Purpose: Epigenetic modulation of gene expression plays an important role in cancer, including leukemia. Furthermore, histone deacetylase inhibitors may induce the reexpression or repression of genes critical for normal hematopoiesis. The purpose of this study was to evaluate the toxicity, pharmacokinetic profile, and selected pharmacodynamic properties of the histone deacetylase inhibitor depsipeptide in patients with myelodysplastic syndromes (MDS) or acute myelogenous leukemia (AML).

Experimental Design: Depsipeptide was administered to MDS or AML patients at a (solid tumor) phase I dose of 18 mg/m2 i.v. on days 1 and 5 every 3 weeks. Toxicities and clinical activity were monitored and pharmacokinetic and pharmacodynamic studies were done.

Results: Twelve patients (nine with AML, three with MDS) received one to five cycles of depsipeptide. The most common grade 3/4 toxicities were febrile neutropenia/infection (five patients), neutropenia/thrombocytopenia (nine patients), nausea (nine patients), and asymptomatic hypophosphatemia (three patients). No clinically significant cardiac toxicity was observed. The best response of 11 assessed patients was one complete remission in a patient with AML, stable disease in six patients, and progression of disease in four patients. Exploratory laboratory studies showed modest but rapid increases in apoptosis and changes in myeloid maturation marker expression. Histone H3 and H4 acetylation levels were evaluated in five patients; no consistent changes were observed.

Conclusion: Depsipeptide therapy can be administered with acceptable short-term toxicity. However, gastrointestinal symptoms and fatigue seem to be treatment-limiting after multiple cycles. Depsipeptide monotherapy has limited clinical activity in unselected AML/MDS patients.

Advances in our understanding of the molecular basis of myeloid malignancies are beginning to translate into new therapeutic approaches. Foremost among these is the observation that epigenetic changes, which may produce or perpetuate the leukemia and myelodysplastic syndrome (MDS) phenotype, are amenable to pharmacologic manipulation. The discovery of aberrant patterns of gene methylation in MDS and leukemia, although still poorly characterized, has provided the rationale for the clinical development of the DNA methyltransferase inhibitors 5-azacytidine and decitabine. These drugs, which are now Food and Drug Administration–approved for treating patients with MDS, can reverse DNA methylation and alter gene expression.

In addition to DNA methyltransferases, other potentially inhibitable enzymatic activities, including histone deacetylases (HDAC), histone methyltransferases, and type II protein arginine methyltransferases, could participate in aberrant epigenetic silencing of gene expression. Of these, only HDACs are susceptible to therapeutic, pharmacologic intervention at this time. HDACs catalyze the removal of acetyl groups from lysine residues in histone tails, leading to a closed chromatin configuration and transcriptional repression (1). Histone acetyltransferases, which promote transcription via acetylation of lysine residue in the histone tails (2), regulate regional histone acetylation with HDACs, which allows for the coordinated expression of genes required for normal hematopoietic cell proliferation and differentiation.

Depsipeptide (NSC 630176, FR901228, Romidepsin) is a bicyclic peptide, originally isolated from Chromobacterium violaceum, which was shown to be an HDAC inhibitor (HDACI) in 1998 (3). Two phase I studies have been done in patients with advanced solid tumors to determine its maximum tolerated dose (MTD). The MTD for a phase I schedule of depsipeptide, given on days 1, 8, and 15 of a 28-day cycle, was 13.3 mg/m2, with asthenia and fatigue representing the dose-limiting toxicities (4). Depsipeptide given to 10 patients with acute myelogenous leukemia (AML) on this weekly schedule produced no clinical responses, although one patient developed tumor lysis syndrome (5). In another ongoing trial, patients with AML receive depsipeptide on this weekly schedule, and transient bone marrow blast responses have been seen in four of seven patients with chromosomal translocations known to recruit HDACs (6). Fatigue, nausea, and vomiting were frequent complications with this dosing schedule in both reported AML trials. When depsipeptide was given to solid tumor patients as a 4-h infusion on days 1 and 5 of a 21-day cycle (7), the MTD was 17.8 mg/m2 due to dose-limiting asthenia and fatigue.

The current study was designed as a pilot to obtain preliminary data for a potential phase II trial using the 21-day solid tumor phase I MTD schedule (MTD of 17.8, rounded up to 18 mg/m2). Because the toxicities seen in the phase I solid tumor studies, including transient thrombocytopenia and fatigue, were likely to be no worse in patients with hematologic malignancies, we chose to study only the MTD. Twelve evaluable patients were enrolled to allow for adequate evaluation of the toxicity of this dose level and schedule, which traditionally requires six patients per level, to obtain confirmatory pharmacokinetics data, and to preliminarily assess select pharmacodynamic variables in patients with MDS or AML.

Eligibility criteria. All patients gave written informed consent, and were required to have a confirmed diagnosis of advanced MDS (FAB subtypes RAEB, RAEBt, CMMoL), relapsed or refractory AML, or newly diagnosed AML, and were ineligible for standard chemotherapy. All patients were ineligible for or they refused allogeneic stem cell transplantation. Chemotherapy, immunotherapy, or radiotherapy was not allowed within 4 weeks prior to starting depsipeptide. Adequate renal function (serum creatinine ≤1.5 mg/dL or urine creatinine clearance ≥60 mL/min), hepatic function (total bilirubin ≤1.5 mg/dL, aspartate aminotransferase and alanine aminotransferase <2 × upper limit of normal), and cardiac function (left ventricular ejection fraction >50%, no cardiac hypertrophy, no NY class III or IV congestive heart disease, no active arrhythmia or requirement for antiarrhythmic therapy) was required. A negative pregnancy test was required of all females with child-bearing potential and pregnant or lactating females were excluded. The protocol was approved by the Memorial Sloan-Kettering Cancer Center and National Cancer Institute Institutional Review Boards.

Trial design. The primary end point was to assess the toxicity of depsipeptide at the solid tumor MTD of 18 mg/m2 on days 1 and 5 of a 21-day cycle in 12 evaluable patients with advanced myeloid malignancies. Secondary end points included clinical efficacy, and pharmacokinetic and exploratory pharmacodynamic studies. Depsipeptide was administered i.v. via central vein catheter over 4 h after the administration of a 32 mg i.v. ondansetron bolus. Patients were treated until either progression of disease (POD) or unacceptable toxicity. Dose reduction to 12 mg/m2 was permitted for significant, but not dose-limiting toxicity.

Toxicity assessments. During treatment, participants were monitored with weekly CBCs, troponin levels, creatinine, and calcium, potassium, and magnesium levels. Serum hepatic transaminase levels were assessed on day 5 of each cycle. Resting multiple gated acquisition scans were done at screening and after each even-numbered cycle. Electrocardiograms and troponin levels were done prior to each depsipeptide infusion, and then 1 and 24 h postinfusion. If no significant electrocardiogram changes were detected during cycle 1, the electrocardiogram and troponin-monitoring schedule was attenuated during subsequent cycles. Toxicity was graded using the National Cancer Institute Common Toxicity Criteria version 2.0. Dose-limiting toxicity was defined as any grade 3 or 4 nonhematologic toxicity attributed to depsipeptide.

Response criteria. For MDS participants, a complete remission was defined as a bone marrow cellularity of >20% with trilineage maturation, <5% blasts, no Auer rods, an absolute neutrophil count of ≥1,500/μL, and a platelet count of ≥100,000/μL, with no circulating blasts or extramedullary leukemia. Partial remission for patients with MDS requires the same criteria as a complete remission, except that the percentage of bone marrow blasts must be reduced by 50% from the pretreatment value. A complete remission for patients with AML required an absolute neutrophil count of ≥1,500/μL, a platelet count of ≥100,000/μL, no circulating blasts or extramedullary leukemia, and a bone marrow cellularity of >20% with trilineage maturation and <5% blasts. Partial remission for patients with AML required the same criteria as a complete remission, except that the percentage of blasts in the bone marrow must be <20%. Patients with AML and MDS who did not achieve at least a partial remission, yet had no evidence of progressive disease, were categorized as having stable disease.

Pharmacokinetics studies. Pharmacokinetics were done on heparinized plasma samples obtained at baseline, at 1, 2, 3, and 4 h after the start of the infusion, and at 15 and 30 min, and 1, 3, and 6 h after the completion of the infusion on day 1 of cycles 1 and 2. Samples were stored at −80°C. Acetonitrile was used to remove proteins in the samples, and an isocratic high-performance liquid chromatography/mass spectrometer method was then used to separate the compound from any potential interference. Levels of depsipeptide were measured by mass spectrometry with a selected positive ion monitoring mode at m/z 541. Standard pharmacokinetic variables were calculated using the WinNonlin software (ver. 5.0.1, Pharsight), and summarized as mean and SD.

Pharmacodynamic studies. Peripheral blood mononuclear cells were obtained for pharmacodynamic studies from patients with leukemia and ≥10% circulating blasts (n = 6) at cycle 1, day 1 (C1D1) prior to the day 1 infusion, at C1D1 6 h after the infusion, at C1D2 24 h after the day 1 infusion, C2D1 (preinfusion), and at C2D2 (24 h after the day 1 infusion). In patients without circulating blasts (n = 6), bone marrow aspirates were obtained at C1D1, prior to the day 1 depsipeptide infusion, and at 24 h after the day 1 infusion during cycles 1 and 2.

Histone acetylation studies. Mononuclear cell fractions were isolated by density centrifugation using Ficoll-Paque, and nuclei were prepared by lysis in buffer containing 10 mmol/L of Tris-HCl (pH 6.5), 50 mmol/L of sodium bisulfite, 1% Triton X-100, 10 mmol/L of MgCl2, 8.6% sucrose, and Dounce homogenization. Histone extraction was done as previously described (8) and, on average, 15 μg of histone proteins were electrophoretically separated on 17.5% SDS-PAGE minigels (Bio-Rad) and transferred to nitrocellulose membranes (Schleicher & Schuell). Hyperacetylated histones were detected by antibodies that specifically recognize the acetylated forms of histone H4 or H3 (Upstate Biotechnology) and visualized by chemiluminescence (Amersham Biosciences). Ponceau staining was done, and total H3 and H4 was detected by antibodies to nonacetylated H3 and H4 (Upstate Biotechnology). Densitometry was done using the MacBas V2.5-1 software program to quantify band intensity, which was expressed as a ratio of acetylated forms to total H3 or H4 protein.

Flow cytometric measurement of apoptosis and differentiation. The degree of apoptosis was evaluated using 7-amino-actinomycin D 7(7-AAD) staining and flow cytometry, in a modification of a method originally described by Philpott et al. (9). Surface membrane antigens were detected on fresh bone marrow or peripheral blood mononuclear cells by direct immunofluorescence staining with the use of FITC-conjugated or phycoerythrin-conjugated monoclonal antibodies that recognize CD16, CD11b, CD33, CD13, HLA-DR, CD34, CD45, and CD14 (Becton Dickinson Biosciences) in conjunction with isotypic controls. Combinations examined included CD33-PE and CD34-FITC, CD33-PE and CD11b-FITC, CD13-PE and CD34-FITC, or CD11b-PE and CD34-FITC. Whole nucleated cell populations were acquired and analyzed on a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson Biosciences).

Patient characteristics. Although 13 patients consented to this treatment protocol between June 2002 and June 2004, 1 patient withdrew consent before receiving therapy. The baseline characteristics of the 12 evaluable patients are summarized in Table 1. Nine patients had either relapsed or refractory AML, and three patients had advanced MDS. The median age was 72 (range, 38-82). The six males and six females had received a median of two prior therapies (range, 0-3). Patients received a median of only one treatment cycle (range, 1-5 cycles). Three patients with stable disease came off-study by withdrawing consent. The other nine patients came off-study for either POD (n = 6), episodes of febrile neutropenia that delayed re-treatment (n = 2), or death (n = 1; see Table 2).

Table 1.

Patient baseline characteristics

Patient no.AgeSexDiagnosisPrior therapyCytogenetics
74 MDS → AML, relapsed (1) Disalcid (clinical trial for MDS) 46,XY, del(5q), del(20q) [12]; 46,XY [3] 
    (2) Ara-C/Bi-Hum195  
74 MDS → AML None 47,XY, del(7q), +21 [11] 
75 MDS → AML, refractory (1) Decitabine 46,XY, del(12p) [18] 
    (2) Ida/Ara-C (no CR)  
51 AML, relapsed (1) Ida/HiDaC (CR) 56,XX,+X,+1,+8,+11x2,+14,+21,+22,1-2mar [cp6]; 46, XY [1] 
    (2) TCD-AlloBMT  
38 AML, refractory (1) Ida/Ara-C 46-47,XX, del(6q), −7,−16, 3 mar [cp20] 
    (2) Ida/Ara-C  
    (3) HiDaC/Mylotarg  
45 AML, second relapse (1) Ida/Ara-C 46,XX [16] 
    (2) Daunoubicin/Ara-C  
76 MDS → AML, relapsed (1) Ida/Ara-C (CR) 46,XX, t(10;21)[6]; 46,XX [14] 
    (2) Ida/Ara-C consolidation ×2  
    (3) MLN518 (FLT3 inhibitor)  
72 RAEBt (1) MLN 518 (FLT3 inhibitor) 47,XY,+9 [2]; 46,XY [18] 
65 MDS → AML, relapsed (1) Ida/Ara-C (no CR) 46, XY [3] 
    (2) Mitoxantrone/Ara-C (CR)  
    (3) Ara-C consolidation ×4  
    (4) Disalcid  
    (5) PKC412 (FLT3 inhibitor)  
10 64 RAEB None 44,XX, del(5q), −6, del(7q),+8, add(12)(p13), +add(19)(p13) [1]; FISH: del(5q), del(7q), +8 
11 71 AML, second relapse (1) Ida/Ara-C (CR) 46, XX, del (11q), der(17)t(3;17) [cp2]; 46,XX [1]. FISH—del(7q), del (11q23) 
    (2) Ida/Ara-C consol ×2  
    (3) Ara-C/Bi-Hum195 (CR)  
12 82 MDS → AML (CR) → RAEB (1) Ara-C/Bi-Hum195 (CR) 46, XY, del (7q) [14]; 47,XY,del(7q) +8 [2]; 46,XY [4] 
Patient no.AgeSexDiagnosisPrior therapyCytogenetics
74 MDS → AML, relapsed (1) Disalcid (clinical trial for MDS) 46,XY, del(5q), del(20q) [12]; 46,XY [3] 
    (2) Ara-C/Bi-Hum195  
74 MDS → AML None 47,XY, del(7q), +21 [11] 
75 MDS → AML, refractory (1) Decitabine 46,XY, del(12p) [18] 
    (2) Ida/Ara-C (no CR)  
51 AML, relapsed (1) Ida/HiDaC (CR) 56,XX,+X,+1,+8,+11x2,+14,+21,+22,1-2mar [cp6]; 46, XY [1] 
    (2) TCD-AlloBMT  
38 AML, refractory (1) Ida/Ara-C 46-47,XX, del(6q), −7,−16, 3 mar [cp20] 
    (2) Ida/Ara-C  
    (3) HiDaC/Mylotarg  
45 AML, second relapse (1) Ida/Ara-C 46,XX [16] 
    (2) Daunoubicin/Ara-C  
76 MDS → AML, relapsed (1) Ida/Ara-C (CR) 46,XX, t(10;21)[6]; 46,XX [14] 
    (2) Ida/Ara-C consolidation ×2  
    (3) MLN518 (FLT3 inhibitor)  
72 RAEBt (1) MLN 518 (FLT3 inhibitor) 47,XY,+9 [2]; 46,XY [18] 
65 MDS → AML, relapsed (1) Ida/Ara-C (no CR) 46, XY [3] 
    (2) Mitoxantrone/Ara-C (CR)  
    (3) Ara-C consolidation ×4  
    (4) Disalcid  
    (5) PKC412 (FLT3 inhibitor)  
10 64 RAEB None 44,XX, del(5q), −6, del(7q),+8, add(12)(p13), +add(19)(p13) [1]; FISH: del(5q), del(7q), +8 
11 71 AML, second relapse (1) Ida/Ara-C (CR) 46, XX, del (11q), der(17)t(3;17) [cp2]; 46,XX [1]. FISH—del(7q), del (11q23) 
    (2) Ida/Ara-C consol ×2  
    (3) Ara-C/Bi-Hum195 (CR)  
12 82 MDS → AML (CR) → RAEB (1) Ara-C/Bi-Hum195 (CR) 46, XY, del (7q) [14]; 47,XY,del(7q) +8 [2]; 46,XY [4] 

Abbreviations: Bi-Hum195, Bismuth-213–labeled HuM195; Ara-C, cytosine arabinoside (1-β-d-arabinofuranosylcytosine); Ida, idarubicin; HiDaC, high-dose Ara-C; TCD, T-cell depleted.

Table 2.

Best response, time on study, and reason for treatment discontinuation

Patient no.DiagnosisBest responseDays on studyCycles receivedReason for treatment discontinuation/comments
MDS → AML, relapsed POD 11 POD (increased BM blast count) 
MDS → AML CR 26 Delay in resuming therapy due to infection. CR noted 57 d after first depsipeptide infusion 
MDS → AML, refractory SD 93 POD (new chloroma) 
AML, relapsed SD 68 Death from infection 
AML, refractory POD 16 POD (increasing peripheral blood blast count) 
AML, relapsed POD 14 POD (progressive chloroma) 
MDS → AML, relapsed SD 51 Withdrawal of consent 
RAEBt SD 88 Withdrawal of consent 
MDS → AML, relapsed SD 28 Withdrawal of consent 
10 RAEB Not assessed 29 Delay in resuming therapy due to infection 
11 AML, relapsed POD 17 POD (increasing peripheral blood blast count) 
12 MDS → AML → MDS SD 86 POD (increased BM blast count) 
Patient no.DiagnosisBest responseDays on studyCycles receivedReason for treatment discontinuation/comments
MDS → AML, relapsed POD 11 POD (increased BM blast count) 
MDS → AML CR 26 Delay in resuming therapy due to infection. CR noted 57 d after first depsipeptide infusion 
MDS → AML, refractory SD 93 POD (new chloroma) 
AML, relapsed SD 68 Death from infection 
AML, refractory POD 16 POD (increasing peripheral blood blast count) 
AML, relapsed POD 14 POD (progressive chloroma) 
MDS → AML, relapsed SD 51 Withdrawal of consent 
RAEBt SD 88 Withdrawal of consent 
MDS → AML, relapsed SD 28 Withdrawal of consent 
10 RAEB Not assessed 29 Delay in resuming therapy due to infection 
11 AML, relapsed POD 17 POD (increasing peripheral blood blast count) 
12 MDS → AML → MDS SD 86 POD (increased BM blast count) 

Abbreviations: POD, progression of disease; SD, stable disease; CR, complete remission; BM, bone marrow.

Toxicity. Two patients (nos. 2 and 10) were removed from the study during cycle no. 1 due to delays in treatment in accordance with the requirements of the protocol; these delays were required to adequately treat episodes of febrile neutropenia. The one death in the study occurred 17 days after the patient received the last infusion of the third cycle of depsipeptide (on day 69 of treatment). This patient was a 51-year-old female who expired from respiratory failure secondary to an uncontrolled infection. Two patients underwent a dose reduction to 12 mg/m2, as allowed by protocol, for an episode of febrile neutropenia in one instance, and the other, for severe hematuria in the setting of thrombocytopenia (both requiring hospital admission). These toxicities did not recur in these patients with continued treatment at the lower dose of 12 mg/m2.

The most common nonhematologic toxicities (grade ≥2) were nausea, vomiting, fatigue, hypocalcemia, and asymptomatic hypophosphatemia (see Table 3). Episodes of grade 3/4 hematologic toxicity were expected in this patient population, and included neutropenia (n = 13), thrombocytopenia (n = 15), and a related episode of infection with neutropenia (n = 1). No patient experienced nonhematologic dose-limiting toxicity. There were no infusion-related vascular complications.

Table 3.

Hematologic and nonhematologic toxicities

Grade
Toxicity1234
Bleeding     
    Hematuria 
    Petechia/purpura 
Cardiovascular     
    EKG changes 
    Sinus tachycardia 
    Hypotension 
Liver/gastrointestinal     
    Nausea 
    Vomiting 
    Anorexia 
    Constipation 
    Dehydration 
    Mouth dryness 
    Elevated alkaline phosphatase 
    Elevated bilirubin 
    Taste disturbance 
Pulmonary     
    Dyspnea 
Constitutional     
    Fatigue 
    Rigors/chills 
    Weight loss 
Pain     
    Back pain 
    Myalgia 
    Headache 
Infection     
    Infection with grade 3/4 neutropenia 
    Fever 
Renal     
    Elevated creatinine 
Metabolic     
    Hypoalbuminemia 
    Hypocalcemia 
Hypokalemia 
    Hypomagnesemia 
    Hyponatremia 
    Hypophosphatemia 
Hematologic     
    Leukopenia 
    Neutropenia 
    Anemia 
    Thrombocytopenia 
Grade
Toxicity1234
Bleeding     
    Hematuria 
    Petechia/purpura 
Cardiovascular     
    EKG changes 
    Sinus tachycardia 
    Hypotension 
Liver/gastrointestinal     
    Nausea 
    Vomiting 
    Anorexia 
    Constipation 
    Dehydration 
    Mouth dryness 
    Elevated alkaline phosphatase 
    Elevated bilirubin 
    Taste disturbance 
Pulmonary     
    Dyspnea 
Constitutional     
    Fatigue 
    Rigors/chills 
    Weight loss 
Pain     
    Back pain 
    Myalgia 
    Headache 
Infection     
    Infection with grade 3/4 neutropenia 
    Fever 
Renal     
    Elevated creatinine 
Metabolic     
    Hypoalbuminemia 
    Hypocalcemia 
Hypokalemia 
    Hypomagnesemia 
    Hyponatremia 
    Hypophosphatemia 
Hematologic     
    Leukopenia 
    Neutropenia 
    Anemia 
    Thrombocytopenia 

There was no clinically significant cardiac toxicity. Transient, asymptomatic electrocardiogram repolarization abnormalities were seen, and in most patients, these individual episodes were minor and deemed “nonspecific.” However, these minor changes generally recurred with re-treatment, indicating that they were likely a treatment effect of depsipeptide. One patient had 2 mm ST segment depressions and T-wave flattening, detected 24 h after the first depsipeptide infusion, which completely resolved within 8 days (patient no. 2). Serial troponin levels were normal during this episode. A repeat echocardiogram showed a left ventricular ejection fraction of >55% with no new wall motion abnormalities, and a thallium stress test showed no ischemic changes or evidence of infarction.

Serial determinations of left ventricular function by multiple gated acquisition or echocardiogram in 10 patients showed no decrement in ejection fraction and no new wall motion abnormalities. Two patients were admitted to other hospitals and died (patient no. 4 died from infection while on study; patient no. 5 died from unknown causes while on another clinical trial) without an off-study evaluation of left ventricular function. No patient experienced an arrhythmia or significant change in QTc interval (defined as QTc of ≥500 ms or an increase of ≥50 ms). Tachycardia was a possible expected effect of depsipeptide, and although mild increases in heart rate were seen across serial electrocardiograms during treatment days (median increase, 15 beats per minute; range, 4-22 beats per minute) to a maximum heart rate of 112 beats per minute, factors such as anemia, dehydration, anxiety, and activity level prior to individual electrocardiograms hampered the interpretation of changes in heart rate between treatment days or scheduled outpatient toxicity evaluations.

Clinical activity. The patients in this study had advanced MDS or AML with significant baseline bone marrow dysfunction. The median percentage of bone marrow blasts prior to therapy was 45%. Half of the patients had a baseline absolute neutrophil count of ≤200 μL. Seven patients had baseline platelet counts of <30,000/μL. One patient (no. 10) was not formally assessed for response. The best response to depsipeptide was characterized as POD in four patients after a median of 15 days, and stable disease in six patients. Of the patients with stable disease, two eventually had POD after 93 and 86 days on study, respectively (see Table 2), and the remaining four patients came off-study with stable disease. One patient (patient no. 2) was shown to be in complete remission at the time of his off-study bone marrow exam 57 days after the patient received a single infusion of depsipeptide at 18 mg/m2, and 32 days after coming off-study. This patient was a 74-year-old male with an untreated MDS that had evolved to AML during the screening period just prior to starting depsipeptide. This patient had recurrent cytopenias 29 days after complete remission was documented, and a repeat bone marrow aspirate 10 weeks after complete remission showed 18% myeloblasts. The patient was re-treated with depsipeptide on a National Cancer Institute compassionate-use protocol, but at a reduced dose of 13 mg/m2 due to the episode of febrile neutropenia and electrocardiogram repolarization abnormalities seen at 18 mg/m2. His disease continued to progress despite three additional cycles of therapy at the lower dose. He expired 170 days after resuming therapy.

Pharmacokinetics data. Pharmacokinetic studies were done on plasma samples from 12 patients who received a total of 15 cycles of depsipeptide at a dose of 18 mg/m2. The results are summarized in Table 4. The two-compartment open model seemed to provide the best fit for our data. In general, the derived pharmacokinetics variables were similar to those reported for patients who received a similar dose (17.8 mg/m2) in an earlier phase I study (7). Data was also collected on two patients who received a dose of 12 mg/m2 during their second cycle; the calculations on this limited data set are not shown.

Table 4.

Calculated pharmacokinetic variables for depsipeptide using two-compartment open model analysis

VariableMean ± SD
Dose (mg/m218 
No. of patients 12 
No. of cycles at dose level 15 
CLtot (L/h/m213.7 ± 9.1 
T1/2 (h) 0.3 ± 0.2 
T1/2α (h) 0.2 ± 0.1 
T1/2β (h) 4.5 ± 8.9 
Cmax (μg/mL) 0.8 ± 0.7 
Vdss (L/m214.8 ± 14.6 
VariableMean ± SD
Dose (mg/m218 
No. of patients 12 
No. of cycles at dose level 15 
CLtot (L/h/m213.7 ± 9.1 
T1/2 (h) 0.3 ± 0.2 
T1/2α (h) 0.2 ± 0.1 
T1/2β (h) 4.5 ± 8.9 
Cmax (μg/mL) 0.8 ± 0.7 
Vdss (L/m214.8 ± 14.6 

Abbreviations: CLtot, systemic clearance; T1/2, half-life; T1/2α, elimination half-life; T1/2β, distribution half-life; Cmax, observed maximum concentration; Vdss, volume of distribution at steady state.

Differentiation and apoptosis studies. Flow cytometric evidence for apoptosis was assessed using 7-AAD staining of serial peripheral blood or bone marrow mononuclear cells. Five of 10 evaluable patients had an increase in 7-AAD staining based on the percentage of 7-AAD uptake increase and scattergram analysis (see Fig. 1), including the patient who had a complete remission (patient no. 2). An increase in 7-AAD staining was also seen when patient no. 2 was re-treated off-study at a dose of 13 mg/m2 (data not shown).

Fig. 1.

Change in 7-AAD expression after depsipeptide infusion. 7-AAD–positive peripheral blood (PB) or bone marrow (BM mononuclear cells prior to and 24 h after first depsipeptide infusion. The Day #1 (D#1) sample was taken within 2 h prior to start of depsipeptide infusion). Line number assignments correspond to patient number (see Table 1).

Fig. 1.

Change in 7-AAD expression after depsipeptide infusion. 7-AAD–positive peripheral blood (PB) or bone marrow (BM mononuclear cells prior to and 24 h after first depsipeptide infusion. The Day #1 (D#1) sample was taken within 2 h prior to start of depsipeptide infusion). Line number assignments correspond to patient number (see Table 1).

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Eleven of 17 paired sample sets were technically evaluable for analysis of depsipeptide-induced differentiation. Population shifts indicating a probable differentiating effect of depsipeptide were seen in two of the three patients (nos. 5 and 11) who had less than maximal CD34/CD13 coexpression at baseline. Patient no. 5 had CD34/CD13 expression that increased to 88% from her baseline of 78%, and patient no. 11 had an increase in CD34/CD13 expression from 33% to 55%.

Histone acetylation studies. Histone H3 and H4 acetylation was assessed by Western blot analysis in five patients with adequate preinfusion and postinfusion paired samples. H3 and H4 acetylation was increased in two patients (nos. 5 and 8), and unchanged in two patients (nos. 3 and 6). One patient (no. 11) had increased H4 acetylation, but no change in H3 acetylation. Unfortunately, changes in histone acetylation could not be assessed in the patient who achieved a complete remission (no. 2) due to an inadequate baseline sample. Western blot and densitometry results for patient no. 5 are shown in Fig. 2.

Fig. 2.

Depsipeptide-induced increase in acetylated histones H3 and H4. Western blot analysis of acetylated and total histones H3 and H4 in peripheral blood mononuclear cells from patient no. 5. Bottom, the corresponding densitometry results, expressed as a fold change from baseline of the ratio of acetylated to total histone H3 and H4.

Fig. 2.

Depsipeptide-induced increase in acetylated histones H3 and H4. Western blot analysis of acetylated and total histones H3 and H4 in peripheral blood mononuclear cells from patient no. 5. Bottom, the corresponding densitometry results, expressed as a fold change from baseline of the ratio of acetylated to total histone H3 and H4.

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The primary objective of this trial was to evaluate the safety of depsipeptide in patients with MDS or AML, using a dose of 18 mg/m2 on days 1 and 8 of a 21-day schedule. In addition, pharmacokinetics and exploratory pharmacodynamic studies were conducted, and clinical activity was monitored. Twelve patients were treated at an initial planned dose of 18 mg/m2. Two of five patients who received more than one cycle were dose-reduced to 12 mg/m2 per protocol allowances to maximize safety in this setting in which the optimal therapeutic dose of depsipeptide is unknown. Depsipeptide was reasonably well tolerated, given the advanced age, number of prior therapies, and severity of baseline marrow dysfunction in this population. Frequent, but acceptable, grade 3/4 hematologic toxicity occurred, and nonhematologic toxicity was consistent with that seen in other depsipeptide trials, including electrolyte abnormalities, gastrointestinal toxicity, and fatigue. Anorexia and fatigue, although not dose-limiting in this study, were common complaints during the first 2 weeks of the 3-week cycle. An indicator of the effect of these chronic, but non–dose-limiting, toxicities is that two of the three patients who withdrew consent with stable disease after multiple cycles of therapy did so because of the effect of these symptoms on their quality of life. It seems that depsipeptide administered at a dose of 18 mg/m2 twice in 3 weeks produced toxicity that was comparable to that seen at a dose of 13 mg/m2 weekly for 3 weeks every 28 days.

There were no clinically significant short-term cardiac toxicities in this trial. Magnesium and potassium supplementation was implemented midway through this trial due to concerns regarding the potential for QTc prolongation (which was not observed). We could not assess the long-term effects of depsipeptide on cardiac function due to the limited survival of the patients entered into this study (median survival from study exit = 108 days).

Pharmacokinetics analyses confirmed the short half-life first reported by Sandor et al. (7). In that study, there was a good fit for both a noncompartmental and a two-compartment model. Our data show a better fit using a two-compartment open model, but this may be explained by difficulties detecting a longer elimination phase as a result of fewer postinfusion time points in our data set.

We observed modest clinical activity in this group of patients with very advanced disease. Progressive disease was documented after a median of only 15 days in six patients who developed progressive disease on study, demonstrating the aggressive nature of their relapsed and refractory disease. Among those with progressive disease, two patients (nos. 3 and 12) remained stable for some time, progressing after four cycles of therapy. Their disease stability may reflect the natural course of their disease or a treatment effect of depsipeptide.

In another ongoing study, patients with AML receive depsipeptide at a dose of 13 mg/m2 weekly × 3 weeks every 28 days, and bone marrow blast responses were reported in patients with chromosomal rearrangements involving the AML1 gene [t(4;21) and t(8;21)] (6). Of relevance is the finding that the AML1-ETO fusion protein, which is generated by the t(8;21), is known to recruit HDACs to its target DNA sequence (10, 11). The patient who achieved complete remission in our study did not have a chromosomal translocation known to recruit HDACs. This patient's sensitivity to depsipeptide, and the responses seen in other patients with specific chromosomal abnormalities, suggests that the schedule of HDACI administration, although important, may be less critical than identifying the subsets of patients likely to benefit from HDAC inhibitory therapy, assuming of course that patients can tolerate therapeutic levels of the HDACI. The complete remission in our patient was documented 8 weeks after he received only one dose of depsipeptide. The basis for this delayed response is unknown, but it argues against a purely cytotoxic effect, and suggests that other mechanisms may be important. For instance, the immunomodulatory effects of HDACIs have been described in preclinical studies (12). Delayed responses are also characteristic of responses seen in patients receiving DNA methyltransferase inhibitors, and the basis for the delayed response with these agents likewise remains unexplained. In another AML study, Byrd et al. reported no clinical responses in 10 patients who received depsipeptide at a dose of 13 mg/m2 weekly × 3 weeks, but tumor lysis was observed in one patient, suggesting at least a transient cytotoxic effect (5).

As a drug with potential transcriptional modulating activity, depsipeptide may trigger alterations in the expression of genes that maintain the aberrant proliferation or differentiation seen in myeloid malignancies. The results of our exploratory analyses of apoptosis and myeloid differentiation suggest that depsipeptide could induce both apoptosis and myeloid immunophenotypic shifts in vivo. The significance of these changes is uncertain, as they were seen both in responding and nonresponding patients. Furthermore, the changes occurred rapidly (within 24 h), yet the one patient who achieved a complete response did so weeks later, making a link to the clinical activity of depsipeptide difficult to establish. Nonetheless, our work extends on the induction of apoptosis by depsipeptide documented in vitro (1315). Despite the difficulty in identifying the precise cell populations undergoing apoptosis, the loss of a high side-scatter cell population in some of our patients (data not shown) suggests the apoptosis of more mature myeloid cells. This would coincide with the early neutrophil nadir seen with depsipeptide treatment in both the solid tumor phase I study (7) and in this study. Multiparameter flow cytometry analysis should be done in future HDACI trials to further characterize the cells in the apoptotic fraction, and to better understand the effects of HDACIs on malignant myeloid cell subsets.

Changes in histone H3 and H4 acetylation were not consistently detected in the five patients studied, which may be related to technical problems or to the possibility that a significant increase in acetylation was not achieved. An increase in overall histone H3 and H4 acetylation does not seem to be sufficient for a clinical response, as the increased histone acetylation seen in other HDACI trials (5) did not correlate with clinical response. The lack of correlation between histone acetylation and clinical activity is not surprising, as transient changes in histone acetylation may not be sufficient to restore gene expression for an adequate period of time. Furthermore, control of gene expression relates not only to alterations in site-specific histone lysine acetylation, but also to changes in arginine and lysine methylation and to changes in the pattern of DNA methylation. Histone reacetylation may not be required for a clinical response to “HDAC” inhibitors, as a wide range of non-histone proteins have been shown to be acetylated by “histone” acetyltransferases. Such non-histone proteins include the tumor suppressor gene p53. Nonetheless, the modest clinical activity of HDACI monotherapy, and the unfolding complexity of transcriptional regulation, suggests that combination therapies will be needed to show significant clinical efficacy in the aggressive myeloid disorders.

Given the in vitro synergy between HDACIs and demethylating agents (15, 16), combinations of these agents are being explored in early clinical trials in patients with MDS and AML (1719). Although we did not see clinically significant cardiac toxicity, concerns regarding the potential cardiac toxicity of depsipeptide alone or in combination with other agents may limit its clinical development (20). Constitutional symptoms were seen in other depsipeptide trials (4, 5, 7), and have also been reported in other HDACI trials in patients with hematologic malignancies (19, 21), suggesting a drug class effect. The constitutional and gastrointestinal symptoms seen in this and other studies with depsipeptide may also limit the future use of this agent in AML and MDS patients at the doses tested in this trial.

Additional studies are required to better understand the clinically relevant biological effects of depsipeptide and other HDACIs in responding patients with leukemia, MDS, and cutaneous T-cell lymphoma. Such an understanding should lead to improved trial design, patient selection, and treatment outcomes.

Grant support: Leukemia and Lymphoma Society Specialized Center of Research grant for the study of myeloid malignancies (S.D. Nimer, V.M. Klimek, and P.P. Pandolfi), and NCI PO1 grant CA 05826 (V.M. Klimek, S.D. Nimer).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Jin Zhang, Ruth Rose, Alina Rzasa-Serrano, and Tony DeBlasio for laboratory support, Maya Kuznetsov for data management, and Stephen Soignet for writing the original protocol. Depsipeptide was provided by the National Cancer Institute Cancer Therapy Evaluation Program.

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