Phase I Trial and Pharmacokinetic Study of High-Dose Clofarabine And

Home , Clofarabine

Phase I trial and pharmacokinetic study of high-dose clofarabine and busulfan and allogeneic stem cell transplantation in adults with high-risk and refractory acute leukemia

SS Farag1,2, LL Wood1,2, JE Schwartz1,2, S Srivastava1,2, RP Nelson Jr1,2, MJ Robertson1,2, R Abonour1,2, A Secrest2, E Cox2, J Baute2, C Sullivan2, K Kane2 and DR Jones3

1Division of Hematology and Oncology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; 2Blood and Bone Marrow Transplantation Program, Indiana University Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA and 3Division of Clinical Pharmacology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

We conducted a phase I trial to determine the maximum Although cyclophosphamide adds significant immunosuppres- tolerated dose (MTD) of clofarabine with high-dose busulfan sion,9 its dose-limiting cardiac toxicity and risks of hemorrhagic followed by allogeneic stem cell transplantation (SCT) in cystitis and hepatic sinusoidal obstruction syndrome remain patients with high-risk and refractory acute leukemia. Patients 6–8,10,11 received intravenous busulfan 0.8 mg/kg every 6 h on days À6 problematic. to À3 and clofarabine 30–60 mg/m2 per day on days À6toÀ2. Recently, the substitution of fludarabine for cyclophospha- Graft-versus-host disease prophylaxis included sirolimus plus mide in combination with targeted oral or intravenous busulfan tacrolimus (days À2to þ 180). A total of 15 patients, median has suggested that disease control equivalent to the BuCy2 age 48 (30–58) years, with acute leukemia that was relapsed and regimen can be achieved with reduced toxicity.12,13 Although refractory (n ¼ 8), primary refractory (n ¼ 6), or in CR2 (n ¼ 1), fludarabine has significant immunosuppression to prevent graft were treated at four clofarabine dose levels: 30 (n ¼ 3), 40 14 (n ¼ 3), 50 (n ¼ 3) and 60 mg/m2 per day (n ¼ 6) with busulfan. All rejection, fludarabine has only modest anti-leukemic activity. engrafted, and the MTD was not reached. Grades 3–4 non- Clofarabine (2-chloro-2-fluoro-deoxy-9-D-arabinofuranosyla- hematological toxicities included vomiting (n ¼ 3), mucositis denine) is a second generation purine nucleoside analogue (n ¼ 9), hand-foot syndrome (n ¼ 1), acute renal failure (n ¼ 1) designed to maintain the favorable qualities of fludarabine and reversible elevation of aspartate aminotransferase/alanine without dose-limiting neurotoxicity.15 In particular, single agent aminotransferase (n ¼ 10). The 1-year event-free survival was clofarabine is active in heavily pre-treated, relapsed and/or 53% (95% confidence interval: 33–86%), and the 1-year overall refractory patients with acute lymphoblastic leukemia and acute survival was 60% (95% confidence interval: 40–91%). Given the 16–18 good tolerability and promising results, we recommend clofar- myeloid leukemia, including older patients with high-risk 19,20 abine 60 mg/m2 per day Â 5 days as a phase II dose in features. In addition, in vitro studies have shown synergistic combination with busulfan (12.8 mg per kg total dose) for activity between clofarabine and alkylating agents.21 We, further study as a myeloablative regimen for allogeneic SCT for therefore, hypothesize that the substitution of clofarabine for high-risk acute leukemia. fludarabine, in combination with high-dose busulfan may be a Leukemia (2011) 25, 599–605; doi:10.1038/leu.2010.319; published online 21 January 2011 well-tolerated preparative regimen with improved anti-leukemic Keywords: allogeneic hematopoietic cell transplantation; activity for patients undergoing allogeneic HSCT. As an initial clofarabine; busulfan; acute leukemia step, we conducted a phase I clinical trial to determine the maximum tolerated dose (MTD) of clofarabine in combination with high-dose busulfan and allogeneic HSCT in patients with high-risk and refractory acute leukemia (ClinicalTrials.gov Identifier: NCT00477542). Introduction

Allogeneic stem cell transplantation (SCT) remains an important Patients and methods curative option for many patients with acute leukemia.1,2 Although reduced-intensity regimens appear to be better Patient and donor eligibility tolerated and allow older patients the opportunity for transplan- Patients were eligible if they had acute myeloid leukemia or tation, recent evidence indicates that dose-intensity is important acute lymphoblastic leukemia that was primary refractory, for disease control in spite of a graft-versus-leukemia effect, 4 3,4 relapsed and refractory ( 5% bone marrow blasts or extra- particularly in patients at high-risk of relapse. The combina- medullary disease), or who were in second or subsequent tion of cyclophosphamide with busulfan (BuCy2) is a commonly complete remission (CR). Patients also had to be 18–60 years used regimen for allogeneic HSCT in patients with acute X 5 6–8 old, have a Karnofsky performance status 70%, a left leukemia, although it is associated with significant toxicity. ventricular ejection fraction 445%, DLCO 450%, creatinine clearance 460 ml/min/1.73 m2, and serum bilirubin, aspartate Correspondence: Dr SS Farag, Division of Hematology and Oncology, aminotransferase and alanine aminotransferase (ALT) levels Department of Internal Medicine, Indiana University School of o2 Â upper limits of normal. Patients were excluded if Medicine, Walther Hall-R3, C414, 980 West Walnut Street, Indianapolis, they had a previous HSCT, central nervous system leukemia, IN 46202, USA. E-mail: [email protected] or if they were seropositive for HIV. Donors were eligible Received 30 August 2010; revised 25 October 2010; accepted if they were related and matched at 5 or 6 of the 6 3 December 2010; published online 21 January 2011 human leukocyte antigen-matched loci (human leukocyte High-dose clofarabine/busulfan in acute leukemia SS Farag et al. 600 antigen-A, -B and -DRB1), or unrelated and 10 of 10 antigen- relapse, or death from any cause; otherwise patients were matched (human leukocyte antigen-A, -B, -C, -DRB1 and censored on the date of last follow-up. Overall survival (OS) was -DQB1) by high-resolution typing. The study was approved by measured from the date of transplantation until death from any the Institutional Review Board of Indiana University. All patients cause, censoring for patients alive on date of last follow-up. and donors gave informed consent. The study was conducted in accordance with the Declaration of Helsinki. Pharmacologic assays and pharmacokinetic analysis Plasma clofarabine samples were obtained on day À6 before infusion, at the end of infusion, and at 2, 3, 4, 5, 6 and 24 h after Study design and treatment plan the start of dosing. In addition, samples were also collected Busulfan was administered at 0.8 mg/kg (lower of actual or ideal immediately before infusion, at the end of infusion and at 6 and body weight) intravenously over 2 h every 6 h on day À7toÀ4 24 h after subsequent doses on days À5toÀ2. Clofarabine was (16 doses), and the dose not adjusted according to pharmaco- extracted from plasma and quantified by high pressure liquid kinetic monitoring. Clofarabine was administered as a daily 1-h chromatography-tandem mass spectrometry (MS/MS) as intravenous infusion for 5 days on days À6toÀ2, with the dose previously described.26 escalated in four successive cohorts: 30, 40, 50 and 60 mg/kg For busulfan, blood was sampled in association with the first per day. On days À6toÀ4, when both drugs were administered and ninth infusion, and in each case collected immediately on the same day, clofarabine was infused 1–2 h before busulfan. before infusion, at the end of infusion, and at 135 and 150 min, Corticosteroids were not used before clofarabine infusion for and at 3, 4, 5 and 6 h after the start of infusion. Busulfan plasma prophylaxis of hand-foot syndrome. Bone marrow or PBSC were levels were assayed using gas chromatography–mass spectro- infused on day 0, and growth factors were not administered. metry, with a dynamic concentration range of 25–4500 ng/ml Sirolimus and tacrolimus were administered for graft-versus-host and coefficient of variation of o8%.27 disease (GvHD) prophylaxis, starting day À3 until day þ 100 to Concentration-time data were analyzed by a non-compart- maintain a serum level of 5–10 ng/ml for both drugs, followed by mental approach using WinNonlin v4.1 (Pharsight, Mountain tapering through day þ 180. All patients received fluconazole, View, CA, USA). The area under the plasma concentration acyclovir and ciprofloxacin prophylaxis. Preemptive treatment curve (AUC) was calculated for busulfan following the first with valganciclovir was instituted if cytomegalovirus activation (AUC ) and ninth (AUC ) doses. The following was detected on plasma monitoring in the first 100 days after 1st dose 9th dose pharmacokinetic parameters were calculated for clofarabine: transplantation. No specific therapy was used for prevention of maximum plasma concentration (C ), terminal elimination sinusoidal obstruction syndrome. max constant (k ), terminal half-life (t ) and AUC from time zero to A standard 3 þ 3 trial design was followed. Three patients el 1/2 the last concentration for each day of infusion, with the average were enrolled on the first clofarabine dose level. If one of the AUC of all doses of clofarabine also calculated (AUC ). three patients experienced dose limiting toxicity (DLT; defined av In addition, the area under the plasma concentration curve below) at a given dose level, three more patients were treated at extrapolated to infinity (AUC -N) was estimated from plasma that dose level. If no DLT was observed in any of the three 0 levels obtained following the first dose (day À6) and the patients or in one of the six patients, the dose was escalated to systemic clearance was calculated from the dose and AUC -N. the next level. If two or more patients experienced DLT, the 0 MTD was exceeded, and three additional patients were to be treated at the next lower dose. All patients assigned to a dose Statistical analysis level were followed until day þ 30 before dose escalation. The primary objective was to determine the MTD of clofarabine when combined with high-dose busulfan for allogeneic HSCT. Secondary objectives were safety and tolerability, response rate, Definitions EFS and OS. Summary statistics were used to describe patient Adverse events were graded using National Cancer Institute’s characteristics, times to engraftment, percent donor chimerism and Common Terminology Criteria for Adverse Events (NCI CTCAE response rates. EFS and OS were estimated using the Kaplan–Meier v3.0). DLT was defined as any grade 3 or 4 non-hematologic method. Safety was monitored by an independent Data Safety toxicity (except alopecia, nausea and vomiting, mucositis, Monitoring Board according to institutional requirements. fatigue and correctable biochemical abnormalities, as these are reversible and commonly observed toxicities following myeloablative chemotherapy) that did not resolve to pgrade Results 2 by day þ 30. The MTD was defined as the dose level at which two out of six patients experienced DLT. Patient characteristics Neutrophil engraftment was defined as recovery of the absolute A total of 15 patients were enrolled between June 2007 and June neutrophil count to 40.5 Â 109/l for 3 consecutive days after 2009. The specific patient characteristics are shown in Table 1. treatment-induced nadir. Platelet engraftment was defined as the The median age was 48 (range: 30–58) years. In total, 13 first day of the platelet count 420 Â 109/l without transfusion for 7 patients had acute myeloid leukemia and two had acute consecutive days. Acute and chronic GvHD were graded lymphoblastic leukemia. All, but one patient had chemother- according to standard criteria.22,23 Chimerism was assessed from apy-refractory disease at the time of transplantation, either whole bone marrow and/or peripheral blood on days þ 30 and primary induction failure (n ¼ 6) or refractory relapse (n ¼ 8). þ 100 using analysis of short tandem repeat loci.24 Patients received a median of 2 (range, 1–3) previous lines of Response was assessed at day þ 30 and was defined treatment. The median percentage of bone marrow blasts was according to recently reported European LeukemiaNet criter- 12% (3–88%), with 6 patients having X25% blasts. In all, 12 ia.25 Relapse was defined by 45% blasts in marrow aspirate or patients also had circulating blasts. One patient received bone development of extramedullary leukemia in patients with marrow from an unrelated donor and 14 received PBSC from previously documented CR. Event-free survival (EFS) was related (n ¼ 9) or unrelated (n ¼ 5) donors. The median measured from the date of transplantation to treatment failure, hematopoietic cell transplantation-specific comorbidity index

Leukemia High-dose clofarabine/busulfan in acute leukemia SS Farag et al. 601 score was 2 (range: 0–4), with speciﬁc comorbidities as shown /kg) 6 in Table 1. 10 Â ( CD34 dose Engraftment morbidity index; All patients had neutrophil engraftment. The median time to an 5 days+etoposide 9

Â absolute neutrophil count 40.5 Â 10 /l was 16 (range: 12–22) source 2 days. The median time to a platelet count 420 Â 109/l was 15 (range: 10–42) days. Only one patient treated at dose level 1 (30 mg/m2 clofarabine) failed to achieve platelet recovery and remained platelet transfusion-dependent. This patient received a VUD PBSC 5.0 VUD PBSC 5.2 VUD BM 0.7 Related PBSC 3.7 Related PBSC 4.9 Related PBSC 2.8 bone marrow graft from an unrelated donor with a CD34 þ cell dose of only 0.7 Â 106/kg. Donor chimerism was almost complete early after transplantation (Table 2). At day þ 30, the median donor chimerism was ) Related PBSC 7.3 2 99% (range: 69–100%). The percentage of donor chimerism remained stable or increased in all, but 1 of 12 patients 66–80%) 66–80%), 66–80%)66–80%) VUD Related PBSC PBSC 11.1 5.2

1 1 1 1 evaluable at day þ 100; median donor chimerism 100% (range: 87–100%). Two patients died of leukemia before day þ 100, 3 days); ALL, acute lymphoblastic leukemia; AML, acute

Â and one patient whose disease failed to go into CR following 2 transplantation had a reduction in donor chimerism associated with disease progression (Table 2). 6 doses; M, male; MEC, mitoxantrone 8 mg/m

Â Toxicity F severe pulmonary Speciﬁc comorbidity Donor Stem cell moderate pulmonary (FEV F F polymyalgia rheumatica The distribution of clinically signiﬁcant grades 3–4 non- hematological toxicities according to clofarabine dose level are shown in Table 3. The most frequent grade 3–4 toxicity was CMI HCT te remission; F, female; HCT CMI, hematopoietic cell transplantation co- asymptomatic and reversible elevation of ALT and aspartate aminotransferase. The median ALT levels from clofarabine administration to day þ 30 are shown in Figure 1a. As shown, the pattern of ALT elevation was very consistent, with ALT level

blasts (%) peaking on days À1 or 0, then falling to baseline by day þ 10 in all patients, and therefore did not constitute DLT. There was no

every 12 h on alternate days signiﬁcant difference in the peak ALT level between different 2 clofarabine dose levels (Figure 1b). A similar pattern and degree of elevation of aspartate aminotransferase was also observed 3 days (patient no. 12 received Idarubicin 12 mg/m (data not shown). In contrast, the bilirubin level and alkaline Â

2 phosphatase levels remained within normal range or increased to grade 1 in all patients (data not shown). Other grades 3–4 toxicities were mucositis, diarrhea, and nausea and vomiting, although these did not occur more frequently with Previous therapies BM clofarabine dose-escalation (Table 1). One patient treated at the 30 mg/m2 dose developed sinusoidal obstruction syndrome documented by liver biopsy, although this resolved by day þ 30 and did not meet criteria for DLT. The same patient also developed diffuse alveolar hemorrhage and grade 3 acute renal insufﬁciency associated with sepsis; both also resolved and did not meet criteria for DLT. One patient at dose level 4 developed thrombotic 5 days; PBSC, peripheral blood stem cells; Prim Ref, primary refractory; Ref Rel, refractory relapse; VUD, volunteer unrelated donor. 12 doses; HiDACx6, cytarabine 1–2 g/m Â 7days and daunorubicin 50 mg/m microangiopathy, which was attributed to sirolimus and tacrolimus Â 2 Â and promptly resolved upon discontinuation of these drugs. 2 time of transplantation Another patient, treated at dose level 4, developed acute renal failure on day þ 14 associated with supratherapeutic levels of -asparaginase, vincristine and daunorubicin; BM, bone marrow; CR, comple L every 12 h tacrolimus, although a contribution by clofarabine could not 2 Sex Disease status at be excluded. As the serum creatinine did not resolved to grade 1 by day þ 30, the event was considered a potential DLT and the 60 mg/m2 cohort was expanded to six patients; no additional renal

(years) toxicity was observed. No further dose-escalation of clofarabine was performed, and a MTD for clofarabine was, therefore, not reached. 9 58 F AML Prim Ref 7+3, MEC 15 2 Moderate pulmonary (FEV 56 48 54 M F AML AML Prim Ref Prim Ref 7+3, HiDACx6, MEC 7+3, HiDACx12 9 12 1 3 Atrial ﬁbrillation Moderate pulmonary (FEV VUD PBSC 9.0 2 58 F ALL Prim Ref HyperCVAD, Asp/VCR/DNR 6 0 3 41 F AML CR2 7+3, HiDACx6, MEC 3 0 8 30 M ALL Ref Rel2 HyperCVAD, Asp/VCR/DNR 9 2 Moderate pulmonary (FEV 5 days+cytarabine 1000 mg/m 15 44 F AML Prim Ref 7+3, HiDACx6 76 3 Severe pulmonary Related PBSC 4.9 1314 48 50 M F AML AML Ref Rel1 Ref Rel1 7+3, HiDACx12 7+3+VP16 25 50 3 Obesity, 0 moderate pulmonary Related PBSC 5.0 1112 47 58 M M AML AML Ref Rel1 Prim Ref 7+3, HiDACx6, MEC 7+3, MEC, Clofarabine 60 7 2 4 Moderate pulmonary Hepatic, mild (bilirubin 1.7x ULN), Related PBSC 5.0 Patient characteristics and outcome UPN Age Â 2 Treatment outcome )

2 Of 14 patients transplanted with active chemotherapy-refractory leukemia, 12 achieved a CR (n ¼ 10) or CRi (n ¼ 2) on day þ 30 Table 1 Cohort (mg/m Abbreviations: 7+3, cytarabine 100 mg/m 30 1 48 M AML Ref Rel2 7+3, HiDACx6, MEC 58 4 Coronary artery disease, diabetes mellitus, 40 4 46 F AML Ref Rel1 7+3, HiDACx6, MEC 83 1 Obesity (body mass index 45 kg/m 60 10 56 M AML Ref Rel1 7+3, HiDACx12 9 4 Atrial ﬁbrillation, severe pulmonary Related PBSC 5.1 HiDACx12, cytarabine 1.5 g/m 50 7 42 F AML Ref Rel1 7+3, HiDACx12 6 4 Severe pulmonary, depression VUD PBSC 7.0 myeloid leukemia; Asp/VCR, DNR, 100 mg/m for an overall response rate of 86%. Two patients with refractory

Leukemia High-dose clofarabine/busulfan in acute leukemia SS Farag et al. 602 Table 2 Patient outcome

Cohort (mg/m2) UPN Disease status Donor chimerism Response (day +30)a Survival (days) Cause of death

Day 30 Day 100

30 1 AML Ref Rel2 99 98 CRi 104 Sepsis 2 ALL Prim Ref 99 98 CR 1140+ F 3 AML CR2 99 100 CR 1067+ F 40 4 AML Ref Rel1 100 100 CR 167 AML 5 AML Prim Ref 69 F Refractory 118 AML 6 AML Prim Ref 99 100 CR 531 cGvHD 50 7 AML Ref Rel1 99 99 CR 868+ F 8 ALL Ref Rel2 93 F Refractory 61 ALL 9 AML Prim Ref 82 87 CR 609 AML 60 10 AML Ref Rel1 96 F CR 170 AML 11 AML Ref Rel1 100 100 CR 593+ F 12 AML Prim Ref 100 100 CR 529+ F 13 AML Ref Rel1 95 99 CR 474+ F 14 AML Ref Rel1 81 F CRi 92 AML 15 AML Prim Ref 99 100 CR 406+ F Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; cGvHD, chronic graft-versus-host disease; CR, complete remission; Prim Ref, primary refractory; Ref Rel, refractory relapse. aResponse evaluation on bone marrow examination on day +30 after transplantation.

Table 3 Clinically significant grades 3–4 toxicity Plasma pharmacokinetics studies and clinical correlations Clofarabine dose level Clofarabine pharmacokinetic parameters are summarized in Table 4. Although substantial interpatient variability was 2 2 2 2 30 mg/m 40 mg/m 50 mg/m 60 mg/m observed, there was a significant linear relationship between (n ¼ 3) (n ¼ 3) (n ¼ 3) (n ¼ 6) 2 2 Cmax (r ¼ 0.43; P ¼ 0.008) and AUC0-N (r ¼ 0.51; P ¼ 0.003) Mucositis 1 3 1 4 and clofarabine dose (Figures 2a and b). No significant Diarrhea 1 1 2 3 differences in other pharmacokinetic parameters were observed Nausea and 1101between dose groups. Significantly, there was no correlation vomiting between Cmax or AUCav and peak transaminase levels (Figures AST/ALT elevation 2 3 2 3 2c and d; data shown only for ALT). Bilirubin elevation 1 0 0 0 Pulmonary 1000 Also, there was no significant difference in the AUC1st dose and hemorrhage AUC9th dose of busulfan with clofarabine dose, indicating Creatinine 1 0 0 1a absence of any significant pharmacokinetic interaction between TMA 0 0 0 1 the two drugs (data not shown). Hepatic SOS 1 0 0 0 Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; TMA, thrombotic microangiopathy; SOS, sinusoidal obstruction syndrome. Discussion aDose limiting toxicity. All other toxicity resolved before day +30 after transplantation. We describe the first study evaluating high-dose clofarabine in combination with busulfan as a myeloablative preparative regimen for allogeneic HSCT, and have shown the feasibility disease, treated at dose levels 2 and 3, respectively, failed to and safety of the combination with clofarabine doses up to achieve CR or CRi (Table 2). Two patients died in remission; one 60 mg/m2 per day for 5 days. treated at dose level 1 died on day þ 104 from gram negative Clofarabine is a newer generation purine nucleoside analogue sepsis and multiorgan failure in the context of grade 3 acute that, like fludarabine, must be converted intracellularly to the GvHD, and another treated at dose level 2 died on day þ 531 of 50-triphosphate form by deoxycytidine kinase to be active.28 chronic GvHD for a total treatment-related mortality of 13%. Clofarabine triphosphate inhibits DNA synthesis and repair Overall, two of 15 patients, both treated at dose level 1, through inhibition of DNA polymerases and ribonucleotide developed acute GvHD; one grade 3 involving skin and gut reductase,29 leading to depletion of intracellular deoxynucleo- (see above), and another grade 1 involving skin only. Four of the tide triphosphate pools, and inhibition of DNA strands elonga- 13 patients surviving beyond 105 days developed extensive tion during synthesis.28,29 Compared with fludarabine stage chronic GvHD. Of 13 patients in CR or CRi at day þ 30, triphosphate, however, the triphosphate of clofarabine is more four relapsed within 6 months of transplantation. After a median active in activation of caspase 9, (see ref. 30) and unlike follow-up of 593 (range: 406–1140) days for surviving patients, triphosphates of other purine analogues, it directly affects the the median EFS and OS are 531 and 609 days, respectively. The mitochondrial membrane leading to release of pro-apoptotic 1-year EFS is 53% (95% confidence interval: 33–86%) and the factors.31 Consistent with this data, clofarabine has shown 1-year OS is 60% (95% confidence interval: 40–91%), with greater anti-leukemia activity compared with fludarabine, and seven patients remaining alive and in ongoing CR beyond 406 to therefore, may be a better agent to combine with high-dose 1140 days post-transplant. busulfan for HSCT.

Leukemia High-dose clofarabine/busulfan in acute leukemia SS Farag et al. 603

800 1600 700 1400 30 mg/m2 (n=3) 600 1200 40 mg/m2 (n=3) P=0.81 500 1000 50 mg/m2 (n=3) 400 800 60 mg/m2 (n=6) 300 600

Median ALT (U/dl) 200 400 100 200 Peak ALT level reached (U/dl) 0 0 -7 -2 3 8 13 18 23 28 30 40 50 60 Day of transplantation Clofarabine (mg/m2)

Figure 1 Change in alanine aminotransferase (ALT) level following high-dose clofarabine and busulfan and allogeneic hematopoietic cell transplantation. (a) Median ALT levels by clofarabine dose level. (b) Relationship between peak ALT level reached and clofarabine dose level. No signiﬁcant relationship between peak ALT level reached and clofarabine dose (P ¼ 0.81; ANOVA).

Table 4 Clofarabine pharmacokinetic parameters

Clofarabine dose level

30 mg/m2 (n ¼ 3) 40 mg/m2 (n ¼ 3) 50 mg/m2 (n ¼ 3) 60 mg/m2 (n ¼ 6)

AUC0-N (h*mg/ml) 6.57±1.35 11.27±3.27 9.80±3.18 23.53±7.95 AUCav (h*mg/ml) 8.15±1.78 11.03±3.03 9.49±3.91 19.97±5.38 Cmax (mg/ml) 1.75±0.52 2.08±0.43 2.34±1.56 4.39±1.72 Cl (l/h) 8.52±2.22 6.99±3.13 9.86±4.58 5.22±2.56 t1/2 (h) 6.09±1.62 3.68±1.22 3.13±1.94 3.89±0.64 À1 kel (h ) 0.12±0.03 0.21±0.08 0.30±0.20 0.18±0.03

Abbreviations: AUC0-N, area under the plasma concentration-time curve from time zero to inﬁnity; AUCav, average AUC of plasma concentration curve from all ﬁve clofarabine doses administered; Cl, clearance; Cmax, maximum concentration; kel, elimination constant; t1/2, elimination half-life. All results expressed as mean±s.d.

6 40 5 35 30 4

g/ml) 25 g/ml)

3 ( 20 (hr* av max 15 C 2

AUC 10 1 P=0.008 5 P=0.003 0 0 30 40 50 60 30 40 50 60 Clofarabine Dose (mg/m2) Clofarabine Dose (mg/m2)

1600 1600

1400 1400

1200 1200

1000 1000

800 800

600 600

400 400 Peak ALT Level (IU/l) Peak ALT Level (IU/l) 200 200 P = 0.93 P=0.67 0 0 0 1 2 3 4 5 6 0 5 10 15 20 25 30 Clofarabine AUC (hr*g/ml) Clofarabine Cmax ( g/ml) av

Figure 2 Clofarabine pharmacokinetics and correlation with hepatotoxicity. (a) Correlation of the maximal concentration of clofarabine (Cmax) after the ﬁrst dose with clofarabine dose; r2 ¼ 0.43; P ¼ 0.008. (b) Correlation of the average area under the plasma concentration-time curve 2 (AUCav) with clofarabine dose; r ¼ 0.51; P ¼ 0.003. (c)Cmax versus peak ALT, P ¼ 0.93; (d) AUCav versus peak ALT, P ¼ 0.67.

In a phase I trial, the MTD of single agent clofarabine in adults In the same trial, however, pharmacologic studies showed there with acute leukemia was deﬁned as 40 mg/m2 per day when was a signiﬁcant dose-dependent increase in sustained inhibi- given for 5 days, with hepatotoxicity being dose limiting.18 tion of replication of leukemic blasts over 24 h, with only the

Leukemia High-dose clofarabine/busulfan in acute leukemia SS Farag et al. 604 highest dose tested, 55 mg/m2 per day, leading to complete Although not the primary endpoint, the clinical outcome of sustainability of inhibition of DNA synthesis until the next patients treated with high-dose clofarabine and busulfan was dose,18 providing rationale for dose-escalation. Importantly, we quite impressive, given the refractory nature of leukemia in all, have shown that in combination with high-dose busulfan, but one patient. Durable remissions were observed in 9 patients, clofarabine may be safely escalated to doses up to 60 mg/m2 per with very encouraging 1-year event-free and OSs of 53 and day, which may be expected to provide superior anti-leukemic 60%, respectively. This compares very favorably with results of activity than lower doses. Although asymptomatic elevation previous studies where the 1-year survival for similar refractory of transaminases was also observed in our study, the change leukemia patients has generally been 25% or less.36,37 It should followed a predictable time course with complete resolution be noted, however, that similar results have been reported in in all patients by day þ 10. Unlike our study, the previously 75 high-risk acute leukemia and myelodysplasia patients using a reported phase I trial in adults with acute leukemia defined the sequential regimen of chemotherapy (FLAMSA) followed by MTD for clofarabine based on grade 3–4 transaminase eleva- reduced-intensity conditioning and allogeneic SCT.38 Although tions regardless of reversibility.18 We contend that if reversibility the clofarabine and busulfan regimen may be simpler to was considered in the definition of DLT, further dose-escalation administer because of its shorter schedule, the relative efficacy of clofarabine may have been feasible. In addition, within the of the two approaches is currently unknown. Nonetheless, our limitations of our sample size, we also did not observe results strongly suggest that high-dose clofarabine and busulfan any relationship between clofarabine dose, Cmax or AUCav should be studied further, particularly in patients with refractory and peak transaminase elevation. This is consistent with a or high-risk disease where more conventional preparative similar observation where elevation of transaminases was not regimens are associated with poor outcomes. related to dose in another phase I trial where doses of We conclude that high-dose clofarabine and busulfan are a clofarabine 30–70 mg/m2 per day for 5 days were administered novel and well-tolerated preparative regimen for allogeneic in combination with high-dose etoposide and cyclophospha- HSCT, and may provide better anti-leukemic activity than currently mide.32 Our data supports further investigation of high-dose used regimens. Further investigation to better assess the efficacy clofarabine, as also suggested by others.33 It should be noted and safety of this regimen, with a recommended phase II dose that in our study clofarabine was used in combination with four of clofarabine of 60 mg/m2 per day for 5 days together with times per day dosing of busulfan, although recent data suggests intravenous busulfan (12.8 mg/kg total dose), is justified. that lower doses of clofarabine may be safely administered in combination with more convenient schedule of once daily dosing of busulfan.34 Conflict of interest Other toxicities, including mucositis and diarrhea, are similar to those previously reported for patients receiving either SF has received research support from Genzyme Inc., and clofarabine or high-dose busulfan. The only potential dose- Honoraria from Genzyme Inc. and Otsuka Pharmaceuticals. limiting toxicity observed was acute renal failure in one patient who received clofarabine 60 mg/m2 per day. Although this Acknowledgements toxicity developed in the setting of high tacrolimus levels, a possible causal relationship to clofarabine could not be This study was supported in part by research funding from excluded necessitating expanding this dose level to six patients. 2 Genzyme Inc. Pharmacoanalytical work was performed by the None of the five additional patients receiving 60 mg/m per day Clinical Pharmacology Analytical Laboratory of the Indiana developed acute renal failure. Despite substantial interpatient University Melvin and Bren Simon Cancer Center and supported variability in Cmax and AUC at any given clofarabine dose, there by Institutional grant P30 CA082709. was no association between plasma pharmacokinetic parameters and toxicity. Although the combination of clofarabine with busulfan appears to be well tolerated, as shown in this trial References and by other using lower doses of clofarabine,34 the combination of clofarabine with cytarabine and anti-thymocyte globulin 1 Moore J, Nivison-Smith I, Goh K, Ma D, Bradstock K, Szer J et al. has recently been reported to be associated with significant Equivalent survival for sibling and unrelated donor allogeneic stem morbidity and mortality in a small study.35 It is likely that the cell transplantation for acute myelogenous leukemia. Biol Blood drugs combined with clofarabine, as well as patient selection, Marrow Transplant 2007; 13: 601–607. significantly affect the tolerability of the regimen. 2 Tomblyn MB, Arora M, Baker KS, Blazar BR, Brunstein CG, Notwithstanding the small sample size, clofarabine appeared Burns LJ et al. Myeloablative hematopoietic cell transplantation for acute lymphoblastic leukemia: analysis of graft sources and to provide sufficient immunosuppression for engraftment of both long-term outcome. J Clin Oncol 2009; 27: 3634–3641. related and unrelated hematopoietic progenitor cells. Myeloid 3 Aoudjhane M, Labopin M, Gorin NC, Shimoni A, Ruutu T, Kolb HJ and platelet engraftment kinetics were similar to those reported et al. Comparative outcome of reduced intensity and myeloabla- using the fludarabine/busulfan regimen.12,13 In addition, high- tive conditioning regimen in HLA identical sibling allogeneic level donor chimerism was achieved in the majority of patients haematopoietic stem cell transplantation for patients older than 50 by day þ 30 and was sustained or increased by day þ 100 in the years of age with acute myeloblastic leukaemia: a retrospective survey from the Acute Leukemia Working Party (ALWP) of the absence of leukemic relapse, indicating that engraftment is European group for Blood and Marrow Transplantation (EBMT). possible even at lower clofarabine doses when combined with Leukemia 2005; 19: 2304–2312. busulfan. On the other hand, when combined with cytarabine 4 de Lima M, Anagnostopoulos A, Munsell M, Shahjahan M, and anti-thymocyte globulin in a reduced intensity regimen, Ueno N, Ippoliti C et al. Nonablative versus reduced-intensity only mixed donor chimerism ranging between 60–75% was conditioning regimens in the treatment of acute myeloid leukemia achieved in three of four evaluable acute myeloid leukemia and and high-risk myelodysplastic syndrome: dose is relevant for long- term disease control after allogeneic hematopoietic stem cell myelodysplasia patients, indicating a lower dose of clofarabine transplantation. Blood 2004; 104: 865–872. 2 (40 mg/m per day Â 5 days) may not be sufficiently immuno- 5 Hartman AR, Williams SF, Dillon JJ. Survival, disease-free survival suppressive in such a regimen.35 and adverse effects of conditioning for allogeneic bone marrow

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