A Phase I Trial of High-Dose Clofarabine, , and and Autologous Peripheral Blood Stem Cell Transplantation in Patients with Primary Refractory and Relapsed and Refractory Non-Hodgkin Lymphoma

Shivani Srivastava,1,3 David Jones,2 Lisa L. Wood,1,3 Jennifer E. Schwartz,1,3 Robert P. Nelson, Jr.,1,3 Rafat Abonour,1,3 Angie Secrest,3 Elizabeth Cox,3 Jay Baute,3 Cheryl Sullivan,3 Kathleen Kane,3 Michael J. Robertson,1,3 Sherif S. Farag1,3

Clofarabine has significant single-agent activity in patients with indolent and aggressive non-Hodgkin lym- phoma and synergizes with DNA-damaging drugs. Treatment, however, may be associated with severe and prolonged myelosuppression. We conducted a phase I trial to determine the maximum tolerated dose (MTD) of clofarabine in combination with high-dose etoposide and cyclophosphamide followed by autologous peripheral blood stem cell transplantation in patients with refractory non-Hodgkin lymphoma (NHL). Patients received clofarabine at 30-70 mg/m2/day on days 26to22 in successive cohorts, in com- bination with etoposide 60 mg/kg (day 28), and cyclophosphamide 100 mg/kg (day 26), followed by filgrastim-mobilized PBSC on day 0. Sixteen patients of median age 57 (range: 32-67) years with diffuse large B cell (n 5 8), follicular (n 5 5), or mantle cell (n 5 3) lymphoma that was either primary refractory (n 5 2) or relapsed and refractory (n 5 14) were treated at 5 clofarabine dose levels: 30 (n 5 3), 40 (n 5 3), 50 (n 5 3), 60 (n 5 3), and 70 mg/m2/day (n 5 4) in combination with etoposide and cyclophosphamide. All patients had grade 4 neutropenia and thrombocytopenia. Grade 3-4 nonhematologic toxicity was evenly distributed across all 5 dose levels, and included (n 5 3), mucositis (n 5 1), (n 5 1), revers- ible elevation of alanine aminotranferease/aspartate aminotransferase (AST/ALT) (n 5 1) or bilirubin (n 5 1), and hemorrhagic cystitis (n 5 1); all resolved by day 130 following transplantation. The MTD was not reached. No treatment-related deaths occurred. At day 130, 13 patients achieved a complete remission (CR) or unconfirmed CR (CRU), and 2 patients achieved a partial response, for an overall response rate of 94%. After a median follow-up of 691 days, the 1-year progression-free survival (PFS) and overall survival (OS) were 63% (95% confidence interval [CI]: 43%-91%) and 68% (95% CI: 49%-96%), respectively. We recommend clofarabine 70 mg/m2/day  5 days as a phase II dose in combination with high-dose etoposide and cyclophosphamide for further testing as a preparative regimen in NHL patients undergoing autologous PBSC transplantation. Biol Blood Marrow Transplant 17: 987-994 (2011) Ó 2011 American Society for Blood and Marrow Transplantation KEY WORDS: Autologous stem cell transplantation, Clofarabine, non-Hodgkins lymphoma

INTRODUCTION From the 1Department of Medicine; 2Department of Pharmacology; and 3Blood and Bone Marrow Transplantation Program, Indi- High-dose and autologous stem cell ana University School of Medicine, Indianapolis, Indiana. transplantation (ASCT) remain the most efficacious Financial disclosure: See Acknowledgments on page 994. Correspondence and reprint requests: Sherif S. Farag, MD, PhD, therapy for patients with relapsed non-Hodgkin Division of Hematology and Oncology, Department of Internal lymphoma (NHL), although only patients with chemo- Medicine, Indiana University School of Medicine, Walther sensitive disease appear to benefit the most [1]. Patients Hall-R3, C414, 980 West Walnut Street, Indianapolis, IN whose disease has never responded to therapy are al- 46202 (e-mail: [email protected]). most never salvaged by ASCT, whereas patients whose Received August 3, 2010; accepted October 12, 2010 Ó 2011 American Society for Blood and Marrow Transplantation relapsed disease is resistant to conventional-dose 1083-8791/$36.00 chemotherapy salvage have only a 15% to 20% chance doi:10.1016/j.bbmt.2010.10.016 of long-term disease-free survival (DFS) with

987 988 S. Srivastava et al. Biol Blood Marrow Transplant 17:987-994, 2011 transplantation [1,2]. The major cause of treatment blood stem cell (PBSC) transplantation in patients failure of ASCT remains relapse, indicating the need with relapsed or refractory NHL. for novel and more effective preparative regimens. The preparative regimens of high-dose etoposide, PATIENTS AND METHODS cyclophosphamide, and either (BCNU), total-body irradiation (TBI), or have been Patient and Donor Eligibility commonly used in patients with relapsed or refractory lymphoma [3-5]. Etoposide and cyclophosphamide Patients were eligible if they had primary refrac- have documented single-agent activity in resistant tory or relapsed and refractory diffuse large B cell lymphoma [6,7], and been shown to be synergistic in (DLBCL), mediastinal B cell, tumor models [8,9]. The extent of single-agent (MCL), or diffuse large cell transformation of an indo- activity of high-dose BCNU or busulfan in lymphoma lent lymphoma, or relapsed or refractory follicular remains undocumented. Furthermore, high-dose lymphoma (FL) with high FL International Prognostic BCNU has been associated with interstitial pneumoni- Index (FLIPI) [23]. Refractory disease was defined as tis (IP) [10] and busulfan with a significant incidence of failure to achieve at least a partial response to either sinusoidal obstruction syndrome (SOS), particularly first-line chemotherapy (primary refractory) or the when combined with high-dose cyclophosphamide or last if given for relapsed disease TBI [11]. Finally, TBI-based regimens have also (relapsed and refractory). Patients were also required to be 18 to 70 years old, have a Karnofsky performance been associated with an increased risk of IP and post- $ transplant myelodysplasia in patients undergoing status of 70%, and have adequate organ function defined by a left ventricular ejection fraction .45%, ASCT for lymphoma [12,13]. . Clofarabine (2-chloro-2-fluoro-deoxy-9-D-arabi- corrected gas transfer (DLCO) 50%, estimated cre- . 2, and serum nofuranosyladenine) is a second-generation, atinine clearance 60 mL/min/1.73 m bilirubin, aminotranferease (AST), and aspartate approved for relapsed or re- aminotransferase (ALT) levels \2 Â upper limits of fractory pediatric acute lymphoblastic leukemia. In ad- normal. Patients were excluded if they had a prior dition to its clinical activity in patients with relapsed autologous or allogeneic transplant, active central and/or refractory acute leukemia [14-16], recent nervous system lymphoma, uncontrolled infection, or studies have also shown significant single-agent activity if they were seropositive for human immunodeficiency in mature lymphoid malignancies, including chronic virus (HIV). Patients could receive only filgrastim- lymphocytic leukemia (CLL) as well as indolent and ag- mobilized PBSC. The study was approved by the insti- gressive NHL [17-19]. Although pharmacodynamic tutional review board of Indiana University-Purdue studies have suggested that higher doses of University Indianapolis. All patients and donors gave clofarabine may be more efficacious with dose- written informed consent. The study was conducted dependent accumulation of the active metabolite in accordance with the Declaration of Helsinki. clofarabine triphosphate in CLL cells [17], severe and prolonged myelosuppression has limited testing of high doses without stem cell support [18,19]. Study Design and Treatment Plan Furthermore, in vitro synergy between clofarabine All patients received the same doses of etoposide and cyclophosphamide has been demonstrated against and cyclophosphamide, whereas the dose of clofara- lymphoid cells, whereby clofarabine triphosphate, bine was escalated in successive cohorts of 3 to 6 which inhibits DNA polymerases and ribonucleotide patients (see below). Etoposide was administered as reductase, impedes DNA damage induced by a single dose of 60 mg/kg (actual body weight) intrave- cyclophosphamide and other DNA damaging agents nously (i.v.) over 4 hours on day 28, and cyclophos- resulting in enhanced apoptotic cell death [20-22]. phamide was administered at 100 mg/kg (lower of Based on its significant antilymphoma activity, po- either actual or ideal body weight) i.v. over 1 hour on tential for dose-response effect, and its synergy with day 26. Clofarabine was administered as a daily the DNA damaging agents such as cyclophosphamide 1-hour i.v. infusion for 5 days on days 26to22, and etoposide, we hypothesized that the combination with the dose escalated in 5 successive cohorts at the of clofarabine, etoposide, and cyclophosphamide may daily dose levels of 30 mg/kg, 40 mg/kg, 50 mg/kg, be an effective, novel preparative regimen for patients 60 mg/kg, and 70 mg/kg. Filgrastim-mobilized with NHL undergoing ASCT, where the limitation of PBSC were infused on day 0. Filgrastim (5 mg/kg/ prolonged myelosuppression would be overcome with day) was then administered from day 11 until neutro- stem cell support. As an initial step in developing this phil recovery to at least 5.0 Â 109/L. PBSC were regimen, we conducted a phase I to deter- mobilized using filgrastim (10 mg/kg/day) for 4 days mine the maximum tolerated dose (MTD) of clofara- and continued until collection was complete. Leuka- bine in combination with high-dose etoposide and pheresis (3-4 blood volumes) was commenced on day cyclophosphamide followed by autologous peripheral 5 and continued daily for a maximum of 4 days until Biol Blood Marrow Transplant 17:987-994, 2011 Phase I High-Dose Clofarabine, Etoposide, and Cyclophosphamide 989

1 a target of .2.0 Â 106/kg CD34 cells were collected from any cause, censoring for patients alive on the date 1 (although CD34 cell doses\2 Â 106/kg were permis- of last follow-up. sible for transplantation if the target could not be reached). PBSC were cryopreserved in 10% DMSO Pharmacologic Assays and Pharmacokinetic according to institutional standards until infusion. Analysis All patients received prophylactic antibiotics using flu- Blood was sampled for assay of clofarabine levels on conazole, acyclovir, and ciprofloxacin. day 26 before infusion of the first dose for baseline A standard 3 1 3 trial design was to be followed. values, at the end of the initial infusion, and at 2, 3, 4, Three patients were enrolled on the first clofarabine 5, 6, and 24 hours after the start of the first dose. In ad- dose level. If 1 of 3 patients experienced dose-limiting dition, samples were also collected immediately prior toxicity (see definition below) at a given dose level, 3 to, at the end of infusion, and at 6 and 24 hours after more patients were to be treated at that dose level. If subsequent doses on day 25 to day 22. Blood samples no dose-limiting toxicity (DLT) was observed in any (5 mL) were collected in green-stopper vacutainer of the 3 patients or in 1 of 6 patients, the dose was esca- tubes containing heparin, immediately placed on ice, lated to the next level. If 2 or more patients experienced plasma separated within 30 minutes by centrifugation DLT, the MTD was exceeded, and 3 additional pa-  (1000 Â g for 10 minutes at 4 C), and then the plasma tients were to be treated at the next lower dose. All  stored in cryovials at 280 C until assay. For assays, patients assigned to a dose level were followed until samples were thawed, and clofarabine was extracted day 130 before dose escalation to the next level began. from plasma and quantified by HPLC-MS/MS. Plasma (100 mL) was buffered to pH 5 7.4 (with 100 mM phos- Definitions phate buffer), internal standard (chloroadenosine) and ethyl acetate were added, then the sample was mixed Adverse events were graded using National Cancer for 20 seconds and then centrifuged at high speed for Institute’s Common Terminology Criteria for Adverse 3 minutes. The upper organic phase was transferred Events (NCI CTCAE v3.0). DLT was defined as any to a new tube, evaporated to dryness, and reconstituted grade 3 or 4 nonhematologic toxicity (except alopecia, with mobile phase prior to injection into the HPLC- nausea and , mucositis, fatigue, and correctable MS/MS (API 4000, Applied Biosystems, Foster City, biochemical abnormalities [eg, electrolyte abnormalities; CA). Analytes were separated with a Zorbax Eclipse glucose elevation] as these are reversible and commonly C8 5 mm, 4.6 Â 50 mm column (Agilent Technologies, observed toxicities following high-dose chemotherapy) Placerville, CA) and gradient mobile phase (acetoni- thatdidnotresolvetograde2orlessbyday130 after trile: 5 mM ammonium acetate), and then detected in transplantation. As profound myelosuppression is an ex- the API 4000 using positive mode. The Q1/Q3 settings pected uniform outcome of high-dose chemotherapy to for clofarabine and chloroadenosine were 304/170 and be mitigated by stem cell support, hematologic toxicity 302/134, respectively. Standard curves were run for was not considered in the definition of DLT. The each batch of plasma samples with a lower limit of MTD was defined as the dose level at which 2 out of 6 pa- quantification of 1 ng/mL. tients experienced DLT. Concentration-time data were analyzed by a noncom- Neutrophil engraftment was defined as the first partmental approach using WinNonlin v4.1 (Pharsight, day of 3 consecutive days where the absolute neutro- Mountain View, CA). The following pharmacokinetic phil count (ANC) recovered to .0.5 Â 109/L after parameters were calculated for clofarabine: maximum conditioning regimen–induced nadir. en- plasma concentration (C ), apparent terminal elimina- graftment was defined as the first day of the of platelet max tion constant (k ), apparent terminal half-life (t ), and count .20 Â 109/L without transfusion for 7 consec- el 1/2 area under the plasma concentration–time curve from utive days. Primary engraftment failure was defined time zero (start of infusion) to the last concentration for as a failure of neutrophil engraftment by day 128. each day of infusion, and the average area under the curve Disease response was evaluated at 30 and 100 days (AUC) following each dose of clofarabine was also calcu- after transplantation, and then every 3 months after lated (AUC ) for each patient. In addition, the area under transplantation for 2 years, and every 6 months for an ad- av the plasma concentration curve extrapolated to infinity ditional year, or until disease progression. Responses (AUC /N) analyzed from plasma levels obtained follow- were assessed according to 1999 NCI International 0 ing the first clofarabine dose (day 26) was also calculated. Workshop NHL response criteria [24]. Progression- The systemic clearance (Cl) was calculated from the dose free survival (PFS) was measured from the date of trans- and AUC /N. plantation to the date of progression of disease, as previ- 0 ously defined [24], or death from any cause; patients not known to have any of these events were censored on the Statistical Analysis date of last follow-up. Overall survival (OS) was mea- The primary objective in this phase I trial was to sured from the date of transplantation to the date death determine the MTD of clofarabine when used in 990 S. Srivastava et al. Biol Blood Marrow Transplant 17:987-994, 2011

Table 1. Patient Characteristics eligible for transplantation on standard protocols. Pa-

N 5 16 tients received a median of 3 (range: 2-6) prior lines of treatment, including an -containing reg- Age, median (range) years 57 (32-67) imen. All patients received PBSC with a median Gender, male (n) 7 1 Â 6 Disease, (n) CD34 cell dose of 1.9 (range: 0.9-3.7) 10 /kg. Diffuse large B cell lymphoma (DLBCL) 8 Follicular lymphoma (FL) 5 Mantle cell lymphoma (MCL) 3 Engraftment Disease status, n All patients had successful neutrophil and platelet Primary refractory 2 First relapsed, refractory 7 engraftment. The median time to reaching an ANC Second relapse, refractory 3 .0.5 Â 109/L was 10 (range: 9-13) days. The median Third relapse, refractory 4 time to achieving a platelet count .20 Â 109/L was Lines of prior treatment, median (range) 3 (2-6) CD34 count, median (range) Â106/kg 1.9 (0.9-3.7) 16 (range: 10-33) days.

Toxicity combination with high-dose etoposide and cyclophos- The distribution of clinically significant grade 1-2 phamide for ASCT. Secondary objectives were to and 3-4 nonhematologic toxicities according to clofara- determine the safety and tolerability of this combina- bine dose level is shown in Table 2. The most com- tion, the response rate, PFS, and OS. Summary monly observed grade 3-4 toxicities were diarrhea statistics were used to describe baseline patient charac- (n 5 3), and nausea and vomiting (n 5 2). Grade 3-4 mu- teristics, times to engraftment, and response rates. PFS cositis, elevation of transaminases, elevation of biliru- and OS were estimated using the Kaplan-Meier bin, and hemorrhagic cystitis occurred each in 1 method. Safety was monitored by an independent patient only. Elevations of AST and ALT were mostly Data Safety Monitoring Board according to institu- grade 1 or 2, and all resolved promptly by day 18in tional requirements. all patients, with no long-term sequelae. The peak ALT and AST levels did not correlate with clofarabine RESULTS dose levels, as shown in Figure 1 (data for AST not shown). Overall, grade 3-4 toxicity did not occur more Patient Characteristics frequently with dose-escalation of clofarabine. As all Sixteen patients were enrolled on study between toxicities resolved by day 130, they did not meet the cri- September 2007 and September 2009. Patient charac- teria for DLT, and the MTD for clofarabine was not teristics are summarized in Table 1. The median age reached. No treatment-related late adverse events was 57 (range: 32-67) years. Eight patients had were observed at the time of last follow-up. DLBCL, 5 had FL (4 of whom with histologic grade Myelosuppression with grade 4 neutropenia and 3), and 3 had MCL. All patients enrolled had thrombocytopenia was observed in all patients. chemotherapy-refractory disease at time of entry into with neutropenia occurred in 9 patients (3 treated in the study; 2 had primary refractory disease, 7 were each of the 30 and 70 mg/m2/day cohorts, and 1 in in refractory first relapse, and 7 in refractory second each of the 40, 50, and 60 mg/m2/day cohorts), with or third relapse. Because of the chemotherapy- bacteremia documented in 4 cases (Staphylococcus epi- refractory nature of their disease, patients were not dermidis [n 5 2], Streptococcus mitis [n 5 1], and

Table 2. Clinically Significant Toxicity*

Clofarabine Dose Level

30 mg/m2 (n 5 3) 40 mg/m2 (n 5 3) 50 mg/m2 (n 5 3) 60 mg/m2 (n 5 6) 70 mg/m2 (n 5 4)

Grade 1-2 Grade 3-4 Grade 1-2 Grade 3-4 Grade 1-2 Grade 3-4 Grade 1-2 Grade 3-4 Grade 1-2 Grade 3-4

Mucositis 2 1 2 0 2 0 3 0 4 0 Diarrhea 3 1 3 0 2 0 2 1 3 1 Nausea and vomiting 3 0 3 1 3 0 2 1 2 0 AST/ALTelevation 2 0 3 0 2 0 1 0 2 1 Bilirubin elevation 3 1 3 0 3 0 2 0 0 0 Alkaline phosphatase 2010100000 elevation Hand-foot syndrome 2 0 0 0 2 0 0 0 1 0 Hemorrhagic cystitis 0 1 0 0 0 0 0 0 0 0 Peripheral neuropathy 1 0 0 0 1 0 1 0 0 0

AST/ALT indicates alanine aminotranferease/aspartate aminotransferase. *All toxicity resolved to at least grade 1 before day +30 after transplantation. Biol Blood Marrow Transplant 17:987-994, 2011 Phase I High-Dose Clofarabine, Etoposide, and Cyclophosphamide 991

a partial response (PR) on day 130 evaluation, for a to- tal response rate of 94% (Table 3). Only 1 patient, treated at the clofarabine 30 mg/m2/day dose level, re- mained refractory to treatment. No treatment-related deaths were observed, with 15 of 16 patients alive at day 1100; 1 patient treated at the 30 mg/m2/day level whose disease failed to respond died of progressive lymphoma on day 162. Of the 15 patients who re- sponded, 6 have had progression of lymphoma at a me- dian of 128 (range: 91-481) days after transplantation. After a median follow-up of 691 (range: 313-1029) days for surviving patients, the 1-year PFS is 63% (95% confidence interval [CI]: 43%-91%) and the 1-year OS is 68% (95% CI: 49%-96%) (Figure 2). Figure 1. Peak ALT level reached during first 30 days following trans- plantation by clofarabine dose level. No significant relationship between Plasma Pharmacokinetics Studies and Clinical peak ALT level reached and clofarabine dose (P 5 .44; ANOVA) was Correlations observed. The clofarabine pharmacokinetic parameters were Escherichia coli [n 5 1]). Other documented episodes of analyzed according to a noncompartment model, and infections included, herpes simplex stomatitis (n 5 2), are summarized in Table 4. Although substantial inter- Clostridium difficile enterocolitis (n 5 3), and BK virus patient variability was observed, there was a statistically 2 5 hemorrhagic cystitis (n 5 1). Although all patients significant linear relationship between Cmax (r .33; 5 2 5 5 received prophylactic antibiotics, the median duration P .02) and AUC0/N (r .67; P .0001), and of antibiotic usage for active treatment of infections the dose of clofarabine administered (Figure 3A and was 10 days (range: 0-24 days). All patients required B). No significant differences in other pharmacoki- transfusions with blood products. The median number netic parameters were observed between the different of units of concentrate transfused was 4 clofarabine dose groups. Significantly, there was no (range: 2-10), and the number of apheresis units of correlation between Cmax, AUC0/N, or AUCav with transfused was 3 (range: 2-8). The median peak ALT or AST levels (data not shown). duration of hospitalization was 21 (range: 19-30) days.

Treatment Outcome DISCUSSION Of the 16 patients transplanted with active We describe the first study, to our knowledge, chemotherapy-refractory lymphoma, 13 patients evaluating high-dose clofarabine in combination with achieved a complete response (CR) or complete re- etoposide and cyclophosphamide as a preparative sponse, unconfirmed (CRU), and 2 patients achieved regimen prior to autologous hematopoietic stem cell

Table 3. Patient Outcome

Clofarabine (mg/m2) UPN Disease Status Lines of Prior Therapy Response* (Day +30) Progression (Day) Alive Cause of Death

30 1 DLBCL Ref Rel 1 3 NR +30 No NHL 2 FL (gr 3) Ref Rel 1 4 CR — Yes — 3 DLBCL Ref Rel 1 2 CRU — Yes — 40 4 FL (gr 3) Ref Rel 1 2 CRU — Yes — 5 FL (gr 3) Ref Rel 3 5 CRU — Yes — 6 MCL Ref Rel 1 3 CRU — Yes — 50 7 DLBCL Ref Rel 2 3 CRU — Yes — 8 FL (gr 2) Ref Rel 2 6 CRU — Yes — 9 DLBCL Ref Rel 3 6 PR +118 No NHL 60 10 DLBCL Prim Ref 2 PR +91 No NHL 11 DLBCL Ref Rel 1 3 CRU +188 No NHL 12 MCL Ref Rel 1 4 CR — Yes — 70 13 DLBCL Prim Ref 3 CR +137 Yes — 14 MCL Ref Rel 2 4 CR — Yes — 15 DLBCL Ref Rel 3 4 CRU +114 No NHL 16 FL (gr 3) Ref Rel 3 3 CRU — Yes — DLBCL indicates diffuse large B cell lymphoma; FL, follicular lymphoma; MCL, mantle cell lymphoma; gr 3, histologic grade 3; CR, complete remission; CRU, complete remission, unconfirmed; PR, partial response; NR, no response; Ref Rel, refractory relapse; Prim Ref, primary refractory. *Response evaluation by computed tomography scans of the chest, abdomen, and pelvis and by bone marrow examination if bone marrow previously involved by lymphoma on day +30 after transplantation. 992 S. Srivastava et al. Biol Blood Marrow Transplant 17:987-994, 2011

Figure 2. (A) PFS and (B) OS for all patients. The 1-year EFS and OS estimates are shown on the respective curves. Figure 3. Clofarabine pharmacokinetics. (A) Correlation of the maxi- transplantation, and have shown the feasibility and mal concentration of clofarabine (Cmax) after the first dose with clofar- safety of the combination with no MTD reached de- abine dose; r2 5 .33; P 5 .02. (B) Correlation of the area under the spite clofarabine doses up to 70 mg/m2/day for 5 days. plasma concentration–time curve (AUC0/N) with clofarabine dose; r2 5 .67; P 5 .0001. Recent reports have indicated that clofarabine has significant single-agent activity in patients with re- lapsed and/or refractory NHL [18,19]. Although abine triphosphate in leukemic blasts with doses be- repeated cycles of low doses of clofarabine (4 mg/m2 yond 30 mg/m2/day, there was a significant dose- for 5 days) have shown activity in NHL [18], pharma- dependent increase in sustained inhibition of replica- codynamic studies suggest that dose escalation of clo- tion of leukemic blasts over 24 hours, with only the farabine may show optimal antineoplastic activity. For highest dose tested of 55 mg/m2/day leading to com- example, in a phase I study in patients with acute leu- plete sustainability of DNA synthesis inhibition kemia testing, doses of 15-55 mg/m2/day for 5 days, until the next dose [16]. Similarly, in CLL patients, pharmacologic studies showed that although there a dose-dependent accumulation of clofarabine triphos- was modest, if any, increase in accumulation of clofar- phate was observed in peripheral blood leukemic

Table 4. Clofarabine Pharmacokinetic Parameters

Clofarabine Dose Level

30 mg/m2 (n 5 3) 40 mg/m2 (n 5 3) 50 mg/m2 (n 5 3) 60 mg/m2 (n 5 3) 70 mg/m2 (n 5 4)

AUCav (h*mg/mL) 11.48 ± 1.32 12.08 ± 1.07 14.92 ± 1.41 22.66 ± 13.58 23.38 ± 7.00 AUC0/N (h*mg/mL) 9.59 ± 2.63 9.66 ± 2.21 14.67 ± 2.09 23.70 ± 8.06 22.98 ± 4.36 Cmax (mg/mL) 2.82 ± 1.24 3.43 ± 0.88 3.15 ± 0.74 3.87 ± 2.43 5.51 ± 1.60 Cl (l/h) 5.80 ± 1.04 8.05 ± 2.15 6.32 ± 1.07 5.02 ± 1.28 3.79 ± 2.35 t1/2 (h) 6.50 ± 1.05 5.66 ± 1.34 4.17 ± 0.38 4.31 ± 0.04 4.04 ± 0.51 21 kel (h ) 0.13 ± 0.02 0.13 ± 0.03 0.17 ± 0.02 0.16 ± 0.01 0.17 ± 0.02

AUC0/N indicates area under the plasma concentration–time curve from time 0 to infinity calculated from levels obtained after the first dose of clo- farabine; AUCav, average AUC for the 5 doses of clofarabine given on days 26to22; Cmax, maximum concentration; Cl, clearance; t1/2, elimination half- life; kel, elimination constant. All results expressed as mean 6 standard deviation. Biol Blood Marrow Transplant 17:987-994, 2011 Phase I High-Dose Clofarabine, Etoposide, and Cyclophosphamide 993 lymphocytes up to the maximum dose tested of 15 mg/ the study in patients who had undergone prior myelo- m2 [17]. Although no study has examined clofarabine ablative therapy and stem cell transplantation within triphosphate accumulation in lymphoma cells relative the year preceding study entry and in the context of to dose, the latter pharmacologic studies do suggest concurrent severe infection [28]. Other toxicities ob- that dose-escalation of clofarabine may achieve optimal served, including mucositis and diarrhea, are similar antilymphoma activity given that clofarabine triphos- to those previously reported for patients receiving clo- phate is the active moiety of the drug, inhibiting farabine or high-dose etoposide and cyclophospha- DNA synthesis and repair [21], directly promoting ap- mide. Therefore, although our data supports further optosis [25], as well as synergizing with DNA-damag- investigation of high-dose clofarabine, as previously ing drugs [22]. When clinically tested in higher doses also suggested by other investigators [29], caution (40 mg/m2/day for 5 days) in patients with heavily pre- should be exercised in patients who have received prior treated NHL, however, severe and prolonged myelo- intensive chemotherapy, particularly those receiving suppression was limiting [19]. These results myeloablative therapy. suggested that high-dose clofarabine could be better The plasma pharmacokinetic parameters of tolerated in such a population if used with stem cell clofarabine in the current study are similar to those pre- support, and in this setting could be tested in combina- viously published [27,30,31]. Despite substantial tion with high-dose etoposide and cyclophosphamide, interpatient variability in Cmax and AUC0/N at any which cause DNA damage and are also know to be ac- given clofarabine dose there was dose-dependent accu- tive in NHL. mulation of the drug in plasma. Furthermore, despite Importantly, we have shown that in combination dose-dependent accumulation of clofarabine, there with high-dose etoposide and cyclophosphamide, was no apparent association between plasma pharmaco- clofarabine may be safely escalated to doses up to kinetic parameters and toxicity. However, as we did not 70 mg/m2/day, which may be expected to provide measure levels of cyclophosphamide and etoposide, we more optimal activity than lower doses. A previous cannot exclude the possibility of any pharmacokinetic phase I study in adult patients with acute leukemia interaction between clofarabine and these drugs. has defined the MTD of single-agent clofarabine Although not the primary endpoint, the clinical (without stem cell support) as 40 mg/m2/day based outcome of patients treated with the combination on grade 3-4 transaminase elevations, although these of high-dose clofarabine, etoposide, and cyclophospha- were completely reversible and asymptomatic [16]. mide was quite encouraging given the chemotherapy- In a recent phase I study of clofarabine and high- refractory nature of the patients treated. The regimen dose busulfan for allogeneic stem cell transplantation was well tolerated, with no treatment-related deaths. in adults with acute leukemia, asymptomatic and Importantly, durable remissions of .1yearwereob- reversible grade 3-4 transaminase elevations were served in 9 patients, including 2 with refractory DLBCL similarly observed although hepatotoxicity was not and 3 with refractory MCL, with overall very encourag- dose dependent in the range of 30-60 mg/m2/day ing 1-year PFS and OS of 63% and 68%, respectively, for 5 days [26]. In the latter study, as transaminase despite the refractory nature of NHL in treated patients. elevations were completely reversible within 10 It should be noted that the majority of patients included days after transplantation, they did not constitute in this study would not otherwise be candidates for DLT [26]. Significantly, in the current study, we ASCT with standard protocols given the advanced and did not observe grade 3-4 AST and ALT elevations chemotherapy-refractory nature of their lymphoma. despite escalating clofarabine to 70 mg/m2/day. Al- This compares favorably with results of previous though the reasons for the absence of significant studies where the reported 1-year survival for similar transaminase elevation in this study are unclear, it patients has generally been 20% or less with more is possible the type and intensity of prior therapy conventional chemotherapy- or TBI-based regimens may influence the severity of transaminitis. In this re- [1,32]. Although the number of patients in the current spect, in a phase I trial of patients with advanced study is too small to make definitive conclusions solid tumors, who may have been less intensively regarding efficacy, the results strongly suggest that the pretreated than refractory leukemia patients, no grade high-dose clofarabine, etoposide, and cyclophospha- 3-4 transaminitis was observed despite clofarabine mide regimen should be studied further to determine doses of up to 129 mg/m2 (given weekly for 3 weeks its relative efficacy, particularly in patients with high- of a 4-week cycle) [27]. Similarly, in a phase I/II trial risk disease where standard preparative regimens are of clofarabine in combination with etoposide and cy- associated with poor outcomes. clophosphamide (given in significantly lower doses We conclude that high-dose clofarabine, etoposide, without stem cell support) in children with relapsed and cyclophosphamide is a novel and well-tolerated pre- and refractory acute leukemia, clofarabine doses of parative regimen for NHL patients undergoing ASCT, 40 mg/m2/day for 5 days resulted in unexpected hepa- with significant antilymphoma activity. Further investi- totoxicity in the form of SOS in the phase II portion of gation of this regimen is justified to better assess its 994 S. Srivastava et al. Biol Blood Marrow Transplant 17:987-994, 2011 efficacy and safety, with a recommended phase II dose of 14. Jeha S, Gaynon PS, Razzouk BI, et al. Phase II study of clofara- clofarabine of 70 mg/m2/day for 5 days in combination bine in pediatric patients with refractory or relapsed acute lym- phoblastic leukemia. J Clin Oncol. 2006;24:1917-1923. with etoposide (60 mg/kg) and cyclophosphamide 15. Kantarjian H, Gandhi V, Cortes J, et al. Phase 2 clinical and (100 mg/kg). pharmacologic study of clofarabine in patients with refractory or relapsed acute leukemia. Blood. 2003;102:2379-2386. 16. Kantarjian HM, Gandhi V, Kozuch P, et al. Phase I clinical and ACKNOWLEDGMENTS pharmacology study of clofarabine in patients with solid and hematologic cancers. J Clin Oncol. 2003;21:1167-1173. Financial disclosure: Pharmacoanalytical work was 17. Gandhi V, Plunkett W, Bonate PL, et al. Clinical and pharma- performed by the Clinical Pharmacology Analytical cokinetic study of clofarabine in chronic lymphocytic leukemia: Laboratory of the Indiana University Melvin and strategy for treatment. Clin Cancer Res. 2006;12:4011-4017. 18. 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