Purpose: To determine the safety, maximum tolerated dose, and pharmacokinetic-pharmacodynamic profile of a histone deacetylase inhibitor, LAQ824, in patients with advanced malignancy.

Patients and Methods: LAQ824 was administered i.v. as a 3-h infusion on days 1, 2, and 3 every 21 days. Western blot assays of peripheral blood mononuclear cell lysates and tumor biopsies pretherapy and posttherapy evaluated target inhibition and effects on heat shock protein-90 (HSP90) client proteins and HSP72.

Results: Thirty-nine patients (22 male; median age, 53 years; median Eastern Cooperative Oncology Group performance status 1) were treated at seven dose levels (mg/m2): 6 (3 patients), 12 (4 patients), 24 (4 patients), 36 (4 patients), 48 (4 patients), 72 (19 patients), and 100 (1 patient). Dose-escalation used a modified continual reassessment method. Dose-limiting toxicities were transaminitis, fatigue, atrial fibrillation, raised serum creatinine, and hyperbilirubinemia. A patient with pancreatic cancer treated at 100 mg/m2 died on course one at day 18 with grade 3 hyperbilirubinemia and neutropenia, fever, and acute renal failure. The area under the plasma concentration curve increased proportionally with increasing dose; median terminal half-life ranged from 8 to 14 hours. Peripheral blood mononuclear cell lysates showed consistent accumulation of acetylated histones posttherapy from 24 mg/m2; higher doses resulted in increased and longer duration of pharmacodynamic effect. Changes in HSP90 client protein and HSP72 levels consistent with HSP90 inhibition were observed at higher doses. No objective response was documented; 3 patients had stable disease lasting up to 14 months. Based on these data, future efficacy trials should evaluate doses ranging from 24 to 72 mg/m2.

Conclusions: LAQ824 was well tolerated at doses that induced accumulation of histone acetylation, with higher doses inducing changes consistent with HSP90 inhibition.

Translational Relevance

Histone deacetylases (HDAC) are validated targets for anticancer therapy, with vorinostat being the first HDAC inhibitor to be Food and Drug Administration approved for the treatment of cutaneous T-cell lymphoma. LAQ824 is a more potent HDAC inhibitor than suberoylanilide hydroxamic acid, which has been shown to inhibit HDAC6 in preclinical studies and abrogate heat shock protein 90 (HSP90) function. HSP90 inhibition may have therapeutic implications for the treatment of many malignancies because many key target proteins, including mutated proteins, are client proteins of this chaperone. This was, to our knowledge, the first clinical trial to evaluate in pharmacodynamic studies HSP90 inhibition by an HDAC inhibitor.

Eukaryotic DNA is wrapped around a core of histone proteins, which have lysine-rich tails (1). Posttranslational modification of the histone NH2-terminal tail region regulates gene expression (24). Histone acetylation can result in transcriptional activation, whereas deacetylation causes gene silencing. This balance is controlled by two enzyme families: histone deacetylases (HDAC) and histone acetyltranferases. There are four distinct groups of HDAC enzyme (57). Class I HDACs (13) are ubiquitous and generally localized to the nuclear compartment of the cell. Class II family members (46, 7, 9, 10) shuttle back and forth between the nucleus and cytoplasm in response to a range of signaling and differentiation pathways in the cell (7). This group of enzymes show tissue specificity. Class III HDACs are homologous to the yeast protein SIR2. Class IV HDACs comprise the more recently discovered HDAC11-related enzymes (6).

Disordered HDAC activity is associated with cancer (810). In tumor cells, HDACs can be aberrantly expressed or inappropriately localized, causing altered gene expression, including silencing of tumor suppressor genes (1116). They are also associated with proteins involved in oncogenesis (10, 17). The pleiotropic function of HDAC enzymes in the regulation of cell growth and their apparent role in supporting the oncogenic phenotype led to this class of enzyme receiving considerable attention as a target for antitumor therapy (18, 19). Many HDAC inhibitors of different structural types have been described (1821), with the hydroxamate vorinostat now registered for treatment of cutaneous T-cell lymphoma (22). The cellular effects of HDAC inhibition include cell cycle arrest at G1 and G2, differentiation, and apoptosis (23, 24), with antitumor activity in vivo (1822) and tumor-selective cytotoxicity observed in proliferating and nonproliferating malignant cells (25). It has been reported that this may be due to the up-regulation of the cell cycle inhibitor p21 in a p53-independent manner (26, 27) with, surprisingly, the transcription of only a limited number of genes being affected by HDAC inhibitors (28, 29). More widespread gene expression changes have, however, recently been described (30) using leukemic rather than solid tumor cell lines.

Rapid hyperacetylation of histones in vitro and in vivo (3134) is a hallmark of HDAC inhibition. Many nonhistone proteins (e.g., α-tubulin; ref. 35), have also been shown to be hyperacetylated in response to HDAC inhibitors. More recently, HDAC6-mediated hyperacetylation of the molecular chaperone heat shock protein 90 (HSP90) following exposure of leukemic and tumor cell lines to HDAC inhibitors has been described (3639). This results in the characteristic molecular signature of HSP90 inhibition (i.e., increased expression of HSP72 and depletion of client proteins; refs. 3739). The therapeutic importance of the increased acetylation of HSP90 and other nonhistone proteins remains to be determined (40, 41).

LAQ824 is a synthetic hydroxamic acid derivative, inhibiting HDACs at nanomolar concentrations. After administration of single i.v. doses to mice, rats, or dogs, LAQ824 was rapidly cleared from plasma in a multiexponential manner and was below the limit of detection after 8 hours. Distribution of [14C]LAQ824-related compounds was largely extravascular, with the highest tissue radioactivity observed in bile, kidney, thyroid, and adrenals. LAQ824 plasma protein binding was 77% in man. Hepatic microsomes metabolism was species dependent, with more rapid clearance in the rat than in the dog or human and largely involved the hydroxamate moiety. This included glucuronidation, reduction to the corresponding amide, and hydrolysis to the carboxylic acid. Other reactions included hydroxylation of the indole moiety and combinations of the above pathways. Preclinically, LAQ824 and its metabolites were excreted rapidly and completely from the body, with 99% of the dose being recovered in excreta within 48 hours. Renal excretion was minor, with the bulk of the dose being excreted with feces via the bile. Preclinical toxicology studies on LAQ824 revealed that the primary target organs for largely reversible toxicity in rats included injection sites, bone marrow, and ovaries. In addition, in dogs, the liver, testes, kidneys, and eyes were found to be target organs at the very highest dose level of 10 mg/kg. Electrophysiologic changes of arterial demand pacing prolongation in rabbit Purkinje fibers in vitro and slightly increased QTc intervals in dogs after repeated dosing at 0.3 to 10 mg/kg/d suggested a potential for LAQ824 to induce electrophysiologic changes. LAQ824 has preclinical antitumor activity in multiple myeloma, acute myelogenous leukemia and lymphoma cell lines, and lung, colon, and breast tumor xenografts (31, 33, 34, 42, 43). A phase I, open-label, dose-escalating study was conducted to determine the safety, tolerability, and recommended phase II dose of i.v. administration of LAQ824 by 3-h i.v. infusion administered once a day for 3 days, every 21 days. Secondary objectives included an evaluation of the pharmacokinetic-pharmacodynamic profile, including histone acetylation in surrogate and tumor tissue and antitumor activity. The effect of LAQ824 administration on the molecular signature of HSP90 was also investigated.

Adult patients with a confirmed histologic diagnosis of advanced solid tumor, for whom there were no standard treatments available, were included. All patients had to have a WHO performance status of ≤2 and be >18 y old. Adequate hepatic and renal function and normal or correctable electrolyte concentrations (magnesium, phosphorus, calcium, and potassium) were requirements. Necessary hematologic parameters were defined as hemoglobin ≥9 g/dL, absolute neutrophil count ≥1.5 × 109/L, and platelets ≥100 × 109/L. Impaired or significant cardiac disease, characterized by left ventricular ejection fraction of ≤45% on multigated acquisition scan, complete left bundle branch block, requirement for cardiac pacemaker, significant ST depression or T-wave inversion in two contiguous leads, congenital long QT syndrome, baseline prolonged QTc (>480 ms), history of either tachyarrhythmias, or significant resting bradycardia were exclusion criteria. Patients on medications associated with QT interval prolongation were excluded. Major surgery and hematopoietic growth factors were not allowed in the 14 d before study entry. Pregnant and breast-feeding females were also excluded. The study was approved by the ethics committee of both centers, and all patients signed an informed consent.

Dosage and dose-escalation schema. This was an open-label, nonrandomized, phase I, dose-escalation study designed to determine the dose-limiting toxicity and maximum tolerated dose of LAQ824 and to characterize the safety, tolerability, biological activity, and pharmacokinetic profile of LAQ824 when administered by 3-h i.v. infusion once daily for 3 consecutive days of each 21-d cycle to patients with advanced solid tumors. A modified continual reassessment method as previously described by O'Quigley et al. (44) was used to determine the maximum tolerated dose. The main modifications from O'Quigley's method have previously been described (45, 46). This modified continual reassessment method was selected to minimize the number of patients treated at potentially subtherapeutic doses of LAQ824 without exposing patients to excess risk of drug-related toxicity. Cohorts of three to four patients were treated with escalating doses of LAQ824 until maximum tolerated dose was reached. No intrapatient dose escalation was permitted. The modified continual reassessment method estimated the maximum tolerated dose by updating estimates of the toxicity rates (the probability of observing a dose-limiting toxicity) for each dose level in the study as patient information became available. Using a model-based evaluation of all available clinical and preclinical data on LAQ824 and initial estimates of the toxicity rates for each dose level, a model for the dose-toxicity relationship was determined. After each cohort of patients was evaluated for at least one course, the dose-toxicity model was updated and, with it, the estimates of the toxicity rates at the next selected dose level. Using this updated information for guidance, the dose level for the next cohort of patients was recommended. The maximal intercohort dose escalation permitted was 100%. The estimated maximum tolerated dose was the dose level that the modified continual reassessment method recommended after a minimum of six patients had been evaluated at that dose level.

Patients continued on their initial dose until the development of unacceptable toxicity or disease progression. Dose escalation between cohorts was done according to the modified continual reassessment method combined with review of available clinical and laboratory data. The evaluable patient population for the determination of maximum tolerated dose consisted of patients who had met the minimum safety evaluation requirements of the study. The minimum safety evaluation requirements were deemed to have been met if the patient received three consecutive daily doses of LAQ824 and all the required safety evaluations had been done during the 21 d following the first dose or the patient experienced a dose-limiting toxicity during cycle 1. Patients who did not meet these minimum study requirements were considered ineligible for the maximum tolerated dose–determining population. Once maximum tolerated dose was established, the cohort was expanded at the recommended phase II dose level to evaluate the safety, tolerability, pharmacokinetic profile, and pharmacodynamic activity of LAQ824. Antitumor efficacy was assessed after two cycles of treatment.

Dose-limiting toxicity. Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria version 2.0. Any grade 3 nonhematologic toxicity (excluding nausea or vomiting responsive to antiemetic treatment, alopecia, alkaline phosphatase elevation), grade 4 neutropenia lasting >7 d, or grade 4 thrombocytopenia of any duration was considered a dose-limiting toxicity. The starting dose of 6 mg/m2/d was one third of the toxic dose low for nonrodent species, which was 1 mg/kg or 20 mg/m2. LAQ824 was supplied as single-use glass ampoules containing 5 mL of 10 mg/mL solution. This was diluted to 150 mL in 5% dextrose in water before administration.

Dose escalation. The modified continual reassessment method provided an estimate of the probability of observing a dose-limiting toxicity at consecutive dose levels. The data were updated after each cohort of patients was evaluated for dose-limiting toxicities for at least one cycle, and from the dose-toxicity model, estimates of the toxicity rates at selected next dose levels calculated. Continually updated information guided the recommended dose escalation for the next cohort. The modified continual reassessment method was chosen to minimize the number of patients treated at potentially subtherapeutic doses without exposing patients to excess risk of drug toxicity. The maximal intercohort dose escalation was 100%, but clinically relevant grade 2 or greater nonhematologic toxicity in more than one patient restricted dose escalation to no more than 50%. Maximum tolerated dose was defined as the highest daily dose of i.v. LAQ824 given for at least one treatment cycle, wherein <33% of patients experienced a dose-limiting toxicity.

Dose modification due to toxicity. Dose modification was undertaken, if the patient experienced a dose-limiting toxicity, to the next lower dose level. The toxicity experienced was required to have resolved to grade ≤1 by the first day of the next scheduled dose of LAQ824; if it had not resolved to this level within 14 d following day 1 of the next cycle, then the patient was discontinued from study. If no toxicity was experienced but the absolute neutrophil count is ≤1.5 × 109/L and/or the platelet count is ≤100 × 109/L for >1 wk but <2 wk after the scheduled 1st day of the next treatment cycle, treatment was resumed but at the next lower dose level when absolute neutrophil count is ≥1.5 × 109/L and the platelet count is ≥100 × 109/L.

Safety and efficacy measures. Pretreatment evaluation included full medical history and physical examination, WHO performance status, full blood count, coagulation screen, biochemistry (including renal, hepatic, and thyroid function), cardiac enzymes (CK-MB and troponin I), urinalysis, multigated acquisition scan, chest X-ray, and 12-lead electrocardiogram. Tumor markers were assessed in appropriate cases. A pregnancy test was obtained in women with child-bearing potential. Imaging included computed tomography scan of thorax, abdomen, and pelvis; radionuclide bone scan; or magnetic resonance image scan as clinically indicated.

On-treatment evaluation included daily studies during the 3 d of treatment. These included full blood count, serum chemistry, and cardiac enzymes as aforementioned. Full blood count and serum chemistry were also done on days 8 and 15. Thyroid function testing was done on day 1 of each cycle. Physical examination was undertaken on day 15 and recording of adverse events and concomitant medication at each visit. Each patient underwent serial electrocardiogram recordings before the administration of the first dose (four sequential electrocardiograms separated by at least 30 min) and then at 15 min, 1, 2, 3, 5, and 7 h postdose in cycle 1. On subsequent days of dosing, only a single electrocardiogram was obtained before administration of LAQ824, unless further electrocardiograms were indicated by either the clinical situation or abnormalities were noted on the predose electrocardiogram. Electrocardiograms postdose were then done as above. An electrocardiogram was obtained on day 8 in each cycle and on days 4 and 5 in cycle 1. After cycle 1, the electrocardiograms at 15 min, 1, and 2 h postdose was not done unless clinically indicated. All electrocardiograms were analyzed by central review, and QTc intervals were calculated using Fridericia's correction formula. Cardiac enzymes were repeated at 3 and 7 h postdose in cycle 1, on days 1 to 3, and at 3 h postdose in subsequent cycles. In addition, troponin I assays was done on day 8 in all cycles. Multigated acquisition scans were repeated before each cycle of treatment being administered. Response Evaluation Criteria in Solid Tumors were used to evaluate response with imaging repeated every 6 wk.

Pharmacokinetic studies. Pharmacokinetic samples were collected during the first cycle of treatment. At the following time points, 3 mL of blood were collected: predose and 1.5, 3, 4, 5, 7, 9, 11, 13, and 15 h postdose on days 1 and 3 of treatment, then at 24 and 48 h following day 3. Additional samples were taken between the 3- and 4-h time points, at 3 h, 5, 20, and 40 min. A pharmacokinetic sample was also taken on days 8 and 15. Plasma concentrations of LAQ824 were determined using high-performance liquid chromatography. All pharmacokinetic calculations were done using noncompartmental models. The area under the plasma concentration curve (AUC) was calculated using the log-linear trapezoidal method. Total body clearance (CL = dose/AUC0-inf) and terminal elimination half-life (t1/2 = 0.693/λz) were calculated, where λz is the terminal disposition rate constant. Volume of distribution at steady state and accumulation ratio [R = (AUC0-24 on day 3) / (AUC0-24 on day 1)] were calculated from the plasma time-concentration profile.

Pharmacodynamic studies. The effect of LAQ824 on histone acetylation and HSP90 associated proteins in mononuclear cells was assessed by Western blotting. Peripheral blood (10-30 mL) was obtained in heparinized tubes at pretreatment, 3, 5, and 7 h postinfusion. Repeated pharmacodynamic sampling was done on day 3 at the same time points as on day 1, followed by sampling at 24 h after last infusion (day 4). Peripheral blood samples were drawn directly into a BD Vacutainer Cell Preparation Tube with sodium heparin (Becton-Dickinson). The BD cell preparation tubes were centrifuged (within 2 h of blood sampling) at 1,800 relative centrifugal force for 30 min at 24°C. The serum was removed to 1 cm above the separated cell layer and the remaining serum and separated cell layer containing mononuclear cells, and platelets were transferred to a 15-mL conical tube. The cells were washed once with 15 mL sterile PBS, centrifuged at 300 relative centrifugal force at 24°C for 15 min, washed again in 10 mL PBS, and centrifuged as before for 10 min. The mononuclear cells were snap frozen on dry ice and stored at −80°C until analysis. The cells were lysed in 50 to 100 μL triple detergent buffer [50 mmol/L Tris (pH 8), 150 mmol/L NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate containing protease inhibitors (Complete Mini Protease Inhibitor cocktail tablets, Roche Diagnostics)] and left on ice for 30 min. To ensure complete lysis, the samples were pipetted 20 times with a P200 Gilson pipette before being centrifuged (14,000 rpm, 4°C, 15 min). Tumor biopsies were obtained before and after treatment (at approximately the time point of maximal surrogate tissue pharmacodynamic effect) with the biopsies being immediately frozen in liquid nitrogen and stored at −80°C until processing. The samples were ground in a freezer mill cooled with liquid nitrogen for 1 min and the powder immediately resuspended in a lysis solution made up of 0.15 mol/L NaCl, 50 mmol/L Tris (pH 7.4), 1 mmol/L EDTA, 1% Triton X-100, 1 mmol/L NaF, 1 mmol/L NaVO3, 1% protease cocktail, 0.1% tosyl-lysine chloromethyl ketone, 1 mmol/L DTT, 0.1% fenvalerate, 0.1% Vphen, and 1 mmol/L phenylmethylsulfonylfluoride. The tumor lysate was homogenized further by repeated pipetting on ice for 20 min and the samples centrifuged (4°C for 10 min at 14,000 rpm) and the supernatant stored at −80°C until required for analysis. Protein concentration was determined with the bicinchoninic acid protein assay reagent (Pierce Biotech).

Western blotting. The lysed samples were separated using the NuPAGE Bis-Tris neutral pH, discontinuous SDS-PAGE electrophoresis system and precast gels (Invitrogen). Protein (25-100 μg) was mixed with 6.25 μL of 4× NuPAGE LDS sample preparation buffer (Invitrogen), 2.5 μL of 10× reducing agent (Invitrogen), and deionized water to bring the total volume to 25 μL and heated for 10 min at 70°C. The samples were loaded onto a 4% to 12% NuPAGE gradient Bis-Tris polyacrylamide precast gel (Invitrogen) and resolved by electrophoresis at 200 V per gel for 35 min using the Novex X-cell II Mini-Cell blotting unit (Invitrogen). NuPAGE MES-SDS running buffer was used with 500 μL of NuPAGE antioxidant added to the inner chamber. The separated proteins were transferred onto polyvinylidene difluoride membranes presoaked in methanol followed by 10 min in transfer buffer (NuPAGE transfer buffer, 0.1% NuPAGE antioxidant, 20% methanol in distilled water) for 1 h at 30 V. The efficacy of protein transfer was confirmed with Ponceau S (Sigma) stain. Membranes were rinsed several times in deionized water and blocked for 1 h in blocking buffer (5% nonfat milk, 0.1% NP40 in PBS) at room temperature to minimize nonspecific antibody binding. The membranes were incubated overnight on an orbital shaker at 4°C with primary antibody diluted in PBS containing 0.1% NP40 and 5% nonfat milk. After incubation, membranes were washed for 30 min with three changes of wash buffer (PBS containing 0.1% NP40). The antibodies used were rabbit anti–acetylated histone H3 or H4 (Upstate Biotechnology UK), rabbit anti-CRAF, rabbit anti–cyclin-dependent kinase 4 (Santa Cruz Biotechnology), mouse anti-HSP72 (Stressgen), and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Chemicon). The membranes were then probed with a horseradish peroxidase secondary mouse or rabbit antibody conjugate (A8924 or A6154, Sigma) diluted in PBS containing 0.1% NP40 and 5% nonfat milk for 1 h. After further washing for 30 min as before, the proteins of interest were detected by enhanced chemiluminescence (GE Bioscience) and visualized by exposure to film (Hyperfilm ECL, Amersham). Densitometry of the tumor lysate blots was used to quantify visualized protein bands using ImageQuant Software (Molecular Dynamics).

Patient demographics.Table 1 summarizes the demographics of the treated patients; 39 patients were recruited into the study and were eligible for assessment of toxicity. Most patients received 2 courses of treatment, whereas two patients treated at 48 and 72 mg/m2 received 21 and 13 consecutive cycles, respectively. Overall, 119 treatment cycles were administered. Four cycles were not fully completed either because of disease progression (two patients) or because of toxicity (one patient developed atrial fibrillation and one patient had QTc prolongation by investigator assessment). The median number of treatment cycles per patient was 2 (range, 1-21). Six patients completed four or more cycles of treatment. One patient treated at 48 mg/m2 had 21 consecutive treatment cycles with no dose reduction throughout treatment. Two patients at the dose level of 72 mg/m2 had 13 and 8 treatment cycles; both had 2 cycles at the initial dose, followed by 11 and 6 cycles at a reduced dose of 48 mg/m2, respectively. The dose was reduced because of non–dose-limiting toxicities, which did not reoccur after dose reduction. Ten patients had only one treatment cycle: seven had the treatment discontinued because of early disease progression, two had dose-limiting toxicity, and one patient withdrew his consent.

Table 1.

Patient demographics

CharacteristicNo. of patients
Total 39 
Age, y  
    Median 53 
    Range 31-69 
No. of assessable courses 116 
    Median/patient 
    Range 1-21 
Sex  
    Male 22 
    Female 17 
ECOG performance status  
    Median 
    Range 0-2 
Tumor type  
    Colon 
    Breast 
    Melanoma 
    Prostate 
    Pancreas 
    Liver 
    Sarcoma 
    Thyroid 
    Renal 
    NSCLC 
    Adrenal 
    Esophagus 
    Mesothelioma 
    Cutaneous T-cell lymphoma 
    SCLC 
    Unknown primary 
    Cholangiocarcinoma 
Previous systemic therapy (no. of regimens)  
    1-2 13 
    ≥3 26 
CharacteristicNo. of patients
Total 39 
Age, y  
    Median 53 
    Range 31-69 
No. of assessable courses 116 
    Median/patient 
    Range 1-21 
Sex  
    Male 22 
    Female 17 
ECOG performance status  
    Median 
    Range 0-2 
Tumor type  
    Colon 
    Breast 
    Melanoma 
    Prostate 
    Pancreas 
    Liver 
    Sarcoma 
    Thyroid 
    Renal 
    NSCLC 
    Adrenal 
    Esophagus 
    Mesothelioma 
    Cutaneous T-cell lymphoma 
    SCLC 
    Unknown primary 
    Cholangiocarcinoma 
Previous systemic therapy (no. of regimens)  
    1-2 13 
    ≥3 26 

Abbreviations: ECOG, Eastern Cooperative Oncology Group; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer.

Safety and tolerability. Overall, minimal toxicity was observed at the first five dose levels (6-48 mg/m2); no dose-limiting toxicity was reported at the 6 and 12 mg/m2 dose levels. Thereafter, an additional cohort was dosed at 24 mg/m2, wherein one patient experienced reversible grade 3 transaminase elevation. Further dose escalation was limited by the modified continual reassessment method to 50% increments. At 36 and 48 mg/m2, four patients were treated in each cohort, and no dose-limiting toxicity was seen. Dose escalation to 72 mg/m2 was then pursued. Dose-limiting toxicities were reported in two of six patients enrolled at 72 mg/m2 with reversible grade 3 transaminitis and reversible grade 3 fatigue in a patient with a history of fatigue before treatment (Table 2). Because this transaminase elevation was rapidly reversible, asymptomatic, and an uncommon event in the study overall and because the dose-limiting fatigue was considered to be at least in part cancer related, along with safety data from a parallel study in patients with leukemia indicating that 72 mg/m2 was well tolerated (23), further dose escalation to 100 mg/m2 was pursued. At this dose level, the first patient, who had advanced pancreatic cancer, developed grade 4 hyperbilirubinemia (conjugated and unconjugated) associated with febrile neutropenia (grade 3; nadir absolute neutrophil count, 0.512) on day 3. The day 3 dose was therefore not administered. The patient then developed thrombocytopenia on day 8 of treatment (platelets decreasing to a nadir of 17) and a decrease in his hemoglobin to 8.4 g/dL, necessitating platelet and blood transfusions. This was followed by an episode of atrial fibrillation on day 13 and then acute renal failure, following which the patient died 18 days after the first infusion. The toxic dose was therefore declared as 100 mg/m2, and no additional patients were treated at this dose level.

Table 2.

Dose-escalation scheme and dose-limiting toxicities in course one

Dose levelDose regimen (mg/m2)nDLT
No 
12 No 
24 Grade 3 transaminase increase 
36 No 
48 No 
72 19 Grade 3 fatigue* 
   Grade 3 transaminitis 
   Grade 3 atrial fibrillation 
   Grade 3 elevated creatinine 
   Grade 3 QTc prolongation 
100 Febrile neutropenia 
   Grade 3 atrial fibrillation 
   Grade 3 hyperbilirubinemia 
   Death 
Dose levelDose regimen (mg/m2)nDLT
No 
12 No 
24 Grade 3 transaminase increase 
36 No 
48 No 
72 19 Grade 3 fatigue* 
   Grade 3 transaminitis 
   Grade 3 atrial fibrillation 
   Grade 3 elevated creatinine 
   Grade 3 QTc prolongation 
100 Febrile neutropenia 
   Grade 3 atrial fibrillation 
   Grade 3 hyperbilirubinemia 
   Death 

Abbreviation: DLT, dose-limiting toxicity.

*

Patient with dose-limiting fatigue had grade 1 fatigue pretreatment.

Dose-limiting toxicity occurred at the expanded cohort; grade 3 QTc prolongation was investigator determined and confirmed by local cardiology assessment but found not to be dose limiting by central cardiology review.

In accordance with the study protocol, 13 additional patients, to a total of 19 patients given 56 courses, were entered to gain information at the recommended dose of 72 mg/m2 (Table 3). During the first cycle, two of these patients were dose reduced because of renal dysfunction, which fully reversed after 13 days, and asymptomatic QTc prolongation of >500 ms as determined by the investigator but subsequently deemed to be <500 ms after central review; both patients tolerated 48 mg/m2 without reoccurrence of these toxicities. An additional patient developed atrial fibrillation at 72 mg/m2; this spontaneously reverted to sinus rhythm after 14 days. The patient developed atrial fibrillation again immediately after rechallenge at a reduced dose of 48 mg/m2, and LAQ824 treatment was then discontinued.

Table 3.

Most common nonhematologic toxicities displayed as a function of dose level and NCI-CTC grade for all treatment courses with number of patients and courses administered at each dose level

ToxicityMax CTC gradeDose levels (mg/m2)
6-12 (7 pts; 17 courses)24-36 (8 pts; 19 courses)48 (4 pts; 26 courses)72 (19 pts; 56 courses)100 (1 pt; 1 course)All doses (39 pts; 119 courses)
Nausea 1-2 16 25 
 3-4 
Vomiting 1-2 13 21 
 3-4 
Fatigue 1-2 11 19 
 3-4 
Headache 1-2 13 
 3-4 
Anorexia 1-2  
 3-4  
Diarrhea 1-2 11 
 3-4 
Constipation 1-2 
 3-4 
Transaminase elevation 1-2 
 3-4 
ToxicityMax CTC gradeDose levels (mg/m2)
6-12 (7 pts; 17 courses)24-36 (8 pts; 19 courses)48 (4 pts; 26 courses)72 (19 pts; 56 courses)100 (1 pt; 1 course)All doses (39 pts; 119 courses)
Nausea 1-2 16 25 
 3-4 
Vomiting 1-2 13 21 
 3-4 
Fatigue 1-2 11 19 
 3-4 
Headache 1-2 13 
 3-4 
Anorexia 1-2  
 3-4  
Diarrhea 1-2 11 
 3-4 
Constipation 1-2 
 3-4 
Transaminase elevation 1-2 
 3-4 

Abbreviations: Pt, patient; NCI-CTC, National Cancer Institute Common Toxicity Criteria.

Both dose-limiting and non–dose limiting toxicities during the first cycle at all dose levels are summarized in Tables 2 and 3. Dose-limiting toxicities included atrial fibrillation, renal failure, fatigue, hyperbilirubinemia, and transaminitis.

Asymptomatic QTc prolongation of >500 ms was noted by the investigators in two patients and confirmed by local cardiology review; however, centralized review (using Fridericia's correction) of all the electrocardiograms done in the study found no electrocardiograms that had a QTc interval (Fridericia's correction) of >500 ms. Nonspecific, asymptomatic, and reversible ST-T-wave changes were also observed. LAQ824 did not affect cardiac function in serial assessments of cardiac function by multigated acquisition scans. Gastrointestinal disturbances, manifesting as nausea and vomiting, were commonly noted during the first course of the trial at higher dose levels when constitutional symptoms such as fatigue and anorexia were also described. Headache was reported by 12 patients but did not exceed grade 2.

Thrombocytopenia was the most common hematologic toxicity (Table 4). This was mostly transient and occurred on or around day 8 and resolved before day 15; however, in those patients that had blood samples taken earlier for clinical indications the resolution of thrombocytopenia was documented within 1 or 2 days. Sustained bone marrow suppression was not a feature of LAQ824 treatment, but at the highest dose (100 mg/m2) administered, febrile neutropenia did occur (Table 4).

Table 4.

Hematologic toxicity following LAQ824 administration

NCI-CTC v2 gradeLAQ824 (mg/m2)
6-12 (n = 7)
24-36 (n = 8)
48 (n = 4)
72 (n = 19)
100 (n = 1)
All doses (n = 39)
No. of patients with toxicity (%)
Thrombocytopenia       
    1 0 (0.0) 0 (0.0) 2 (50.0) 7 (36.8) 0 (0.0) 9 (23.1) 
    2 1 (14.3) 1 (12.5) 0 (0.0) 4 (21.1) 0 (0.0) 6 (15.4) 
    3 0 (0.0) 1 (12.5) 0 (0.0) 4 (21.1) 1 (100.0) 6 (15.4) 
    4 0 (0.0) 0 (0.0) 1 (25.0) 2 (10.5) 0 (0.0) 3 (7.7) 
Neutropenia       
    1 1 (14.3) 2 (25.0) 1 (25.0) 3 (15.8) 0 (0.0) 7 (17.9) 
    2 0 (0.0) 1 (12.5) 0 (0.0) 4 (21.1) 0 (0.0) 5 (12.8) 
    3 0 (0.0) 0 (0.0) 0 (0.0) 2 (10.5) 0 (0.0) 2 (5.1) 
    4 0 (0.0) 0 (0.0) 0 (0.0) 2 (10.5) 1 (100.0) 3 (7.7) 
Lymphopenia       
    1 2 (28.6) 3 (37.5) 0 (0.0) 2 (10.5) 0 (0.0) 7 (17.9) 
    2 3 (42.9) 4 (50.0) 2 (50.0) 9 (47.4) 0 (0.0) 18 (46.2) 
    3 0 (0.0) 0 (0.0) 2 (50.0) 6 (31.6) 1 (100.0) 9 (23.1) 
    4 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 
Anemia       
    1 4 (57.1) 1 (12.5) 1 (25.0) 5 (26.3) 0 (0.0) 11 (28.2) 
    2 1 (14.3) 3 (37.5) 1 (25.0) 8 (42.1) 1 (100.0) 14 (35.9) 
    3 1 (14.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (2.6) 
    4 0 (0.0) 0 (0.0) 1 (25.0) 0 (0.0) 0 (0.0) 1 (2.6) 
NCI-CTC v2 gradeLAQ824 (mg/m2)
6-12 (n = 7)
24-36 (n = 8)
48 (n = 4)
72 (n = 19)
100 (n = 1)
All doses (n = 39)
No. of patients with toxicity (%)
Thrombocytopenia       
    1 0 (0.0) 0 (0.0) 2 (50.0) 7 (36.8) 0 (0.0) 9 (23.1) 
    2 1 (14.3) 1 (12.5) 0 (0.0) 4 (21.1) 0 (0.0) 6 (15.4) 
    3 0 (0.0) 1 (12.5) 0 (0.0) 4 (21.1) 1 (100.0) 6 (15.4) 
    4 0 (0.0) 0 (0.0) 1 (25.0) 2 (10.5) 0 (0.0) 3 (7.7) 
Neutropenia       
    1 1 (14.3) 2 (25.0) 1 (25.0) 3 (15.8) 0 (0.0) 7 (17.9) 
    2 0 (0.0) 1 (12.5) 0 (0.0) 4 (21.1) 0 (0.0) 5 (12.8) 
    3 0 (0.0) 0 (0.0) 0 (0.0) 2 (10.5) 0 (0.0) 2 (5.1) 
    4 0 (0.0) 0 (0.0) 0 (0.0) 2 (10.5) 1 (100.0) 3 (7.7) 
Lymphopenia       
    1 2 (28.6) 3 (37.5) 0 (0.0) 2 (10.5) 0 (0.0) 7 (17.9) 
    2 3 (42.9) 4 (50.0) 2 (50.0) 9 (47.4) 0 (0.0) 18 (46.2) 
    3 0 (0.0) 0 (0.0) 2 (50.0) 6 (31.6) 1 (100.0) 9 (23.1) 
    4 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 
Anemia       
    1 4 (57.1) 1 (12.5) 1 (25.0) 5 (26.3) 0 (0.0) 11 (28.2) 
    2 1 (14.3) 3 (37.5) 1 (25.0) 8 (42.1) 1 (100.0) 14 (35.9) 
    3 1 (14.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (2.6) 
    4 0 (0.0) 0 (0.0) 1 (25.0) 0 (0.0) 0 (0.0) 1 (2.6) 

Pharmacokinetic analysis. A characteristic plasma concentration time curve for a patient at 72 mg/m2 is presented in Fig. 1A. Mean plasma concentration-time curves for each dose level are presented in Fig. 1B. The analyses excluded two patients with outlier values treated at 12 mg/m2 (15-fold higher than mean) and 72 mg/m2 (100-fold lower than mean) described in Table 5; no apparent explanation for these outlier values was identified. Patients with higher exposure did not seem to have a higher incidence of dose-limiting toxicity or other significant toxicity. Overall, mean pharmacokinetic parameters from all patients treated at all dose levels are presented in Table 5. Cmax and AUC increased proportionally with increasing dose. There was moderate interpatient variability. The elimination half-life of the terminal phase (t1/2λz) ranged between 6 and 15 hours. An ∼1.5-fold drug accumulation at steady state was observed following once daily dosing for 3 days. The patient who died following treatment at 100 mg/m2 did not complete his pharmacokinetic sampling but did not seem to have unusually high exposure.

Fig. 1.

A, typical plasma pharmacokinetic profile for a patient treated with LAQ824 in patients at 72 mg/m2 on days 1 and 3 after daily 3-h i.v. infusion on days 1, 2, and 3. B, mean plasma concentrations and pharmacokinetic parameters of LAQ824 in patients on day 3 following 3-h i.v. infusion on days 1 to 3.

Fig. 1.

A, typical plasma pharmacokinetic profile for a patient treated with LAQ824 in patients at 72 mg/m2 on days 1 and 3 after daily 3-h i.v. infusion on days 1, 2, and 3. B, mean plasma concentrations and pharmacokinetic parameters of LAQ824 in patients on day 3 following 3-h i.v. infusion on days 1 to 3.

Close modal
Table 5.

Noncompartmental mean pharmacokinetic parameter estimates for LAQ824 for days 1 and 3 for each cohort, excluding two outlier patients; the data for the two outlier patients are described separately below

Dose (mg/m2)DaynTmax (h)Cmax (ng/mL)AUC0-24 (ng·h/mL)AUC0-inf (ng·h/mL)t1/2 el (h)RA
2.500 75.233 240.667 240.667 8.220 1.024 
 2.500 76.433 254.657 254.657 7.167  
12* 1.500 151.667 467.590 504.187 13.683 1.073 
 2.000 155.333 504.260 561.070 12.867  
24 1.500 331.000 971.290 971.290 14.267 0.949 
 2.000 276.333 833.883 833.883 27.640  
36 1.875 455.250 1,323.695 1,323.695 11.775 1.105 
 2.250 516.750 1,561.588 1,561.588 8.318  
48 1.500 472.500 1,291.417 1,291.417 8.697 1.356 
 1.500 586.750 1,732.307 1,732.307 12.355  
72* 15 2.000 824.867 2,314.122 2,393.195 8.294 1.199 
 13 1.846 994.992 2,988.678 3,478.263 13.213  
100 3.000 2,500.000 9,331.410 9,331.410 10.130  
         
Pharmacokinetic parameters in outliers
 
        
Subject ID
 
Dose (mg/m2)
 
Day
 
Tmax (h)
 
Cmax (ng/mL)
 
AUC0-24 (ng·h/mL)
 
AUC0-inf (ng·h/mL)
 
t1/2 el (h)
 
RA
 
Outlier 1 12 1.5 4,070 4,730.65 4,736.2 5.81 NA 
  1.5 3,190 5,466.12 5,513.01 20.19 1.1555 
Outlier 2 72 1.5 51,200 58,370.83 58,393.64 3.71 NA 
  1.5 33,600 41,411.94 41,481.87 4.27 0.7104 
Dose (mg/m2)DaynTmax (h)Cmax (ng/mL)AUC0-24 (ng·h/mL)AUC0-inf (ng·h/mL)t1/2 el (h)RA
2.500 75.233 240.667 240.667 8.220 1.024 
 2.500 76.433 254.657 254.657 7.167  
12* 1.500 151.667 467.590 504.187 13.683 1.073 
 2.000 155.333 504.260 561.070 12.867  
24 1.500 331.000 971.290 971.290 14.267 0.949 
 2.000 276.333 833.883 833.883 27.640  
36 1.875 455.250 1,323.695 1,323.695 11.775 1.105 
 2.250 516.750 1,561.588 1,561.588 8.318  
48 1.500 472.500 1,291.417 1,291.417 8.697 1.356 
 1.500 586.750 1,732.307 1,732.307 12.355  
72* 15 2.000 824.867 2,314.122 2,393.195 8.294 1.199 
 13 1.846 994.992 2,988.678 3,478.263 13.213  
100 3.000 2,500.000 9,331.410 9,331.410 10.130  
         
Pharmacokinetic parameters in outliers
 
        
Subject ID
 
Dose (mg/m2)
 
Day
 
Tmax (h)
 
Cmax (ng/mL)
 
AUC0-24 (ng·h/mL)
 
AUC0-inf (ng·h/mL)
 
t1/2 el (h)
 
RA
 
Outlier 1 12 1.5 4,070 4,730.65 4,736.2 5.81 NA 
  1.5 3,190 5,466.12 5,513.01 20.19 1.1555 
Outlier 2 72 1.5 51,200 58,370.83 58,393.64 3.71 NA 
  1.5 33,600 41,411.94 41,481.87 4.27 0.7104 

NOTE: t1/2 el, elimination half-life; RA, accumulation ratio or AUC0-24 day 3/AUC0-24 day 1.

Abbreviation: NA, not applicable.

*

Excluding 2 outliers treated at 12 and 72 mg/m2.

Pharmacodynamic studies. Histone acetylation, as a mechanistic biomarker of response, was evaluated by Western blot analysis of peripheral blood mononuclear cell (PBMC) lysates from 32 patients. In addition, a paired tumor biopsy was obtained for biomarker assessment from 1 patient (72 mg/m2) with cutaneous T-cell lymphoma. Increased histone acetylation, compared with predose samples, was not observed in post-LAQ824 samples from the first cohort of patients (6 mg/m2). A robust increase in the extent and duration of histone acetylation was observed with increasing dose of LAQ824 (Fig. 2A). All patients treated with LAQ824 at 12 mg/m2 or above with samples assessable for histone acetylation showed an increase in histone acetylation. Consistently increased acetylation of histones compared with predose levels was observed at doses ≥24 mg/m2. This effect was maintained for 24 hours at doses ≥36 mg/m2 and for 48 hours after last LAQ824 infusion at doses ≥72 mg/m2 (Table 6).

Fig. 2.

Pharmacodynamic studies. A, the duration and extent of histone H3 acetylation with increasing dose of LAQ824 were observed in patient PBMC. Samples shown are from patients included in cohorts 3 (24 mg/m2), 4 (36 mg/m2), and 6 (72 mg/m2). B, Western blots of PBMC samples from patients included in cohorts 5 (48 mg/m2) and 6 (72 mg/m2) showing the molecular signature of HSP90 inhibition with an associated increase in histone acetylation (seen on day 3 in patient treated at 48 mg/m2). Pre–day 1 sample was not available. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, Western blots showing similar patterns of increased histone H3 acetylation and the molecular signature of HSP90 inhibition in PBMC and tumor samples from a patient with cutaneous T-cell lymphoma treated with 72 mg/m2 LAQ824. Expression of the HSP90 client protein cyclin-dependent kinase 4 (CDK4) was also reduced in the tumor sample.

Fig. 2.

Pharmacodynamic studies. A, the duration and extent of histone H3 acetylation with increasing dose of LAQ824 were observed in patient PBMC. Samples shown are from patients included in cohorts 3 (24 mg/m2), 4 (36 mg/m2), and 6 (72 mg/m2). B, Western blots of PBMC samples from patients included in cohorts 5 (48 mg/m2) and 6 (72 mg/m2) showing the molecular signature of HSP90 inhibition with an associated increase in histone acetylation (seen on day 3 in patient treated at 48 mg/m2). Pre–day 1 sample was not available. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, Western blots showing similar patterns of increased histone H3 acetylation and the molecular signature of HSP90 inhibition in PBMC and tumor samples from a patient with cutaneous T-cell lymphoma treated with 72 mg/m2 LAQ824. Expression of the HSP90 client protein cyclin-dependent kinase 4 (CDK4) was also reduced in the tumor sample.

Close modal
Table 6.

Summary of observed biomarker effects in PBMC for all cohorts

CohortDose (mg/m2)↑AcH3 ↓CRAF ↑HSP72↑AcH3 ↓CRAF↑AcH3 ↑HSP72↑AcH3 aloneSample sets available/suitable for all markers
0/3 0/3 0/3 0/3 3/3 
12 0/1 0/1 1/1 0/1 1/4 
24 1/4 1/4 2/4 0/4 4/4 
36 1/4 0/4 1/4 2/4 3/4 
48 1/4 0/4 2/4 1/4 4/4 
72 5/19 3/19 7/19 4/19 16/19 
100 — — — — 0/1 
CohortDose (mg/m2)↑AcH3 ↓CRAF ↑HSP72↑AcH3 ↓CRAF↑AcH3 ↑HSP72↑AcH3 aloneSample sets available/suitable for all markers
0/3 0/3 0/3 0/3 3/3 
12 0/1 0/1 1/1 0/1 1/4 
24 1/4 1/4 2/4 0/4 4/4 
36 1/4 0/4 1/4 2/4 3/4 
48 1/4 0/4 2/4 1/4 4/4 
72 5/19 3/19 7/19 4/19 16/19 
100 — — — — 0/1 

NOTE: Column 3 shows the number of patients with increased acetylation and the molecular signature of HSP90 inhibition. Columns 4 and 5 show the numbers with increased acetylation and either decreased CRAF or increased HSP72. Column 6 shows those with increased acetylation alone. Eight patient sample sets were not suitable or available for assessment of all markers.

During recruitment to the study, evidence emerged to suggest that HDAC inhibitors are able to inhibit the activity of the molecular chaperone HSP90 (3639). Following exposure to an HSP90 inhibitor, client proteins such as CRAF and cyclin-dependent kinase 2 are depleted with a concomitant increase in HSP72 levels (40). This specific molecular signature has been observed in patients treated with the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (41). The PBMC samples collected from patients in this LAQ824 phase I trial were therefore interrogated for the presence of changes in CRAF and HSP72 consistent with HSP90 inhibition. In eight patients, a pattern of response in CRAF and HSP72 expression consistent with inhibition of HSP90 was observed following treatment with LAQ824 (Fig. 2B). Changes in CRAF and HSP72 consistent with HSP90 inhibition were first observed at a dose of 24 mg/m2. However, this effect was not as robust as increased histone acetylation. Four patients had an isolated reduction in CRAF without a clear pattern of response in HSP72. Thirteen patients showed increased expression of HSP72 concurrent with increased acetylation without an observed effect on CRAF. One patient was negative for CRAF (Table 6). In a patient with cutaneous T-cell lymphoma, it was possible to compare mechanistic end points in the surrogate tissue with that in tumor tissue. The LAQ824-associated effects of H3 acetylation, CRAF depletion, and increased expression of HSP72 observed in PBMCs were consistent with those observed in tumor tissue (Fig. 2C).

Clinical efficacy and recommended dose for future efficacy studies. No responses were documented. One patient with hepatocellular carcinoma treated at 48 mg/m2 completed 21 full treatment cycles and achieved prolonged disease stability (14 months) with a 10-fold reduction in α-fetoprotein. Stable disease but of lesser duration was also observed in two patients in the 72 mg/m2cohort who received 13 (fibrosarcoma) and 8 (papillary carcinoma of the thyroid) treatment cycles. Most patients discontinued treatment after completing one or two treatment cycles because of disease progression. Overall, based on the safety, pharmacokinetic, and pharmacodynamic data, future efficacy trials should evaluate doses ranging from 24 to 72 mg/m2.

This study aimed to define a safe and biologically active dose of LAQ824 that could be taken forward into efficacy trials. The toxicity of the patient in the 100 mg/m2 cohort and subsequent toxicities seen in the 72 mg/m2 cohort in the expansion phase led us to believe that the recommended phase II dose is unlikely to be >72 mg/m2. This is similar to the predicted maximum tolerated dose from preclinical toxicology studies. Further dose escalation was stopped after the fatal event in the one patient at 100 mg/m2.

The most commonly observed toxicities of fatigue, anorexia, myelosuppression and nausea, vomiting, and diarrhea are common to HDAC inhibitors and seem to be a class effect (1821). The thrombocytopenia and neutropenia occurred by day 8 and resolved quickly, usually within 3 days and certainly by 7 days. Hypokalemia did not feature prominently with LAQ824.

Common electrocardiographic manifestations included reversible, asymptomatic, nonspecific ST-segment and T-wave changes as previously described with other HDAC inhibitor. Uncomplicated QTc prolongation was described during this study by investigator assessment and local cardiology review in two patients but was deemed to not amount to dose-limiting toxicity (>500 ms) by central cardiology review using Fridericia's correction (QTcF) after evaluating large numbers of electrocardiograms. These findings are in keeping with difficulties interpreting electrocardiographic data in patients treated with HDAC inhibitor because of drug-induced flattening of the ST-T waves. Nevertheless, central tendency analysis of mean changes in QTcF showed a dose-related increase in QTcF on day 3 of dosing that reached 14 ms at the 72 mg/m2 cohort. There are many implications from these observations, taking into account data from other HDAC inhibitor trials (21, 47). Mild QTc prolongation and nonspecific ST-T-wave changes have been observed in studies involving vorinostat, LBH589, depsipeptide, PXD101, and MGCD0103 (47). Our experience with numerous HDAC inhibitors in clinical trials shows that gastrointestinal toxicity of nausea, vomiting, and diarrhea, possibly with renal derangement, can cause electrolyte imbalance predisposing patients to cardiac rhythm changes (47). This can be difficult to separate from the direct cardiac effects of the HDAC inhibitor. With more stringent patient selection, with the rapid management of gastrointestinal toxicity, by ensuring high normal levels of potassium and magnesium before dosing, and by avoiding the concurrent administration of other QTc prolonging drugs, the incidence of such electrocardiographic changes may be minimized. Finally, the challenges of conducting appropriate real-time electrocardiographic monitoring in early clinical trials of novel anticancer drugs are shown in this trial, wherein differing QTc interpretations were acquired from the central cardiology reviewer and a local expert cardiologist who has a specific academic interest in drug-induced QTc prolongation. This indicates that interobserver variation can significantly affect QTc assessment.

Overall, our experience in this study and in trials of other i.v. administered hydroxamate HDAC inhibitor, including PXD-101 (belinostat), suggests that gastrointestinal toxicity is less common and less frequently dose limiting with i.v. administration compared with oral administration. This may be a significant advantage of i.v. administration of these agents, which can also result in higher drug concentrations that can potentially lead to an increased pharmacodynamic effect and improved antitumor activity. A possible disadvantage of i.v. administration and higher peak drug concentrations may, however, be an increased risk of other effects, particularly cardiac toxicities.

The pharmacodynamic analysis aspect of the study showed increased histone acetylation in a dose-dependent manner, starting from as low as 12 mg/m2. Certainly from 24 mg/m2 onwards, consistent rapid acetylation of histone H3 was observed in PBMC samples. In the paired samples of tumor tissue obtained before and after LAQ824 administration, comparable enhanced histone acetylation was observed. Histone hyperacetylation has been widely used as a mechanistic marker of HDAC inhibition, but this effect does not seem to be predictive of response (3235). Recently, an acetylation site in the middle domain of HSP90, which regulates its chaperone function, has been identified (36). LAQ824 has been shown to induce acetylation of HSP90 in cell lines and client protein depletion (37, 38). Depletion of CRAF and increased expression of HSP72, consistent with the inhibition of HSP90 chaperone function, were observed in more than a third of patients with assessable PBMC effects following LAQ824. These pharmacodynamic effects occurred at higher dose levels than those seen for histone acetylation and than those observed after direct HSP90 inhibition by the geldanamycins 17-N-allylamino-17-demethoxygeldanamycin and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (41). The inhibition of HSP90 by HDAC inhibitor is thought to be due to acetylation of the molecular chaperone resulting in a conformational change. However, HDAC inhibitors have also been shown to directly acetylate HSP72 (48). This interaction with the HSP90 pathway at multiple levels is a possible explanation for the inconsistent effects observed. The observation that HSP72 increases in tandem with acetylation in some patients without depletion of CRAF may indicate a stress response. The therapeutic implications of HSP90 inhibition by HDAC inhibitors remain to be determined. To support this, the prospective further evaluation of HSP90 inhibition in tumor biopsies or circulating tumor cells is now required in future HDAC inhibitor trials.

Although no responses were identified in this study, prolonged disease stabilization was observed in three patients lasting 14, 9, and 6 months. Antitumor activity with HDAC inhibitors in clinical trials has been mostly associated with lymphoma and hematologic malignancies with little activity described in epithelial tumors, although modest antitumor activity has been reported in castration-resistant prostate cancer (49). Nonetheless, because preclinical studies indicate that HDAC inhibitors can be synergistic with a number of cytotoxic agents, including fluoropyrimidines and platinums, as well as radiation therapy, further clinical evaluation of these combinations in solid tumors is warranted. Such clinical trials must consider how to best combine HDAC inhibitors with these agents. I.v. administered HDAC inhibitors using administration schedules such as the one used in this trial should be considered to potentially optimize tolerability and antitumor efficacy. However, a single agent approach for this class of drug may be most effective using a more protracted schedule, as recommended for the approved agent vorinostat. Finally, because LBH589 is closely related structurally to LAQ824 and is more potent with better bioavailability, the further development of this enhanced compound is now under way.

J.S. de Bono and S. Kaye have served as consultants on Novartis advisory boards. J.S. de Bono, S. Kaye, P. Workman, W. Aherne, R. Kristeleit, S. Pacey, and P. Fong were employed by the Institute of Cancer Research, which has a commercial interest in HDAC inhibitors under development by Chroma Therapeutics. W. Aherne, P. Workman, and J.S. de Bono have been consultants to Chroma Therapeutics. The authors received research funding from Novartis Therapeutics for these trials. P. Atadja and J. Scott are employed by Novartis.

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.

Note: J. S. de Bono and R. Kristeleit are joint first authors.

1
Kornberg RD. Chromatin structure: a repeating unit of histones and DNA.
Science
1974
;
184
:
868
–71.
2
Jenuwein T, Allis CD Translating the histone code.
Science
2001
;
293
:
1074
–9.
3
Strahl BD, Allis CD. The language of covalent histone modifications.
Nature
2000
;
403
:
41
–5.
4
Marmorstein R. Protein modules that manipulate histone tails for chromatin regulation.
Nat Rev Mol Cell Biol
2001
;
2
:
422
–32.
5
Gregoretti IV, Lee YM, Goodson HV. Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis.
J Mol Biol
2004
;
338
:
17
-31.
6
Voelter-Mahlknecht S, Ho AD, Mahlknecht U. Chromosomal organization and localization of the novel class IV human histone deacetylase 11 gene.
Int J Mol Med
2005
;
1
:
589
–98.
7
Bertos NR, Wang AH, Yang XJ. Class II histone deacetylases: structure, function and regulation.
Biochem Cell Biol
2001
;
79
:
243
–52.
8
Cress WD, Seto E. Histone deacetylases, transcriptional control and cancer.
J Cell Phys
2000
;
184
:
1
–16.
9
Wade PA. Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin.
Hum Mol Genet
2001
;
10
:
693
–8.
10
Glozak MA, Seto E. Histone deacetylases and cancer.
Oncogene
2007
;
26
:
5420
–32.
11
Halkidou K, Gaughan L, Cook S, et al. Up-regulation and nuclear recruitment of HDAC1 in hormone refractory prostate cancer.
Prostate
2004
;
59
:
177
–89.
12
Zhu P, Martin E, Mengwasser J, et al. Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis.
Cancer Cell
2004
;
5
:
455
–63.
13
Zhang Z, Yamashita H, Toyama T, et al. HDAC6 expression is correlated with better survival in breast cancer.
Clin Cancer Res
2004
;
10
:
6962
–8.
14
Osada H, Tatematsu Y, Saito H, et al. Reduced expression of class II histone deacetylase genes is associated with poor prognosis in lung cancer patients.
Int J Cancer
2004
;
112
:
26
–32.
15
Dhordain P, Lin RJ, Quief S, et al. The LAZ3 (BCL6) oncoprotein recruits a SMRT/mSin3A/histone deacetylase containing complex to mediate transcriptional repression.
Nucleic Acids Res
1998
;
26
:
4645
–51.
16
Pasqualucci L, Bereschenko O, Niu H. Molecular pathogenesis of non-Hodgkin's lymphoma: the role of Bcl-6.
Leuk Lymphoma
2003
;
44
Suppl 3:
S5
–12.
17
Magnaghi-Jaulin L, Groisman R, Naguibneva I, et al. Retinoblastoma protein represses transcription by recruiting a histone deacetylase.
Nature
1998
;
391
:
601
–5.
18
Kristeleit R, Stimson L, Workman P, Aherne W. Histone modification enzymes: novel targets for cancer drugs.
Expert Opin Emerg Drugs
2004
;
9
:
135
–54.
19
Arts J, de Schepper S, Van Emelen K. Histone deacetylase inhibitors: from chromatin remodeling to experimental cancer therapeutics.
Curr Med Chem
2004
;
10
:
2343
–50.
20
Marchion D, Munster P. Development of histone deacetylase inhibitors for cancer treatment.
Expert Rev Anticancer Ther
2007
;
7
:
583
–98.
21
Kristeleit R, Fong P, Aherne GW, de Bono J. Histone deacetylase inhibitors: emerging anticancer therapeutic agents?
Clin Lung Cancer
2005
;
7
Suppl 1:
S19
–30.
22
Duvic M, Vu J. Vorinostat: a new oral histone deacetylase inhibitor approved for cutaneous T-cell lymphoma.
Expert Opin Investig Drugs
2007
;
16
:
1111
–20.
23
Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase inhibitors.
Nat Rev Drug Discov
2006
;
5
:
769
–84.
24
Peart MJ, Tainton KM, Ruefli AA, et al. Novel mechanisms of apoptosis induced by histone deacetylase inhibitors.
Cancer Res
2003
;
63
:
4460
–71.
25
Burgess A, Ruefli A, Beamish H, et al. Histone deacetylase inhibitors specifically kill non-proliferating tumor cells.
Oncogene
2004
;
23
:
6693
–701.
26
Huang L, Sowa Y, Sakai T, Pardee AB. Activation of the p21WAF1/CIP1 promoter independent of p53 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through the Sp1 sites.
Oncogene
2000
;
19
:
5712
–9.
27
Gui CY, Ngo L, Xu WS, Richon VM, Marks PA. Histone deacetylase (HDAC) inhibitor activation of p21WAF1 involves changes in promoter-associated proteins, including HDAC1.
Proc Natl Acad Sci U S A
2004
;
101
:
1241
–6.
28
Glaser KB, Staver MJ, Waring JF, Stender J, Ulrihch RG, Davidsen SK. Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines.
Mol Cancer Ther
2003
;
2
:
151
–63.
29
Mitsiades CS, Mitsiades NS, McMullan CJ, et al. Transcriptional signature of histone deacetylase inhibition in multiple myeloma: biological and clinical implications.
Proc Natl Acad Sci U S A
2004
;
101
:
540
–5.
30
Chambers AE, Banerjee S, Chaplin T, et al. Histone acetylation-mediated regulation of genes in leukaemic cells.
Eur J Cancer
2003
;
39
:
1165
–75.
31
Atadja P, Gao L, Kwon P, et al. Selective growth inhibition of tumor cells by a novel histone deacetylase inhibitor, NVP-LAQ824.
Cancer Res
2004
;
64
:
689
–95.
32
Plumb JA, Finn PW, Williams RJ, et al. Pharmacodynamic response and inhibition of growth of human tumor xenografts by the novel histone deacetylase inhibitor PXD101.
Mol Cancer Ther
2003
;
2
:
721
–8.
33
Atadja P, Hsu M, Kwon P, Trogani N, Bhalla K, Remiszewski S. Molecular and cellular basis for the anti-proliferative effects of the HDAC inhibitor LAQ824.
Novartis Found Symp
2004
;
259
:
249
–66; discussion 266–8, 285–8.
34
Weisberg E, Catley L, Kujawa J, et al. Histone deacetylase inhibitor NVP-LAQ824 has significant activity against myeloid leukemia cells in vitro and in vivo.
Leukemia
2004
;
18
:
1951
–63.
35
Glaser KB, Li J, Pease LJ, et al. Differential protein acetylation induced by novel histone deacetylase inhibitors.
Biochem Biophys Res Commun
2004
;
325
:
683
–90.
36
Scroggins BT, Robzyk K, Wang D, et al. An acetylation site in the middle domain of HSP90 regulates chaperone function.
Mol Cell
2007
;
12
:
151
–9.
37
Bali P, Pranpat M, Bradner J, et al. Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors.
J Biol Chem
2005
;
280
:
26729
–34.
38
Bali P, Pranpat M, Swaby R, et al. Activity of suberoylanilide hydroxamic acid against human breast cancer cells with amplification of her-2.
Clin Cancer Res
2005
;
11
:
6382
–9.
39
Yu X, Guo ZS, Marcu MG, et al. Modulation of p53, erbB1, erbB2 and raf-1 expression in lung cancer cells by depsipeptide FR901228.
J Natl Cancer Inst
2002
;
94
:
504
–13.
40
Maloney A, Workman P. HSP90 as a new therapeutic target for cancer therapy: the story unfolds.
Expert Opin Biol Ther
2002
;
2
:
3
–24.
41
Banerji U, O'Donnell A, Scurr M, et al. Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino, 17-demethoxygeldanamycin in patients with advanced malignancies.
J Clin Oncol
2005
;
23
:
4152
–61.
42
Catley L, Weisberg E, Tai YT, et al. NVP-LAQ824 is a potent novel histone deacetylase inhibitor with significant activity against multiple myeloma.
Blood
2003
;
102
:
2615
–22.
43
Leyton J, Alao JP, Da Costa M, et al. In vivo biological activity of the histone deacetylase inhibitor LAQ824 is detectable with 3′-deoxy-3′-[18F] fluorothymidine positron emission tomography.
Cancer Res
2006
;
66
:
7621
–9.
44
O'Quigley J, Pepe M, Fisher L. Continual reassessment method: a practical design for phase I clinical trials in cancer.
Biometrics
1990
;
46
:
33
–48.
45
Faries D. Practical modifications of the continual reassessment method for phase I clinical trials.
J Biopharm Stat
1994
;
4
:
147
–64.
46
Goodman SN, Zahurak ML, Piantadosi S. Some practical improvements in the continual reassessment method for phase I studies.
Stat Med
1995
;
14
:
1149
–61.
47
Molife R, Fong P, Scurr M, Judson I, Kaye S, de Bono J. HDAC inhibitors and cardiac safety.
Clin Cancer Res
2007
;
13
:
1068
–9.
48
Wang Y, Wang SY, Ahang XH, et al. FK228 inhibits HSP90 chaperone function in K562 cells via hyperacetylation of Hsp70.
Biochim Biophys Res Commun
2007
;
356
:
998
–1003.
49
Molife R, Patterson S, Riggs C, et al. Phase II study of FK228 in patients with metastatic hormone refractory prostate cancer (HRPC). Proc of Prostate ASCO 2006;A217.