PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Clin Cancer Res. Author manuscript; available in PMC Jan 29, 2010.
Published in final edited form as:
PMCID: PMC2813676
NIHMSID: NIHMS169568
A Phase I and Pharmacokinetic Study of the Oral Histone Deacetylase Inhibitor, MS-275, in Patients with Refractory Solid Tumors and Lymphomas
Lia Gore,1 Mace L. Rothenberg,2 Cindy L. O'Bryant,1 Mary Kay Schultz,1 Alan B. Sandler,2 Denise Coffin,3 Candice McCoy,3 Astrid Schott,4 Catherine Scholz,3 and S. Gail Eckhardt1
1University of Colorado Cancer Center, Aurora, Colorado
2Vanderbilt-Ingram Cancer Center, Nashville, Tennessee
3Bayer Healthcare, Seattle, Washington
4Bayer Schering Pharma AG, Berlin, Germany
Requests for reprints: Lia Gore, University of Colorado Health Sciences Center at Fitzsimons, Pediatrics, Mail Stop 8302, P.O. Box 6511, Aurora, CO 80045. Phone: 303-724-4011; Fax: 303-724-4015; lia.gore/at/uchsc.edu.
Purpose
To evaluate the toxicity profile, pharmacologic, and biological properties of 3-pyridylmethyl N-{4-[(2-aminophenyl)carbamoyl]benzyl}carbamate (MS-275), a histone deacetylase inhibitor, when administered orally on three different dosing schedules.
Experimental Design
Patients with advanced solid malignancies and lymphomas were treated on three dose schedules: once every other week, twice weekly for 3 weeks every 28 days, and once weekly for 3 weeks every 28 days. First-cycle plasma pharmacokinetics and peripheral blood mononuclear cell histone acetylation were determined.
Results
Twenty-seven patients received ≥149 courses of treatment. Hypophosphatemia and asthenia were dose limiting on the weekly and twice-weekly dosing schedules; there was no dose-limiting toxicity on the every other week schedule. Pharmacokinetic variables revealed dose-dependent and dose-proportional increases. Two of 27 patients showed partial remissions, including one patient with metastatic melanoma who had a partial response and has remained on study for >5 years. Six patients showed prolonged disease stabilization. Levels of histone H3 and H4 acetylation in peripheral blood mononuclear cells increased qualitatively but with a high degree of interpatient variation.
Conclusions
MS-275 is well tolerated at doses up to 6 mg/m2 every other week or 4 mg/m2 weekly for 3 weeks followed by 1 week of rest and results in biologically relevant plasma concentrations and antitumor activity. Twice-weekly dosing was not tolerable due to asthenia, and further evaluation of this schedule was halted. The recommended dose for further disease-focused studies is 4 mg/m2 given weekly for 3 weeks every 28 days or 2 to 6 mg/m2 given once every other week.
Aberrant gene expression is a hallmark of cancer. Gene expression is modified by a variety of molecular mechanisms via multiple pathways. One such mechanism is the acetylation and deacetylation of histones by histone acetylases and histone deacetylases (HDAC). Although increased histone acetylation is generally associated with increased levels of gene expression possibly due to freer access of transcription factors to the histone-associated DNA, the expression of specific genes may be either up-regulated or down-regulated by histone acetylation due to effects on upstream regulators of that gene's expression. HDAC may also affect gene expression by direct association with proteins such as transcription factors (1) and allow reexpression of abnormally silenced genes, which are deacetylated in malignancy.
Inhibition of HDAC has been shown to affect the expression of proteins involved in cellular proliferation. For example, the cyclin-dependent kinase inhibitor p21WAF1/CIP1, a tumor suppressor protein, has been shown to be up-regulated in cancer cell lines treated with trichostatin A, suberoylanilide hydroxamic acid, 3-pyridylmethyl N-{4-[(2-aminophenyl)-carbamoyl]benzyl}carbamate (MS-275), and other HDAC inhibitors (2). p21WAF1/CIP1 is induced by p53, which is often defective or inactive in tumor cells. Up-regulation of p21WAF1/CIP1 results in reduced cyclin-dependent kinase activity, thus limiting progression of cells through the cell cycle and proliferation (3).
MS-275 is a synthetic small-molecule benzamide derivative that has been shown to inhibit HDAC activity in vitro (2). MS-275 induces the expression of the tumor suppressors p21WAF1/CIP1 and gelsolin in cell lines regardless of the p53 status of the cells. It has been shown to have an antiproliferative effect in cell lines, which correlates with p21WAF1/CIP1 expression in the cells but not with gelsolin expression. MS-275 can up-regulate the expression of the transforming growth factor receptor II gene, which could lead to increased activity of transforming growth factor-β, another tumor suppressor (4, 5). At high doses, MS-275 can induce apoptosis in leukemia and lymphoma cell lines (6, 7).
In vitro, MS-275 has been shown to have antiproliferative activity in human breast, colon, lung, myeloma, ovary, pancreas, prostate, and leukemia cell lines (1, 2, 4, 5, 811). Antiproliferative activity has also been shown in vivo in xenograft models (1, 2, 810).
This study was designed to determine the maximum tolerated dose and dose-limiting toxicity (DLT) of MS-275 in patients with advanced solid malignancies. Other objectives included determination of the pharmacokinetics of MS-275; measurement of histone acetylation in peripheral blood mononuclear cells (PBMC) before, during, and after MS-275 treatment; characterization of the adverse events and changes in laboratory variables induced by MS-275; and observation of any preliminary evidence of tumor response in patients treated with MS-275.
Patient selection
Patients of both genders and any ethnic group with a pathologically confirmed malignancy that was metastatic or unresectable or for whom standard therapy did not exist were eligible. Additional criteria included Eastern Cooperative Oncology Group performance status ≤22, with no more than 210% of average body weight loss within the previous 2 months; life expectancy >3 months; age ≥218 years; the ability to swallow intact drug tablets; normal organ and bone marrow function including absolute neutrophil count ≥21,500/μL, platelets ≥2,100,000/μL, creatinine ≤21.52 × 2 times upper limit of normal or measured creatinine clearance ≥260 mL/min/1.73 m2, total bilirubin ≥21.5 times upper limit of normal, and aspartate aminotransferase/alanine aminotransferase ≤22.5 times upper limit of normal. Exclusion criteria were known history of HIV infection; history of allergic reaction to compounds similar chemically or biologically to MS-275; concurrent breastfeeding; patients meeting criteria for suspected Gilbert's syndrome; chemotherapy, radiotherapy, vaccines, immunotherapy, and hormone therapy (with the exception of gonadotropin-releasing hormone agonists for prostate cancer) within 4 weeks of entry (6 weeks for nitrosoureas or mitomycin Cor agents known to cause prolonged marrow suppression and 2 weeks for palliative external radiotherapy) or during the study; acute or chronic gastrointestinal conditions that might predispose to poor drug absorption; concomitant use of corticosteroids (except when administered for refractory nausea/vomiting) or valproic acid; known brain metastasis; or uncontrolled intercurrent illness that would limit compliance with study requirements. Patients gave written informed consent according to federal and institutional guidelines before treatment, and the study was conducted in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization Guideline for Good Clinical Practice.
Dosage and drug administration
MS-275 was provided by Berlex Laboratories as 0.1, 1, and 5 mg tablets and was administered orally, with doses rounded to the nearest 0.1 mg. Patients had fasted a minimum of 6 h at the time of dosing, although water and analgesic medications were permitted. All patients received study drug under direct observation by the site personnel for all doses in cycle 1. Date and time of dosing were recorded. For all other doses and cycles, patients recorded the date and time each dose was taken in a study-specific diary. A new diary was issued to the patient at the start of each cycle and the completed diary for the prior cycle was returned.
The starting doses were (a) MS-275 2 mg/m2 once every 2 weeks (part 1); (b) 2 mg/m2 twice weekly (with at least 2 days between doses) for 3 weeks followed by 1 week of rest (part 2); or (c) 4 mg/m2 once weekly for 3 weeks followed by 1 week of rest (part 3). Successive cohorts of three patients were enrolled in increasing increments of 2 mg/m2 in parts 1 and 2 and in 1 mg/m2 increments in part 3 until DLT was noted in the first course. DLT was defined as (a) any grade 4 hematologic toxicity; (b) any grade ≥3 nonhematologic toxicity that represented at least a two-grade increase over baseline (except for alopecia and nausea/vomiting without maximal symptomatic and prophylactic treatment); (c) any grade 2 nonhematologic toxicity, except alopecia that was intolerable to the patient or of concern to the investigator, interrupted the dosing cycle, or failed to resolve to grade <1 or baseline at the time the next treatment cycle was scheduled to begin. During part 2, grade 3 hypophosphatemia was considered a DLT if it persisted despite adequate phosphate supplementation. There was no intrapatient dose escalation.
If one of the first three patients at a dose level experienced DLT, then up to three additional patients (total up to six patients) were enrolled at that dose level. If more than two patients at a dose level experienced DLT, dose escalation was halted. The maximum tolerated dose was defined as the highest dose at which no more than one of six patients experienced DLT in the first course.
Dose adjustments for toxicity
Interval toxicities had to resolve to grade 1 or baseline before proceeding with further treatment. Patients could receive successive courses of treatment until they withdrew consent, exhibited progressive disease, or failed to resolve drug-related toxicity within 4 weeks of the start of the next course or if the investigator believed discontinuation of treatment was in their best interest. For any patient experiencing toxicity, supportive and prophylactic care was instituted according to best clinical practice. In part 1, patients who experienced grade 2 toxicity, which prevented starting the next treatment on time, could resume treatment at a lower dose level or 50% to 75% dose reduction when the toxicity had resolved to grade <1. In parts 2 and 3, for grade 2 toxicity, treatment was held until the toxicity had resolved to grade <1. If grade 2 toxicity persisted and/or was not responsive to supportive care measures or prevented the patient from starting the next treatment cycle, a 25% dose reduction was made for subsequent cycles.
Any patient with any grade 3 nonhematologic toxicity or grade 3 or 4 hematologic toxicity was not retreated until the toxicity had resolved to grade <1 or baseline, and retreatment was reduced to 75% of the original dose for the remainder of his/her participation in the study. No additional dose reductions were permitted. Any patient with grade 4 nonhematologic toxicity was withdrawn from the study.
Pretreatment and follow-up studies
Pretreatment evaluations included height and weight, a 24-h urine collection for creatinine clearance, blood chemistries, complete blood count/differential, and a serum pregnancy test (if appropriate). Baseline adverse events and concomitant medications were recorded. On-study and end-of-study evaluations included physical exams, performance status evaluations, urinalysis, complete blood count/differential, coagulation studies, serum chemistries, and electrocardiograms. Tumor measurements and radiologic measurements were done at baseline and every 6 weeks thereafter and assessed by the Response Evaluation Criteria in Solid Tumors (12). Confirmatory measurement of response of complete or partial remission was done 4 weeks after the initial studies documenting response. In the case of stable disease, follow-up measurements must have met criteria at least once after study entry at a minimum interval of 6 weeks.
Pharmacokinetic and pharmacodynamic sampling
Peripheral blood (5 mL) was collected in sodium heparin tubes for analysis. In part 1, samples were obtained at 0, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84, 96, 120, and 168 h after the first dose of MS-275. In part 2, samples were collected at 0, 0.5, 1, 1.5, 2, 4, 8, 12, 24, 48, and 72 or 96 h after the first and sixth doses. In part 3, samples were collected at 0, 0.25, 0.5, 1, 1.5, 2, 4, 8, 24, 48, and 96 h after the first and third doses and 168 h after the third dose. Pre-dose trough plasma samples were also drawn on day 1 of subsequent even-numbered cycles in part 1.
The plasma concentration of MS-275 was measured using a validated, quantitative liquid chromatography-mass spectrometry method on a 1100 LC-MSD (Agilent Technologies) by the Pharmacokinetics Division of Schering. In brief, the samples were centrifuged at 2,000 × g for 10 min at room temperature within 30 min of collection. Plasma was separated and stored at −18°C to −25°C and then extracted with ethyl acetate. The organic layer was separated and evaporated under a nitrogen stream, and the residue was reconstituted in the mobile phase. An aliquot was then subjected to standard chromatography-mass spectrometry analysis. Variables were determined using the TOPFIT program (Thomae).
In part 1, 5 mL peripheral blood was collected in sodium heparin tubes in cycle 1 pretreatment and 6, 12, 24, 48, 72, and 96 h after treatment for pharmacodynamic analysis. Samples were also drawn before dosing at the start of cycle 2 and every even-numbered cycle thereafter. Immunocytochemical analysis of histone acetylation was done using a modification of the method of Warrell et al. (13). Briefly, PBMC were purified by density-gradient centrifugation using Histopaque tubes (Sigma-Aldrich). Cells were then pelleted onto glass slides by cytocentrifugation, fixed using 95% ethanol and 5% glacial acetic acid, and permeabilized with 0.2% Triton X-100. Nonspecific binding was blocked by a 1-h incubation with 1% bovine serum albumin-PBS at 4°C. Slides were stained for acetylated histone H3 and H4 using a polyclonal rabbit antibody (Upstate Biotechnology) at a stock concentration of 1 mg/mL diluted 1:150 in PBS and incubated for 1 h at 4°C. After washing twice in PBS, slides were incubated with a Cy3-conjugated goat anti-rabbit secondary antibody (Molecular Probes) at a stock concentration of 20 mg/mL diluted 1:200 in PBS for 1 h at 4°C and mounted using SlowFade mounting media (Molecular Probes).
Pharmacokinetic and pharmacodynamic analyses
The concentration of MS-275 in plasma samples was calculated using a reference calibration curve. Control specimens were run in parallel using human plasma samples spiked with known amounts (0.5, 1, 3, 40, and 80 ng/mL) of MS-275 and subjected to the same assay conditions as the patient samples. The mean interassay values were 94% to 108% for accuracy and 3.1% to 8.3% for the coefficient of variation.
The terminal disposition rate constant (λz) was calculated by regression analysis of the perceivable linear part of the curve in a semilogarithmic plot (where λz was the slope of the regression line). The corresponding terminal half-life (t1/2) was calculated by t1/2 = ln2 / λz. Individual half-lives were not accepted if the time range covered by the perceivable linear part of the curve was <2 half-lives. The area under the plasma concentration-time curve (AUC0-tlast) was calculated according to the linear trapezoidal rule. The total AUC0-∞ value was calculated by the formula: AUC0-∞ = AUC0-tlast + Ctlast / λz. The total oral clearance (CL/F) was obtained from the ratio of the dose (D) and the corresponding AUC value as CL/F = D / AUC, where F was the absolute bioavailability. Accumulation ratios were calculated from the ratio of AUC (0–48 h) and AUC (0–168 h) values for parts 2 and 3 based on the differences between the sixth and first and the third and first doses, respectively.
For pharmacodynamic analysis, digital pictures were taken using a ×632 or ×1,002 objective lens at ×6,302 or ×10,002 magnification, respectively, using an Axiphot microscope equipped with a CCD camera (Carl Zeiss). Staining intensity was analyzed in at least 20 PBMC in at least two fields per slide using image analysis software (Openlab, Improvision).
General
Twenty-seven patients, whose characteristics are listed in Table 1, received a total of ≥149 courses of MS-275 on three different dosing schedules (every other week, twice weekly, and weekly). All patients had received prior treatment for their tumors, and all but one had metastatic disease at study entry. The total numbers of patients assigned to each dose level, numbers of courses, and dose escalation scheme are depicted in Table 2. The median number of weeks on study was 7.5 (range, 2–250) in part 1, 8 (range, 5–30) in part 2, and 8 (range, 2–12) in part 3.
Table 1
Table 1
Patient characteristics
Table 2
Table 2
Dosing administration and escalation
The original schedule (part 1) for this study was developed based on observations of DLT in a study being conducted at the National Cancer Institute (NCI), where the first two patients treated on a daily schedule experienced dose-limiting abdominal/epigastric pain, supraventricular tachycardia, transaminase elevation, hypotension, hypoalbuminemia, and hypophosphatemia (14). As such, this study started dosing at 2 mg/m2 every other week. No first-cycle DLT were observed, no patient withdrew due to adverse events, and no maximum tolerated dose was established with this initial treatment schedule at doses from 2 to 6 mg/m2 every other week. Therefore, the protocol was amended to evaluate a twice-weekly dosing schedule (part 2), where patients received MS-275 starting at 2 mg/m2 twice weekly for 3 weeks followed by 1 week of rest (4-week cycle).
Although only one first-cycle DLT (grade 3 asthenia) was noted with twice-weekly dosing, moderate toxicity was observed, with grade 3 hypophosphatemia in two patients, grade 3 hyponatremia in one patient, and grade 2 aspartate aminotransferase elevation in one patient, which precluded on-time dosing. Therefore, the dosing frequency was changed to weekly dosing for 3 weeks followed by 1 week of rest (part 3) with a starting dose of 4 mg/m2. On this schedule, DLT was noted in two of five patients at the 5 mg/m2 cohort (grade 3 asthenia and hypophosphatemia in a patient receiving optimized supportive replacement). Therefore, the maximum tolerated dose with this dose schedule was determined to be 4 mg/m2 weekly. Weekly administration for 3 of 4 weeks (4-week cycle) was selected as the most frequent dosing that would allow chronic continuous exposure to MS-275 with an acceptable toxicity profile.
Dose reductions and modifications
Dose reductions of 25% were required in five patients due to adverse events, with a median dose intensity of 92% of planned cycles. The first patient's dose (part 1) was reduced from 2 to 1.5 mg/m2 every other week due to grade 2 thrombocytopenia in cycle 1. To date, this patient has received >120 cycles of therapy at this reduced dose level and has not had recurrence of thrombocytopenia grade >1. Another patient's dose was reduced from 2 to 1.5 mg/m2 twice weekly (part 3) in cycle 10 due to grade 2 elevation in creatinine. This patient was subsequently removed from study before cycle 11 due to disease progression.
Two patients required reduction from 4 to 3 mg/m2 weekly: one patient in cycle 2 due to grade 2 leukopenia and thrombocytopenia and grade 3 anemia and asthenia. This patient received one dose at this reduced level and then was removed from study due to persistent grade 2 anemia, thrombocytopenia, and neutropenia. Transfusions were refused due to religious beliefs, but the patient accepted darbepoetin alfa every 3 weeks. At the 30-day follow-up visit, anemia remained stable at grade 2. A second patient in this cohort required dose reduction at the end of cycle 1 due to grade 3 hypophosphatemia. This patient had metastatic pancreatic cancer with baseline grade 1 hypophosphatemia and grade 2 hypocalcemia. Oral phosphorus supplementation was instituted on cycle 1, day 1, and the patient experienced grade 2 hypophosphatemia on cycle 1, days 8 and 15, when the supplements were increased. One week later (cycle 1, day 22), the serum phosphorus worsened to grade 3, and increased supplementation was given; within 3 days of the increased supplementation, the phosphorus level returned to normal. By cycle 1, day 29, however, the patient had recurrence of grade 3 hypophosphatemia, thus meeting criteria for DLT. The patient did not have further grade ≥2 hypophosphatemia but was removed from study due to disease progression after cycle 2.
Doses for two patients required reduction from 5 to 3.75 mg/m2 weekly: one due to grade 2 thrombocytopenia and grade 3 neutropenia (nadir absolute neutrophil count 720/mm3) and one due to grade 3 hypophosphatemia. The patient with neutropenia and thrombocytopenia received one dose at 3.75 mg/m2, however, due to persistent grade 2 neutropenia, was removed from study due to inability to continue dosing. At 30-day follow-up, the patient continued to have grade 2 neutropenia. The second patient on the 5 mg/m2 dosing level who required dose reduction had metastatic colon cancer with baseline grade 1 hypophosphatemia. Grade 3 hypophosphatemia was observed on cycle 1, day 2, and oral phosphorus supplements were instituted immediately. The patient was monitored daily, and the hypophosphatemia improved to grade 2 on cycle 1, day 3. On cycle 1, day 8, when due for the next dose, the patient again had asymptomatic grade 3 hypophosphatemia. Increased oral supplementation was instituted, the hypophosphatemia was corrected to normal, and the patient received the cycle 1, day 8 dose after a 1-week delay at a reduced dose. Recurrent grade 3 hypophosphatemia was noted on cycle 1, day 15, and thus considered DLT due to persistent toxicity despite maximal supportive care. With additional increased oral supplementation, the patient's serum phosphorus level returned to normal.
Hematologic toxicity and laboratory abnormalities
The numbers of cycles with hematologic toxicities are listed in Table 3. Overall, hematologic toxicities were rare and none was dose limiting. Grade 3 hemoglobin occurred in 5 of 149 cycles (3%): 1 on the 2 mg/m2 twice-weekly schedule and 4 in the 5 mg/m2 weekly cohort. Grade 3 or 4 neutropenia was seen in 6 of 149 cycles (4%): 2 at 2 mg/m2 and 1 at 4 mg/m2 every other week and in 3 on the 5 mg/m2 weekly schedule. No grade 3 or 4 thrombocytopenia was observed. There was no relationship between the development of myelosuppression and prior chemotherapy or radiation therapy, and none of the episodes was complicated by fever, serious infection, or bleeding.
Table 3
Table 3
Number of courses with hematologic toxicity by dose level in all courses
Nonhematologic toxicity
Nonhematologic toxicity is shown in Table 4. The most common adverse events were nausea and asthenia [occurring in 48 and 47 of 149 cycles (32%), respectively]. The second most common event, anorexia, occurred in 23 of 149 cycles (15%). The most common grade 3 or 4 event was asthenia, occurring in 7 of 149 cycles (5%).
Table 4
Table 4
Nonhematologic toxicities of MS-275 in all courses
Asymptomatic hypophosphatemia was common and constituted DLT in two patients. The hypophosphatemia was not associated with other toxicities such as renal insufficiency or other electrolyte imbalances, and no serious complications were observed. One patient with metastatic colon cancer also experienced concurrent grade 3 hyponatremia during an episode of massive abdominal ascites and dehydration associated with rapid tumor progression and decreased oral intake. This patient had no prior history of renal disease, electrolyte wasting, or prior chemotherapy treatment known to contribute to electrolyte wasting. This patient's dehydration and electrolyte abnormalities resolved within 72 h of therapeutic paracentesis, i.v. fluid, and colloid administration.
Patients who had grade ≥2 hypophosphatemia underwent analysis of urine and serum electrolytes, were given daily oral phosphorus supplements, and were monitored at least weekly until stabilization of serum phosphorus levels to within normal range.
With other HDAC inhibitors, including the hydroxamic acid derivatives, there has been concern for cardiac rhythm disturbances and myocardial infarction, including prolonged QTc intervals and T- and ST-wave abnormalities, particularly based preclinical models. In this study, electrocardiograms were required at baseline, through cycle 1, at the beginning of cycle 2, and at the end of study participation for the every other week dosing and at baseline and as clinically indicated on the twice-weekly and weekly schedules. MUGA scans were done at baseline, before cycles 2 and 4, and every 6 weeks after cycle 4 on the every other week schedule and at baseline and as clinically indicated in the twice-weekly and weekly treatment groups. Eighteen patients had electrocardiograms after the baseline evaluation: 10 on every other week dosing, 3 on twice-weekly dosing, and 5 on weekly dosing schedules. Ten patients completed serial MUGA scanning: 6 on every other week dosing and 2 each on twice-weekly and weekly dosing schedules. No significant electrocardiograms or MUGA abnormalities attributed at least possibly related to MS-275 were noted.
Antitumor activity
Two patients on the every other week schedule had confirmed partial responses. A 68-year-old patient with metastatic melanoma refractory to prior surgery and biochemotherapy had disappearance of nodal metastases and remains on study for >5 years, with only one subcentimeter lymph node in the neck evident on scan and physical exam. This patient was treated at the lowest dose level and required a 25% dose reduction early in treatment due to grade 2 thrombocytopenia. A 26-year-old patient with Ewing's sarcoma metastatic to the lungs, who had received four prior combination chemotherapy regimens, surgery, and lung irradiation, had a 25% reduction in the size of a solitary pulmonary nodule, thus meeting partial response criteria according to WHO criteria but stable disease according to Response Evaluation Criteria in Solid Tumors. This patient remained on study for 1 year before eventually experiencing growth of the pulmonary nodule back to the original size at study entry. Additionally, two patients in part 1 (one with rectal adenocarcinoma and one with colon cancer), one patient in part 2 (melanoma), and four patients in part 3 (one each with non-small cell lung carcinoma, prostate cancer, melanoma, and leiomyosarcoma) had prolonged disease stabilization, ranging from 45 days to 10 months.
Pharmacokinetics and pharmacodynamics
A summary of the pharmacokinetics of MS-275 is presented in Table 5. In part 1, with every other week dosing, the maximum plasma concentrations (Cmax) and the early part of the AUC were poorly characterized, because the first sampling point was taken after tmax. A plasma concentration × time curve following single doses of 2, 4, and 6 mg/m2 is shown in Fig. 1.
Table 5
Table 5
Pharmacokinetic variables of MS-275
Fig. 1
Fig. 1
First dose mean plasma concentration μ time profiles in patients administered 2, 4, or 6 mg/m2 MS-275 (arithmetic mean ± SD).
On all dosing schedules, maximum plasma levels of MS-275 were reached within 1 h after single and repeated dose administration. Mean Cmax after the first administration increased almost dose-proportionally. MS-275 plasma concentrations declined to ~4% of Cmax within 4 h after single administration in parts 2 and 3 of the study, where the Cmax was well defined, indicating a rapid distribution into tissues. This initial distribution phase was followed by a slower secondary disposition phase. However, terminal half-life values could not be determined appropriately in most patients, because the perceivable linear part of the curve was <2 half-lives. Thus, the calculated terminal half-life values, as well as AUCand apparent clearance values, were only estimated and may be further considered as approximate reference values. The terminal half-life values were estimated to range between 60 and 150 h, irrespective of the dose or schedule, when the perceivable linear part of the curve could be followed for a relatively long time (up to ~168 h post-dose).
The accumulation of the drug in plasma following once-weekly and twice-weekly dosing schedules was evaluated to a limited extent due to deviations from the dosing schedule such as skipped doses and dose reductions due to toxicity. Based on the limited data, plasma drug concentrations appeared to increase continuously up to the last dose in cycle 1, indicating that steady state was not achieved in the twice-weekly treatment cohort. There was no clear indication of drug accumulation following once-weekly treatment.
Pharmacodynamic assessment of histone H3 and H4 acetylation status in PBMCwas determined by immunocytochemical analyses based on a method developed at the NCI (15). An example image of PBMC staining for histone H3 before and 24 h after treatment with MS-275 from a patient treated at the 4 mg/m2 every other week dose level is shown in Fig. 2. Although the levels of histone acetylation generally appeared qualitatively to increase after MS-275 administration, the quantitative results showed a large degree of variation. Because of the variability of the results, quantitation of a dose-response to MS-275 and correlation of the pharmacodynamics of MS-275 with response to treatment was not done.
Fig. 2
Fig. 2
Histone H3 hyperacetylation of PBMC in a patient treated at 4 mg/m2 every other week.
The HDAC are a large family of enzymes that catalyze the removal of acetyl groups from substrate proteins and thereby regulate their function and activity (16, 17). Several lines of evidence suggest a role for histone acetylation in tumorigenesis. First, overexpression of HDACor mutation or deletion of their counterparts, the histone acetylases has been observed in a variety of human cancers. In addition, several proteins that have been implicated in tumorigenesis, including p53, BCL6, Hsp90, Stat3, and E2Fs, are regulated by acetylation. Finally, several oncogenic proteins and fusion proteins interact with HDACto mediate inappropriate repression of target genes, and restoration of aberrant gene expression has been shown to be critical in the control of cancer cell proliferation.
Preclinical and early clinical data support the potential utility of a variety of HDAC inhibitors for treatment of human cancers. Suberoylanilide hydroxamic acid was well tolerated and both hematologic (22% overall response rate) and solid tumor responses (12% OR rate) were observed (18). This compound (also known as Zolinza or vorinostat, Merck & Co.) has recently been approved for use in cutaneous T-cell lymphoma based on strong phase II findings (19). Similarly, depsipeptide has shown early promise in the treatment of refractory chronic and acute leukemias (2022). Frequent DLT include myelosuppression, nausea, vomiting, dehydration, diarrhea, anorexia, fatigue, asthenia, and electrolyte disturbances. Histone acetylation in PBMC and tumor biopsies was detected following treatment and pharmacokinetic data indicate that serum concentrations of HDAC inhibitors can be achieved well in excess of the concentrations that are necessary for cytodifferentiation in vitro (18, 22).
MS-275 is unique among HDAC inhibitors in clinical development, in that it inhibits class I HDAC(HDAC 1 and 3) more than class II (HDAC 4), displays a unique in vitro cytotoxicity profile in the NCI COMPARE algorithm, inhibits tumor cell growth in nude mice comparable with or better than conventional cytotoxic agents such as 5-fluorouracil, and has relatively limited clinical toxicity, thus making it an attractive agent for development in humans (2).
This phase I study was done to evaluate the toxicity and pharmacokinetic profiles of MS-275 on three different schedules: one dose every other week, twice-weekly dosing for 3 weeks followed by 1 week of rest, and weekly dosing for 3 weeks followed by 1 week of rest. No DLT were noted on the every other week dosing schedules at doses ranging from 2 to 6 mg/m2. On the twice-weekly dosing schedule, patients were treated at 2 mg/m2, and grade 3 hypophosphatemia and hyponatremia and grade 2 transaminitis (aspartate aminotransferase) were noted. Although these toxicities were not dose limiting, they resulted in dose delays and omissions in the first cycle, which were felt to preclude further dose escalation on this schedule.
For patients treated on the weekly administration schedule, 4 mg/m2 was felt to be tolerable. Patients treated at 5 mg/m2 experienced grade 3 asthenia and hypophosphatemia (despite adequate oral phosphorus supplementation). Overall, hypophosphatemia occurred frequently and may be a class effect, as it has been seen with several other HDACinhibitors, although the mechanism remains undefined. In this study, grade 2 and 3 hypophosphatemia was noted in all patients treated on the twice-weekly schedule, whereas it was less frequent on the other schedules.
No hematologic DLT were observed in this study. However, the rules for dose reduction in the case of hematologic toxicities were relatively conservative, requiring dose reduction after any cycle with grade 3 or 4 toxicity. There did not appear to be any relationship between prior therapy received and specific toxicities or DLT noted. Patients who were more heavily pretreated did not experience toxicities or DLT at a higher rate than did patients who had less treatment before study entry.
No significant cardiac events, electrocardiogram changes, or MUGA abnormalities were noted in this study. Byrd et al. noted few cardiac effects with depsipeptide given to patients with refractory acute and chronic lymphoid leukemias (22), whereas another study of depsipeptide in patients with refractory solid tumors identified frequent, reversible ST-T wave changes, and one patient treated was reported to have grade 4 atrial fibrillation (23). In the NCI study with MS-275, dose-limiting supraventricular tachycardia was noted on the daily × 28 schedule, but no symptomatic cardiac events were noted in patients who received alternate-week MS-275 (14).
Pharmacokinetic studies revealed that MS-275 was rapidly absorbed under fasting conditions reaching a tmax within 60 min of dosing for all schedules. Thereafter, concentrations declined biphasically, with a fast elimination phase within 4 h and a slow terminal elimination phase with distribution into tissues. All patients receiving 4 mg/m2 had a detectible plasma drug level 168 h after dosing. Patients who received 6 mg/m2 (n = 4) attained a Cmax with a larger coefficient of variation between patients (285%) than in either 2 or 4 mg/m2 dose levels. Similar to the 4 mg/m2 dose level, biphasic elimination was noted, with faster clearance within 4 h, and two patients having detectible levels 168 h after dosing. Terminal half-life values could not be determined in the majority of patients, as the perceivable linear portion of the curve was <2 half-lives. Thus, the calculated t1/2 values, AUC, and clearance were estimated and can only been seen as general reference values. Similar to the study of Ryan et al., the terminal t1/2 values for MS-275 were estimated to be 60 to 150 h. This was noted regardless of the dose and schedule when the perceivable part of the curve could be followed for a relatively long time (168 h).
On the every other week administration schedule, the Cmax and tmax samples were likely underestimated with the first time-point collection at 1 h following administration as evidenced by the shorter time to maximal concentrations noted in the cohorts where earlier sampling was done (0.25 and 0.5 h for the weekly and twice-weekly cohorts). In these latter cohorts, Cmax values were observed within 0.5 h in 13 of 17 patients (76%).
Due to toxicity in patients, and subsequent holding of doses on the twice-weekly and weekly administration schedules, some pharmacokinetic variables could not be completed. This deviation from the planned schedule may have led to an underestimate of the exposure of MS-275 following repeated administration. The individual terminal half-life values could not be determined appropriately in all patients, because the time interval used for half-life estimation was too long. Thus, the mean terminal half-life values and the mean AUCand apparent clearance were estimated. For two of six patients in whom individual terminal half-lives could be determined, the values were slightly shorter compared with the values for patients in whom t1/2 was estimated (63.4 and 31.5 h, respectively, compared with 52–150 h for the population estimates). In the 5 mg/m2 weekly cohort, all patients held the second and/or third dose due to toxicity, and some patients required dose reduction. As a result, deviations prevented accurate estimation of the exposure of MS-275, and descriptive statistics were not calculated for this cohort.
Accumulation of MS-275 in plasma following weekly and twice-weekly administration was evaluated to a limited extent due to deviation form the dosing schedules required for toxicity. Based on the limited data, plasma concentrations of MS-275 appeared to increase continuously up to the last dose in cycle 1, indicating that no steady state was achieved in each cycle of the twice-weekly treatment. Conversely, there was no indication of drug accumulation on the weekly schedule.
Finally, it is difficult to evaluate dose-proportionality due to variability in the Cmax and AUC0–48 h and the small patient numbers investigated over a range of doses and schedules. The Cmax and the early part of the AUC were poorly defined, because the first sampling point was taken after the tmax on the every other week schedule. On this schedule, there appear to be dose-dependent but overproportional increases in Cmax and AUC0–48 h. However, when the twice-weekly and weekly schedules were evaluated, the Cmax and AUC0–48 h values increased almost dose-proportionally after single administration of 2 to 5 mg/m2.
Compared with another study of MS-275 in patients with refractory solid tumors and lymphoma (14), the pharmacokinetic results are consistent, although direct comparison of the PK variables is limited, because MS-275 was administered under nonfasting conditions in that study. In common, however, are the observations of large interpatient variability and dose dependency of systemic exposure as measured by the AUC. This study suggests that the Cmax increased in a dose-dependent manner. Also similarly, hyperacetylation of histone H3 and H4 was seen in PBMCof patients treated with MS-275, but the amount of hyperacetylation was variable.
Objective responses were only observed in patients receiving the every other week dosing schedule, but the numbers of patients enrolled were too small to determine whether this dosing regimen was truly more efficacious. There did not appear to be any relationship between dose or pharmacokinetic variables and confirmed response or prolonged stabilization of disease. The patient with the metastatic melanoma who experienced a confirmed partial response and who remains on study for >5 years, in fact, was treated at the lowest dose level and required a 25% dose reduction for grade 2 thrombocytopenia after cycle 2. It is indeed intriguing that responses were noted at the lower doses and on the every other week schedule, suggesting that maximal doses and frequent dosing are not required with MS-275, and increasing the attractiveness of such a regimen to patients as well. In other studies of MS-275 in patients with a variety of refractory solid tumors, there appears to be a trend that responding patients were often treated in the lower-dose cohorts, thus suggesting that dose escalation of this agent may not be necessary (NCI Advisory Panel, November 2005).
Overall, MS-275 was well tolerated when administered every other week at doses up to 6 mg/m2 and at doses up to 4 mg/m2 weekly for 3 weeks every 28 days. Either dosing schedule of MS-275 could be considered for further disease-directed studies and should be determined based on individual goals of the studies, biological correlative findings, and the pharmacologic rationale for combinations of agents considered. Based on a single patient in this study who has received MS-275 for 5 years on the every other week schedule, it appears to be well tolerated in chronic, long-term use to date. Further longitudinal observations in additional patients will be critical to follow over time.
Acknowledgments
Grant support: National Cancer Institute and University of Colorado Cancer Center NIH K12 Clinical Oncology Scholars Award CA-86913 (L. Gore).
1. Hess-Stumpp H. Histone deacetylase inhibitors and cancer: from cell biology to the clinic. Eur J Cell Biol. 2005;84:109–21. [PubMed]
2. Saito A, Yamashita T, Mariko Y, et al. A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors. Proc Natl Acad Sci U S A. 1999;96:4592–7. [PubMed]
3. Boulaire J, Fotedar A, Fotedar R. The functions of the cdk-cyclin kinase inhibitor p21WAF1. Pathol Biol (Paris) 2000;48:190–202. [PubMed]
4. Lee BI, Park SH, Kim JW, et al. MS-275, a histone deacetylase inhibitor, selectively induces transforming growth factor β type II receptor expression in human breast cancer cells. Cancer Res. 2001;61:931–4. [PubMed]
5. Park SH, Lee SR, Kim BC, et al. Transcriptional regulation of the transforming growth factor β type II receptor gene by histone acetyltransferase and deacetylase is mediated by NF-Y in human breast cancer cells. J Biol Chem. 2002;277:5168–74. [PubMed]
6. Lucas DM, Davis ME, Parthun MR, et al. The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells. Leukemia. 2004;18:1207–14. [PubMed]
7. Rosato RR, Almenara JA, Grant S. The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIP1/WAF11. Cancer Res. 2003;63:3637–45. [PubMed]
8. Jaboin J, Wild J, Hamidi H, Khanna C, Kim CJ, Robey R. MS-275, an inhibitor of histone deacetylase, has marked in vitro and in vivo antitumor activity against pediatric solid tumors. Cancer Res. 2002;62:6108–15. [PubMed]
9. Wei Y, Qian DZ, Ren M, et al. In vivo real-time imaging of transcriptional activation of the RARβ gene promoter by the histone deacetylase inhibitor MS-275 in a prostate cancer model. Proceedings of the AACR; Orlando, FL. 2004.
10. Qian D, Wang XF, Ren MQ, et al. The histone deacetylase inhibitor MS-275 inhibits prostate tumor growth. Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; Boston, MA. 2003.
11. Kato Y, Qian DZ, Ryu B, et al. Restoration of transforming growth factor (TGF) β signalling pathway in human prostate carcinoma LNCaP cell line by the histone deacetylase inhibitor MS-275. Proceedings of the AACR; Orlando, FL. 2004.
12. Therasse P, Arbuck SG, Eisenhauer EA, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst. 2000;92:205–16. [PubMed]
13. Warrell RP, Jr., He LZ, Richon V, Calleja E, Pandolfi PP. Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J Natl Cancer Inst. 1998;90:1621–5. [PubMed]
14. Ryan QC, Headlee D, Acharya M, et al. Phase I and pharmacokinetic study of MS-275, a histone deacetylase inhibitor, in patients with advanced and refractory solid tumors or lymphoma. J Clin Oncol. 2005;23:3912–22. [PubMed]
15. Hwang K, Acharya MR, Sausville EA, et al. Determination of MS-275, a novel histone deacetylase inhibitor, in human plasma by liquid chromatography-electrospray mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2004;804:289–94. [PubMed]
16. Zhang Y, Reinberg D. Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev. 2001;15:2343–60. [PubMed]
17. Fischle W, Wang Y, Allis CD. Histone and chromatin cross-talk. Curr Opin Cell Biol. 2003;15:172–83. [PubMed]
18. Kelly WK, Richon VM, O'Connor O, Curley T, MacGregor-Curtelli B, Tong W. Phase I clinical trial of histone deacet ylase inhibitor: suberoylanilide hydroxamic acid administered intravenously. Clin Cancer Res. 2003;9:3578–88. [PubMed]
19. Duvic M, Talpur R, Ni X, et al. Phase 2 trial of oral vorinostat (suberoylanilide hydroxamic acid, SAHA) for refractory cutaneous T-cell lymphoma (CTCL) Blood. 2007;109:31–9. [PubMed]
20. Kano Y, Akutsu M, Tsunoda S, et al. Cytotoxic effects of histone deacetylase inhibitor FK228 (depsipeptide, formally named FR901228) in combination with conventional anti-leukemia/lymphoma agents against human leukemia/lymphoma cell lines. Invest New Drugs. 2007;25:31–40. [PubMed]
21. Kawano T, Horiguchi-Yamada J, Iwase S, et al. Depsipeptide enhances imatinib mesylate-induced apoptosis of Bcr-Abl-positive cells and ectopic expression of cyclin D1, c-Myc or active MEK abrogates this effect 2004. Anticancer Res. 24:2705–12. [PubMed]
22. Byrd JC, Marcucci G, Parthun MR, et al. A phase 1 and pharmacodynamic study of depsipeptide (FK228) in chronic lymphocytic leukemia and acute myeloid leukemia. Blood. 2005;105:959–67. [PubMed]
23. Sandor V, Bakke S, Robey RW, et al. Phase I trial of the histone deacetylase inhibitor, depsipeptide (FR901228, NSC 630176), in patients with refractory neoplasms. Clin Cancer Res. 2002;8:718–28. [PubMed]