Phase I Clinical and Pharmacokinetic Study of Kahalalide F AdministeredWeekly as a 1-Hour Infusion to Patients with Advanced SolidTumors

A dose-escalation, phase I study evaluated the safety, pharmacokinetics, and efficacy of a weekly 1-h regimen of kahalalide F, a cyclic depsipeptide isolated from the marine mollusk Elysia rufescens , in adult patients with advanced solid tumors and no standard treatment available.  Patients received an i.v. 1-h infusion of kahalalide F once weekly until disease progression or unacceptable toxicity. The starting kahalalide F dose was 266 Ag/m, and dose escalation proceeded based on the worst toxicity found in the previous cohort.  Thirty-eight patients were enrolled at three Spanish institutions and received onceweekly kahalalide F1-h infusions at doses between 266 and1,200 Ag/m. Dose-limiting toxicities consisted of transient grade 3/4 increases in transaminase blood levels. The maximum tolerated dose for this kahalalide F schedule was 800 Ag/m, and the recommended dose for phase II studies was 650 Ag/m. No accumulated toxicity was found. One patient with malignant melanoma had unconfirmed partial response, one patient with metastatic lung adenocarcinoma had minor response, and six patients with different types of metastatic solid tumors had stable disease for 2.8 to 12.7 months. The noncompartmental pharmacokinetics of this kahalalide F schedulewas linear and showedanarrowdistribution and short body residence.The transaminitis associated with kahalalide F was dose dependent. Themaximum tolerated dosewas 800 Ag/m. Dose-limiting toxicities withweekly kahalalide F1-h i.v. infusions were transient grade 3/4 increases in blood transaminase levels, and 650 Ag/m was declared the recommended dose for phase II studies. This schedule showed a favorable safety profile and hints of antitumor activity. Kahalalide F is a moderately soluble cyclic depsipeptide isolated from the marine mollusk Elysia rufescens (1, 2). Its chemical structure, elucidated using spectroscopic and physicochemical methods, consists of a cyclic region and a lateral NH2-terminal chain (3). Preclinical studies showed that kahalalide F is a COMPARE (National Cancer Institute)-negative compound that induces strong cytotoxic activity against solid tumor cell lines in vitro. Half maximal inhibitory concentration (IC50) values ranging from 200 nmol/L to 10 Amol/L were reported for kahalalide F against colon, central nervous system, melanoma, prostate, and breast cancer cell lines of human origin and, in general, against tumor cell lines overexpressing the Her2/neu gene, with hormone-independent prostate cancer cells being the most sensitive to kahalalide F (4). The cytotoxic effects of kahalalide F were confirmed in vitro using human tumor colony-forming units isolated from surgically removed samples of breast, colon, kidney, non–small cell lung, ovary, prostate, stomach, and uterine cancer (5). Kahalalide F was effective against several cell lines with strong multidrug resistance (e.g., PC-3 prostate, CACO-2 colon, UO-31 renal, or MCF7 breast) and against cell lines resistant to topoisomerase II inhibitors. No significant protection against cytotoxicity induced by kahalalide F was found with ectopic overexpression of the multidrug resistance protein, MDR1, or with inhibition of protein synthesis or caspase-dependent apoptosis (6). In vivo, total kahalalide F doses of 140 and 210 Ag/kg administered i.p. once daily for 5 days resulted in treatment-to-control ratios of 15% and 5%, respectively, in athymic mice with human PC-3 prostate cancer samples implanted i.p. using hollow silastic fibers. Antitumor activity was also found against other human cell lines xenografted into athymic mice, including breast cancer (treatment-to-control ratio of 22%), non–small cell lung (treatment-to-control ratio of 37%), and colon cancer (treatmentto-control ratios of 41-47%). The feasibility of using multicourse Cancer Therapy:  Institut Catala' d’Oncologia and Hospital Vall d’Hebron, Barcelona, Spain; and Hospital Universitario 12 de Octubre and PharmaMar R&D, ColmenarViejo, Madrid, Spain Received 9/18/07; revised11/14/07; accepted11/18/07. 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 with18 U.S.C. Section1734 solely to indicate this fact.  Izquierdo is currently an employee of PharmaMar. Requests for reprints:Beatriz Pardo, Institut Catala' d’Oncologia, Av. GranVia s/n, km. 2.7, 08907-L’Hospitalet de Llobregat, Barcelona, Spain. Phone: 34932607744; , 2008 1116 Research. on April 19, 2017.  The primary mechanism of action of kahalalide F remains to be elucidated, although multiple membrane-associated targets have been found that may be related to its hydrophobic nature, and evidence suggests that lysosomes may be a likely target. Incubation with kahalalide F even for short periods of time quickly induced loss of mitochondrial membrane potential and lysosomal integrity, severe cytoplasmic swelling and vacuolization, irregular clumping of chromatin within the cell nucleus, and finally, cell death in human breast and prostate cancer cells. These effects were independent of caspase activation and were not associated with DNA degradation or cell cycle block (6). Biologically relevant concentrations of kahalalide F induced similar effects, together with detachment from substrate and migration of large vacuoles from the cell periphery to a perinuclear location, both in normal kidney cells and in human epithelioid cervical carcinoma cells. Neither the cytoskeleton nor the morphology of the Golgi apparatus and the endoplasmic reticulum were affected by kahalalide F (8). In vitro tests have identified a cell cycle block in G0-G1 in several tumor cell lines sensitive to kahalalide F (IC50 values in the range of 1 Amol/L), including prostate, cervical, colon, head and neck, and non–small cell lung (7). Other studies found that kahalalide F also inhibits Her2/neu transmembrane tyrosine kinase activity, blocks the epidermal growth factor receptor, and inhibits transforming growth factor a gene expression (9), all of which are growth factors that control the tyrosine kinase class I subfamily that normally mediates signal transduction in the Ras signaling pathway. Several lines of evidence point to ErbB3 as a major determinant of kahalalide F action. The sensitivity to kahalalide F in a panel of tumor cell lines (including non–small cell lung, breast, ovarian, and hepatic carcinomas) significantly correlated with ErbB3 protein expression levels, whereas no correlation was found with the levels of epidermal growth factor receptor, ErbB2, and ErbB4. In contrast to the general down-regulation of ErbB family members observed after long-term exposure to kahalalide F, a 4-h treatment with kahalalide F resulted in the selective down-regulation of ErbB3 in a sensitive, high ErbB3expressing cell line; moreover, ectopic expression of ErbB3 increased the kahalalide F sensitivity of a resistant cell line. The PI3K/Akt signaling pathway coupled to ErbB receptors was also affected by kahalalide F treatment. Kahalalide F decreased phosphorylated Akt levels, and the ectopic expression of a constitutively active Akt mutant reduced kahalalide F cytotoxicity in a sensitive cell line. Moreover, kahalalide F induced the redistribution of p27, a marker for Akt activity, from the cytosol to the nucleus (10). The toxicity profile of kahalalide F has been evaluated in mice, rats, and rabbits. Single i.v bolus injections of kahalalide F resulted in reversible alterations in liver function and, at doses greater than the MTD of 250 Ag/kg, reversible nephrotoxicity and potentially fatal neurotoxicity in rodents (7). Fractionated administration of kahalalide F for 5 days did not result in drugrelated mortality, histopathologic lesions, or clinical signs of toxicity in mice or rats (11). A previous phase I clinical trial evaluated kahalalide F given daily for 5 days in cycles of 3 weeks in patients with advanced androgen-resistant prostate cancer. The dose-limiting toxicities (DLT) were reversible, and asymptomatic grade 3/4 increases in blood levels of transaminases. No other relevant toxicities, including severe hematologic toxicity, were observed, and the recommended dose was 560 Ag/m (12). The aim of this phase I study was to establish the MTD, and the recommended dose of kahalalide F administered as a weekly i.v. 1-h infusion to adult patients with advanced solid tumors. Materials andMethods This dose-escalating phase I study was conducted in three Spanish centers: the Institut Català d’Oncologia, the Vall d’Hebron University Hospital (both in Barcelona), and the Hospital Universitario 12 de Octubre (Madrid). The study was conducted in accordance with the Good Clinical Practice requirements and the Declaration of Helsinki. The Independent Ethics Committees of the three centers approved the study protocol, and all patients were required to give their written informed consent before being entered into the study.  The primary objective was to determine the MTD and the recommended dose for phase II studies of kahalalide F weekly i.v. 1-h infusion administered to adult patients with advanced solid tumors. The secondary objectives were to determine the safety and toxicity profile, and the corresponding DLTs, of kahalalide F; to study the pharmacokinetics of kahalalide