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(CANCER RESEARCH 33, 651-658, April 1973]

Evidence for an Aldehyde Possessing Alkylating Activity as thePrimary Metabolite of Cyclophosphamide1

N.E.SIadek2

Department of Pharmacology1. University of Minnesota, Minneapolis, Minnesota 55455

SUMMARY

Two-dimensional thin-layer chromatography was used toresolve cyclophosphamide metabolites present (a) in blood andurine of cyclophosphamide-treated rats and (b) after incubation of cyclophosphamide with hepatic microsomes, a hepatic9000 X g supernatant fraction, or a model oxygenase system.

Incubation of cyclophosphamide with rat hepatic micro-somes yielded a single metabolite (aldophosphamide) capableof alkylating 4-(/>-nitrobenzyl)pyridine. This metabolite couldbe trapped as the semicarbazone by the addition ofsemicarbazide to the incubation mixture. Aldophosphamidewas unstable to incubation at 37Â°for 44 hr, degrading to

two metabolites capable of alkylating 4-(p-nitrobenzyl)pyri-dine, whereas the semicarbazone derivative was relativelystable under the same conditions. Enzymatic action in thehepatic cytosol converted aldophosphamide to carboxyphos-phamide.

One hr after cyclophosphamide injection, carboxyphos-phamide and aldophosphamide were present in approximatelyequal amounts in blood. Urine collected for 3 hr aftercyclophosphamide injection contained large amounts ofcarboxyphosphamide and small amounts of aldophosphamide.

Aldophosphamide was also produced by mouse microsomesand by a model oxygenase system.

INTRODUCTION

Cyclophosphamide (Chart 1, I) is an antineoplastic agentthought to exert its therapeutic effect via the alkylation ofDNA. However, cyclophosphamide itself exerts minimalalkylating activity, does not exert a cytostatic effect in vitro,and must be activated in vivo to exert its biological effect.Reports from this and other laboratories indicate that theactivation of cyclophosphamide is catalyzed by the samehepatic microsomal mixed-function oxidase system that isfunctional in the oxidative metabolism of many other drugs(5-8,10,13,23-25).

The identity of the active metabolite(s) has not yet beenelucidated, although considerable effort has been expended inthis direction (1-7, 13-19, 21, 22, 26, 28). Bakke et al.(2-4) found 11 resolvable (Sephadex LH-20 column chroma

tography) radioactive compounds in the urine of sheep givenring- and side chain-labeled cyclophosphamide p.o. One ofthese compounds, representing approximately 45% of the totalurinary radioactivity, was identified as carboxyphosphamide(Chart 1, IV). Similarly, Struck et al. (28) isolated andidentified carboxyphosphamide as the major urinary metabolite in dogs given cyclophosphamide i.v. They also reportedthat 25 to 30% of the total radioactivity in human urinefollowing cyclophosphamide administration could be attributed to carboxyphosphamide.

Although carboxyphosphamide appears to be the majorurinary metabolite of cyclophosphamide, it probably is notthe active cytotoxic metabolite since it is noncytotoxic orminimally cytotoxic to human epidermoid cells in vitro (28),to leukemia LI210 cells in vivo (28), or to a tumor implantedon the chorioallantois of chick embryos (21).

Hill et al. (15, 17) reported (a) that the initial metaboliteresulting from hepatic microsomal enzyme action could befurther converted to carboxyphosphamide by an enzyme inthe hepatic cytosol, as well as by purified aldehyde oxidase(EC 1.2.3.1) and a commercial preparation of NAD-linkedaldehyde dehydrogenase (EC 1.2.1.3); (b) that conversion ofthe initial metabolite to carboxyphosphamide could beinhibited by substrates or inhibitors of aldehyde oxidase; (c)that the initial metabolite was highly toxic to humanepidermoid carcinoma No. 2 and leukemia LI 210 cells; and(d) that the combination of cyclophosphamide with aldehydesthat are substrates for aldehyde oxidase is more toxic toleukemia L1210 cells than is cyclophosphamide alone.

A scheme (Chart 1) describing the major pathway ofcyclophosphamide metabolism in which the acid, carboxyphosphamide, is formed through the intermediacy of a

.ring-opened aldehyde, aldophosphamide3 (Chart 1, III), has

been suggested (17, 19, 27, 28) and is strongly supported bythe previously considered data. Moreover, Struck and Hill (27)have synthesized aldophosphamide and have found that it is

'This research was supported by USPHS Grant GM 15477. This isPaper 4 in the series on "Cyclophosphamide Metabolism."

*Research Career Development Awardee of the National CancerInstitute, USPHS (1-K04-CA70383-01).

Received September 15, 1972;accepted December 22, 1972.

3The trivial name, aldophosphamide, has been assigned to compound

III, Chart 1, by Hill et al. (17). The name aldophosphamide is used inthis paper to denote both aldophosphamide (Chart 1, III) and thehemiaminal tautomer of aldophosphamide, 4-hydroxycyclophospha-mide (Chart 1, II). The 2 tautomers presumably exist in equilibrium,but the constant describing this equilibrium is not known. Theequilibrium constant may be such that, whereas III is formed throughthe intermediacy of II, II converts to III so rapidly and completely thatfor all practical purposes it does not exert a biological effect and cannotbe detected. At the other extreme, the equilibrium constant could behighly favorable to II, although this would seem less likely. In anyevent, the investigations described here cannot distinguish between thering-opened tautomer, III, and the cyclized tautomer, II.

APRIL 1973 651

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toxic to leukemia L1210 cells in vivo. However, the presenceof aldophosphamide (or, more generally, of a cyclophosphamide metabolite that is an aldehyde and that possessesalkylating activity) in blood and/or urine following cyclophosphamide administration has yet to be demonstrated. Ourexperiments were undertaken (a) to provide more evidence ofthe formation of such a metabolite by hepatic microsomalmixed-function oxidase action on cyclophosphamide, (b) todetermine whether such a metabolite is present in bloodand/or urine following cyclophosphamide administration, and(c) to determine whether such a metabolite can be formedwith a model oxygenase system. After establishing thepresence of such a metabolite, we performed additionalexperiments to determine its metabolic fate.

MATERIALS AND METHODS

Animalsand Dosage Schedules. MaleHoltzmanrats(230 to270 g; The Holtzman Co., Madison, Wis.) and femaleSwiss-Webstermice (23 to 27 g; Simonsen Laboratories, WhiteBear, Minn.) were used in all experiments and were fed astandard chow diet ad libitum.

PhÃ©nobarbitalsodium (Merck and Co., Inc., Rahway, N. J.),40 mg/kg, was injected i.p. in a volume of about 0.5 ml of0.9% NaCl solution every 24 hr (into rats) or every 12 hr (intomice) for 5 days before sacrifice or injection of cyclophosphamide (a gift from Dr. W. A. Zygmunt, Mead-JohnsonResearch Center, Evansville, Ind.).

Tissue Preparation, Hepatic 9000 X g Supernatant andMicrosomal Cyclophosphamide Metabolism, and Preparationof Metabolites for 2-Dimensional TLC.4 All animals were

pretreated with phÃ©nobarbitaland sacrificed between 8 and 9a.m. Hepatic 9000 X g supernatant and microsomal fractionswere obtained as previously described (23).

The incubation mixture was as described previously (23)except that (a) the concentration of cyclophosphamide was2.76 mM; (b) a 9000 X g supernatant fraction or a microsomalfraction obtained from 500 mg wet liver was used perincubation flask; (c) the mixture was incubated for 20 min; (d)semicarbazide hydrochloride, 37.5 /umoles/reaction flask, wasused in some of the experiments; and (e) nicotinamide, 4.0mM, and NAD+, 2.0 mM, were added when the 9000 X^

supernatant fraction was used. After incubation, the reactionwas stopped and deproteinized by the addition of 2 ml of a5.5% ZnSO4-7H2O solution to the reaction mixture, followedby the addition of 2 ml of a 4.5% Ba(OH)2-8H2O solution.The mixture was transferred to glass centrifuge tubes andcentrifuged at 9000 Xgmax for 15 min in a refrigerated (4Â°)

Lourdes Model LRA centrifuge (Rotor 9RA). Appropriatealiquots of the resultant clear supernatant fraction wereremoved and (a) assayed for alkylating activity as describedpreviously (23), (b) assayed for the presence of aldehydes, (c)

'The abbreviations used are: TLC, thin-layer chromatography; NBP,5% 4-(p-nitrobenzyl)pyridine in acetone; nor-HN2, bis(2-chloroethyl)-amine hydrochloride.

652 CANCER RESEARCH VOL. 33



Cyclophosphamide Metabolism

spotted for 2-dimensional TLC, and (d) incubated at 37Â°for

44 hr with shaking in air, after which assays a through c wererepeated.

Metabolism of Cyclophosphamide with a Model OxygenaseSystem and Preparation of Metabolites for 2-DimensionalTLC. The model oxygenase system used in these experimentswas essentially that described by Udenfriend et al. (29). Thecomplete reaction mixture contained cyclophosphamide, 20AmÃ³les; ascorbic acid, 100 jumÃ³les; EDTA disodium, 25jumÃ³les;semicarbazide hydrochloride, 45 timÃ³les;and ferroussulfate, 15 /umoles (omitted in control flasks), in 0.1 M sodiumacetate buffer, pH 5.8. The total volume of the reactionmixture was 6 ml. In some experiments, semicarbazidehydrochloride was omitted. The reaction was started by theaddition of ferrous sulfate. Additional ascorbic acid (10 mg ofdry powder) was added to each flask 45 min after theinitiation of incubation. After the mixtures were incubatedwith shaking (120 oscillations/min) at 37Â°in open 25-ml

Erlenmeyer flasks in a Dubnoff metabolic shaker for 2 hr, thereaction flasks were placed in ice water. Without furthertreatment, appropriate aliquots were removed, assayed foralkylating activity, and spotted for 2-dimensional TLC.

Preparation of Cyclophosphamide Metabolites Obtainedfrom Rat Blood for 2-Dimensional TLC. Phenobarbital-pretreated rats were anesthetized with ether, a dorsal incisionwas made, and the blood supply to both kidneys was occludedby ligation. One hr later, 2.6 ml of 0.9% NaCl solution(control rats) or 100 mg of cyclophosphamide dissolved in 2.6ml of 0.9% NaCl solution were injected i.p. One hr afterinjection, the abdominal cavities were opened under etheranesthesia, and 5-ml blood samples were removed from theaortic artery. The blood samples were placed into glasscentrifuge tubes containing 10 ml of 0.01 M phosphate buffer,pH 7.4. Five ml of a 5.5% ZnSO4 -7H2O solution were added,followed by 5 ml of a 4.5% Ba(OH)2 -8H2O solution, and the

mixture was centrifuged as before. Appropriate aliquots of theresultant clear supernatant fraction were removed, assayed foralkylating activity and for the presence of aldehydes, andspotted for 2-dimensional TLC.

Preparation of Cyclophosphamide Metabolites Obtainedfrom Rat Urine for 2-Dimensional TLC. Phenobarbital-pre-treated rats were given injections i.p. of 2.6 ml of 0.9% NaClsolution (control rats) or of 100 mg of cyclophosphamidedissolved in 2.6 ml of 0.9% NaCl solution. The rats wereplaced in restraining cages and 1-ml urine samples werecollected. After dilution with water, the samples werecentrifuged as before for removal of any solids present, andappropriate aliquots of the resultant clear supernatant fractions were removed, assayed for alkylating activity and for thepresence of aldehydes, and spotted for 2-dimensional TLC.

Aldehyde Assay. An adaptation of the method of Dickinsonand Jacobsen (11) was used to estimate relative aldehydecontent. Purpald (4-amino-3-hydrazino-5-mercapto-l,2,4-tri-azole) (Aldrich Chemical Co., Inc., Milwaukee, Wis.), 100mg/ml 1.0 N sodium hydroxide solution, was added to anequal volume of test material. After 3 hr, during which time itwas thoroughly aerated, the mixture was placed in a cuvetwith a light path of 1 cm, and readings were made at 530 nmin a Gilford 2400 spectrophotometer. Appropriate blanks were

determined and subtracted in all experiments. As expected,cyclophosphamide, 4-ketocyclophosphamide and carboxy-phosphamide (4-Ketocyclophosphamide and carboxyphospha-mide were gifts of Dr. R. F. Struck, Southern ResearchInstitute, Birmingham, Ala.) did not give a positive test withthis assay. Results are expressed as formaldehyde equivalentsper ml of undiluted test material. The lower limit of sensitivitywas about 1 nmole of formaldehyde per ml.

Two-Dimensional TLC. From 5 to 100 ,ul of the testsolution were applied to 1 corner of a flexible, 20- x 20-cm,200-/um-thick, poly-(ethylene terephthalate) sheet coveredwith a 100-jum-thick layer of silica gel (Eastman Kodak Co.,Rochester, N. Y.), and the chromatogram was developed in 2dimensions. The solvent systems finally adopted for routineuse were 10% methanol in chloroform, for development in the1st dimension, and 1-butanol:glacial acetic acid:water (3:1:1),for development in the 2nd dimension. After development, thechromatograms were sprayed with a mixture of 1 part 0.2 Macetate buffer, pH 4.0, and 4 parts NBP reagent. Thechromatograms were placed in an oven (180â€”200Â°F) for 15

min, after which they were sprayed with 1.0 N NaOH solution.Compounds capable of alkylating the NBP reagent appeared asbrilliant violet-blue spots; this color was not stable to light anddisappeared within minutes, making anything other thanrelative quantitation impossible. The limit of sensitivity, withnor-HN2 as a standard, was 0.003 /umole.

RESULTS

Experiments were designed to quantitate alkylating activityand aldehyde concentration after cyclophosphamide activationin vivo and in vitro (Table 1) and to estimate relative amountsof individual metabolites, capable of alkylating NBP, with theaid of 2-dimensional TLC (Table 2).

After incubation of cyclophosphamide with microsomesobtained from the livers of male rats, the incubation mixturecontained 0.47 jumÃ³leof nor-HN2 equivalents and 0.10 /Â¿moleof formaldehyde equivalents per ml (Table 1). Two-dimensional TLC of the incubation mixture revealed the presence of2 blue-violet spots (Table 2, A and L). Spot A is probablycyclophosphamide and, possibly, 4-ketocyclophosphamide(Chart 1, V), since the authentic compounds migrate to thisposition in the system used. Thus, only 1 major metabolitecapable of reacting with the NBP reagent was present. Thisobservation is in agreement with those of Connors et al. (9),who reported the formation of a single, chloroform-extracta-ble radioactive metabolite after incubating cyclophosphamide-32? with rat hepatic microsomes. This metabolite was

relatively unstable and highly cytotoxic when bioassayed in aWalker 256 tumor cell system. Metabolite L is not carboxy-phosphamide, because carboxyphosphamide is not formed bymicrosomal enzymes (17), and authentic carboxyphosphamidemigrates with RF's identical to those of Metabolite K in the

system used.Cyclophosphamide was next incubated with rat hepatic

microsomes in the presence of semicarbazide, the rationalebeing that, since the previous experiment suggested theformation of an aldehydic metabolite, semicarbazide might

APRIL 1973 653



N. E. Sladek

Table 1Alkylating activity and aldehyde concentration following cyclophosphamide activation

in vitro and in vivoAssays for alkylating activity and aldehyde content were conducted as described in "Materials and

Methods."

Source of cyclophosphamideSemimetabolitescarbazideMouse

microsomalincubationmixture+Mouse

microsomalincubationmixture,ageddRat

microsomalincubationmixture+Rat

microsomalincubationmixture,agedd+Rat

9000 X gsupernatantincubationmixture+Rat

bloodRaturineModel

oxygenase system+Alkylating

activity"(nmoles/ml)0.78

Â±0.03C0.83Â±0.020.45+0.030.47+0.010.44

Â±0.010.28+0.010.41

Â±0.060.73Â±0.050.67Â±0.051.14Â±

0.0183.28Â±8.500.32

Â±0.020.27Â±0.02Aldehyde

content6(Mmoles/ml)0.20

Â±0.010.11+0.010.21Â±0.010.10

+0.030.05Â±0.020.10

Â±0.020.01Â±0.010.09Â±0.010.07Â±0.010.07Â±0.016.44Â±0.55eeAldehydecontent0

:alkylating

activity"%2613472111362121068

Â°Expressed as AmÃ³lesof nor-HN2 equivalents per ml of incubation mixture, blood, or urine.

Expressed as AmÃ³lesof formaldehyde equivalents per ml of incubation mixture, blood, or urine.c Mean Â±S.E. of 3 animals or experiments.d Metabolites were prepared and then incubated at 37Â°,with shaking, for 44 nr as described in

"Materials and Methods."e Could not determine accurately.

trap it to form the corresponding semicarbazone (Chart 1, VI).Following incubation, the mixture contained 0.44 jumÃ³leofnor-HN2 equivalents and 0.05 //mole of formaldehydeequivalents per ml (Table 1). The amount of nor-HN2equivalents present after incubation in the presence ofsemicarbazide is essentially the same as that present afterincubation in the absence of semicarbazide, suggesting thatsemicarbazide does not inhibit hepatic microsomal mixed-function oxidase activity and that any semicarbazone derivative of the NBP-reactive metabolite which might be formed hasan extinction coefficient in the NBP assay similar or identicalto that of the parent compound. Alternatively, it may meanthat less (more) of the metabolite is formed and that thesemicarbazone derivative fortuitously has an extinction coefficient that is proportionately greater (lower), such that thenet effect is to give the same amount of nor-HN2 equivalents.The amount of formaldehyde equivalents present was only50% of that present after incubation in the absence ofsemicarbazide, indicating that some of the aldehydic metabolite was indeed trapped by semicarbazide as the semicarbazone. Further proof of this was provided by 2-dimensionalTLC of the incubation mixture, which revealed the appearanceof a major new metabolite, H, and the almost completedisappearance of Metabolite L (Table 2), indicating thatMetabolite L is indeed an aldehyde (because much of itappears to be trapped by semicarbazide) and that it is capableof alkylation (because it reacts with the NBP reagent). At thetime, we could not determine whether Metabolite L wasaldophosphamide, but the data were certainly consistent withthis idea. The argument could be made that, since semi

carbazide is present during incubation of cyclophosphamidewith microsomes, it may block the synthesis of Metabolite Land open up a new pathway so that Metabolite H could beformed. This was not the case, as revealed by the followingexperiment. Cyclophosphamide was incubated with micro-somes in the absence of semicarbazide. After incubation, themixture was deproteinized and then incubated with semicarbazide. The incubation mixture was submitted to 2-dimensional TLC before and after its incubation with semicarbazide.Metabolites L and H were the major metabolites before andafter incubation with semicarbazide, respectively (data notpresented).

To determine whether nonenzymatic degradation of metabolite L occurs, we incubated cyclophosphamide with rathepatic microsomes in the absence of semicarbazide. Theincubation mixture was then deproteinized, after which it wasincubated for an additional 44 hr, with shaking, at 37Â°.

Approximately 40% of the nor-HN2 equivalents were lostunder these conditions, but no loss of formaldehyde equivalents occurred (Table 1). Two-dimensional TLC revealed thepresence of 2 new metabolites, J and N, and the disappearanceof Metabolite L (Table 2). In addition, metabolites that arealdehydes but that do not possess alkylating activity may alsobe formed, e.g., acrolein (1). Such metabolites would not bedetected on the TLC plate with the NBP assay. Nonenzymaticformation of carboxyphosphamide was not detected in theseexperiments. Further conclusions based on these experimentsare difficult to make because the relative extinction coefficients of Metabolites J, N, and L in the NBP and aldehydeassays are unknown.




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APRIL 1973 655



N. E. Sladek

To determine whether the semicarbazone derivative, H, wasas sensitive to nonenzymatic degradation as Metabolite L, weincubated cyclophosphamide with rat hepatic microsomes inthe presence of semicarbazide. As before, the incubationmixture was deproteinized, after which it was incubated for anadditional 44 hr, with shaking, at 37Â°.Less than 10% of the

nor-HN2 equivalents were lost under these conditions, andconversion of aldehyde to semicarbazone was nearly complete,since little or no aldehyde was detectable (Table 1). In theTLC experiments, Metabolite L was not detectable, MetaboliteJ was only barely detectable, Metabolite N gave intermediatecolor intensity, and Metabolite H gave by far the most intensespot (Table 2). Thus, the semicarbazone derivative, H, ofMetabolite L appears to be more stable to incubation at 37Â°

than Metabolite L itself.All of the experiments thus far described were repeated

with microsomes obtained from the livers of female mice tosee whether any species differences in the metabolism ofcyclophosphamide could be detected. The data obtainedshowed some quantitative differences but little or noqualitative differences (Tables 1 and 2). As has been reportedpreviously (23), mouse hepatic microsomes metabolize cyclophosphamide at a faster rate than do rat hepatic microsomes(Table 1). An additional metabolite, D, was present in barelydetectable amounts when freshly prepared metabolites werechromatographed. Hill et al. (17) were also able to detect 2metabolites after the incubation of cyclophosphamide withmouse hepatic microsomes. Another barely detectable metabolite, C, was observed when these metabolites were "aged"

(Table 2). Metabolites C and D may not be detectable whenrat hepatic microsomes are used because of the lower rate ofcyclophosphamide metabolism observed with these preparations.

Incubation of cyclophosphamide with rat hepatic 9000 X gsupernatant fraction in the absence of semicarbazide resultedin the production of 0.73 jumÃ³leof nor-HN2 equivalents and0.09 Â¿miÃ³leof formaldehyde equivalents per ml of incubationmixture (Table 1). Thus, while the use of 9000 X gsupernatant fraction resulted in the production of morenor-HN2 equivalents relative to that obtained with a comparable amount of microsomes (a characteristic of hepaticmicrosomal mixed-function oxidation), the ratio, Â¿/molesofformaldehyde equivalents/jumole of nor-HN2 equivalent, wasmuch less (0.12 versus 0.21) when 9000 X g supernatantfraction was used as the source of enzyme. A probableexplanation is that microsomal mixed-function oxidase activity results in the oxidation of cyclophosphamide to analdehyde and that this aldehyde is further oxidized to thecorresponding acid by aldehyde oxidase and/or NAD-linkedaldehyde dehydrogenase ; the latter enzymes are known to bepresent in hepatic cytosol but apparently are not present inmicrosomes (12, 17). Such a pathway has been proposed byseveral investigators (17, 19, 27, 28), and Hill et al. (17), afterhaving identified the end product of aldehyde oxidase actionas carboxyphosphamide, have suggested that the aldehydicmetabolite is aldophosphamide. If, in our experiments,Metabolite L was indeed aldophosphamide, as was suggestedby its aldehydic nature coupled with its ability to alkylate theNBP reagent, incubation of cyclophosphamide with 9000 X g

supernatant fraction should have resulted in the formation ofMetabolite L, followed by partial or total conversion(depending on the kinetic constants of the 2 reactions) ofMetabolite.L to carboxyphosphamide (Metabolite K). Suchwere the results obtained. Two-dimensional TLC of metabolites obtained by incubating cyclophosphamide with 9000X g supernatant fraction for 20 min revealed the presence of 2metabolites, K and L (Table 2). Incubation for longer periodsof time, e.g., 1 hr, resulted in greater quantities of MetaboliteK and diminished quantities of Metabolite L (data notpresented). Incubation of cyclophosphamide with rat hepaticmicrosomes under otherwise identical conditions (i.e., nicotin-amide and NAD* were included in the incubation mixture)

resulted in the production of Metabolite L only (data notpresented). No metabolites of cyclophosphamide could bedetected after the incubation of cyclophosphamide with a105,000 X g supernatant fraction. Thus it would appear thatMetabolites K and L are indeed carboxyphosphamide andaldophosphamide, respectively.

Incubation of cyclophosphamide with rat hepatic 9000 X gsupernatant fraction in the presence of semicarbazide yielded0.67 RimÃ³le of nor-HN2 equivalents and 0.07 .umole offormaldehyde equivalents per ml of incubation mixture (Table1). Two-dimensional TLC revealed that the major metabolitewas Metabolite H, presumed to be the semicarbazide derivativeof aldophosphamide. Small amounts of Metabolites L and K,presumed to be aldophosphamide and carboxyphosphamide,respectively, were also detected (Table 2). These observationssuggest that semicarbazide traps most of the aldophosphamideas the semicarbazone, preventing its conversion to carboxyphosphamide.

Experiments were next designed to determine whetheraldophosphamide and/or carboxyphosphamide could be detected in blood and urine following cyclophosphamideadministration.

Cyclophosphamide, 400 mg/kg, was injected i.p. into malerats 1 hr after renal occlusion. One hr after cyclophosphamideinjection, the blood contained 1.14 Â¿/moles of nor-HN2equivalents and 0.07 /umole of formaldehyde equivalents perml (Table 1). Two-dimensional TLC revealed the presence ofMetabolites K and L (Table 2). Of the 2, Metabolite K(carboxyphosphamide) was usually present in slightly greaterquantities than Metabolite L (aldophosphamide). The incubation of deproteinized rat blood (obtained from cyclophos-phamide-treated male rats) with semicarbazide consistentlyresulted in the formation of small amounts of Metabolite H(aldophosphamide semicarbazone) (data not presented).

In other experiments, cyclophosphamide, 400 mg/kg, wasinjected i.p. into male rats and 1 ml of urine was collected(collection period, about 3 hr). The collected urine contained83.28 Â¿/molesof nor-HN2 equivalents and 6.44 Â¿/molesofformaldehyde equivalents per ml (Table 1). Two-dimensionalTLC revealed (a) that Metabolite K (carboxyphosphamide)was the major metabolite present, (b) that Metabolite L(aldophosphamide) was usually present in only relatively smallquantities, and (c) that several metabolites were present, inrelatively small quantities, that had not been detected in bloodor after in vitro cyclophosphamide metabolism (Table 2). Theincubation with semicarbazide of urine obtained from cyclo-




Cyclophosphamide Metabolism

phosphamide treated male rats, consistently resulted in theformation of relatively small amounts of Metabolite H(aldophosphamide semicaibazone) (data not presented).

We incubated cyclophosphamide in a model oxygenasesystem in an attempt to simulate hepatic microsomalmixed-function oxidase action. In the absence of semicarba-zide, 2 metabolites, B and L, present in approximately equalamounts, were detected following incubation and 2-dimen-sional TLC (Table 2). When the incubation mixture containedsemicarbazide, Metabolite B could not be detected, MetaboliteL (aldophosphamide) was present in relatively small quantities,and the major metabolite was H (aldophosphamide semicarbazone) (Table 2). Thus the model oxygenase system metabolized cyclophosphamide much as did the hepatic microsomalmixed-function oxidase system.

DISCUSSION

In these experiments, cyclophosphamide metabolites capable of alkylation were quantitated by their ability to alkylateNBP spontaneously. nor-HN2 was used as a standard, and allconcentrations of alkylating activity are expressed in terms ofnor-HN2 equivalents. This assay does not reveal the number orrelative amounts of metabolites formed that are capable ofalkylation. Furthermore, these metabolites may possess extinction coefficients different from that of nor-HN2 and fromeach other. Thus, the estimates of alkylating activity reportedherein are more meaningful on a relative basis and may besomewhat in error on an absolute basis. On the other hand, itis probable that these metabolites have extinction coefficientsquite similar to that of nor-HN2 and to each other, e.g., theextinction coefficient for HN2 (nitrogen mustard), determinedby the assay procedure described, is 85% of that for nor-HN2(N. E. Sladek, unpublished observations).

Aldehyde concentrations are expressed in terms of formaldehyde equivalents. Again, this assay cannot determine thenumber or relative amounts of metabolites with aldehydicfunctional groups that are formed, and the estimates ofaldehyde concentration reported herein are probably moremeaningful on a relative basis than on an absolute basis, sincethe extinction coefficient of the aldehyde(s) measured neednot be the same as that of formaldehyde or of each other (ifmore than 1 aldehyde is present), e.g., the extinctioncoefficient of acrolein in the aldehyde assay used is only 0.06that of formaldehyde.

Although the various ratios, AmÃ³les of formaldehydeequivalents:jumole of nor-HN2 equivalent, obtained in thepresent experiments have relative significance, they may nothave significance in an absolute sense, /.e., a ratio of 0.2 shouldnot be construed to mean that 20% of the metabolites withalkylating activity are also aldehydes, although this could bethe case.

Two-dimensional TLC was used to resolve cyclophosphamide and its metabolites. Cyclophosphamide and itsmetabolites were detected by the NBP test, which is specificfor compounds capable of alkylating NBP. While this assay isuseful for qualitative testing, difficulties are encountered whenquantitation is attempted, since the color complex formed isnot stable and the extinction coefficient of each of themetabolites capable of alkylation is not known but need not

be the same as described above. For these reasons, theamounts of NBP-detectable cyclophosphamide metabolitesresolved by 2-dimensional TLC were only crudely andrelatively approximated. Also, any metabolites that may beformed but that are incapable of reacting with NBP reagentwould not have been detected.

These experiments, together with evidence from otherlaboratories (17, 19, 27, 28), strongly support the conclusionthat the scheme presented in Chart 1 is descriptive of themajor pathway of cyclophosphamide metabolism. Thus, itwould appear that activation of cyclophosphamide (to acytotoxic agent) occurs when it is oxidized to aldophosphamide by a mixed-function oxidase of the hepatic endoplasmicreticulum, and that aldophosphamide is inactivated (as acytotoxic agent) by aldehyde oxidase and/or NAD-linkedaldehyde dehydrogenase, which oxidize(s) aldophosphamideto carboxyphosphamide. If correctly interpreted, our experiments predict that cytotoxic activity per unit nor-NH2equivalent should be greatest after the incubation of cyclophosphamide with hepatic microsomes, intermediate in blood(after cyclophosphamide administration), and lowest in urine(after cyclophosphamide administration). Experimentationconfirmed this prediction (manuscript in preparation).

Exception to the proposed pathway has been taken byNorpoth et al. (21), who electrophoretically compared asynthetic specimen of carboxyphosphamide with the majorurinary metabolite obtained from rats administered cyclophosphamide, and concluded that the 2 were not identical. Theseinvestigators postulated that the major cyclophosphamidemetabolite might result from enzymatic O-dealkylation ratherthan 7V-dealkylation, although positive evidence of O-dealkyla-tion was not reported. Moreover, Struck et al. (28) investigated the aforementioned discrepancy extensively, in part, byrepeating the experiments of Norpoth et al. (21), andconcluded that carboxyphosphamide was indeed the majorurinary metabolite of cyclophosphamide.

Alarcon and Meienhofer (1) have demonstrated thatmicrosomal oxidation of cyclophosphamide generates acroleinand have suggested that this cytotoxic aldehyde mightparticipate in the antitumor activity of cyclophosphamide.These findings are not inconsistent with the scheme presentedhere, since aldophosphamide could conceivably serve as theintermediate in the enzymatic or nonenzymatic production ofacrolein. The mechanism by which cyclophosphamide exertsits cytotoxic effect has yet to be conclusively demonstratedand, conceivably, acrolein could play a major role. However,the experiments of Lane and Yancey (20), who developed aline of leukemia LI210 cells resistant to cyclophosphamidecytotoxicity and found these cells to be resistant to otheralkylating agents but sensitive to antimetabolites, suggest thatthe major mechanism of cyclophosphamide cytotoxicityinvolves alkylation. Also, Struck and Hill (27) reportedevidence that aldophosphamide itself could account for theantitumor activity of cyclophosphamide (although the possibility that aldophosphamide is converted to acrolein, whichthen exerts the cytotoxic effect, cannot be excluded).Evidence that aldophosphamide could be nonenzymaticallydegraded at 37Â°was provided by our experiments although the

breakdown products were not identified.The model oxygenase system used in these experiments

APRIL 1973 657



N. E. Sladek

appears to yield aldophosphamide and may prove to be usefulin generating large quantities of this compound. However, oneof the problems with this approach in our laboratory has beenthat, at maximum, only about 8% of cyclophosphamide isconverted to metabolite, and not all of the metaboliteproduced is aldophosphamide. The relative instability ofaldophosphamide may make attempts to isolate it (after invitro synthesis or after cyclophosphamide injection) insufficient quantities for chemical identification, extremelydifficult. On the other hand, the semicarbazone derivative ofaldophosphamide appears to be much more stable and mayfacilitate attempts at positive chemical identification of whatappears to be aldophosphamide. Such an approach is currentlybeing undertaken in this laboratory.

ACKNOWLEDGMENTS

The author gratefully acknowledges the technical assistance of MissBarbara Carpenter.

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1973;33:651-658. Cancer Res N. E. Sladek Primary Metabolite of CyclophosphamideEvidence for an Aldehyde Possessing Alkylating Activity as the

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