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

The paucity of effective therapies for the treatment of MDS³ and resistant subsets of AML mandates the development of new therapeutic strategies for these disorders. The impetus to use “differentiating” agents in MDS arises from the clinical observation that bone marrows in MDS are hypercellular, with aberrant differentiation and concomitant bone marrow failure. Similarly, in resistant AML, agents that promote functional hematopoiesis might enable patients to survive with their disease. Differentiating agents have at least three potential roles in the treatment of myeloid neoplasms: (a) terminal differentiation of a malignant clone to clonal extinction, as in retinoic acid induction of acute promyelocytic leukemia (1); (b) enforced clonal differentiation leading to functional but clonal hematopoiesis; and (c) prolongation of remission duration in patients with AML or MDS with residual disease after chemotherapy through suppression of proliferation of the malignant clone.

We recently completed a Phase I study of 7-day continuous infusions of the aromatic fatty acid PB in patients with MDS and selected patients with AML. (2) In vitro, PB induces differentiation, inhibits proliferation of AML cell lines and primary leukemic cells (3, 4), and inhibits CFU-L production from bone marrow specimens from patients with MDS (4). In the ML-1 myeloid leukemia cell line, PB-induced differentiation is associated with induction of p21^WAF1/CIP1 expression, hypophosphorylation of retinoblastoma protein, and arrest in the G₁ phase of the cell cycle (3). At least some of the pharmacodynamic effects of PB appear to be because of its ability to inhibit HDAC (5); histone acetylation contributes importantly to regulation of gene transcription (6). The MTD of PB administered as a 7-day continuous infusion was 375 mg/kg/day; higher doses were 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).

Although steady-state concentrations achieved in this study were lower than we had targeted, sustained HI in neutrophils and platelet counts were documented in several patients, and transient improvement in hemograms were frequent (2). In addition, peripheral blood blasts were cleared in several patients treated on this study. In vitro, prolonged exposure to lower concentrations of PB led to pharmacodynamic effects comparable with briefer exposures to higher concentrations (3). In acute promyelocytic leukemia, the best clinical model of successful differentiation therapy, prolonged exposure to all-trans-retinoic acid is required to effect remission (1). Therefore, we studied the feasibility and efficacy of prolonged exposure to continuous i.v. infusion of PB in a cohort of patients with resistant myeloid neoplasms.

Patients were recruited from referrals to the Division of Hematological Malignancy at The Johns Hopkins Oncology Center. Previously treated or untreated patients age ≥18 years with any French-American-Beinsh subset of MDS were eligible; however, patients in whom blasts were not increased were required to have some hematological abnormality potentially benefiting from therapy: transfusion-dependence, granulocyte count <1,000/μl, or platelet count <50,000/μl. Most recent chemotherapy administration was required to be ≥1 month before enrollment.

Patients with AML were eligible if they had relapsed or if they were untreated but were deemed to be poor candidates medically for AML induction chemotherapy because of age, comorbidity, poor-risk cytogenetics, or because they had refused chemotherapy. Patients with AML were required to have WBC count <30,000/μl, stable for at least 2 weeks, and be deemed unlikely to require cytotoxic therapy during the duration of the trial.

All of the patients were required to meet the following eligibility criteria: Zubrod performance status ≤2, absence of active infections at the time of study entry, serum creatinine <2 mg/dl, total serum bilirubin <2.5 mg/dl, no clinical evidence of CNS or pulmonary leukostasis, disseminated intravascular coagulation, CNS leukemia, or negative serum β- human chorionic gonadotropin (female patients of reproductive age). Administration of hematopoietic growth factors must have been discontinued 3 weeks before protocol entry and was prohibited while on study. All of the patients gave written informed consent approved by the Institutional Review Board under guidelines of the Department of Health and Human Services.

PB is manufactured by Elan Pharmaceutical Research Corporation (Gainesville, GA). The drug was supplied through the Cancer Therapy Evaluation Program of the Division of Cancer Treatment and Diagnosis in the National Cancer Institute as 50 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.

After registration, patients had prestudy blood testing and bone marrow aspiration (see below). Patients or family members were instructed in the use of continuous ambulatory infusion pumps (CADD-plus; SIMS/Deltec, St. Paul, MN). Seven-day supplies of PB were distributed to patients. Patients or their family members changed the liter bag of PB daily; all of the used bags were returned to the pharmacy for verification of administration. PB was administered through an indwelling central venous catheter (Hickman, Groshong, or port). Twelve weeks of therapy was planned; patients responding to PB could continue to receive the drug as long as they continued to show improvement.

All of the patients received 375 mg/kg/day of PB, the MTD defined previously. Two dosing schedules were studied. In the first schedule (7/14), patients received 7 days of PB, followed 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 weeks of drug holiday). In the second schedule (21/28), patients received 21 consecutive days of PB infusion followed by a 7-day drug holiday. This schedule was repeated for a total of three cycles (12 weeks). Accrual was planned at each schedule until 6 patients were evaluable for a full 12-week period of observation for toxicity at each schedule.

An infusional schedule was considered tolerable if no more than 1 of 6 evaluable patients had grade 3 or 4 nonhematological toxicity. As in the previous study, dose-limiting hematological toxicity for MDS was defined as severe aplasia (neutrophils <500/μl; platelets <25000/μl if they were higher pretreatment), which lasted >21 days. Dose-limiting hematological toxicity for relapsed AML was defined as prolonged aplasia in the absence of disease progression if the neutrophil and platelet counts were sufficiently higher before therapy, as defined above.

Despite the observation of asymptomatic alterations in uric acid metabolism in the previous Phase I trial of a 7-day PB infusion, allopurinol was not administered. Administration of hematopoietic growth factors was prohibited.

All of the patients had the following studies obtained before starting treatment: complete blood count with differential WBC count, platelet and reticulocyte count, serum chemistry profile, prothrombin time, partial thromboplastin time, fibrinogen degradation products, fibrinogen, determination of fetal RBCs (F-cells) and reticulocytes (F-reticulocytes; see below), Wright’s stained bone marrow aspirate, and bone marrow biopsy. Karyotype analysis was performed on aspirated marrow. In addition, marrow aspirate was collected for bone marrow correlative studies (see below) and FISH (see below). Electrocardiogram and chest radiograph were also obtained. Blood tests were repeated weekly while on study; bone marrow was repeated at the conclusion of weeks 6 and 12; F-cells and F-reticulocytes were quantified monthly. Patients continuing to receive PB beyond the first 12 weeks had bone marrow aspirates repeated every 3 months while on study; these were studied for morphology only.

Responses were graded according to Cancer and Leukemia Group B criteria (7) as in the previous study (2). This included a category of HI, defined as a ≥50% restoration of the deficit from normal in one or more peripheral blood cell lines but insufficient to meet criteria for partial remission or complete remission, or a ≥50% decrease in packed red blood cell or platelet transfusion requirements. HI constituted a remission for the purpose of follow-up.

Because pharmacokinetic analysis in the previous Phase I study showed no evidence of induction of PB metabolism over time, limited plasma sampling was performed in the present study to demonstrate maintenance of PB steady-state concentrations over prolonged exposures. Samples were obtained weekly during the first 12 weeks of study; this sample was to be drawn before completing the 7-day infusion (7/14 schedule) or before completing the seventh infusion per week (21/28 schedule). Plasma PB, PA, and PAG concentrations were determined in all of the blood specimens by a modification of a validated high-performance liquid chromatography method (8) as described (2).

Bone marrow mononuclear cells were isolated by density gradient centrifugation (specific gravity < 1.077 g/dl; Ficoll-Hypaque; Pharmacia, Piscataway, NJ) from heparinized bone marrow aspirates. Clonogenic assays were performed as in the previous study (2). CFU-L were scored on days 5–7 of culture; CFU-GM were scored on day 14.

CD34+ cells were isolated from bone marrow mononuclear cells using immunomagnetic beads ((Dynabeads; Dynal, Inc., Lake Success, NY) and removed from the beads using Detachabead (Dynal, Inc.). All of the isolation procedures were performed according to the manufacturer’s instructions.

Two-color immunofluorescence was performed as described previously (4, 9). Bone marrow mononuclear cells were stained to examine changes in the progenitor cell fraction and the mature myeloid compartment. The following antibodies were used: CD34 (HPCA-2; Becton Dickinson, Mountain View, CA) and HLA-DR (CR3/43; Dako, Carpinteria, CA), and CD14 (Tuk4; Dako) and CD15 (C3D-1; Dako). Isolated CD34+ cells were stained for the following antigens: CD13 (Leu-M7; Becton Dickinson), CD33 (WM-54; Dako), HLA-DR, CD38 (Leu-17;, Becton Dickinson), and glycophorin (GA-R2; PharMingen, San Diego, CA). C-kit expression was measured after binding of phycoerythrin-labeled Steel Factor (R and D Associates, Minneapolis, MN). Fluorochrome-labeled isotype-matched controls were purchased from Dako.

Cell cycle analysis and apoptosis were performed in isolated CD34+ cells using bromodeoxyuridine incorporation and terminal deoxynucleotidyl transferase-mediated nick end labeling assays, respectively, as in the previous study (2).

As in the previous study, all of the initial samples underwent standard cytogenetic analysis. In those patients with abnormalities amenable to FISH, this methodology was used to monitor subsequent samples (2).

Erythrocytes and reticulocytes containing fetal hemoglobin (F-cells and F-reticulocytes) were measured in peripheral blood as described previously (10).

Patient characteristics are listed in Table 1. To accrue 6 patients on each dose schedule fully evaluable for 12 weeks of toxicity, 13 patients [4 MDS and 9 AML (3 progressed from MDS)] were treated on the 7/14 schedule and 10 patients on the 21/28 schedule [5 MDS and 5 AML (2 progressed from MDS)]. The median age was 69 (48–76) in the first schedule and 62 (54–79) in the latter schedule. Eight patients in each cohort had clonal cytogenetic abnormalities; 4 patients in each group were previously treated, all with intensive 1-β-d-arabinofuranosylcytosine-based chemotherapy.

All of the patients were successfully instructed in the operation of the CADD pumps; all of the dispensed bags of PB were completely infused and returned empty by the patients. No patient chose to discontinue the study because of the inconvenience of the infusion schedule. Toxicities are compiled in Table 2.

Neurocortical toxicity, identical to the dose-limiting toxicity seen previously in the 7/28-day Phase I study (2) occurred in 1 patient. This 68-year-old man with MDS developed CNS toxicity during week 6 of study. Unlike the patients with CNS toxicity in the previous study, serum ammonia was not elevated at the time of CNS toxicity. As with patients in the previous study, CNS toxicity completely reversed within 48 h of cessation of PB infusion. Similar to the previous patients, this toxicity met National Cancer Institute Common Toxicity criteria for grade 2 and was clinically dose-limiting. This patient was not rechallenged with PB because of patient preference. No other patient developed significant neurocortical toxicity; however, 5 of 13 patients noted fatigue while receiving PB.

Ten of 13 patients treated were hospitalized at least once for granulocytopenic fever. One patient developed grade 2 hypersensitivity skin reaction requiring cessation of PB infusion. This resolved within 2 weeks; subsequently PB was restarted with concurrent use of hydroxyzine and no significant recurrence of the skin eruption. As in the previous trial, asymptomatic hypocalcemia was noted in 7 patients; hyperuricemia developed in 9. Three patients noted new or increased peripheral edema; 1 noted urinary frequency while receiving PB infusion. The spouses of 2 patients noted a subtle odor about the patient during PB infusion (this could be detected by the study nurse but not principal investigator). One patient developed hypercholesterolemia while on study. Intermittent mild nausea was noted by 3 patients. None of these toxicities required discontinuation of study drug administration.

Seven patients did not complete 12 weeks of planned PB administration. These included the patient with CNS toxicity, 2 patients who developed granulocytopenic fevers and reduced performance status making additional protocol participation unfeasible (days 26 and 57 of study), 3 with progressive disease (days 34, 64, and 73 of study), and 1 who declined additional therapy because of personal preference (day 58).

No patient developed neurocortical toxicity on this schedule; 5 of 10 patients noted fatigue while receiving PB infusion. Four of 10 patients were hospitalized for granulocytopenic fever. One neutropenic patient developed grade 1 mucositis and diarrhea. Asymptomatic hypocalcemia was noted in 2 of 10 patients; 3 of 10 developed hyperuricemia. One patient developed peripheral edema. A transient skin eruption was also noted in 1 patient. One patient with relapsed AML developed severe biopsy-documented Sweet’s syndrome, associated with high grade fevers; this required treatment with corticosteroids and cessation of PB therapy. No other significant toxicities were observed.

Three of 10 patients did not complete the planned 12 weeks of therapy. The patient who developed Sweet’s syndrome was taken off-study on day 33 because of declining performance status. One patient with AML went off-study on day 11 because of progressive disease. One patient declined additional therapy on day 30 for personal reasons.

Because reduction in the fractional excretion of uric acid had been noted in several patients in the previous study, this measurement was studied on day 1 and day 8 of the first week of PB infusion in 12 patients. The results are shown in Table 3. The fractional excretion of uric acid on day 8 was <90% of day 1 in 8 patients (range 15- 127%; median 73%). The mean day 8/day 1 percentage was 69 ± 0.09 (P < 0.001 testing hypothesis that the percentage was different from 100%; Student’s t test).

Serum ammonia values were measured weekly because of the concurrence of hyperammonemia and neurocortical toxicity in the previous study. No patient developed abnormally high serum ammonia.

For the 7/14 dose schedule, PB end-of-infusion concentrations showed significant variability among patients; although there was a slight trend toward increasing concentrations with time, this was not statistically significant (Table 4). Median end-of-infusion PB plasma concentration was 0.4 mm (mean, 1.2; range 0.03–6.42 mm); 45% of values were >0.5 mm (Table 4). PB concentrations at the end of cycle 5 were inordinately low compared with the other cycles; the reasons for this are unclear. Examination of end-of-infusion PB concentrations by individual patient showed no trends to either accumulation or catabolism of drug over time. End-of-infusion PA concentrations appeared relatively constant over time (median 0.83 mm, mean 0.92; Table 4). PAG concentrations were constant throughout the 12 weeks (Table 4). Serial pharmacokinetic data on the 21/28 schedule were available in 5 patients. End-of-infusion PB concentrations were highly variable but similar to those on the 7/14 schedule (median 0.43 mm, range 0.13- 3.39 mm; Table 5). Examination of end-of-infusion PB concentrations by individual patient showed no trends to either accumulation or catabolism of drug over time. End-of-infusion PA concentrations were constant over time, although median plasma concentration was lower than that of the 7/14 schedule (0.23 versus 0.83 mm; Table 5). The median (range) of PA concentrations at cycles 1 and cycle 2 were 0.29 (0.11–0.74) mm and 0.25 (0.08–0.89) mm, respectively. Two subjects had 53% and 51% decreased PA concentration on week 3 compared with week 1. The other 3 subjects showed increased or unchanged PA concentrations over time. The mean PA concentration at week 3 was lower than week 1; however, the difference was not statistically significant (0.239 ± 0.1 mm versus 0.354 ± 0.331 mm; P = 0.4). PAG concentrations were constant over time.

No patient on this schedule achieved measurable response. Five patients developed progressive disease [4 AML, 1 MDS (RAEB-t)]; 4 were stable [2 AML, 2 MDS (RAEB)]; and four were nonevaluable because of inadequate length of treatment [3 AML, 1 MDS (RAEB)]. Nonsustained changes in hematopoiesis were seen in 4 patients. Three patients developed transient increases in platelet counts [39,000–83,000 (AML); 55,000–76,000 (AML); 98,000 to 198,000/μl (RAEB-t)]. The duration of elevation in each case was 7–14 days. One patient with AML evolved from MDS developed a progressive increase in monocytes with concomitant decrease in bone marrow CD34 + cells; peripheral blood monocytes increased from 13 to 60% and CD34+ cells decreased from 62.8 to 30.8 at week 6. This patient did not complete study because of geographic relocation, discontinuing enrollment on day 58. One patient with progressive AML (WBC 1,600–23,000/μl) demonstrated a simultaneous increase in neutrophil production (ANC 48–908/μl).

Seven patients had circulating blasts at the time of study entry; none cleared the blasts while on study.

No patient achieved a complete remission or partial remission. Two patients developed HI, both based on improvement in platelet counts. One was a 55-year-old man with recurrent MDS (RA) after induction therapy for AML, which arose from long-standing MDS. This patient sustained platelet counts in the 20–30,000/μl range, having required platelet transfusions to maintain platelets >10,000/μl before protocol entry. Platelet transfusion independence developed after the first 4-week cycle of PB administration and was sustained for 10 months of PB therapy. At the request of the patient, the infusion schedule was successively changed during his 12 months of protocol enrollment after the initial 12-week administration. From months 4–6, PB was administered for 14 days followed by a 14-day rest period. After this, from months 6–12, he received PB on a 7/28 day schedule. On decline of his platelet counts, his PB infusion was increased again to 14/28 days for 2 months, with no subsequent response.

HI was also documented in a 60-year-old woman with secondary MDS (RA) attributed to a previous autologous bone marrow transplant for non-Hodgkin’s lymphoma. This heavily alloimmunized patient converted from three times weekly platelet transfusion requirements to platelet transfusion independence, which lasted for 3 months. She became platelet transfusion-dependent again after her fourth cycle of PB and was taken off-study.

Mean end-of-infusion plasma PB concentrations in the 2 patients who achieved HI were 1.6 ± 0.2 and 0.67 ± 0.3 mm. Whereas these values were both above the median, there was no statistically significant difference between the combined mean of end-of-infusion PB concentrations for these 2 patients and the nonresponding patients treated on this schedule.

Three patients developed progressive disease while on study [2 AML, 1 MDS (RAEB-t)]; 4 patients had stable disease [2 AML, 2 MDS (RA)]; and 1 was not evaluable because of inadequate time on study (AML). In addition to the patients with HI, 2 patients developed nonsustained improvement in ANC (200–3000 and 80–390/μl). The patient who developed Sweet’s syndrome had a decrease in bone marrow blasts of 33% (60–40% on bone marrow differential count) at the time of removal from study. This was accompanied by differentiating myeloid cells showing both granulocytic and monocytic maturation not present at study initiation. On that date, WBC had increased from 1800 to 3400/μl; ANC had increased from 216 to 595/μl. On the next day, after a single dose of G-CSF (5 μg/kg, administered because of ongoing fevers), WBC was 4900/μl; after a second dose of G-CSF, WBC was 5300/μl and ANC was 2600/μl.

Four patients had circulating blasts on study entry. Three of these developed progressive disease; none cleared the blasts.

Quantification of CD34+ bone marrow cells was successful at all three of the time points in 6 patients; data were available at week 0 and week 6 in 4 additional patients. CD34+ cells increased over time in 6 patients, were unchanged in 2 patients, and decreased in 2 patients (46–37% and 63–33%). The latter patient had progressive decline in blasts, with increased number of monocytes (see above). This patient also had progressive increase in CD14+ cells (12–30%) and CD15 + cells (18–57%). No other patient developed significant increases in CD14+ cells; however, CD15+ cells increased in 2 other patients (4–18% and 7–24%).

Quantification of CD34+ bone marrow cells was successful at all three of the time points in 6 patients; data were available at week 0 and week 6, or week 0 and week 12 in 5 additional patients. One patient had progressive increase in CD34+ cells; the others were unchanged. Of the 2 patients with HI, 1 patient had a small trend to increase in CD34+ cells (0.5% week 0; 1.3% week 6; and 6% week 12); the second patient had a hypocellular marrow, and adequate cells were not available for CD34+ quantification. CD14+ cells did not increase in any patient; however, progressive increase in CD15+ cells was noted in 4 patients (2–13%, 4–22%; 45–67%; 21–35%; and 11–30%). This group included 1 patient with HI.

The results of clonogenic assays were variable. CFU-GM increased over time in 3 patients treated on the 7/14 schedule; no patient in this cohort had increased CFU-L on serial bone marrow sampling. Two of 7 evaluable patients demonstrated progressive increases in both CFU-GM and CFU-L while receiving PB on the 21/28 schedule. The patient with HI who had evaluable CFU had increased CFU-L at week 6 but decreased CFU-L at week 12 [compared with baseline (CFU results reported per 100,000 cells; baseline, 89.7 ± 11; week 6, 143 ± 13; week 12, 17 ± 3; P < .01 week 12 versus baseline)]. CFU-GM changed in parallel in this patient (46 ± 5; 71 ± 7; 15 ± 3; P < 0.05 week 12 versus baseline).

The median percentage of isolated CD34+ cells in S phase at baseline was 4% (range 0–11; n = 16). The corresponding numbers at weeks 6 and 12 were 2.7 (range 0–10; n = 14) and 3.4 (range 0–22; n = 7). No consistent pattern of change of percentage of S phase CD34+ cells was seen in either dose schedule. Baseline apoptosis among isolated CD34+ cells, assessed using a terminal deoxynucleotidyl transferase-mediated nick end labeling technique, was 0% (range 0–4; n = 16). The corresponding numbers at weeks 6 and 12 were 0 (range 0–3; n = 14) and 0 (range 0–1; n = 5). No consistent pattern of change of percentage of apoptotic CD34+ cells was seen in either dose schedule. The sample size for these studies diminished over time because of patient attrition and bone marrow samples which were inadequate for analysis.

Sequential data on percentage of reticulocytes and erythrocytes containing fetal hemoglobin (F-reticulocytes and F-cells, respectively) were available in 8 patients treated on the 7/14 schedule and 5 patients on the 21/28 schedule. F-reticulocytes and/or F-cells increased over time in 1 of 4 of patients on the 7/14 schedule who completed 12 weeks of therapy (F-reticulocytes, 0.7–13% and F-cells, 6.6–19.7%) and 2 of 4 fully evaluable patients on the 21/28 schedule (F-reticulocytes, 0–10% and 9.3–24%; F-cells 4.7–5.3% and 5.4–9.5%). The first-noted patient in this group was one of the patients who achieved HI.

Serial samples were evaluated for changes in percentage clonal cells in 7 patients on the 7/14 schedule and 4 patients on the 21/28 schedule (Table 6). Percentage of clonal cells increased over time in 5 patients receiving PB on the 7/14 day schedule, was stable in 1 patient and decreased from 50 to 0% in the seventh patient (monitored using metaphase cytogenetics). Among the 4 patients studied on the 21/28 schedule (all using metaphase cytogenetics), percentage of clonal cells declined in 1 patient and increased or were stable in the other 3. Neither of the 2 patients treated on the 21/28 schedule who developed HI had clonal cytogenetic abnormalities.

The current study was designed to explore the feasibility of long-term administration of the putative differentiating agent sodium PB as a continuous infusion through ambulatory infusion pumps. The previous Phase I study of 7-day continuous infusion PB showed a narrow therapeutic window: at the MTD, plasma concentrations were lower than those initially targeted, whereas higher dose rates regularly induced neurocortical toxicity through nonlinear accumulation of the metabolite PA (2). Despite the lower-than-targeted plasma concentrations at the MTD, clinical responses were seen; in addition, the concentrations achieved were within the range of PB concentrations that inhibit HDAC in vitro (5). Because of the narrow therapeutic window, a single dose was administered in the present study of two prolonged dosing schedules.

Both schedules were well tolerated, with a single episode of dose-limiting neurocortical toxicity among the 23 patients studied as the most serious complication. Mild fatigue was experienced with greater frequency. Not surprising in this group of granulocytopenic patients with indwelling catheters, neutropenic fever was common; no patient developed sepsis. Decreased fractional excretion of uric acid with increased serum uric acid was again documented in this cohort of patients as was asymptomatic hypocalcemia. Home administration of PB was accepted by most patients and all were successful in self-administration or administration of PB by a family member. No patient discontinued study because of inconvenience of administration.

Few objective responses were achieved. Two patients developed platelet transfusion independence, 1 sustained for ∼1 year. Nonsustained improvement in platelet count and neutrophil counts were observed as in the previous study. The development of progressive monocytosis with decreasing bone marrow CD34+ cells in 1 patient and the development of severe Sweet’s syndrome, associated with decreasing bone marrow blast cells and a remarkably rapid (1-day) neutrophil response to G-CSF administration, are provocative as potential examples of myeloid differentiation in response to PB.

Pharmacokinetic analysis was necessarily limited by the end-of-infusion sampling performed as well as the variability of the PB concentration data. However, no evidence of progressive PA accumulation nor declining PB concentration was documented. The median PB concentration of 0.4 μm throughout the 7/14 schedule and 0.43 throughout the 21/28 schedule, similar with the previous Phase I steady state value of 0.3 mm, demonstrates that this concentration, which effectively induces histone acetylation in leukemic cell lines, mononuclear cells, and primary leukemic blasts, can be sustained.

As in the initial Phase I study, changes in CFU-L and CFU-GM were variable. The single responding patient evaluable for changes in CFU showed declining CFU-L and CFU-GM despite increased platelet counts. As in the 7/28 day schedule studied previously, the current schedules enabled increases in production of hemoglobin F-bearing RBCs in several patients. Switching of globin gene expression by butyrate analogues appears to be because of HDAC inhibition (11, 12); thus, the successful induction of fetal hemoglobin expression by these agents may imply successful inhibition of HDAC (see below). It is noteworthy that although the percentage of clonal cells increased in most patients studied, PB administration was associated with a suppression of clonal cells in 2 patients. Both of these patients were studied using metaphase cytogenetics; this finding may be attributed to a cell cycle-inhibitory effect of PB (with concomitant inability to detect the clonal cells) rather than true clonal suppression.

No consistent changes were seen in the proliferative or apoptotic percentage of the CD34 compartment during PB therapy. It should be noted that bone marrow was necessarily sampled intermittently (baseline, weeks 6 and 12), and early changes may have been missed. It is now recognized that the inhibition of proliferation by PB is associated with induction of expression of p21^WAF1/CIP1 (3); because this trial was initiated before the possibility of a mechanistic role for this gene product was demonstrated, its expression was not monitored in the trial.

Recent reports have shown that expression of a wide variety of genes regulating differentiation and proliferation, including retinoid-responsive genes and retinoblastoma gene, is regulated in part through changes in histone conformation regulated in turn through acetylation (13). Histone acetylation is regulated through local recruitment of histone acetyltransferases and HDACs. An increasing number of leukemia-associated fusion genes inhibit gene transcription through recruitment of HDACs (6). Histone deacetylation contributes to the transcriptional repression of genes of which the promoters are methylated at CpG sites through the corepressor protein MeCP2, which binds to methylated CpG sites and recruits HDACs (14). Sequential administration of DNA methyltransferase inhibitors and HDAC inhibitors (including PB) has led to the synergistic re-expression of genes silenced through methylation (15). HDAC inhibitors synergize with retinoids (5, 16, 17) and render retinoid-resistant acute promyelocytic leukemia cells retinoid sensitive in vitro and in vivo (18). Thus, HDAC inhibitors may be exploited clinically to alter the malignant phenotype (19). Unfortunately, this trial was initiated before the recent recognition of the importance of chromatin remodeling in transcriptional regulation and the significance of HDAC as a potential target for PB, precluding the in vivo monitoring of changes in histone acetylation in this trial. Whereas a variety of more potent HDAC inhibitors are in early stages of preclinical and clinical development, the ability to sustain HDAC-inhibitory concentrations of PB with minimal toxicity make this a logical choice for early stage clinical development of HDAC inhibitors in myeloid malignancies.

We thank Julitta Krawiec and Yvette Freeman for outstanding data management; Drs. Michael Carducci and Sharyn Baker for helpful discussions; Abbie Mays and Vicky Smith for invaluable technical support; Kariann Johnson for expert assistance with manuscript preparation; and Targon Pharmaceuticals for support for data management.