Phase I and Pharmacokinetic Study of Aplidine, a New Marine Cyclodepsipeptide in Patients With Advanced Malignancies
http://www.100md.com
《临床肿瘤学》
the Department of Medicine, Institute Gustave-Roussy, Villejuif, France
PharmaMar R&D, Colmenar Viejo, Madrid, Spain
ABSTRACT
PURPOSE: To establish the safety, pharmacokinetic parameters, maximum-tolerated dose, and recommended dose of aplidine, a novel marine cyclodepsipeptide, in patients with advanced cancer.
PATIENTS AND METHODS: Using a modified Fibonacci method, we performed a phase I and pharmacokinetic study of aplidine administered as a 24-hour intravenous infusion every 2 weeks.
RESULTS: Sixty-seven patients received aplidine at a dose ranging from 0.2 to 8 mg/m2. Dose-limiting myotoxicity corresponding to grade 2 to 3 creatine phosphokinase elevation and grade 1 to 2 myalgia and muscle weakness occurred in two of six patients at 6 mg/m2. No cardiac toxicity was observed. Electron microscopy analysis showed the disappearance of thick filaments of myosin. Grade 3 muscle toxicity occurred in three of 14 patients at the recommended dose of 5 mg/m2 and seemed to be more readily reversible with oral carnitine (1 g/10 kg). Therefore, dose escalation was resumed using carnitine prophylactically, allowing an increase in the recommended dose to 7 mg/m2. Other toxicities were nausea and vomiting, diarrhea, asthenia, and transaminase elevation with mild hematologic toxicity. Aplidine displayed a long half-life (21 to 44 hours), low clearance (45 to 49 L/h), and a high volume of distribution (1,036 to 1,124 L) with high interpatient variability in plasma, whereas in whole blood, clearance ranged from 3.0 to 6.2 L/h. Minor responses and prolonged tumor stabilizations were observed in patients with medullary thyroid carcinoma.
CONCLUSION: Muscle toxicity was dose limiting in this study. Recommended doses of aplidine were 5 and 7 mg/m2 without and with carnitine, respectively. The role of carnitine will be further explored in phase II studies.
INTRODUCTION
Didemnins and tamandarins are high molecular weight macrolide cyclodepsipeptides with antiviral and cytotoxic properties. Didemnins were isolated from marine tunicates that use these molecules to alter predator-prey relations in the marine ecosystem.1 On the basis of potent antiproliferative effects against a variety of human tumor models, didemnin B was the first ascidiacea cytotoxin to undergo phase I/II clinical trials in patients with cancer. This drug displayed dose-limiting toxicities (DLTs) consisting of nausea/vomiting, hepatic dysfunction, and neuromyotoxicity at higher doses in phase I and II trials when administered as a 30-minute infusion.2 Moderate to severe muscle weakness and elevated creatine phosphokinase (CPK) and aldolase levels were associated with necrosis and type II muscle fiber atrophy, but no inflammation was found in muscle biopsy specimens.3 Cardiac toxicity has also been reported with didemnin B.4 Although responses have been reported with didemnin B, especially in heavily pretreated non-Hodgkin's lymphoma patients, the safety profile (cumulative fatigue and neuromuscular and cardiac toxicity) precluded the administration of multiple cycles; thus, the clinical development was stopped. A search for derivatives with lower toxicity and better therapeutic indexes was initiated.
Aplidine (Fig 1) is a second-generation didemnin isolated from the Mediterranean tunicate Aplidium albicans, which inhibits protein and DNA synthesis and induces apoptosis and a G1 cell-cycle arrest in cancer cells independently of p53 and MDR expression.5 The antiproliferative proprieties of aplidine may result from its binding to several key enzymes including elongation factor 1-alpha, ornithine decarboxylase, and palmitoyl protein thioesterase 1, the latter being involved in the lipidation process of several signaling proteins. In addition, aplidine was shown to inhibit flt-1 gene expression encoding vascular endothelial growth factor receptor-1, to decrease vascular endothelial growth factor secretion,6 and to induce apoptosis in cancer cells.7
In vitro screening of a panel of human cancer cell lines performed at the National Cancer Institute showed that aplidine has a unique cytotoxic profile and demonstrated that aplidine was more active than didemnin B against a broad spectrum of tumor types, including melanomas and non–small-cell lung, prostate, ovarian, and colorectal cancers, with 50% inhibitory concentrations in the range of 0.18 to 0.45 nmol/L. No bone marrow toxicity was seen in experimental models, and the maximum-tolerated doses (MTDs) in the mouse and rat were 3.75 and 3.42 g/m2/mo, respectively. In rats, aplidine displayed rapid clearance, and its antitumor activity in mice bearing human xenografts was maximal when the drug was administered by continuous intravenous infusion or via the intraperitoneal route.8 These preclinical pharmacology data provided the rationale for dose-dense administration of aplidine via intravenous continuous infusion for 24 hours and for targeting a sustained threshold plasma level of approximately 0.5 nmol/L in patients.
The objectives of this study were to determine the MTD and recommended dose of aplidine when administered as a 24-hour infusion repeated every 2 weeks, to describe the DLTs of this schedule, to assess its pharmacokinetic profile, and to document any antitumor activity in patients with advanced malignancies.
PATIENTS AND METHODS
Patient Selection
Patients who were entered onto this study met the following criteria: histologically or cytologically confirmed diagnosis of a solid tumor or non-Hodgkin's lymphomas refractory to standard therapy or for which no standard therapy existed; age 18 years; life expectancy 3 months; WHO performance status 2; no history of a previous malignancy; no chemotherapy, hormonal therapy, immunotherapy, or radiotherapy within 4 weeks before treatment with aplidine (6 weeks in case of previous treatment with nitrosoureas, mitomycin, or extensive radiotherapy); no prior bone marrow transplantation or intensive chemotherapy with stem-cell support during the previous 6 months; no radiation therapy involving more than 20% of bones; adequate hepatic function defined as serum bilirubin less than 25 μmol/L (< 1.5 mg/dL), transaminases and alkaline phosphatases 3.0x the upper limit of normal ( 5x upper limit of normal in case of documented liver metastases); adequate bone marrow function, including an absolute neutrophil count 1,500/μL, platelets 100,000/μL, and hemoglobin 10.0 g/dL; adequate renal function with calculated creatinine clearance (Cockroft formula) 50 mL/min; prothrombin within normal limits; a psychosocial state compatible with participation; signed informed consent according to institutional and national guidelines; and no medically uncontrolled disease. Patients were excluded if they were pregnant or breast feeding (men and women of child-bearing potential were required to use a reliable method of contraception), had symptomatic brain metastases/leptomeningeal tumor involvement, or had concomitant active bacterial infection.
In addition to these criteria, patients had to meet the following criterion specifically designed based on previous data collected with didemnin B: adequate cardiac function defined as New York Heart Association class I (cardiac disease without resulting limitation of physical activity provided that ordinary physical activity did not cause undue dyspnea, fatigue, palpitation, or angina pain). Left ventricular ejection fraction 45% with normal ECG was also mandatory at study entry. Patients with a history of arrhythmia, myocardial infarction, and/or uncontrolled angina pectoris during the last 6 months were not included in this study. The use of concomitant therapeutic anticoagulants was not permitted. Other exclusion criteria included any pre-existing motor or sensory neurotoxicity grade 2, current or previous history of liver dysfunction caused by chronic hepatitis and/or cirrhosis, a history of hypersensitivity reaction to any drug formulated in polyethylated castor oil, and participation in any other clinical trial with another investigational drug.
Pretreatment and Follow-Up Examinations
A complete medical history, physical and neurologic examination, WHO performance status, CBC with differential and platelet count, electrolytes and creatinine, serum calcium, magnesium, phosphorus, urea, uric acid, hepatic function tests, glucose, total protein, albumin, bilirubin, AST, ALT, alkaline phosphatase, prothrombin time, and urinalysis were performed at baseline and repeated twice weekly (except for prothrombin time and urinalysis, which were performed weekly). CPK and aldolase were assessed at baseline, immediately before each infusion, and the day after each infusion. An ECG, a cardiac ultrasound with left ventricular ejection fraction, and a chest x-ray were obtained within 7 days before receiving aplidine. Toxicity was evaluated by clinical and biologic examination on a weekly basis and graded using the National Cancer Institute Common Toxicity Criteria.9 Additional laboratory tests and/or an increase in the frequency of monitoring were permitted to document acute drug-related toxicity until recovery.
To be assessable for response, patients had to have measurable disease, as defined by WHO criteria, and had to have completed two courses of aplidine before reassessment. Tumor measurements were obtained at baseline and after every two cycles of aplidine. Responses were assessed according to WHO criteria.10
Drug Administration
Aplidine was manufactured and formulated for intravenous injection by PharmaMar (Madrid, Spain). The drug was supplied in ready-to-use 15-mL glass vials containing 500 μg of aplidine as lyophilized powder, which was diluted with a 2-mL solution containing 15%/15%/70% polyethylated castor oil/ethanol/water to obtain a final concentration of 0.5 mg/mL for injection. The reconstituted solution was stable for 48 hours at room temperature and under ambient light. The appropriate volume of this stock solution was added to 100 mL of isotonic saline to yield the required dose. Aplidine was administered as a 24-hour intravenous infusion using rate-controlled electronic pumps every 2 weeks through a central intravenous line. Patients had to have recovered to at least grade 1 toxicity before receiving the next dose of the drug. If the patient had not recovered by the time of the next infusion, a rest week was proposed. Patients requiring deferral of treatment for more than 2 weeks because of aplidine-related toxicity were withdrawn from this study.
Dose-Escalation Procedure
On the basis of toxicologic studies in rodents, the dose of 0.4 mg/m2/mo (approximately one tenth of the MTD) administered as 0.2 mg/m2 every 2 weeks was selected as the starting dose for this trial; the dose was then escalated according to a modified Fibonacci scheme. A minimum of three patients were included at each dose level. DLT was defined as grade 4 neutropenia lasting 5 days or associated with temperature 38.5°C, grade 4 thrombocytopenia, or grade 3 nonhematologic toxicity (including hepatotoxicity and myotoxicity). Emesis, alopecia, and hypersensitivity reactions were not considered as DLTs. If no or minimal (including grade 1) toxicity was observed, doses were escalated in 50% to 100% steps; otherwise, if grade 2 toxicity (other than alopecia, anemia, and untreated nausea and vomiting) occurred, doses were escalated in 20% to 30% steps. The results of the pharmacokinetic analyses were taken into consideration for subsequent dose escalation.
The MTD was initially defined based on the occurrence of DLT during the first 2 months of treatment (up to the fifth infusion). During the course of the study, occurrence of DLT at cycle 3 in one patient prompted us to redefine the MTD based on the occurrence of DLT at any cycle. If DLT was observed in any of the patients entered at a specific dose level, an additional three patients were enrolled at that dose level. The dose at which DLT occurred in two or more patients was defined as the MTD. The recommended safe dose for further study was defined as the dose level immediately below the MTD. At least 12 patients, of whom six patients had to have received at least 2 months of treatment (four infusions), were required to define the recommended dose.
Plasma and Urine Pharmacokinetic Sampling and Assay
A pharmacokinetic profile of aplidine was obtained after the first infusion. Venous blood samples were collected from different infusion sites into tubes containing lithium-heparin anticoagulant immediately before the start of the infusion (time 0); at 3, 6, 8, and 12 hours during the infusion; at the end of the infusion (24 hours); and at the following times thereafter: at 15, 30, and 45 minutes and at 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, and between 120 and 168 hours after infusion. The sample was immediately centrifuged, and the resulting plasma was stored at –20°C before analysis. A pretreatment sample of urine was collected. Thereafter, three separate urine samples were collected according to the time interval after aplidine (0 to 24 hours and 24 to 48 hours) and stored at 4°C. Each container was thoroughly mixed, and its total volume was recorded. Two duplicate samples of 10 mL from each container were stored at –20°C before analysis.
The concentration of aplidine in whole blood, plasma, and urine was measured using high-performance liquid chromatography coupled to a mass spectrometer. Aplidine concentrations in biologic fluids were assayed using a method described elsewhere.11 The lower limit of quantification of aplidine was 0.25 ng/mL. Plasma concentration and whole blood versus time profiles were generated, and both observed and calculated variables were reported. These variables included area under the curve from time zero to infinity (AUCinf), maximum concentration (Cmax), clearance, terminal half-life (t1/2), median residual time (MRT), and volume of distribution (Vss). Statistical analysis was performed using the SAS PROC MIXED procedure (SAS Institute, Cary, NC). The level of statistical significance was set at = .05, and CI criteria for assessment of dose proportionality were calculated according to the Smith et al12 method. Pharmacokinetic and pharmacodynamic interactions were performed using both Pearson and Spearman correlation in SAS.
RESULTS
General
A total of 67 patients with a variety of advanced malignancies were entered onto the study. One patient who experienced early tumor progression before receiving aplidine was not assessable for toxicity. Patient characteristics (n = 66) are listed in Table 1. All but one patient had received prior chemotherapy, with a median number of three prior regimens (range, zero to seven regimens). A total of 161 cycles of aplidine were administered at doses ranging from 0.2 to 8 mg/m2 (Table 2). The median number of courses administered per patient was two (range, one to seven courses).
Dose Escalation
Dose escalation from 0.2 to 4 mg/m2 was not associated with acute and/or delayed DLT. One patient receiving aplidine 1.6 mg/m2 experienced thrombocytic microangiopathy, which was later considered unrelated to aplidine but led us to increase the cohort from three to six patients. Three patients entered at 5 mg/m2 had no DLT, so the dose level of 6 mg/m2 was explored. At this dose level, at day 15 of cycle 2 (15 days after the third injection), one of the first three patients experienced grade 3 muscle toxicity with grade 3 CPK elevation (with approximately a seven-fold increase in the aldolase baseline value), grade 2 myalgia, grade 2 muscle weakness, and grade 3 asthenia. This prompted us to increase the cohort from three to six patients. Another patient treated at 6 mg/m2 presented at day 27 of cycle 3 (13 days after the sixth injection) with grade 2 CPK elevation lasting for more than 15 days. Although this DLT occurred after the period that was classified for the initial definition of MTD, the severity of this delayed toxicity was later considered as part of the MTD. Meanwhile, dose escalation had been resumed, and four patients had already been treated at 7 mg/m2; one of these patients developed grade 4 dose-limiting neutropenia.
The occurrence of muscle DLT in two of six patients at 6 mg/m2 prompted us to stop dose escalation and define dose levels 6 mg/m2 as the MTD. The cohort of patients treated at 5 mg/m2 was further extended to define the recommended dose. Among a total of 14 patients, three (21%) developed DLT, which consisted of grade 3 to 4 myotoxicity (two patients) during cycle 2 at days 19 and 21 after the third injection and grade 3 transaminase elevation at cycle 1 (one patient).
Aplidine myotoxicity, as characterized by muscle cramps, pain, weakness, and/or increases in CPK, was found to be closely related to the symptoms described in the adult form of carnitine palmitoyl transferase deficiency type 2, which is a genetic disease treated with L-carnitine.13 On the basis of preclinical data showing that aplidine inhibits palmitoyl thiosterase, an enzyme related to the carnitine palmitoyl transferase family, we proposed L-carnitine to treat and prevent aplidine-induced effects on muscles. The dose of 1g/10 kg L-carnitine used in this study was based on doses classically used in carnitine deficiency.14
Dose escalation was reinitiated from 6 mg/m2 of aplidine associated with an oral solution of L-carnitine at a dose of 1 g/10 kg (three times a day) from the first day of treatment up to at least 4 weeks after the last aplidine injection. Four patients, instead of three patients, received aplidine 6 mg/m2 with carnitine (one patient was not assessable for safety because of early tumor progression), and no DLT was reported. Dose escalation was pursued at aplidine 7 mg/m2 plus carnitine in three patients without DLT, and the dose was further escalated to aplidine 8 mg/m2 plus carnitine. Of the four patients treated at this dose level, one patient developed DLT consisting of grade 3 asthenia and grade 1 fever (flu-like syndrome) at cycle 1, and another patient developed grade 3 to 4 myotoxicity at cycle 2. Because two of four patients experienced DLT, this dose level was considered the MTD, and the subsequent patients were treated at 7 mg/m2. Of a total of 12 patients treated at aplidine 7 mg/m2 plus carnitine, two patients (16%) experienced grade 3 to 4 myotoxicity, both at cycle 2. On the basis of toxicity during the first two cycles, the recommended doses were 5 and 7 mg/m2 without and with carnitine, respectively.
Myotoxicity
Muscle toxicity was reported when doses 5 mg/m2 of aplidine were administered. In the first cohort without carnitine, muscle toxicity consisting of myalgia, muscle weakness, and/or CPK elevation was the main relevant adverse effect observed with aplidine (Table 3). Grade 1 to 2 myalgia occurred in 12 patients (26.0%) and 17 cycles (14.5%). Grade 1 to 2 muscle weakness was documented in four patients (8.7%) during four cycles (3.4%). Elevated CPK and aldolase levels were reported in 10 patients (21.7%) and 15 cycles (12.8%), reaching grade 3 to 4 in three patients (6.5%). Clinical symptoms typically started 7 to 10 days after the third injection of aplidine (middle of cycle 2), with muscle pain predominantly in the shoulders, back, and thighs. Pain was followed by muscle weakness and an increase in CPK (Fig 2A), occurring within 7 to 15 days after the onset of myalgia (ie, 15 to 21 days after the third injection of aplidine). Among patients with myotoxicity who had a muscle biopsy, light microscopy showed either normal features or minimal necrosis (type II diffuse fiber atrophy). There was no evidence of rhabdomyositis. Electron microscopy analysis showed unspecific accumulation of glycogen and autophagocytic vacuoles, and the major change was the disappearance of thick filaments of myosin (Fig. 2B). Importantly, aplidine-induced muscle toxicity was not associated with myocardic and/or diaphragmatic myotoxicity. In the first patients presenting with severe muscle toxicity, a complete recovery occurred within 2 months after discontinuation of aplidine. In the following patients, the treatment was postponed; active physiotherapy and L-carnitine administered orally at a dose of 1 g/10 kg reduced the delay of reversibility to 4 to 6 weeks (Fig 3). In the second cohort of patients treated prophylactically with carnitine, severe myotoxicity was observed in only three (15%) of 20 patients receiving higher doses of aplidine ranging from 6 to 8 mg/m2 (Tables 2 and 3).
Other Toxicities
Grade 1 to 2 asthenia was observed in 44 patients (65.7%) during 89 cycles (89%) and was severe in only four patients who also experienced muscle toxicity. GI symptoms included grade 1 to 2 nausea and vomiting in 54 patients (80.6%) and grade 1 to 2 diarrhea in 12 patients (17.9%). One patient presented with grade 3 fever concomitantly with asthenia without documentation of infection at the highest dose level. Other toxicities included grade 3 elevation of transaminases without evidence of hepatic metastasis in one patient and grade 1 to 2 local pain at the injection site in two patients. Hematologic toxicity was limited to rare episodes of asymptomatic neutropenia in 4.5% of patients and 1.7% of cycles, whereas grade 1 to 2 thrombocytopenia was reported in 10.6% of cycles. Asymptomatic grade 3 lymphopenia occurred in 19.4% of patients (11.2% of cycles).
Pharmacokinetics
A pharmacokinetic analysis was performed in 48 patients during the first infusion of cycle 1 (Table 4). Aplidine concentrations were near or less than the lower limit of quantification for doses less than 0.8 mg/m2, thus prohibiting adequate quantification.
The pharmacokinetic analysis indicates that aplidine has a relatively long half-life, low clearance, and a high volume of distribution in plasma (Fig 4). In plasma and whole blood, mean half-life values ranged from 21 to 44 hours. Aplidine mean clearance values in plasma ranged from 45 to 49 L/h, with Vss values ranging from 1,036 to 1,124 L; whereas in whole blood, clearance ranged from 3.0 to 6.2 L/h with high interpatient variability, and mean Vss values ranged from 102 to 145 L. As described earlier, whole-blood aplidine concentrations were frequently higher than concentrations in plasma for patients who had measurement of concentrations in both media. When comparisons were made between blood and plasma results at the 6 mg/m2 dose level, mean whole-blood Cmax and AUCinf values were approximately 144 ng/mL and 5,430 ng · h/mL, respectively, compared with values of approximately 32 ng/mL for Cmax and 1,113 ng · h/mL for AUCinf in plasma.
Aplidine area under the curve calculated to the last time point (AUClast), AUCinf, and Cmax values generally increased with the dose. Dose proportionality of aplidine in plasma or whole blood was not proven, most probably because of the limited sample size for all dose levels and high interpatient variability. Compartmental analysis showed that plasma profiles best fit a first-order two-compartment model.
Urinary excretion of aplidine was low, with a mean recovery of 4.16% in 48 hours, and was less than 15% in all patients. There was no correlation between patient characteristics at study entry and pharmacokinetic results.
Antitumor Activity
Among 66 patients enrolled, no objective response was observed. Fifteen patients had stable disease lasting 2 to 16 months as their best response. Among the patients, eight (treated at 5, 6, and 7 g/m2 in three, three, and two patients, respectively) with documented tumor progression in the 3 months preceding study entry had tumor stabilization for 6 months. Interestingly, six patients with endocrine tumors (medullary thyroid carcinoma, bronchial carcinoid tumor, and malignant pheochromocytoma in four, one, and one patient, respectively) experienced a clinical benefit and prolonged disease stabilization (> 6 months) associated with a relevant decrease in their tumor markers.
DISCUSSION
We report here the results of a large-scale phase I study using the marine compound aplidine administered in a 24-hour infusion every 2 weeks. Dose escalation greater than 5 g/m2 revealed dose-limiting myotoxicity consisting of myalgia and muscle weakness followed by an elevated CPK level. Myotoxicity occurred mainly at high doses and during prolonged exposure and affected patients with high, sustained plasma aplidine concentrations. Indeed, the three patients showing severe myotoxicity had t1/2 in excess of 44 hours compared with a median t1/2 of 25.8 hours after a 24-hour infusion. Aplidine-induced muscle toxicity resembles the toxicity extensively described in alcohol- and some congenital-induced proximal myopathies. At microscopic analysis, aplidine seems to affect fast-twitch type II muscle fibers, which are particularly vulnerable to the imbalanced metabolism of skeletal muscle proteins.15 Although the precise mechanism of toxicity remains unknown, aplidine inhibits thioesterase I, a palmitoyl protein16 closely related to carnitine palmitoyl transferase 2, which is a muscle enzyme the deficiency of which leads to congenital myopathy in children.13 Another possible mechanism of toxicity may involve the interaction with elongation factors, given that impairment in translation efficiency is known to decrease muscle protein synthesis.17
Interestingly, the use of carnitine seems to facilitate recovery from myotoxicity and allowed us to pursue further aplidine treatment after toxicity. An extensive in vitro program conducted in a broad panel of solid tumors and leukemia has shown that carnitine does not interact with aplidine cytotoxicity in cancer cells and ruled out any tumor protective effect.18,19 The prophylactic use of high-dose carnitine enabled us to increase the recommended dose of aplidine without severe myotoxicity but with a slight increase in grade 1 to 2 diarrhea (Table 3), which is a toxicity known to be also related to carnitine.
Other digestive toxicities were frequent but remained mild to moderate (grade 1 to 2 nausea and vomiting in 80.6% of the patients and 61.5% of cycles). Transient hepatotoxicity was observed in only two patients at doses 5 mg/m2. Hematologic toxicity of aplidine was low because only three episodes of asymptomatic grade 3 to 4 neutropenia were documented in three patients and no grade 3 to 4 thrombocytopenia occurred. Although grade 1 to 3 lymphopenia was frequently reported, it was not associated with any clinical meaningful immunosuppression.
During the course of the study, patient plasma levels of aplidine were found to be lower than expected from animal pharmacokinetic data. Therefore, we decided to explore both plasma and whole-blood concentrations in humans for doses of 5 and 6 mg/m2. We found major differences between plasma and whole-blood concentrations, suggesting RBC count partitioning of aplidine in human. Interestingly, interpatient clearance variability of aplidine in plasma was lower than that of whole-blood clearance. This led us to favor further evaluation of whole-blood pharmacokinetic data at doses of more than 6 mg/m2. Considering the high molecular weight of aplidine, our results suggest the adsorption of aplidine on erythrocytes and/or other blood cells, making possible variability in the release of aplidine from blood cells in individuals. Furthermore, we found that the degrees of variability between body surface area- (BSA) normalized and fixed dosing were low, suggesting that no improvement in variability in whole blood would be expected from normalizing the dose based on BSA. Pharmacokinetic modeling will have to be performed to determine the clinical consequences of this possible blood-cell partitioning and the variability of BSA-normalized versus fixed dosing of aplidine.
The recommended doses in our study are 7 and 5 mg/m2 with and without prophylactic carnitine, respectively. On the basis of our data and those of other phase I studies, the toxicity profile of aplidine is henceforth well defined at the recommended doses (transient, mild to moderate asthenia, nausea and vomiting, myalgia, and CPK and/or transaminase elevation without hematologic toxicity or alopecia). When several schedules are compared, aplidine toxicity seemed, at least in part, dependent on the duration of exposure. Protracted infusions of aplidine were reproducibly associated with myotoxicity, which was also reported as the DLT in another phase I trial using a 24-hour weekly infusion, 3 consecutive weeks out of 4 weeks.20 In contrast, shorter duration infusions of 1 to 3 hours21-23 were associated with severe transaminitis with sparse myotoxicity. One case of fatal renal, hepatic, and cardiac failure has also been reported at the highest dose level using the 3-hour schedule.21 Regardless of the schedule, dose-intensities of 24-hour schedules (ranging from 10 to 11 mg/m2/4 wk) compare favorably with the dose-intensities of 1- to 3-hour schedules (8 to 10 mg/m2/4 wk) at the recommended dose.
Although antitumor activity was not the primary objective of this study, six patients with endocrine tumors (four with medullary thyroid carcinoma) benefited from aplidine treatment, with long-term stabilization of their disease lasting 6 to 16 months and including one minor response of approximately 40% in a patient with bronchial carcinoid tumor. Within the entire phase I program, 10 patients with advanced medullary thyroid carcinoma received aplidine. All but one patient were symptomatic at baseline, with pain (n = 7), diarrhea (n = 3), and an elevated median carcinoembryonic antigen value of 363 ng/mL at entry (range, 5.4 to 7,828 ng/mL). Symptoms improved in four patients. Carcinoembryonic antigen decreased in five of seven assessable patients (reductions of 30%, 45%, 45%, 48%, and 70%). A partial response and two incidents of tumor shrinkage of 20% and 27% were documented in three of five patients with measurable disease, suggesting that aplidine is able to yield a clinical benefit in patients with advanced medullary thyroid cancer. Activity observed in other neural crest–derived tumors (malignant pheochromocytoma and carcinoid tumors) also warrants further clinical and mechanistic research using aplidine.
In summary, dose-limiting myotoxicity consisting of type II fiber atrophy requires careful clinical and biologic monitoring during treatment with high doses of aplidine. The role of carnitine warrants further exploration to improve the safety of aplidine in future trials. At the recommended doses, the toxicity profile, including the lack of hematotlogic toxicity, makes aplidine potentially combinable with other cytotoxic or targeted compounds. Aplidine allowed prolonged periods ( 6 months) of drug administration in patients with evidence of a clinical benefit. On the basis of our data, this schedule was selected for further phase II clinical development.
Authors' Disclosures of Potential Conflicts of Interest
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Acknowledgment
We gratefully acknowledge the nursing staff of the Department of Medicine (GARD unit) for their assistance and Lorna Saint-Ange for editing at the Institute Gustave-Roussy.
NOTES
Supported in part by PharmaMar, Madrid, Spain.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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PharmaMar R&D, Colmenar Viejo, Madrid, Spain
ABSTRACT
PURPOSE: To establish the safety, pharmacokinetic parameters, maximum-tolerated dose, and recommended dose of aplidine, a novel marine cyclodepsipeptide, in patients with advanced cancer.
PATIENTS AND METHODS: Using a modified Fibonacci method, we performed a phase I and pharmacokinetic study of aplidine administered as a 24-hour intravenous infusion every 2 weeks.
RESULTS: Sixty-seven patients received aplidine at a dose ranging from 0.2 to 8 mg/m2. Dose-limiting myotoxicity corresponding to grade 2 to 3 creatine phosphokinase elevation and grade 1 to 2 myalgia and muscle weakness occurred in two of six patients at 6 mg/m2. No cardiac toxicity was observed. Electron microscopy analysis showed the disappearance of thick filaments of myosin. Grade 3 muscle toxicity occurred in three of 14 patients at the recommended dose of 5 mg/m2 and seemed to be more readily reversible with oral carnitine (1 g/10 kg). Therefore, dose escalation was resumed using carnitine prophylactically, allowing an increase in the recommended dose to 7 mg/m2. Other toxicities were nausea and vomiting, diarrhea, asthenia, and transaminase elevation with mild hematologic toxicity. Aplidine displayed a long half-life (21 to 44 hours), low clearance (45 to 49 L/h), and a high volume of distribution (1,036 to 1,124 L) with high interpatient variability in plasma, whereas in whole blood, clearance ranged from 3.0 to 6.2 L/h. Minor responses and prolonged tumor stabilizations were observed in patients with medullary thyroid carcinoma.
CONCLUSION: Muscle toxicity was dose limiting in this study. Recommended doses of aplidine were 5 and 7 mg/m2 without and with carnitine, respectively. The role of carnitine will be further explored in phase II studies.
INTRODUCTION
Didemnins and tamandarins are high molecular weight macrolide cyclodepsipeptides with antiviral and cytotoxic properties. Didemnins were isolated from marine tunicates that use these molecules to alter predator-prey relations in the marine ecosystem.1 On the basis of potent antiproliferative effects against a variety of human tumor models, didemnin B was the first ascidiacea cytotoxin to undergo phase I/II clinical trials in patients with cancer. This drug displayed dose-limiting toxicities (DLTs) consisting of nausea/vomiting, hepatic dysfunction, and neuromyotoxicity at higher doses in phase I and II trials when administered as a 30-minute infusion.2 Moderate to severe muscle weakness and elevated creatine phosphokinase (CPK) and aldolase levels were associated with necrosis and type II muscle fiber atrophy, but no inflammation was found in muscle biopsy specimens.3 Cardiac toxicity has also been reported with didemnin B.4 Although responses have been reported with didemnin B, especially in heavily pretreated non-Hodgkin's lymphoma patients, the safety profile (cumulative fatigue and neuromuscular and cardiac toxicity) precluded the administration of multiple cycles; thus, the clinical development was stopped. A search for derivatives with lower toxicity and better therapeutic indexes was initiated.
Aplidine (Fig 1) is a second-generation didemnin isolated from the Mediterranean tunicate Aplidium albicans, which inhibits protein and DNA synthesis and induces apoptosis and a G1 cell-cycle arrest in cancer cells independently of p53 and MDR expression.5 The antiproliferative proprieties of aplidine may result from its binding to several key enzymes including elongation factor 1-alpha, ornithine decarboxylase, and palmitoyl protein thioesterase 1, the latter being involved in the lipidation process of several signaling proteins. In addition, aplidine was shown to inhibit flt-1 gene expression encoding vascular endothelial growth factor receptor-1, to decrease vascular endothelial growth factor secretion,6 and to induce apoptosis in cancer cells.7
In vitro screening of a panel of human cancer cell lines performed at the National Cancer Institute showed that aplidine has a unique cytotoxic profile and demonstrated that aplidine was more active than didemnin B against a broad spectrum of tumor types, including melanomas and non–small-cell lung, prostate, ovarian, and colorectal cancers, with 50% inhibitory concentrations in the range of 0.18 to 0.45 nmol/L. No bone marrow toxicity was seen in experimental models, and the maximum-tolerated doses (MTDs) in the mouse and rat were 3.75 and 3.42 g/m2/mo, respectively. In rats, aplidine displayed rapid clearance, and its antitumor activity in mice bearing human xenografts was maximal when the drug was administered by continuous intravenous infusion or via the intraperitoneal route.8 These preclinical pharmacology data provided the rationale for dose-dense administration of aplidine via intravenous continuous infusion for 24 hours and for targeting a sustained threshold plasma level of approximately 0.5 nmol/L in patients.
The objectives of this study were to determine the MTD and recommended dose of aplidine when administered as a 24-hour infusion repeated every 2 weeks, to describe the DLTs of this schedule, to assess its pharmacokinetic profile, and to document any antitumor activity in patients with advanced malignancies.
PATIENTS AND METHODS
Patient Selection
Patients who were entered onto this study met the following criteria: histologically or cytologically confirmed diagnosis of a solid tumor or non-Hodgkin's lymphomas refractory to standard therapy or for which no standard therapy existed; age 18 years; life expectancy 3 months; WHO performance status 2; no history of a previous malignancy; no chemotherapy, hormonal therapy, immunotherapy, or radiotherapy within 4 weeks before treatment with aplidine (6 weeks in case of previous treatment with nitrosoureas, mitomycin, or extensive radiotherapy); no prior bone marrow transplantation or intensive chemotherapy with stem-cell support during the previous 6 months; no radiation therapy involving more than 20% of bones; adequate hepatic function defined as serum bilirubin less than 25 μmol/L (< 1.5 mg/dL), transaminases and alkaline phosphatases 3.0x the upper limit of normal ( 5x upper limit of normal in case of documented liver metastases); adequate bone marrow function, including an absolute neutrophil count 1,500/μL, platelets 100,000/μL, and hemoglobin 10.0 g/dL; adequate renal function with calculated creatinine clearance (Cockroft formula) 50 mL/min; prothrombin within normal limits; a psychosocial state compatible with participation; signed informed consent according to institutional and national guidelines; and no medically uncontrolled disease. Patients were excluded if they were pregnant or breast feeding (men and women of child-bearing potential were required to use a reliable method of contraception), had symptomatic brain metastases/leptomeningeal tumor involvement, or had concomitant active bacterial infection.
In addition to these criteria, patients had to meet the following criterion specifically designed based on previous data collected with didemnin B: adequate cardiac function defined as New York Heart Association class I (cardiac disease without resulting limitation of physical activity provided that ordinary physical activity did not cause undue dyspnea, fatigue, palpitation, or angina pain). Left ventricular ejection fraction 45% with normal ECG was also mandatory at study entry. Patients with a history of arrhythmia, myocardial infarction, and/or uncontrolled angina pectoris during the last 6 months were not included in this study. The use of concomitant therapeutic anticoagulants was not permitted. Other exclusion criteria included any pre-existing motor or sensory neurotoxicity grade 2, current or previous history of liver dysfunction caused by chronic hepatitis and/or cirrhosis, a history of hypersensitivity reaction to any drug formulated in polyethylated castor oil, and participation in any other clinical trial with another investigational drug.
Pretreatment and Follow-Up Examinations
A complete medical history, physical and neurologic examination, WHO performance status, CBC with differential and platelet count, electrolytes and creatinine, serum calcium, magnesium, phosphorus, urea, uric acid, hepatic function tests, glucose, total protein, albumin, bilirubin, AST, ALT, alkaline phosphatase, prothrombin time, and urinalysis were performed at baseline and repeated twice weekly (except for prothrombin time and urinalysis, which were performed weekly). CPK and aldolase were assessed at baseline, immediately before each infusion, and the day after each infusion. An ECG, a cardiac ultrasound with left ventricular ejection fraction, and a chest x-ray were obtained within 7 days before receiving aplidine. Toxicity was evaluated by clinical and biologic examination on a weekly basis and graded using the National Cancer Institute Common Toxicity Criteria.9 Additional laboratory tests and/or an increase in the frequency of monitoring were permitted to document acute drug-related toxicity until recovery.
To be assessable for response, patients had to have measurable disease, as defined by WHO criteria, and had to have completed two courses of aplidine before reassessment. Tumor measurements were obtained at baseline and after every two cycles of aplidine. Responses were assessed according to WHO criteria.10
Drug Administration
Aplidine was manufactured and formulated for intravenous injection by PharmaMar (Madrid, Spain). The drug was supplied in ready-to-use 15-mL glass vials containing 500 μg of aplidine as lyophilized powder, which was diluted with a 2-mL solution containing 15%/15%/70% polyethylated castor oil/ethanol/water to obtain a final concentration of 0.5 mg/mL for injection. The reconstituted solution was stable for 48 hours at room temperature and under ambient light. The appropriate volume of this stock solution was added to 100 mL of isotonic saline to yield the required dose. Aplidine was administered as a 24-hour intravenous infusion using rate-controlled electronic pumps every 2 weeks through a central intravenous line. Patients had to have recovered to at least grade 1 toxicity before receiving the next dose of the drug. If the patient had not recovered by the time of the next infusion, a rest week was proposed. Patients requiring deferral of treatment for more than 2 weeks because of aplidine-related toxicity were withdrawn from this study.
Dose-Escalation Procedure
On the basis of toxicologic studies in rodents, the dose of 0.4 mg/m2/mo (approximately one tenth of the MTD) administered as 0.2 mg/m2 every 2 weeks was selected as the starting dose for this trial; the dose was then escalated according to a modified Fibonacci scheme. A minimum of three patients were included at each dose level. DLT was defined as grade 4 neutropenia lasting 5 days or associated with temperature 38.5°C, grade 4 thrombocytopenia, or grade 3 nonhematologic toxicity (including hepatotoxicity and myotoxicity). Emesis, alopecia, and hypersensitivity reactions were not considered as DLTs. If no or minimal (including grade 1) toxicity was observed, doses were escalated in 50% to 100% steps; otherwise, if grade 2 toxicity (other than alopecia, anemia, and untreated nausea and vomiting) occurred, doses were escalated in 20% to 30% steps. The results of the pharmacokinetic analyses were taken into consideration for subsequent dose escalation.
The MTD was initially defined based on the occurrence of DLT during the first 2 months of treatment (up to the fifth infusion). During the course of the study, occurrence of DLT at cycle 3 in one patient prompted us to redefine the MTD based on the occurrence of DLT at any cycle. If DLT was observed in any of the patients entered at a specific dose level, an additional three patients were enrolled at that dose level. The dose at which DLT occurred in two or more patients was defined as the MTD. The recommended safe dose for further study was defined as the dose level immediately below the MTD. At least 12 patients, of whom six patients had to have received at least 2 months of treatment (four infusions), were required to define the recommended dose.
Plasma and Urine Pharmacokinetic Sampling and Assay
A pharmacokinetic profile of aplidine was obtained after the first infusion. Venous blood samples were collected from different infusion sites into tubes containing lithium-heparin anticoagulant immediately before the start of the infusion (time 0); at 3, 6, 8, and 12 hours during the infusion; at the end of the infusion (24 hours); and at the following times thereafter: at 15, 30, and 45 minutes and at 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, and between 120 and 168 hours after infusion. The sample was immediately centrifuged, and the resulting plasma was stored at –20°C before analysis. A pretreatment sample of urine was collected. Thereafter, three separate urine samples were collected according to the time interval after aplidine (0 to 24 hours and 24 to 48 hours) and stored at 4°C. Each container was thoroughly mixed, and its total volume was recorded. Two duplicate samples of 10 mL from each container were stored at –20°C before analysis.
The concentration of aplidine in whole blood, plasma, and urine was measured using high-performance liquid chromatography coupled to a mass spectrometer. Aplidine concentrations in biologic fluids were assayed using a method described elsewhere.11 The lower limit of quantification of aplidine was 0.25 ng/mL. Plasma concentration and whole blood versus time profiles were generated, and both observed and calculated variables were reported. These variables included area under the curve from time zero to infinity (AUCinf), maximum concentration (Cmax), clearance, terminal half-life (t1/2), median residual time (MRT), and volume of distribution (Vss). Statistical analysis was performed using the SAS PROC MIXED procedure (SAS Institute, Cary, NC). The level of statistical significance was set at = .05, and CI criteria for assessment of dose proportionality were calculated according to the Smith et al12 method. Pharmacokinetic and pharmacodynamic interactions were performed using both Pearson and Spearman correlation in SAS.
RESULTS
General
A total of 67 patients with a variety of advanced malignancies were entered onto the study. One patient who experienced early tumor progression before receiving aplidine was not assessable for toxicity. Patient characteristics (n = 66) are listed in Table 1. All but one patient had received prior chemotherapy, with a median number of three prior regimens (range, zero to seven regimens). A total of 161 cycles of aplidine were administered at doses ranging from 0.2 to 8 mg/m2 (Table 2). The median number of courses administered per patient was two (range, one to seven courses).
Dose Escalation
Dose escalation from 0.2 to 4 mg/m2 was not associated with acute and/or delayed DLT. One patient receiving aplidine 1.6 mg/m2 experienced thrombocytic microangiopathy, which was later considered unrelated to aplidine but led us to increase the cohort from three to six patients. Three patients entered at 5 mg/m2 had no DLT, so the dose level of 6 mg/m2 was explored. At this dose level, at day 15 of cycle 2 (15 days after the third injection), one of the first three patients experienced grade 3 muscle toxicity with grade 3 CPK elevation (with approximately a seven-fold increase in the aldolase baseline value), grade 2 myalgia, grade 2 muscle weakness, and grade 3 asthenia. This prompted us to increase the cohort from three to six patients. Another patient treated at 6 mg/m2 presented at day 27 of cycle 3 (13 days after the sixth injection) with grade 2 CPK elevation lasting for more than 15 days. Although this DLT occurred after the period that was classified for the initial definition of MTD, the severity of this delayed toxicity was later considered as part of the MTD. Meanwhile, dose escalation had been resumed, and four patients had already been treated at 7 mg/m2; one of these patients developed grade 4 dose-limiting neutropenia.
The occurrence of muscle DLT in two of six patients at 6 mg/m2 prompted us to stop dose escalation and define dose levels 6 mg/m2 as the MTD. The cohort of patients treated at 5 mg/m2 was further extended to define the recommended dose. Among a total of 14 patients, three (21%) developed DLT, which consisted of grade 3 to 4 myotoxicity (two patients) during cycle 2 at days 19 and 21 after the third injection and grade 3 transaminase elevation at cycle 1 (one patient).
Aplidine myotoxicity, as characterized by muscle cramps, pain, weakness, and/or increases in CPK, was found to be closely related to the symptoms described in the adult form of carnitine palmitoyl transferase deficiency type 2, which is a genetic disease treated with L-carnitine.13 On the basis of preclinical data showing that aplidine inhibits palmitoyl thiosterase, an enzyme related to the carnitine palmitoyl transferase family, we proposed L-carnitine to treat and prevent aplidine-induced effects on muscles. The dose of 1g/10 kg L-carnitine used in this study was based on doses classically used in carnitine deficiency.14
Dose escalation was reinitiated from 6 mg/m2 of aplidine associated with an oral solution of L-carnitine at a dose of 1 g/10 kg (three times a day) from the first day of treatment up to at least 4 weeks after the last aplidine injection. Four patients, instead of three patients, received aplidine 6 mg/m2 with carnitine (one patient was not assessable for safety because of early tumor progression), and no DLT was reported. Dose escalation was pursued at aplidine 7 mg/m2 plus carnitine in three patients without DLT, and the dose was further escalated to aplidine 8 mg/m2 plus carnitine. Of the four patients treated at this dose level, one patient developed DLT consisting of grade 3 asthenia and grade 1 fever (flu-like syndrome) at cycle 1, and another patient developed grade 3 to 4 myotoxicity at cycle 2. Because two of four patients experienced DLT, this dose level was considered the MTD, and the subsequent patients were treated at 7 mg/m2. Of a total of 12 patients treated at aplidine 7 mg/m2 plus carnitine, two patients (16%) experienced grade 3 to 4 myotoxicity, both at cycle 2. On the basis of toxicity during the first two cycles, the recommended doses were 5 and 7 mg/m2 without and with carnitine, respectively.
Myotoxicity
Muscle toxicity was reported when doses 5 mg/m2 of aplidine were administered. In the first cohort without carnitine, muscle toxicity consisting of myalgia, muscle weakness, and/or CPK elevation was the main relevant adverse effect observed with aplidine (Table 3). Grade 1 to 2 myalgia occurred in 12 patients (26.0%) and 17 cycles (14.5%). Grade 1 to 2 muscle weakness was documented in four patients (8.7%) during four cycles (3.4%). Elevated CPK and aldolase levels were reported in 10 patients (21.7%) and 15 cycles (12.8%), reaching grade 3 to 4 in three patients (6.5%). Clinical symptoms typically started 7 to 10 days after the third injection of aplidine (middle of cycle 2), with muscle pain predominantly in the shoulders, back, and thighs. Pain was followed by muscle weakness and an increase in CPK (Fig 2A), occurring within 7 to 15 days after the onset of myalgia (ie, 15 to 21 days after the third injection of aplidine). Among patients with myotoxicity who had a muscle biopsy, light microscopy showed either normal features or minimal necrosis (type II diffuse fiber atrophy). There was no evidence of rhabdomyositis. Electron microscopy analysis showed unspecific accumulation of glycogen and autophagocytic vacuoles, and the major change was the disappearance of thick filaments of myosin (Fig. 2B). Importantly, aplidine-induced muscle toxicity was not associated with myocardic and/or diaphragmatic myotoxicity. In the first patients presenting with severe muscle toxicity, a complete recovery occurred within 2 months after discontinuation of aplidine. In the following patients, the treatment was postponed; active physiotherapy and L-carnitine administered orally at a dose of 1 g/10 kg reduced the delay of reversibility to 4 to 6 weeks (Fig 3). In the second cohort of patients treated prophylactically with carnitine, severe myotoxicity was observed in only three (15%) of 20 patients receiving higher doses of aplidine ranging from 6 to 8 mg/m2 (Tables 2 and 3).
Other Toxicities
Grade 1 to 2 asthenia was observed in 44 patients (65.7%) during 89 cycles (89%) and was severe in only four patients who also experienced muscle toxicity. GI symptoms included grade 1 to 2 nausea and vomiting in 54 patients (80.6%) and grade 1 to 2 diarrhea in 12 patients (17.9%). One patient presented with grade 3 fever concomitantly with asthenia without documentation of infection at the highest dose level. Other toxicities included grade 3 elevation of transaminases without evidence of hepatic metastasis in one patient and grade 1 to 2 local pain at the injection site in two patients. Hematologic toxicity was limited to rare episodes of asymptomatic neutropenia in 4.5% of patients and 1.7% of cycles, whereas grade 1 to 2 thrombocytopenia was reported in 10.6% of cycles. Asymptomatic grade 3 lymphopenia occurred in 19.4% of patients (11.2% of cycles).
Pharmacokinetics
A pharmacokinetic analysis was performed in 48 patients during the first infusion of cycle 1 (Table 4). Aplidine concentrations were near or less than the lower limit of quantification for doses less than 0.8 mg/m2, thus prohibiting adequate quantification.
The pharmacokinetic analysis indicates that aplidine has a relatively long half-life, low clearance, and a high volume of distribution in plasma (Fig 4). In plasma and whole blood, mean half-life values ranged from 21 to 44 hours. Aplidine mean clearance values in plasma ranged from 45 to 49 L/h, with Vss values ranging from 1,036 to 1,124 L; whereas in whole blood, clearance ranged from 3.0 to 6.2 L/h with high interpatient variability, and mean Vss values ranged from 102 to 145 L. As described earlier, whole-blood aplidine concentrations were frequently higher than concentrations in plasma for patients who had measurement of concentrations in both media. When comparisons were made between blood and plasma results at the 6 mg/m2 dose level, mean whole-blood Cmax and AUCinf values were approximately 144 ng/mL and 5,430 ng · h/mL, respectively, compared with values of approximately 32 ng/mL for Cmax and 1,113 ng · h/mL for AUCinf in plasma.
Aplidine area under the curve calculated to the last time point (AUClast), AUCinf, and Cmax values generally increased with the dose. Dose proportionality of aplidine in plasma or whole blood was not proven, most probably because of the limited sample size for all dose levels and high interpatient variability. Compartmental analysis showed that plasma profiles best fit a first-order two-compartment model.
Urinary excretion of aplidine was low, with a mean recovery of 4.16% in 48 hours, and was less than 15% in all patients. There was no correlation between patient characteristics at study entry and pharmacokinetic results.
Antitumor Activity
Among 66 patients enrolled, no objective response was observed. Fifteen patients had stable disease lasting 2 to 16 months as their best response. Among the patients, eight (treated at 5, 6, and 7 g/m2 in three, three, and two patients, respectively) with documented tumor progression in the 3 months preceding study entry had tumor stabilization for 6 months. Interestingly, six patients with endocrine tumors (medullary thyroid carcinoma, bronchial carcinoid tumor, and malignant pheochromocytoma in four, one, and one patient, respectively) experienced a clinical benefit and prolonged disease stabilization (> 6 months) associated with a relevant decrease in their tumor markers.
DISCUSSION
We report here the results of a large-scale phase I study using the marine compound aplidine administered in a 24-hour infusion every 2 weeks. Dose escalation greater than 5 g/m2 revealed dose-limiting myotoxicity consisting of myalgia and muscle weakness followed by an elevated CPK level. Myotoxicity occurred mainly at high doses and during prolonged exposure and affected patients with high, sustained plasma aplidine concentrations. Indeed, the three patients showing severe myotoxicity had t1/2 in excess of 44 hours compared with a median t1/2 of 25.8 hours after a 24-hour infusion. Aplidine-induced muscle toxicity resembles the toxicity extensively described in alcohol- and some congenital-induced proximal myopathies. At microscopic analysis, aplidine seems to affect fast-twitch type II muscle fibers, which are particularly vulnerable to the imbalanced metabolism of skeletal muscle proteins.15 Although the precise mechanism of toxicity remains unknown, aplidine inhibits thioesterase I, a palmitoyl protein16 closely related to carnitine palmitoyl transferase 2, which is a muscle enzyme the deficiency of which leads to congenital myopathy in children.13 Another possible mechanism of toxicity may involve the interaction with elongation factors, given that impairment in translation efficiency is known to decrease muscle protein synthesis.17
Interestingly, the use of carnitine seems to facilitate recovery from myotoxicity and allowed us to pursue further aplidine treatment after toxicity. An extensive in vitro program conducted in a broad panel of solid tumors and leukemia has shown that carnitine does not interact with aplidine cytotoxicity in cancer cells and ruled out any tumor protective effect.18,19 The prophylactic use of high-dose carnitine enabled us to increase the recommended dose of aplidine without severe myotoxicity but with a slight increase in grade 1 to 2 diarrhea (Table 3), which is a toxicity known to be also related to carnitine.
Other digestive toxicities were frequent but remained mild to moderate (grade 1 to 2 nausea and vomiting in 80.6% of the patients and 61.5% of cycles). Transient hepatotoxicity was observed in only two patients at doses 5 mg/m2. Hematologic toxicity of aplidine was low because only three episodes of asymptomatic grade 3 to 4 neutropenia were documented in three patients and no grade 3 to 4 thrombocytopenia occurred. Although grade 1 to 3 lymphopenia was frequently reported, it was not associated with any clinical meaningful immunosuppression.
During the course of the study, patient plasma levels of aplidine were found to be lower than expected from animal pharmacokinetic data. Therefore, we decided to explore both plasma and whole-blood concentrations in humans for doses of 5 and 6 mg/m2. We found major differences between plasma and whole-blood concentrations, suggesting RBC count partitioning of aplidine in human. Interestingly, interpatient clearance variability of aplidine in plasma was lower than that of whole-blood clearance. This led us to favor further evaluation of whole-blood pharmacokinetic data at doses of more than 6 mg/m2. Considering the high molecular weight of aplidine, our results suggest the adsorption of aplidine on erythrocytes and/or other blood cells, making possible variability in the release of aplidine from blood cells in individuals. Furthermore, we found that the degrees of variability between body surface area- (BSA) normalized and fixed dosing were low, suggesting that no improvement in variability in whole blood would be expected from normalizing the dose based on BSA. Pharmacokinetic modeling will have to be performed to determine the clinical consequences of this possible blood-cell partitioning and the variability of BSA-normalized versus fixed dosing of aplidine.
The recommended doses in our study are 7 and 5 mg/m2 with and without prophylactic carnitine, respectively. On the basis of our data and those of other phase I studies, the toxicity profile of aplidine is henceforth well defined at the recommended doses (transient, mild to moderate asthenia, nausea and vomiting, myalgia, and CPK and/or transaminase elevation without hematologic toxicity or alopecia). When several schedules are compared, aplidine toxicity seemed, at least in part, dependent on the duration of exposure. Protracted infusions of aplidine were reproducibly associated with myotoxicity, which was also reported as the DLT in another phase I trial using a 24-hour weekly infusion, 3 consecutive weeks out of 4 weeks.20 In contrast, shorter duration infusions of 1 to 3 hours21-23 were associated with severe transaminitis with sparse myotoxicity. One case of fatal renal, hepatic, and cardiac failure has also been reported at the highest dose level using the 3-hour schedule.21 Regardless of the schedule, dose-intensities of 24-hour schedules (ranging from 10 to 11 mg/m2/4 wk) compare favorably with the dose-intensities of 1- to 3-hour schedules (8 to 10 mg/m2/4 wk) at the recommended dose.
Although antitumor activity was not the primary objective of this study, six patients with endocrine tumors (four with medullary thyroid carcinoma) benefited from aplidine treatment, with long-term stabilization of their disease lasting 6 to 16 months and including one minor response of approximately 40% in a patient with bronchial carcinoid tumor. Within the entire phase I program, 10 patients with advanced medullary thyroid carcinoma received aplidine. All but one patient were symptomatic at baseline, with pain (n = 7), diarrhea (n = 3), and an elevated median carcinoembryonic antigen value of 363 ng/mL at entry (range, 5.4 to 7,828 ng/mL). Symptoms improved in four patients. Carcinoembryonic antigen decreased in five of seven assessable patients (reductions of 30%, 45%, 45%, 48%, and 70%). A partial response and two incidents of tumor shrinkage of 20% and 27% were documented in three of five patients with measurable disease, suggesting that aplidine is able to yield a clinical benefit in patients with advanced medullary thyroid cancer. Activity observed in other neural crest–derived tumors (malignant pheochromocytoma and carcinoid tumors) also warrants further clinical and mechanistic research using aplidine.
In summary, dose-limiting myotoxicity consisting of type II fiber atrophy requires careful clinical and biologic monitoring during treatment with high doses of aplidine. The role of carnitine warrants further exploration to improve the safety of aplidine in future trials. At the recommended doses, the toxicity profile, including the lack of hematotlogic toxicity, makes aplidine potentially combinable with other cytotoxic or targeted compounds. Aplidine allowed prolonged periods ( 6 months) of drug administration in patients with evidence of a clinical benefit. On the basis of our data, this schedule was selected for further phase II clinical development.
Authors' Disclosures of Potential Conflicts of Interest
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Acknowledgment
We gratefully acknowledge the nursing staff of the Department of Medicine (GARD unit) for their assistance and Lorna Saint-Ange for editing at the Institute Gustave-Roussy.
NOTES
Supported in part by PharmaMar, Madrid, Spain.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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