Purpose: Sodium phenylbutyrate (PB) demonstrates potent differentiating capacity in multiple hematopoietic and solid tumor cell lines. We conducted a Phase I and pharmacokinetic study of PB by continuous infusion to characterize the maximum tolerated dose, toxicities, pharmacokinetics, and antitumor effects in patients with refractory solid tumors.

Patients and Methods: Patients were treated with a 120-h PB infusion every 21 days. The dose was escalated from 150 to 515 mg/kg/day. Pharmacokinetics were performed during and after the first infusion period using a validated high-performance liquid chromatographic assay and single compartmental pharmacokinetic model for PB and its principal metabolite, phenylacetate.

Results: A total of 24 patients were enrolled on study, with hormone refractory prostate cancer being the predominant tumor type. All patients were evaluable for toxicity and response. A total of 89 cycles were administered. The dose-limiting toxicity (DLT) was neuro-cortical, exemplified by excessive somnolence and confusion and accompanied by clinically significant hypokalemia, hyponatremia, and hyperuricemia. One patient at 515 mg/kg/day and another at 345 mg/kg/day experienced this DLT. Toxicity resolved ≤12 h of discontinuing the infusion. Other toxicities were mild, including fatigue and nausea. The maximum tolerated dose was 410 mg/kg/day for 5 days. Pharmacokinetics demonstrated that plasma clearance of PB increased in a continuous fashion beginning 24 h into the infusion. In individuals whose Vmax for drug elimination was less than their drug-dosing rate, the active metabolite phenylacetate accumulated progressively. Plasma PB concentrations (at 410 mg/kg/day) remained above the targeted therapeutic threshold of 500 μmol/liter required for in vitro activity.

Conclusion: The DLT in this Phase I study for infusional PB given for 5 days every 21 days is neuro-cortical in nature. The recommended Phase II dose is 410 mg/kg/day for 120 h.

Differentiation therapy for epithelial malignancies may potentially alter tumor growth and progression, slow or inhibit metastases, inhibit angiogenesis, and/or effect response to other forms of therapy (1, 2, 3, 4, 5). Nonretinoid agents such as hexamethylenebisacetamide and sodium butyrate have been evaluated clinically, but sustained systemic levels required for activity have not been achieved because of toxicity and/or lack of a suitable formulation (6, 7, 8, 9). Sodium PB,3 an aromatic fatty acid, is a lead candidate as a cancer differentiating agent and a histone deacetylase inhibitor (10, 11, 12, 13). Sodium PB is the precursor to PA, and both compounds are potent differentiating agents in vitro(10, 11, 12, 13, 14, 15, 16, 17, 18). PA is formed by β-oxidation of PB (19). PB is Food and Drug Administration approved for children and adults with hyperammonemia associated with urea cycle disorders (recommended dose of 13 g/m2/day) and has been used for adult patients with hyperammonemia secondary to high-dose chemotherapy for leukemia and transplant therapies (19, 20, 21, 22). PB is also under investigation for cystic fibrosis and adrenal leukodystrophy.

Preclinical studies have looked at the ability of these drugs to alter gene expression and promote differentiation (23, 24, 25). In various tumor model systems, PA/PB can arrest cells in G1-G0 with induction of p21WAF1 and other cdk-2-associated cell cycle checkpoint proteins, alter expression of invasion products such as urokinase-plasminogen activator (UPA), induce apoptosis, inhibit telomerase, and increase MHC class I expression at concentrations of 500-2500 μmol/liter PB (23, 24, 25, 26, 27, 28, 29, 30, 31). Delay in tumor progression has been noticed in prostate and malignant glioma models (12, 16, 17). It should be noted that tumor markers, such as PSA, might not be the most accurate measurements of progressive disease in patients treated with PB, as a rise in tumor markers may signal cell differentiation rather than disease progression. Proposed mechanisms of action to elicit these effects include modification of lipid metabolism, activation of the peroxisome proliferator activator receptor, maintenance of histone acetylation through inhibition of histone deacetylase, inhibition of protein isoprenylation, and/or glutamine starvation (10, 27, 28, 29, 30, 31). There may be other cellular and molecular effects of PB that contribute to its antitumor activity.

In addition to all of the potential cellular and molecular effects, PB is minimally toxic in children with urea cycle disorders and hemoglobinopathies (31). Serum concentrations of PB have been reported in the range (500–2500 μmol/liter) required for in vitro effects of growth arrest and differentiation. Preclinical data from our laboratory and others suggest PB may be more potent and produce more molecular effects than PA (12). PB has activity similar to butyrate (induction of apoptosis and histone acetylation) that PA may not possess. For these reasons, Phase I studies of PB were initiated.

The purpose of this Phase I trial was to determine the MTD and toxicity profile of PB administered i.v. as an 120-h infusion every 21 days and to describe the pharmacokinetic behavior of this novel agent in patients with refractory solid tumors.

Patient Eligibility

The protocol was approved by the Joint Committee on Clinical Investigation of the Johns Hopkins University School of Medicine. Male and female patients ≥18 years of age with a histologically or cytologically documented diagnosis of cancer (solid tumor or lymphoma) refractory to conventional therapeutic modalities were eligible. All patients enrolled onto the study had progressing, evaluable, or measurable disease. Patients were required to have an Eastern Cooperative Oncology Group performance status of 0, 1, or 2 and a life expectancy of ≥3 months. Patients were not to have had major surgery, radiotherapy, or chemotherapy in ≤28 days and were to be fully recovered from the toxicity of prior therapy. Patients with prostate cancer were allowed to continue on a leutenizing hormone releasing hormone (LHRH) agonist. A 4–6-week period of antiandrogen (flutamide and bicalutamide) withdrawal and evidence of PSA progression was required of all men on an antiandrogen before entry. Patients that previously received suramin were eligible if their suramin level was <50 μg/ml. Adequate organ function was required at study entry: WBC > 2,000 or absolute neutrophil count (ANC) > 1,500/mm3, platelets > 100,000/mm3, and hemoglobin > 9 g/dl, a serum creatinine < 2 mg/dl, total bilirubin < 1.5, and aspartate aminotransferase/alanine aminotransferase < 1.5 × the upper limit of normal, left ventricular ejection fraction (LVEF) by MUGA or echocardiogram > 40% and no history of congestive heart failure (CHF) requiring hospitalization, or uncontrolled hypertension (diastolic blood pressure (BP) >110 mmHg) and a forced expiratory volume (FEV)1 > 1.5 liter/min. Patients with obstructive uropathy were eligible if obstruction was relieved by nephrostomy or other appropriate intervention. Patients were required to give informed consent with understanding of the investigational nature of the treatment and its potential risks. Patients were ineligible if they had an active infectious process, including HIV or viral hepatitis; a malignant brain tumor, or known central nervous system metastases even if treated previously and not clinically active; an active seizure disorder; or a baseline dementia with mini-mental score < 23 (32). Pregnant or nursing females were ineligible. Patients with requirement for steroids for any reason, including previous suramin therapy, were excluded because of concerns about potential interference with the mechanism of action of PB.

Pretreatment Evaluation

Patients were assessed before receiving the first dose of PB with a complete history, including prescription and nonprescription drug history, physical exam with a thorough neurological examination, including a mini-mental status examination (32), and performance status assessment. Documentation of evaluable or measurable disease was performed ≤4 weeks of initiation of therapy. Pretreatment laboratory evaluations included a complete blood count with platelet and differential, chemistry panel (including electrolytes, creatinine, bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, uric acid, total protein, albumin, calcium, phosphorous, and magnesium), partial thromboplastin time and prothrombin time, urinalysis, human chorionic gonadotropin (if woman of childbearing age), EKG, MUGA or echocardiogram for LVEF, spirometry with lung volumes, chest roentgenogram (or chest computed tomography if part of the tumor assessment), appropriate tumor marker (i.e., PSA, CA-125, CA15-3, and CA19-9), and a suramin level, if the patient had been treated previously with suramin.

Evaluation During Therapy

Patients were monitored with laboratory and clinical evaluations weekly by an investigator or research nurse while on study. The history and physical was performed at the beginning of each 5-day infusion. Patients were also seen as needed for additional toxicity assessment. Toxicity was graded using the NCI common toxicity criteria, version 2.0. Tumor markers were assessed monthly. Evaluation of measurable and evaluable disease was performed after each two cycles of therapy. Documentation of performance status was required with all visits. Complete response was defined as the disappearance of all clinical evidence of active tumor and symptoms for ≥1 month. Partial response required a >50% decrease in the sum of the products of the perpendicular tumor diameters of all measurable lesions for ≥4 weeks without simultaneous increase in the size of any lesion or appearance of any new lesion. Progressive disease was defined as a >25% increase in the size of any measurable lesion or the appearance of any new lesion. Stable disease represented a response less than a partial response or growth less than progressive disease. Changes in tumor markers did not factor in to the definition of response, e.g., a rising PSA did not define progression. Changes in PSA are merely descriptive.

Treatment Plan

The Investigational Drug Branch of the NCI received phenylbutyric acid, sodium salt (National Service Center number 657802; EL532) from Elan Pharmaceutical Research Corp., Gainesville, GA. PB was formulated in 50-ml glass vials containing 40% viscous solution of sodium PB in sterile water (400 mg/ml). The sodium content of one vial was 4.9 grams of sodium. PB was provided for the patients on this study by the Division of Cancer Treatment, NCI.

Patients were admitted to the General Clinical Research Center of the Johns Hopkins Hospital for each course of therapy. PB was administered as a continuous i.v. infusion (centrally or peripherally) for 120 h every 21 days. A course was defined as the 21-day cycle, consisting of the 5-day infusion and 16 days of rest. The infusional starting dose was 150 mg/kg/day for 5 days, a dose below that shown previously to be safe in children with urea cycle disorders. Subsequent dose levels were 225, 285, 345, 410, and 515 mg/kg/day for 5 days. Doses could be escalated within individual patients if 3 patients at the next higher dose level had received therapy for >1 month without DLT. Patients remained on study until evidence of progressive disease, severe DLT, or patient request to discontinue therapy. DLT was defined using the NCI common toxicity criteria, as neurotoxicity ≥ grade 2 or any other toxicity ≥ grade 3. A registered nutritionist met with every patient to discuss salt intake and to provide recommendations of lowering sodium intake while receiving PB.

Cohorts of 4 patients were initially enrolled at each dose level to fully evaluate the central nervous system toxicity and to gather more pharmacokinetic data. If no patients in a cohort experienced a DLT during the initial 3-week observation period, the dose was escalated, and the next cohort was commenced. If 1 of 4 patients experienced a DLT, 2 additional patients were added at that dose level. If 2 patients experienced a DLT at any dose level, no additional patients were enrolled at that dose level, and an additional 2 patients were entered at the next lower dose level. Dose escalation could occur once all 4 patients in a given cohort were treated, and at least 2 patients had completed one course (21 days) with the 3rd and 4th patients ≥1 week into therapy without meeting criteria for the MTD. Although the study was not designed to escalate doses within patients, intrapatient dose escalation was allowed. A patient who received at least two courses of treatment without toxicity (except alopecia) greater than grade II at a given dose level could have their subsequent treatments at the next higher dose level. Before such an escalation could occur, 2 PB-naïve patients must have completed at least one course (22 days) at the next higher dose level without any DLT. The MTD was defined as that dose level at which consistent, definable toxicity occurs that is reversible and does not subject patients to excessive risk or discomfort.

Patients who received ≥1 day of PB were assessable for toxicity, but only those patients who received one full 21-day course of PB were assessable for response. Patients for whom a drug was held because of DLT still had to complete the full 21-day observation period. Any patient who experienced DLT and recovered fully from that toxicity could resume PB at the next lower dose level; however, for MTD determination, patients were included only at their initial, higher dose level. Patients who did not recover fully from DLT ≤14 days or had a DLT at a subsequent lower dose were taken off study. The study was defined as complete for a given patient if the patient did not recover fully from a DLT ≤14 days or had a DLT at a subsequent lower dose. Patients were also removed from the study if they had evidence of progressive disease or if the patient requested to be taken off the study.

Recognizing that the recommended Phase II dose is often one dose level below the MTD, the recommended Phase II dose was to be dictated by toxicities, patient tolerability, and plasma levels achieved. An objective of this study was to achieve a range of plasma levels that may be clinically relevant as dictated by preclinical models.

Pharmacokinetic Study

Pharmacokinetic Sampling.

Drug disposition studies were performed during the first course of treatment. Blood specimens were collected before the start of the infusion, at 0.5, 1, 2, 4, 6, 12, 24, 36, 48, 60, and 96 h during the infusion and just before completion of the infusion (120 h). Postinfusion specimens were obtained at 0.5, 1, 2, 4, 8, and 24 h as measured from the end of the infusion. Urine specimens (every 24 h) were collected between 0 and 24 h and between 96 and 120 h as measured from the start of the infusion.

Analytic Assay.

Plasma PB, PA, and PG concentrations were determined in all blood specimens by reverse-phase high-performance liquid chromatography assay. These methods have not been published previously. Plasma (200 μl) containing compound was transferred to a microcentrifuge tube, followed by the addition of 50 μl of 10% perchloric acid (Sigma Chemical Co., St. Louis, MO) for protein extraction. The samples were vortexed and centrifuged (International Equipment Co., Needham Heights, MA) at 4°C at 8500 rpm for 10 min. After centrifugation, 150 μl of supernatant was added to 5 μl of super-saturated potassium bicarbonate solution for neutralization and centrifuged again at 4°C at 8500 rpm for 10 min. All of the supernatant was transferred to an autosampler vial for analysis. The chromatographic apparatus consisted of a Hewlett Packard Series II 1090 Liquid Chromatographic (Hewlett Packard Corp., Palo Alto, CA) with an autosampler compartment, solvent delivery system, and a diode-array UV absorbance detector with a resolution of 2 nm. The absorbance wavelength was 208 nm (bandwidth, 10 nm) with a reference wavelength of 400 nm (bandwidth, 80 nm). Mobile phase A consisted of 100% deionized water (Milli-QUV Plus; Millipore Corp., Bedford, MA) with 0.005 m phosphoric acid (Sigma Chemical Co.) buffer. Mobile phase B consisted of 100% high-performance liquid chromatography grade acetonitrile (J. T. Baker, Phillipsburg, NJ) with 0.005 m phosphoric acid buffer, and mobile phase C was identical to mobile phase A. All mobile phases were run at a flow rate of 1 ml/min for a run time of 45 min at the gradient profile listed on Table 1.

A Waters Nova-Pak C18 guard column was placed in line before the analytical column. The samples were injected onto a reverse-phase (Waters Bond-Pak C18, 3.9 × 300 mm; Millipore Corp) column, which was maintained at 60°C. PA, PB, and PG had retention times of ∼18.2, 31.4, and 10.2 min, respectively. Chromatographic peak area was used for quantitation by linear regression analysis. The lower limit of detection for the assay was 5 μg/ml. Quality control samples were assayed at concentrations of 15, 35, and 85 μg/ml, and the inter-day coefficients of variation were 11% for PA, <7% for PB, and <11% for PG. One set of quality control samples were placed before the calibration standards, before the patient samples, and then immediately after the patient samples were assayed. Samples from 1 patient were assayed with each analytical run. Each analytical run was equivalent to ∼26 h; autosampler stability for 35 h demonstrated that metabolites are stable for this period of time with a <10% change (loss or gain) in concentration. The lower limit of quantitation was 5 μg/ml for PA and PG and 10 μg/ml for PB. PG concentrations were determined in the urine specimens, and the amount of PG excreted during the urine collection period was calculated as concentration × urine volume. The accuracy of the assay was 93–98% with precision of the assay to ≤1–4%.

Pharmacokinetic Methods.

The plasma PB disposition curves were analyzed in two ways: (a) individual subject disposition curves were fit by nonlinear regression (PCNONLIN; Scientific Consulting, Apex, NC) using a pharmacokinetic model consisting of a single distribution compartment and a first-order elimination process that increases in magnitude in an exponential fashion beginning 24 h after the institution of drug therapy, i.e., clearance rate(t > 24 h) = clearance rate(t = 0)×exp[kinc×(t − 24 h)], where kinc is the rate constant of the exponential process; and (b) the data were analyzed using a pharmacokinetic model consisting of a single distribution compartment and a Michaelis-Menten-type saturable elimination process in which Vmax increases in magnitude in an exponential fashion beginning 24 h after the institution of drug therapy. For this model, the disposition curves were analyzed by nonlinear mixed effect modeling (P-Pharm; MicroPharm International, Champs-sur-Marne, France). With this software, population parameter values were estimated using an EM-type iterative algorithm, and individual subject parameter values were estimated by MAP Bayesian fitting.

The plasma PA and PG disposition curves were also analyzed by nonlinear mixed effect modeling. In the pharmacokinetic models used, PA was modeled as arising from PB via the Michaelis-Menten-type saturable elimination process for PB (Fig. 1). PG was modeled as arising from PA via a Michaelis-Menten-type saturable process. For both species, it was assumed that biotransformation was complete. PA was modeled as distributing in a single compartment with elimination solely via the process that gives rise to PG. The Vmax of this elimination process was treated as increasing in an exponential fashion beginning 24 h after the institution of drug therapy. PG was modeled as distributing in a single compartment with first-order elimination. In one model, the elimination was treated as time invariant, and in another, it was modeled as increasing in an exponential fashion beginning 24 h after the institution of drug therapy.

Urine clearance rates of PG were calculated as the amount of PG excreted in the urine during the period of urine collection divided by the area under the PG plasma disposition curve during the same period of time. The areas were determined using a linear trapezoidal rule.

Patients (24) were enrolled at six dose levels. Patient characteristics are listed in Table 2. A total of 89 full cycles of treatment were administered. All patients were evaluable for toxicity and response assessment. The majority of patients were men with advanced hormone refractory prostate cancer. Over half of the patients had received previous chemotherapy. One patient with renal cell cancer had had no previous therapy, and 4 men with prostate cancer received PB as their first therapy in the hormone refractory state. The median number of courses per patient was four (range, one to eight) for an average time on trial of 84 days.

Patients (21) were withdrawn from therapy secondary to progressive disease documented by radiograph or symptoms. One patient refused additional therapy after six cycles for personal reasons despite continued stable disease. Another patient discontinued therapy secondary to toxicity during his first cycle of therapy at 410 mg/kg/day. Lastly, 1 patient had toxicity at two different dose levels despite dose reduction and met criteria for withdrawal secondary to toxicity. This patient had a 10% decline in his PSA and subjective improvement in bone pain over the two cycles that he received therapy.

Three patients had their dose escalated during the course of therapy. Two of these patients remained on the study for a total of eight cycles. In fact, 1 of these patients had his dose increased twice from 150 mg/kg/day (dose level 1) to 285 mg/kg/day (dose level 3). Two patients had their dose reduced secondary to toxicity at their starting dose during cycle 1. The starting doses for these patients were 345 and 515 mg/kg/day. No patient at 410 mg/kg/day had their dose reduced or escalated and was determined to be the recommended Phase II dose. Table 3 describes all grades 3 and 4 toxicities during the study period.

Neuro-cortical Toxicity.

DLT was neuro-cortical in nature. One patient on 515 mg/kg/day experienced grade 3 neuro-cortical toxicity ≤48 h of the start of drug infusion. The toxicity was exemplified by excessive somnolence and confusion and was accompanied by metabolic changes consisting of grade 4 hypokalemia (2.1 mmol/liter), grade 3 hyponatremia (128 mmol/liter), grade 2 hypocalcemia (7.9 mg/dl), and grade 4 hyperuricemia (15.2 mg/dl). The metabolic nadirs occurred 4 days after initiation of infusion. The patient also had grade 3 nausea before the decline in mental status. Full recovery and return to baseline clinical state occurred ≤10–12 h after discontinuation of the drug at approximately the same time as the electrolyte abnormalities returned to normal values after aggressive repletion. The patient received a second cycle at the next lower dose and experienced a similar toxicity syndrome on rechallenge. An additional patient developed grade 3 neuro-cortical toxicity at the 345 mg/kg/day dose. This was not accompanied by electrolyte abnormalities and occurred at 96 h into the infusion. The symptoms resolved 10–12 h after discontinuing the drug. A 3rd patient developed grade 2 neuro-cortical toxicity with only grade 1 hyponatremia and hypocalcemia at 410 mg/kg/day. This patient was not rechallenged at a lower dose because he developed obstructive urinary symptoms and was taken off study for progression of disease. Of note, plasma ammonia levels in the grade 3 toxicity patients remained in the normal range.

Patients with nausea had more sedating effects from antiemetics, such as the phenothiazines (prochloperazine). This observation led to the use of nonsedating antiemetics such as the serotonin 5-HT3 receptor antagonists, ondansetron and granisetron. Patients had less sedation and minimal emesis with this change in antiemetic.

Hematological Toxicity.

No dose-limiting hematological toxicity was noted. Minor decreases in hematocrit were noted over the course of therapy. No platelet toxicities were noted in any patient. One patient had a transient grade 3 neutropenia (absolute neutrophil count of 600) noted at dose level 1 that resolved in 2 days. No other declines in total white cell count or in subpopulations were noted.

Nonhematological Toxicity.

Patients experienced mild grade 2 nausea at the higher dose levels, in particular, the 410 mg/kg/day dose. Fatigue was dose related as well. Grade 2 fatigue was noted in 1 patient at 345 mg/kg/day and 2 patients at 410 mg/kg/day. Grade 3 fatigue was noted in 2 of 7 patients receiving 410 mg/kg/day infusions. This degree of fatigue was not felt to be drug related but rather attributable to tumor progression. In fact, the 2 patients with grade 3 fatigue did not receive a rechallenge with PB as they were taken off study for progressive disease. Of note, 7 (38%) of the patients with bony metastatic PCA experienced a flare in bone pain that started on discontinuation of the infusion and persisted for 48 h. Only the 2 patients with grade 3 neuro-cortical toxicity developed the characteristic odor of PA. The ability to detect the odor also resolved promptly with discontinuation of the study drug.

Pharmacokinetics and Pharmacodynamics.

Of 24 patients enrolled in the study, 1 patient had incomplete blood sampling for pharmacokinetic data analysis, and 2 patients did not have adequate plasma sample volumes to allow determination of PA and PG concentrations. These 3 patients were excluded from the pharmacokinetic data analysis.

In the majority of the patients, the plasma PB disposition curves were characterized by a rise to an apparent plateau achieved by 4–6 h into the infusion. However, a number of the subjects had curves that showed a substantial decline in PB concentrations starting around 24 h into the infusion and continuing throughout the remainder of the infusion. This pattern was seen in 0 of 4 patients 150 mg/kg/day, 0 of 4 patients at 225 mg/kg/day, 1 of 4 patients at 285 mg/kg/day, 3 of 4 patients at 345 mg/kg/day, 5 of 7 patients at 410 mg/kg/day, and 0 of 1 patient at 515 mg/kg/day. This pattern suggested that the plasma clearance of PB increased in a continuous fashion beginning 24 h into the infusion. Fig. 2 demonstrates the PB disposition pattern for 2 patients, 1 at 225 mg/kg/day and the other at 515 mg/kg/day. This feature was incorporated in the pharmacokinetic models used to describe the disposition curves.

Individual subject PB disposition curve analysis using a pharmacokinetic model with first-order elimination showed an inverse relationship between clearance rate and dose, suggesting that a model with saturable elimination would be more appropriate. In the model that was used in the population pharmacokinetic analysis of PB disposition, elimination was modeled as being Michaelis-Menten in character. The population pharmacokinetic estimates for PB disposition are listed in Table 4. The mean kinc value of 0.0029/h equates to a 1.32-fold increase in Vmax by 120 h into the infusion.

The population pharmacokinetic estimates for PA and PG disposition are listed in Table 4. The rate of increase in Vmax for PA elimination is less than that for PB; the mean kinc value of 0.0009/h equates to a 1.09-fold increase by 120 h into the infusion. Using the estimates of mean and SD to calculate the population distribution of Vmax for PA, 92% of the population would be expected to have Vmax values less than the highest dosing rate in this study, 91.35 μmol/h/kg (410 mg/day/kg); 49% would be expected to have Vmax values less than a dosing rate of 76.87 μmol/h/kg (345 mg/day/kg); and 9% would be expected to have Vmax values less than a dosing rate of 63.5 μmol/h/kg (285 mg/day/kg). In any individual whose Vmax is less than his or her drug-dosing rate, PA can be expected to accumulate progressively. Such a zero-order pattern of metabolite accumulation was seen in 1 of 4 subjects receiving the drug at a dosing rate of 76.87 μmol/h/kg and in 4 of 6 subjects receiving the drug at a dosing rate of 91.35 μmol/h/kg.

PG is eliminated exclusively in the urine so direct urine drug clearance measurements should equal the systemic clearance rate for the metabolite. Two 24-h urine collections were performed during the infusion, one during the first 24 h of the infusion and one during the final 24 h. Excluding two urine collections in which the calculated urine clearance rates were unphysiologically large, the mean urine clearance rate for the 0–24-h collection period is 116 ml/min (SD, 62 ml/min), and for the 96–120-h collection period, it is 136 ml/min (SD, 97 ml/min). The coefficient of correlation for paired values is 0.84. A paired t test indicated that the increase in the mean value of the urine clearance rate between the first and second urine collections is not significant (P = 0.10, two-tailed test). Previous studies have shown that PB is almost completely metabolized to PA via β-oxidation and that PA undergoes a ∼99% conversion to PG. The urine concentrations of PB and PA in these studies were negligible and were not measured in this trial, as the predominant form excreted in the urine is PG.

The estimated mean systemic clearance rate of PG, expressed in unscaled terms, is 147 ml/min (SD, 85 ml/min). The mean value is somewhat larger than the urine clearance rates measured during both urine collection periods but not strikingly so, and the individual subject systemic clearance rates are highly correlated with their urine clearance rates; the correlation coefficient is 0.84 for both urine collection periods.

Fig. 3 demonstrates cumulative disposition curves for all evaluable patients on dose levels 2–5. The highest peak PB concentrations were noted at the 410 mg/kg/day dose, with all but 1 patient maintaining a plasma concentration >500 μmol/liter throughout the duration of the infusion. This concentration is a therapeutic threshold required for in vitro activity. PA concentrations across the dose levels tested can reach ≥1000 μmol/liter throughout the infusion.

Response Data.

No complete responses were noted. Two patients remained on therapy with clinically stable disease for 168 days (dose level 1 and dose level 4; Table 5). Three other patients remained on therapy for a total of six cycles. Of these 3 patients, 1 discontinued therapy at his own request in the setting of clinically stable disease. Another patient in this group had a persistent resolution of para-aortic adenopathy noted after the second cycle of therapy but had progression on bone scan with the presence of new bony lesions after six cycles of therapy (126 days). Average time on therapy for the entire cohort was 84 days.

Many of the PCA patients had relief from pain during the infusion, which lasted through the off-therapy period. Declines in opiod use and improvement in pain ratings were noted but not fully characterized or consistently captured prospectively as this data were not an intended end point of this study. One patient had successful removal of a chronic indwelling urinary catheter after two cycles of PB. He remained on study for a total of eight cycles, despite a rising PSA.

Tumor Marker and Cytokine Response.

Preclinically, it was suggested that PA and PB can increase PSA production/secretion of the LNCaP cell line in vitro(33). Eighteen of the 19 patients with prostate cancer had complete profiles of PSA over the course of therapy. Nearly 90% of the patients with PCA had a rise in their PSA (range, 15–114%) during the 5-day infusion. Three of 18 patients had stabilization (no increase or decline of <10%) of their PSA while on study. Four patients had their PSA rise <50% over the course of therapy. The remaining 11 had persistently rising PSAs during the treatment period. In four cases, the PSA dramatically rose (50–93%) in the 1-month period after the discontinuation of therapy. The significance of a rising PSA is uncertain and may point to the differentiating properties of PB or progressive disease. Of note, all 4 men with non-PCA diagnoses had no change in their PSA while on therapy.

Differentiation as a therapeutic approach in advanced malignancies has undergone a resurgence of interest in clinical development, in part attributable to the success of all-trans-Retinoic Acid in acute promyelocytic leukemia (34, 35). Solid tumor oncology continues to explore the role of differentiation agents as secondary preventive agents and as adjuncts to interferon in cervical carcinoma, squamous cell carcinoma of the skin, and renal cell carcinoma (36, 37). The clinical development of nonretinoid differentiating agents has also spurred additional interest in differentiation therapy (38, 39).

This study of sodium PB was based on preclinical laboratory studies demonstrating cell growth arrest and differentiation in multiple solid tumor and hematopoietic cell lines and animal models (10, 11, 12, 13, 14, 15, 16, 17). The molecular effects of PB are noted at concentrations of >500 μmol/liter as a single agent and at 100 μmol/liter when added in combination with retinoids (25). Pharmacokinetic data and plasma concentrations obtained from clinical studies of PB in children with urea cycle disorders had shown previously that these levels were readily achievable (22). This Phase 1 clinical trial has demonstrated the safety of PB in patients with solid tumors. A similar trial of PB in patients with acute myelogenous leukemia and myelodysplasia at our institution has successfully administered the infusion in the outpatient setting without much difficulty.4

The dose limiting neuro-cortical toxicity of excessive somnolence and confusion occurred suddenly and was noticed readily by family and staff. The metabolic changes of hypokalemia, hyponatremia, hypocalcemia, and hyperuricemia reversed with supportive measures on discontinuation of the study drug. The neuro-cortical symptoms resolved promptly, and the patients returned to their baseline mental status before discharge. This toxicity syndrome may correlate with a rapid rise in PA plasma concentration, secondary to an individual patient’s Vmax being less than his or her dosing rate. The pharmacodynamics explain why on rechallenge, the patients at lower doses had symptoms recur that were delayed in time as compared with the onset during the initial pharmacokinetically monitored cycle (Fig. 3). The metabolic changes are also explained by rapid accumulation of a weak acid formed by β-oxidation of PB to PA. The variability in individual handling of the drug suggests observation over the first cycle of therapy is appropriate. At least 1 patient at each dose level from ≥345 mg/kg/day developed neuro-cortical toxicity, but no clinical or laboratory feature predictive of toxicity was identified.

The population estimates for mean Vmax and km for the elimination of PA are different from those found by Thibault et al(40, 41). Thibault reported a time-dependent increase in the Vmax of PA when it is administered by prolonged infusion. In this report, the Vmax of the PA elimination process was treated as increasing in an exponential fashion beginning 24 h after the institution of PB drug therapy. We obtained Vmax and km values of 77 μmol/h/kg and 62 μmol/liter, respectively, whereas Thibault et al. report values of 129 μmol/h/kg (24.1 mg/h/kg) and 561 μmol/liter (105 μg/ml). This report describes the elimination pattern of PA after the administration of PB and its subsequent β-oxidation to PA. Although PB was initially thought to be a prodrug for PA, laboratory data suggest PB has biological activity more potent than PA. The pharmacodynamic evaluation and Phase II assessment of PB will determine the clinical usefulness of these compounds. Synergy of PB and PA in combination has been difficult to show in preclinical models;5 however, PB administration on this schedule at the 410 mg/kg/day dose generates PB and PA concentrations in the low range required for in vitro activity.

Phase II studies of PB with time to progression or lack of progression end points and evaluation of any “clinical benefit response” are rational. As many of the molecular effects of PB occur in ≤2–3 days of continuous exposure, the described, intermittent schedule was modeled as if PB was a cytotoxic agent. It is not known what the best schedule is for differentiating agents. It may be that an intermittent schedule may not be the optimal schedule. Chronic therapy seems intuitively to be more appropriate.

A Phase I trial of chronic oral administration of sodium PB in refractory solid tumor malignancies has been completed. The study demonstrated that the oral formulation has an excellent bioavailability of 78% for all dose levels, and the concentration which has shown biological activity in vitro, 0.5 mmol/liter, was achieved at all dose levels. No persistent resolution of para-aortic adenopathy or complete response was seen, but 7 patients had stable disease for >6 months while on the drug, despite progressive disease at study entry (42). Agents like PB, vitamin D, and the retinoids have demonstrated preclinical ability to increase PSA secretion and/or expression (33, 43, 44, 45). Trials of these agents can merely describe changes in PSA in the setting of disease stabilization defined by clinical symptoms, bone scans, and other radiographic studies. The rises in PSA in response to therapy described here and with similar agents may run counter to traditional belief that a rising PSA suggests tumor progression.

PB is now entering clinical trials in combination with other agents to demonstrate potentially synergistic effects. One combination involves PB and 13-cis-retinoic acid. Preclinical studies on multiple cancer cell lines have shown that the combination of these agents has a significant inhibitory effect on both the tumor and vascular compartments, suggesting an antiangiogenic effect as well. Additionally, both agents have distinct activity at steroid nuclear receptors, such as the human peroxisome proliferator-activated receptor α and γ, which regulates transcription of genes involved in peroxisomal and mitochondrial pathways of lipid metabolism (46, 47).

PB is also in trial with 5-azacytidine, a DNA methyltransferase inhibitor. Dense methylation of gene promoter regions in cancer cells and histone deacetylation of key genes act as synergistic layers of epigenetic gene silencing. Preclinical studies have demonstrated that the reversal of dense methylation followed by histone deacetylase inhibition can result in robust gene expression of target genes. This principle forms the basis of an ongoing Phase I trial involving this combination of agents in patients with refractory solid tumor malignancies and heme malignancies (48).

In summary, the clinical expectations of differentiation therapy are those of low toxicity, disease stabilization, and symptom amelioration, either as a single agent or in combination with other therapies. The Phase I results of PB, a novel nonretinoid differentiating agent, suggest additional clinical and laboratory evaluation of PB is warranted. At the recommended Phase 2 dose of 410 mg/kg/day by continuous infusion, PB is tolerable, with some variability, and may be responsible for improvement in clinical symptoms and for disease stabilization in a subset of treated patients.

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.

        
1

Supported by UO-1 CA 70095, American Society of Clinical Oncology Young Investigator Award; NIH-K08-CA-69164; NIH-R01-CA75525 (to M. A. C.); Aegon Scholarship in Oncology (to J. G.), General Clinical Research Center N01-CM07302 from the National Cancer Institute.

                
3

The abbreviations used are: PB, phenylbutyrate; PA, phenylacetate; PG, phenylacetylglutamine; PSA, prostate-specific antigen; MTD, maximum tolerated dose; NCI, National Cancer Institute; DLT, dose-limiting toxicity; PCA, prostate cancer.

        
4

S. Gore, personal communication.

        
5

M. A. Carducci, R. Pili, unpublished data.

Fig. 1.

Single compartment model of PB disposition. PB is β-oxidized to PA, and glutamine binds PA to form PG, which is cleared in the urine. The disposition model for these three compounds is defined by a specific Vmax and km for the disposition of one compound to another.

Fig. 1.

Single compartment model of PB disposition. PB is β-oxidized to PA, and glutamine binds PA to form PG, which is cleared in the urine. The disposition model for these three compounds is defined by a specific Vmax and km for the disposition of one compound to another.

Close modal
Fig. 2.

Individual disposition curves of PB, PA, and PG for two subjects at varying dose levels. At two distinct dose levels, PB rapidly reaches peak concentration within 4–6 h of infusion initiation. PB concentrations slowly decline over the 120-h infusion period. PA, at lower doses of PB infusion, maintains a steady concentration throughout the infusion. However, when an individual’s Vmax is less than his/her drug-dosing rate, the PA accumulates progressively. This rapid PA accumulation is associated with dose-limiting neuro-cortical toxicity. There was no kinetically significant change in the urine clearance rate of PG.

Fig. 2.

Individual disposition curves of PB, PA, and PG for two subjects at varying dose levels. At two distinct dose levels, PB rapidly reaches peak concentration within 4–6 h of infusion initiation. PB concentrations slowly decline over the 120-h infusion period. PA, at lower doses of PB infusion, maintains a steady concentration throughout the infusion. However, when an individual’s Vmax is less than his/her drug-dosing rate, the PA accumulates progressively. This rapid PA accumulation is associated with dose-limiting neuro-cortical toxicity. There was no kinetically significant change in the urine clearance rate of PG.

Close modal
Fig. 3.

Cumulative disposition curves of PB and PA for all evaluable patients (dose levels 2–5). PB concentrations increase with increasing dose and can be maintained at >500 μmol/liter in most subjects at the recommended Phase II dose, 410 mg/kg/day. PA concentrations increase with increasing doses of PB. Four patients demonstrate rapid PA accumulation (>2000 μmol/liter) at the highest dose levels.

Fig. 3.

Cumulative disposition curves of PB and PA for all evaluable patients (dose levels 2–5). PB concentrations increase with increasing dose and can be maintained at >500 μmol/liter in most subjects at the recommended Phase II dose, 410 mg/kg/day. PA concentrations increase with increasing doses of PB. Four patients demonstrate rapid PA accumulation (>2000 μmol/liter) at the highest dose levels.

Close modal
Table 1

Gradient profile for HPLC

Time (min)A (%)B (%)C (%)
0.00 47.5 5.0 47.5 
20.00 38.5 23.0 38.5 
23.00 38.5 23.0 38.5 
35.00 27.5 45.0 27.5 
35.01 5.0 90.0 5.0 
38.00 5.0 90.0 5.0 
38.01 47.5 5.0 47.5 
45.00 47.5 5.0 47.5 
Time (min)A (%)B (%)C (%)
0.00 47.5 5.0 47.5 
20.00 38.5 23.0 38.5 
23.00 38.5 23.0 38.5 
35.00 27.5 45.0 27.5 
35.01 5.0 90.0 5.0 
38.00 5.0 90.0 5.0 
38.01 47.5 5.0 47.5 
45.00 47.5 5.0 47.5 
Table 2

Patient characteristics

Total patientsa 24 
Male 23 
Female 
Diagnosis  
 Prostate, hormone-refractory 19 
 Kidney 
 Melanoma 
 Endometrium 
Performance status  
 0 
 1 18 
 2 
Prior therapy  
 Chemotherapy, XRT, hormone 
 XRT,b hormone 
 Hormone 
 Chemotherapy, hormone 
 Chemotherapy, radiation 
 Immunotherapy only 
 Chemotherapy, immunotherapy 
No therapy 
Total patientsa 24 
Male 23 
Female 
Diagnosis  
 Prostate, hormone-refractory 19 
 Kidney 
 Melanoma 
 Endometrium 
Performance status  
 0 
 1 18 
 2 
Prior therapy  
 Chemotherapy, XRT, hormone 
 XRT,b hormone 
 Hormone 
 Chemotherapy, hormone 
 Chemotherapy, radiation 
 Immunotherapy only 
 Chemotherapy, immunotherapy 
No therapy 
a

Age: range, 48–80, median, 66.

b

XRT, X-ray therapy.

Table 3

No. of patients with grade 3 or 4 toxicity by dose level

150 mg/kg/d225 mg/kg/d285 mg/kg/d345 mg/kg/d410 mg/kg/d515 mg/kg/d
Neurocortical     1a 
Hypokalemia      1a 
Hyperuricemia      1a 
Nausea      1a 
Neutropenia 1b      
Fatigue    2c   
150 mg/kg/d225 mg/kg/d285 mg/kg/d345 mg/kg/d410 mg/kg/d515 mg/kg/d
Neurocortical     1a 
Hypokalemia      1a 
Hyperuricemia      1a 
Nausea      1a 
Neutropenia 1b      
Fatigue    2c   
a

Toxicities occurred in the same patient.

b

Lasted 2 days only.

c

Felt to be related to disease progression rather than drug.

Table 4

Population pharmacokinetic estimatesa

MeanSD
PB (n = 20)   
 volume of distribution, liter/kg 0.169 0.043 
Vmax, μmol/h/kg 148 23 
 kinc, 1/h 0.0029 0.0015 
 km, μmol/liter 546 159 
PA (n = 19)   
 volume of distribution, liter/kg 0.720 0.562 
Vmax, μmol/h/kg 77 10 
 kinc, 1/h 0.0009 0.0006 
 km, μmol/liter 62 28 
Phenylacetylglutamine (n = 19)   
 volume of distribution, liter/kg 0.281 0.130 
 clearance rate, ml/min/kg 1.83 1.15 
MeanSD
PB (n = 20)   
 volume of distribution, liter/kg 0.169 0.043 
Vmax, μmol/h/kg 148 23 
 kinc, 1/h 0.0029 0.0015 
 km, μmol/liter 546 159 
PA (n = 19)   
 volume of distribution, liter/kg 0.720 0.562 
Vmax, μmol/h/kg 77 10 
 kinc, 1/h 0.0009 0.0006 
 km, μmol/liter 62 28 
Phenylacetylglutamine (n = 19)   
 volume of distribution, liter/kg 0.281 0.130 
 clearance rate, ml/min/kg 1.83 1.15 
a

Kinc, rate constant of increase in Vmax after 24 h.

Table 5

Duration of therapy by dose

No. of cycles
123468
150 mg/kg/day (n = 4)    
225 mg/kg/day (n = 4)     
285 mg/kg/day (n = 4)    
345 mg/kg/day (n = 4)   
410 mg/kg/day (n = 7)   
515 mg/kg/day (n = 1)      
Total (n = 24) 
No. of cycles
123468
150 mg/kg/day (n = 4)    
225 mg/kg/day (n = 4)     
285 mg/kg/day (n = 4)    
345 mg/kg/day (n = 4)   
410 mg/kg/day (n = 7)   
515 mg/kg/day (n = 1)      
Total (n = 24) 

We thank Mark Kruszewski, O. Michael Colvin, Stephanie Porter, Mary Duerr, Sharyn D. Baker, and the Oncology Pharmacy for their contributions to this manuscript.

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