Cellular Pharmacokinetics of -Deoxyadenosine Nucleotides ... · Table 2 Pharmacokinetic parameters of C dA after interm ittent (2 h) an d continuous iv. infusion Intracellular CdAN

Vol. 1, 385-390, April 1995 Clinical Cancer Research 385

Cellular Pharmacokinetics of ‘ -Deoxyadenosine

Nucleotides: Comparison of Intermittent and Continuous

Intravenous Infusion and Subcutaneous and Oral

Administration in Leukemia Patients’

Jan Liliemark2 and Gunnar Juliusson

Departments of Clinical Pharmacology and Oncology, Karolinska

Hospital, S-104 01 Stockholm [J. L.], and Division of Clinical

Hematology and Oncology, Department of Medicine, Huddinge

Hospital, Huddinge [G. J.], Sweden

ABSTRACT2-Chloro-2’-deoxyadenosine (CdA) is a new purine nu-

cleoside analogue with major activity in lymphoproliferative

diseases. Its intracellular nucleotides, in particular the 5’-

triphosphate, are thought to be the pharmacologically active

metabolites. The present study was undertaken to elucidate

the cellular pharmacokinetics of these active metabolites in

leukemia patients during CdA treatment The concentra-tions of CdA in plasma and of CdA nucleotides (CdAN) in

leukemic cells were measured by liquid chromatography in

69 patients with chronic lymphocytic, acute myeloid, andhairy cell leukemia after intermittent and continuous i.v.

infusion, s.c. injection, and p.o. administration. The t112 of

CdAN during the first dose interval was 13.8 h (n = 67),

while after the last dose the t112 was 327 h (n 8). The area

under the concentration versus time curve was similar after

intermittent and continuous infusion, 268.3 and 2378 �i�si/h,

respectively (n = 7) The area under the concentration

versus time curve after p.o. administration (024 mg/kg) was

slightly lower than that after intermittent infusion (0.12

mg/kg), 120.6 versus 188.8 psi/h (P < 0.05, n = 7). However,

when all p.o. administrations (n 16) were compared withall 2-h infusions in other patients with chronic lymphocyticleukemia (n = 32), there was no significant difference (149.6versus 168.6 p�s/h). The cellular concentration of CdAN was

320 times higher than the plasma concentration of CdA, but

there was no correlation in individual patients (r� 0.02,

n 69). The t512 of CdAN was significantly shorter in

patients with acute leukemias (9 h) compared to those with

chronic lymphocytic (12.9 h) and hairy cell leukemias (15.1

h). The area under the concentration versus time curve ofCdAN in leukemic cells from the 11 patients with hairy cell

leukemia given CdA s.c. was in the same range (179.8 p�M/h)

Received 9/28/94; accepted 12/22/94.

1 This work was supported by grants from the Swedish Cancer Foun-

dation (2409-B92-O2XBP), the Children Cancer Foundation of Sweden,

the Jenny Foundation, and the National Society for Cancer and Traffic

Victims.

2 To whom requests for reprints should be addressed, at Department ofClinical Pharmacology, Karolinska Hospital, S-104 01 Stockholm, Swe-

den.

as in patients with chronic lymphocytic leukemia. The re-

tention of CdAN in leukemic cells supports intermittent

administration. The lack of correlation between cellular and

plasma drug concentrations indicates that plasma drug con-

centrations are not useful for individualization of dose.

INTRODUCTION

CdA3 is a new purine nucleoside analogue which has

recently emerged as the drug of choice for HCL (1-3). The

results of treatment of other lymphoproliferative diseases (4-6)

as well as of AML (7) are also very promising, making it one of

the most interesting new anticancer drugs developed during the

last decade. CdA is resistant to deamination by adenosine

deaminase due to protonization at N-7 (8). Intracellularly CdA

is phosphorylated by deoxycytidine kinase to its 5’-monophos-

phate. Its nucleotides (CdAN), presumably the 5’-triphosphate,

are thought to be the active metabolites by inhibiting DNA

polymerase 1� (9) and possibly DNA repair, causing DNA sin-

gle-strand breaks. The DNA damage activates poly(ADP-ribo-

sylation) which consumes NAD. The depletion of NAD and

ATP is shown to be important for the toxic effects of CdA in

vitro (10). An alternative mechanism of action is mediated

through DNA fragmentation and apoptosis (1 1). Early studies

on the pharmacokinetics of CdA revealed a rapid distribution of

CdA from plasma and undetectable levels 2 h after administra-

tion (12). On the basis of these studies, continuous infusion was

used as the mode of administration of choice. Later it was shown

that there is a prolonged elimination phase (13, 14) and that the

AUC of plasma CdA is similar after intermittent and continuous

infusion (13). The metabolism of CdA in tumor cells has been

studied in vitro. The retention of the CdA nucleotides was short

in these studies based on wash-out experiments (15, 16). The

present study was undertaken in order to describe the cellular

pharmacokinetics of its active metabolite, the CdAN, in leuke-

mic cells in patients in vivo.

PATIENTS AND METHODSPatients and Treatment. CdA was produced at the

Foundation for Diagnostics and Therapy (Warsaw, Poland) by

Dr. Zygmunt Kazimierczuk (17). The doses and concentrations

stated in this study where calculated using the extinction coef-

ficient 15 AU/mM (18, 19). Sixty-nine leukemia patients were

3 The abbreviations used are: CdA, 2-chloro-2’-deoxyadenosine; HCL,hairy cell leukemia; AML, acute myeloid leukemia; CdAN, CdA nude-

otides; AUC, area under the concentration versus time curve; CLL,chronic lymphocytic leukemia.

Research. on February 1, 2021. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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100�

10�

1�

.1�

.01#{149}

.001�

E

z

4.)

386 Cellular CdA Pharmacokinetics

Table I Patients and treatments

2h�+ 2h�+

2-h

i.v. infusion

Continuous

infusion”

s.c.

injection” poP 2 hC + poP

continuous

infusion’

continuous

infusionc + p.o.” Total-�------

CLL

HCLAML

24

8”

2 11

10 6 7 1 48

13

8

Total 32 2 11 10 6 7 1 69

(1 0.6 mg/kg/course (7 days).

I, � .2 mg/kg/course (5 days).

( 0.6 mg/kg/course (5 days).

‘I 1 . 1 mg/kg/course (5 days).

I

0 6 12 18 24

Time, h

Fig. I The concentration of CdA in plasma (lower part of the panel)

and the concentration of CdA nucleotides (CdAN) in leukemic cells

(tipper part of the panel) in the first 16 patients with CLL given a 2-h

infusion of CdA (0.12 mg/kg).

treated with CdA and the pharmacokinetics was monitored

during one to three treatment courses as outlined in Table 1. All

of the patients had normal renal and liver function. The study

was approved by the local ethics committee and by the Swedish

Drug Products Agency. All patients gave their oral informed

consent to participate in the study.

Sampling Procedure. Blood was sampled in heparinized

tubes at the end of the 2-h infusion and at 1 , 2, 4, 8, 20, and 22 h

postinfusion. In some patients, samples were taken once or

twice daily after the infusion on day 5. During continuous

infusion, samples were taken daily during infusions and once or

twice daily after the end of infusion. In selected patients, sam-

ples were taken every 4-6 h during the first 2 days of infusion

to delineate the drug accumulation during continuous infusion.

After s.c. infusion, samples were taken at 30 mm and at 1, 2, 4,

8, 20, and 24 h postinjection.

Determination of Drug Concentrations. The concen-

tration of CdA in plasma was determined with HPLC as de-

scribed previously (20).

The concentration of CdAN in leukemic cells was deter-

mined as follows: Leukemic cells were isolated on Lymphoprep

and washed twice with PBS (8.1 g NaC1, 0.22 g KC1, and 1.14 g

Na,HPO�/liter H20 at pH 7.4). The total cell volume in each

sample was calculated using the cell number and median cell

volume as determined on a Coulter Multisizer (Coulter Elec-

tronics, Luton, United Kingdom). The nucleotides were ex-

tracted twice with 2.5 ml 60% methanol in H20. After evapo-

ration, the cell extracts were reconstituted in I ml Tris buffer (40

mM Tris, 40 mM NaCl, 40 mr�i MgCl2, 40 p.g/ml BSA, pH 7.5)

and incubated with 5 units of alkaline phosphatase (Grade II;

Boehringer Mannheim, Mannheim, Germany) for 2 h at 37#{176}C.

With this procedure >95% of endogenous 5’-triphosphate

nucleotides are degraded while >95% of added CdA is recov-

ered. The CdA was extracted with ethylacetate and the amount

of CdA was determined with HPLC as for plasma concentra-

tions. The intracellular concentration of CdAN was calculated

by dividing the the amount of CdA in the alkaline phosphatase-

treated extracts with the total cell volume of the samples.

Pharmacokinetic Calculations. The AUC of plasma

CdA was calculated according to the trapezoid rule. The AUC

and t112 of cellular CdAN was calculated using logarithmic

regression assuming a monophasic elimination from the peak

concentration. The AUC of CdAN from the start of infusion to

the peak was calculated using the trapezoid rule. The t112 of CdA

in plasma was calculated with logarithmic regression using

determinations from the last three time points. To enable a

comparison to other patients, the AUC of CdA and CdAN in

HCL and AML patients was normalized for dose by assuming a

linear relationship between dose, plasma AUC, and AUC for

CdAN in leukemic cells in individual patients within the dose

interval 0.085-0.22 mg/kg.

Statistical Calculations. Student’s t test for paired sam-

ples was used to assess the difference between the AUC of

CdAN after iv. and p.o. administration in nine patients where

both routes of administration were used. Student’s t test for

unpaired samples was used to test the difference of the AUC of

CdAN between the 16 patients treated with CdA p.o. and 32

other patients treated i.v. An ANOVA was made to assess the

differences in pharmacokinetic parameters between different

diagnoses. Linear and logarithmic regression analyses were used

to assess the correlation between parameters for plasma CdA

and intracellular CdAN pharmacokinetics. All statistical calcu-

lations were made using the StatView SE + Graphics software



L)

E

z

Ia

4)

C..)

Ia

1,000

100 -

10

5-

2-

1

I CdA 0.6mg/kg, 120 hI

I I � #{149} I I

�CdA0.12mg/kg, 2h]

! ! !0

I I I I I I I

192 240

Clinical Cancer Research 387

Table 2 Pharmacokinetic parameters of C dA after interm ittent (2 h) an d continuous iv. infusion

Intracellular CdAN Plasma CdA

ti,2 (h)

AUC (nM X h)

t�,,�(h)

2 hAUC (p.M X h) 2 h

2 h ci.” Day 1 Day 5 ci.Patient 2 h ci. Day 1

MeanSDCVn

268.3 237.8 15.363.1 108.8 4.8

0.24 0.46 0.317 7 7

33.118.3

0.55

4

32.29.60.30

6

589.1 654.8188.1 313.7

0.32 0.48

6 7

11.33.3

0.29

4

a c.�., contin uous infusion, CV, coefficient of variation.

10,000 -

48 96 144

Time, h

Fig. 2 The concentration of CdA in plasma (lower part of the panel) and the concentration of CdA nucleotides (CdAN) in leukemic cells (upper

part ofthe panel) in one patient with CLL given 0.6 mg/kg CdA as 2-h infusions (0) and 4 weeks later as continuous infusion (#{149})of the same dose

during 5 days.

(Abacus Concepts Inc., Berkeley, CA) on a Macintosh SE

computer (Apple, Inc.)

RESULTS

Only very low concentrations of CdA (<3% of CdAN)

were found in cell extracts before treatment with alkaline phos-

phatase, indicating that the CdA measured after alkaline phos-

phatase treatment represents CdAN.

Intermittent i.v. Infusion The pharmacokinetics ofplasma CdA and cellular CdAN in the first 16 patients treated is

shown in Fig. 1. There was a monophasic elimination of CdAN

from leukemic cells during one dose interval in contrast to CdA

in plasma which showed a biphasic elimination. The t112 of

CdAN during the first dose interval after 2-h infusions, s.c.

injections, or p.o. administration was 13.8 � 10.3 h (mean �

SD; n = 67) while after the fifth day the t112 was more than

twice as long (Table 2). This difference is probably due to a

biphasic elimination (Fig. 2) which is not appreciated during the

first 24 h after infusion.

Comparison of Cellular CdAN and Plasma CdA Phar-

macokinetics. The peak and trough concentrations of CdAN

in leukemic cells after a 2-h infusion, with a dose interval of

24 h (n = 38), were 10.7 ± 7.1 and 2.4 ± 1.3 p.M, respectively.

The corresponding values for CdA in plasma was 1 1 1 .9 ± 45.7

and 7.9 ± 6.7 nM. The difference between peak and trough

concentrations was therefore 30.0 ± 36.5 times for CdA in



Table 3 Correlations between plasma and cellular pharmacokinetic

parameters of CdA

600

500

�4O0

�300

� 200LI

100

0

r2=0.02

0 #{176} �.25

019 0 0

0

0

0

00

0

0


AUC t112

r2p r2 p

CLLHCL

AML

(ii = 48)(n = 13)(n = 8)

0.010.080.20

0.500.340.26

0.0010.280.07

0.880.090.56

Total (n = 69) 0.02 0.25 0.05 0.08

plasma but only 5.1 ± 3.4 times for CdAN in leukemic cells.

The cellular concentration of CdAN was 104 ± 7 and 511 ±

453 times higher than the plasma CdA concentration at the peak

and trough, respectively. When the plasma and cellular AUC

was compared after 2-h intermittent infusion in 44 patients, the

cellular AUC of CdAN was 318 ± 194 times higher than

the plasma AUC of CdA. There was no correlation between the

AUC or ti!2 of plasma CdA (terminal t112) and cellular CdAN in

any of the analyzed patient categories (Table 3 and Fig. 3).

During continuous infusion the steady-state concentration of

CdA in plasma was 27.3 ± 13.1 n�t and of CdAN in leukemic

cells, 9.9 ± 4.5 p.M. Thus the mean difference between plasma

CdA and cellular CdAN during continuous infusion was 422 ±

199 times. The pattern of plasma and cellular pharmacokinetics

during intermittent and continuous infusion in one patient is

shown in Fig. 2. The AUC for CdAN was similar after inter-

mittent 2-h and continuous iv. infusions (Table 2).

p.o. Administration. The AUC of CdAN in leukemic

cells after p.o. administration (0.24 mg/kg) was slightly lower

than that after intermittent infusion (0.12 mg/kg; Table 4). When

all p.o. administrations (a = 16) were compared with 2-h

infusions in all other patients with CLL (n 32), there was no

significant difference in the AUC of CdAN (149.6 ± 101.7

versus 168.6 ± 91.2).

CdA and CdAN Pharmacokinetics according to Diag-nosis The AUC and t112 of plasma CdA and cellular CdAN

after normalization for dose in different diagnoses are shown in

Table 5. There were no statistically significant differences in

plasma AUC. The ti!2 of plasma CdA in AML patients was,

however, shorter than that in CLL patients. Both the AUC and

the ti!2 of CdAN in patients with AML was significantly shorter

than those of both CLL and HCL patients. The AUC and ti!2 of

CdA in plasma and CdAN in leukemic cells from 1 1 patients

with HCL were similar to those in patients with CLL.

DISCUSSION

CdA is one of the most important new anticancer drugs

developed during the last decade. Its therapeutic potential in

lymphoproliferative disorders is very promising (4-6) and it has

rapidly become the drug of choice for HCL (1-3). We have

previously delineated the plasma pharmacokinetics of this drug

(13, 14). The intracellular metabolism has been studied previ-

ously in vitro in both cell lines and in leukemic cells from

patients with acute myeloid leukemia (15). In contrast, no data

on the cellular metabolism in vivo have been published except a

preliminary report of the present study (21). The present study

delineates the cellular pharmacokinetics of CdAN in patients

0

c�00 oo��00 0

0 � �

200 400 600 800 1000 1200 1400 1600

Plasma AUC, nMh

Fig. 3 The relationship between the AUCs of plasma CdA and intra-cellular CdAN in 69 patients with CLL, HCL, and AML treated withCdA 0.085-0.22 mg/kg iv. or s.c. or 0.24 mg/kg p.o.

with CLL, AML, and HCL during intermittent and continuous

i.v. infusions as well as s.c. and p.o. administration.

The method used in this study to determine the intracellular

concentration of CdAN does not separate CdA from CdAN. In

contrast to the CdAN, the nucleoside CdA traverses the cell

membrane quite readily and most of the intracellular CdA is

probably lost during the washing procedure. We have shown

here that the intracellular concentration of CdA and CdA nude-

otides is several hundred times higher than the plasma concen-

tration of CdA. Thus, if it is assumed that the extracellular and

intracellular concentration of CdA is similar, the nucleoside

represents only a very small part of the total intracellular drug

concentration. This assumption is supported by the very small

amount of CdA found in cell extracts before treatment with

alkaline phosphatase. Thus, practically all of the measured in-

tracellular drug concentration represents CdAN. Our chromato-

graphic method easily separates CdA and 2-chloroadenosine

(22). Thus ribonucleotide metabolites of CdA are not measured

as CdAN.

The intracellular CdAN concentrations measured in the

present study represent a mixture of the 5’-monophosphates,

5’-diphosphates, and 5’-triphosphates. In vitro studies, using

radioactively labeled drug, have shown that the major intracel-

lular metabolite is actually the monophosphate (15, 16). Thus, it

seems that the first step in the bioactivation, phosphorylation by

deoxycytidine kinase, is not the rate-limiting step. The same

relationship is seen with other nucleoside analogues, e.g. , azido-

thymidine (23). The triphosphate, considered to be the active

metabolite, is therefore probably only a minor part of the intra-

cellular metabolites. This has to be taken into consideration

when evaluating the present study. However, it can be assumed

that the CdA 5’-monophosphate and S’-diphosphate serve as a

substrate pool for furher phosphorylation to the 5’-triphosphate.

Therefore, there is probably a direct relationship between the

intracellular concentration of CdA 5’-triphosphate and total

CdAN. The determination of the low CdA 5’-triphosphate con-

centrations in leukemic cells in vivo requires a very sensitive

methodology which is not yet available. It is therefore important

to develop more specific methods which can quantitatively

extract and separate the cellular metabolites of CdA. Such

development is ongoing.



Clinical Cancer Research 389

Table 4 Pharmacokinetic parameters of CdA after p.o. and iv. infusion

Intracellular CdAN Plasma CdA

AUC t1/2 AUC ti!7

(p.M X h) (h) (nM X h) (h)

Patient iv. p.o. iv. p.o. iv. p.o. iv. p.o.

Mean 188.8” 120.7” 10.9 12.1 759.3 656.3 9.6 10.0

SD 68.8 48.2 2.2 3.8 361.3 256.0 1.3 1.4

CV 0.36 0.40 0.20 0.32 0.48 0.39 0.14 0.14

n 7 7 7 7 6 6 6 4

a p < 0.05.

b CV, coefficient of variation.

Table 5 Pharmacokinetic parameters of CdA and CdAN in different diagnoses

Plasma CdA Leukemic cell CdAN

AUC ti!7 AUC ti!)

CLL� (n 38) 572.7 ± 244.3 10.0 ± 5.0” 165.5 ± 84.8 12.9 ± 5.2

HCLC (n 11) 610.5 ± 181.1 10.3 ± 7.0 179.8 ± 74.2 15.1 ± 5.3

AMLd (n 8) 453.2 ± 308.7 5.3 ± 2.6” 89.6 ± 56.8e 9.0 ± 3.fIa 2-h iv. infusion.

hp < 005,

C � injection, AUC normalized for dose (0.085 mg/kg).

d 2-h iv. infusion normalized for dose (0.22 mg/kg).

e p < o.os toward AUC for CLL or HCL.

fp < 0,05 toward t�,2 for CLL or HCL.

Previous plasma pharmacokinetic data showed that the

AUC was similar after intermittent and continuous iv. infusion

(13). The long terminal t112 of CdA resulted in a trough level of

CdA in plasma between the infusions that was only slightly

lower than the steady-state level during continuous infusion.

The present data showing even longer half-lives of intracellular

metabolites further support the use of intermittent infusions.

Furthermore, the AUC of intracellular metabolites was also

similar after the two modes of administration. The bioavailabil-

ity of CdA administered p.o. is 50% (14). The AUC of intra-

cellular metabolites was slightly lower when 0.24 mg/kg was

given p.o. compared to 0.12 mg/kg as a 2-h intravenous infu-

sion. However, this was seen in a small number of patients and

does not preclude the use of p.o. administration when repeated

courses are administered. The promising clinical activity of p.o.

CdA in CLL also supports this mode of administration (24).

Although promising, the therapeutic results in CLL pa-

tients (4, 6) are far from as impressive as the responses in HCL

patients, where 1 week of treatment is sufficient to induce

complete remission in 85% of the patients (1-3). It is therefore

interesting to note that the intracellular concentrations of CdA

metabolites in leukemic cells from patients with HCL is not

higher than that in CLL patients. In contrast, in AML patients

both the AUC and t112 of CdAN is lower than in the indolent

diseases. However, due to the large interindividual variability of

both plasma and cellular pharmacokinetics, there is a great

overlap between the diagnosis groups. Therefore, the conclu-

sions drawn are valid for groups of patients but not necessarily

for every single patient. Such interindividual variability is seen

also with other antimetabolites (25). Three of the eight patients

with AML previously received 1 -�3-D-arabinofuranosylcytosine

and could have developed a biochemical resistance to both

drugs through an impairment of the deoxycytidine kinase activ-

ity. However, neither AUC nor t112 of CdAN is lower in these

three patients as compared to the five patients who were naive

to nucleoside analogues. It has previously been shown that the

ti!2 of 1 -�3-o-araninofuranosylcytosine S ‘ -triphosphate in leuke-

mid cells in vivo is also shorter in AML patients compared to

CLL patients (26). It is therefore possible that CdA should be

administered twice daily in patients with AML to achieve an

optimal therapeutic result.

Thus, the less impressive clinical response seen in AML (7)

compared to the more indolent lymphoproliferative disorders

might be partly explained by intracellular pharmacokinetic dif-

ferences while the outstanding sensitivity of HCL to the action

of CdA probably is due to some event beyond the bioactivation

of CdA.

Although there are data indicating that the response in CLL

patients correlates with the activity of the metabolizing enzyme,

deoxycytidine kinase (27, 28), no correlation has been seen

between the metabolism of CdA in vitro and cytotoxic effects in

cell lines or in patients (15, 16). However, there are also large

differences in the plasma pharmacokinetics of CdA between

patients which might obscure any correlation between in vitro

metabolism and in vivo effects. The very short retention of CdA

metabolites in leukemic cells from patients with AML in vitro

(t112 � 1.5 h; Refs. 15 and 16) compared to the ti!2 of CdA

metabolites in leukemic cells in vivo (t112 - 9.0 h) shown in this

study illustrates this problem. In fact, the retention of CdA

metabolites in leukemic cells from patients with CLL in vitro is




4 V. Gandhi, personal communication.

also much shorter4 than what we show here in vivo. Further-

more, the longer ti!) seen when a prolonged sampling was made

after the end of a treatment course indicates that there might be

a biphasic elimination and an even slower elimination phase of

CdAN from leukemic cells in vivo.

The lack of correlation between plasma CdA and cellular

CdAN concentrations is not surprising. The intracellular CdAN

concentration is a product of the plasma CdA concentration and

the activity of intracellular metabolizing enzymes, both phos-

phorylating and dephosphorylating. The activity of these en-

zymes is highly variable among patients (27, 28). However, it

remains to be shown whether intracellular concentrations of

CdA metabolites are important for the clinical effects of CdA.

ACKNOWLEDGMENTS

We thank Birgitta Pettersson for her skillful and reliable technical

assistance throughout this study and the staff at the Department of

Medicine at Huddinge Hospital for the blood sampling.

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1995;1:385-390. Clin Cancer Res J Liliemark and G Juliusson in leukemia patientsintravenous infusion and subcutaneous and oral administrationnucleotides: comparison of intermittent and continuous Cellular pharmacokinetics of 2-chloro-2'-deoxyadenosine

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Cellular Pharmacokinetics of -Deoxyadenosine Nucleotides ... · Table 2 Pharmacokinetic parameters of C dA after interm ittent (2 h) an d continuous iv. infusion Intracellular CdAN