural studies showed that CCNU and CCNU+ADR induced the same degree of cholestasis. With the administered dose, CCNU is not nephrotoxic, ADR induces a nephrotic syndrome and ADR+CCNU appeared more nephrotoxic. With ADR, visceral epithelial foot process fusion was seen on day 15 and tubulo-interstitial lesions and glomerulosclerosis on day 60. With ADR+CCNU fusion of the foot process was seen on day 4, glomerular vacuolation on day 8, tubulo-interstitial alterations on day 15 and glomerulosclerosis on day 60. For both ADR and ADR+CCNU wrinkling and thickening of the basement membrane of proximal tubular cells were seen on day 60. Lipid mesangial overload was seen with ADR and was more intense with ADR+CCNU on day 60. CCNU hepatoxicity modifies the excretion of ADR and the predominantly renal excretion of ADR seems to induce earlier renal alterations in ADR+CCNU-treated rats. This study supports the concept that lipid mesangial overload may play an important role in chronic progressive glomerulosclerosis and thus the ADR+CCNU combination appears to be an interesting model in which to study these relationships.This work was supported by INSERM-CNAMTS and ROGER BELLON Company grants     

Increased cytotoxicity of an unusual DNA topoisomerase II inhibitor compound C-1305 toward HeLa cells with downregulated PARP-1 activity results from re-activation of the p53 pathway and modulation of mitotic checkpoints

Our previous studies have shown that murine fibroblast cells, in which PARP-1 gene was inactivated by gene disruption, are extremely sensitive to triazoloacridone compound C-1305, an inhibitor of DNA topoisomerase II with unusual properties. Here, we show that pharmacological inhibition of PARP-1 activity by its inhibitor compound NU1025, sensitizes human cervical carcinoma HeLa cells to compound C-1305 compared to treatment with drug alone. Cytotoxic effect of drug/NU1025 of other topoisomerase II inhibitors varied depending on the dose of PARP-1 inhibitor. Increased cytotoxicity of topoisomerase II inhibitor/NU1025 combinations was attributable to the re-activation of the p53 pathway in drug-treated HeLa cells. This lead to a more stringent cell cycle checkpoint control during G2 and M and enhanced cell death by mitotic catastrophe induced by drug/NU1025 combinations. Interestingly, treatment of HeLa cells with NU1025 alone also increased p53 expression. This effect is, at least in part, related to the inhibition of proteasome activity by drug treatments. Together, our results show that concomitant inhibition of topoisomerase II and PARP-1 leads to the synergistic cytotoxic effect toward tumor cells that may be important for combination therapies with NU1025 and topoisomerase II inhibitors. We also confirmed our earlier work and show the important role of PARP-1 activity in the maintenance of the G2 arrest induced by DNA damaging drugs. Finally, based on our studies we propose that NU1025 and possibly other inhibitors of PARP-1 may be used as non-genotoxic agents to activate p53 in tumor cells with non-functional p53 pathways.     

Inhibition by phenothiazine derivatives of the adenylate cyclase of amphibian oocytes

The adenylate cyclase activity of membranes of Xenopus laevis oocytes and follicle cells was affected by the presence of 2-chloro-10-(3-aminopropyl)phenothiazine (CAPP) and two other antipsychotic drugs, fluphenazine and penfluridol. CAPP, at concentrations of 10 and 100 microM, had opposite effects on the activation of the oocyte adenylate cyclase by effectors that act through the G/F regulatory subunit. Under these conditions, the drug stimulated the activation by fluoride and drastically inhibited the activation by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and by cholera toxin and GTP. The activity of the catalytic subunit measured in the presence of either Mn2+ or forskolin was not affected by 100 microM CAPP. however, concentrations of this drug above 100 microM inhibited the adenylate cyclase activated by fluoride or by forskolin and also inhibited the activity of a calmodulin-independent cyclic nucleotide phosphodiesterase present in the same oocyte membrane preparation. Oocyte adenylate cyclase has been shown previously to be inhibited by the hormone progesterone. The inhibitory effect of CAPP is additive to that measured with the hormone, indicating that these compounds act through different mechanisms. CAPP did not modify the concentration of Gpp(NH)p required to yield half-maximal activation and, although the drug inhibited more strongly at lower concentrations of Gpp(NH)p, saturating amounts of the guanine nucleotide did not reverse completely the inhibition caused by CAPP. The effects of these antipsychotic drugs on oocyte adenylate cyclase did not require the presence of free Ca2+ and were not altered by the addition of exogenous calmodulin and calcium.     

Antitumour imidazotetrazines--IV. An investigation into the mechanism of antitumour activity of a novel and potent antitumour agent, mitozolomide (CCRG 81010, M & B 39565; NSC 353451)

8-Carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4-(3H )-one- mitozolomide (CCRG 81010, M & B 39565, NSC 353451) is a potent inhibitor of the growth of a number of experimental tumours and can potentially decompose to give either an isocyanate or the monochloroethyltriazene (MCTIC). In vitro CCRG 81010 is not cross-resistant with the bifunctional alkylating agents against the Walker carcinoma. To investigate the mechanism of the antitumour activity of CCRG 81010 a comparison has been made with BCNU and MCTIC on precursor incorporation into macromolecules in TLX5 mouse lymphoma cells. Whereas BCNU produces a rapid and extensive inhibition of both (methyl 3H) thymidine and [5-3H]uridine incorporation into acid-insoluble material, neither CCRG 81010 or MCTIC have an early effect on precursor incorporation. Inhibition of precursor uptake is also not produced by concentrations of 2-chloroethylisocyanate that inhibit intracellular glutathione reductase activity. The potential carbamoylating activity of CCRG 81010 has also been assessed by comparing its effect with that of BCNU and 2-chloroethyl isocyanate on enzymes known to be inhibited by carbamoylation. Such enzymes, glutathione reductase, chymotrypsin and gamma-glutamyltranspepidase are not inhibited by CCRG 81010 under conditions where BCNU and 2-chloroethyl isocyanate show complete inhibition of enzyme activity, suggesting an absence of carbamoylating species. The results suggest that the most likely antitumour metabonate produced from CCRG 81010 is the triazene MCTIC.     

Antagonism of the Acute and Long–term Biochemical Effects of 4–Chloroamphetamine on the 5–HT Neurones in the Rat Brain by Inhibitors of the 5–Hydroxytryptamine Uptake

Abstract The effects of H 102/09 ((Z)–3–(4–bromophenyl)–N, N–dimethyl–3–(3–pyridyl)allylamine dihydrochloride) and chlorimipramine, two inhibitors of the membrane 5–hydroxytryptamine (5–HT) uptake, on the acute and long–term effects of 4–chloroamphetamine on the 5–HT neurones in rats were examined. The acute effect determined as the decrease in the brain level of 5–HT was antagonized by both compounds. The long–term effects determined as the decreases in 5–hydroxyindoles and the synaptosome accumulation of ¹⁴–C–5–HT were antagonized by a single injection of H 102/09 (20 mg/kg intraperitoneally) immediately before or shortly after the 4–chloroamphetamine administration. Chlorimipramine, 40 mg/kg intraperitoneally, had no antagonizing effect but on repeated injections of 40 mg/kg intraperitoneally before and four times with 3 hours' intervals after 4–chloroamphetamine, a partial antagonism of the longterm decrease in 5–HT was obtained. Combined with proadifen (SK & F 525 A) chlorimipramine partially antagonized the long–term effect. Two monoamine oxidase inhibitors (pheniprazine and clorgyline), pretreatment of the rats with p–chlorophenylalanine (2 × 300 mg/kg intraperitoneally) or proadifen alone (40 mg/kg intraperitoneally injected before and four times with 3 hours' interval) had no antagonistic effect on the long–term decrease in 5–HT. The accumulation of ³H–chloroamphetamine in synaptosomes of the hypothalamus midbrain region was slightly decreased by high concentrations of H 102/09 at 37° and 0°. H 102/09 decreased slightly but significantly the ratio between the ³H–chloramphetamine in the synaptosomal fraction and the supernatant as obtained when the rats were killed 2 hours after the injection. The increased efflux of ³H–5–HT from synaptosomes in vitro produced by 4–chloroamphetamine, ouabain and low external Na⁺ concentrations was in all cases inhibited by H 102/09 at 2.5 × 10^–6 M. Although the antagonism of the acute effect of 4–chloroamphetamine seems to be related to inhibition of the membrane 5–HT transport mechanism the antagonism of the long–term, irreversible effect produced by 4–chloroamphetamine may also involve an additional as yet unknown mechanism.