Originally published as JCO Early Release 10.1200/JCO.2005.08.004 on October 3 2005

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Fishing for New Drugs

Cancer Research Unit, Newcastle General Hospital, Newcastle upon Tyne, United Kingdom

Natural products have provided a rich source of biologically active compounds and have contributed several of our most active anticancer drugs. For example, doxorubicin is a bacterial product, while paclitaxel is extracted from the yew tree, Taxus brevifolia. Many marine organisms have been found to contain pharmacologically active novel toxic compounds and have possibly evolved these as a defense mechanism against predators. Historically, extracts from marine organisms have been used more with the intention of poisoning for political advantage than for therapeutic purposes. The following excerpt shows that the Romans were well aware of the potential of organisms found in the sea: "Domitian (81-96) was accused (probably wrongly) of poisoning with sea-hare his brother Titus (who died unexpectedly at the age of 42, probably of malaria), and Agricola, governor of Britain (Tac. Agr. 43)."¹ So far, a limited number of compounds discovered in marine organisms have undergone clinical trials with the more charitable intention of improving the treatment of cancer. Among these are dolastatins, ecteinastatins, and didemnins. A phase I trial of dehydrodidemnin-B (aplidine) is reported in this issue.²

Didemnin B was originally reported in 1981 by Rinehart et al³ as a compound extracted from the Caribbean tunicate, Trididemnum solidum. Didemnin B was shown to have antiviral activity as well as anticancer activity. It was particularly potent, with IC50s reported in cell culture in the region of 10 nmol/L. It also extended the survival of mice bearing the P388 leukemia at µg doses.³ Didemnin B underwent extensive clinical evaluation in the late 1980s and 1990s. The main toxicities reported were anaphylactic reactions (possibly attributable to the polyoxyethlated castor oil [Cremophor EL] used in the formulation) and extreme lassitude,⁴ though neuromuscular toxicity was observed at an early stage.⁵ Phase II trials were performed in non-Hodgkin's lymphoma,⁶ anaplastic astrocytoma and glioblastoma multiforme,⁷ malignant melanoma,⁸ squamous carcinoma of the cervix,⁹ non–small-cell lung cancer,¹⁰ myeloma,¹¹ epithelial ovarian cancer,¹² colorectal cancer,¹³ breast cancer,¹⁴ and renal cell carcinoma.¹⁵ Anaphylactic reactions and severe fatigue were frequently reported, and several studies also noted a myopathic picture, with muscular cramps and elevated plasma aldolase levels. In addition, cardiac toxicity was reported.⁶ For the most part, little was seen in the way of antitumor activity, although complete and partial responses were seen in non-Hodgkin's lymphoma. Although these results were on the whole discouraging, the novelty of the mechanism of action of the class of compounds and the high potency in experimental models maintained interest. In 1996, dehydrodidemnin B (aplidine) was shown to be more potent than didemnin B against a variety of cell lines.¹⁶ Further in vitro data showed preferential activity in human tumor cell lines compared with normal human cell lines, and in vivo activity was seen in prostate, gastric, breast, and colon models.¹⁷

The mechanism of action of the didemnins, and aplidine in particular, has been the subject of several investigations. Early studies showed that didemnin B caused a cell cycle arrest at G1-S in the cell cycle and implicated prolactin stimulated ornithine decarboxylase activity in the action of the drug.¹⁸ There are clearly effects on protein synthesis. Ahuja et al¹⁹ established a rank order for in vitro protein synthesis for a number of didemnins, and the same rank order was found for cytotoxicity toward MCF-7 cells. GTP-dependent binding of didemnins to the elongation factor EF-1 and binding to palmitoyl protein thioesterase 1 have been documented, and are believed to be the mechanisms by which didemnins inhibit protein synthesis.²⁰ Subsequently more focused studies have been performed that throw light on the mechanism of action of aplidine. García Fernández et al²¹ showed that aplidine caused apoptosis in cultured cells and that this was associated with the early induction of oxidative stress, followed by persistent activation of JNK and P38 MAPK, and a biphasic activation of ERK resulting in caspase activation and PARP cleavage. Inhibition of JNK prevented the proapoptotic action of aplidine, suggesting that JNK is central to the activity of the drug. Cuadrado et al²² used several genetically deficient cell lines to further identify critical targets of aplidine. Mouse embryonic fibroblast cells deficient for src, yes, and fyn, and those lacking all P38 MAPK isoforms were just as sensitive as wild-type cells, while those lacking jnk1 and jnk2 were much less sensitive. In addition, cells lacking or having mutated c-jun, downstream targets of JNK, showed an intermediate sensitivity. These observations lend further support to a central role for JNK in the action of aplidine in these cell lines. There are also a number of interesting data on the activity of aplidine in leukemia models. Broggini et al²³ have shown in MOLT4 leukemia cells that aplidine inhibits vascular endothelial growth factor (VEGF) secretion, thus blocking the VEGF/VEGF receptor-1 autocrine loop. This phenomenon was subsequently shown to occur in lymphoblastic leukemia cell lines and also in primary cultures of cells from relapsed ALL patients, at concentrations lower than those achieved clinically.²⁴ Selective apoptosis in human leukemia cell lines has been reported to be induced by aplidine through a Fas/CD95 and mitochondrial mediated route.²⁵ Further, cross-resistance with other commonly used drugs for leukemia (except for podophyllotoxins) was not found in a number of clinical samples from childhood leukemia.²⁶

While the mechanism of action of aplidine is not fully understood, it is clearly different from known agents and favors clinical development of this class of agents. Phase I studies have been reported in abstract form for a number of different schedules comprising: (1) 24-hour infusion once a week; (2) 1-hour infusion daily x 5 repeated every 3 weeks; (3) 1-hour infusion weekly; and (4) 24-hour infusion every 2 weeks. Most of these studies were incomplete at the time of reporting, but the 24-hour infusion every 3 weeks protocol had documented muscle cramps with elevations in creatine phosphokinase at a dose of 6,000 µg. In this issue, this phase I study is reported in full, with an account of the use of carnitine to ameliorate the muscular toxicity. Faivre et al² started by conducting a fairly conventional phase I study of aplidine, but on approaching toxic dose levels, encountered myalgia with creatine phosphokinase elevation. This unusual toxicity required some reworking of the normal criteria for maximum tolerated dose and dose-limiting toxicity; but in addition, these investigators recognized a similarity between the signs and symptoms induced by aplidine and the described form of adult carnitine palmitoyl transferase deficiency, type 2. Carnitine palmitoyl transferase 2 is related to palmitoyl protein thioesterase 1, a known target of didemnins.²⁰ This observation prompted a trial of the use of L-carnitine to alleviate the muscular toxicities of aplidine and to permit a further dose escalation. While statistical analysis of a phase I trial is always problematic, this is a large trial of 67 patients, and the data presented are persuasive that there is an effect of L-carnitine in reducing the severity and duration of the muscle toxicity for a given dose of aplidine.

The clinical development of anticancer drugs is seldom straightforward, and many of the most widely used drugs at the present have started their careers looking distinctly unpromising. Early trials of cisplatin showed severe and life-threatening toxicities, particularly renal toxicity, and the drug only began to succeed in clinical development after the introduction of hydration and diuresis. Paclitaxel could easily have been discarded on the basis of severe hypersensitivity reactions and only modest signs of activity in the phase I program. More recently, the large-scale clinical development of pemetrexed was made possible by the observation that nutritional levels of folate supplementation greatly reduced the incidence of life-threatening toxicities.²⁷ The determination of the clinical investigators to overcome the problems encountered with new agents is a major prognostic and predictive factor for their likely success. Faivre et al are to be congratulated on their lateral thinking and imaginative approach to an unusual clinical problem—that of drug-induced myopathy. This trial sets a background from which the potential clinical role of aplidine can be explored.

Whatever the clinical future of aplidine, we are now in a position to explore it. The novel, if only partly understood, mechanism of action remains as intriguing as ever. The observations in this study have already thrown some light on the potential mechanism for one of the toxicities. Clinical studies with new anticancer drugs have a habit of improving our knowledge of tumor biology in general. The recent observations that response to gefitinib in non–small-cell lung cancer is associated with certain EGFR mutations has done much to increase knowledge of the factors associated with the pathogenesis and progression of lung cancer.²⁸ The fascinating observation that six patients with neuroendocrine tumors in the current trial of aplidine experienced prolonged disease stabilization deserves further exploration as suggested by the authors. It is possible that further clinical studies with aplidine, in addition to defining its clinical role, may also provide additional leads for novel new drug development.

Author's Disclosures of Potential Conflicts of Interest

The author indicated no potential conflicts of interest.

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