[CANCER RESEARCH 32, 658-665, April 1972]

Enzymatic Metabolism of Cyclophosphamide and Nicotine andProduction of a Toxic Cyclophosphamide Metabolite1

Donald L. Hill, W. Russell Laster, Jr., and Robert F. Struck

Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama 35205

SUMMARY

Cyclophosphamide is converted by enzymes of mouse liverinto two metabolites. Production of the first(aldophosphamide), which is uncharged, requires TPNH, isinhibited by CO, and is accomplished predominantly by themicrosomal fraction. With the microsomal enzyme, the Km forCyclophosphamide is 0.5 mM; nicotine, atropine, ephedrine,apomorphine, and cocaine are potent inhibitors.Phénobarbital, cytochrome c, 2-diethylaminoethy 1-2,2-diphenylvalerate, and some steroid hormones also inhibit thereaction. Aldophosphamide is very toxic, as judged byinhibition of clone formation of human epidermoid carcinomaNo. 2 cells and by toxicity to L1210 leukemia cells.

The initial metabolite is further converted to2 -carboxyethyl 7V,A^-bis-(2-chloroethyl)phosphorodiamidate(carboxyphosphamide) by an enzyme in the soluble portion ofthe cell. This enzyme can be replaced by purified aldehydeoxidase (aldehyde: oxygen oxidoreductase, EC 1.2.3.1).Carboxyphosphamide, which has little or no antitumor effect,is much less toxic to clone formation of human epidermoidcarcinoma No. 2 cells and to LI210 cells. Administration ofpyridoxal, which could saturate the endogenous aldehydeoxidase and thus delay the production ofcarboxyphosphamide, in combination with Cyclophosphamideincreases the life-span of mice implanted with LI210 cells.

The metabolic conversion of nicotine to cotinine by liverproceeds in the same manner as Cyclophosphamide oxidation.Nicotine is also oxidized by an amine oxidase to nicotine1'-oxide.

Lung homogenates accomplish the initial oxidation of bothCyclophosphamide and nicotine but do not metabolize theproducts further. Kidney homogenates contain the amineoxidase producing nicotine 1'-oxide. Several other tissues are

not active in the metabolism of either Cyclophosphamide ornicotine.

INTRODUCTION

Cyclophosphamide (2-[bis(2-chloroethyl)amino] tetra-hydro-2//-l,3,2-oxazaphosphorine 2-oxide), a widely usedantitumor agent (10, 20, 36, 37, 40), has little cytotoxic oralkylating activity until it is acted upon by an enzyme of livermicrosomes (4, 6). Following this metabolic step, cytotoxic

1This work was supported by Contract PH43-66-29 withChemotherapy. National Cancer Institute, NIH.

Received September 24, 1971 ¡acceptedDecember 17, 1971.

alkylating materials appear in the serum, urine, and bile oftreated animals (4, 16). The major urinary metabolite ofCyclophosphamide is 2-carboxyethyl 7V,./V-bis(2-chloro-ethyl)phosphorodiamidate (1, 35). Other serum and urinarymetabolites, present in small amounts, are bis-2-chloroethyl-amine, 2-chloroethylaziridine, hydracrylic acid (4, 5, 30), and4-ketocyclophosphamide (2- [bis(2-chloroethyl)amino] tetra-hydro-2/M,3,2-oxazaphosphorin-4-one 2-oxide) (1, 14, 35).None of these metabolities can account for the cytotoxic orantitumor effects of Cyclophosphamide (16,35).

We report here a characterization of the enzymes involvedin conversion of Cyclophosphamide to its major excretoryform. We studied the metabolism of nicotine as a model forthis process. With biological assays, we find that theintermediate in the conversion of Cyclophosphamide to itsmajor metabolite possesses substantial toxicity. A preliminaryreport of some of these results has been given (13).

MATERIALS AND METHODS

Ring-labeled (6-14 C)cyclophosphamide (0.51 //Ci/linole)was a gift from the Mead-Johnson Research Center, Evansville,Ind., and was supplied to us by Dr. G. P. Wheeler. It wasfurther purified by column chromatography onDEAE-Sephadex A-25 and was found to contain no detectableimpurities on paper chromatography or by mass spectrometry.Nicotine-methyl-14 C Ç>98% pure by paper chromatography)

was purchased from Amersham/Searle Corp., Des Plaines, 111.Cotinine was synthesized by the method of Pinner (27) andnicotine 1'-oxide as described by Papadopoulos (26).

Aldehyde dehydrogenase and xanthine oxidase were purchasedfrom Sigma Chemical Corporation, St. Louis, Mo.

Mass spectra were obtained with a Hitachi high-resolution,double-focusing mass spectrometer (RMU-6-D-3).

Alkylating activity was detected by spraying paperchroma tograms with a 1% solution of4-{/?-nitrobenzyl)pyridine in acetone. The chromatograms wereheated in an oven at 100°for 3 min and then sprayed with 3%

KOH in ethyl alcohol. Alkylating substances appeared as bluespots.

Human epidermoid carcinoma No. 2 cells were maintainedin culture and grown as clones as described previously (18).Procedures for antitumor tests with in vivo L1210 cells havebeen described (32).

Microsomes and mitochondria were prepared from the liversof female DBA/2 mice, except as noted. The mice, weighingabout 25 g, were killed by cervical dislocation; the livers were

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Metabolism of Cyclophosphamide and Nicotine

excised, cooled, and homogenized in 3 volumes to 0.25 Msucrose. The homogenate was centrifuged at 900 X g for 5min. The supernatant was removed and centrifuged at 9,000 Xg for 15 min to obtain the mitochondrial fraction. Microsomeswere prepared from the resulting supernatant by centrifugingat 100,000 X g for 45 min and were washed with 0.25 Msucrose.

Aldehyde oxidase2 was purified to near homogeneity from

rabbit liver by the method of Rajagopalan and Handler (29).The specific activity was 2.5 units/mg, with a unit beingdefined as the amount of enzyme producing a change at 300nm of 1 absorbance unit/min at 25° with TV'-methyl-

nicotinamide as substrate. The enzyme preparation had thecharacteristic absorption spectrum reported for aldehydeoxidase (28).

The standard reaction mixture for cyclophosphamideoxidation contained the following: microsomes equivalent to40 mg of liver; TPNH, 1.0 jumóle;sodium-potassium phosphatebuffer (pH 7.3), 6.9 Amóles; cyclophosphamide-I4C, 88

nmoles; and water to a final volume of 175 pi. The reactionwas initiated by addition of the microsomal preparation andwas stopped by streaking a 50-^(1portion on paper strips or byadding 100 p\ of ethyl alcohol prior to streaking on paper.Incubation of the preparation was for 30 min in an open tubeat 37°.Substrate and metabolites were separated by paper

chromatography with isopropyl alcohol: NH3:H20 (80:5:15,by volume) followed by scanning with a Packard 7201radiochromatogram scanner.

For nicotine oxidation, the standard reaction mixturecontained the following: microsomes equivalent to 40 mg ofliver; TPNH, 0.4 /¿mole;sodium-potassium phosphate buffer(pH 7.3), 6.9 jumóles;nicotine-methyl-14 C (0.51 juCi/jitmole),

0.18 /imole; and water to a final volume of 175 ¿d.Incubation was for 15 min at 37°.The reaction was started,stopped, and assayed as for cyclophosphamide-14C.

For the kinetic experiments, the initial velocity of thereactions was determined, and the optimum amount ofmicrosomes was used. The presence of excess microsomesinhibited the reactions. pH 7.3 was optimal for the oxidationof both cyclophosphamide and nicotine.

Protein was determined by the method of Lowry et al. (22).

RESULTS

Production of Metabolites. Cyclophosphamide-14 C was

converted by mouse liver microsomes to a product (MetaboliteA) which remained near the origin (RF 0.02) in the isopropylalcohol :NH3:H20 paper Chromatographie system (Chart 1)and which had alkylating ability as judged by reaction with4-(/j-nitrobenzyl)pyridine. Metabolite A did not migrate onelectrophoresis at pH 6 or 9. The formation of this compoundwas accompanied by the production of Metabolite C (RF0.85), which was always present at 15 to 20% of Metabolite A.Metabolite C probably represented another form of MetaboliteA, for both were converted to Metabolite B (RF 0.57) by anenzyme in the soluble portion of mouse liver (Chart 1).Metabolite B was formed quantitatively at the expense ofMetabolites A and C.

lUUU*

5OOu01000a

5OOuaIt

A1V i j i -.Vsio ;'o 304bB

°K

. ,/x«A|10 ZO 3O *

?f)0.C

f|/

rAM _ ^x , yVIOZO TO i-

500

250

10 20C«ntlm«ter«

30 <IO

'Aldehyde:oxygen oxidoreductase, EC 1.2.3.1.

Chart 1. Paper chromatography of cyclophosphamide and nicotinemetabolites, a, microsomal metabolism of cyclophosphamide-1 4C (D,unchanged substrate); b, metabolism of cyclophosphamide-1 4C in the

presence of microsomes plus 100,000 X g supernatant; c, microsomalmetabolism of nicotine-14 C (f, unchanged substrate); d, metabolism ofnicotine-14 C in the presence of microsomes plus 100,000 X g

supernatant. Standard conditions of assay were used, except that 50 julof 100,000 X g supernatant (equivalent to 16.7 mg of liver) werepresent in b and d.

Mouse liver microsomes converted nicotine-methyl-14 C into

2 metabolites, 1 of which (M) remained near the origin (RF0.02) and another (N) of which appeared as a double peakwith RF values of 0.48 and 0.43. Addition of the supernatantfraction to this system quantitatively converted Metabolite Mto Metabolite O (RF 0.75) but had no effect on Metabolite N(Chart 1).

To saturate the enzyme(s) producing Metabolite M andMetabolite A, 5.7 mM TPNH was required, but for productionof Metabolite N only 2.3 mM TPNH was necessary. In neithercase did the saturation curve follow Michaelis-Menten kinetics.DPNH was 30% as effective as TPNH in the production ofMetabolite N, but was less than 10% as effective as TPNH forproduction of Metabolite A or Metabolite M.

The kinetic values for formation of Metabolites A fromcyclophosphamide and M and N from nicotine are in Table 1.Cyclophosphamide had a K,,, of 0.5 mM, and nicotine was acompetitive inhibitor of the reaction with a K¡of 0.5 mM. Forthe reverse experiment, the production of nicotine MetaboliteM, nicotine had a K^ of 1.9 mM, and cyclophosphamideinhibited with a K¡of 13.6 mM. For production of nicotineMetabolite N, nicotine had a K,,, of 1.3 mM, butcyclophosphamide was not inhibitory. The inhibitionconstants for atropine, ephedrine, apomorphine, cocaine,tremorine, and TV-methylpyrrolidine in each of the reactionsystems are included in Table 1. Each of the inhibitors inTable 1, except ephedrine, contains a 5- or 6-memberedheterocyclic ring, as do nicotine and cyclophosphamide.Ephedrine has an alkyl amino group. The oxidation ofcyclophosphamide was substantially inhibited by testosterone,

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D. L. Hill, W. R. Laster, Jr., and R. F. Struck

Table IKinetic values for inhibition of production of cydophosphamide and nicotine metabolitesThe values are averages of 2 or more separate determinations.

CydophosphamideNicotineAtropineEphedrineApomorphineCocaineTremorinejV-MethylpyrrolidineMetabolites

A +C(0.5)°0.5

Comp2.0Comp2.0Comp0.4Noncomp0.7

Noncomp>10.0>

15.0Metabolite

M(mM)13.6

Comp6(1.9)6.6

Comp>15.01.2

Comp1.7Comp>15.0>15.0Metabolite

N>20.0(1.3)2.2

Comp7.0Comp2.7Comp0.3

Comp10.0Comp0.9

Comp

0 Values in parentheses are Michaelis-Menten constants; other values are inhibition

constants.Comp, competitive inhibition; Noncomp, noncompetitive inhibition.

Table 2Inhibition of cydophosphamide metabolism by phénobarbital,

cytochrome c, and sterolsStandard conditions of assay were used. The ethyl alcohol was used

to dissolve the sterols. The values are averages for 3 separateexperiments, the results of which varied less than 8% in the relativevalues.

AdditionsNonePhénobarbital

(0.85mM)Cytochromec (17MM)Ethyl

alcohol (0.3M)Ethylalcohol (0.3 M) +hydrocortisone(0.17

mM)Ethylalcohol (0.3 M) +prednisolone(0.17

mM)Ethylalcohol (0.3 M) +testosterone(0.17

mM)Metabolites

A + C(nmoles)41.834.013.927.023.021.714.1Relative

values(%)100813065555234

but only slightly inhibited by prednisolone, hydrocortisone, orphénobarbital.Cytochrome c was a potent inhibitor (Table 2).

The oxidation of both cydophosphamide and nicotine wasinhibited by carbon monoxide (Table 3). However, theenzyme(s) producing Metabolites A and M were more sensitivethan that catalyzing the formation of Metabolite N. All ofthese reactions were dependent upon the presence of oxygen.The standard reaction system for cydophosphamide oxidationwas inhibited 50% by 30 ßM2-diethylaminoethyl-2,2-diphenylvalerate. The inhibition did not followMichaelis-Menten kinetics.

Cell fractionation studies showed that 76% of thecyclophosphamide-metabolizing activity present in the 900 Xg supernatant of liver homogenates was in the microsomalfraction. The remainder was in the mitochondria. Washing themitochondria 3 times in 0.25 M sucrose did not reduce theiractivity.

The Effect of Aldehyde Oxidase. The enzyme of thesupernatant fraction responsible for the conversion ofcydophosphamide Metabolites A and C to Metabolite B andfor the conversion of nicotine Metabolite M to Metabolite 0was selectively inhibited by aldehydes and by p-hydrox-vmercuribenzoate, sodium arsenite, and potassium cyanide.

Table 4 shows the inhibition of formation of cydophosphamide Metabolite B by these agents, all of which are eithersubstrates or inhibitors of aldehyde oxidase. None of thecompounds, at these concentrations, had a strong inhibitoryeffect on the production of Metabolite A. In fact, theformation of Metabolite A was stimulated by acetaldehyde at80 mM and by sodium arsenite at 8 mM. These concentrationsgave maximal stimulation. Sodium arsenale at 20 mM had noeffect on either reaction. The supernatant fraction could bereplaced by purified rabbit liver aldehyde oxidase. Thereactions, both for cydophosphamide Metabolites A and Cand nicotine Metabolite M, proceeded as with the 100,000 X gsupernatant and were blocked by the substrates and inhibitorslisted in Table 4. In the presence of DPN*, a commercial

preparation of aldehyde dehydrogenase, accomplished theconversion of Metabolites A and C to B. Commercial xanthineoxidase was not effective.

Identification of Metabolites. The fact that Metabolites Aand M served as substrates for aldehyde oxidase indicated thatthey were either aldehydes or potential aldehydes. Likewise,the products of these reactions were either carboxylic acids orclosely related compounds. Cydophosphamide Metabolite Bwas isolated in quantity from a reaction mixture scaled up100-fold by chromatography on DEAE-Sephadex A-25 (35).

This product was identical to the major urinary metaboliteof dogs and to synthetic 2-carboxyethylAf,7V-bis-2-(chloroethyi)phosphorodiamidate, as determined bycochromatography in 7 solvent systems widely differing incomposition and pH and by electrophoresis in 2 systems.Following chemical methylation, the liver metabolite had amass spectrum identical to those for the previouslycharacterized, methylated urinary metabolite and methylated2-carboxyethyl jV,7V-bis(2-chloroethyl)phosphorodiamidate

(35).

As a product of aldehyde oxidase, nicotine Metabolite 0could have been either y^S-pyridy^^y-methylaminobutyricacid or cotinine, both of which are reported metabolites ofnicotine (17, 24). From a scaled-up reaction mixturecontaining aldehyde oxidase and depleted of nicotine byprolonged incubation, Metabolite 0 could be extracted fromthe alkaline reaction mixture by a single treatment withchloroform. No other metabolite appeared in this fraction in

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Metabolism of Cyclophosphamide and Nicotine

Table 3Inhibition of cyclophosphamide and nicotine oxidation by carbon monoxide

The standard reactions were scaled up 5-fold for this experiment. Incubation wasaccomplished in tubes equipped with a side arm and reservoir. The incubation mixture,complete except for enzyme in the reservoir, was flushed with gas for 10 min. At this time theside arm was sealed and the reaction initiated by tipping in the enzyme. The control values wereas follows: Metabolites A + C, 91 nmoles; Metabolite M, 53 nmoles; Metabolite N, 40 nmoles.When this experiment was repeated, the relative values did not change more than 5%.

Conditions95%

N2 :5%O285%N2:5%O2:10%CO100%N2Metabolites

A +C100

3428Relative

values(%)Metabolite

M100

1422Metabolite

N100

5822

Table 4Inhibition of the reaction converting cyclophosphamide

Metabolite A to Metabolite BA TPNH-generating system replaced TPNH of the standard reaction

mixture. The system consisted of 2.5 jumólesof nicotinamide, 75nmoles of TPN+, and 2.5 jumólesof glucose 6-phosphate. Supernatant(10,000 X g) from 40 mg of liver replaced the microsomal fraction.Incubation was for 60 min. Control values are 8.6 nmoles forMetabolite A and 6.3 nmoles for Metabolite B. The concentrations arethose which gave greatest inhibition.of formation of Metabolite Bwithout strongly affecting the formation of Metabolite A. A number ofconcentrations were tested for each inhibitor.

% of control

InhibitorNoneAcetaldehydeBenzaldehydep-Hydroxymercuri-benzoateSodium

arsenitePotassiumcyanideConcen

tration(mM)8070.2810MetaboliteA1001159494121101MetaboliteB1001722391725

quantity. Mass spectral analysis showed a molecular ion oîm/e176 and characteristic peaks of m/e 78 and 98. The majorpeaks of the spectrum were the same as for an authenticsample of cotinine. The compound was further identified bycochromatography with cotinine in 8 paper Chromatographiesystems.

The appearance of nicotine Metabolite N as a double peakin the isopropanol-NH3-H20 Chromatographie system wassimilar to that reported for nicotine 1'-oxide in an alkaline

solvent system (39). The metabolite was prepared in ascaled-up reaction mixture that contained a low level ofTPNH, for low concentrations of this cofactor retard theformation of Metabolite M. Metabolite N was identified asnicotine 1'-oxide by cochromatography with the synthetic

compound in 8 paper Chromatographie systems.Tissue Distribution of Enzymes Oxidizing

Cyclophosphamide and Nicotine. A survey for the ability tooxidize cyclophosphamide of a number of tissues gave theresults in Table 5. Under conditions of this assay, only liverand lung homogenates had activity for the oxidation ofcyclophosphamide. The lung tissue is not capable of

converting Metabolite A to the carboxylic acid derivative B.Lung homogenates catalyze the formation of Metabolite Mfrom nicotine but are unable to convert M to cotinine. Kidneyhomogenates produce only nicotine 1'-oxide (N) from

nicotine.Biological Evaluation of Cyclophosphamide Metabolite A.

Our tests indicated that Metabolite A was toxic, as judged byinhibition of clone formation of cultured human epidermoidcarcinoma No. 2 cells (Chart 2). This type of experimentinvolved using the microsomal system to generate varyingamounts of Metabolite A and, after Millipore filtration, addingthe preparations to the cloning bottles. Fifty % of MetaboliteA was bound to the microsomes, for half of the total wasremoved by Millipore filtration. The compound remaining insolution inhibited clone formation by 50% at 0.2 jug/ml. On amolar basis, this toxicity is 4-fold greater than nitrogenmustard in this system. Addition of aldehyde oxidase to aportion of the preparation producing 98% inhibition of cloneformation reduced toxicity to the point that clone formationwas 70% of control. This was the toxicity expected for anequivalent amount of 2-carboxyethyl7V,7V-bis(2-chloroethyl)phosphorodiamidate (35).

The method used for testing the toxicity of the initialoxidation products of cyclophosphamide to LI210 leukemiacells involved using the microsomal system to generate theproducts, diluting the preparation to the appropriateconcentration, and adding LI210 cells at a final concentrationof 4 X IO6 cells/ml for an exposure period of 1 hr at 35°.The

cells were washed with 0.9% NaCl solution and then injectedinto BDFi mice at 2 X 10s cells/mouse with 10 animals ineach group. Their life-span was observed. The results ofvarying the reaction components are in Table 6. The initialmetabolites of cyclophosphamide (A + C) increased thelife-span, and removal of these products by aldehyde oxidasereturned the value to that of the controls. Some 30-daysurvivors were found in the groups given injections of cellsexposed to higher levels of the initial metabolites.

Unchanged cyclophosphamide had little or no effect on thecells. Only 15% or less of the total cyclophosphamide used inthese experiments was oxidized. The remaining 85% (40jug/ml) had little effect on the median day of death of theanimals, as shown with the combination of microsomes,cyclophosphamide, and aldehyde oxidase. Exposure of LI 210cells in vitro to 300 /Kg/ml cyclophosphamide for 24 hr results

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D. L. Hill, W.R. Laster, Jr., and R. F. Struck

Table 5Metabolism of cydophosphamide and nicotine by various tissues

Standard reaction conditions were used, except that incubation was for 30 min and that900 X g supernatants of the homogenized tissues replaced the microsomal fraction. The valuesare averages of 2 separate experiments.

Cydophosphamide Nicotine

TissueLiverKidneyLungSpleenMuscleBrainIntestineLI

2 10 solidtumorCa755cultured cellsTotalmetabolites(nmoles/mg

protein)24<

25<2<2<

2<2<2<

2TotalmetabolitesMetabolites

(nmoles/formedmgprotein)A,

B268A

9<2<2<2<2<2<2MetabolitesformedM,

N,ONM,

N

100

ao

ö 60

40

ao

+ Aldehyd« Oxida««

^5Jr 0.3 048MttobolltM t,ug/mi)

Chart 2. Inhibition of clone formation of H.Ep. 2 cells bycydophosphamide Metabolites A + C. The systems to which aldehydeoxidase was added and those from which TPNH was omitted containedno detectable Metabolite A or C. Values on abscissa, ¿ig/mlofmetabolites present in the cloning bottle.

in little or no cell kill (L. J. Wilkoff, personal communication).We determined that cydophosphamide was not altered byaldehyde oxidase.

The reduced toxicity to cloning cells caused by removal ofthe initial oxidation products with aldehyde oxidase and thestrong toxicity of these products of L1210 cells made thealdehyde a strong candidate for the active form ofcydophosphamide. To test this idea we administeredcydophosphamide in combination with pyridoxal, acompound which could possibly saturate aldehyde oxidase andallow an accumulation of the initial oxidation products. Dosesof cydophosphamide at 300 or 200 mg/kg administered i.p.15 min after 380, 255, or 167 mg/kg of pyridoxal to femaleBDF[ mice bearing LI 210 ascites cells moderately increasedthe life-span in each case to an extent greater thancydophosphamide alone. The additional elongation of

life-span ranged up to +154%, with an average increase of 53%.The increase was noted when either IO7 or IO6 L1210 cells

were implanted 24 hr before treatment. Pyridoxal alone hadno effect on life-span.

DISCUSSION

As metabolites of nicotine, cotinine and its ring-openedform [7-(3-pyridyl)--y-methylaminobutyric acid] have beenfound in the urine of dogs and humans (2, 24), Nicotine isconverted by liver microsomes, in the presence of TPNH andÛ2, to a product which reacts with aldehyde reagents (17).Cyanide inhibits its further oxidation. Our results show thataldehyde oxidase, which is inhibited by cyanide, is the enzymeresponsible for the production of cotinine. Nicotine l'-oxide is

present in the urine of cats injected with nicotine (39). Threeother nicotine metabolites, nornicotine, demethylcotinine, andpyridylacetic acid (39), could not be detected in our studiesbecause the labeled carbon atom of nicotine-methyl-14C

would not appear in these products.The results of our studies, along with those of others (17,

24), indicate that nicotine is metabolized to cotinine by thepathway outlined in Chart 3. Nicotine can be oxidized by themixed-function amine oxidase to nicotine l'-oxide or by the

cytochrome P-450-linked oxidase to2-hydroxy-5-(3' -pyridyl)-l-methylpyrrolidine, which

apparently exists primarily as an aldehyde. Aldehyde oxidaseconverts this compound to cotinine. We find no evidence forthe open-ring form of cotinine [7-(3-pyridyl)^y-methylamino-butyric acid], which perhaps is formed in urine as adegradation product.

Norpoth et al. (25) suggest that the metabolism ofcydophosphamide might be similar to that of nicotine, andour results confirm that similarities exist. For bothcompounds, the carbon atom adjacent to a nitrogen of asaturated ring is the site of oxidation by a cytochromeP-450-linked enzyme, and the products are further oxidized tocompounds with carbonyl groups (Charts 3 and 4). Forcydophosphamide, the initial product(4-hydroxycyclophosphamide) apparently exists in

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Table 6Toxicity of the initial metabolites of Cyclophosphamide to L12IO cells

The results of 2 separate experiments are given here. For the 1st experiment, microsomesfrom livers of female BDF, mice were used.

Components of reactionsystem0.9%

NaCl solutioncontrol(noincubation)0.9%

NaCl solutioncontrol(1hrincubation)MicrosomesMicrosomes

+CyclophosphamideMicrosomes+CyclophosphamideMicrosomes+CyclophosphamideMicrosomes+ aldehydeoxidaseMicrosomes+ Cyclophosphamide+aldehyde

oxidaseA

+Cin

cellsuspension0,00,00,0-,3.65.7,--,7.20,00,0MODfor10mice07.5,8.0'

8.0,9.08.0,9.0-,11.514.0,--,15.08.0,9.58.0,9.030-daysurvivors0,00,00,0-,o1,-—

,60,00,0

a MOD, median day of death for those dying.

TPNH. Oz,P450-Linked

Oxidase

Nicotine

TPNH AmineOxidase

ICH,

'Nicotine I-Oxide

(Metabolite fi)

COOH

CH,

HOCH— N o TPNH.Oz, \H—N 0

/ * s \ *u P450-Llnked / v rf°CHjS 2NPX Oxidase .A,V'"\.

,/\CHj—¿�0N(CH2CH2CI)2CyclophosphamideV /\XHjj—ONN(CH2CH2CI)2

(Metabolite C)?»

"N-y-(3-Pyridyl)-y-

MethylamlnobulyricAcidChart 3. Metabolism of nicotine to cotinine and nicotine l'-oxide.

CH,Cotinine

(Metabolite Q)

equilibrium with the open-ring aldehyde [2-formylethyl7V,jV-bis(2-chloroethyl)phosphorodiamidate], for which wepropose the trivial name "aldophosphamide." From the

present data, we cannot decide whether Metabolite A orMetabolite C is the open-ring form; since the aldehyde group ismore likely to bind to components of the reaction mixture orto paper and remain at the origin on chromâtography,Metabolite A may be the aldehyde. Both forms of the initialmetabolite are converted by aldehyde oxidase to2 -carboxyethyl Af,7V-bis(2-chloroethyl)phosphorodiamidate(Metabolite B), for which we propose the trivial name"carboxyphosphamide." This product may spontaneously

cyclize to form 4-ketocyclophosphamide, a metabolitepreviously isolated from urine (1, 14).

ONXCOH

CH2

IÃŽ

\

NHZ 0

\S

x\AldehydeOxidase

CH

\

H2_ 0 N(CH2CHZCI)2Corboxyphosphamide

(Metabolite B)

/\CHj— 0 N(CH.CHXI),

Aldophosphamiae(Metabolite A)?

V/ \0 N(CH2CH2CI)2

4-Ketocyclophosphamide

Chart 4. Metabolism of Cyclophosphamide.

The Kj,, for oxidation of Cyclophosphamide found in ourstudies, 0.5 mM, is similar to that reported for female miceand female rats, 0.68 mM (33) and 0.94 mM (31). That for theenzyme of male rats is higher at 1.4 mM (33) or 7 mM (6). TheVmax of our enzyme, 1.5 jumoles/g/hr, is lower than thatpreviously reported, 9.7 /Limóles/g/hr(33). Kinetic values mayvary with the strain of mice used. Although the kineticsindicate that nicotine is a competitive inhibitor ofCyclophosphamide oxidation, the fact that the Kj for nicotineMetabolite M is not equal to the Km for Cyclophosphamideoxidation means that something other than simple competitiveinhibition is involved.

Inhibition of Cyclophosphamide metabolism by CO hasbeen previously noted (6). Such inhibition implies theinvolvement of cytochrome P-450 in the oxidative process.The lower sensitivity to CO of the reaction converting nicotine

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D. L. Hill, W.R. Laster, Jr., and R. F. Struck

to nicotine 1'-oxide implies that 2 different enzymes are

involved. Further support for the existence of 2 enzymescomes from the fact that there are differing requirements forreduced pyridine nucleotides for the formation of thealdehydes as compared to nicotine l'-oxide. Formation ofnicotine l'-oxide very likely is catalyzed by the

mixed-function amine oxidase in mammalian liver microsomes(15). Tremorine and jV-methylpyrrolidine do not inhibit theformation of cyclophosphamide Metabolites A and C ornicotine Metabolite M. These compounds may be substratesonly for the amine oxidase. The other inhibitors listed in Table1 may be substrates for both the amine oxidase and for theP-450-linked oxidase.

Inhibition of the activation of cyclophosphamide bytestosterone, prednisolone, and cytochrome c has been notedpreviously (6, 12). Testosterone and prednisolone aresubstrates for the microsomal enzyme system utilizingcytochrome P-450 (7, 12), and cytochrome c interferes withthe microsomal oxidation of drugs by interrupting the flow ofelectrons along the electron chain (23).2-Diethylaminoethyl-2,2-diphenylvalerate inhibits a number ofmicrosomal enzymes (9) and is reported as an inhibitor ofcyclophosphamide metabolism in intact animals (4, 11, 38).

Brock and Hohorst (5) report that rat microsomes are 4times as active (expressed as ¿/molesof product/g liver) asmitochondria in metabolizing cyclophosphamide. Cohen andJao (6) find a ratio of 5:1. Our results for the mouse (3:1)are in close agreement with these reports.

Our finding that enzymes capable of catalyzing theoxidation of cyclophosphamide are present in liver and lungtissue is in agreement with results of Brock and Hohorst (4).These investigators also report slight activity in renal cortextissue and in the Jensen tumor but can find no activity inspleen, testis, adrenal cortex, urinary bladder mucosa, musclefibers, Yoshida tumor, or Walker tumor. Brock et al. (3) findthe cyclophosphamide-activating enzyme in human liver andconclude that there are no basic qualitative differences in theoxidative process between man and rat. Sladek (33) cannotfind any activity in thymus, adrenal, kidney, or Walker 256carcinosarcoma cell fractions. In contrast, Kondo andMuragishi (19) report activation in liver and kidney of mice;bone marrow of rabbits; liver, bone marrow, and tumors ofhumans; solid Yoshida sarcoma; and solid Ehrlich carcinoma.Ascites forms of the solid tumors are inactive in their system,which is based on inhibition of growth of HeLa cells in thepresence of tissue slices and cyclophosphamide. Althoughother tissues may metabolize cyclophosphamide to a smallextent, the liver is the major site, for hepatectomy greatlyreduces the amount of alkylating material in the serum (4).

The experiments in Chart 2 and Table 6 show that theinitial products of cyclophosphamide oxidation are toxic totumor cells in in vitro tests. For inhibition of clone formation,the products are more than 300 times as effective ascyclophosphamide, about 20 times as active as4-ketocyclophosphamide, and about 5 times as toxic asMetabolite B (35). The toxicity is greatly reduced whenaldehyde oxidase converts the initial metabolites tocarboxyphosphamide, a compound which has little or noantitumor activity (25, 35). Further, the potentiating

antitumor effect of pyridoxal with cyclophosphamide can beexplained by competition between pyridoxal andaldophosphamide for the inactivating enzyme. Apparently,more aldophosphamide is made available for antitumoractivity.

There are only a few reports regarding the antitumoractivity of metabolites of cyclophosphamide. Tochino et al.(38) state, in abstract form, that each of 2 uncharacterizedmetabolites isolated from rat urine have as much antitumoractivity as nitrogen mustard against Yoshida sarcoma cells invitro. Liss et al. (21) report that exposure to thecyclophosphamide transformation products at 1 Mg/mlcompletely eliminates the transplantability of Yoshida ascitestumor cells. They do not mention how the transformationproducts were prepared or characterized. An indication of acyclophosphamide metabolite with a potent antitumor effectcomes from a report by Connors et al. (8), who show thatincubation of cyclophosphamide with rat liver microsomes anda TPNH-generatingsystem produces a toxic product. Exposureof Walker tumor cells to this material and subsequent injectionof the cells plus microsomes into animals gives an increasedsurvival time as compared to several controls. Sladek (34) hasperformed a similar experiment. We think that the antitumoraffect of cyclophosphamide metabolites in these studies and inours is due to aldophosphamide, either as the free aldehyde oras its cyclized form.

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1972;32:658-665. Cancer Res   Donald L. Hill, W. Russell Laster, Jr. and Robert F. Struck  Production of a Toxic Cyclophosphamide MetaboliteEnzymatic Metabolism of Cyclophosphamide and Nicotine and

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