[CANCER RESEARCH 33, 2810 2815, November 1973]

/V-Demethylation of the Antineoplastic AgentHexamethylmelamine by Rats and Man1

John F. Worzalla, Bruce M. Johnson, Guillermo Ramirez, and George T. Bryan2

Division of Clinical Oncology, University of Wisconsin Medical School, Madison, Wisconsin 53706

SUMMARY

The metabolism and physiological distribution of hexa-methylmelamine (HMM), an effective human anticancerchemotherapeutic agent, was studied in cancer patientsand in rats. Administration of HMM-methyl-14C to twopatients p.o. was followed by the prompt appearance ofrespiratory MCO2 within 1 hr, accumulating to 9% of ad

ministered dose in 6 hr. After 72 hr, 29% of the radioactivity was recovered in the urine, and 0.5% was recoveredin the feces. In rats, p.o. and i.p. administration of HMM-14C led to the recovery of 13 and 30%, respectively, ofthe dose as 14CO2 in 24 hr. After 72 hr, 58% of the p.o.dose of radioactivity was recovered, 16% as 14CO2,38% in the urine, and 4% in the feces; while in the sameinterval, 80% of the i.p. dose was recovered, 33% as14CO2, 42% in the urine, and 5% in the feces. No un-metabolized HMM was detected in urine.; instead fourdistinct compounds could be chromatographically separated. The major urinary metabolites in patients and ratsexhibited identical Chromatographie and spectroscopicproperties. Use of gas chromatography and mass spectro-metry led to the identification of the major urinary metabolites as /V2,Ar4,yV6-trimethylmelamine, Af2,Ar4-dimethyl-melamine, monomethylmeiamine, and melamine. Thus, N-demethylation appears to be of major significance in themetabolism of HMM in man and in rats. HMM and itsmetabolites did not demonstrate significant alkylating activity, as evidenced by their failure to react with 4-(p-nitrobenzyl)pyridine.

INTRODUCTION

HMM3 has exhibited slight but definite tumor-inhibit

ing properties against Crocker mouse Sarcoma 180 (12)and Walker rat Carcinosarcoma 256 (20). However, theseanimal screening tests showed that the tumor-inhibitingproperties of HMM were inferior to those of 2 structurally

'Supported in part by Grants CA-13290 and CA-06749-ll from theNational Cancer Institute, USPHS. Presented in part at the 62nd AnnualMeeting of the American Association for Cancer Research, Inc., Chicago.111..April 1971 (37).

2Career Development Awardee of the National Cancer Institute (I-K4-CA-8245-05).

3The abbreviations used are: HMM, hexamethylmelamine [2,4,6-tris(dimethylamino)-i-lriazine] (NSC-13875); TEM, triethylenemelamine[2,4,6-tris(l-aziridinyl)-i-triazine]; DEGS, diethylene glycol succinate;N BP, 4-(p-nitrobenzyl)pyridine.

Received April 27, 1973; accepted August 6, 1973.

similar s-triazine compounds, TEM and trimethylolmela-mine [2,4,6-tris(methylolamino)-i-triazine] (12, 20, 21).

Clinical trials with HMM were initiated by Wilson andde la Garza (36) and by Louis et al. (24). Phase II studieswith HMM were conducted, and the response rate of patients demonstrated HMM to be worthy of further clinical study especially for lung carcinoma. The clinicalliterature for HMM has been recently reviewed by Blumet al. (8).

Although HMM has been used in several clinical trialswith cancer patients, information concerning the mechanism of action of this drug and its metabolism is sparse.Studies in our laboratory with HMM have included thedevelopment of an ion-exchange method for the isolationand quantitation of HMM (10) and an investigation ofthe plasma and urinary levels of HMM and its metabolitesin treated cancer patients (11). This paper describes themetabolism of HMM-methyl-14C and reports the identification of the human and rat urinary metabolites ofHMM.

MATERIALS AND METHODS

Chemicals. Unlabeled HMM was provided by the Clinical Branch, Collaborative Research, National Cancer Institute, USPHS. Samples of the other methylmelaminecompounds were generously provided by Dr. A. B.Borkovec of the Entomology Research Division of theUnited States Department of Agriculture, Beltsville, Md.Melamine (Aldrich Chemicals, Milwaukee, Wis.) wasrecrystallized from water. TEM was synthesized (38) fromcyanuric chloride (2,4,6-trichloro-i-triazine, EastmanOrganics, Rochester, N. Y.) and ethylenimine (K and KLaboratories, Plainview, N. Y.).

The synthesis of HMM-methyl-14C was based on amethod used by Kaiser et al. (22). To a flask containing 3mmoles (554 mg) of cyanuric chloride and 75 ml of acetone were added 8.7 mmoles (710 mg) of dimethylaminehydrochloride (Aldrich Chemicals) and 0.3 mmole (24 mg,0.5 mCi) of dimethylamine-14C hydrochloride (New Eng

land Nuclear, Boston, Mass.). After the addition of 8 mlof 2 N NaOH solution, the reaction mixture was refluxedfor 3 hr, and the acetone was then removed by distillation.The remaining aqueous solution was allowed to cool, andthe HMM was extracted with chloroform. After washingwith water, the chloroform was evaporated, and the HMMwas recrystallized twice from methanol, yielding 260 mg(41%) of crystalline HMM-methyl-14C, m.p. 170-172°;

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—¿�p";

literature (22), 172 174°.The identity of the crystals was

confirmed by comparison of the infrared and UV spectrawith those of authentic HMM.

Radioactivity measurements were performed on a Nuclear-Chicago liquid scintillation counter as previouslydescribed (25). The specific activity of the HMM-14C wascalculated to be 0.92 ¿iCi/mg(2.04 x IO6dpm/mg). The

radiopurity of HMM was determined to be greater than98.5% by thin-layer and ion-exchange chromatography.

Administration and Collection Procedures. Patientsselected for study were those accepted for Phase II orPhase III clinical cancer chemotherapy trials with HMM.Two patients were given HMM-methyl-MC, dissolved in

a small portion of 6 N HC1 and added to orange juice (pH2.3), p.o. in the morning. Patient 1 received 110 mg (98.6MCi) of HMM-14C. Patient 2 received 93 mg (81.9 MCi)of HMM-MC together with 112 mg nonradioactive HMM.Patients were fitted with face masks (30), and their expired breath was monitored for I4CO2 by bubbling itthrough a solution consisting of 1 part redistilled etha-nolamine and 4 parts methanol (2). Aliquots of theseethanolamine solutions were taken for counting. TheseCO2 collections were made during the 1st 54 min of eachhr for the 1st 6 hr after the administration of the HMM-methyl-MC. Twenty-four-hr urine and fecal sampleswere collected from patients for 3 days. Aliquots of theurine samples were taken for counting, and the samplesthen treated as described (11). Fecal samples were frozenuntil analysis; after thawing, they were homogenized inwater and digested with nitric acid (27, 28). After digestion, an aliquot was counted by liquid scintillation. Plasmasamples were centrifuged, protein was precipitated withsulfosalicylic acid (31), and an aliquot of the plasma wascounted.

Sprague-Dawley male rats (Sprague-Dawley, Inc.,Madison, Wis.) weighing 180 to 250 g were used throughout the experiments. Four rats were each given 1 ml ofHMM-'4C solution containing 24 mg of unlabeled HMMand 1 mg of HMM-I4C (2.04 x IO6 dpm) by stomachtube. They were immediately placed in glass metabolismcages, where their CO2, urine, and feces were collected for72 hr. Three rats were also given 1 ml of the HMM-'"Csolution, except that they received it i.p. They were alsoplaced in metabolism cages for 72 hr. The 14CO2was collected by bubbling through ethanolamine:methanol solutions, and these CO2 trapping solutions were changed andcounted at 1,3, 6, 12, and every 12 hr thereafter. For urine,the 24-hr samples were acidified to 0.1 N with HC1 andtaken to a volume of 50 ml. An aliquot was counted, and theurine samples were stored under refrigeration. Fecal samples were collected as 24-hr samples. They were homogenized in water, and an aliquot was counted.

The distribution of HMM-14C in the tissues of ratswas studied to determine whether there were any areasof localization of radioactivity. Four rats were given I ml ofthe HMM-methyl-14C solution by stomach tube, and theywere placed in the metabolism cages for 24 hr, where theCO2, urine, and feces were collected. At the end of 24 hr,the rats were sacrificed with anesthesia. The various tis-

N-Demethylation of Hexamethylmelamine

sues were removed and placed in flasks containing ali-quots of a solution consisting of 1000 ml of 2 N KOH, 500ml of 95% ethanol, and 200 ml of toluene (6). The flaskswere heated at 80°for 1 hr, diluted with 95% ethanol to

known volume, and aliquots were counted.Ion-Exchange Chromatography of Urine Samples. The

ion-exchange chromatography procedures used in theanalysis of urine samples for the recovery of HMM metabolites have been described previously (10, 11). Continuous gradient elution chromatography was carried outover a period of 18 hr utilizing a gradient of increasingHC1 concentration. The effluent was routed through a flowcell in a Beckman DB-G spectrophotometer and was monitored at 242 and 260 nm with the aid of a Beckman DBprogrammer and recorder. The effluent was collectedin 5-ml portions using an automatic fraction collector.

Identification of Urinary Metabolites by Gas Chroma-tography-Mass Spectrometry. Gas Chromatographiemethods to separate methylmelamines have been reported (13, 16). The column used by Cincotta and Fein-land (16) did not separate all the isomers, so DEGS columns similar to those used by Chang et al. ( 13) were utilizedin these experiments. The samples were prepared for gaschromatography by the following method. Rats weregiven 10 mg of unlabeled HMM i.p., and 24-hr urines werecollected. The urines were acidified (0.1 N) with HC1 andapplied to 7-cm resin beds of Dowex 1 (Cl~) and rinsedwith 100 ml 0.1 N HC1. This eluate was applied to 7-cmresin beds of Dowex SOW (H+), and the columns wererinsed with 200 ml of 2.4 N HC1. The 2.4 N effluents werediscarded, and then the columns were treated with 200 mlof 6 N HC1. These 6 N fractions were evaporated todryness in vacuum, and the residues were treated with two2-ml portions of concentrated NH4OH and freeze driedafter each treatment. The freeze-dried material was dissolved in 0.5 ml of 2-methoxyethanol, and the samples werethen ready for gas chromatography.

A Tracor MT-220 gas Chromatograph equipped withan alkali flame ionization detector was used. The detectorwas modified through the use of an RbSCvCsCl (3:1)pellet placed over the exit tip of the detector (1). A 60-cmglass column (4-mm inside diameter) containing 15%DEGS on Chromosorb W (60 to 80 mesh) was used. Thecolumn was well conditioned at 230°.The carrier gas was

nitrogen (120 ml/min); hydrogen (40 ml/min) and air(275 ml/min) were used for the detector. Column temperature was programmed at 9°/min from 150 to 245°and

held for 4 min.For the gas chromatography:mass spectrometry experi

ments, a Hewlett-Packard 5700A gas Chromatographcoupled to a Hewlett-Packard 5930 mass spectrometersystem was used with a 70-cm aluminum column (3-mminside diameter) packed with 15% DEGS on ChromosorbW. Helium was used as a carrier gas (50 ml/min), and thecolumn temperature was 240°.Mass spectra were recorded

at 70 eV and 30 ma trap current with the transfer linesat 240°and source at 200°.

Test for Alkylating Activity. Solutions of HMM, theother methylmelamines, TEM, and appropriate blanks

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Worzalla, Johnson, Ramirez, and Bryan

were assayed for alkylating activity using the NBP test(18). To a tube were added 2 ml of 0.25 mM solutions ofthe test compounds in methanol, 1 ml of 0.05 Mpotassiumhydrogen phthalate buffer (pH 4.2), and 1 ml of 5% NBPsolution (7). The tubes were placed in a 70°water bath for2 hr and then transferred to an ice bath. After addition ofmethanol to a total volume of 5 ml, 3 ml of 0.01 N KOH in80% methanol were added. The tubes were mixed and readimmediately in a Bausch and Lomb Spectronic 20 spec-trophotometer at 600 nm. Solutions of the methylmela-mines equimolar in methyl groups to the 0.25 mM HMMsolution were also assayed in the same manner.

RESULTS

Distribution of ll\1\1-\U(h>l- ( in Humans andRats. Recovery of radioactivity in CO2, urine, and fecesafter administration of HMM-'4C to 2 cancer patients isshown in Table 1. Analysis of plasma levels of radioactivity 1 hr after administration revealed plasma levels of3480 and 2680 dpm/ml for Patients 1 and 2. Radioactivitylevels in plasma dropped steadily, but at 24 hr the plasmastill contained 440 and 320 dpm/ml, respectively.

Recovery of excreted radioactivity after administrationof HMM-14C to rats is shown in Table 2. The results of a

study to determine the fate of radioactivity in varioustissues of the rat after p.o. administration of HMM-14Care shown in Chart 1. There were no tissues which showedan especially high localization of radioactivity.

Table IRecovery of radioactivity after administration of HMM-methyl-l4C

to 2 cancer patients

% of total dose ofradioactivityUrine

onDayPatient

1"Patient 2"C02(6

hr) 127.0

22.4 4.810.5 24.5 3.93-day

3total1.7

28.91.0 29.4Feces

(3days)0.4

0.7

' Received 110 mg (219 x 10«dpm) HMM p.o.' Received 205 mg (182 x 10«dpm) HMM p.o.

»2.4

Chart 1. Tissue distribution of radioactivity in rats after HMM-methyl-"C. Data expressed as dpm/mg of tissue 24 hr after the p.o. administration of 2.04 x IO6dpm HMM-methyl-14C. Numbers represent

average ±S.D. for 4 animals.

Urinary Excretion of HMM Metabolites. The urinary excretion patterns of HMM and its metabolites wereexamined to determine whether the metabolism of HMMin rats was similar to that observed in humans. Stepwiseelution on ion-exchange columns (10) was performed onurines of rats that had received HMM-MC, and thefractions were assayed for radioactivity. When the acidified urines (0.1 N) were added to Dowex 1 (Cl ) columnsand eluted with 0.1 N HC1, 98% of the radioactivity wasrecovered. This 0.1 N eluate was applied to Dowex 50W(H+) columns and eluted with stepwise gradients of HC1.The normality of each step of HC1 and the recovery ofradioactivity in that fraction are as follows: 0.1 N, 29%;0.5 N, 3%; 1.0 N, 1%; 2.4 N, 2%, and 6 N, 55%. The 6 Nfraction with 55% of the activity also contained all theUV-absorbing metabolites.

Gradient elution chromatography (11) was carried outon the 6 N fraction obtained from the stepwise elution. Theeffluent was monitored for UV absorption, and 4-ml fractions were assayed for radioactivity. Radioactive peakscoincided almost exactly with the spectrophotometricpeaks (see Chart 2) except that Peak I contained verylittle radioactivity. A comparison of the UV absorptionof the typical urinary excretion patterns of humans (11)

Table 2Recovery of radioactivity after administration of HMM-melhyl-"C to rats

%" of total dose of radioactivity (2.04 x IO6dpm)

p.o.administration"Day1

233-day totalCO13.2

±2.1 ±0.7 ±

16.1 ±23.7

0.90.13.5Urine23.6

13.10.9

37.6±4.9

±3.7±0.2±2.1Feces2.3

±1.91.8 ±1.30.2 ±0.14.4 ±2.3Total

CO2urineand

feces39.2

±9.717.0 ±4.61.9 ±0.358.1

±4.7CO,30.4

±1.21.6 ±O.I0.9 ±0.4

32.9 ±1.2i.p.

administration'Urine36.6

4.41.0

42.0±

2.8±0.5±0.2±

2.5Feces3.9

±2.90.6 ±0.30.5 ±0.45.0 ±2.2Total

CO 2urine and

feces70.9

±1.96.6 ±0.72.3 ±0.4

79.8 ±1.30

Average ±S.D.' Four animals.' Three animals.

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N-Demethylation of Hexamethylmelamine

and of rats (Chart 2) showed qualitative and quantitativesimilarities. Cochromatography of corresponding metabolite peaks from the urines of humans and rats confirmed that the peaks represented the same compounds inboth species. Compounds II and III were most abundantin both rat and human urines.

Identification of Urinary Metabolites. Gas Chromatographie analysis of rat urines showed 4 major urinary metabolite peaks plus 3 minor peaks (see Chart 3). Injectionof a standard solution containing melamine and the 9methylmelamine homologs gave the chromatogram shownin Chart 4. The retention times of the major urinary metabolites were found to be identical to those of known samples of yV2,N4,Ar6-trimethylmelamine, A^/VMimethyl-

melamine, monomethylmelamine, and melamine. The minor peaks had retention times similar to pentamethylmela-mine, N2,N2,/V4,;V6-tetramethylmelamine, and N2,N\N4-

trimethylmelamine.Mass spectra of the rat urinary metabolites of HMM

were obtained as they eluted from the gas Chromatograph,and these spectra were then compared to mass spectra ofknown melamine and methylmelamine compounds. Thisgave positive identification of the major urinary me-

IOOO

URINARY EXCRETION PATTERNSOOf AFTER ADMINISTRATION

HMM-METHYL-'*C TO RATS

20O 400 600

EFFLUENT VOLUME (ML)

Chart 2. Rat urinary excretion pattern from Dowex SOW (H*) ion-

exchange column utilizing an increasing gradient of HC1. After administration of HMM-methyl-uC, 24-hr urines were prepared and chroma-

tographed as described in text. /, melamine; //, monomethylmelamine;///, yV2,/V4-dimethylmelamine; IV, yVJ,Af4,A"-trimethylmelamine.

CELUOIo

+I IO

RETENTION TIME, MINUTES

Chart 3. Gas chromatogram of rat urine after the administration ofHMM. Twenty-four-hr urine samples were prepared and treated as described in text. PMM. pentamethylmelamine; tetra-MM, tetramethylmelamine; tri-MM, trimethylmelamine; DA/A/, dimethylmelamine;MMM, monomethylmelamine.

IIin

<rluQ<r

I

801 234567

RETENTION TIME, MINUTES

Chart 4. Gas chromatogram of a standard solution containing 500 ngof the methylmelamines plus melamine. PMM, pentamethylmelamine;telra-MM, tetramethylmelamine; tri-MM, trimethylmelamine; DA/A/,

dimethylmelamine; A/A/A/, monomethylmelamine.

tabolites (Charts 2 and 3) as N2,N4,/VMrimethylmela-mine (IV), /V2,yV"-dimethylmelamine (III), monomethylmelamine (II), and melamine (I). The minor urinary metabolites were identified as pentamethylmelamine,W2,W2,W4,Ar6-tetramethylmelamine, and N2,N\N'-

trimethylmelamine.Test for Alkylating Activity. The NBP alkylation test

(7, 18) was performed on equimolar solutions of melamine,all methylmelamines, and TEM. The test was also repeated on samples of all the methylmelamines which wereequimolar in methyl groups to HMM. The absorbancevalues ranged from 0.00 to 0.02 for all the samples exceptTEM, which had an absorbance of 0.68. Thus, TEM wasthe only compound to demonstrate significant alkylatingactivity in this test.

DISCUSSION

Structural similarities between HMM and TEM makeit of interest to compare the metabolism of these 2 chemicals. Smith et al. (33) reported that the metabolism ofTEM containing a 14C-label in the ethylenimino moietyprovided "comparatively little radioactivity" in the ex

haled CO2 of mice. In contrast, 16 to 33% of the radioactivity from administration of HMM-methyl-14C to ratswas found as 14CO2. Nadkarni et al. (26) reported that in

mice TEM was metabolized, and 72 to 88% was excretedin urine as cyanuric acid. The identification of the majorhuman and rat urinary metabolites of HMM as melaminecompounds shows that HMM differs metabolically fromTEM.

Quantitatively, the amounts of HMM metabolites weredetermined (13) from radioactivity measurements ofmetabolite peaks separated by gradient elution ion-exchange chromatography. In rats, at least 64% of the HMMadministered could be accounted for as methylmelaminesin the 1st 24-hr urine; 4% as /V2,yV4,yV6-trimethylmela-mine, 33% as yV2,jV4-dimethylmelamine, and 27% as

monomethylmelamine. This does not include melamine,which could not be determined from radioactivity measurements because of the absence of any methyl-14C

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Worzalla, Johnson, Ramirez, and Bryan

groups. However, monitoring UV absorption during gradient elution chromatography gave an estimate of theamount of melamine (4%), since the UV absorption is verysimilar for these melamine compounds.

HMM is metabolically very active in rats as evidencedby the fact that no unchanged HMM could be detected byion exchange or gas chromatography. The identification ofurinary metabolites led to the formulation of a metabolicpathway for HMM (see Chart 5) which follows the solidarrows. It is interesting to note the presence in the urine ofmajor quantities of only 1 of 2 possible structural isomersof tetramethylmelamine, trimethylmelamine, and di-methylmelamine. From the pattern of metabolites observed, it appears that a dimethylamino group may be N-demethylated preferentially to a monomethylamino group(15).

These experiments demonstrated that in rats greaterthan 64% of HMM was excreted as metabolites containingan intact j-triazine ring, suggesting the metabolic stabilityof j-triazines in mammalian systems (4, 5). In the stepwisegradient elution of urines on ion-exchange columns, 29%of the radioactivity was found in the 0.1 N HC1 fraction,but this fraction did not contain UV-absorbing material.The metabolites in this fraction were not identified.

The metabolism of HMM in humans is very similar tothat in rats. HMM was /V-demethylated, and 9% of theradioactivity from HMM-methyl-14C was excreted asMCO2 within 6 hr. The major urinary metabolites werethe same as those observed in rats. The peak plasma levelof radioactivity in patients occurring 1 hr after administration of HHM-methyl-14C differs from the peak level ofUV-absorbing metabolites (11) which occurs at 2 to 3 hr.This discrepancy may be explained by the metabolism ofHMM which involves /V-demethylation; there could be anincrease in the concentration of UV-absorbing metabolites,together with a decrease in plasma radioactivity, as a result of an increase in the concentration of metabolites containing fewer methyl-14C groups.

HMM is an effective chemosterilant for house flies (14),and the metabolism of HMM in male house flies has beenstudied by Chang et al. (13). They reported that the principal metabolic transformation of HMM in flies involved./V-demethylation and identified pentamethylmelamine,N2,N2,Af4,N6-tetramethylmelamine, /V2,/V4,./V6-trimeth-ylmelamine, and /V2,/V2,jY4-trimethylmelamine as

"»V H'V "»V

Hf CH, Ni,!*,»f y lATETR»-»«

»*^V

KM

Õ**

K. M

Chart 5. Suggested pathway for the metabolism of HMM in humansand rats. PMM, pentamethylmelamine; letra-MM, tetramethylmelamine;tri-MM, trimethylmelamine; DMM, dimethylmelamine; MMM, mono-

methylmelamine.

metabolites. They were also unable to detect any HMMor ^V2,Ar2,A'4,yV4-tetramethylmelamine. Thus, it would

appear that similarities exist between humans, rats, andflies in their metabolism of HMM.

The mechanism of action for the antineoplastic properties of HMM is unknown. For cancer chemotherapy, thedosage level of HMM is approximately 200 times asgreat as that of TEM. Philips and Thiersch (29) also reported that the 50% lethal dose of HMM in rats and micewas approximately 100 times greater than for TEM. Thesefacts suggest that HMM has a different mechanism ofaction than its structural analog, TEM, which is an alkylat-ing agent (33). Borkovec and others (9, 13, 17, 23) haveattempted to learn the basis for the sterilizing activity ofHMM in flies, and they have stated (9, 23) that HMMdoes not act as an alkylating agent in the generally accepted meaning of the term. The negative results of HMMand its metabolites with the NBP alkylation test in theseexperiments, while TEM gave a positive result, also suggest that HMM should not be classified as an alkylatingagent at this time, contrary to previous suggestions (36).

However, the clinical spectrum and toxicity of HMM resemble those of alkylating agents (8). Thus, the possibilitythat intermediates in the metabolism of HMM or unidentified metabolites might possess alkylating activity cannot be dismissed. Cyclophosphamide, for example, showsvery little reaction with NBP (32), but its active form isbelieved to be an alkylator.

There is evidence that certain s-triazines can function aspyrimidine antimetabolites (34, 35). Heere and Donnelly(19) reported that HMM exhibited greater inhibition ofuptake of DNA and RNA precursors than of protein precursors in Ehrlich ascites tumor cells in vitro. Somediaminodihydro-j-triazines act as folie acid antagonists (3).Thus, it is evident that further experiments are requiredto determine the mechanism of action of the antineoplastic properties of H M M.

ACKNOWLEDGMENTS

We thank D. M. Lee and B. D. Kaiman for expert technical assistance,Dr. J. L. Skibba and E. Myers for assistance with the collection of samples from patients, and K. Deighton and S. Pertzborn for assistance withthe manuscript preparation.

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NOVEMBER 1973 2815

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1973;33:2810-2815. Cancer Res   John F. Worzalla, Bruce M. Johnson, Guillermo Ramirez, et al.   Hexamethylmelamine by Rats and Man

-Demethylation of the Antineoplastic AgentN

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