doi:10.1182/blood-2003-03-0925Prepublished online June 5, 2003;   

 Keating, William Plunkett and Emil J FreireichFrancis Giles, Stefan Faderl, Susan O'Brien, Sima Jeha, Jan Davis, Zeev Shaked, Adam Craig, Michael Hagop M Kantarjian, Varsha Gandhi, Jorge Cortes, Srdan Verstovsek, Min Du, Guillermo Garcia-Manero, refractory or relapsed acute leukemiaPhase II clinical and pharmacology study of clofarabine in patients with

(4217 articles)Neoplasia   � (3716 articles)Clinical Trials and Observations   �

Articles on similar topics can be found in the following Blood collections

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

digital object identifier (DOIs) and date of initial publication. theindexed by PubMed from initial publication. Citations to Advance online articles must include

final publication). Advance online articles are citable and establish publication priority; they areappeared in the paper journal (edited, typeset versions may be posted when available prior to Advance online articles have been peer reviewed and accepted for publication but have not yet

Copyright 2011 by The American Society of Hematology; all rights reserved.20036.the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by    

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

PHASE II CLINICAL AND PHARMACOLOGY STUDY OF CLOFARABINE IN

PATIENTS WITH REFRACTORY OR RELAPSED ACUTE LEUKEMIA

Hagop Kantarjian1, Varsha Gandhi1,2, Jorge Cortes1, Srdan Verstovsek1, Min Du2,

Guillermo Garcia-Manero1, Francis Giles1, Stefan Faderl1, Susan O’Brien1, Sima Jeha1,

Jan Davis1, Zeev Shaked3, Adam Craig3, Michael Keating1,

William Plunkett1,2, Emil J Freireich1

From the Departments of Leukemia1 and Experimental Therapeutics2, M.D. Anderson

Cancer Center, Houston, Texas; and ILEX Company3, San Antonio, Texas.

Address Correspondence: Hagop Kantarjian, M.D., Department of Leukemia, Box 428,

The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston,

TX 77030. Phone: 713-792-7026;

Fax: 713-794-4297; Email: hkantarj@mdanderson.org

Supported in part by grants CA57629 and CA32839 and FD-R-00212 from the National

Cancer Institute, Department of Health and Human Services and FD-R-002127 from the

Food and Drug Administration.

Key Words: Clofarabine, acute myelogenous leukemia, salvage, pharmacology

Word Count: 4,122

Copyright (c) 2003 American Society of Hematology

Blood First Edition Paper, prepublished online June 5, 2003; DOI 10.1182/blood-2003-03-0925 For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

2

ABSTRACT

In a phase II study, 62 patients with relapsed and refractory acute myeloid leukemia

(AML; N=31), myelodysplastic syndrome (MDS; N=8), chronic myeloid leukemia in

blastic phase (CMLBP; N=11) and acute lymphocytic leukemia (ALL; N=12) received

clofarabine 40 mg/m2 IV over 1 hour daily x 5, every 3 to 6 weeks. Twenty patients

(32%) achieved complete response (CR), 1 had a partial response (PR), and 9 (15%)

had CR but without platelet recovery (CR-p), for an overall response rate of 48%. In

AML, responses were noted in 2/11 patients (18%) in first salvage with short first CR (≤12 months), in 7/8 (87%) patients with longer first CR, and in 8/12 patients (67%) in

second or subsequent salvage. Responses were observed in 4/8 patients with high-risk

MDS (50%), in 7/11 (64%) with CML-BP, and in 2/12 (17%) with ALL. Severe reversible

liver dysfunction was noted in 15% to 25%. After the first clofarabine infusion,

responders accumulated more clofarabine triphosphate in blasts compared to non-

responders (median 18 vs 10 µM; p=0.03). This increased only in responders (median,

1.8-fold; p=0.008) after the second clofarabine infusion. In summary, clofarabine is

active in acute leukemias and MDS; cellular pharmacokinetics may have prognostic

significance.

Word Count: 198

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

3

INTRODUCTION

Nucleoside analogues have been a rich source of many highly effective agents in the

treatment of leukemias and other hematologic cancers.(1-8) Deoxyadenosine analogues,

such as fludarabine, cladribine, or deoxycoformycin have been active against

lymphoproliferative disorders including chronic lymphocytic leukemia (CLL), indolent

lymphomas, Waldenstrom macroglobulinemia and hairy cell leukemia without untoward

toxicity.(7, 8) Of particular interest is the activity of these agents in acute leukemia, albeit

at dose levels associated with prohibitive extramedullary toxicities (9-11). These were

dose limited by frequent serious renal and neurologic toxicities.

A series of 2-halo-2’-halo- 2’-arabinosyladenine analogues including clofarabine (Cl-F-

ara-A; 2-chloro-2’-fluoro-deoxy-9-β-D-arabinofuranosyladenine) were developed as a

rational extension of the experience with fludarabine and chlorodeoxyadenosine, in an

attempt to improve the drug efficacy and minimize extramedullary toxicities (12) (Figure

1). The 2-(F, Cl, or Br)-2’-F-ara-A analogues (but not the 2’-Cl or 2’-Br analogues) were

found to be potent inhibitors of cell growth. These analogues were synthesized

because of the noted anti-tumor activity of 2-halodeoxyadenosine analogues, and the

appearance of 2-F-adenine (a toxic compound with no anti-tumor selectivity) in animals

treated with fludarabine.(13, 14) which resulted from cleavage of the glycosidic bond by

the bacterial purine nucleoside phosphorylase.(15,16) Substitution of a fluorine at the 2’

carbon in the arabino configuration, made these derivatives highly resistant to

phosphorolysis by bacterial purine nucleoside phosphorylase.(17, 18) Similar to

fludarabine and cladribine, clofarabine was resistant to deamination by adenosine

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

4

deaminase and required intracellular phosphorylation by deoxycytidine kinase to the

cytotoxic triphosphate form.(18,19) Both fludarabine and cladribine inhibit DNA synthesis;

however, fludarabine triphosphate does this by terminal incorporation into DNA while

cladribine triphosphate particularly inhibited ribonucleotide reductase. Clofarabine

triphosphate has the favorable properties of both congeners and affects both DNA

elongation and ribonucleotide reductase.(18, 20-23)

Although clofarabine had been synthesized in the 1980s, there was little interest in its

development by pharmaceutical companies because of the availability of other

nucleoside analogues, its perceived small market potential, and lack of compelling

evidence for activity outside the lymphoproliferative disorders. In 1993, we initiated the

development of clofarabine at M.D. Anderson. Clofarabine was produced in quantities

large enough for animal toxicology studies and for phase I human studies. Studies in

mice established the LD10 of clofarabine at 75 mg/kg/d=225 mg/m2 IV over 1 hour daily

x 7; the dose-limiting toxicity was gastrointestinal. Limited dog toxicology studies

demonstrated lack of toxicities at clofarabine doses of 7.5 mg/Kg IV over 1 hour daily x

5 = 150 mg/m2 IV over 1 hour daily x 5 (unpublished data). This resulted in the initiation

of a phase I study of clofarabine in solid and hematologic cancers in 1998. The MTD of

clofarabine on a daily x 5 schedule were 2 and 4 mg/m2 in solid tumors and

lymphoproliferative disorders with myelosuppression as dose-limiting toxicity (DLT). In

acute leukemia reversible severe hepatotoxicity defined the non-myelosuppressive DLT,

and suggested a clofarabine dose of 40 mg/m2 daily for 5 days as a reasonable

schedule for phase II acute leukemia studies. Among the 32 evaluable patients in this

group, two complete responses (CR), and 3 marrow CRs were observed. (24)

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

5

This encouraging clinical response rate in this heavily pretreated study group, tolerable

toxicity profile, and associated pharmacokinetic and pharmacodynamic parameters (24)

led us to investigate clofarabine in acute leukemia. In the current report, we summarize

our clinical and pharmacological experience in the phase II study of clofarabine in

patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome

(MDS), acute lymphocytic leukemia (ALL), and chronic myelogenous leukemia in blastic

phase (CML-BP).

PATIENTS AND METHODS

Study Group

Patients with a diagnosis of AML, MDS, ALL, or CML-BP whose disease relapsed or

was refractory to frontline and/or salvage therapy were eligible. Informed consent was

obtained according to institutional guidelines. Other eligibility criteria included: 1) age

15 years or older; 2) performance status 0 to 2 (ECOG scale); and 3) adequate renal

(creatinine 2 mg/dl or less), hepatic (bilirubin 2 mg/dl or less), and cardiac functions.

Drug and Other Chemicals

Clofarabine for clinical use was initially prepared by Ash Stevens Inc, Detroit, MI and

formulated for injection by the University of Iowa Pharmaceutical Services, Ames, IA.

Subsequently, production of bulk drug was conducted by Delmar Chemicals, Lasalle,

Quebec. For cellular pharmacokinetics, high-pressure liquid chromatography (HPLC)

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

6

standards, clofarabine 5’-triphosphate was synthesized by Sierra Biochemicals (Tucson,

AZ). All other chemicals were reagent grade.

Therapy

Clofarabine was administered at 40 mg/m2 IV over 1 hour daily for 5 days every 3-6

weeks depending on marrow anti-leukemic response and recovery of counts.

Subsequent courses of induction chemotherapy were given at the same dose schedule

if toxicity was grade 1 or less, at 1 dose level reduction (30 mg/m2) if grade 2

extramedullary toxicity was noted, and at 2 dose level reductions (50% of dose, 20

mg/m2) if grade 3-4 extramedullary toxicity was observed and associated with favorable

response. Dose level reductions were also implemented for severe (-1 level) and life

threatening infections (-2 levels). Patients who did not achieve an objective response

after 2 courses of induction therapy were taken off study.

Pretreatment evaluation included history, physical exam, complete blood counts (CBC),

differentials and platelets counts and marrow aspiration and cytogenetic analysis.

Follow up studies included CBC, differential and platelet counts 2-3 times weekly, and

serum chemistries weekly until response. Marrow aspiration was done on day 14 to 21

of therapy then every 1-2 weeks as indicated to document response. Patients with

marrow studies showing no evidence of leukemia but with persistent cytopenias were

allowed time to recover their counts to document best response. Growth factors were

allowed as indicated by the clinical condition. In patients with persistent or increasing

marrow leukemic infiltrates on serial studies, a subsequent induction course was given

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

7

even with persistent cytopenia. Patients received routine antiemetics prophylaxis during

chemotherapy. Prophylactic antibiotic prophylaxis was given (usually ciprofloxacin or

levaquin, and fluconazole) at the discretion of the treating physician.

Patients achieving remission were offered consolidation therapy at 1 dose level

reduction or the last tolerable induction dose schedule, whichever was lower. Up to 6

courses of consolidation chemotherapy could be offered every 28 days upon recovery

of myelosuppression. However, consolidation therapy could be modified at the

discretion of the treating physician as judged to be in the best patient interest.

Response Criteria

A complete remission (CR) required normalization of the marrow (5% or less blasts in a

normocellular marrow) and peripheral counts, a granulocyte count above 109/L and a

platelet count of 100 x 109/L or above. A partial response was as for CR but with

persistence of 6% to 25% marrow blasts. A hematologic improvement (HI) had similar

criteria as for CR, but without recovery of the platelet counts to 100 x 109/L or above.

This is also called marrow CR with low platelets or CRp.

A partial response in MDS required improvement in at least 2 parameters: reduction of

marrow blasts by 50% or more and to less than 10%; increase of platelet counts by

100% and to above 10 x 109/L; increase of granulocyte count by 100% and to above

0.5 x 109/L.

In CML-BP, a complete hematologic response (CHR) was similar to CR. A partial

hematologic (PHR) was as for CHR but with persistence of few immature peripheral

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

8

cells (less than 5% myelocytes + metamyelocytes) or with persistent but 50% or more

reduced splenomegaly or thrombocytosis. A return to second chronic phase (second

CP) required disappearance of accelerated or blastic phase features.

Clinical Pharmacology

Blood samples were obtained from 29 patients (CML-BP = 10, ALL = 6, AML = 12, MDS

= 1) who agreed and signed the written informed consent for blood drawing for

pharmacological determinations. Samples were collected from patients based on three

criteria: signed consent from patients, number of circulating leukemia cells, and

laboratory preparedness. Samples were obtained prior to therapy for baseline values,

at the end of the initial infusion and at several times thereafter, including 24 hours after

start of therapy. For some patients, samples were also obtained after the second

infusion and daily for 5 days prior to and at the end of clofarabine infusion in some

patients. All samples (10 ml) were collected in green stopper vacutainer tubes

containing heparin and 1 µM deoxycoformycin (obtained from the NCI, Bethesda, MD)

to inhibit potential deamination of clofarabine by adenosine deaminase. The tubes were

immediately placed in an ice-water bath and transported to the laboratory. Control

studies have demonstrated that normal and leukemia cells are stable under these

conditions with respect to size and membrane integrity. The cellular nucleotide content

is stable for at least 15 hours under these conditions.(25)

Plasma Pharmacology

The plasma was removed after centrifugation of peripheral blood samples and stored at

-70oC until analyses were done. Human plasma samples (100 µL) containing

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

9

clofarabine were spiked with cladribine as the internal standard. Samples were

precipitated, evaporated, and reconstituted with mobile phase. The samples were

analyzed by reversed phase high performance liquid chromatography using a tandem

quadrupole mass spectrometer by a modified previously described procedure(26) and

analyzed at MicroConstants, Inc. (San Diego, CA). Authentic clofarabine standard was

used for assay validation, and for identification and quantitation of the nucleoside in

plasma. The assay has a sensitivity range from 10 to 5000 ng/mL and the CV for

accuracy and precision was less than 7%.

Cellular Pharmacology

Cell pellets from blood samples were diluted with phosphate-buffered saline, and

mononuclear cells were isolated using Ficoll-Hypaque density-gradient step-gradient

centrifugation procedures described previously.(26) A Coulter electronics channelizer

(Coulter Corporation, Hialeah, FL) was used to determine the mean cell volume. After

being washed with phosphate-buffered saline, cells were processed for nucleotide

extraction. Normal nucleotides and clofarabine triphosphate were extracted from cells

using standard procedures with HClO4. Triphosphates were separated on an anion-

exchange Partisil-10 SAX column (Waters Corporation, Milford, MA) using HPLC as

previously described.(27) The intracellular concentration was calculated and expressed

as the quantity of nucleotides contained in the extract from a given number of cells of a

determined mean volume. This calculation assumes that nucleotides are uniformly

distributed in total cell water. In general, the lower limit of quantitation of this assay was

about 1 pmol in an extract of 2 x 107 cells, corresponding to a cellular concentration of

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

10

about 0.2 µM. For many samples as much as 8 x 107 cell-equivalents were analyzed to

detect and quantitate the peak.

Statistical Considerations

Survival was calculated from date of start of therapy until death. Remission duration

was calculated from the date of initial response until disease recurrence. Survival and

remission duration were plotted by the method of Kaplan and Meier. Toxicity was

graded according to the NCI Common Toxicity Criteria, Version 2.0.(28)

Exponential decay of clofarabine in plasma was obtained using the Prism software

program (GraphPad Software, Inc., San Diego, CA). Comparison of clofarabine

triphosphate values in responders and non-responders was done by student’s unpaired

1-tailed t-test.

RESULTS

Study Group

A total of 62 patients were treated on study. Their characteristics are shown in Table 1.

The median age was 54 years (range 19 to 82 years). Thirty-nine patients had AML or

high risk MDS; 25 of them were in first salvage: first CR duration of less than 12

months in 17 patients, and more than 12 months in 8 patients. Karyotypic studies

showed unfavorable chromosomal abnormalities in 9. Eleven patients had Ph-positive

CML in blastic phase, and 12 had ALL.

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

11

Response and Outcome

Overall, 20 patients (32%) achieved complete response, 1 had a PR (2%) and 9 (15%)

had hematologic improvement or CRp, for an overall objective response rate of 48%

(Table 2). Response by disease category and by subset analysis are shown in Table 3.

In AML, 13 of 31 patients achieved CR (42%) and 4 had CRp (13%) for an overall

objective response rate of 55%. Response rates were higher in patients with longer first

CR durations. In MDS, 4 of 8 patients (50% had objective responses (2 CRs, 2 CR- p).

Among the 39 patients with AML or MDS, the objective response rate by karyotype was:

diploid or favorable - 9 CR + 2 CRp/21 (52%); unfavorable – 6 CR + 4 CRp/18 (56%).

Among the 15 patients with AML or MDS achieving CR, 7 had chromosomal

abnormalities at the start of therapy (Patients 1, 4, 6, 11, 12, 13, and 19; Table 4). Six

of them were analyzed at CR by cytogenetic studies: all 6 showed reappearance of a

normal karyotype. In CML blastic phase, 7 patients (58%) achieved objective

responses: 4 CHR, 2 CRp, 1 PR. Suppression of Ph-positive cells to 0% was also

noted in 3 patients, lasting for 1 to 3 months. Pertinent details of responding patients

are shown in Table 4. Patient 7 (Table 4) had previously received clofarabine on the

Phase I studies and achieved a CR lasting for 9 months. Upon relapse, she was

offered clofarabine therapy again on this phase II study, and achieved a CR lasting for

5.5 months.

Response duration and survival are shown in Figure 2. Among the 20 patients

achieving CR, 16 achieved CR after 1 course and 4 required 2 or more courses to

achieve CR. Among the 10 patients achieving PR or CR-p, 1 required 2 or more

courses to achieve the response.

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

12

Side Effects

Common side effects with clofarabine have included transient liver dysfunction, skin

rashes and palmoplantar erythrodysesthesia, and mucositis. Side effects by grade are

shown in Table 5. Although grade 3-4 liver dysfunction was common, it was transient,

starting around day 5-6 of therapy and subsiding by day 10 to 15.

Myelosuppression-associated complications included febrile episodes in 45/62 patients

(73%): fever of unknown origin in 14 patients (23%), and documented infections in 31

patients (50%). Some patients had multiple febrile episodes in any particular course.

While 5 patients died during induction (2 early deaths, 3 induction deaths; Table 2),

none was related to drug toxicities. All deaths were attributed to leukemia or to

myelosuppression associated complications.

Plasma Pharmacokinetics

Our previous Phase I study suggested that there was a dose-proportionality between

plasma concentration of clofarabine at dose range of 2 mg/m2 to 55 mg/m2 (24). At 40

mg/m2, the median plasma clofarabine level was 1.5 µM (n =7). Among the 29 patients

studied in the current trial, data after end of first infusion of clofarabine were available

from 25 patients. There was heterogeneity for accumulation of plasma clofarabine; the

median value was 1.0 µM (range 0.26 – 1.94 µM). To determine the variability in

elimination kinetics of the drug from plasma, clofarabine levels were quantified at

different time points after infusion. As presented in Figure 3, for 5 patients, clofarabine

appeared to be eliminated in a biphasic manner with a faster kinetics during the first 6

hours followed by a slower kinetics up to 24 hours. Because the data points are sparse,

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

13

elimination half-lives were not calculated. Nonetheless, there was a measurable

clofarabine concentration at 24 hours. Among the 26 patients, the median

concentration of plasma clofarabine at 24 hours was 0.038 µM (range 0.013 – 0.11).

When applied to leukemia cell lines in vitro such concentrations caused greater than

50% loss of clonogenicity.(22).

Cellular Pharmacokinetics

Similar to plasma clofarabine levels, concentration of clofarabine triphosphate (the

cellular cytotoxic metabolite) measured in circulating leukemia blasts varied among

patients; the median value was 15 µM with a range of <1 µM to 44 µM (n =29). The

variability in the level of clofarabine triphosphate appeared similar in different

diagnoses, with a <1 – 18 µM in ALL (n = 6), 6 – 26 µM in CML (n = 10), and 5 – 44 µM

in AML (n = 12). The concentration of cellular clofarabine triphosphate is a combination

of the phosphorylation of drug to its triphosphate level and the cellular retention of the

triphosphate. Therefore, we sought to determine if there was a relationship between the

levels of plasma clofarabine and the concentration of the triphosphate (Figure 4). As

shown in Figure 4, there was heterogeneity for both the cellular levels of clofarabine

triphosphate and plasma levels of clofarabine. However, linear and non-linear

regression analyses suggested no relationship between these two parameters.

Retention of clofarabine triphosphate was measured after the end of the first infusion.

The intracellular clofarabine triphosphate value was increased about 10% in each case

for up to 2 hours (Figure 5), suggesting that the cells were still phosphorylating the

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

14

available plasma clofarabine (see Figure 3). The limited data points 4 hours after the

end of infusion indicated that clofarabine triphosphate was maintained in the leukemia

cells with an elimination half-life of 24 hours or more.

These elimination profiles indicated that, in many patients, there would be residual

triphosphate at 24 hours when a second infusion of clofarabine was administered. In

such situations, the intracellular clofarabine triphosphate value would increase with

each infusion. To test this, clofarabine triphosphate values were quantified after each

infusion of clofarabine for 3 to 4 days (Figure 6). As shown in two patients, after the first

infusion, the intracellular level of clofarabine triphosphate accumulated to about 30 µM.

Because of a differential elimination profiles of the analog triphosphate, the levels of

clofarabine triphosphate following subsequent daily infusions varied in each patient. In

one patient, levels increased marginally and, after a total of 4 infusions, the value was

40 µM (Figure 6A). In contrast, the elimination kinetics of clofarabine triphosphate was

slower in the next patient (Figure 6B) resulting in about 65 µM of the analog

triphosphate after 3 infusions. These data suggested an incremental increase in

clofarabine triphosphate concentration that was related to its rate of elimination.

Because the two patients in Figure 6 responded differently to clofarabine treatment:

non-responder (Figure 6A) and achiever of complete remission (Figure 6B), we studied

whether there were differences in the intracellular pharmacokinetics of clofarabine

triphosphate in responders versus non-responders. Of the 29 patients studied for

pharmacokinetic analyses, 16 were responders (CR or CRp), 11 were non-responders,

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

15

and 2 were early deaths. First, the median triphosphate value at the end of infusion

(eoi) of clofarabine on the first day (one hr after start of therapy; day 1 eoi in Figure 7A,

B) was higher in responders (18 µM, range, 5 – 44 µM, n = 16) compared to non-

responders (10 µM , range <1 – 23 µM, n = 11; p = 0.032). Second, the 24-hour value

(23 hours after the end of the first clofarabine infusion or just prior to the second

infusion) in patients who responded to therapy was higher (11 µM, range, <1 – 44 µM, n

= 15) than in non-responders (5 µM, range, <1 – 20 µM, n = 10; p = 0.039). Third, as

illustrated for individual patients, there was a trend for an increase in the end of infusion

value from d1 to d2 in responders (Figure 7A) compared with non–responders (Figure

7B). Overall, the median concentration of clofarabine triphosphate at the end of second

infusion (25 hr after start of therapy) was 30 µM (range <1 – 67, n = 15) for patients who

responded to therapy and 9 µM (range <1-23 µM, n = 10) for those who failed the

treatment (P = 0.015). Finally, when plotted for the fold- increase in clofarabine

triphosphate value, responders had a median 1.8-fold increase compared to non-

responders who did not increase the concentration of clofarabine triphosphate from day

1 to day 2 (Figure 7C, p = 0.008). Taken together, these data suggested an overall

greater accumulation, longer duration of analog triphosphate retention, and incremental

increase in clofarabine triphosphate during the 5-day therapy in patients who benefited

from clofarabine treatment.

DISCUSSION

In this phase II study of clofarabine, given at 40 mg/m2 daily for 5 days per course in

acute leukemia, we observed a significant antileukemic activity in AML, MDS, and CML-

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

16

BP, and modest activity in ALL. Overall, 30 of the 62 patients (48%) achieved either

CR, CRp, or PR. In AML and high risk MDS, the CR rates were 43% and 29%,

respectively with an objective response rate of about 50%. In CML-BP 7 of 11 patients

treated (64%) experienced objective responses. In ALL, 2 of 12 patients (17%)

responded. The experience in AML was particularly impressive with response rates of

87% and 67% respectively in patients with long first CR duration or those treated with

clofarabine as second or subsequent salvage therapy.

In addition to these encouraging response rates, the toxicity profile of clofarabine was

noteworthy. Side effects were tolerable and mostly reversible. Severe but transient

liver dysfunction was noted in 15% to 25%, while skin rashes and hand-foot syndrome

were observed in 10% to 15% of patient population. Myelosuppression associated

complications were similar to other salvage programs in leukemia. Of importance, is the

activity of clofarabine in acute leukemia at doses not associated with severe

extramedullary side effects, particularly neurotoxicity.

In the Phase I study of fludarabine in acute leukemia (9, 10), doses of 125 mg/m2 or more

given intravenously (IV) over 1 hour daily for 5 days were associated with severe

neurotoxicities including mental changes, coma and blindness. Interestingly, 4 of 9

patients with acute leukemia treated at these doses achieved complete remission.(9)

However, because of the severe and fatal extramedullary toxicities, fludarabine was

subsequently developed at dose schedules of 20 to 30 mg/m2 IV daily for 5 days in

phase II studies and demonstrated marked activity in CLL and other lymphoproliferative

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

17

disorders. Similar renal and nervous system toxicities were observed with cladribine (11)

in the acute leukemia studies in adults, although this treatment is efficacious in pediatric

acute leukemias.29 Our results showed that, in contrast to these other deoxyadenosine

analogues, clofarabine was active in acute leukemia at doses well tolerated by patients.

Cellular pharmacokinetic studies suggested that the accumulation and especially

retention of clofarabine triphosphate were favorable in leukemia cells of responding

patients. For instance, there was a 1.8-fold increase in the level of clofarabine

triphosphate in the blasts of responders after the second infusion (Figure 7). This

demonstrates that leukemia blasts have an additional capacity to accumulate the active

triphosphate. Indeed, after 5 days of therapy, a gradual and daily increase of the

clofarabine triphosphate was observed in blasts of a responder (Figure 6). Prospective

validation of this relationship between clinical response and cellular pharmacokinetics

may form a rational basis for the design of alternative dose-schedules and combinations

with other active agents.

Purine nucleoside analogs have been highly effective in indolent lymphocytic leukemias.

(5-7) In these hematological malignancies, there was also a prolonged retention of

analog triphosphates. For example, the elimination half-lives of ara-G triphosphate,

fludarabine triphosphate, and cladribine triphosphate were >24, >24, and 10 hours,

respectively (6, 30, 31). This positive feature may in part be associated with the favorable

response in indolent leukemias. However, in circulating leukemia blasts of patients with

relapsed acute leukemias, the elimination half-life of fludarabine triphosphate was much

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

18

faster (t1/2 = 7 hours).(32) The peak levels of triphosphate at the maximum tolerated

dose was a median 40 µM. (32) With these kinetic profiles, the pharmacodynamic effect

will be greatly diminished prior to the subsequent fludarabine dose. For cladribine

triphosphate, the information in acute leukemia was very limited. Nonetheless, the

accumulation of triphosphate in blasts either after continuous infusion or bolus

administration was less than 5 µM.(34, 35) Thus incremental daily increases of

clofarabine triphosphate and its prolonged retention, in comparison to other

deoxyadenosine nucleoside analogues, may account in part for its beneficial clinical

effect in acute leukemias.

In a phase II pediatric study,(36) 22 evaluable children were treated at our institution: 5

(23%) achieved CR and 8 (36%) had an objective response. Phase II multi institutional

studies are currently ongoing in both adult and pediatric acute leukemia (37,38). The

results from these studies should be available in final forms in the near future.

An important extension of the current trial is the rational future combination of

clofarabine based on laboratory results and clinical experience. Both clofarabine and

cytarabine are active in adult acute leukemia, and target two different DNA sites.(39) The

pharmacokinetic profile of clofarabine is favorable compared to other deoxyadenosine

analogs, and in the preclinical settings, pre-treatment with clofarabine increases cells

ability to accumulate ara-CTP through a biochemical modulation strategy. (40) Hence,

one promising approach is to sequentially combine clofarabine with cytarabine, as had

been done with fludarabine and cladribine.(33,35) We are currently testing the

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

19

biochemical modulation strategy in circulating leukemic cells from patients receiving

such combination therapy with clofarabine and cytarabine. Another approach is to use

clofarabine as an inhibitor of excision DNA repair elicited by DNA damaging agents (41).

This could be done in a combination of clofarabine with anthracyclines or alkylating

agents.

In summary, clofarabine is the first deoxyadenosine analogue which has promising

activity in adult acute leukemia at doses that are well-tolerated. A favorable

pharmacokinetic profile of clofarabine triphosphate was associated with response.

Mechanism-based combinations of clofarabine with other active agents are planned

based on its toxicity profile, pharmacokinetic properties, and clinical activity.

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

20

Table 1. Characteristics of the study group (62 patients).

Parameter Category No. (%)

• Age (years) ≥60 23 (38)

• Diagnosis AMLSalvage 1; CRD1<12 mos

CRD1≥12 mosSalvage ≥ 2

31 (50)11 (18)8 (13)

12 (19)

MDSSalvage 1, CRD1<12 mosSalvage ≥ 2

8 (13)6 (10)2 (3)

CML – blastic phaseSalvage 1Salvage ≥ 2

11 (18)7 (11)4 (6)

ALLSalvage 1Salvage ≥ 2

12 (19)4 (6)

8 (13)

• Karyotype AML – MDSfavorablediploid

unfavorable

1 (2)20 (32)18 (29)

CML- Ph onlyPh ± other

4 (6)7 (11)

ALL Ph ± otherdiploid

2 (3)1 (2)

other 9 (15)

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

21

Table 2. Overall Response (62 patients).

Response No. (%)

Complete response

Partial response

20 (32)

1 (2)

Hematologic improvement (CR-p) 9 (15)

Early death (within 2 weeks) 2 (3)

Induction death 3 (5)

Primary resistance 11 (18)

Secondary resistance 16 (26)

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

22

Table 3. Response by disease category, salvage number and duration of first CR.

No. (%)

DiseaseSalvage No.

CRD1(Mos.)

No. treated CR CR-p/PR Overall

AML First ≤12 11 2 (18) 0 2 (18)>12 8 6 (75) 1 (13) 7 (87)

≥Second ≤12 4 2 (50) 1 (25) 3 (75)>12 8 3 (38) 2 (25) 5 (63)

MDS First 6 2 (33) 1 (17) 3 (50)≥Second 2 0 (0) 1 (50) 1 (50)

CML blastic phase First 7 3 (43) 1 (14) 4 (57)≥Second 4 1 (25) 1/1 (50) 3 (75)

ALL 12 1 (8) 1 (8) 2 (17)

CRD1 – Duration of first complete response

.

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

23

Table 4. Characteristics of responders.

Patient Age/Gender Diagnosis SalvageCRD1; CRD2; CRD3 (mos) Prior therapy Karyotype

Response/Duration

(mos)1

63/M AML 2 3.5; 5 Thalidomide x 2 deletion 5q CR/7.5+2

61/M AML 1 10.5 CAT + ATRA diploid CR/53

73/F AML 1 13.5 IA diploid CR/24

53/M AML 1 0 FA + mylotarg multiple CR/45

32/F AML 1 29 CAT + GCSF diploid CR/10.5+6

65/M AML 1 32 CAT + GCSFt( 10;11)

(p13;q21) CR/5.57

75/F AML 3 0; 5; 9 CAT; mylotarg; clofarabine

diploid CR/5.5

860/M AML 1 16 IA diploid CR/8+

922/M AML 1 30 IA diploid CR/11+

1058/M AML 2 18; 18

CAT + GCSFtroxatyltopotecan diploid CR/4

1146/M AML 2 24; 22

IA; FAM;MUD SCT in CR

t(11;19) +other CR/3.5+

1274/M AML 2 11; 0 IA; Gelonin

t(9;11), deletion 7q-, trisomy 8 CR/7

1358/M AML 1 32.5 IA trisomy 8 + other CR/2.5+

F

or personal use only. by guest on June 7, 2013.

bloodjournal.hematologylibrary.org

From

24

Patient Age/Gender Diagnosis SalvageCRD1; CRD2; CRD3 (mos) Prior therapy Karyotype

Response/Duration

(mos)14

34/M CML-BP 1 7 *imatinib Ph + otherCR/7 (died in

CR)15

46/M CML-BP 1 NA*IFN + LDAC;

imatinibDouble Ph, trisomy 8,

isochromosome 17CR/3 (died in

CR)16

67/M CML-BP 2 3 HCVAD + imatinib Ph CR/717

46/M CML-BP 1 NA None Ph CR/6+18

61/F MDS 1First Rx for

MDS†COAP; IFN + LD

AC; 9NC diploid CR/5+19

70/M MDS 1 12 IA t(5;12) CR/220

61/F AML 2 8; 7IA; Mylotarg +

ara-C t(8; 21) CR-p/3.521

57/F AML 2 27; 30CAT + ATRA; lipo

DNR + HDAC t(3;21)+ other CR-p/222

28/F AML 2 12; 6.5IA; HDAC; MUD

in CR diploid CR-p/4.523

76/M AML 1 20.5 IA trisomy 8; deletion 20q-

CR-p/1

2439/M CML-BP 2 0

*IFN + Hydrea; allo SCT;

IA + imatinibPh; trisomy 8 CR-p/5

2537/F CML-BP 1 NA

*PEG IFN + LDAC; imatinib; Schering 66336

Ph + other CR-p/5

2660/F MDS 2 0

DNR + HDAC; Mylotarg

chromosome 5 and 7 abnormalities CR-p/1.3

2782/F MDS 1 9 IA

chromosome 5 and 7 abnormalities CR-p/2

F

or personal use only. by guest on June 7, 2013.

bloodjournal.hematologylibrary.org

From

25

Patient Age/Gender Diagnosis SalvageCRD1; CRD2; CRD3 (mos) Prior therapy Karyotype

Response/Duration

(mos)28

31/M ALL 1 0 VAD Ph - monosomy 7 CR/429

23/M ALL 3 20.5; 3; 0CALGB ALL

therapy; allo SCT; IA;, asparaginase

+ VP

multiple CR-p/1

3042/M CML-BP 2 14 imatinib Ph PR/1

* Therapy for chronic phase CML; + prior history of Ph-negative CML

Abbreviations: M = male; F = female; AML = acute myeloid leukemia; CML-BP = chronic myeloid leukemia in blastic

phase; MDS = myelodysplastic syndrome; ALL = acute lymphoid leukemia; CRD1 = duration of first complete response; CAT

= cyclophosphamide, ara-C, topotecan; ATRA = all transretinoic acid; IA = idarubicin + high-dose ara-C; BID FA =

fludarabine + ara-C; GCSF = granulocyte colony stimulating factor; FAM = fludarabine, ara-C, mitoxantrone; MUD SCT =

matched unrelated donor stem cell transplantation; IFN = interferon alpha; LD AC = low-dose ara-C; HCVAD =

hyperfractionated cyclophosphamide, vincristine, Adriamycin, dexamethasone; allo SCT = allogeneic stem cell transplant;

DNR = daunorubicin; VP = vincristine + prednisone

F

or personal use only. by guest on June 7, 2013.

bloodjournal.hematologylibrary.org

From

26

Table 5. Side effects (62 patients).

Side EffectGrade 1-2

No. (%)Grade 3-4

No. (%)

A. Non-Hematologic• Liver dysfunction

elevated bilirubinelevated transaminases

22 (37)31 (50)

9 (15)15 (24)

• Skin rashes 23 (37) 6 (10)• Palmoplantar erythrodysesthesia 5 (8) 7 (11)• Mucositis 9 (15) 0• Diarrhea 13 (21) 0• Nausea/vomiting 40 (65) 2 (3)• Other 42 (69) 7 (11)

B. Hematologic• Median time to recovery (20 patients in CR)

→platelets > 100 x 109/L→granulocytes > 109/L

31 days28 days

• No. (%) febrile episodes→unknown origin→documented infections

• bacterial/sepsis

19 (31)

16 (26)

• fungal 3 (5)• pneumonia (includes fungal) 12 (19)• mixed 3 (5)• minor/other 4 (7)

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

27

References

1. Mayer RJ, Davis RB, Schiffer CA, et al. Intensive post remission

chemotherapy in adults with acute myeloid leukemia. Cancer and Leukemia

Group B. N Engl J Med 331:896-903, 1994.

2. Fosså A, Santoro A, Hiddemann W, Truemper L, Niederle N, Buksmaui S,

Bonadonna G, Seeber S, Nowrousian M. Gemcitabine as a single agent in

the treatment of relapsed or refractory aggressive non-Hodgkin’s lymphoma.

J Clin Oncol 17:3786-3792, 1999.

3. Silverman LR, Demakos EP, Bercedis L, et al. A randomized controlled trial

of azacitidine in patients with myelodysplastic syndrome: a study of the cancer

and leukemia group B. J Clin Oncol, 20:2429-2440, 2002.

4. Wijermans P, Lübbert M, Verhoef G, Bosly A, Ravoet C, Andre M, Ferrant A.

Low-dose 5-aza-2´-deoxycytidine, a DNA hypomethylating agent, for the

treatment of high-risk myelodysplastic syndrome: a multicenter phase II study

in elderly patients. J Clin Oncol 18:956-962, 2000.

5. Goodman GR, Burian C, Koziol JA, Saven, A. Extended follow-up of patients

with hairy cell leukemia after treatment with cladribine. J Clin. Oncol. 21:891-

896, 2003.

6. Gandhi, V., Plunkett, W., Weller, S., et al. Evaluation of the Combination of

Nelarabine and Fludarabine in Leukemias: Clinical Response,

Pharmacokinetics and Pharmacodynamics in Leukemia Cells. J. Clin. Oncol.,

19:2142-2132,2001.

7. Keating MJ, O'Brien S, Lerner S, et al. Long-term follow-up of patients with

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

28

chronic lymphocytic leukemia (CLL) receiving fludarabine regimens as initial

therapy. Blood, 92:1165-1171, 1998.

8. Estey E, Thall P, Andreeff M et al. Use of granulocyte colony-stimulating

factor before, during, and after fludarabine plus cytarabine induction therapy

of newly diagnosed acute myelogenous leukemia or myelodysplastic

syndromes: comparison with fludarabine plus cytarabine without granulocyte

colony-stimulating factor. J Clin. Oncol. 12:671-678, 1994.

9. Warrell R, Berman E. Phase I and II study of fludarabine phosphate in

leukemia: therapeutic efficacy with delayed central nervous system toxicity. J

Clin Oncol 4:74-79, 1986.

10. Spriggs DR, Stopa E, Mayer RJ, Schoene W, Kufe DW. Fludarabine

phosphate (NSC 312878) infusions for the treatment of acute leukemia:

phase I and neuropathological study. Cancer Res 46: 5953-5958, 1986.

11. Vahdat L, Wong ET, Wile MJ, Rosenblum M, Foley KM, Warrell RP Jr.

Therapeutic and neurotoxic effects of 2-chlorodeoxyadenosine in adults with

acute myeloid leukemia. Blood 84: 3429-3434, 1994.

12. Montgomery JA, Shortnacy-Fowler A T, Clayton SD, Riordan JM, Secrist JA

III. Synthesis and biologic activity of 2'-fluoro-2-halo derivatives of 9-β-D-

arabinofuranosyladenine. J Med Chem; 35:397-401, 1992.

13. Avramis, V.I. and Plunkett, W. 2-Fluoro-ATP: a toxic metabolite of 9-β-D-

arabinofuranosyl-2-fluoroadenine. Biochem Biophys Res Comm 113:35-43,

1983.

14. Avramis, V.I. and Plunkett, W. Metabolism of 9-β-D-arabinofuranosyl-2-

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

29

fluoroadenine 5'-phosphate by mice bearing P388 leukemia. Cancer Drug

Delivery 1:1-10, 1993.

15. Huang, P. and Plunkett, W. Phosphorolytic cleavage of 2-fluoroadenine from

9-β-D-arabinofuranosyl-2-fluoroadenine by Escherichia coli: a pathway for 2-

fluoro-ATP production. Biochem Pharm 36:2945-2952, 1987.

16. Parker WB, Allan PW, Hassan AE, Secrist JA 3rd, Sorscher EJ, Waud WR.

Antitumor activity of 2-fluoro-2'-deoxyadenosine against tumors that express

Escherichia coli purine nucleoside phosphorylase. Cancer Gene Ther 10:23-

9, 2003.

17. Carson DA, Wasson DB, Esparza LM, Carrera CJ, Kipps TJ, Cottam HB.

Oral antilymphocyte activity and induction of apoptosis by 2-chloro-2'-arabino-

fluoro-2'deoxyadenosine. Proc Natl Acad Sci USA; 89:2970-2974, 1992.

18. Parker WB, Shaddix SC, Chang CH, et al. Effects of 2-chloro-9-(2-deoxy-2-

fluoro-β-D-arabinofuranosyl) adenine on K562 cellular metabolism and the

inhibition of human ribonucleotide reductase and DNA polymerases by its 5’-

trisphosphate. Cancer Res 51:2386-2394, 1991.

19. Parker WB, Shaddix SC, Rose LM., et al. Comparison of the mechanism of

cytotoxicity of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine, 2-

chloro-9-(2-deoxy-2-fluoro-β-D-ribofuranosyl)adenine, 2-chloro-9-(2-deoxy-

2,2-difluoro-β-D-ribofuranosyl)adenine in CEM cells. Mol Pharmacol 55:515-

520, 1999.

20. Plunkett, W. Gandhi V. Purine and Pyrimidine Nucleoside Analogs. Cancer

Chemotherapy and Biological Response Modifiers, Annual 19, Giaccone G,

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

30

Schilsky R, Sondel P, Eds. Elsevier Science, B.V., Amsterdam, pp 21-45,

2001.

21. Parker WB, Bapat AR, Shen JX, Townsend AJ, Cheng YC. Interaction of 2-

halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases,

DNA primase, and ribonucleotide reductase. Mol Pharmacol 34:485-91,

1988.

22. Xie C, Plunkett W. Metabolism and actions of 2-chloro-9-(2-deoxy-2-fluoro-β-

D-arabinofuranosyl)adenine in human lymphoblastoid cells. Cancer Res;

55:2847-2852, 1995.

23. Xie KC, Plunkett W. Deoxynucleotide pool deletion and sustained inhibition of

ribonucleotide reductase and DNA synthesis after treatment of human

lymphoblastoid cells with 2-chloro-(2-deoxy-fluoro-β-D-

arabinofuranosyl)adenine. Cancer Res; 56:3030-3037, 1996.

24. Kantarjian H, Gandhi V, Kozuch P, et al. Phase I clinical and pharmacology

study of clofarabine (CI-F-ara-A; 2 chloro-2’-fluoro-deoxy-9-beta-D-

arabinofuranosyladenine) in patients with solid and hematologic cancers. J

Clin Oncol. 21:1167-1173, 2003.

25. Plunkett W, Hug V, Keating M, et al: Quantitation of 1-ß-D-

arabinofuranosylcytosine 5'-triphosphate in the leukemic cells from bone

marrow and peripheral blood of patients receiving 1-ß-D-

arabinofuranosylcytosine therapy. Cancer Res 40:588-591, 1980.

26. Gandhi V, Chen W, Ayres M, et al. Plasma and cellular pharmacology of 8-

chloro-adenosine in mice and rats. Cancer Chemother Pharmacol. 50:85-94.

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

31

2002.

27. Rodriguez CO Jr, Plunkett W, Paff, MT, et al: A high-pressure liquid

chromatography method for the determination and quantitation of

arabinosylguanine triphosphate and fludarabine triphosphate in human cells.

J. Chromatography B 745:421-430, 2000.

28. National Cancer Institute Common Toxicity Criteria. Version 2, 1998.

29. Krance RA, Hurwitz CA, Head DR, et al. Experience with 2-

chlorodeoxyadenosine in previously untreated children with newly diagnosed

acute myeloid leukemia and myelodysplastic diseases. J Clin Oncol.

19:2804-2811, 2001

30. Gandhi, V., Kemena, A., Keating, M.J., and Plunkett, W. Cellular

pharmacology of fludarabine triphosphate in chronic lymphocytic leukemia

cells during fludarabine therapy. Leukemia Lymphoma 10:49-56, 1993.

31. Albertioni F, Lindemalm S, Reichelova V, et al. Pharmacokinetics of cladribine

in plasma and its 5'-monophosphate and 5'-triphosphate in leukemic cells of

patients with chronic lymphocytic leukemia. Clin Cancer Res ;4:653-658,

1998.

32. Kemena, A., Gandhi, V., Shewach, D., Keating, M. J., and Plunkett, W.

Inhibition of fludarabine metabolism by arabinosylcytosine during therapy.

Cancer Chemother. Pharmacol. 31:193-199, 1992.

32. Gandhi, V., Estey, E., Keating, M. J. and Plunkett, W. Fludarabine potentiates

metabolism of arabinosylcytosine in patients with acute myelogenous

leukemia during therapy. J. Clin. Oncol. 11:116-124, 1993

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

32

33. Crews, K.R., Gandhi, V., Srivastava, D.K., et al. An Interim Analysis of a

Continuous Infusion versus a Short Daily Infusion of Cytarabine Given in

Combination with Cladribine for Pediatric Acute Myeloid Leukemia. J. Clin.

Oncol.,20:4217-4224, 2002

34. Gandhi, V., Estey, E., Keating, M.J., Chucrallah, A., and Plunkett, W.

Chlorodeoxyadenosine and arabinosylcytosine in patients with acute

myelogenous leukemia: Pharmacokinetic, pharmacodynamic, and molecular

interactions. Blood 87:156-164, 1996.

35. Jeha, S, Gandhi V, Chan KW, et al. Phase I study of Clofarabine (Clofarex)

in pediatric leukemia. Blood. 100:86a, 2002.

36. Foran J, Wetzler M, Kantarjian H, et al. A phase II, open-label study of

Clofarex in adult patients with refractory or relapsed acute myelogenous

leukemia. Blood. 100:271b, 2002.

37. Jeha S, Steinherz P, Altman A, et al. A phase II, open-label study of

Clofarex in pediatric patients with refractory or relapsed acute-myelogenous

or lymphoblastic leukemia. Blood. 100:271b, 2002.

38 Gandhi V., Huang, P., Chapman, A. J., et al. Incorporation of fludarabine-

and arabinosylcytosine-5'-triphosphates by DNA polymerase α: affinity,

interaction, and consequences. Clin. Cancer Res., 3:1347-1355, 1997

39 Cooper, T., Nowak, B., and Gandhi, V. Biochemical modulation of cytarabine

triphosphate by clofarabine. Proc. AACR, 44: 142, 2003.

40 Yamauchi, T., Nowak, B., Keating, M.J., and Plunkett, W. DNA repair initiated

in chronic lymphocytic leukemia lymphocytes by 4-

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

33

hydroperoxycyclophosphamide is inhibited by fludarabine and clofarabine.

Clin. Cancer Res.,7:3580-3589, 2001.

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

34

FIGURE LEGENDS

Figure 1: Structures of fludarabine, cladribine, and clofarabine.

Figure 2: Response duration and survival with clofarabine.

Figure 3: Plasma pharmacokinetics after first infusion of clofarabine from 5 patients.

Blood samples were collected prior to, at the end of infusion, and at indicated

times on the first day of therapy. Clofarabine levels were measured in plasma as

described.

Figure 4: Relationship between plasma levels of clofarabine and cellular

accumulation of clofarabine triphosphate at the end of first infusion. Blood

samples obtained at the end of infusion from 25 patients were processed to

analyze both plasma levels of clofarabine and clofarabine triphosphate in

leukemia cells.

Figure 5: Accumulation and elimination profiles of clofarabine triphosphate in 3

patients. Blood samples obtained at different times after first infusion of

clofarabine were processed to isolate leukemia cells and to analyze the

concentration of clofarabine triphosphate in these cells.

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

35

Figure 6: Incremental accumulation of clofarabine triphosphate in 2 representative

patients. Pretreatment and end of infusion samples were obtained from two

patients after several infusions of clofarabine to quantitate intracellular levels of

triphosphate. Rectangular bars on the abscissa, clofarabine infusions.

Figure 7: Clofarabine triphosphate pharmacokinetics in responders and non-

responders. The analogue triphosphate levels were compared at the end of

infusion [eoi] on day 1 (d1, eoi) and day 2 (day 2, eoi) in patients who were

responders (Fig. 7 A) or nonresponders (Fig. 7 B). The increase in clofarabine

triphosphate value was determined in these patients after calculating and plotting

the ratio of clofarabine triphosphate on day 2 versus day 1 (Fig. 7 C).

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

36

Fludarabine Cladribine Clofarabine

Figure 1

N

N N

N

O-O3PO

HO

F

HO

N

N N

NNH2

OHO

HO

N

N N

NNH2

OHO

HO

ClCl

F

NH2

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

37

Figure 2

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

38

Figure 3

0 6 12 18 240.01

0.1

1

10

Hours

Clo

fara

bine

,µM

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

39

Figure 4

0.0 0.5 1.0 1.5 2.0 2.5

0

10

20

30

40

Clofarabine, µM

Clo

fara

bine

trip

hosp

hate

, µM

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

40

Figure 5

0 6 12 18 24

0

10

20

30

Hours

Clo

fara

bine

tri

phos

phat

e, µ

M

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

41

A B

Figure 6

0 12 24 36 48 60 72

0

10

20

30

40

50

60

70

Hours

Clo

fara

bine

tri

pho

spha

te, µ

M

0 12 24 36 48 60 72

0

10

20

30

40

50

60

70

Hours

Clo

fara

bine

tri

pho

spha

te, µ

M

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

42

A B

Figure 7

d1-eoi d2-eoi0

10

20

30

40

50

60

Clo

fara

bin

e tr

iph

osp

hat

e,µµ µµM

d1-eoi d2-eoi0

10

20

30

40

50

60

Clo

fara

bin

e tr

iph

osp

hat

e,µµ µµM

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom

43

C

Figure 7

0

1

2

3

Respond Fail

d2/d

1 C

lofa

rabi

ne t

riph

osph

ate

For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom