[CANCER RESEARCH 46, 5518-5523, November 1986] Reduced Toxicity and Enhanced Antitumor Effects in Mice of the lonophoric Drug Valinomycin When Incorporated in Liposomes1 Sayed S. Daoud and R. L. Juliano Department of Pharmacology, University of Texas Medical School, Houston, Texas 77225 ABSTRACT severely impair membrane function in tumor cells might well have useful chemotherapeutic potential. Indeed, some of the Valinomycin (NSC 122023) is a cyclic depsipeptide antibiotic with effects of anthracyclines have been attributed to their actions potassium selective ionophoric activity. This drug has been reported to on plasma membrane or mitochondria! membrane function (3). display antitumor effects but its utilization has been limited by its extreme toxicity. Here we report that the incorporation of valinomycin into However, none of the current clinically useful antitumor agents multilamellar liposomes composed of dimyristoyl phosphatidyl cho- acts primarily at the membrane level (1). Perhaps one reason )ine:cholesterol:phosphatidyl serine (10:4:1 Mratio) results in a profound for this is that membrane active drugs are likely to be quite reduction in toxicity with maintainence of antitumor efficacy. Thus the toxic to those tissues and organs whose physiological functions median lethal dose (LDW) for i.p. administered valinomycin (VM) in rely on ion gradients and membrane potentials. CS7BL/6 x DBA/2 mice is 1.7 mg/kg whereas the I IK,, for liposome lonophores comprise a class of agents which act primarily on incorporated valinomycin (MVL-VM) is in excess of 50 mg/kg. In like cellular membranes (4-6); there are two basic types of iono- manner, the LDWfor i.v. administered VM is 0.18 mg/kg where the LDM phore. Mobile ionophores bind monovalent or divalent cations for MI. V-VM preparations passed through a (l.6-um filter is greater than with various degrees of specificity. The cation-ionophore com 10 mg/kg. The antitumor efficacies of i.p. administered VM or MLV- plex then can diffuse across cell membranes; well-known ex VM against i.p. P388 mouse leukemia were similar in multiple dose formats using doses below the maximal tolerated dose for VM. However, amples of mobile ionophores include the neutral cyclic depsi since MLV-VM was substantially less toxic than VM, the liposomal peptide agent valinomycin and negatively charged carboxylic drug also produced significant (170% median survival time of treated ionophores of the monensin type. Channel forming ionophores mice/median survival time of untreated control) antitumor effects when partition into membranes and create water filled pores which administered as a single dose at levels above the maximal tolerated dose can allow the diffusion of cations, anions, and small polar for free VM; single doses of free VM at the maximal tolerated dose were metabolites; well-known examples of channel formers include ineffective in this context. In experiments with i.v. inoculated P388 polyene antibiotics such as amphotericin B, which is used in leukemia, MLV-VM but not free VM, displayed antitumor activity therapy of fungal infections, as well as the linear polypeptide (144% median survival time of treated mice/median survival time of untreated control) when administered i.v. at equitoxic doses. Thus the antibiotic gramacidin A. Channel forming and mobile types of use of a lipid vesicle drug carrier system permits a reduction in the ionophores have been reported to affect a variety of membrane toxicity of valinomycin with maintainence or enhancement of antitumor related activities in mammalian cells. This would include ac activity against i.p. or i.v. P388 leukemia. tions on ion gradients and potentials in plasma membranes (4, 5), protein synthesis (7), glycoprotein secretion (8, 9), endocy- tosis (10, 11), mitochondrial membrane potential (12, 13), INTRODUCTION energy metabolism (14, 15, 16), and cell proliferation (17). It The cytotoxic actions of most current anticancer drugs are seems then that ionophores are likely to have important cyto due to their effects on the biosynthesis, repair, replication, or toxic effects on neoplastic cells. However, a major obstacle to stability of DNA. Thus, alkylating agents, purine and pyrimi- the use of ionophores as anticancer agents relates to their dine analogues, antifolates, and intercalating agents such as toxicities to critical organ systems such as the central and actinomycin D and anthracyclines, all primarily effect DNA, in peripheral nervous tissues, the heart and the kidney. For ex both tumor and normal cells. As a result, these agents all have ample, the carboxylic ionophores are known to profoundly a similar, predictable, spectrum of toxicities primarily involving perturb the cardiovascular system (6, 18), while amphotericin cell populations undergoing rapid DNA synthesis such as those B is extremely toxic to the renal system (19). If one could devise found in the bone marrow, gastrointestinal tract, testes, and a means of reducing the toxicity of ionophoric drugs while skin (1). Agents which display dose limiting toxicities at other maintaining therapeutic actions, then this might open the path tissue sites, for example, the antitubulin drug vincristine, which to developing ionophores as useful entities for cancer treatment. is neurotoxic, provide a valuable adjunct to cancer chemother The first indication that one could diminish the toxicity of apy since their use in multidrug combinations permits enhanced an ionophore with retention of therapeutic activity came from tumor cell killing without substantial additional toxicity to studies conducted by our laboratory (20, 21) and by others (22) rapidly dividing cell populations (2). Thus the development of of amphotericin B incorporated in liposomes. The liposomal agents which act against cellular targets other than DNA can formulation of amphotericin B proved far less toxic to mam conceivably lead to drugs which provide effective antitumor malian organ systems than the conventional form of the drug therapy and whose toxicities are distinct from most currently while maintaining full activity for therapy of systemic fungal available chemotherapeutic substances. infections. This valuable improvement in the therapeutic index The plasma membrane as well as intracellular membranes of amphotericin B seems to stem from a profound alteration in are important loci for drug action, since membranes play a vital the relative affinity of the drug for fungal membranes as op role in cellular organization and regulation. Thus drugs which posed to mammalian membranes caused by incorporation of the drug into the lipid vesicles (21, 23). Received 4/30/86; revised 7/22/86; accepted 7/28/86. The costs of publication of this article were defrayed in part by the payment We have now sought to extend the concept of modulating of page charges. This article must therefore be hereby marked advertisement in ionophore toxicity using liposomes to the arena of antitumor accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by NIH grant CA36840 and by an award from the Elsa Pardee drugs and have attempted to reduce the toxicity of the po Foundation (R. L. .1.). tassium specific cyclic peptide antibiotic valinomycin 5518 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1986 American Association for Cancer Research. ANTITUMOR EFFECTS OF VALINOMYCIN IN LIPOSOMES (NSC 122023) by incorporating this agent in a liposomal drug Preparation of Liposomes. Multilamellar lipid vesicles (liposomes) carrier. Valinomycin is known to perturb ion gradients across containing VM were prepared by mixing 100 mg of total lipid in both the plasma membrane (4) and the mitochondrial mem chloroform solution with 10 mg of VM in ethanol solution and then brane (24), with the latter effect perhaps being more important bringing the sample to dryness under vacuum using a Buchi rotary evaporator. The molar ratios of the various lipids used are given in the in terms of cytotoxicity (12). Valinomycin can also perturb legends to Figs. 1-4 and in Tables 1 and 2. Liposomes were formed by membrane structure and function in ways unrelated to its cation adding SOml of 0.2 M Tris-Cl buffer, pH 7.4, and dispersing the solids carrying ability; for example, it has been reported to alter fatty by vortex agitation. As much as possible of the nonincorporated VM acid (25) and phosphatidyl inositol (26) turnover as well as was removed by sedimenting the vesicles at 10,000 rpm in a Beckman cellular proliferation and ATP levels (27) independent of in J-21 centrifuge and resuspending the pellet in IS ml Tris buffer. This duced potassium fluxes. Valinomycin was chosen for study washing procedure was repeated at least 3 times. The resulting pellet largely because several workers have previously reported that was then diluted with Tris buffer to the required volume. Previous this ionophore has some degree of antitumor activity (28, 29, experience suggests that this procedure gives rise to multilamellar vesicles in the 1 to 2 ¡¡msizerange (34). This was confirmed by negative 30). In addition, very recent work by Kleuser et al. (31) has stain electron microscopy.1 In some cases the liposomes were sequen shown that valinomycin can be selectively toxic to transformed tially extruded through 2, 1, and 0.6 urn Nuclepore filters under cells in vitro. In a series of experiments which foreshadowed nitrogen pressure as described by Hope et al. (3S). This gives rise to a the present work in some ways, Repta (32) reported an attempt more uniform liposome population with a mean diameter approxi to use a commercially available fat emulsion (Intralipid) as a mately equal to the filter pore size. The amount of VM entrapped in carrier for valinomycin. However, in contrast to the experi MLV was determined by colorimetrie assay as described above. ments presented below using valinomycin in liposomes, the fat Acute Toxicity. B6D2Ft male mice, 18-22 g were treated by the i.p. emulsion failed to reduce the toxicity of valinomycin in vivo.