One of the first so-called “alkylating―agents tobe introduced as a chemotherapeutic drug formalignant tumors was the nitrogen mustardderivative, mechlorethamine (Mustargen, methylbis(@-chloroethyl)amine, HN@). Many biologicaland clinical effects of this material have been investigated in recent years, and a great deal of information has been accumulated (8). Skipper et at.(6) and more recently Nadkarni et at. (4) havestudied certain aspects of the physiological disposition of mechiorethamine in mice and rats.

The present report gives the details of thespecific fluorometnic determination of mechlorethamine in biological materials with a procedurewhich may have application for other “alkylating―agents. The results of studies on the biologicaldisposition of the drug are summarized.

MATERIALS AND METHODSE8timation of nwchlorethamine in bidogical ma

terials.—To a 35-mi. centrifuge tube' were added5 ml. of ice-cold or refrigerated aqueous solution,plasma, or urine, and 10 ml. of ice-cold chloroformcontaining 10 per cent methanol (by volume).Base was added to the above mixture as follows:for saline solutions (the saline solutions or standards were prepared by making all dilutions inrefrigerated 0.9 per cent sodium chloride, pH 7),

* This project was supported by USPHS Grant No.

CY-2992of the National Institutes of Health.I Glass-stoppered, 35-ml. pyrex centrifuge tubes with

tapered bottoms were used throughout.

Received for publication November 16, 1959.

0.@ml. of 0.05 N NaOH; for plasma, 0.1 ml. of@ N NaOH; and for urine, appropriate small

volumes of 0.5—1.0N NaOIJ.2 It was essential towork rapidly during this period of preparation ofthe samples particularly after the addition of thebase. The samples were kept chilled in an icewater bath at all times and placed on the shakeras soon as possible. To insure stability of the drugand reliability of determinations, the routinepreparation of the standard was as follows:Mechlorethamine hydrochloride was stored in awax-sealed brown bottle which was kept in adesiccator except for the actual weighing of thedrug. Standard stock solutions containing 1.0mg/mi of the drug were prepared in cold 0.9 percent saline which had been adjusted to a pH ofexactly 7.0 by the addition of a small amount ofNaOH. These stock solutions were found byanalysis to be stable for a period of several weeks,although in practice the standard was preparedfresh each week.

The above mixture, after addition of base, wasshaken for 45 mm. on an International shakermachine set at a frequency of @.80-300/min. and

2 The addition of the base to the respective solutions wasnecessary to bring the pH to approximately 10 to allow for

extraction of the mechiorethamine base. If the bufferingcapacity of the biological samples was different from thatindicated above, then the appropriate amount of base wasadded. Since urine samples vary in buffering capacity, analiquot of a given urine sample was titrated to determine theamount of base to be added to other aliquots of the same urinesample to be analyzed for drug content. Thus, the exactvolume of base to be added to the specific urine sample wasdetermined in advance.

518

The Fluorometric Estimation of Mechiorethamine(Mustargen) and Its Biological Disposition

in the Dog*

L. B. MELLETTANDL. A. WOODS

(Department of Pharmacology, Univervity of Michigan Medical School, Ann Arbor, %fich.)

SUMMARY

A specific method of estimation is described for mechlorethamine which has application for a number of other alkylating agents. Experiments were performed on bloodlevels, urinary excretion, and the tissue distribution of mechlorethamine in dogs. Thedrug rapidly disappeared from plasma, and less than 0.01 per cent was excreted in theurine. The concentration of the drug in tissues was very low, the maximum beingobserved in bone marrow.

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MELLETT @mWooDs—Bidogical Disposition of Mechlorethaniine 519

with the use of the bottle holders which havebeen previously described (7), care being taken tokeep the shaker away from a room heater of anysort. After the shaking period, the samples werecentrifuged for 5 mm., and as much of the aqueouslayer (upper) as possible was removed with awater aspirator without loss of organic solvent.For saline atandard@ and urine: the sample wasshaken twice by hand for 1 mm. with 5 ml. of0.1 M pH 10 borate buffer, after each wash thebuffer being removed by aspiration followingcentrifugation. For plasma: as much of the organicsolvent as possible was transferred to a cleancentrifuge tube without transfer of the proteinprecipitate. The samples were shaken twice byhand for 1 mm. with 5 ml. of 0.1 M pH 10 boratebuffer. After each wash the samples were centrifuged and the buffer removed by aspiration. Analiquot of 7 ml. (or less, the exact amount beingrecorded) of the chloroform-methanol extract fromsaline, urine, or plasma was transferred to a cleancentrifuge tube containing 5 ml. of 0.1 N HC1.3The mixture was shaken on the mechanical shakerfor 10 mm., centrifuged, and the organic solventlayer (lower) removed by aspiration. Additionalmanipulation was required for plasma as follows:the acid extract was centrifuged for 5 mm. atquite high speed, a white precipitate appearing atthe bottom of the tube. Five ml. of ethylenedichionide (EtCl,)4 was added, the mixture shakengently by hand and centrifuged, and the EtC!2removed by aspiration.

An aliquot of 4.5 ml. (or less, the exact amountbeing noted, and with enough additional stock 0.1N HC1 to bring the volume up to 4.5 ml.) of the

0.1 N HC1 extract from saline, urine, or plasmasamples was transferred to a clean centrifuge tubecontaining@ ml. of 0.@ per cent p-phenylphenol,'3 ml. of 0.1 M pH 10 borate buffer, and 0.5 ml. of0.9 N NaOH.6 The p-phenylphenol mixture washeated for 30 mm. in an actively boiling waterbath and then cooled to room temperature. Tenml. of purified ethylene dichioride (EtCl,) and 3ml. of 0.5 N NaOH were added and the mixture

3 All bottles and solutions were kept ice-cold.

4 The EtCh used for the extraction was purified by passage

over alumina and silica gel as described by Woods et at. (9)and then shaken with@ volume of 1 N HC1 by hand for 1 mm.and separated by centrifugation.

SThe reagent was prepared by adding 3.5 ml. of 0.5 N NaOHand about 5 ml. of distilled water to 200 mg. of the phenol. Themixture was heated until the phenol dissolved and the resultingsolution diluted to 100 ml. This reagent was stable for severalweeks.

S It must be determined by titration with a pH meter that

0.5 ml. of this NaOH solution, when mixed with exactly 4.5 ml.of 0.1 N HCI, gives a solution having a pH of approximately 10.

shaken mechanically for 30 mm., centrifuged, andthe aqueous layer (upper) removed by aspiration.The EtCl2 extract was shaken by hand for 1 mm.4 times with 5 ml. of 1 N NaOH. The mixture wascentrifuged and the aqueous layer (upper) removed by aspiration after each wash.

Seven ml. of EtC!2 extract was transferred to aclean centrifuge tube containing 5 ml. of 1.0 N HCI.The mixture was shaken for 10 mm., centrifuged,and the EtCl2 layer (lower) removed by aspiration. A sufficient quantity of the acid extract wastransferred to the appropriate cuvettes, and thefluorescence was determined in the AmincoBowman Spectrophotofluorometer at an activation wave length of 285 mj@ and a fluorescencewave length of 340 mz.

Parallel standards of the mechlorethamine insaline having concentrations in the range of thesubstances to be analyzed were carried throughwith each set of determinations. The results wereused to estimate the drug concentration in the unknown samples. Reagent controls were also runroutinely in duplicate with each set of analysis.

For low concentrations of mechlorethamine,the final extraction from the EtC!2 into 0.1 N HCIcould be performed with 1 ml. or less of the acid.Micro-cells could then be employed to determinethe fluorescence in the spectrophotofluorometer.With smaller volumes of acid, as little as 0.0@j@gofdrug/mi could be estimated. The relationship between concentration and fluorescence was reasonably linear up to 10 pig/mi. Concentrations greaterthan 10.0 @g/mlhad to be diluted before analysisto bring them within a readable range on thespectrophotofluorometer. The reliability at thelowest concentration (0.0@ j@g/ml) was approximately 115 ± @4per cent (S.D.) and 99 ± 13 percent (S.D.) at higher concentrations (@.O—10.0@tg/mi) of the drug in plasma and urine.

The specificity of the method for unchangedmechlorethamine cannot be assayed by the usualbuffer-solvent distribution studies because of therapid destruction of the drug in alkaline pH. Thedemonstration of specificity was approached onthe basis of kinetics as developed by Golumbicet a!. (@)and utilized by Hunt and Philips (3) intheir studies on the acute pharmacology ofmechlorethamine hydrolysis products. For thispurpose concentrations of the drug were preparedin 0.16 M NaHCO3 and maintained at 30 ±0.5°C.for varying intervals in a Dubnoff apparatus. Incubation of mechlorethamine in this mixture for

@0mm. resulted in 90 pen cent conversion to thechlorimine according to Hunt and Philips. A similan incubation performed in our laboratory resulted in a 75 per cent loss of mechiorethamine

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5@0 Cancer Research Vol. @0,May 1960

(Chart @).Incubation of the above mixture for16.5 hours resulted in theformation of the hydroxyimine to an extent of 4@ per cent of the originaldrug concentration according to Hunt andPhilips. A similar incubation performed andanalyzed according to the fluonimetnic proceduregave blank readings. Pure methy1bis(@-dihydroxyethyl)amine assayed by the fluorimetnicprocedure gave blank values. From these studies itwould appear that the method is quite specific forunchanged mechlorethamine with the chlonimineinterfering to the extent of about 10—iSper cent.The hydroxyimine, and the methyldiethanolamine hydrolysis products do not interfere in theanalysis.

Tissues were prepared by homogenizing 3-gm.samples in 1@ ml. of saline. Two 5-rn!. aliquotswere removed and frozen immediately for subsequent analysis. Extreme care was taken to keepthe tissues in the homogenizing tubes cold duringthe homogenization. The amount of base (1.0—1.5 N NaOH) required to increase the pH of S ml.of the tissue homogenate to approximately 10 wasdetermined on aliquots of homogenized tissueother than those to be analyzed Subsequently themethod of estimation was that described above forsaline solutions. Tissue recoveries with added concentrations of 0.5 sg/gm or ml of tissue, or fluid,were about 85 ±15 per cent (S.D.) with theexception of bone marrow and bile, which gaverecoveries in the range of 40—50per cent.

In vitro studies.—Solutions of mechiorethaminewere incubated in a Dubnoff shaker at 30°and38°C. in 0.16 M NaHCO3, plasma, and wholeblood. At the end of the incubation period thesamples were chilled to 0°C. or frozen on solidCO2 to prevent any further chemical changes inthe drug. The samples were then analyzed according to the procedure outlined above.

In other experiments mechlorethamine wasadded to whole blood previously chilled to 4°C.The plasma was separated at this temperatureby centrifugation and analyzed for drug content.In parallel experiments drug was added to wholeblood at 38°C., and it was then chilled to 4°C.The plasma was separated and analyzed as before.In other studies the formed elements were separated from the plasma, washed 3 times with saline,resuspended in saline or plasma containing knownamounts of mechlorethamine, and allowed toincubate for 10 mm. at@ C. The formed elements were again separated, and the amount ofdrug associated with the cells and in the suspending medium was estimated.

Animal experinwnts.—Doses of 1—3mg/kg ofmechlorethamine HC1 were administered intra

venously in 1.0 per cent solution to female dogsweighing 15—@5kg. Blood samples were obtainedas soon after injection as possible and as frequently as possible, with oxalate used as an anticoagulant. The blood samples were immediatelycooled in an ice bath and aliquots pipetted toclean centrifuge tubes and frozen promptly onsolid CO2. In three experiments on dogs the plasmawas separated from the red cells by spinning thechilled blood samples in a refrigerated centrifuge.Determinations of drug levels in simultaneoussamples of whole blood and plasma were then performed. All samples were frozen immediately andkept in a freezer until analysis.

Urine samples were obtained with the aid ofan indwelling Foley-bag catheter@ placed in theurinary bladder. Immediately after collection ofthe samples, aliquots were pipetted into centrifugetubes which were then placed on solid CO2. Thefrozen urine samples were stored in a freezer untilanalysis.

For the obtaining of tissue samples, animalswere anesthetized with ether and exsanguinated asquickly as possible. Tissues were removed immediately and frozen on solid CO2. Immediatelyprior to homogenization the tissues were thawedjust enough to allow the weighing of the samples.Aliquots of the homogenized tissue were pipettedinto clean centrifuge tubes, which were frozen onsolid CO2. The frozen samples were kept in afreezer until analysis. In two animals the drugcontent was assayed in lymph obtained by cannulation of the thoracic duct. In both animals thedrug level in the lymph was appreciably lowerthan the corresponding plasma levels.

Plasma, blood, urine, or tissue homogenatesamples contained in the centrifuge tubes wereallowed to warm until the material was an icymush and were then carried through the method ofestimation as indicated above. Duplicates ortniplicates of each individual biological samplewere analyzed for content of mechlorethamine.

Most dogs which received the dose of 3 mg/kgof mechlorethamine died in 5—10days of the toxiceffects of the drug.

RESULTS

As illustrated in Chart 1, the initial bloodsample (3—4 mm. after intravenous administration of 3 mg/kg of mechlorethamine) indicated avery rapid disappearance of the drug. Theseinitial levels were in the range of 0.15—0.20 @zg/mlof whole blood. An even distribution of the drugin all tissues of the body, which could hardlyoccur in 4 mm., would provide a concentration of3 j@g/ml. Thus, it is apparent that the rate of

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MELLETT AND WooDs—Biological Disposition of Mechiorethamine 5@21

destruction in blood must be extremely rapid.That a large fraction of the destruction is duesimply to the incubation of the drug with bloodor plasma at body temperature is indicated by thedata from in vitro incubation studies shown inChart @.Approximately 90 per cent of mechiorethamine disappeared in 4 mm.

Of particular interest in Chart 1 is the observation that the simultaneous plasma level is alwayslower than the blood level. This would seem toindicate that a substantial fraction of the administered drug is associated with the formed elements. To insure that these results were indeed notartifacts resulting from the additional manipulation for plasma, further experiments were performed in which drug was added to whole bloodand to plasma at 4°or at 38°C. The whole blooddrug mixture was centrifuged at 4°C. and theplasma separated and analyzed as usual. The results of these experiments are summarized in

0 4 8 $2 6 20 24 28 32 36 40 44 48

TIME (MINUTES)

CHART 1.—Siniultaneous whole blood and plasma levels ofmechiorethamine in dogs after an intravenous dose of 3.0

mg/kg.

Table 1. At 4°and at 38°C. the amount of drugrecovered from the plasma separated from wholeblood-drug mixture was less than the recovery ofdrug from plasma alone. This would again indicatethat a substantial fraction of the unchanged drugin blood is associated with the formed elements.It can be demonstrated that the binding of thedrug in or on the formed elements was weak by thenearly quantitative recovery of drug added tochilled whole blood (90 ±10 per cent) andanalyzed immediately.

In other in vitro studies in which separated cellswere washed with pH 7.4 saline and then resuspended in saline or plasma containing mechiorethamine and incubated at@ C. for 10 mm., the

cells separated from plasma or from saline showedapproximately the same drug content (Table 1).From all of these data one can conclude that substantially more of the unchanged mechiorethamineis associated with the formed elements of theblood than with plasma.

On the basis of the results in plasma, one wouldexpect that urinary excretion of unchanged drugwould be very low, and indeed that is the case, asshown in Table @.The recovery of the administered dose was less than 0.01 pen cent. The results on urinary excretion indicate almost cornplete destruction of the drug in the animal. It isquite likely, of course, that' some of the inter

CHART 2.—Concentrations of mechlorethamine in wholeblood and plasma after various times of incubation at 30°and38°C. and after incubation in 0.16 M NaHCO, at 30@C.

mediate hydrolysis products of mechiorethaminesuch as the chlorimine or the hydroxyiminederivatives appear both in plasma and in urine andmight persist for a considerable period of time.However, the method of estimation utilized aboveis quite specific for the mechlorethamine and consequently indicates rapid and nearly completedestruction of the original drug.

The results on tissue distribution 15 mm. afterthe intravenous administration of 3.0 mg/kg ofmechlorethamine are summarized in Table @.Ingeneral the tissue concentrations were quite low,the highest being observed in bone marrow.

DISCUSSIONIt is obvious that mechiorethamine is rapidly

destroyed when administered intravenously tolaboratory animals. Nadkarni et at. (4) reportedthat over 90 per cent of the radioactivity ofmethyl-C'4-labeled mechlorethamine was removedfrom the blood within 30 sec. when mice were in

BLOODPLASMA —__ ——

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RECOVFRY OF MFCHLOREThAMINE FROM PLASMA SEPARATFD

FROM WHOLE BLOOD-DRUG MIXTURESRecovaa@

OF MECIILORETHAMINEFROMPLASMA•SALINS.ANDFORMED ELEMENTS FROM MIXTURES OF DRUG WITH THE FORMED

ELEMENTS IN PLASMA OR SALINE AT 25°C.Concentration

in @&g/mloforiginal whole

blood orplasmaTemperature

in‘C.of blood or

plasma attime ofmixingRecovery

of drugin plasma(pg/mi)Concentra

tion in pg/miof salineor plasma

mixturePer

cent recovery of@ drug from

cells, saline,orplasmaRe@very

of drug fromplasma separatedfrom whole blood5

Recovery of drug fromplasma alone2.0

2 .0

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cells resuspended in salinedrug mixturet

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DocNO.AMOUNT

RECOVEREDINURINE IN 8 HOURS

(Per cent(jig)of dose)Doc

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DISTRIBUTIONOF MZCUWRETRAMINE(pa/GM) IN DOOR15 MIX.AFTERADMINJBTRATION5

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5@2@2 Cancer Research

jected. Of the remaining 10 per cent, the majoramount was present in plasma. Skipper et a!. (6)have also studied th@ metabolism of methyl-C'4-labeled mechioretharnine in mice and found thatthe specific activity of the blood serum was higherthan that of the cells. The results given in this report indicate that substantially more of the unchanged mechlorethamine is associated with theformed elements than with plasma. It is importanthere to keep in mind the differences in the analytical technics involved. Both Nadkarni andSkipper assayed radioactivity, whereas the fluorimetric procedure used in the present study is ameasure of alkylating property and has beendemonstrated to be quite specific for unchanged

mechlorethamine. It is quite reasonable to assumethat the differences in methodology and/or animal species would explain the variations betweenour findings and those of the previous investigators.

Skipper et at. (6) reported that between 10 and18 per cent of the methyl carbon was oxidized tocarbon dioxide and exhaled. They were able to recover between 5 and 10 per cent of the radioactivity in urine. This is in contrast to our findings ofless than 0.01 per cent of unchanged mechiorethamine recovered from the dog. Smith et at. (7)have studied the urine by means of chrornatographic analysis after the administration ofmethyl-C'4-mechlorethamine and have found at

TABLE 1

In Vitro RECOVERY OF MECRLORETHAMJNE

Recovery was determined from plasma separated from whole blood-drug mixtures, and from plasma, saline, and formed elements from mixtures of drug with the formed elements in plasma or saline.

* Whole blood at 4° C. was mixed with mechiorethamine to give a concentration of 2.0 ,@g/ml, centrifuged at this temperature,

and the plasma separated, frozen, and then analyzed. Blood or plasma to which drug had been added at 38°C. was chilled to40 C. and then centrifuged and treated as before.

t Cellswereseparated from wholeblood, washed3 times with pH 7.4 saline,and resuspendedin saline containing 2.0 @g/mlof mechlorethamine. After standing for 10 mm. at 25@C. the mixture was centrifuged, and the saline and cells were separatedand analyzed.

@ Cells washed S times with saline were resuspended in plasma containing 2.0 @g/mlof mechlorethamine. This mixture wasthen treated in a manner identical with the cell-saline mixture above.

TABLE 2

URINARY RECOVERY AND TIssuE DISTRIBUTION OF MECELORETRAMINE IN Doos

AFTER THE INTRAVENOUS ADMINISTRATION OF 3.0 MG/KG

* The figures in the table are the averaged results of duplicate determinations on each tissue.

t Correctedfigureson basisof 40-50 per cent recoveryof drugaddedto tissue.

Vol. @0,May 1960

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MELLETT AND WooDs—Biological Disposition of Mechlorethamine 5@23

least eight labeled products. Some of these wereprimary or secondary amines and were not identifled further. Smith et a!. (7) further concluded thattracer studies with N-methyl-C'4-labeled mechlorethamine may lead to results that do notnecessarily represent the uptake or distribution ofthe whole mechlorethamine molecule. This, ofcourse, does not imply that radioisotopic methodsare not useful in these areas particularly whencombined with specific chemical and carriertechnics. Radioisotopic methods would be especially useful in determining the mechanism of actionof alkylating agents and in establishing the extentof fixation of these agents to tissues and tissuecomponents.

In studying the distribution of radioactivity ofmethyl-C'4-mechlorethamine in mice Skipper et a!.(6) found radioactivity in all tissues. The intestine was consistently the tissue of highestactivity. The kidney also showed appreciableactivity. One of their most interesting observations was the low activity of bone marrow. In ourstudies we have consistently found the bone marrow to contain the highest amount of unchangedmeclilorethamine. It would appear that thisdiscrepancy must again be explained on the basisof differences in methodology and/or species.

Hunt and Philips (3) have pointed out thatonly the chlorirnine, the hydroxyimine andmethyldiethanolamine have any appreciable existence in mildly alkaline solution. Only the chlorimine possesses alkylating properties comparable tothe parent mechlorethamine. Thus, if alkylationis indeed the mechanism by which the drug produces its biological effects, then the unchangedmechlorethamine and the chlorimine represent thegreatest potential for introducing biological responses.

Price (5) has discussed the mechanism ofalkylation as a first order nucleophilic substitutionin which the reaction proceeds in two steps, Step Iis a rate-controlling ionization of the R-Y bond ofthe alkylating agent.

I. R-Y relatively R@ solv. + ye solv.slow

The reaction is facilitated by a polar solventthat solvates and stabilizes the resulting ions. Thesecond step is a rapid and indiscriminate reactionof the resulting alkyl carbonium ion.

II. R@+ Xe !@X@RX (where X is the biologicallast

material which is alkylated)

In the presence of water alone where there is adeficiency of reactants the reactive alkyl carbonium ion (Re) proceeds to the formation of the

relatively stable chlorimine. However, because ofthe rapidity of step II and the excess of reactantsin the vascular compartment, i.e., protein, water,etc., relatively little of the chlorimine is likely tobe formed. Thus, where R-Y is rnechlorethamineand R@' is the alkyl carbonium ion, one can prediet on the basis of charge and solubility properties that mechiorethamine would penetrate cellsmore readily than the carbonium ion and subsequently could undergo step II within the cell, thusalkylating intracellular components. This conceptof differences in distribution of mechlorethamineand the carbonium ion receives further supportfrom the many clinical experiments demonstratingthat intra-arterial administration of mechlorethamine has an action confined primarily to the areasupplied by the particular artery. On the otherhand, the delayed vomiting which is observedwith this drug probably represents an action ofone of the conversion products (chlorimine, hydroxyimine). The physiological mechanism andsite of action of mechlorethamine emesis has beenstudied by Borrison et a!. (1) in cats and dogs andapparently represents the interplay of both centraland peripheral factors.

ACKNOWLEDGMENTSThe authors greatly acknowledge the technical assistance

of Miss Beverly Waterman and Miss Rita Czewski in carryingout the experiments as reported in this communication and ofDr. William Baker for assistance in performing certain experiments.

REFERENCES

1. BORRISON, H. L.; Bn@nr, E. D.; and ORKAND, R. K.Emetic Action of Nitrogen Mustard (Mechiorethaminehydrochloride) in Dogs and Cats. Am. J. Physiol., 192: 410—16,1958.

2. GoLuasBic, C. ; FRUTON, J. S. ; and BERGMANN,M. ChemicalReactions of Nitrogen Mustard Gases. I. The Transformstions of Methyl-Bis ($-chloroethylamine) in Water. J.Org. Chem., 11:518—35, 1946.

3. H@mrr,C., and PHIups, F. S. The Acute Pharmacology ofMethyl-Bis (2-chloroethyl) amine (HN2). J. Pharmacol. &Exper. Therap., 95: 131—44,1949.

4. N@.uic&m@i,M. V.; GOLDENTHAL,E. I.; and SarrH, P. K.Observation on the Rapid Disappearance of Radioactivityfrom Blood after Intravenous Triethylene Melamine-C'4.Proc. Am. Assoc. Cancer Research, 2: 136, 1956.

5. Piucz, C. C. Fundamental Mechanisms of Alkylation. Ann.N.Y. Acad. Sc., 68:663-68, 1958.

6. SKIPPER,H. E.; BENNETT,L. L.; and LANOMAN,W. H.Over-all Tracer Studies with C―-labeled Nitrogen Mustardin Normal and Leukemic Mice. Cancer, 4: 1025—27,1951.

7. SMITH, P. K.; N@u@uu@i, M. V.; Tnasss, E. G.; and DAmSON, C. Distribution and Fate of Alkylating Agents. Ann.

N.Y. Acad. Sc., 68:884-50, 1958.8. WHITELOCK, 0. V., St. (ed.) Comparative Clinical and Bio

logical Effect of Alkylating Agents. Ann. N.Y. Acad. Sc.68:657—1266,1958.

9. WOODS, L. A.; CocmN, J.; FORNETELD, E. J.; MCMAHONF. G.; and SERVERS,M. H. The Estimation of Amines inBiological Materials with Critical Data for Cocaine andMescaline. J. Pharmacol. & Exper. Therap., 101: 188—99,1951.

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1960;20:518-523. Cancer Res   L. B. Mellett and L. A. Woods  and Its Biological Disposition in the DogThe Fluorometric Estimation of Mechlorethamine (Mustargen)

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