Carlo Nisi

Lysosomal enzyme inhibitors and antimetastatic activity in the mouse

Abstract Saccharo- 1,4 -lactone, a β-glucuronidase inhibitor, and three protease inhibitors did not affect primary tumour growth when tested against Lewis lung carcinoma in mice. The number of lung metastases was significantly reduced by aprotinin only. The inactivity of leupeptin and pepstatin seems to indicate that cathepsin B and D are not involved in metastasis formation. These results also indicate that aprotinin, a broad spectrum protease inhibitor, reduces tumour dissemination, in addition to the tumour invasion already reported by others.

Investigation on the oxidative N-demethylation of aryl triazenes In vitro

Abstract The oxidative N -demethylation of several p -substituted phenyl triazenes has been determined in vitro under carefully controlled conditions. Three of these compounds had been reported not to be appreciably demethylated, one of them still possessing antitumour activity. The per cent demethylation figures obtained for the tested compounds ranged from about 20 to 60 per cent and was not dependent on the nature of the solvent used to keep the drug in solution. The enzymatic reaction proved to be strongly inhibited by diazonium cations either generated by spontaneous hydrolysis of the drug or added to the incubation mixture. The reaction is also inhibited at various degrees by the monomethyl derivatives produced by demethylation of the dimethyl triazenes. The inhibition by diazonium and monomethyl derivatives can not account for the observed time-course of demethylation, which occurs for all the tested compounds only in the first minutes of incubation. The possible occurrence of other reaction(s), competing with the demethylation process, the impossibility of correctly expressing kinetic parameters for demethylation, and the possible relevance of these observations for the activity of triazenes in vivo , are pointed out.

Antitumor and antimetastatic effects of benzenoid triazenes in mice bearing lewis lung carcinoma

Summary Three parasubstiouted dimethyl-phenyltriazenes have been examined for their differential effects on primary tumor growth and lung metastasis formation in mice bearing Lewis lung carcinoma. At equitoxic dosages, the effects on the primary tumor ranged from marked depression (DM-NO 2 ) to none (DM-CH 3 ); those of DM-CONH 2 falling inbetween. All these compounds sharply reduced the number of lung colonies, the effects of DM-CH 3 being slightly more pronounced in terms of number of animals free of lung secondaries at sacrifice. In the case of DM-NO 2 , the depression of metastasis formation is attributed to cytotoxic effects, evident on the subcutaneous tumor. DM-CH 3 , on the contrary, possesses selective antimetastatic properties, since it is devoid of any effect on the growth of the primary subcutaneous implant. The activity of these compounds correlates with their half-life time of hydrolysis to aryl-diazonium cations. The three corresponding monomethyltriazenes were less active than the parent dimethyl derivatives. This suggests that microsomal oxidative N-demethylation of dimethyltriazenes to monomethyltriazenes plays a marginal role for the generation of the in vivo active species. This is further indicated by the high activity possessed by DM-COO-, another possible metabolite of DM-CONH 2 .

Selective antimetastatic effects of n-diazoacetylglycine derivatives in mice.

Abstract Three N -diazoacetylglycine derivatives and two N -diazoacetylglycylglycine derivatives were examined for their differential effects on subcutaneous tumor growth and lung metastasis formation in mice bearing Lewis lung carcinoma. None of the tested compounds caused marked inhibition of primary tumor growth, even at maximum tolerated dosages. However, the two N -diazoacetylglycine and N -diazoacetylglycylglycine amide derivatives respectively caused a dramatic and a moderate inhibition of lung metastasis formation. The lack of correlation between inhibition of subcutaneous tumor growth and formation of lung secondaries indicates that the two amide derivatives possess selective antimetastatic properties.

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.

Mechanism of the antimetastatic action of N-diazoacetylglycinamide in mice bearing lewis lung carcinoma

Abstract N -diazoacetylglycinamide (DGA) sharply reduced the formation of spontaneous pulmonary metastases in mice bearing subcutaneous Lewis lung carcinoma. At the same time, it caused no significant and parallel inhibition of primary tumor growth. The inhibition of metastasis formation, thus, appears to be due to selective antimetastatic effects, different from cytotoxicity for tumor cells. This is supported by the low effectiveness of the late treatment of mice having spontaneous metastases, and also by the absence of significant effects on the formation of artificial metastases obtained by i.v. injection of tumor cells. The lack of any cytotoxic effect for tumor cells localized in the lungs and subcutaneously was also shown by examining the effects of the treatment with DGA on the fractional incorporation of 3 H -TdR in tumor cells. A proposed mechanism for the antimetastatic effects of DGA is the inhibition of tumor cell detachment from the primary implant and their access to the blood stream. This mechanism is also consistent with the fact that DGA caused the greatest reduction of spontaneous metastasis formation when administered at the same time as the peak of dissemination of tumor cells in the blood stream.

Observations on the “in vitro” oxidative N-demethylation of 4-(3,3-dimetryl-1-triazeno)-imidazole-5-carboxamide (DIC, NSC 45388)

Abstract The time course of the oxidative N-demethylation of DIC shows that this process levels off within the first five minutes of incubation. Similar results had been previously obtained with p-substituted aryl dimethyl triazenes, and were attributed to the existence of side metabolic reactions, competing with and subtracting the substrate for demethylation. These data indicate the need of a more detailed examination of the “in vitro” microsomal metabolism of DIC and of the role of possible metabolites other than its monomethyl derivative for the “in vivo” activity of this drug.

High-performance liquid chromatographic assay for the determination of p-(3,3-dimethyl-1-triazeno)benzoic acid in mouse plasma

A reproducible and sensitive method is described for assaying p-(3,3-dimethyl-1-triazeno)-benzoic acid (pCOOH-DMT) and for identifying the N-desmethyl metabolite, p-(3-methyl-1-triazeno)benzoic acid (pCOOH-MMT) using high-performance liquid chromatography. The method measures concentrations as low as 1.25 nmol/ml of plasma. Extraction efficiency of internal standard or of added triazenes averages 88% and the coefficient of variation of the method is less than 10%. pCOOH-DMT is stable at room temperature at pH 7.4, whereas pCOOH-MMT undergoes rapid decomposition (half-life 6 min). pCOOH-MMT is more stable in an albumin-containing solution or in plasma, but not in boiled 9000 g mouse liver. After 80 min incubation with a 9000 g mouse liver fraction and reduced nicotinamideadenine dinucleotide phosphate, only 24% pCOOH-DMT was metabolized. Plasma pharmacokinetic studies in mice treated with 200 mg/kg intraperitoneally showed that the potassium salt of pCOOH-DMT has a half-life of 67 min.

Antineoplastic action ofp-(3-methyl-1-triazeno)benzoic acid potassium salt, a monomethyl derivative of the antimetastatic compound DM-COOK

SummaryThe antitumor and antimetastatic effects ofp-(3-methyl-1-triazeno)benzoic acid potassium salt (MM-COOK) as compared with those of the parent 3,3-dimethyl derivative (DM-COOK) were examined using Lewis lung carcinoma, MCa mammary carcinoma of the CBA mouse and TLX5 lymphoma. Similarly to DM-COOK, MM-COOK reduces metastasis formation and significantly prolongs the survival of mice bearing the Lewis lung carcinoma when given at a daily dose corresponding to one-half that of DM-COOK. Unlike DM-COOK, MM-COOK exhibits significant cytotoxicity to metastatic foci and pronounced inhibition of primary tumor development. MM-COOK also causes cytotoxic effects on TLX5 lymphoma cell growing in the peritoneal cavity, even when used at low doses. The antimetastatic effects observed in mice bearing MCa mammary carcinoma are unrelated to the inhibition of primary tumor growth and are more likely due to the selection of clones endowed with lower metastatic ability. It appears that MM-COOK exhibits the same antineoplastic activity as DM-COOK, but the former does so at a lower daily dose and produces interesting cytotoxic effects other than those reflecting its antimetastatic properties. It thus seems to be a valid alternative to DM-COOK, in view of the possible introduction of newer aryltriazenes into clinical use.