Impact of Prolonged Infusions of the Putative Differentiating Agent Sodium Phenylbutyrate on Myelodysplastic Syndromes and Acute Myeloid Leukemia1

Vol. 8, 963–970, April 2002 Clinical Cancer Research 963

Advances in Brief Impact of Prolonged Infusions of the Putative Differentiating Agent Sodium Phenylbutyrate on Myelodysplastic Syndromes and Acute Myeloid Leukemia1

Steven D. Gore,2 Li-Jun Weng, William D. Figg, ule developed hematological improvement. Prolonged infu- Suoping Zhai, Ross C. Donehower, George Dover, sions of PB are well tolerated making this an attractive platform for the clinical investigation of HDAC inhibition. Michael R. Grever, Constance Griffin, Louise B. Grochow, Anita Hawkins, Introduction Kathleen Burks, Yelena Zabelena, and The paucity of effective therapies for the treatment of Carole B. Miller MDS3 and resistant subsets of AML mandates the development The Johns Hopkins Oncology Center [S. D. G., L-J. W., R. C. D., of new therapeutic strategies for these disorders. The impetus to M. R. G., C. G., A. H., K. B., and C. B. M.], Department of use “differentiating” agents in MDS arises from the clinical Pediatrics, Johns Hopkins School of Medicine, Baltimore, Maryland 21231 [G. D], and Clinical Pharmacokinetic Section, National Cancer observation that bone marrows in MDS are hypercellular, with Institute, Bethesda, Maryland 20892 [W. D. F., L. B G., and S. Z.] aberrant differentiation and concomitant bone marrow failure. Similarly, in resistant AML, agents that promote functional hematopoiesis might enable patients to survive with their dis- Abstract ease. Differentiating agents have at least three potential roles in The aromatic fatty acid sodium phenylbutyrate (PB) the treatment of myeloid neoplasms: (a) terminal differentiation promotes cytostasis and differentiation in a wide variety of of a malignant clone to clonal extinction, as in retinoic acid tumor types; among several molecular activities, inhibition induction of acute promyelocytic leukemia (1); (b) enforced of histone deacetylase (HDAC) may account for many of its clonal differentiation leading to functional but clonal hemato- pharmacodynamic effects. A Phase I study demonstrated poiesis; and (c) prolongation of remission duration in patients promising preliminary evidence of clinical activity in acute with AML or MDS with residual disease after chemotherapy myeloid leukemia and myelodysplastic syndrome; however, through suppression of proliferation of the malignant clone. plasma concentrations achieved at the maximum tolerated We recently completed a Phase I study of 7-day continuous dose were less than those targeted based on in vitro studies. infusions of the aromatic fatty acid PB in patients with MDS and Because prolonged exposure to suboptimal concentrations of selected patients with AML. (2) In vitro, PB induces differen- PB in vitro led to pharmacodynamic changes similar to a tiation, inhibits proliferation of AML cell lines and primary more brief exposure to higher concentrations, a study of the leukemic cells (3, 4), and inhibits CFU-L production from bone feasibility of prolonged administration of sodium PB was marrow specimens from patients with MDS (4). In the ML-1 performed. Selected patients with acute myeloid leukemia myeloid leukemia cell line, PB-induced differentiation is asso- and myelodysplastic syndrome were treated with sodium PB ciated with induction of p21WAF1/CIP1 expression, hypophos- as a continuous i.v. infusion via ambulatory infusion pump. phorylation of retinoblastoma protein, and arrest in the G1 phase Sequential cohorts were treated for 7 consecutive days out of of the cell cycle (3). At least some of the pharmacodynamic 14 or with 21 consecutive days out of 28. Prolonged infusions effects of PB appear to be because of its ability to inhibit HDAC were well tolerated; dose-limiting central nervous system (5); histone acetylation contributes importantly to regulation of toxicity developed in 1 of 23 patients treated. End-of-infu- gene transcription (6). The MTD of PB administered as a 7-day sion plasma concentrations were maintained within a range continuous infusion was 375 mg/kg/day; higher doses were sufficient to inhibit HDAC. Two patients on the 21/28 sched- associated with encephalopathy because of nonlinear accumulation of its metabolite PA (2). At the MTD, median steady state

concentration of PB was 0.3 mM, less than the ED50 for differentiation and cytostasis in vitro (1–2 mM; Ref. 3) but well within the concentration range which induces acetylation of histones (5). Received 5/17/01; revised 11/8/01; accepted 11/16/01. Although steady-state concentrations achieved in this study The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked were lower than we had targeted, sustained HI in neutrophils advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by Grants RO1 CA67803 and CA 15396. S. D. G. is the recipient of a Scholar Award for Clinical Research from The Leukemia and Lymphoma Society of America. Targon Pharmaceuticals 3 The abbreviations used are: MDS, myelodysplastic syndrome; AML, provided support for data management. Presented in part at the annual acute myeloid leukemia; PB, phenylbutyrate; MTD, maximum tolerated meeting of The American Society of Hematology, December 1998. dose; PA, phenylacetate; CNS, central nervous system; FISH, fluores- 2 To whom requests for reprints should be addressed, at Johns Hopkins cence in situ hybridization; HI, hematological improvement; PAG, Oncology Center, 1650 Orleans Street, Room 288, Baltimore, MD phenylacetylglutamine; CFU-L, leukemia colony-forming unit; CFU- 21231-1000. Phone: (410) 955-8781; Fax: (410) 614-1005; E-mail: Ste- GM, colony-forming unit, granulocyte-macrophage; G-CSF, granulo- ven.Gore @jhu.edu. cyte colony-stimulating factor; HDAC, histone deacetylase. Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2002 American Association for Cancer Research. 964 Prolonged Infusions of Sodium Phenylbutyrate

and platelet counts were documented in several patients, and supplies of PB were distributed to patients. Patients or their transient improvement in hemograms were frequent (2). In family members changed the liter bag of PB daily; all of the addition, peripheral blood blasts were cleared in several patients used bags were returned to the pharmacy for verification of treated on this study. In vitro, prolonged exposure to lower administration. PB was administered through an indwelling concentrations of PB led to pharmacodynamic effects compara- central venous catheter (Hickman, Groshong, or port). Twelve ble with briefer exposures to higher concentrations (3). In acute weeks of therapy was planned; patients responding to PB could promyelocytic leukemia, the best clinical model of successful continue to receive the drug as long as they continued to show differentiation therapy, prolonged exposure to all-trans-retinoic improvement. acid is required to effect remission (1). Therefore, we studied All of the patients received 375 mg/kg/day of PB, the MTD the feasibility and efficacy of prolonged exposure to continuous defined previously. Two dosing schedules were studied. In the i.v. infusion of PB in a cohort of patients with resistant myeloid first schedule (7/14), patients received 7 days of PB, followed neoplasms. by 7 days of drug holiday. This schedule was repeated for a total of 12 weeks (total of 6 weeks of PB infusion alternating with 6 Patients and Methods weeks of drug holiday). In the second schedule (21/28), patients received 21 consecutive days of PB infusion followed by a Patients 7-day drug holiday. This schedule was repeated for a total of Patients were recruited from referrals to the Division of three cycles (12 weeks). Accrual was planned at each schedule Hematological Malignancy at The Johns Hopkins Oncology until 6 patients were evaluable for a full 12-week period of Ն Center. Previously treated or untreated patients age 18 years observation for toxicity at each schedule. with any French-American-Beinsh subset of MDS were eligible; An infusional schedule was considered tolerable if no more however, patients in whom blasts were not increased were than 1 of 6 evaluable patients had grade 3 or 4 nonhematological required to have some hematological abnormality potentially toxicity. As in the previous study, dose-limiting hematological benefiting from therapy: transfusion-dependence, granulocyte toxicity for MDS was defined as severe aplasia (neutrophils Ͻ ␮ Ͻ ␮ count 1,000/ l, or platelet count 50,000/ l. Most recent Ͻ500/␮l; platelets Ͻ25000/␮l if they were higher pretreat- Ն chemotherapy administration was required to be 1 month ment), which lasted Ͼ21 days. Dose-limiting hematological before enrollment. toxicity for relapsed AML was defined as prolonged aplasia in Patients with AML were eligible if they had relapsed or if the absence of disease progression if the neutrophil and platelet they were untreated but were deemed to be poor candidates counts were sufficiently higher before therapy, as defined medically for AML induction chemotherapy because of age, above. comorbidity, poor-risk cytogenetics, or because they had re- Despite the observation of asymptomatic alterations in uric fused chemotherapy. Patients with AML were required to have acid metabolism in the previous Phase I trial of a 7-day PB Ͻ ␮ WBC count 30,000/ l, stable for at least 2 weeks, and be infusion, allopurinol was not administered. Administration of deemed unlikely to require cytotoxic therapy during the duration hematopoietic growth factors was prohibited. of the trial. All of the patients were required to meet the following eligibility criteria: Zubrod performance status Յ2, absence of Study Parameters active infections at the time of study entry, serum creatinine All of the patients had the following studies obtained Ͻ2 mg/dl, total serum bilirubin Ͻ2.5 mg/dl, no clinical evi- before starting treatment: complete blood count with differential dence of CNS or pulmonary leukostasis, disseminated intravas- WBC count, platelet and reticulocyte count, serum chemistry cular coagulation, CNS leukemia, or negative serum ␤- human profile, prothrombin time, partial thromboplastin time, fibrino- chorionic gonadotropin (female patients of reproductive age). gen degradation products, fibrinogen, determination of fetal Administration of hematopoietic growth factors must have been RBCs (F-cells) and reticulocytes (F-reticulocytes; see below), discontinued 3 weeks before protocol entry and was prohibited Wright’s stained bone marrow aspirate, and bone marrow bi- while on study. All of the patients gave written informed con- opsy. Karyotype analysis was performed on aspirated marrow. sent approved by the Institutional Review Board under guide- In addition, marrow aspirate was collected for bone marrow lines of the Department of Health and Human Services. correlative studies (see below) and FISH (see below). Electro- cardiogram and chest radiograph were also obtained. Blood tests were repeated weekly while on study; bone marrow was re- Treatment peated at the conclusion of weeks 6 and 12; F-cells and F- PB is manufactured by Elan Pharmaceutical Research Cor- reticulocytes were quantified monthly. Patients continuing to poration (Gainesville, GA). The drug was supplied through the receive PB beyond the first 12 weeks had bone marrow aspirates Cancer Therapy Evaluation Program of the Division of Cancer repeated every 3 months while on study; these were studied for Treatment and Diagnosis in the National Cancer Institute as 50 morphology only. ml of a 40% viscous solution of PB in sterile water (400 mg/ml). To administer this drug as a continuous infusion, the total daily dose was diluted in one liter of sterile D5W, USP. Response Criteria After registration, patients had prestudy blood testing and Responses were graded according to Cancer and Leukemia bone marrow aspiration (see below). Patients or family members Group B criteria (7) as in the previous study (2). This included were instructed in the use of continuous ambulatory infusion a category of HI, defined as a Ն50% restoration of the deficit pumps (CADD-plus; SIMS/Deltec, St. Paul, MN). Seven-day from normal in one or more peripheral blood cell lines but

Table 1 Patient characteristics 7/14 schedule 21/28 schedule Number Median Range Number Median Range Patients 13 10 Age 69 (48–76) 62 (54–79) Diagnosis: MDS 4 5 RA 0 4a RAEB 3 RAEB-t 1 1a AML 9 5 WBC 1200 (500–2700) 2000 (1000–12800) Absolute neutrophic count 160 (0–748) 467 (0–3888) Hematocrit 28.7 (24.7–33.8) 27.9 (19.3–42.3) Platelet 47 (16–98) 20 (4–198) Blast % 1 (0–76) 0 (0–41) Clonal cytogenetics 8 8 Previous therapy Yes 4 4 No 9 6 a Two patients with RA and one with RAEB-t had chemotherapy-induced MDS.

insufficient to meet criteria for partial remission or complete used: CD34 (HPCA-2; Becton Dickinson, Mountain View, CA) remission, or a Ն50% decrease in packed red blood cell or and HLA-DR (CR3/43; Dako, Carpinteria, CA), and CD14 platelet transfusion requirements. HI constituted a remission for (Tuk4; Dako) and CD15 (C3D-1; Dako). Isolated CD34ϩ cells the purpose of follow-up. were stained for the following antigens: CD13 (Leu-M7; Becton Dickinson), CD33 (WM-54; Dako), HLA-DR, CD38 (Leu-17;, Pharmacokinetic Studies Becton Dickinson), and glycophorin (GA-R2; PharMingen, San Because pharmacokinetic analysis in the previous Phase I Diego, CA). C-kit expression was measured after binding of study showed no evidence of induction of PB metabolism over phycoerythrin-labeled Steel Factor (R and D Associates, Min- time, limited plasma sampling was performed in the present neapolis, MN). Fluorochrome-labeled isotype-matched controls study to demonstrate maintenance of PB steady-state concen- were purchased from Dako. trations over prolonged exposures. Samples were obtained Cell Cycle Analysis and Determination of Apoptosis. weekly during the first 12 weeks of study; this sample was to be Cell cycle analysis and apoptosis were performed in isolated drawn before completing the 7-day infusion (7/14 schedule) or CD34ϩ cells using bromodeoxyuridine incorporation and ter- before completing the seventh infusion per week (21/28 sched- minal deoxynucleotidyl transferase-mediated nick end labeling ule). Plasma PB, PA, and PAG concentrations were determined assays, respectively, as in the previous study (2). in all of the blood specimens by a modification of a validated Cytogenetics and FISH. As in the previous study, all of high-performance liquid chromatography method (8) as described (2). the initial samples underwent standard cytogenetic analysis. In those patients with abnormalities amenable to FISH, this meth- Bone Marrow Correlative Studies odology was used to monitor subsequent samples (2). Clonogenic Assay. Bone marrow mononuclear cells Fetal Hemoglobin. Erythrocytes and reticulocytes con- were isolated by density gradient centrifugation (specific gravity taining fetal hemoglobin (F-cells and F-reticulocytes) were Ͻ 1.077 g/dl; Ficoll-Hypaque; Pharmacia, Piscataway, NJ) from measured in peripheral blood as described previously (10). heparinized bone marrow aspirates. Clonogenic assays were performed as in the previous study (2). CFU-L were scored on Results days 5–7 of culture; CFU-GM were scored on day 14. CD34 Isolation. CD34ϩ cells were isolated from bone Patients Treated marrow mononuclear cells using immunomagnetic beads Patient characteristics are listed in Table 1. To accrue 6 ((Dynabeads; Dynal, Inc., Lake Success, NY) and removed patients on each dose schedule fully evaluable for 12 weeks of from the beads using Detachabead (Dynal, Inc.). All of the toxicity, 13 patients [4 MDS and 9 AML (3 progressed from isolation procedures were performed according to the manufac- MDS)] were treated on the 7/14 schedule and 10 patients on the turer’s instructions. 21/28 schedule [5 MDS and 5 AML (2 progressed from MDS)]. Flow Cytometric Determination of Differentiation. The median age was 69 (48–76) in the first schedule and 62 Two-color immunofluorescence was performed as described (54–79) in the latter schedule. Eight patients in each cohort had previously (4, 9). Bone marrow mononuclear cells were stained clonal cytogenetic abnormalities; 4 patients in each group were to examine changes in the progenitor cell fraction and the previously treated, all with intensive 1-␤-D-arabinofuranosylcy- mature myeloid compartment. The following antibodies were tosine-based chemotherapy.

Table 2 Toxicitya 7/14 21/28 National Cancer Institute toxicity grade 0 1234 0 1234 Nausea/vomiting 10 3 10 Mucositis 13 9 1 Diarrhea 13 9 1 Liver: Alkaline phosphatase 9 1 Bilirubin 9 Transaminases 13 10 Skin 11 2 8 1 1 CNS: Motor 8 5 5 5 Cortical 10 2 1 10 Hypocalcemia 6 4 3 8 2 Fever 10 1 2 8 2 Other: Hyperuricemia 9 3 Edema 3 1 Odor 2 Urinary frequency 1 Hypercholesterolemia 1 a No Ͼgrade 0 toxicities were found in categories of renal, cardiac, hemorrhagic, or alopecia.

Feasibility and Toxicities Seven patients did not complete 12 weeks of planned PB All of the patients were successfully instructed in the administration. These included the patient with CNS toxicity, 2 operation of the CADD pumps; all of the dispensed bags of PB patients who developed granulocytopenic fevers and reduced were completely infused and returned empty by the patients. No performance status making additional protocol participation un- patient chose to discontinue the study because of the inconven- feasible (days 26 and 57 of study), 3 with progressive disease ience of the infusion schedule. Toxicities are compiled in (days 34, 64, and 73 of study), and 1 who declined additional Table 2. therapy because of personal preference (day 58). 7/14 Schedule. Neurocortical toxicity, identical to the 21/28 Day Schedule. No patient developed neurocortical dose-limiting toxicity seen previously in the 7/28-day Phase I toxicity on this schedule; 5 of 10 patients noted fatigue while study (2) occurred in 1 patient. This 68-year-old man with MDS receiving PB infusion. Four of 10 patients were hospitalized for developed CNS toxicity during week 6 of study. Unlike the granulocytopenic fever. One neutropenic patient developed patients with CNS toxicity in the previous study, serum ammo- grade 1 mucositis and diarrhea. Asymptomatic hypocalcemia nia was not elevated at the time of CNS toxicity. As with was noted in 2 of 10 patients; 3 of 10 developed hyperuricemia. patients in the previous study, CNS toxicity completely reversed One patient developed peripheral edema. A transient skin erup- within 48 h of cessation of PB infusion. Similar to the previous tion was also noted in 1 patient. One patient with relapsed AML patients, this toxicity met National Cancer Institute Common developed severe biopsy-documented Sweet’s syndrome, asso- Toxicity criteria for grade 2 and was clinically dose-limiting. ciated with high grade fevers; this required treatment with This patient was not rechallenged with PB because of patient corticosteroids and cessation of PB therapy. No other significant preference. No other patient developed significant neurocortical toxicities were observed. toxicity; however, 5 of 13 patients noted fatigue while receiving Three of 10 patients did not complete the planned 12 weeks PB. of therapy. The patient who developed Sweet’s syndrome was Ten of 13 patients treated were hospitalized at least once taken off-study on day 33 because of declining performance for granulocytopenic fever. One patient developed grade 2 hy- status. One patient with AML went off-study on day 11 because persensitivity skin reaction requiring cessation of PB infusion. of progressive disease. One patient declined additional therapy This resolved within 2 weeks; subsequently PB was restarted on day 30 for personal reasons. with concurrent use of hydroxyzine and no significant recur- Hyperuricemia and Hyperammonemia. Because re- rence of the skin eruption. As in the previous trial, asympto- duction in the fractional excretion of uric acid had been noted in matic hypocalcemia was noted in 7 patients; hyperuricemia several patients in the previous study, this measurement was developed in 9. Three patients noted new or increased peripheral studied on day 1 and day 8 of the first week of PB infusion in edema; 1 noted urinary frequency while receiving PB infusion. 12 patients. The results are shown in Table 3. The fractional The spouses of 2 patients noted a subtle odor about the patient excretion of uric acid on day 8 was Ͻ90% of day 1 in 8 patients during PB infusion (this could be detected by the study nurse but (range 15- 127%; median 73%). The mean day 8/day 1 percent- not principal investigator). One patient developed hypercholes- age was 69 Ϯ 0.09 (P Ͻ 0.001 testing hypothesis that the terolemia while on study. Intermittent mild nausea was noted by percentage was different from 100%; Student’s t test). 3 patients. None of these toxicities required discontinuation of Serum ammonia values were measured weekly because of study drug administration. the concurrence of hyperammonemia and neurocortical toxicity

Table 3 Fractional excretion of uric acida ease [4 AML, 1 MDS (RAEB-t)]; 4 were stable [2 AML, 2 MDS Serum uric acid Fractional excretion of uric acid (RAEB)]; and four were nonevaluable because of inadequate length of treatment [3 AML, 1 MDS (RAEB)]. Nonsustained Day 8/Day 1 changes in hematopoiesis were seen in 4 patients. Three patients Patient Day 1 Day 8 Day 1 Day 8 (%) developed transient increases in platelet counts [39,000–83,000 204 3.9 11.8 6.2 0.9 15.2 (AML); 55,000–76,000 (AML); 98,000 to 198,000/␮l (RAEB- 202 4.4 7.8 9.7 2.6 26.4 308 6.6 10 12.9 5.6 43.6 t)]. The duration of elevation in each case was 7–14 days. One 209 7.5 9.8 6.8 3.1 45.6 patient with AML evolved from MDS developed a progressive 208 6.1 7.3 8.4 4.1 48.4 increase in monocytes with concomitant decrease in bone mar- 301 4.5 5.9 7.3 5.4 73.5 row CD34 ϩ cells; peripheral blood monocytes increased from 210 5.7 5.9 9.7 7.7 79.8 13 to 60% and CD34ϩ cells decreased from 62.8 to 30.8 at 203 5.2 5.4 10.7 9.0 84.0 206 5.5 5.2 7.2 6.6 91.1 week 6. This patient did not complete study because of geo- 207 3.7 5.7 8.2 8.0 97.6 graphic relocation, discontinuing enrollment on day 58. One 306 3.4 4.1 13.4 17.0 127.4 patient with progressive AML (WBC 1,600–23,000/␮l) dem- a Uric acid was measured in serum and in 24-h urine collections on onstrated a simultaneous increase in neutrophil production days 1 and 8. Data are arrayed according to increasing Day 8:Day 1 ratio (ANC 48–908/␮l). of fractional excretion of uric acid. Patient numbers beginning with 2 Seven patients had circulating blasts at the time of study were treated on the 7/14 schedule; patient numbers beginning with 3 entry; none cleared the blasts while on study. were treated on the 21/21 schedule. 21/28 Schedule. No patient achieved a complete remission or partial remission. Two patients developed HI, both based in the previous study. No patient developed abnormally high on improvement in platelet counts. One was a 55-year-old man serum ammonia. with recurrent MDS (RA) after induction therapy for AML, which arose from long-standing MDS. This patient sustained Pharmacokinetic Analysis platelet counts in the 20–30,000/␮l range, having required For the 7/14 dose schedule, PB end-of-infusion concentra- platelet transfusions to maintain platelets Ͼ10,000/␮l before tions showed significant variability among patients; although protocol entry. Platelet transfusion independence developed af- there was a slight trend toward increasing concentrations with ter the first 4-week cycle of PB administration and was sus- time, this was not statistically significant (Table 4). Median tained for 10 months of PB therapy. At the request of the patient, end-of-infusion PB plasma concentration was 0.4 mM (mean, the infusion schedule was successively changed during his 12 1.2; range 0.03–6.42 mM); 45% of values were Ͼ0.5 mM (Table months of protocol enrollment after the initial 12-week admin- 4). PB concentrations at the end of cycle 5 were inordinately low istration. From months 4–6, PB was administered for 14 days compared with the other cycles; the reasons for this are unclear. followed by a 14-day rest period. After this, from months 6–12, Examination of end-of-infusion PB concentrations by individual he received PB on a 7/28 day schedule. On decline of his platelet patient showed no trends to either accumulation or catabolism of counts, his PB infusion was increased again to 14/28 days for 2 drug over time. End-of-infusion PA concentrations appeared months, with no subsequent response. relatively constant over time (median 0.83 mM, mean 0.92; HI was also documented in a 60-year-old woman with Table 4). PAG concentrations were constant throughout the 12 secondary MDS (RA) attributed to a previous autologous bone weeks (Table 4). Serial pharmacokinetic data on the 21/28 marrow transplant for non-Hodgkin’s lymphoma. This heavily schedule were available in 5 patients. End-of-infusion PB con- alloimmunized patient converted from three times weekly plate- centrations were highly variable but similar to those on the 7/14 let transfusion requirements to platelet transfusion independ- schedule (median 0.43 mM, range 0.13- 3.39 mM; Table 5). ence, which lasted for 3 months. She became platelet transfu- Examination of end-of-infusion PB concentrations by individual sion-dependent again after her fourth cycle of PB and was taken patient showed no trends to either accumulation or catabolism of off-study. drug over time. End-of-infusion PA concentrations were con- Mean end-of-infusion plasma PB concentrations in the 2 stant over time, although median plasma concentration was patients who achieved HI were 1.6 Ϯ 0.2 and 0.67 Ϯ 0.3 mM. lower than that of the 7/14 schedule (0.23 versus 0.83 mM; Table Whereas these values were both above the median, there was no 5). The median (range) of PA concentrations at cycles 1 and statistically significant difference between the combined mean cycle 2 were 0.29 (0.11–0.74) mM and 0.25 (0.08–0.89) mM, of end-of-infusion PB concentrations for these 2 patients and the respectively. Two subjects had 53% and 51% decreased PA nonresponding patients treated on this schedule. concentration on week 3 compared with week 1. The other 3 Three patients developed progressive disease while on subjects showed increased or unchanged PA concentrations over study [2 AML, 1 MDS (RAEB-t)]; 4 patients had stable disease time. The mean PA concentration at week 3 was lower than [2 AML, 2 MDS (RA)]; and 1 was not evaluable because of week 1; however, the difference was not statistically significant inadequate time on study (AML). In addition to the patients with (0.239 Ϯ 0.1 mM versus 0.354 Ϯ 0.331 mM; P ϭ 0.4). PAG HI, 2 patients developed nonsustained improvement in ANC concentrations were constant over time. (200–3000 and 80–390/␮l). The patient who developed Sweet’s syndrome had a decrease in bone marrow blasts of 33% (60– Clinical Outcomes 40% on bone marrow differential count) at the time of removal 7/14 Schedule. No patient on this schedule achieved from study. This was accompanied by differentiating myeloid measurable response. Five patients developed progressive dis- cells showing both granulocytic and monocytic maturation not

Table 4 End-of-infusion plasma concentrations of PB, PA, and PAG throughout treatment; 7/14 schedulea Cycle 12 3 456 PB Mean 834.9 1249.4 1314.5 1587.6 248.6 4382.5 SD 900.3 2059.4 1122.4 1451.3 184.5 2887.1 n 10811472 PA Mean 838.1 977.7 959.8 1098 594.5 1637 SD 988.8 788.2 688.7 668.8 485.6 36.8 n 10711572 PAG Mean 586.3 684.9 770.5 721.6 541.6 681 SD 217.7 361.3 249.2 287.3 197.3 215 n 10711572 a Plasma sampling was performed at the end of each week of infusion. Plasma concentrations are all ␮M.

Table 5 End-of-infusion plasma concentrations of PB, PA, and PAG throughout treatment; 21/28 schedulea Cycle 1 Cycle 2 (week) (week) 123567 PB Mean 300 313.5 700.8 1177.2 963.7 1110.8 SD 230.5 184.6 781.6 1376.8 770.9 968.8 n 225 53 4 PA Mean 354.3 431.5 239.2 341.4 401.3 277.3 SD 330.9 43.1 100.4 331.2 397.9 160.5 n 335 53 4 PAG Mean 379.7 595.5 558 477.8 451 538.5 SD 120.4 210 353.5 137.3 140.9 184.1 n 325 53 4 a Plasma sampling was done at the end of each week of infusion. Plasma concentrations are all ␮M.

present at study initiation. On that date, WBC had increased HI, 1 patient had a small trend to increase in CD34ϩ cells (0.5% from 1800 to 3400/␮l; ANC had increased from 216 to 595/␮l. week 0; 1.3% week 6; and 6% week 12); the second patient had On the next day, after a single dose of G-CSF (5 ␮g/kg, a hypocellular marrow, and adequate cells were not available for administered because of ongoing fevers), WBC was 4900/␮l; CD34ϩ quantification. CD14ϩ cells did not increase in any after a second dose of G-CSF, WBC was 5300/␮l and ANC was patient; however, progressive increase in CD15ϩ cells was 2600/␮l. noted in 4 patients (2–13%, 4–22%; 45–67%; 21–35%; and Four patients had circulating blasts on study entry. Three of 11–30%). This group included 1 patient with HI. these developed progressive disease; none cleared the blasts. Clonogenic Assay Bone Marrow Correlative Studies The results of clonogenic assays were variable. CFU-GM increased over time in 3 patients treated on the 7/14 schedule; no Differentiation patient in this cohort had increased CFU-L on serial bone 7/14. Quantification of CD34ϩ bone marrow cells was marrow sampling. Two of 7 evaluable patients demonstrated successful at all three of the time points in 6 patients; data were progressive increases in both CFU-GM and CFU-L while re- available at week 0 and week 6 in 4 additional patients. CD34ϩ ceiving PB on the 21/28 schedule. The patient with HI who had cells increased over time in 6 patients, were unchanged in 2 evaluable CFU had increased CFU-L at week 6 but decreased patients, and decreased in 2 patients (46–37% and 63–33%). CFU-L at week 12 [compared with baseline (CFU results re- The latter patient had progressive decline in blasts, with in- ported per 100,000 cells; baseline, 89.7 Ϯ 11; week 6, 143 Ϯ creased number of monocytes (see above). This patient also had 13; week 12, 17 Ϯ 3; P Ͻ .01 week 12 versus baseline)]. progressive increase in CD14ϩ cells (12–30%) and CD15 ϩ CFU-GM changed in parallel in this patient (46 Ϯ 5; 71 Ϯ 7; cells (18–57%). No other patient developed significant in- 15 Ϯ 3; P Ͻ 0.05 week 12 versus baseline). creases in CD14ϩ cells; however, CD15ϩ cells increased in 2 other patients (4–18% and 7–24%). 21/28. Quantification of CD34ϩ bone marrow cells was Cell Cycle Analysis and Apoptosis successful at all three of the time points in 6 patients; data were The median percentage of isolated CD34ϩ cells in S phase available at week 0 and week 6, or week 0 and week 12 in 5 at baseline was 4% (range 0–11; n ϭ 16). The corresponding additional patients. One patient had progressive increase in numbers at weeks 6 and 12 were 2.7 (range 0–10; n ϭ 14) and CD34ϩ cells; the others were unchanged. Of the 2 patients with 3.4 (range 0–22; n ϭ 7). No consistent pattern of change of

Table 6 Percentage clonality Discussion Percent clonal cells The current study was designed to explore the feasibility of FISH/metaphase Week long-term administration of the putative differentiating agent Abnormality cytogenetics Schedule studied (F/C)a 06 12 sodium PB as a continuous infusion through ambulatory infusion pumps. The previous Phase I study of 7-day continuous 7/14 ϪY F 38 83 off protocol infusion PB showed a narrow therapeutic window: at the MTD, del (5) C 11 100 off protocol plasma concentrations were lower than those initially targeted, Ϫ7 F 65 78 off protocol whereas higher dose rates regularly induced neurocortical tox- Ϫ 7 F 32 39 54 icity through nonlinear accumulation of the metabolite PA (2). Ϫ7 F 23 56 off protocol ϩ8 F 31 16 28 Despite the lower-than-targeted plasma concentrations at the i14 C 50 62.5 0 MTD, clinical responses were seen; in addition, the concentra- 21/28 tions achieved were within the range of PB concentrations that ϩ 2C05535inhibit HDAC in vitro (5). Because of the narrow therapeutic ϩ8 C 12.5 0 0 ϩ8 C 100 100 80 window, a single dose was administered in the present study of Ϫ7 C 100 100 ndb two prolonged dosing schedules. a F refers to samples in which clonality was monitored using FISH. Both schedules were well tolerated, with a single episode C refers to samples in which clonality was monitored using metaphase of dose-limiting neurocortical toxicity among the 23 patients cytogenetics. studied as the most serious complication. Mild fatigue was b nd, not done. experienced with greater frequency. Not surprising in this group of granulocytopenic patients with indwelling catheters, neutropenic fever was common; no patient developed sepsis. De- creased fractional excretion of uric acid with increased serum percentage of S phase CD34ϩ cells was seen in either dose uric acid was again documented in this cohort of patients as was schedule. Baseline apoptosis among isolated CD34ϩ cells, as- asymptomatic hypocalcemia. Home administration of PB was sessed using a terminal deoxynucleotidyl transferase-mediated accepted by most patients and all were successful in self- nick end labeling technique, was 0% (range 0–4; n ϭ 16). The administration or administration of PB by a family member. No corresponding numbers at weeks 6 and 12 were 0 (range 0–3; patient discontinued study because of inconvenience of admin- n ϭ 14) and 0 (range 0–1; n ϭ 5). No consistent pattern of istration. ϩ change of percentage of apoptotic CD34 cells was seen in Few objective responses were achieved. Two patients de- either dose schedule. The sample size for these studies dimin- veloped platelet transfusion independence, 1 sustained for ϳ1 ished over time because of patient attrition and bone marrow year. Nonsustained improvement in platelet count and neutro- samples which were inadequate for analysis. phil counts were observed as in the previous study. The development of progressive monocytosis with decreasing bone mar- Fetal Hemoglobin row CD34ϩ cells in 1 patient and the development of severe Sequential data on percentage of reticulocytes and eryth- Sweet’s syndrome, associated with decreasing bone marrow rocytes containing fetal hemoglobin (F-reticulocytes and F- blast cells and a remarkably rapid (1-day) neutrophil response to cells, respectively) were available in 8 patients treated on the G-CSF administration, are provocative as potential examples of 7/14 schedule and 5 patients on the 21/28 schedule. F-reticulo- myeloid differentiation in response to PB. cytes and/or F-cells increased over time in 1 of 4 of patients on Pharmacokinetic analysis was necessarily limited by the the 7/14 schedule who completed 12 weeks of therapy (F- end-of-infusion sampling performed as well as the variability of reticulocytes, 0.7–13% and F-cells, 6.6–19.7%) and 2 of 4 fully the PB concentration data. However, no evidence of progressive evaluable patients on the 21/28 schedule (F-reticulocytes, PA accumulation nor declining PB concentration was docu- 0–10% and 9.3–24%; F-cells 4.7–5.3% and 5.4–9.5%). The mented. The median PB concentration of 0.4 ␮M throughout the first-noted patient in this group was one of the patients who 7/14 schedule and 0.43 throughout the 21/28 schedule, similar achieved HI. with the previous Phase I steady state value of 0.3 mM, demon- strates that this concentration, which effectively induces histone Clonality acetylation in leukemic cell lines, mononuclear cells, and pri- Serial samples were evaluated for changes in percentage mary leukemic blasts, can be sustained. clonal cells in 7 patients on the 7/14 schedule and 4 patients on As in the initial Phase I study, changes in CFU-L and the 21/28 schedule (Table 6). Percentage of clonal cells in- CFU-GM were variable. The single responding patient evalu- creased over time in 5 patients receiving PB on the 7/14 day able for changes in CFU showed declining CFU-L and schedule, was stable in 1 patient and decreased from 50 to 0% CFU-GM despite increased platelet counts. As in the 7/28 day in the seventh patient (monitored using metaphase cytogenet- schedule studied previously, the current schedules enabled in- ics). Among the 4 patients studied on the 21/28 schedule (all creases in production of hemoglobin F-bearing RBCs in several using metaphase cytogenetics), percentage of clonal cells de- patients. Switching of globin gene expression by butyrate ana- clined in 1 patient and increased or were stable in the other 3. logues appears to be because of HDAC inhibition (11, 12); thus, Neither of the 2 patients treated on the 21/28 schedule who the successful induction of fetal hemoglobin expression by these developed HI had clonal cytogenetic abnormalities. agents may imply successful inhibition of HDAC (see below). It

is noteworthy that although the percentage of clonal cells in- 2. Gore, S. D., Weng, L-J., Zhai, S., Figg, W. D., Donehower, R. C., creased in most patients studied, PB administration was associ- Dover, G. J., Grever, M., Griffin, C. A., Grochow, L. B., Rowinsky, ated with a suppression of clonal cells in 2 patients. Both of E. K., Zabalena, Y., Hawkins, A. L., Burks, K., and Miller, C. B. Impact of the putative differentiating agent sodium phenylbutyrate on myelo- these patients were studied using metaphase cytogenetics; this dysplastic syndromes and acute myeloid leukemia. Clin. Canc. Res., 7: finding may be attributed to a cell cycle-inhibitory effect of PB 2330–2339, 2001. (with concomitant inability to detect the clonal cells) rather than 3. DiGiuseppe, J. A., Weng, L-J., Yu, K. H., Fu, S., Kastan, M. B., true clonal suppression. Samid, D., and Gore, S. D. Phenylbutyrate-induced G1 arrest and No consistent changes were seen in the proliferative or apoptosis in myeloid leukemia cells: structure-function analysis. Leu- apoptotic percentage of the CD34 compartment during PB ther- kemia (Baltimore), 13: 1243–1253, 1999. apy. It should be noted that bone marrow was necessarily 4. Gore, S. D., Samid, D., and Weng, L-J. Impact of the putative differentiating agents sodium phenylbutyrate and sodium phenylacetate sampled intermittently (baseline, weeks 6 and 12), and early on proliferation, differentiation, and apoptosis of primary neoplastic changes may have been missed. It is now recognized that the myeloid cells. Clin. Canc. Res., 3: 1755–1762, 1997. inhibition of proliferation by PB is associated with induction of 5. Yu, K. H., Weng, L-J., Fu, S., and Gore, S. D. Augmentation of expression of p21WAF1/CIP1 (3); because this trial was initiated phenylbutyrate-induced differentiation of myeloid leukemia cells using before the possibility of a mechanistic role for this gene product all trans-retinoic acid. Leukemia (Baltimore), 13: 1258–1265, 1999. was demonstrated, its expression was not monitored in the trial. 6. Redner, R. L., Wang, J., and Liu, J. M. Chromatin remodeling and Recent reports have shown that expression of a wide vari- leukemia: new therapeutic paradigms. Blood, 94: 417–428, 1999. ety of genes regulating differentiation and proliferation, includ- 7. Silverman, L. R., Holland, J. F., Weinberg, R. S., Alter, B. P., Davis, R. B., Ellison, R. R., Demakos, E. P., Cornell, C. J., Jr., Carey, R. W., ing retinoid-responsive genes and retinoblastoma gene, is regu- Schiffer, C., and et al. Effects of treatment with 5-azacytidine on the in lated in part through changes in histone conformation regulated vivo and in vitro hematopoiesis in patients with myelodysplastic syn- in turn through acetylation (13). Histone acetylation is regulated dromes. Leukemia, 7 (Suppl. 1): 21–29, 1993. through local recruitment of histone acetyltransferases and 8. Piscitelli, S. C., Thibault, A., Figg, W. D., Tompkins, A., Headlee, HDACs. An increasing number of leukemia-associated fusion D., Lieberman, R., Samid, D., and Myers, C. E. Disposition of phenylbutyrate and its metabolites, phenylacetate and phenylacetylglutamine. genes inhibit gene transcription through recruitment of HDACs J. Clin. Pharmacol., 35: 368–373, 1995. (6). Histone deacetylation contributes to the transcriptional re- 9. Loken, M. R., Shah, V. O., Dattilio, K. L., and Civin, C. I. Flow pression of genes of which the promoters are methylated at CpG cytometric analysis of human bone marrow: II. Normal B lymphocyte sites through the corepressor protein MeCP2, which binds to development. Blood, 70: 1316–1324, 1987. methylated CpG sites and recruits HDACs (14). Sequential 10. Dover, G. J., Brusilow, S., and Charache, S. Induction of fetal administration of DNA methyltransferase inhibitors and HDAC hemoglobin production in subjects with sickle cell anemia by oral inhibitors (including PB) has led to the synergistic re-expression sodium phenylbutyrate. Blood, 84: 339–343, 1994. of genes silenced through methylation (15). HDAC inhibitors 11. Ishiguro, K., and Sartorelli, A. C. Coinduction of embryonic and adult-type globin mRNAs by sodium butyrate and trichostatin A in two synergize with retinoids (5, 16, 17) and render retinoid-resistant murine interleukin-3-dependent bone marrow-derived cell lines. Blood, acute promyelocytic leukemia cells retinoid sensitive in vitro 92: 4383–4393, 1998. and in vivo (18). Thus, HDAC inhibitors may be exploited 12. Perrine, S. P., Faller, D. V., Swerdlow, P., Miller, B. A., Bank, A., clinically to alter the malignant phenotype (19). Unfortunately, Sytkowski, A. J., Reczek, J., Rudolph, A. M., and Kan, Y. W. Stopping this trial was initiated before the recent recognition of the the biologic clock for globin gene switching. Ann. N. Y. Acad. Sci., 612: 134–140, 1990. importance of chromatin remodeling in transcriptional regula- 13. Luo, R. X., and Dean. D. C. Chromatin remodeling and transcription and the significance of HDAC as a potential target for PB, tional regulation. J. Natl. Can. Inst., 91: 1288–1292, 1999. precluding the in vivo monitoring of changes in histone acety- 14. Nan, X., Ng, H-H., Johnson, C. A., Laherty, C. D., Turner, B. M., lation in this trial. Whereas a variety of more potent HDAC Eisenman, R. N., and Bird, A. Transcriptional repression by the methyl- inhibitors are in early stages of preclinical and clinical devel- CpG-binding protein MeCP2 involves a histone deacetylase complex. opment, the ability to sustain HDAC-inhibitory concentrations Nature (Lond.), 393: 386–389, 1998. of PB with minimal toxicity make this a logical choice for early 15. Cameron, E. E., Bachman, K. E., Myohanen, S., Herman, J. G., and stage clinical development of HDAC inhibitors in myeloid Baylin, S. B. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet., 21: malignancies. 103–107, 1999. 16. Breitman, T. R., and He, R. Combinations of retinoic acid with Acknowledgments either sodium butyrate, dimethyl sulfoxide, or hexamethylene bisacet- We thank Julitta Krawiec and Yvette Freeman for outstanding data amide synergistically induce differentiation of the human myeloid leukemia cell line HL60. Canc. Res., 60: 6268–6273, 1990. management; Drs. Michael Carducci and Sharyn Baker for helpful discussions; Abbie Mays and Vicky Smith for invaluable technical 17. Lin, R. J., Nagy, L., Inoue, S., Shao, W., Miller, W. H., Jr., and Evans, R. M. Role of the histone deacetylase complex in acute promy- support; Kariann Johnson for expert assistance with manuscript prepa- elocytic leukaemia. Nature (Lond.), 391: 811–814, 1998. ration; and Targon Pharmaceuticals for support for data management. 18. Warrell, R. P., Jr., He, L-Z., Richon, V., Calleja, E., and Pandolfi, P. P. Therapeutic targeting of transcription in acute promyelocytic References leukemia by use of an inhibitor of histone deacetylase. J. Natl. Can. 1. Warrell, R. P., Frankel, S. R., Miller, W. H., Jr., Scheinberg, D. A., Inst., 90: 1621–1625, 1998. Itri, L. M., Hittelman, W. N., Vyas, R., Ndreeff, M., Tafuri, A., 19. Gore, S. D., and Carducci, M. A. Modifying histones to tame Jakubowski, A., Gabrilove, J., Gordon, M. S., and Dmitrovsky, E. cancer: clinical development of sodium phenylbutyrate and other his- Differentiation therapy of acute promyelocytic leukemia with tretinoin tone deacetylase inhibitors. Expert Opin. Investig. Drugs, 9: 2923–2934, (all-trans-retinoic acid). N. Engl. J. Med., 324: 1385–1393, 1991. 2000.

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2002 American Association for Cancer Research. Impact of Prolonged Infusions of the Putative Differentiating Agent Sodium Phenylbutyrate on Myelodysplastic Syndromes and Acute Myeloid Leukemia

Steven D. Gore, Li-Jun Weng, William D. Figg, et al.

Clin Cancer Res 2002;8:963-970.

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