Pentamethylmelamine Research Articles

Absorption and metabolism of hexamethylmelamine and pentamethylmelamine in rat everted perfused gut segments: correlation with in-vivo data.

The intestinal oxidative metabolism of hexamethylmelamine (HMM) and pentamethylmelamine (PMM) has been studied in microsomes, isolated mucosal cells and intestinal perfused segments. (sub) Cellular systems revealed an almost equal Km (53-65 microM) and Vmax (5.6-7.0 nmol min-1 g-1 intestine) for both compounds. Detailed studies in everted intestinal perfused segments, showed that HMM is metabolized to a far greater extent than PMM (e.g. 11-times, at 80 microM substrate concentration) while PMM transport was 3 times greater than the transport of unchanged HMM. Only when perfused segments were used as an in-vitro tool was a good correlation observed between the in-vivo and in-vitro rate of intestinal metabolism of HMM and PMM. It is concluded that this is due to preservation of structural integrity of the mucosa for both absorptive and metabolic processes.

Labelled compounds of interest as antitumour agents – VIII. Synthesis of 2H‐isotopomers of pentamethylmelamine and of a potential prodrug thereof

AbstractTreatment of 2‐chloro‐4,6‐(dimethylamino)‐1,3,5‐triazine with trideuteromethylamine gave 4,6‐(dimethylamino)‐2‐trideuteromethyl‐1,3,5‐triazine, an isotopomer of the experimental anticancer agent pentamethylmelamine (PMM). Mitsunobu coupling with 1,2‐dimethyl‐3‐hydroxymethyl‐5‐methoxy‐indole‐4,7‐dione gave 1,2‐dimethyl‐3‐(N‐(4,6‐bis(dimethylamino)‐1,3,5‐triazin‐2‐yl)‐N‐trideuteromethylaminomethyl)‐5‐methoxyindole‐4,7‐dione. This conjugate is a potential reductively triggered prodrug of PMM. Copyright © 2002 John Wiley & Sons, Ltd.

Pharmacokinetics and metabolism of hexamethylmelamine in mice bearing renal cell tumors.

The pharmacokinetics of hexamethylmelamine (HMM) and its main metabolites hydroxymethylpentamethylmelamine (HMPMM), pentamethylmelamine (PMM), and 2,2,4,6, tetramethylmelamine (2,2,4,6 TetrMM) were studied in renal cell (RC) tumor tissues and plasma of CDF1 mice that had received IP bolus injections of the maximally tolerated dose (200 mg/kg) of HMM. HMM, PMM, and 2,2,4,6 TetrMM concentrations determined in RC tissues were much higher than the plasma values, as indicated by the pharmacokinetic parameters (Cmax and AUC). On the other hand, very low levels of HMPMM, generally considered to be a potentially active antitumor compound, were detected in the target tissues, whereas this hydroxylated metabolite was stable and easily determined in plasma. High HMM concentrations in RC tissues could correlate with the high sensitivity of the tumor to this drug. However, the behavior of HMPMM remains unclear; related hypotheses are presented in this paper.

Preclinical toxicology, pharmacokinetics and formulation of N2,N4,N6-trihydroxymethyl-N2,N4,N6-trimethylmelamine (trimelamol), a water-soluble cytotoxic s-triazine which does not require metabolic activation.

N2,N4,N6-Trihydroxymethyl-N2,N4,N6-trimethylmelamine (Trimelamol) is a water-soluble synthetic s-triazine which, unlike hexamethylmelamine (HMM) and pentamethylmelamine (PMM), does not require metabolic activation. The physico-chemical characteristics of Trimelamol were studied with the aim of overcoming the problems of chemical instability, low solubility and polymerisation which had hindered the development of the drug for clinical use. Trimelamol had similar activity to PMM against the murine PC6 plasmacytoma, but enhanced activity with respect to PMM against the Walker 256 carcinosarcoma in the rat, a species which metabolizes PMM less efficiently. Pharmacokinetic studies in mouse, rat and man did not show the major species differences characteristic of PMM. The drug exhibited similar toxicity to PMM against rodents, but had virtually no neurotoxicity. The potential advantages of Trimelamol over previously tested melamines are discussed.

Low central nervous system penetration of N2,N4,N6,-trihydroxymethyl-N2,N4,N6,-trimethylmelamine (Trimelamol): A cytotoxic s-triazine with reduced neurotoxicity

Trimelamol (N2,N4,N6-trihydroxymethyl-N2,N4,N6-trimethylmelamine) is an analogue of pentamethylmelamine (PMM). In early clinical trials PMM failed to show significant anti-tumour activity in man which was attributed to poor metabolic activation. Trimelamol does not require activation and is therefore expected to be more active in man. PMM caused dose-limiting emesis and sedation whereas Trimelamol is much less neurotoxic in rodents. The relative penetration of PMM and Trimelamol into mouse brain has therefore been examined. Mice receiving PMM at 90 mg kg-1 i.p. were found to have high concentrations of the drug in the CNS compared to plasma (mean brain/plasma ratio 1.04) whereas animals receiving Trimelamol had consistently low CNS concentrations (mean brain/plasma ratio 0.08). This difference did not correlate with plasma protein binding which is greater for PMM (68.2%) than for Trimelamol (17.5%). However, it does appear to be related to lipophilicity. In Phase I clinical trial Trimelamol has proved much less emetic than PMM and causes no acute sedation. It is likely that this reduction in toxicity may be explained by the relatively poor ability of Trimelamol to enter the CNS.

Central side effects of pentamethylmelamine: Biochemical and behavioural studies

The central side effects of pentamethylmelamine (PMM), an antitumoral agent, were studied on brain neurotransmitters from the biochemical and behavioural points of view. PMM causes a dose-related reduction in the body temperature and motility of mice. 100 mg/kg of PMM lowers the levels of noradrenaline (NA) and raises 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in the telencephalon. A similar dose increased striatal levels of dopamine (DA) metabolites, homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), at earlier times (30 min), reducing their levels at 2 hr. These effects disappear at longer times (4 hr). No changes were observed in the levels of 3-methoxytyramine (3-MT), the extraneuronal metabolite of DA. The serotonin metabolite 5-hydroxyindolacetic acid (5HIAA) was almost not affected. PMM and its metabolites do not displace [ 3H]-spiroperidol from mouse striatal binding sites. These data show that some of the neurological effects induced by PMM are associated with changes in the metabolism and/or release of brain catecholamines but are not mediated by direct action on DA receptors.

Studies of the mode of action of antitumour triazenes and triazines—V. The correlation of the in vitro cytotoxicity and in vivo antitumour activity of hexamethylmelamine analogues with their metabolism

Experiments were conducted to ascertain whether the antitumour activity of hexamethylmelamine analogues correlated with their in vitro cytotoxicity and metabolism. Two analogues, namely pentamethylmelamine (PMM) and 2,2,4,4-tetramethylmelamine (TMM), and hexamethylmelamine (HMM) itself were shown to be active towards the murine ADJ/PC6A (PC6) plasmacytoma; another three, 2-chloro-4,6-bis(dimethylamino)-1,3,5-triazine (CBDT), 2,4-bis-(dimethylamino)-6-hydrazino-1, 3,5-triazine (HBDT) and 2,4,6-trimethylmelamine (TriMM) were inactive against the same tumour. The cytotoxicity of these compounds was examined against a PC6 tumour cell line in vitro. In the absence of liver microsomal activation only CBDT proved to be significantly cytotoxic at a concentration of 5 mM. In the presence of murine liver microsomes the three active antitumour agents were all cytotoxic at this concentration whereas HBDT and TriMM remained non-toxic. The degree of cytotoxicity correlated with the extent of metabolism for these analogues. The products of biotransformation of these compounds were stable precursors of formaldehyde (presumably N-hydroxymethyl intermediates) (FP) rather than formaldehyde itself. After injection of these 6 compounds to Balb/c mice the levels of FP generated in the plasma were markedly greater for the three active antitumour agents than for the inactive analogs. No free formaldehyde was detected in the plasma after administration of any of the compounds. These results suggest that for these compounds in vitro cytotoxicity correlates with in vitro biotransformation and their antitumour activity correlates with plasma levels of FP generated by metabolism in vivo.

Triethylenemelamine: An initiator of two-stage carcinogenesis in mouse skin which lacks the potential of a complete carcinogen

The dorsal skin of female CD-1 mice was treated with triethylenemelamine (TEM) to determine whether this agent acted either as a complete carcinogen or as an initiator of carcinogenesis. A dose of 0.01–1.0 μmol of TEM applied once a week for 32 weeks to the skin of the backs of mice did not produce any detectable tumors. A dose of 2.5 μmol applied once a week over the same period produced only a single papilloma in a group of 20 mice. However, when mice were treated with a single dose of 1 μmol of TEM followed by promotion with 12- O-tetradecanoylphorbol-13-acetate (TPA) twice a week, 88% of the mice produced papillomas after 28 weeks. Using the same protocol, a single application of hexamethylmelamine (HMM), pentamethyl-melamine (PMM), or melamine followed by promotion with TPA had no significant tumor initiating activity. These data suggest that TEM acts primarily as an initiator of two-stage carcinogenesis.

Distribution, metabolism, and irreversible binding of hexamethylmelamine in mice bearing ovarian carcinoma.

The covalent binding of hexamethylmelamine (HMM) and its metabolites was studied in liver, tumor, blood, kidney, spleen, lung, brain, heart, and small intestine after a single IP injection of 2,4,6-14C-hexamethylmelamine (50 mg/kg) to C57Bl/6J female mice bearing 20-day-old M5076/73A ovarian cancer. Covalent binding to tissue macromolecules was measured 2, 10, and 40 h after injection of the drug. At 2 h liver and small intestine showed the highest levels of irreversibly bound metabolites, the lowest being found in brain and heart. Except in the small intestine, where a decrease was observed between 2 and 10 h, the level of covalent binding was constant up to 40 h. HMM metabolism was also studied. Tissue distribution of pentamethylmelamine (PMM), 2,2,4,6-tetramethylmelamine (TMM), and 2,4,6-trimethylmelamine (TriMM) was determined at the three times considered. At 2 h the drug was already extensively metabolized, TriMM being the major metabolite among those determined.

Hexamethylmelamine and pentamethylmelamine: an update.

An updated review of the anticancer agents hexamethylmelamine (HMM) and its water-soluble analog pentamethylmelamine (PMM) is presented. Severe gastrointestinal and hematologic toxicity have limited the use of HMM drug combinations in ovarian cancer. Combinations involving HMM, cyclophosphamide, cisplatin, and doxorubicin in advanced ovarian cancer have resulted in only moderate response rates, with little to no change in median survival of previously treated patients. HMM now is being studied in previously untreated patients with advanced disease, in combination with these agents. In lung cancer, HMM continues to be a part of intensive and other regimens for the treatment of small-cell and non-small-cell carcinoma, although the value of the HMM is yet to be determined. Future trials have been recommended to determine whether HMM has a role in the treatment of endometrial and prostatic carcinomas. Five phase I studies of PMM have demonstrated severe, dose-limiting gastrointestinal and central nervous system toxicities. Thus, this agent may offer little advantage over HMM. Further phase I studies, with different PMM dosage schedules, are necessary before phase II studies can be recommended.

Specific determination of pentamethylmelamine and its demethylated metabolites in human plasma by high pressure liquid chromatography.

Pentamethylmelamine (PMM) and its demethylated metabolites tetra-N2246, tri-N246 and tri-N224 have comparable relative antitumor potencies in animal systems. We have developed a sensitive and specific high pressure liquid chromatography (HPLC) assay for these compounds in the plasma of patients receiving pentamethylmelamine, a cytotoxic S-triazine derivative now undergoing clinical trials. The method involves analysis of an ethyl acetate extract of plasma by a high pressure liquid chromatograph containing a reverse-phase column and ultraviolet detection at 240 nm. The mobile phase is acetonitrile and KH2PO4 buffer (pH 5.5); gradient elution over 25 min yields relative retention times to hexamethylmelamine as internal standard of 0.36, 0.40, 0.54 and 0.70 for tri-N246, tri-N224, tetra-N2246 and PMM. The lower limit of sensitivity for each is 300 ng/ml. Pharmacokinetic studies on four patients receiving a 24-h infusion of PMM (doses 900-3000 mg/m2) showed a mean terminal half-life of PMM of 101 +/- 28 (SD) min, a mean volume of distribution of 2036 +/- 75 ml/kg, and a metabolic clearance rate of 14.2 +/- 3.7 (ml/min)/kg. Mean areas under the plasma-vs-time curves of tri-N246, tri-N224 and tetra-N2246 were 126%, 41% and 56% of PPM, demonstrating extensive formation of these metabolites in man.

Heritable translocation test in mice with triethylenemelamine (TEM) and ergotamine

The dorsal skin of female CD-1 mice was treated with triethylenemelamine (TEM) to determine whether this agent acted either as a complete carcinogen or as an initiator of carcinogenesis. A dose of 0.01–1.0 μmol of TEM applied once a week for 32 weeks to the skin of the backs of mice did not produce any detectable tumors. A dose of 2.5 μmol applied once a week over the same period produced only a single papilloma in a group of 20 mice. However, when mice were treated with a single dose of 1 μmol of TEM followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) twice a week, 88% of the mice produced papillomas after 28 weeks. Using the same protocol, a single application of hexamethylmelamine (HMM), pentamethyl-melamine (PMM), or melamine followed by promotion with TPA had no significant tumor initiating activity. These data suggest that TEM acts primarily as an initiator of two-stage carcinogenesis.

Influence of ascites on the pharmacokinetics of hexamethylmelamine and N-demethylated metabolites in ovarian cancer patients

Levels of hexamethylmelamine (HMM) and its metabolites pentamethylmelamine (PMM), N 2 N 2 N 4 N 6 tetramethylmelamine (TMM) and N 2 N 4 N 6 trimethylmelamine (TriMM) were determined in plasma of 31 patients with advanced ovarian cancer receiving HMM. In 14 of them with ascites the drug metabolites PMM, TMM and TriMM were assayed in this fluid. Both in plasma and in ascites a GLC method with nitrogen detection was applied after extraction with n-hexane and ethyl acetate at pH 11.5. Plasmatic HMM T/2α and T/2β were respectively 47 ± 6 and 403 ± 37 min. Mean AUC o→ 12 hr values ±S.E.M., expressed in μ g/ml × min, were 312 ± 95 for HMM, 117 ± 32 for PMM, 260 ± 50 for TMM and 311 ± 82 for TriMM. AUCs of HMM, PMM, TMM and TriMM in ascites calculated up to 6 hr were 78 ± 13, 31 ± 7, 60 ± 12 and 129 ± 38 μg/ml × min respectively. The presence of ascites, the patient's age, the concomitant administration of adriamycin and cyclophosphamide and fatness did not appear to influence the kinetic behaviour of HMM and metabolites. HMM was not detectable in urine and PMM, TMM and TriMM accounted for less than 1% of the HMM dose.

Comparison of DNA Damage by Methylmelamines and Formaldehyde<xref ref-type="fn" rid="FN2">2</xref>

The cytoxicity and DNA damaging activity of S9-activated hexamethylmelamine (HMM) and pentamethylmelamine (PMM) were compared with suspected active metabolites in mouse leukemia L1210 cells. The presence of semicarbazide hydrochloride did not alter the cytotoxicity of S9-activated HMM and PMM or that of their hydroxylated analogs monomethylolpentamethylmelamine (MPM) and trimethyloltrimethylmelamine (TTM), which have been suggested as active metabolites. Following treatment of L1210 cells with high concentrations of activated HMM and PMM, there were no DNA single-strand breaks or interstrand cross-links observed by DNA alkaline elution and only a low frequency of DNA-protein cross-links. Formaldehyde (FA) at nonlethal concentrations caused far greater DNA-protein cross-linking. In contrast, the polyfunctional TTM produced both DNA-protein cross-linking and DNA interstrand cross-linking. The cytotoxicities of HMM and PMM were found unlikely to be related to extracellular or intracellular release of FA.

Studies of the metabolism of N-methyl containing anti-tumour agents : 14CO 2 breath analysis after administration of 4C-Labelled N-methyl drugs, formaldehyde and formate in mice

The 14CO 2; content of the breath was analysed after administration of the following N- 14 CH 3 labelled drugs to mice: aminopyrine, hexamethylmelamine (HMM), pentamethylmelamine (PMM), procarbazine and caffeine. Except for aminopyrine, the 14CO 2; exhalation rate plots declined monophasically with half lives of 91 min ([ 14C]-HMM), 97 min ([ 14C]-PMM), 68 min ([ 14C]procarbazine) and 92 min ([ 14C]caffeine). The 14CO 2 exhalation rate peaked rapidly after aminopyrine administration and declined bi-phasically with an initial t 1 2 of 15 min and a terminal t 1 2 of 126 min. The 14CO 2; plots after both [ 14C]-HMM and [ 14C]aminopyrine were influenced by pre-treatment of mice with proadifen. Pretreatment with phenobarbitone shortened the t 1 2 of the 14CO 2 appearance rate after [ 14C]HMM by 24% but did not change the 14CO 2 curve after administration of [ 14C] aminopyrine. The 14CO 2 exhalation rate plots after administration of H 14CHO and H 14COOH were virtually identical with that obtained after [ 14C]aminopyrine and not influenced by either proadifen or phenobarbitone pretreatment. The 14CO 2 exhalation rate profile obtained on metabolism of [ 14C]aminopyrine in mice thus appears to be determined by the rate of the oxidation of formaldehyde or formate to CO 2. Only 24% of the label injected with the N-methyl moieties of [ 14C]HMM and 21% of the label in [ 14C]procarbazine were exhaled as 14CO 2, whereas 49% of the N- 14CH 3 in [ 14C]aminopyrine were metabolized to 14CO 2. It remains to be determined whether this difference and the difference in the shapes of the 14CO 2 exhalation profiles obtained with the cytotoxic N- 14CH 3 drugs as compared to [ 14C]aminopyrine, are related to the biochemical processes mediating their antineoplastic activity.

Dose-dependent pharmacokinetics of PMM in the rat.

Concentrations of pentamethylmelamine (PMM) and some metabolites were determined in plasma of rats treated with 10 and 50 mg PMM/kg IV. The areas under the plasma levels curve after these doses were 241 and 1,827 mu g/ml X min; plasma clearances were 0.042 and 0.027 l . kg(-1) . min(-1), respectively. These data suggest that PMM pharmacokinetics is dose-dependent in the rat. The N-demethylated metabolite levels were not proportional to the administered dose.

Time dependence of the in vitro cytotoxicity of hexamethylmelamine and its metabolites.

The cytotoxicity of hexamethylmelamine (HMM) and its metabolites pentamethylmelamine (PMM), N,2,2,4,6-tetramethylmelamine (TMM) and hydroxymethylpentamethylmelamine (HMPMM) and of the alkylating agent triethylenemelamine (TEM) were studied on a cell line derived from a human ovarian cancer, by measuring [3H]TdR uptake. After 24 h of incubation all the tested compounds inhibited [3H]TdR uptake, but only at a concentration of 100 micrograms/ml. However, after 120 h incubation, concentrations of 0.1--10 micrograms/ml resulted in highly significant cytotoxicity. HMPMM and TEM were the most active and their effect was not reversed 72 h after their removal. In our in vitro system no metabolism of HMM was observed.

In vitro cytotoxicity of the methylmelamines

Hexamethylmelamine (HMM) and a number of its derivatives are toxic to PC6 plasmacytoma cells in vitro. However, there is no correlation between in vitro cytotoxicity and activity against this tumour in vivo. N-Methylolmelamines are significantly more toxic than HMM itself. These compounds break down to release formaldehyde which is itself a highly cytotoxic agent. Pentamethylmonomethylolmelamine rapidly inhibits the growth of PC6 cells in culture, whereas HMM and pentamethylmelamine (PMM) require prolonged contact with the cells in order to exert a cytotoxic effect. HMM and its metabolites are also toxic to a number of other cell lines. The toxicity of the N-methylols to cultured L1210 leukaemia and Walker 256 ascites cells appears to be due entirely to formaldehyde release, whereas in PC6 cells the methylols appear to be acting more selectively.