Cancer Treatment Reviews (1983) I0 (Supplement A), t7-24

2 - C h l o r o a c e t a l d e h y d e : a m e t a b o l i t e o f c y c l o p l i o s p h a m l d e i n t h e r a t

I. C. S h a w , M . I. G r a h a m a n d A. E. M . M c L e a n

Laborato 9, of Toxicolo~,, Department of Clinical Pharmacologr, Universi(v College London, London WC1, U.K'.

I n t r o d u c t i o n

T h e metabol i sm of cyc lophosphamide ( C P ) t has been extensively s tudied and a general metabo l ic p a t h w a y p roposed (2) (Figure 1). In this p a t h w a y N-dech lo roe thy la t i on ~Jf c y c l o p h o s p h a m i d e to yield d e c h l o r o e t h y l - C P is suggested as a l~onenzymic react ion, fol lowing initial hydroxy la t ion , and resul t ing in the l ibera t ion of ch loroace t - a ldehyde. T h e format ion o fdech lo roe thy l -CP as a metabol i te o f C P has been shown by o the r workers in a var ie ty o f an ima l species, inc lud ing the mouse (8) and the sheep (3). C h l o r o a c e t a l d e h y d e has, however , not been posit ively ident i f ied as a p roduc t o f C P metabo l i sm, a l t h o u g h the p a t h w a y has been pos tu la ted on a n u m b e r of occasions (9).

C h l o r o a c e t a l d e h y d e , like acrole in [ ano the r metabo l i t e o f C P ( I )], is a react ive molecule ; it readi ly forms a t r imer in solut ion (10) and so its isolation is difficult. Since it is very react ive, ch lo roace t a ldehyde is p r o b a b l y direct ly cyto toxic at low concen t ra t ions (6).

Recen t ly there has been m u c h interes t in the use o f C P at very high dose levels (50 m g / k g / d a y on each o f 4 successive days) (7) for the t r e a t m e n t o f small-cell c a r c i n o m a of the b ronchus and ova r i an cancer . At such dose levels the toxic side effects o f CP are a ma j o r const ra int . O n e o f the most i m p o r t a n t toxic side-effects at h igh dose levels is h a e m o r r h a g i c cystitis, t h o u g h t to be caused by the l iberat ion o fac ro le in (5), a me tabo l i t e of ClP wh ich is spon taneous ly fo rmed in the b ladder . T h e inc idence of haema ' .u r ia in pa t ien ts receiving high dose C P has been grea t ly r educed by the i n t roduc t ion of 2 - m e r c a p t o e t h a n e s u l p h o n i c acid (mesna)~ wh ich forms a chemica l c o m p o u n d wi th acrolein in the b l a d d e r (4), so r educ ing the b l a d d e r toxicity.

C h l o r o a c e t a l d e h y d e m a y also have b l adde r associated toxicity, since it is, in cer ta in respects, s t ruc tura l ly ana logous to acrolein; s imiiarly this s t ruc tu ra l ana logy migh t pe rmi t its reac t ion wi th mesna a n d so result in the abol i t ion o f any possible toxicity to the u r i na r y b ladder .

In the p resen t p a p e r ev idence is presented tha t c h l o r o a c e t a t d e h y d e is a u r ina ry

"~ Endoxana~ (Boehringer Ingelheim Hospital Division, U.K. and Eire) and Endoxan@ (Asta-Werke AG, F.R.G.).

~: Uromitexan® (Boehringer Ingelheim Hospital Division, U.K. and Eire) and Holoxan® (Asta-Werke AG, F.R.G.).

0305-7372]83] 10A0017 + 08 $03.00/0 © 1983 Academic Press I no. (London) Limited

17

18 1. C. SHAW E T AL.

~ ' - , f 0

o

CP

( k /P,~H * ~ ' - - 0 "NCHzCHzCt

dechloroethyl - CP

* H + CICH2C--" 0

chloroacetaldehyde

l hydroxy - CP 0 H ~ N ~ p - ,'0

~.... 0 t ~'N(CH2CH zCI) z

keto- CP

HO H / ~ N ~ p I 0 ~ . 0

- H f ~ _ . 0 / ~"N(CHzCH2CI) 2 ~ 0 =CCH2CHzOPN(CHzCHzCI) 2

• l NH 2

aidophosphamide

0

HO ~ PN(CH2CH2CI) z I NH 2

phosphoromide - M

+, HN(CH2CH2CI) 2

o

HOCCH2CH2OPN(CH2CH 2CI) 2 I NH 2

corboxyphosphomide

+ H

CH z = CHC --" 0 0

acrolein

nornitrogen- M

Figure 1. The metabolism ofcyclophosphamide showing the postulated production ofchloroacetaldehyde from CP with the consequettt liberation ofdechloroethyl-CP. Position of the radioactive labels used in this study are shown

by l'*C = O; 3H = *; CP = cyelophosphamide; M = mustard.

2-CHLOROACETALDEHYDE 1N THE RAT 19

metabolite of CP and preliminary experiments on its reaction with other CP metabolites, in the urine, in the presence and absence of mesna are described.

M a t e r i a l s a n d m e t h o d s

Wistar rats of approximate body weight 300 g were used. For metabolic studies the animals were housed in glass metabolic cages to facilitate the separate collection of urine and faeces. Urine was collected over dry ice to minimize evaporation of volatile metabolites and possibly prevent in vitro changes which might result during the urine collection period.

Chemicals

[14C-ring C-4]-CP was purchased from New England Nuclear, W Germany, [3H- chloroethyl]-CP was custom synthesized by Amersham International and nonlabelled CP was a gift from WB Pharmaceuticals Ltd, Braeknell. All other chemicals were ofanalytieal reagent grade.

Animal experiments

Two rats were given (i.v.) a mixture of [14C-ring C-4]-CP, [3H-chloroethyl]-CP and unlabelled CP (total dose = 50 mg/kg; 12/ICi 3H plus 2/tCi ~aC). Urine was collected over dry ice for 18 h.

Analysis of urine

The urine was thawed and an aliquot (0.5 ml) immediately reacted with 2,4-dinitrophenyl- hydrazine (2,4-DNPH) (0.5 ml sat in 2u HC1). The reaction mixture was extracted with diethyl ether, the organic layer separated and evaporated to dryness and the residue dissolved in methanol (0.2 ml) and subjected to thin-layer c]~romatography (tlc).

Thin-layer chromatography

Aliquots (0.1 ml) of the methanolic solution (see above) were applied as bands to Camlab Sil G (Camlab Ltd, Cambridge) tlc plates. The plates were developed over 7 cm in chloroform then divided into 2 mm bands, scraped and the silica extracted with methanol (1 ml). The A350 nm (i.e. the absorption maximum of both 2,4-DNPH and 2,4,- dinitrophenylhydrazone) and the radioactive (14C and 3H separately) content of the methanolic extracts were determined.

Preparation of 2,4-dinitrophel~lhydrazones of acrolein and chloroacetaldehyde

Acrolein 2,4-dinitrophenylhydrazone was prepared by simple reaction ofacrolein (0.5 ml) with 2,4-DNPH (2 ml sat in 2M HCI), the reaction mixture was diethyl ether extracted and the extract treated as previously described (see analysis of urine).

Chloroaeetaldehyde was prepared by boroh ",ride reduction ofchloroacetic acid (2 ml chloroacetic acid (25% aq) was added to I'~aBH4 (50% in 41vl NaOH)) ; the 2,4- dini t rophenylhydrazone derivative was then formed in the ether phase following ex traction

20 1. C. SHAW E T AL.

ofthe reaction mixture with diethyl ether (5 mi) and agitation with 2,4-DNPH ( 1 ml sat in 2M HCI). "Fhe organic phase was removed, evaporated to dryness and treated as before (see analysis of urine).

R e s u l t s

The CP given to the animals was either labelled in the oxazaphosphorine ring with 14C or the chloroethyl sidechains with 3 H , therefore metabolites containing 14C must have originated from the oxazaphosphorine ring, while metabolites containing 3 H m u s t have originated from the chloroethyl sidechain of CP (Figure 1 ).

The tic ofurine samples following treatment with 2,4-DNPH demonstrated tile presence of several compounds (Plate 1), and by the use of authentic chloroacetaldehyde-2,4- dinitrophenylhydrazone and acrolein-2,4-dinitrophenylhydrazone it was possible to demonstrate co-chromatography ofone of the high Rfspots with chloroacetaldehyde 2,4- dinitrophenylhydrazone (RF = 0.88).

Scintillation counting of 2 mm portions of the tlc plates demonstrated the presence of a single large '4C peak at the chromatographic origin (Figure 2 (b)) and several peaks of all radioactivity (Figure 2 (a)). The large 3H peak which ran at Rf0.88 (Figure 2 peak C) co- chromatographed with chloroacetaldehyde-2,4-dinitrophenylhydrazone. This peak was therefore thought to have originated from [aH]-chloroacetaldehyde originally present in the urine sample. The peak constituted some 38% (mean of duplicate values) of the total urinary tritium.

When the urine sample was stood overnight at +4°C with an aliquot of the urine then being treated with 2,4-DNPH followed by extraction and chromatography as before (see analysis of urine), the peak of aH radioactivity at high Rf disappeared (Figure 3). This linding suggests that the peak's component molecule is chemically unstable (or reactive).

E ¢1. C~

5 0 0 -

2 5 0 -

B

0-5 ..... p

(o) C

-"u't-.q ,,r'l n ~ , . ~ I:0 0 0 :5 I:0

Rf Rf

(b)

Figure 2. Levels of (a) aH and (b) 14C along a tic plate of 2,4-DNPH-treated urine from a rat given [aH- chloroethyl]-CP plus [x'*C C-4, ring]-CP. Peak A comprises a number of urinary metabolites containing both t4C and all; peak B was unidentified, but is possibly a reaction product of peak C (see Figure 4). I t contained

predominantly aH radioactivity. Peak C contained only 3H and was identified as chloroacetaldehyde.

Plate 1. The tic plate of 2,4-DNHP-treated urine from a rat given cyclophosphamide. Spot 1 --- unreacted 2,4- DNPH; spot 2 co-ehromatographed with authentic chloroaeetaldehyde-2,4-dinitrophenylhydrazone; spot 3 was

unidentified, but occurred in some control urine samples.

(Facing p. 20)

2 - C H L O R O A C E T A L D E H Y D E IN T H E R A T 21

± ,'9

E Q

16-

14-

I0 -

8 -

2 -

, ' l 3H:14C = 21.0

t I t !

; ,, ,^, • ,, / ':

~q t

0 I0 20 30 4 0 50 TIc section (turn)

i

. . . . . . . . . . t . . . . . . . . . . . .

60 70

- 8

-5 § - I

K

- 3 E

Figure 3. Levels of ~H radioac t iv i ty a long tic plates of 2 , 4 - D N P H - t r c a t e d ur ine from [aH-chlolx~cthyl]-Cl ' plus [ t '*C-C-4 r ing] -CP-doscd rats. T h e ur ine sal~aple was e i ther treated with 2 ,4 -DNPH im m ed ia t e ly ( . . . . ) o r allowed to s t and overn igh t ( + 4"C) ( . . . . . . ). T h e high R f p e a k (C) had a 3H : 14C rat io of 21.0 a n d d i sappeared on

s tand ing . Peak (3 was though t to be ch loroace ta ldehyde .

These observations are in accord with the suggestion that the molecule is chloroacetaldehyde.

Further experiments on the disappearance of the peak of 3H radioactivity from urine samples following their incubation at 37°C showed that on disappearance of the high Rf peak of3H, a lower Rf3H peak appeared. The peaks were quantitated in terms of the total urinary tritium; the high Rf peak of :~H radioactivity present in urine samples prior to standing (Figure 4) constituted 5.9% of the total urinary tritium, following standing tile peak diminished to 2.2% (Figure 4). A new peak of 3H appeared (Figure 4 peak 2) after standing the urine at 37"C, this peak accounted for 11.0% of the total urinary 3H; it contained no 14C. The presence ofmesna in the urine during this experiment had no effect on the phenomenon.

D i s c u s s i o n

Experimental evidence has been provided in support of the hypothesis that chloroacet- aldehyde is a metabolite of CP (Figure 5). The N-dealkylation of CP has been suggested to be spontaneous (2), however, the involvement ofcytochrome P450 in this type of oxidative dealkylation is certainly a possibility. The high chemical reactivity ofchloroacetaldehyde would presumably prevent its existing in the free form in the cell or circulatory system for any length of time, this would particularly be the case when proteins were present, since it is likely that chloroacetaldehyde alkylates proteins and would thus be removed from the biological system. The presence of chloroacetaldehyde in the urine suggests that it is produced at a site near to the bladder, perhaps the kidney. T h e production of chloroacetaldehyde in the kidney might be spontaneous or via the active cytochrome P450

22 1. C. S H A W E T AL.

E

1500-

I000-

500-

11.0%

C 5.9%\. ;,

" * ¢ ' 2 , 2 %

i !' | !

', , % :

, '~'-~m" ~ 7 "i . . . . I0 20 50 40 50 60

TIc section (mm, l

Figure 4. l.evels of 3H along a tic of 2 ,4 -DNPH-t rea ted urine before ( . . . . ) and ariel" ( - - ~ ) the urine has incubated at 37"C for 31) min. Peak C is chloroaceta ldehyde; peak B is tile postulated react ion produc t between chloroacc ta ldehyde and ano ther ur inary metabol i te of CP conta ining only 31|. Peak A comprises a mixture o f the remaining ur inary metabol i tes of CP. T h e values assigned to each peak refer to the propor t ion of the total ur inary

3I-I accounted for by the respective peaks of radioact ivi ty .

H N

f - - ~ .,.,.0 ~ _ 0 zp'~ N - CH2CH ::,CI I

CH2CH 2 CI

cyclophosphomide

CHO

I CH2C I

chloroocetoldehyde H N p 0

~ - - - ~ ~NHCH2CH2CI

monodechloroethyl- cyclophosphomide

Figure 5. T h e l iberat ion of ch loroaceta ldehyde from cyc lophosphamide with the consequent product ion of d ichloroethyl-CP. T h e react ion is possibly cytochrome P450 mediated.

2 - C H L O R O A C E T A L D E H Y D E IN T H E RAT 23

CHO (Q) [

CHECl

~ k HCl

CHO

I CHa

I Cl-- CH 2 CH}~ ~N-- CHzCH2CI

(c)

4- NH (CH2CH2Cl) 2 (b)

Figure 6. The postulated reaction between chloroacetaldehyde (a) and nornitrogen mustard (b) yielding N,N- dichloroethylaminoethylacetatdehyde.

system presen t in the k idney cells. O n the o the r h a n d s p o n t a n e o u s p r o d u c t i o n of c h l o r o a c e t a l d e h y d e , e i ther from CPper se or d ich loroe thyl metabo l i t es o f C P present in the urine, m i g h t occur . I r respect ive o f the m e c h a n i s m of c h l o r o a c e t a l d e h y d e p roduc t ion , the o u t c o m e is the same, name ly tha t ch lo roace ta ldehyde , a toxic, react ive subs tance is released into the u r ina ry b ladder . T h e s i tua t ion is ana logous to the acrolein p rob lem. M e s n a m i g h t therefore be i m p o r t a n t in de toxi fy ing c h l o r o a c e t a l d e h y d e by a m e c h a n i s m s imilar to tha t p roposed for acrolein (4). For this reason expe r imen t s were carr ied out to inves t iga te the pu ta t ive react ion be tween mesna and ch lo roace t a ldehyde . In these expe r imen t s c h t o r o a c e t a l d e h y d e was shown to react not wi th mesna , bu t wi th a n o t h e r me tabo l i t e o f C P present in the urine. In this reac t ion tile or iginal 5 . 9 % of the total u r ina ry 3H presen t in the ch lo roace t a ldehyde peak d imin i shed on incuba t ion , bo th in the presence a n d absence o f m e s n a , to abou t 2 .2%, thus 3.7~/o h a d been conve r t ed to s o m e t h i n g else. At tile same t ime a peak of 3H (con ta in ing no 14C) which cons t i tu t ed 11.0% of tile total u r ina ry t r i t ium a p p e a r e d on the c h r o m a t o g r a m . I f it is a s sumed that the la l ter peak was fo rmed f rom the ch lo roace t a ldehyde lost f rom the h igh R f peak, and from the fact that c h l o r o a c e t a l d e h y d e conta ins a single 3H a tom, this result suggests tha t the ch loroace ta l - d e h y d e reac ted in the ur ine with a me tabo l i t e of CP which theoret ica l ly con t a ined 2 3H a toms a n d no x4C (i.e. 1 ch lo roacc t a ldehyde molecule (one 3H a tom) = 3 .7% plus t d i c h l o r o e t h y l - C P me tabo l i t e ( two 3H a toms) = 7 . 4 ~ ; total 3H con t en t therefore theoret i - cally = 11.1%). Since tile molecule with which c h l o r o a c e t a l d e h y d e appea r s to react has lost the C-4 14C a t o m while re ta in ing bo th 3H a toms it mus t be e i ther p h o s p h o r a m i d e m u s t a r d or n o rn i t r o g e n mus t a rd , the la t ter is the most likely c a n d i d a t e on pure ly chemica l g r o u n d s (F igure 6). These results, a l t h o u g h p re l iminary , p rov ide a m e c h a n i s m tbr the remova l o f c h l o r o a c e t a l d e h y d e , a toxic me tabo l i t e of CP, f rom the ur ine and thus the toxic effects o f c h l o r o a c e t a l d e h y d e u p o n the b l a d d e r m a y not mani fes t themselves.

Summary

C h l o r o a c e t a l d e h y d e has been shown to be a u r i na ry me tabo l i t e o f c y c l o p h o s p h a m i d e in the rat. I t was ident i f ied in the ur ine of rats g iven a mix tu re of [ r ing C-4, 14(2]-

24 I. C. SHAW E T AL.

cyclophosphamide and [chloroethyl-SH]-cyclophosphamide as its 2,4-dinitrophenyl- hydrazone derivative by its 3H label and by its co-chromatography with chemically synthe- sised chloroacetaldehyde.

The reaction of chloroacetaldehyde with another urinary metabolite ot" cyclophos- phamide is discussed and a nornitrogen mustard adduct postulated. The toxicity of chloroacetaldehyde is also discussed particularly in relation to the urinary bladder.

Acknowledgements

The authors are grateful to the Wellcome Trust and WB Pharmaceuticals Ltd for financiM support and to Miss Claire O'Brien for technical assistance.

R e f e r e n c e s

I. Alarcon, R. A. & Meienhofer, .]. (1971) Formation of the cytotoxic aldehyde acrolein during in vivo degradation ofcyclophosphanaide..Vature 223: 250-252.

2. Alhcrts, D. S., Peng, Y. M., Ghcn, H. S. & Struck, R. F. (1978) Effect of phenobarbital on plasma levels of eyclophosphamide and its metabolites ill the mouse. Br. o7. Cancer 38:316-324.

3. Bakke, J. E., Fell, V.J., Fjclstul, C. E. & Thaker, E.J. (I 972) Metabolism ofc)'clophosphamidc by sheep. O7. Agric. Food Chem. 20: 384-388.

4. Brock, N. (1980) Konzeption und wirkmechanismus yon Uromitexan. Beitrage zur Onkologie 5:1 I 1. 5. Cox, P .J . (1979) Gyclophosphamide cystitis--identification of acrolcin a.s the causative agent. Biochem.

PharmacoL 28: 2045-2049. 6. Lawrence, W. H., Dillingham, E. O., Turner, J. E. & Autian, J. (1972) Toxicity profile of ehloroacetal-

dehydc. ,7. Pharm. ScL 61: 19-25. 7. Souhami, R. 1.., Harper, P. G., Linch, D., Trask, G., Goldstone, A. H., Tobias,J., Spiro, S. G , Geddes, D. M.

& Richarcls,J. D. M. (1982) High dose cyclophosphamide with at, tologous marrow transplantation as initial treatment of small cell carcinoma of the bronchus. Cancer Chemother. Pharmacol. 8:31-34.

8. Struck, R. F., Kirk, M. G., Wilt, M. H. & Laster, W. R. (1975) Isolation and mass spectral identification of blood metabolites of cyclophosphamide: evidence for phosphoramidc mustard as the biologically active metabolite. Biomed. Alass Spectrom. 2: 46-52.

9. Whitehousc, M. W., Beck, F.J. & Kaccna, A. (1974) Some (pharmacological) properties of chloroacetat- dehyde, an oxidation product and potential metabolite ofcyctophosphamide. Agents Acts. 4: 34-43.

10. Zief, M. & Schramm, (3. H. (1964) Ghloroethylene oxide. Chem. Ind. 16: 660--661.