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STUDIES ON BIOCHEMICAL AND PHARMACOLOGICAL EFFECTS.I • .

OF CYCLOPHOSPHAMIDE AND PHOSPHORODIAMIDIC ACID

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STUDIES ON BIOCHEMICAL AND PHARMACOLOGICAL EFPECTS

-OF CYCLOFHOSPHAMIDE AND PHOSPHORODIAMIDIC ACID

<oJ

'-,

/By ,

YVONNE H. ADIWINATA SURYA, B.Sc.

A Thesis

Submitted to the School 'of Graduate Studies

in Yartial Pulfilment of the Requirements

for the Degree

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Master of Science•

McMaster University

September, 1976

YVONNE H. ADIWINATA SURYA 1977

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MASTER OF SCIENCE (1976)(Biochemis try)

McMASTER. UNIVERSITYHamil ton, Ontario

TITLE: Studies on B~ochemical and Pharmacological Effects of Cyclophos­phamide and Phosphorodismidic Acid.

AUTHOR: Yvonne Il. Adilo/inata Sury,!, B.Sc. (Sim"!' Fraser University)

SUPERVISOR: Dr. B.L. Ilillcoat; Professor, Department of Biochemistryl

NUMBER OF PAGEs': vi t, 72..SCOPE AND CONTENTS: . Recent evidence sugges ts that phosphorodiamidic acid

is formed from cyclophosphamide in~ and in vitro and may be the active- .form of chis drug ..

We have studied the effect of phosphorodiamidit. acid and cyclo-

Iphosphamide on the grO\olth of the mouSe leukemic cell lines LM4 and LS2 in

culture. On this basis, phosphorodiam1dic acid Io/as at least 100 times more•

·potent than cyclophosphamide. in inhibiting grO\olth. It also produced

enlargement of cells, an effect no.t seen Io/ith' cyclophosphamide.

•Although tlo/O metaL.clites of cyclophosphamide,. phosphoro-

diamidic add and· acrolein Io/ere isolated and identified. attempts to.measure these metabolites accurately Io/ere unsuccessful. Consequently

the'stoichiometry of th~ conversion could not be determined.

Phosphorodiamidic acid significantly increased the amount of

cross-linked ItlA after incubation with intad LM4 cells or isolated nuclei

from these cells. Cyclophosphamide had'a similar effect only in the

i~ated nuclei.

These findings 'strengthen the proposed role of phosphorodiamidic

.add as the active me tabollte of cyclophosphamide.

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TABLE OF CONTENTS

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Page , .Chapte,r 1 Introduction • 1

Chapter 2 Reagents and Method... 18

C!lapter" 3 Results 34v

Chapter 4 Discussion 59

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LIST OF FIGURES /

Figure

1. Scheme of metabolic activation of cyclophosphamidc·.

2. Mechanism of action of alkylating agents.

3. Thc rear-tion and trapping flasks uscd.in microsomalincubation of cyclophosphamide;

4. Growth curvc of LS2 and LM4 cells.

5. Effect of varying concentrations of cyclophosphamideand phosphorodismidic scid On LM4 cells.

6. Effect of varying concentrations of cyclophosphamidcand phosphorodiamidic acid on LS2 cells.

7. Effect of cyclophosphamide at var10us stages of LM4cell growth.

8. Effcct of phosphorodiamidic acid at various stages ofLM4 cell growth.

.9. Effect of cyclophosphamidc at various stages of LS2ce 11 grow tl:1.

10. Effect of phosphorodismtdic acid at various stages ofLS2 cell growth. ~

11. Effcct of cyclophosphamide and phosphorodiamidi~ acid atvarious tfnie exposurcs.

12. Mass spectral data of monomethyl derivative ofphosphorodismtdic acid.

13. Mass spectral data of dimethyl derivative of phosphoro­diamidic add.

. .14. Mass spectral data of trimethyl der~vattve Cl.l phosphoro­

,dismtdic acid •.

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biological activityand nitrogen mustsrds,

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LIST OF TABLES

Data of chemical reactivity andof cyclophosphamide metabolitescompounds.

~Microsomal incubation of cyclophosphamide.

Amount of materials produced during microsomal incubationof cyclophosphamide. .

P~rcentage of DNA extracted to the upper phase by biphasicpartition system.

Eskimation of cross li2king of DNA from intact cellsi~ubated with I x 10- M phosphorodiamidic acid and1 x IO-3M cyclophosphamide.

Estimation of cross linking of DNA from nuclei' incubationwith I x 10-4M phosphorodiamidic acid and 1 x la-3Mcyclophosphamide.

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ACKNOWLEDCEMENTS

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1 wish to express my special appreciation, to th~ following

people for their contributions to this thesis:

Dr. B.L. Hillcoat and Dr. J. Roscnfeld for their aupport and

guidancc during both the preparation of thia manuscript and thc

/invcstigationupon which it is hased. /

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Dr. P. McCulloch and Dr. D. McCalla for 'many hclpfuL suggestions

and commen ts •

Dr. V.Y. Taguchi for many helpful suggestions snd discussion,

aa wcll as his help with the glass blowing work.

'Ms. L. Winger for typing the manuscript.

My husband, Kris, for his constant help and cncouragcment

throughout thc study.(,

1 am grateful to McMaster Universitv and thc Medical Research

Council of Canada for financial support throughout the coursc of thisI

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1. INtRODUCTION

1.1. Chemical properties and biological action of alkylating- agents

Alkylating agents are highly reactive chemical compounds capable.---- ,"/ + --- ."

of oubstituting an alkyl group (Ie. R-CH2-CH2) for hydrogen .atolllfl of many

organic compoundo .. There are two classes of alkylating agents, mono"'

. functional and polyfunctional. Monofunctional alkylating agents have

only one active alkyl group, wDile the polyfunctional alkyl;ding agento

have -two or more. In general, the ,poly functional agento h'ave greater

antItumor activity than the monofunctional (Cline and' Haokell', 1975).

The differen.ces in acti~ity among the variouo alkr,.la.tj-ng agenta

apparently relate to differencea in aboorptlon,rate C)f metabolism and

,tiosue affini y rather than to a baoic difference ·In theIr mode of action,

(Cline and aaKell, 1975). All undergo. 0 rrongly electrophilic chemIcal

)

reactions' thro the formation of carbonium ion intermediates or of

transition complexeo ~ith the target moleculeo. Hence there are two

generally accepted basic mecilanisms of alkylation, .first order nucleo-

philic substitution (5n l ) and second order nucleophilic substitution (5n2

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(Warwick,1963). Many groups such ao the phosphate, amino,- sulfhydryl,

hydroxyl, carboxyl and imidazole groups present in biological moleculeo

can be alkylated and oeveral'otudies suggest that the reaction of

alkylating agents with DNA relates to their cytotoxic effects (Van Durn,

1969; Wheeler, 1973 and Calabreoi and Parks, 1976).

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1.2. Toxicological properties of cyclophosphamide

Hundreds of compounds bearing potential alkylnting groups have

been synthesized and evaluated in on effort to obtain grea,tar'spacificity

against malignant as compared to normal tissues. ' Alkylating agants

such as bis-(2 chloroethyl)-amine (A) are toxIc tJJboth normal and tumor/

tissues. Friaoman and Seligman (1954) madegC;eries of phosphamide

derivatives of (A) in an attempt to lower the toxIcity of the compound

while its chemotherapeutic potency was preserved. Arnold and Bourseaux

(1958)' synthesized cyclophosphamick, 2- [bis- (2~oroethYl)-amino J-2H-'

1,3,2,oxazaphosphorine,2-oxide, which has little cytotoxic or alkylating

activity in vitro, and which requires activation by an enzyme of liver, -

'microsomes (Cohen and Jao, 1970).

(A) bis-(2-chloroethyl)-amine (B) cyclophosphamide

Cyclophosphamide ia an effective antitumor agent by both oral and

intravenoua routes of administration. It is moderately soluble'in

and can be readily extracted from aqueous solution into lipid ~olvents.

L 3. Me tabolic conversion of cyclophos,phamide

1.3.1. !ticrosomal activation in vitro: rhe nature of activation of'

cyclophosphaoide Whereby it exerts its cytotoxic action has been,

extensively investigated. Arnold and Bourseaux (1958) designed cyclo-

phosphamide, hoping that tumor cells may contain a phoaphorodiamldaae

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enzyme to hydrolyze the P-N linkage, thu8 11betntln'g n nnctlve nlkylntf,nR •"

IIP~c1011. -I!owever, activation hall bean IIhovn to,\ccur primn:lly in the

liver rather than in the tumor till.uOll. - J 'i"

Liver homoRonll.teli (Foley et Ill, 1961.-f or iAollltud l.1ver m1.cro-

RomeR (Cohen ,!nd Jao, 1970; Connon ct ,~i, 1970), when'IIuppl1rld wHh

reduced nicotinamide adenine dinucleotide phollphate nnd oXYKen J.!! Y-U!!'.,\ .. '

will tranAform cyclophollphamide to 1l1kyllltinR and cytotoxic me tllbol1 tllH •

Sladek (1971)' studied extensively the cn1.ymlltic cO\lvcrAlon of cyc1ophonphll-,

mide to alky11lting mctabolit~n a«d found thIn en1.ymlltic Ilctivity loclllizcd~ . . .

in~the liver microsomal frllction.

The inrtinl oxidative R'tep in cyclophosphllmlde mctllboHllm 1H due'.

to the microaomal'mixed-function ox-ida8e aylltcm., Sladek (19.72) Ilnd Ohlrll

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inhibited the

ahowed that'pretrelltmcnt with agentH'which Rtlmuillted or, '

, "~ate of production of hepatic microRomnl mIxed-function

'l' oxidnae markedly influenced the metabolic convernion of cyclophoRphamidel;'

'to active alky1ating agenta •. The microaomn1 mixed-func,tion~ ,

conve r ted cyclophonphamide in to I.-h~droxycy c'topho;lph 11m! de.

oxidaae ayRtcm

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evidence for generation of 4-hydroxycyclophoRphamide in' vitro hllR he en,-.--provided recently by Connors et aI, (1974). Sladek (1973) reported

evidence demonstrating the generation of an alkylllting Ilfdehyde from

cyclophoaphamide in~ and in vitro in bio1ogiclll nnd chemical nyatcms,

and Struck (1974) haa ponitively characterized. nldophosphamldc 08 ItA

semicarbazone derivative. The compounds, 2-carboxyethy1-N,N,bis~

(2-chloroethyl)-phosphoroaiamidate (or carboxycyclophosphamide) and

4-ketocyclophosphamide have been identi~ied as metabolic products of

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cyclophosphamide in men .~ dogs (Struck et aI, 19711. Alarcon and

Meinhofer (1971) identified acrolein as a product of the microsomal

incubation of cyclophosphamide and N,N-bis(2-chloroethyl)-phosphoro-

diamidate (or phosphorodiamidic acid) has also been identified both

in vitro' (Colvin' et aI, 1973; Connors et aI, 197~) and i.!!. vivo

(Struck et aI, 1975; Fenselau et aI, 1975). From the evidence available," ' ,

a series 'of biotransformation reactions was post~lated as shown in

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Fig. 1.

1.3.2.

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Identification of active metabolites:

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The main difficulty in

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clarifying the metabolism of cyclophosphamide was the assay, isolation

and identification of the metabolites as well a's their synthesis. Only

after a sufficient quantity of the various metaboJites was available inI)

pure form did the pharmacologic characterization ana e~aluation become

possible,

BecauSe of their instability, special efforts were necessary

for the chemical synthesis of the primary activated metabolites, the

tautomeric,compounds 4-hydroxycyclophosphamide and aldophosphamide.

Takamizawa et aI, (1973) succeeded in synthesizing 4-hydroperoxycyclo~

phosphamide, the reduction of which permitted the preparation of 4-hydroxy~', '

cyclophosphamide. Friedman et aI, (1963) synthesized phosphorodiamidic

acid but aldophosphamide has not yet been obtained in a pure form."

Cyclophosphamide and its metabolites are characterized'by-determining their chemical and biological activities in vitro and their

pharmacotherapeutic properties in vivo. Their chemical reactivity can

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\\ Figure 1: Scheme of metabolic activation of cyclophosphamide from

Connors.. et al (19 74b).

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01\ O-CHZ./ "-

M.P" CHZ." / -NH-CHZ

CYClOP HOSPHA MIDE

+CHZ= CH-CHO

ACROLEIN

AlDOPHOSPHAMI DE

CARBOXY­PHOSPHAMIOE

o o-Il/

M.P",

NHZ

P HOSPHORO-

DI A 1111 DI C ACI0

o 0lI/o-cHZ", II/o-CHZ",

M.P" CHZ ;:.==~l M.P CH z" /' ~ "'" /NH-CH NHZ CHO

\.OH

4 frlYDROXY­

CYCLOPHOSPHAMIDE

I; KETO-

C YC lOPHOSPHA MIDE

1o 0 - CHz

11/ "-M.P . NHZ

. "" .. NH-C/

IIo

frbe assessed by means of the [4-(p-nitrobenzyl») pyridine assay, and by

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their behavior in aqueous solution where the rate of liberation of chloride

ion from the Z-chloroethy~group is taken as a measur7 for chemical

reactivity. Their cytotoxic potency is ~ssessed by comparing the

concentration' which kill 50% of cancer cells after incubation in cell

culture (EC 50). Their therapeutic effect in vivo is measured by

determining the LD50

/CD50

ratio (50% lethal dose/50? curative'dose)

against tumor cells in intact animals. Table 1 contains data of chemical

reactivity and biological activity of cyclophosphamide metabolites and

nitrogen mustards compounds (Brock, 1976). Preliminary studies by

Haddock et 'aI, (1966) and Connors et al, (1974b) indicate that phosphorodiamidic

acid is an active metabolite. Phosphorodiamidic acid has been shown to have

higher toxicity at much lower concentrations than cyclophosphamde. Table 1

shows that phosphorodiamidic acid indeed has intense alkylating activity but

its cytotoxic activity in only 2.5 CU/umole. One cytotoxic unit (CU) is that

amount of a cytotoxic compound which produces survival of 50% of animals after

inoculation wi th 'tumor cells. On the other h.aru!-£ydroxycyc'lophosphamide had

highcytD<oxic activity, 63 CU/mole, and showed a similar CD50

index to cyclo­

phosphamide, but weak alkylating activity. Voelcker et 31, (1974) showed that

only after cleavage of the phosphoric ester bond of 4-hydroxycyclophosphamide to

form phosphorodiamidic acid and acrolein, that it was strongly alkylating. The

4-hydroxy derivative of 5,5 dimeEhylcyclophosphamide has been isolated (Cox

.et aI, 1976a) and has low toxicity to both normal and tumor tissue. This

compound cannot release phosphorodiamidic acid by B-elimination due to

the gemdimethyl group at C5

, suggesting the importance of the release of

phosphorodiamidic acid and acrolein during the activation step."