Luis Lopez-Lazaro

Phase I and Pharmacokinetic Study of Aplidine, a New Marine Cyclodepsipeptide in Patients With Advanced Malignancies

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/m(2). 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/m(2). 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/m(2) 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/m(2). 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/m(2) without and with carnitine, respectively. The role of carnitine will be further explored in phase II studies.

Phase I Clinical and Pharmacokinetic Study of Kahalalide F in Patients with Advanced Androgen Refractory Prostate Cancer

Purpose: The purpose is to determine the maximum tolerated dose, profile of adverse events, and dose-limiting toxicity of Kahalalide F (KF) in patients with androgen refractory prostate cancer. Furthermore, the pharmacokinetics after KF administration and preliminary antitumor activity were evaluated. KF is a dehydroaminobutyric acid–containing peptide isolated from the marine herbivorous mollusk, Elysia rufescens. Experimental Design: Adult patients with advanced or metastatic androgen refractory prostate cancer received KF as an i.v. infusion over 1 hour, during five consecutive days every 3 weeks. The starting dose was 20 μg per m2 per day. Clinical pharmacokinetics studies were done in all patients using noncompartmental analysis. Prostate-specific antigen levels were evaluated as a surrogate marker for activity against prostate cancer. Results: Thirty-two patients were treated at nine dose levels (20-930 μg per m2 per day). The maximum tolerated dose on this schedule was 930 μg per m2 per day. The dose-limiting toxicity was reversible and asymptomatic Common Toxicity Criteria grade 3 and 4 increases in transaminases. The recommended dose for phase II studies is 560 μg per m2 per day. Pharmacokinetics analysis revealed dose linearity up to the recommended dose. Thereafter, a more than proportional increase was observed. Elimination was rapid with a mean (SD) terminal half-life (t1/2) of 0.47 hour (0.11 hour). One patient at dose level 80 μg per m2 per day had a partial response with a prostate-specific antigen decline by at least 50% for ≥4 weeks. Five patients showed stable disease. Conclusions: KF can be given safely as a 1-hour i.v. infusion during five consecutive days at a dose of 560 μg per m2 per day once every 3 weeks.

Clinical pharmacology of the novel marine-derived anticancer agent Ecteinascidin 743 administered as a 1- and 3-h infusion in a phase I study

Ecteinascidin 743 (ET-743) is an anticancer agent derived from the Caribbean tunicate Ecteinascidia turbinata. In the present article, the pharmacokinetics and pharmacodynamics of ET-743 are described within a phase I study. Forty patients with solid tumors initially received ET-743 as a 1-h i.v. infusion every 21 days at nine dose levels (50–1100 μ g/m2). The maximal tolerated dose (MTD) was 1100 μ g/m2, with thrombocytopenia and fatigue as dose-limiting toxicities (DLTs). As this MTD was substantially lower than in parallel phase I studies, dose escalation continued using a prolonged, 3-h infusion. Thirty-two patients were entered at five dose levels (1000–1800 μ g/m2). The MTD was 1800 μ g/m2 with pancytopenia and fatigue as DLTs. The recommended phase II dose was 1650 μ g/m2 given over 3 h at which 12 patients were treated. Pharmacokinetic monitoring was performed for both treatment schedules. Non-compartmental pharmacokinetic parameters at the recommended dose with the 3-h infusion were (mean value±SD): clearance 87±30 l/h and mean elimination half-life 26±7 h. Pharmacokinetics were linear at the dose range tested with this schedule. The percentage decrease in platelets, white blood cells and neutrophils correlated with the area under the plasma concentration versus time curve (AUC), dose and maximal plasma concentration (Cmax). Hepatic toxicity increased with dose, AUC and Cmax. Administration of 1650 μ g/m2 ET-743 over 3 h seemed clinically feasible; pharmacokinetics were linear with this schedule. Hepatic and hematological toxicities correlated with exposure to ET-743.

Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man.

Ecteinascidin 743 (ET-743) is a potent anti-tumoral agent of a marine origin. It is currently being tested in phase II clinical trials using a 3-weekly 24-h i.v. infusion of 1500 μg/m2 and 3-h infusions of 1650 μg/m2. Knowledge of the metabolism of ET-743 is, however, still scarce. In the present study, a qualitative chromatographic discovery of metabolites of ET-743 in man is reported. ET-743 and its demethylated analog ET-729 were incubated at 37°C in the presence of enzyme systems, pooled human microsomes, pooled human plasma and uridine 5′-diphosphoglucuronyltransferase, respectively, in appropriate media. Reaction products were investigated chromatographically using photodiode array and ion spray-mass spectrometric detection (LC-MS). The main reaction products in microsomal incubations of ET-743 resulted from a remarkable breakdown of the molecule. In plasma the drugs were deacetylated, and the transferase did actually yield a glucuronide of both ET-743 and ET-729. In contrast, screening of urine, plasma and bile, collected from patients treated with ET-743 at the highest dose levels, using a sensitive LC-MS assay, did not result in detection of ET-729 and metabolites which were generated in vitro. The urinary excretion of ET-743 in man was lower than 0.7% of the administered dose for a 24-h infusion.

Trabectedin (YondelisTM, formerly ET-743), a mass balance study in patients with advanced cancer

Trabectedin (YondelisTM, formerly ET-743) is an anti-cancer drug currently undergoing phase II development. Despite extensive pharmacokinetic studies, the human disposition and excretory pathways of trabectedin remain largely unknown. Our objective was to determine the mass balance of trabectedin in humans. To this aim, we intravenously administered [14C]trabectedin to 8 cancer patients, followed by collection of whole blood, urine and faeces samples. A 24-h infusion was administered to 2 patients, whereas the other 6 patients received a 3-h infusion. Levels of total radioactivity and unchanged trabectedin were determined and used for calculation of pharmacokinetic parameters. No schedule dependency of pharmacokinetic parameters was observed apart from Cmax. Plasma and whole blood concentrations of [14C]trabectedin related radioactivity were comparable. Only 8% of the plasma exposure to [14C]trabectedin related compounds is accounted for by trabectedin, indicating the importance of metabolism in trabectedin elimination. Trabectedin displays a large volume of distribution (±1700 L), relative to total radioactivity (±220 L). [14C]trabectedin related radioactivity is mainly excreted in the faeces (mean: 55.5% of the dose). Urinary excretion accounts for 5.9% of the dose on average resulting in a mean overall recovery of 61.4% (3-h administration schedule). The excretion of unchanged trabectedin is very low both in faeces and in urine (< 1% of dose). In conclusion, trabectedin is extensively metabolised and principally excreted in the faeces.

Development of a lyophilized parenteral pharmaceutical formulation of the investigational polypeptide marine anticancer agent kahalalide F.

Kahalalide F is a novel antitumor agent isolated from the marine mollusk Elysia rufescens; it has shown highly selective in vitro activity against androgen-independent prostate tumors. The purpose of this study was to develop a stable parenteral formulation of kahalalide F to be used in early clinical trials. Solubility and stability of kahalalide F were studied as a function of polysorbate 80 (0.1%–0.5% w/v) and citric acid monohydrate (5–15 mM) concentrations using an experimental design approach. Stabilities of kahalalide F lyophilized products containing crystalline (mannitol) or amorphous (sucrose) bulking agents were studied at +5°C and +30°C ±60% relative humidity (RH) in the dark. Lyophilized products were characterized by infrared (IR) spectroscopy and differential scanning calorimetry (DSC). Recovery studies after reconstitution of kahalalide F lyophilized product and further dilution in infusion fluid were carried out to select an optimal reconstitution vehicle. It was found that a combination of polysorbate 80 and citric acid monohydrate is necessary to solubilize kahalalide F. Lyophilized products were considerably less stable with increasing polysorbate 80 and citric acid monohydrate concentrations, with polysorbate 80 being the major effector. A combination of 0.1% w/v polysorbate 80 and 5 mM citric acid monohydrate was selected for further investigation. Lyophilized products containing sucrose as a bulking agent were more stable compared to the products containing mannitol. The glass transition temperature of the sucrose-based product was determined to be + 46°C. The amorphous state of the product was confirmed by IR analysis. A solution composed of Cremophor EL, ethanol, and water for injection (5%/5%/90% v/v/v CEW) kept kahalalide F in solution after reconstitution and further dilution with 0.9% w/v sodium chloride (normal saline) to 1.5 μg/m. A stable lyophilized formulation was presented containing 100 μg of kahalalide F, 100 mg sucrose, 2.1 mg citric acid monohydrate, and 2 mg polysorbate 80 to be reconstituted with a vehicle composed of 5%/5%/90% v/v/v CEW and to be diluted further using normal saline.

Chemical and enzymatic stability of a cyclic depsipeptide, the novel, marine-derived, anti-cancer agent kahalalide F.

Kahalalide F is a cyclic depsipeptide isolated from the Hawaiian mollusk Elysia rufescens. This compound is under present phase I clinical investigations as an anti-tumor drug. The role of possible metabolic reactions of this drug in (pre-)clinical investigations has not yet been explored. The first results for kahalalide F in this field of research are given in this paper. The chemical degradation of kahalalide F was investigated under acid, neutral and alkaline conditions using high-performance liquid chromatography with ultraviolet detection. The half-lives at 80°C were 1.1, 20 and 8.6 h at pH 0, 1 and 7, respectively. At 26°C and pH 11, the half-life was 1.65 h. At pH 7 and 11, only one reaction product of kahalalide F was observed, kahalalide G, the hydrolyzed lactone product of kahalalide F. At pH 0 and 1, additional reaction products emerged. Metabolic conversion of kahalalide F was tested in vitro using three different enzyme systems based on pooled human microsomes, pooled human plasma and uridine 5′-diphosphoglucuronyl transferase, respectively. The incubated samples were analyzed using the same chromatographic technique as for the degradation samples. Biotransformations were not observed under these conditions and, therefore, it is concluded that kahalalide F is a metabolically stable drug.

Plitidepsin Has a Cytostatic Effect in Human Undifferentiated (Anaplastic) Thyroid Carcinoma

Undifferentiated (anaplastic) thyroid carcinoma is a highly aggressive human cancer with very poor prognosis. Although there have been a few studies of candidate treatments, the fact that it is an infrequent tumor makes it very difficult to design clinical trials. A strong association has been observed between undifferentiated thyroid carcinoma and TP53 mutations in numerous molecular genetic and expression studies. Plitidepsin (Aplidin, PharmaMar, Madrid, Spain) is a novel anticancer compound obtained from a sea tunicate. This compound has been reported to induce apoptosis independently of TP53 status. We investigated the actions of plitidepsin in human thyroid cancer cells. In initial experiments using primary cultured cells from a differentiated (papillary) carcinoma, we found that 100 nmol/L plitidepsin induced apoptosis, whereas lower doses were cytostatic. Because our aim was to study the effects of plitidepsin at clinically relevant concentrations, subsequent experiments were done with a dosage regimen reflecting plasma concentrations observed in previously reported clinical trials: 100 nmol/L for 4 hours, followed by 10 nmol/L for 20 hours (4100/2010 plitidepsin). This plitidepsin dosage regimen blocked the proliferation of a primary undifferentiated/anaplastic thyroid carcinoma culture obtained in our laboratory and of a commercial cell line (8305C) obtained from an undifferentiated thyroid carcinoma; however, it did not induce apoptosis. The proportion of cells in the G1 phase of the cell cycle was greatly increased and the proportion in the S/G2-M phases greatly reduced, suggesting that plitidepsin blocks G1-to-S transition. Levels of the cyclin D1/cyclin-dependent kinase 4/p21 complex proteins were decreased and, in line with this, the levels of unphosphorylated Rb1 increased. The decrease in cell cycle proteins correlated with hypoacetylation of histone H3. Finally, we did experiments to assess how rapidly tumor cells return to their initial pretreatment proliferative behavior after 4100/2010 plitidepsin treatment. Cells from undifferentiated tumors needed more than 3 days to recover logarithmic growth, and after 7 days, cell number was still significantly lower than in control cultures. 4100/2010 plitidepsin inhibited the growth in soft agar. Together, our data show that plitidepsin is able to block in vitro cell cycle progression at concentrations similar to serum concentrations observed in vivo, and that this effect is persistent for several days after plitidepsin removal. Whether plitidepsin will prove to be clinically useful in the treatment of undifferentiated thyroid cancers remains to be established. However, our results raise the possibility that plitidepsin might be effective alone or in combination with radiotherapy and/or other drug treatments.

Trabectedin (ET-743, Yondelis™) is a substrate for P-glycoprotein, but only high expression of P-glycoprotein confers the multidrug resistance phenotype

SummaryTrabectedin (ET-743, Yondelis™) is a novel anticancer drug currently undergoing phase II and III investigations. There are various and conflicting reports whether trabectedin is a substrate for P-glycoprotein (P-gp), an important factor in drug disposition and multi-drug resistance (MDR). We have now unambiguously shown that trabectedin is a P-gp substrate by investigating vectorial transport over monolayers of LLC-PK1 pig kidney and Madine-Darby Canine kidney (MDCK) cells and the mdr1a and/or MDR1 transfected subclones. We further characterized the cytotoxic effects and cellular accumulation of trabectedin in these cell lines as well as in a panel of other cell lines with high or moderate expression levels of P-gp. Trabectedin displayed the typical MDR phenotype only in highly P-gp expressing cell lines, but not in cell lines with expression levels more closely conforming to clinical samples, suggesting that P-gp will not confer resistance to trabectedin in cancer patients.

Determination of Aplidin®, a marine-derived anticancer drug, in human plasma, whole blood and urine by liquid chromatography with electrospray ionisation tandem mass spectrometric detection

A sensitive and highly specific liquid chromatographic method with electrospray ionisation tandem mass spectrometric detection (LC-ESI-MS/MS) is reported for the determination in human plasma, whole blood and urine of Aplidin (APL), a novel depsipeptide derived from the tunicate Aplidium albicans with a potent cytotoxic activity under investigation in clinical studies. Didemnin B was used as internal standard and, after protein precipitation with acetonitrile and liquid-liquid extraction with chloroform, APL was separated by liquid chromatography using a reversed-phase C18 column and a linear gradient of acetonitrile in water (both containing 0.5% formic acid). Detection was performed using a turboionspray source operated in positive ion mode and by multiple reaction monitoring (MRM; m/z 1111 --> 295 for APL and m/z 1113 --> 297 for didemnin B). The method was linear (r > or = 0.9933) over the range 1-250 ng/ml, with intra- and inter-batch precision and accuracy below 12.2% (except at LLOQ < or = 15.4%) for both plasma and urine. Recoveries were moderate, ranging from 54 to 70% in plasma and blood, and from 46 to 60% in urine, for both APL and didemnin B. The LOD was 0.25 ng/ml for both matrices. APL resulted stable in the different matrices at least for 6 h (both at room temperature and 37 degrees C), after freeze and thaw cycles and long term storage at -20 degrees C. The method allowed demonstrating that APL is in a dynamic equilibrium between plasma and blood cells. Moreover, the method was successfully applied to the pharmacokinetic study of Aplidin in cancer patients.