Among the varieties of alkylating agents introduced for the chemotherapy of neoplasms, thesubstance N,N',N―-triethylenethiophosphoramide(thioTEPA) has demonstrated clinical usefulnessin the temporary palliation of certain cancers (9,1@, 15). The drug is administered by variousroutes clinically and has been demonstrated to beeffective per os. However, information on plasmalevels, urinary excretion, and fate after oral orparenteral administration has not been available.

a This project was supported by USPHS Grant No. CY2992 of the National Institutes of Health.

Receivedfor publication November 16, 1959.

Preliminary experiments indicated that thioTEPA is converted to N,N',N―-triethylenephosphoramide (TEPA). Methodology was thereforedeveloped for the estimation of thioTEPA andTEPA separately and in mixtures. This cornmunication reports the details of the methods ofestimation of these materials and the results ofstudies on physiological disposition.

MATERIALS AND METHODS

ESTIMATION OF DRUGS IN BIoLooIc@i MA@rm@L&Ls

ThioTEPA.—Five ml. of urine or 0.9 per centsaline, mixed with 0.@5 ml. of 0.5 M pH 7.4

5@24

The Comparative Physiological Disposition ofThioTEPA and TEPA in the Dog*

L. B. MELLETT AND L. A. WOODS

(Deparirnera of Pharmacology, University of Mwhigan Medical School, Ann Arbor, Mich.)

SUMMARY

A sensitive, specific fluorometric method for the determination of N,N',N―-triethylenethiophosphoramide (thioTEPA) and N,N',N―-triethylenephosphoramide(TEPA), separately and in the presence of each other, has been developed. The percentage recovery of thioTEPA or TEPA added to plasma and urine in the range of0.05—0.@ j@g/ml was 95 ± @0per cent, and at levels of 0.@ @g—@.O.ig/ml it was 90 ± 1@

per cent.After the intravenous or oral administration of thioTEPA to dogs the presence of

TEPA in the urine was demonstrated both by analytical procedures and by means ofpaper chromatography. After 3.0 mg/kg intravenously, the percentage excretion ofTEPA was 8—13per cent of the administered dose of thioTEPA. After 6.0 mg/kgorally, the percentage excretion of TEPA was 13—15per cent. The plasma concentrationcurves of thioTEPA after 3.0 mg/kg intravenously fell rapidly to a level of 0.@ @tg/miin@ hours. On the other hand, maximal plasma levels (1.@—1.4pg/mi) of TEPA werenot reached until 90 minutes to@ hours. On the basis of information on plasma levelsand urinary excretion of thioTEPA, at least 50 per cent of the administered dose wasabsorbed.

Prior administration of $-diethylaminoethyldiphenyl propyl acetate HC1 (SKF5@5A) delayed the conversion of thioTEPA to TEPA. This delay in conversion wasaccompanied by an increased toxicity in mice and dogs.

When TEPA was administered to dogs in a dose of 3.0 mg/kg intravenously, theplasma levels were @—4times the levels of thioTEPA after intravenous administration.The urinary recovery of TEPA was @4—34per cent of the administered dose.

The stability of thioTEPA and TEPA in various media was investigated. There wasno significant difference between the two drugs upon incubation in plasma, whole blood,or various buffers. Both drugs were stable in alkaline medium (pH 8.4) and were rapidlydestroyed in acid medium (pH 4.@).

Tissue distribution of thioTEPA could not be determined, because the drug wasalmost completely destroyed when in contact with tissue. TEPA, on the other hand,could be determined and displayed a marked affinity for bone marrow as comparedwith other tissues.

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MELLETT AND WooDs—Physiological Disposition of ThiOTEPA and TEPA

phosphate buffer, or 3 ml. of plasma mixed with0.5 ml. of 15 per cent tetrasodium edathamil( Versene),' were shaken for 40 min. in a centrifuge

tube2 with 15 ml. of thiophene-free benzene. Thetube was then centrifuged, and 10 ml. of the benzene extract was pipetted into a clean centrifugetube containing@ ml. of a solution of @3-naphthol5(1.@5 mg/mi) in benzene (prepared daily). Themixture was evaporated to dryness in a boilingwater bath in a fume hood, the evaporation requiring approximately 15—@Omm. The centrifugetube was then placed in a wax bath (Fischer'sbath wax) heated to 1@5—1SO@C. for a period of @Omin.Fourml. of@NHClwasusedthwashdownthe wall of the centrifuge tube to remove as muchof the sublimate as possible. The acid mixture wasthen heated in a boiling water bath for 15 mm.One ml. of 10 N NaOH was added to the warmmixture and the tube shaken quickly@ times withcare to avoid the stopper's being blown out. Thetube was heated in the boiling water bath for@mm. The mixture was cooled to room temperature,and 10 ml. of ethylene dichioride (EtC!2)4 added.The mixture was shaken for @0min., centrifuged,and the aqueous layer (upper) removed by siphonbig. The EtCI2 was shaken twice with 5 ml. of0.5 N NaOH, the mixture being centrifuged and

the aqueous layer (upper) removed by aspirationafter each washing. Then 9 ml. of the EtC!2 extractwas transferred with a clean graduated 10-mi.pipette to a clean centrifuge tube containing@ ml.of 1 N HC1, and the mixture was shaken for 10mm., centrifuged and the EtCl2 layer (lower) removed by aspiration. The acid extract was shakenby hand with 15 ml. of pure EtC125 for @0sec., afterwhich the mixture was centrifuged and the EtCl2removed by aspiration. As much of the acid cx

1 This solution was prepared by dissolving the proper

amount of disodium Versene in distilled water and adding thecalculated amount of NaOH to give the tetrasodium Versene.Commercial solutions of tetrasodium Versene gave very highreagent blanks in the TEPA procedure.

S Unless otherwise indicated, all extractions and reactionswere performed with 35-ml., glass-stoppered, pyrex centrifugetubes with a tapered bottom. The bottles were shaken mechanically with an International shaker machine set at a frequency of 280-390 oscillations/mm with the use of the shakingblocks described by Woods el al. (14).

3 @9-naphthol was sublimed at 15—20 mm. of Hg pressure

and stored in small, colored bottles in a desiccator.

4 The EtC!2 used for the extraction was purified by passage

over alumina and silica as described by Woods et at. (14) andthen shaken with@ volume of 1 N HC1 by hand for 1 mm. andseparated by centrifugation.

S The EtCh used to wash this acid extract was purified bypassage over alumina and silica gel as above and then shakenby hand respectivelywith@ volumesof 1 N NaOH, 1 N HG,and finallydistilled water.

tract as possible was transferred to the 10-mmsquare cuvettes and the fluorescence determinedin an Aminco-Bowman spectrophotofluorometerat an activation wave length of @90mjs andfluorescence wave length of 355 mgi. The sensitivity of the machine was adjusted to read 30 percent transmission at a meter multiplier setting of1.0 with a standard aqueous solution of @.0@g/mlof $-naphthol in distilled water.6 The slit arrangement in the spectrophotofluorometer was thatrecommended for maximum sensitivity.

Parallel standards of thioTEPA with concentrations falling in the range of the unknownsamples were always carried through the procedure with the unknown samples. The concentration in the unknown was determined by a directproportionality. Saline solutions, or drug-freeurine or plasma, were carried through the procedure to serve as reagent blanks, The parallelstandards were always prepared fresh by dilutionsof a stock solution of 1 mg/mi of thioTEPA in 0.9per cent saline solution which was stored in a refrigerator. The dilutions were made in saline. Thestock solution was prepared by dissolving thecontents of seven 15.0-mg. vials which containedsodium bicarbonate as a buffering substance anddiluting to appropriate volume. The vials werekept sealed and refrigerated prior to use. It wasfound that standards so prepared were moresuitable than those made from bulk thioTEPA,which although kept cold and desiccated showedchanges in solubility over a period of severalmonths. These changes were characterized by incomplete solution in water and benzene, and consequently gave low readings when carried throughthe method of estimation by fluorescence as described above. However, the clear supernatantliquid obtained by centrifuging the milky suspensions of the bulk thioTEPA in water gave 100 percent values with the thiosulfate titration assayprocedure.7 It would appear that bulk thioTEPAwas being converted to an insoluble substance notcontaining radicals which could be titrated withthiosulfate and to a completely water-soluble

6 A stock solution of 1 mg/kg of the j9-naphthol was prepared just before use by dissolving the phenol in the equivalentamount of NaOH. This solution was diluted accurately to the2 ,sg/ml concentration.

7 This technic involved the addition of excess sodium thiosulfate to an aliquot of the thioTEPA solution, with theaddition of one drop of methyl orange solution and titrationwith 0.05 N HG to the end point of pinkish-orange color witha side by side comparison of pH 4 buffer in a similar volumecontaining one drop of methyl orange. The end-point colorshould remain for at least 10 sec. The solution was then allowed to stand for 30 mm. and 4 drops of phenolphthaleinindicator added and the excess acid back titrated with 0.05 NNaOH to the first pink color remainingfor 30 sec.

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MEDIUMPERCENTAGE

LOSS

IN SO MINUTES

ThioTEPATEPAWhole

blood, pH 7.8Plasma, pH 7.80.16@ NaHCO,, pH 8.40.1 M acetate buffer, pH 4.20.1 M phosphate buffer,PH 7.460

450

95

6860

300

95

72

5@26 Cancer Research Vol. @0,May 1960

material which did contain the ethylenimine configuration. One can speculate that the insolublematerial was sulfur in colloidal form and that thewater-soluble material might be TEPA. The stocksolutions of 1 mg/mi of thioTEPA in saline notedabove were frequently checked by titration according to the thiosulfate procedure.

The determinations on plasma, urine, etc., wereperformed in duplicate or triplicate since oneanalysis in fifteen or twenty was considerablybeyond the variation of the method.

The recovery of known amounts of thioTEPAadded to plasma and urine in the range of 0.05—0.@ @g/mlwas 95 ± @0per cent (standard deviation), and at levels of@ @g/ml it was 90 ± 1@per cent.

TABLE 1

STABILITY OF CONCENTRATIONS OF 1.0 @aG/ML OF Tm0TEPA* AND TEFA INcuBATED AT 38°C. IN A DUBNOFF APPARATUSFOR 30 MINUTES

cedure was identical with that employed forthioTEPA. The recovery of known TEPA addedto plasma and urine was somewhat less satisfactory (88 ±15 per cent) than that noted forthioTEPA.

When both thioTEPA and TEPA were to beestimated simultaneously, aliquots of the samesample were carried through both the proceduresfor thioTEPA and for TEPA noted above. Theprocedure for TEPA estimated both thioTEPAand TEPA and therefore was equated to totalalkylating agent. The procedure for thioTEPAwas specific for thioTEPA, and accordingly thedifference between the two values was TEPA.Known mixtures of thioTEPA and TEPA inaqueous saline were carried through these procedures with adequate recovery.

Specificity of estiination.—Evaluation of thespecificity of estimation of thioTEPA and TEPAwas difficult since the usual buffer distributionstudies were not possible. However, aqueous solutions of TEPA analyzed with the procedure forthioTEPA gave blank readings since the TEPAwas not extracted from aqueous solution by benzene. Aqueous solutions of thioTEPA or TEPAhydrolyzed by acid gave blank readings in theabove procedures for the two drugs, indicatingthat the ethylenimino group is necessary eitherfor extraction or, more probably, for reaction withthe @9-naphthol.

Dog urine which showed the presence of thioTEPA and TEPA by the above procedures wasextracted with 10 per cent methanol in chloroformand the organic solvent extract evaporated tosmall volume under reduced pressure and spotsapplied on paper strips (Whatman No. 1). Paperchromatograms were developed with two separatesolvent systems—n-butanol saturated with 1 percent ammonia and acetone-water (80:@0, v/v)—,sprayed with 0.5 per cent ninhydrmn solution in nbutanol and heated for several hours at 45°C.Two spots only were obtained from the urine extract. The R, values were identical with parallelknown thioTEPA and TEPA : n-butanol andammonia, thioTEPA 0.84, TEPA 0.68; acetonewater, thioTEPA 0.80, TEPA 0.90.

STABILITY OF THIOTEPA IN V@t.mous

MEDIA AT 88°C.

The stability of thioTEPA and TEPA invarious media incubated in a Dubnoff apparatusat 38°C. is shown in Table 1. The media employedwere whole blood, plasma, 0.16 M NaHCO3 (pH8.4), 0.1 M acetate buffer (pH 4.1), and 0.1 M

8 Reagent grade methanol and chloroform were used with

out further purification.

S Determinations for each compound were

performed in sets of 8.

It was not possible to adequately recover thioTEPA which had been added to tissues eventhough tissues were homogenized, cooled in ice,and then mixed with a suitable buffer and benzeneas described above with the addition of thethioTEPA being made just before the mixture wasplaced on the shaker for the purposes of extraction.A large number of enzyme inhibitors was added tothe tissue homogenates without satisfactory recovery of the added thioTEPA. The conclusionwas drawn that the drug was reacting chemicallyand almost instantaneously with substances derived from intracellular structures. Recoverieswere somewhat satisfactory from brain (60—100per cent) but from kidney, liver, muscle, andspleen were in the range of 0—30per cent.

TEPA.—The initial extraction of TEPA fromaqueous solutions, urine, plasma, blood, or tissueswas performed with 15 ml. of 10 per cent methanol(by volume) in chloroform.8 Otherwise the pro

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MELLETT AND WOODS—Physiological Disposition of T/iiOTEPA and TEPA 5@7

0.6

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phosphate buffer (pH 7.4). In all instances the concentration of alkylating agent in the medium was1 @g/mlat the beginning of incubation. It is alsoevident that both drugs are quite stable in alkaline medium (0.16 M NaHCO3, pH 8.4) and Unstable in acid medium (0.1 M acetate buffer, pH4.@). This is in marked contrast to certain otheralkylating agents such as mechlorethamine (Mustargen), which is stable in acid but is rapidly destroyed in alkaline medium.

BINDING TO PLASMA PROTEINS

The binding of thioTEPA and TEPA to plasmaproteins of separated dog plasma was determinedby ultracentrifugation procedures according to themethod of Taylor et at. (11). The centrifugationwas carried out at 7°C. to minimize destruction ofdrug. The original concentration of drug in theplasma was @.0gig/mi, and the pH of plasma was7.8. All determinations were performed in quadruplicate. The binding of both drugs is approximately equal and falls in the range of 0—10percent.

ANIMAL EXPERIMENTS

Plasma levels and urinary excretion in dogs.—Female mongrel dogs were used routinely exceptfor tissue distribution and tissue recovery experiments. In the latter case, mixed sexes were employed. Food was removed from the cage 1@hoursbefore the experiment was scheduled to begin.ThioTEPA or TEPA was administered in a doseof 3 mg/kg intravenously or 6 mg/kg of thioTEPA by stomach tube. In no instances wereacute pharmacological effects observed with thesedoses. About one-half of the animals died in @—3weeks because of action of the alkylating agent onbone marrow. Plasma samples were obtained for 8hours and urine samples for @4hours. The drugcould not be detected in the respective samplesafter the times indicated. Urine samples were collected by an indwelling bladder catheter for thefirst 6—8hours. All blood samples were cooled immediately on ice and then centrifuged, the plasmabeing pipetted into clean centrifuge tubes andfrozen on solid carbon dioxide. The urine sampleswere frozen immediately on solid carbon dioxide.The samples were kept in a deep freeze untilanalysis was performed, at which time they wereallowed to melt spontaneously and were placed onthe shaker machine at a time when there was still asmall amount of ice present in the mixtures.

For experiments involving tissue distribution,appropriate samples of the tissue or organs wereremoved as quickly as possible and frozen on solidcarbon dioxide after anesthetization of the animalwith ether or sodium pentobarbital and subse

quent exsanguination. The tissues were stored in afreezer until analysis.

Effect of pretreatment of SKF 525A9 (@-diethylaminoethyldiphenyl propyl acetate HC1) on theconversion ofthioTEPA to TEPA in dogs.—Plasmalevels and urinary excretion of thioTEPA andTEPA in female dogs were determined after theintravenous administration of 3.0 mg/kg ofthioTEPA following pretreatment of the animalswith either @0mg/kg of SKF 5@5A (a knownmetabolic inhibitor ; Cooper et at. [3]; Murphy andDubois [5]) 1 hour prior to thioTEPA administration or 50 mg/kg of SKF 5@5A @0mm. prior to theadministration of the alkylating agent. The dosesof SKI? 5@5A were administered subcutaneously.

CHART 1.—Time-concentration plasma curves for thio-.TEPA and TEPA in a typical experiment in the dog after theintravenous injection of S mg/kg of thioTEPA.

Modification of the toxicity of thiOTEPA in miceby prior admini4ration of SKF 51?5A.—Mterpreliminary determination of toxicity dosage, twogroups of twenty albino mice were injected with 30mg/kg of thioTEPA intraperitoneally. In onegroup of twenty mice, @0mg/kg of SKF 5@5A wasadministered intraperitoneally 45 min. prior to thethioTEPA. These animals were observed daily,and survival times were recorded.

RESULTSPlasma levels and urinary excretion of IhiOTEPA

and TEPA following the intravenous admini4ration of 3 mg/kg of thioTEPA.—The plasma concentration curves for thioTEPA and TEPA in atypical experiment are illustrated in Chart 1. ThethioTEPA rapidly disappeared from the plasma,reaching levels of 0.@pig/mi @2hours after administration of the drug, the most rapid disappearanceoccurring during the first 60 mm. On the other

S Kindly supplied by Dr. Paul Mattis of Smith, Kline, andFrench Laboratories, Philadelphia, Pa.

DOG 2

0

(3.0 MG/KG. IV)

THIO-TEPA •.--•TEPA S S

I 2 3 4 5 6

HOURS

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DcxiNO.PERCENTAGE

RECOVERY AFTER

I.V. ADMDIIRTRATION

As AsThioTEPA TEPADoe

NO.PERCENTAGE

RECOVERY AFTER

ORAL ADMDOS

TRATION

As AsThioTEPATEPA‘FT-I

T'F.2TT-30.3

0.40.313

88TT-4

‘FT-S0.7 0.515 13

Cancer Research Vol. @0,May 1960

hand, the maximal plasma concentration ofTEPA was not reached until 90 mm. to@ hoursafter the administration of the thioTEPA. TheTEPA disappeared from the plasma at a somewhat slower rate than did the thioTEPA. Theseresults are certainly suggestive of the fact thatthioTEPA is converted to TEPA more rapidlythan the latter drug can be metabolized. The concentration of thioTEPA in lymph obtained bycannulation of the thoracic duct was studied inone animal. The concentrations were @0per centless than those found in simultaneous samples inplasma.

The urinary recovery of thioTEPA andTEPA from the animals receiving 3.0 mg/kg ofthioTEPA intravenously is summarized in Table2. More than 99.5 per cent of the thioTEPAwas destroyed. Although the urine samples werecollected for @4hours, most of the thioTEPA

TABLE 2

that at least 50 per cent of the thioTEPA is absorbed after oral administration but probably notas much as 100 per cent.

The percentage recovery of thioTEPA andTEPA from the urine of dogs receiving 6 mg/kgof thioTEPA orally was in the same range as theurinary values after 3 mg/kg of thioTEPA intravenously (Table @).

Plasma levels and urinary excretion of TEPA after

DOG5(6.0 MG/KG.

THIO-TEPAORALLY)I- - -‘SS S

PERCENTAGE URINARY RECOVERY IN 24 HouRs OF Two.TEPA AND TEPA IN Doos AFFER INTRAVENOUS AD.MINISTRATION OF S MG/KG OR ORAL ADMINISTRATION

OF 6 MG/KG OF Tm0TEPA

. 0 1 2 3 4 5 6

HOURS

CHART 2.—Time-concentration plasma curves for thinTEPA and TEPA in a typical experiment in the dog after theoral administration of 6 mg/kg of thioTEPA.

DOGS IAND 2-———3.0 MG/KG. IV.

4

-j

ccUia-

UiI-which was recovered was found in the urine ob

tamed during the first@ or 3 hours after intravenous administration. A small but significant percentage of the administered thioTEPA was excreted as TEPA by the dog.

Plasma levels and urinary excretiOn of thiOTEPAand TEPA after oral administration of 6 mg/kg ofIhioTEPA.—Plasma levels of thioTEPA andTEPA in a typical experiment are shown in Chart

@.Significant levels of thioTEPA in plasma after

oral administration of 6 mg/kg were found for alonger period of time than after the intravenousinjection of 8 mg/kg of the thioTEPA. The maximal concentrations of TEPA occurred in approximately@ hours. Following the collection of theinitial sample of plasma, the pattern of the plasmalevels of thioTEPA and TEPA after the 6 mg/kgof thioTEPA orally were quantitatively quitesimilar to the plasma curves after 3 mg/kg ofthioTEPA intravenously. This would indicate

HOUR

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CHART &—Time-concentration plasma curves for TEPA intwo dogs after the intravenous injection of S mg/kg of TEPA.

the injection of3.O mg/kg of TEPA intravenously.—The plasma concentrations in two animals aregiven in Chart 3. The values for TEPA were @—4times greater in these animals than the levels ofTEPA after the intravenous administration of thesame dose of thioTEPA. Indeed, the total plasmaconcentration of alkylating agents was muchhigher after TEPA administration than afterthioTEPA administration.

The urinary recovery of TEPA in these animalswas @4per cent and 34 per cent, respectively,

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TISSUE OR FLUID5CoNCawra@TIoN

INPG/GM OR ML

Dog T-3 DogT-4Bile

FatLungSkeletal muscleHeartSpleenBonemarrowtBrain0.5

0.50.30 .30.503.30.80.3

0.40.40.20.704.80.5

No. dogsPretreatmentAv. percentagerecovery3None9.6(8.0—13.0)@220

mg/kg SKF 525A,1 hour prior to thio.

TEPA10

.4(5.7—15.2)2SOmg/kgSKF52SA,

20 mm. prior to thioTEPA8.1(5.9—10.4)

MELLETT AND WOODS—Physiological Disposition of ThiOTEPA and TEPA 5@9

values which represent a threefold increase in recovery of the TEPA as compared with the dogswhich were given thioTEPA.

Tissue distri&zdion of TEPA.—The results oftissue distribution studies after intravenous administration of 3.0 mg/kg of TEPA are summarized in Table 3. From these results it isapparent that the drug had a marked affinity forbone marrow as compared with other tissues (andas compared with thioTEPA in bone marrow).

Plasma levelsand urinary excretiOnof thiOTEPAand TEPA after pretreatment with SKF 5@5A.—The results of studies on plasma levels of TEPA

TABLE 3

TIssuE DISTRIBUTION OF TEPA INDOGS 15 MINUTES AFFER THE INTRAVENOUS ADMINISTRATION OF3 MG/KG OF Tins DRUG

The results of urinary recovery of TEPA afterthioTEPA administration in these animals aresummarized in Table 4.

ModificaEon of thiOTEPA to@ricity in mice byprior administration of ISKF 59@5A.—Thesurvivaltimes of mice after 30 mg/kg of thioTEPA withand without pretreatment with 40 mg/kg of SKF5@5A are shown in Chart 5. It is apparent fromthese data that SKF 5@5A eiihanced the toxicity

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CHART 4.—Averaged time-concentration plasma curves of

TEPA after 3.0 mg/kg of thioTEPA with no pretreatment andwith pretreatment with 20 mg/kg of SKF 525A 1 hour prior tothioTEPA administration, and with 50 mg/kg of SKF 5%5A 20mm. prior to thioTEPA administration.

TABLE 4

AVERAGE PERCENTAGE URINARY RECOVERY OF TEPAFROM FEMALE DoGS IN 24 HouRs WITR AND WITHOUT SEP 525A PRETREATMENT AFTER 3.0 MG/KGTmoTEPA INTRAVENOUSLY

NO PRETREATMENT20 MGM./KGM. SKF 525AI HOUR PRIOR TO THIO-TEPA50 MGM./KGM.SKF 525A - —

@ 20 MINUTES PRtOR TO THIO-TEF@.

TIME (HOURS)

5 Intestine, liver, and kidney gave

variable blanks from animal to animaland therefore gave unreliable figures inthe distribution studies. The recovery ofdrug added to other tissues was 90 ±20pei- cent. Each figure in the table represents the average of duplicate determinations.

t Althoughthe percentage recoveryofthioTEPA added to bone marrowwasunsatisfactory (25—40per cent), two animalsinjected intravenously with S mg/kg ofthioTEPA showedconcentrationsin bonemarrow of 0.8 @tg/gm15 minutes afterdrug administration. (This figure is corrected to 100 per cent recovery value.)

after the administration of thioTEPA with andwithout pretreatment with SKF 5@5A are shownin Chart 4. The curves are each the results ofaveraged duplicate determinations on two dogs.The administration of either 20 mg/kg subcutaneously 1 hour prior to thioTEPA or 50 mg/kg

@0minutes prior to thioTEPA resulted in lowerplasma concentrations of TEPA. There was a significant delay in the appearance of TEPA in theplasma as compared with the untreated animals,but also a slower disappearance of TEPA fromplasma after SKF 5@5A.

a Range is given in parentheses.

of thioTEPA. In the group of animals which received only thioTEPA the survivors were inexcellent condition at the end of @0days andshowed no signs of succumbing to the drug effects.This would also appear to be true of the dog, sincea 100 per cent mortality rate was observed inanimals pretreated with SKF 5@5A vs. 50 per centin untreated animals at 3.0 mg/kg.

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530 Cancer Research Vol. @0,May 1960

DISCUSSIONFrom the foregoing results differences between

the biological stability of thioTEPA and TEPAare apparent. These differences result in part fromthe individual physiocochemical properties of thetwo drugs, which in turn undoubtedly influencethe pathways of drug metabolism in the dog.TEPA is extremely soluble in water, whereasthioTEPA has limited solubility in water. Thelipide solubility is reversed, ThioTEPA beingmore lipide-soluble than TEPA. The stability ofboth drugs in whole blood or plasma (or in variousbuffer mixtures pH 8.4, 7.4, 4.@) is not significantly different. On the other hand, if one comparesthe rate of disappearance of both substances fromdog plasma after the intravenous injection of 3.0mg/kg (Charts 1 and 3), it is apparent that the

@ ‘ 30 MGM./KGMOF THIO-TEPAI I (NOPRETREATMENT).--—.I ‘@40MGM./KGM.OFSKF5254I k 45 MIN.PRIORTO S •‘@‘%@OMGM./KGM.OFTHIO-TEPA

2 4 6 8 0 2 4 (6 8 20

c:@@Ys OF SURVIVAL

CHART 5.—Survival times in mice given 30 mg/kg thio

TEPA with and without pretreatment with 40 mg/kg of SKF525A.

decay curve for TEPA is much slower than thatfor thioTEPA.

On the basis of the amounts of thioTEPA andTEPA recovered in urine after the intravenous administration of each respective substance, it is evident that TEPA (@4—34per cent recovery) is considerably more resistant to metabolic degradationthan is thioTEPA (less than 0.5 per cent recovery). The results of in vitro studies on the attempted recovery of drug added to tissues furthersubstantiate this conclusion. It was not possible torecover thioTEPA adequately (0—30per cent)from most tissue homogenates even under conditions where stability should have been maximal.In contrast, recoveries for TEPA added to manytissue homogenates was satisfactory (90 ± @0percent).

Brodie and Hogben (1) have indicated thatlipide solubiity is a limiting factor in urinaryexcretion of drugs, i.e., that the more highly

lipide-soluble materials are more slowly excretedthan are materials of high water solubility. Theresults with thioTEPA and TEPA would seem toconform to this suggestion.

Craig and Jackson (@) have demonstrated biological stability of TEPA in rats. They have reported that 80—90per cent of P32-labeled TEPAradioactivity is excreted in @4hours. The bulk ofthis material was excreted as unchanged TEPAalong with some inorganic phosphate. In contrastto the findings of Craig and Jackson, Nadkarniet a!. (6, 7) have reported that 60—80per cent of theradioactivity of TEPA-P32 administered to micewas recovered in the urine in @4hours. Of thisamount of radioactivity 80 per cent correspondedto inorganic phosphate, and the remainder was anintermediate between TEPA and its final hydrolytic product. No unchanged TEPA was found.

Our findings more closely parallel those ofCraig and Jackson, although there is a quantitative difference between the manner in whichTEPA is metabolized by the dog and the rat. Onthe basis of the report of Nadkarni and co-workers, one must conclude that the mouse presents aconsiderable degree of species variation for themetabolic conversion of TEPA or that differencesin methodology account for the variation in results.

Murphy and DuBois (5) have shown that theirreversible anticholinesterases of the organicthiophosphate family are inactive or weaklyactive in vitro. They have demonstrated that anenzymatic conversion of the thio-derivative to thecorresponding oxy-compound occurs in vivo andthat it is the oxy-derivative that is highly active.This conversion has been demonstrated to occurin vitro with liver homogenates.

Maller and Heidelberger (4) have studied themetabolism of N-(3-oxapent.amethylene)-C'4-N',N―-diethylenethiophosphoramide (OPSPA) andalso the @32derivative. By carrier technics andchromatographic procedures they have shown thatOPSPA is metabolized in rats and humans tomorpholine, inorganic phosphate, and chiefly toN- (3-oxapentamethylene)- C'4-N',N―-diethylenephosphoramide (MEPA). They concluded thatsince J\IEPA was present in appreciable amountsin blood, tissues, and urine, whereas OPSPA wasnot, it appeared likely that the tumor inhibitionobserved was due to metabolically producedMEPA rather than to the administered OPSPA.The metabolic alterations of thioTEPA would appear to be analogous to the changes occurring withorganic thiophosphate anticholinesterase agentsand OPSPA.

It is not possible to relate the toxicity (or anti

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MELLETT AND WooDs—Physiological Disposi&n of ThIOTEPA and TEPA 531

tumor activity) specifically to thioTEPA orTEPA. In our experiments with normal mice theadministration of SKF 5@2I5Aprior to administration of thioTEPA resulted in an increased toxicityand a shorter survival time when compared with agroup of animals receiving only thioTEPA, mdieating that metabolic blockade or delay of desulfurization increases toxicity.

Murphy and DuBois (5) have also demonstratedthat many factors such as sex, age, etc., are involved in the extent of the conversion of the thiocompounds to the oxy-derivatives. These andother factors may account for the fact that wefound no substantial difference in the urinary output of TEPA in animals receiving thio-TEPA withand without pretreatment with SKF 5@5A (Table4). However, the data on plasma levels in theseanimals (Chart 4) indicate a long delay in theappearance of TEPA in the plasma. It would appear from these data that the effects of SKF 5@25Aare to produce a delay in the metabolism of thioTEPA to TEPA rather than a complete blockade.(This would seem to substantiate the idea oflimited urinary excretion of thioTEPA because oflipide solubility. It is also noteworthy that the recovery of thioTEPA in the urine does not varygreatly from controls in these animals.)

It is of interest that desulfurization also occurswith the thio-barbiturates as shown by Williamsand Shideman (13) and Raventos (10). Whetherthe enzyme systems involved in these variousprocesses of sulfur removal are identical remainsto be demonstrated, but many of them requirediphosphopyridine nucleotide (DPN) and 02.

The results of our studies on the tissue distribution of TEPA in dogs are somewhat at vanance with the findings of Nadkarni et al. (7) inmice and Craig and Jackson (@)in rats. Nadkarniet at. have shown some selective distribution ofradioactivity, after TEPA-P32 administration, inthe gastrointestinal tract, bone, spleen, lung,liver, and kidney. Craig and Jackson, also utilizing P32-labeled TEPA in rats, showed accumulation of radioactivity in kidney, liver, and spleen.In our results the only tissue showing affinity forthe drug was the bone marrow. Very probablythe explanation for the divergence of these findingsis the difference in methodology. Both Nadkarniet al. and Craig and Jackson assayed radioactivityin the tissue. The assaying of radioactivity as suchis not a specific measure of the concentration ofthe drug in the tissue particularly in the light ofmetabolic studies which indicate an extensive conversion of the drug to inorganic phosphate. On theother hand, the fluorescent procedure outlinedherein is a direct measure of alkylating properties

of the drug and provides greater specificity ofestimation of drug. It is also quite possible thatvariation among the species (and dosage) mightaccount for some of the differences in the resultson distribution. Nadkarni ci at. (8) recently reported their findings on the fate of TEPA in thehuman which tend to confirm their earlier studiesin the mouse.

ACKNOWLEDGMENTS

The authors are indebted to Miss Beverly Waterman andMiss Rita Czewski for invaluable technical assistance and toDrs. Bernard Sehacter and Howard Bond, Cancer Chemotherapy National Service Center, for providing supplies ofdrugs.

ADDENDUM

Recently, Craig et at., utilizing Pn@labeled thioTEPA,have shown that the drug is converted to TEPA in the rat, dog,and rabbit, whereas the mouse seems to carry the conversionof the drug to inorganic phosphate. This finding confirms the

observation presented here for the dog (A. W. Craig, B. W.Fox, and H. Jackson, Metabolic Studies of Pn@labelled tnethylene-thiophosphoramide, BiOchem. Pharinacol., 3:42—50,1959).

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1960;20:524-532. Cancer Res   L. B. Mellett and L. A. Woods  TEPA in the DogThe Comparative Physiological Disposition of ThioTEPA and

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