Reduced Toxicity and Enhanced Antitumor Effects in Mice of the Lonophoric Drug Valinomycin When Incorporated in Liposomes1

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[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), endocytosis (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

(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 Â¡Â¡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. In or i.v. (tail vein) routes with free VM suspension or liposomal VM. Six this communication we present data which suggest that one can mice were given injections for each dose and 4-8 doses were adminis markedly reduce the toxicity of valinomycin in mice by incor tered for each form. A straight line relating dose and lethality was porating the drug in liposomes. Further, valinomycin incorpo drawn and the I I>-,â€žwasdetermined graphically. Therapeutic Assay. On day 0, B6D2F> male mice were inoculated rated in liposomes of appropriate composition displays sub i.p. with IO6P388 leukemia cells or i.v. with 10* cells. There were 6-8 stantial antitumor activities against the P388 mouse leukemia mice in each drug treatment group and 10-12 mice in vehicle treated inoculated i.p. or i.v. control groups. On day 1, the various treatment schedules began as indicated in Figs. 1-4 and Tables 1 and 2. The mortality was monitored daily and the increase in life span ('.'/'(') was used as an index to MATERIALS AND METHODS assess antitumor effectiveness (36). The percentage of increase in host Reagents. Valinomycin (lot 74Ã•-4018),CH2 and PS (lot 45Ã•-8425) life span (%T/Q = T/C x 100, where T = the median survival time of the treated mice and C= median survival time of the untreated control. were purchased from Sigma Chemical Co. (St. Louis, MO). Egg yolk lecithin, DMPC, dimyristoylphosphatidyl glycerol, dioleoylphosphati- No results of therapy are reported in which death attributed to drug toxicity exceeded 15% in the treated group (i.e., one early death was dyl choline, and dipalmitoylphosphatidyl choline were purchased from allowed among a group of 8 mice). Avanti Polar Lipids Inc. (Birmingham, AL). Lipid purity was confirmed by thin layer chromatography on silica coated plates. All other chemi cals were reagent grade. RESULTS Animals and Tumors. DBA/2 and CS7BL/6 x DBA/2 (hereafter called B6D2F,) mice were supplied by The Jackson Laboratory (Bar Valinomycin Assay. The 36-membered valinomycin ring is Harbor, ME). The P388 mouse leukemia, obtained from Dr. W. Plun- devoid of any chromophoric moiety, which makes its direct kett (M. D. Anderson Hospital and Tumor Institute, Houston, TX) detection using spectrophotometric methods rather difficult. was maintained in ascites form by weekly implantation of IO6 cells in On the other hand, VM has the ability to form lipid soluble DBA/2 mice. The experimental inocula of leukemia cells were injected complexes with K+ ion which can be extracted, along with either i.p. (1 x in" cells) or i.v. (1 x 10s cells) into male B6D2F, mice counter aniÃ³n, from the aqueous phase into an organic solvent that weighed between 18 and 22 g at the beginning of the experiments. phase. If the aniÃ³n moiety has chromophoric character, it is Valinomycin Assay. The method of Suzuki et al. (33) for the quanti tative determination of macrotetrolide antibiotics was modified for then possible to determine the amount of VM by measuring valinomycin determination. This assay is based on the formation of an the amount of the extracted aniÃ³n. In this case, potassium organic soluble complex of valinomycin and potassium picrate. Briefly, picrate was selected due to the excellent chromophoric charac an aliquot of the unknown sample (100 ^1) is dissolved in ethylene ter of picrate aniÃ³n. There is a linear relation between the dichloride (15 ml) and then shaken for 5 min with potassium picrate amount of VM and the absorbance at 380 nm in the range from (10 ml). The lower organic layer is then separated and dried over 1-100 ng (data not shown). Using a standard curve, the amount anhydrous sodium sulfate for 30 min. The absorbance of the dried of VM can thus be determined in unknown samples. organic layer is determined at 380 nm using an 1 Kli Ultrospec II Toxicity Effects in Non-tumor-bearing Mice. Table 1 shows spectrophotometer. The concentration of VM in the unknown sample the incorporation of VM into 8 compositions of MLV lipo is determined from a standard curve of known amounts of valinomycin. somes. The 8 formulations were also evaluated, in preliminary The potassium picrate solution is prepared by dissolving picric acid (100 mg) in I N KOH (10 ml) and then diluting to 100 ml with distilled fashion, in terms of toxicity to mice. It is clear from this table water. that negatively charged liposomes composed of DMPC:CH:PS Formulation of Free Valinomycin. An ethanolic stock solution of (10:4:1) could dramatically reduce VM toxicity as compared to valinomycin (1 mg/ml) was diluted in Tris-Cl buffer (0.2 \u pH 7.4). the other types of liposomes tested. This composition is the Samples for injection were diluted so that the ethanol concentration one chosen for all further studies reported here. The dose- did not exceed 5%. The material for injection was slightly turbid and mortality curves for DMPC:CH:PS (10:4:1) liposomes contain is henceforth termed "VM-Suspension." ing valinomycin (MLV-VM) in B6D2F, mice are illustrated in 2The abbreviations used are: CH, cholesterol; VM, valinomycin; MLV-VM, Fig. 1. In Fig. \A the LD5o of free VM given i.p. is shown to be liposomal valinomycin; DMPC, dimyristoylphosphatidyl choline; PS, phospha 1.7 mg/kg, while as much as 50 mg/kg of MLV-VM can be tidyl serine; MTD, maximum tolerated dose; MLV, multilamellar liposomes; I.I).,,, median lethal dose. 3 H. J. Krause, personal communication. 5519

Table 1 Incorporationoftalinomycin in MVLliposomeswithdifferentlipid injection of the drug; (b) mice bearing i.v. P388 leukemia compositions received the drug i.v. or (c) mice bearing i.v. leukemia received Valinomycinwas administered i.p. in liposomal form using different lipid compositions. Mice were checked daily and the LDMin each case was noted. the drug i.p. Groups of 3-4 miceeach were used for preliminaryestimates of The results of the experiments in which both P388 cells, and LDÂ» drug, and/or drug incorporated liposomes were injected i.p. are ratioEggCompositionand M incorporation>95>9560>9580>956555Approximate(mg/kg)(i.p.)1.5626426>501.7shown in Figs. 2-4. To evaluate the chemotherapeutic potency PCDMPCDMPCdimyristoylphosphatidylglycerol of VM in free form or when incorporated in DMPC/CH/PS liposomes, groups of 8 mice each were inoculated i.p. with IO6 (7:3)DMPC:CH(2:1)Dioleoylphosphatidyl P388 leukemic cells, and then daily treatment was started on day 1 for 5 days with different doses of free VM or MLV-VM cholineDipalmitoylphosphatidyl (Fig. 2). Free VM or MLV-VM up to 0.2 mg/kg/day produced cholineDMPC:CH:stearylamine (5:4:1)DMPC:CH:PS( only marginal ant humor activity. A dose of 0.4 mg/kg/day of 10:4:1)FreeVM% free VM increased the life span of the mice by 40%. The same dose given in liposomal form produced an increase in life span of 80%, thus suggesting a somewhat greater potency for MLV- 100- â€”o.-â€”ooâ€”-oâ€”--oâ€”oo-ji VM in this model. Since MLV-VM was far less toxic than free VM we explored 80- antitumor effects of single doses of MLV-VM at levels in excess of the MTD for free VM. Thus, single injections of MLV-VM 060- exhibit dose-dependent antitumor activity, as illustrated in Fig. 'i 3, with a maximum degree of effectiveness (%T/C = 170) at 4 Â¿i40 â€¢ mg/kg. It is also clear from Fig. 3 that free VM at maximum N tolerated dose (0.4 mg/kg) does not produce any significant 20

20 p r 180 0.1 1 10 50 o VM-MLV Dose (mg/kg) â€¢VM-SUSP.

- 140 16 100â€¢80-060â€¢Â£Â¿?40

.â€”â€¢O"" I/I Untreated Control Susp.OVM-MLV 12 filtered)Â»VM-MLV(0.6 urn (unfiltered)1 10 -20-o

.05 0.1 0.2 0.3 0.4 â€¢0â€¢*Â«â€¢*1â€¢VM Dose (mg/kg/doy) Fig. 2. Antitumor activity of free or liposomal valinomycin against P388 1

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1986 American Association for Cancer Research. ANTITUMOR EFFECTS OF VALINOMYCIN IN LIPOSOMES antitumor activity against mice bearing P388 cells when given rated into liposomes, that the liposomal form of valinomycin as single dose. Thus one can achieve significant effects with is considerably less toxic than the free drug, and that liposomal single doses of MLV-VM but not with free VM. valinomycin displays activity against the P388 mouse leukemia The role of the treatment regimen is illustrated in Fig. 4. when the tumor is inoculated either i.p. or i.v. This study Treatment with VM liposomes was started on days 1, 3, or 5 constitutes an important departure from previous studies using post-1'388 implantation and continued for up to 5 days in each lipid vesicles as drug carriers in 2 distinct ways: (a) in contrast case. At the early stage of the disease, MLV-VM liposomes to other studies of liposome encapsulated antitumor drugs, we given once (4 mg/kg), once every 12 h (2 mg/kg) for 1 day, or are not trying to improve upon the efficacy of a currently daily (0.8 mg/kg) for 5 days, produced comparable positive accepted antitumor agent; rather we are attempting to develop results (%T/C values of 158, 146, and 170%, respectively). an approach which will permit the utilization of a broad class Although the maximum increase in life span was seen with of agents (ionophores) which are not now used in cancer ther daily drug treatment, there was about a 10% loss in average apy; (b) we have chosen to utilize, in conjunction with lipo mouse body weight suggesting some toxicity. A statistically somes, drugs which act primarily on cell membranes and which significant (P < 0.005) increase in median survival time was have strong affinities for lipid bilayers; by this means we hope obtained with all 3 dose regimens for the early treatment group to permit the ready formulation of such lipophilic agents and (Student's t test). However, no improvement in survival resulted possibly to be able to modulate their undesirable side effects by when i.p. treatment with liposomal VM was delayed until day manipulating the composition of the liposomal drug carrier. To 3 or 5 after tumor implantation (Fig. 4); thus, i.p. MLV-VM a considerable degree our goals have been realized in the case was ineffective in late stages of the disease. of liposomal valinomycin. When i.v. P388 leukemia was treated by i.p. VM or i.p. Valinomycin is easily incorporated into lipid vesicles (Table MLV-VM no significant antitumor activity was observed at 1). Based on our previous studies with lipophilic drugs (37), it drug levels up to the MTD for each case (data not shown) seems likely that valinomycin is intercalated into the bilayers suggesting that the i.p. drug did not have access to the tumor of the multilamellar liposomes used in this study rather than cells. The effects of i.v. administered drug against i.v. tumor being entrapped in the internal aqueous compartments (37, 38). were also evaluated. We elected to use doses equivalent to 25% The association of the drug with the vesicle seems fairly stable of the MTDs for free VM or MLV-VM given i.v. (Fig. 2B). since, after several washes with buffer, a constant amount of This was done so as to assure an adequate margin of safety for drug, ranging from 50 to greater than 95% of the initial amount, multiple dose administration. Mice bearing i.v. leukemia and remains affiliated with the liposomes. At this juncture, however, treated with i.v. MLV-VM at 25% of the MTD per injection due to technical problems with the valinomycin assay, we have had an improvement in the survival index when multiple doses not evaluated the stability of liposomal valinomycin in biolog were used (Table 2). Administration of free VM at 25% of ical fluids such as blood plasma; this may turn out to be quite MTD/injection had no effect on survival with any schedule different from stability in buffer. tested. Thus MLV-VM but not free VM had substantial effects The composition and physical form of the liposomes used against i.v. P388 leukemia when the 2 forms were compared at seem to have important effects on the toxicity of liposomal equitoxic doses. valinomycin. Thus both cholesterol and presence of anionic lipid (PS) seem to be important for reducing the toxicity of the DISCUSSION drug (Table 1). Formulations containing both of these lipids produce a greater than 50-fold reduction in toxicity as compared In this report we demonstrate that valinomycin, a potassium to 2- to 6-fold reductions for other liposome formulations selective cyclic peptide ionophore, can be efficiently incorpo- tested. Our choice of the DMPC/CH/PS formulation for fur ther study was partly arbitrary since it is not clear at this time -2 if further reductions in drug toxicity could be attained using -1 other formulations. The DMPC/CH/PS preparation was sim u o ply the most promising in terms of reduced toxicity of the O limited number of preparations screened in our initial study. + 1 Toxicity by the i.v. route but not i.p. toxicity seems to be strongly affected by the average size of the liposomal popula +2 tion; presumably this may relate to differences in the blood 18 r 170 clearance rates and organ distributions of differently sized p<0.005 liposome populations (39). 16- o Liposomal valinomycin, given in low multiple doses, is at TJ - 135 least as potent as free valinomycin against i.p. implanted P388 14- tumor. Thus incorporation of the drug in liposomes seem to reduce toxicity to critical host organs but does not reduce direct 12- antitumor effectiveness. Since liposomal valinomycin is consid erably less toxic than free drug, we were able to treat mice with 10 single large doses of liposomal valinomycin and attain substan Days Days Days 1-5 3-8 5-10 tial antitumor effects against i.p. P388; free valinomycin at the maximal tolerated dose was ineffective in this context. Thus TREATMENT SCHEDULE one can increase the therapeutic index for valinomycin in the Fig. 4. Role of disease stage in treatment with liposomal valinomycin. Treat i.p. P388 system through use of a liposomal carrier; this is ment with liposomal valinomycin via the various schedules indicated was started accomplished primarily by reducing the toxicity to the host either 1, 3, or 5 days after i.p. inoculation of B6D2F, mice with P388 leukemic cells. Liposome composition, DMPC:CH:PS (10:4:1). D, untreated control; m, with no loss of antitumor effect. single; S, every 12 h/day; B. daily. W, weight; MST, median survival time. The studies shown in Fig. 4 indicate one of the limitations of 5521

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1986 American Association for Cancer Research. ANTITUMOR EFFECTS OF VALINOMYCIN IN LIPOSOMES Table 2 Therapeutic effect of i.v. administered valinomycin on i.v. P-3S8 leukemia Both free and liposomal (DMPC:CH:PS) valinomycin were used at 25% of their MTDs. (mg/kg)Treatment Valinomycin total dose (days)VM survival time scheduleSingle suspension0.045 suspension9(8)Â» suspension100

Daily (1-9) 0.405 22.50 9(8) 13(8) 100 144138 Every 12 h x 3 0.270VM-MLV2.50 15.00Median 9(8) 12.5(8)%T/CÂ°VM 100VM-MLV100 Untreated controlVM 9(12)VM-MLV9(8) " Relative percentages of median survival of test and control animals. b Numbers in parentheses, number of B6D2F, mice. the use of i.p. administered liposomal valinomycin. In these swered in this system. For example, we have no information on studies good antitumor effect was obtained when treatment the basis for the cellular toxicity to P388 cells of liposomal commenced 1 day after tumor inoculation; however, when valinomycin and whether it is the same as for free drug. Further therapy was delayed until 3 or 5 days postinoculation only we have no clear insights into the mechanism of the host toxicity marginal effects were observed using several different treatment of either free or liposomal valinomycin. Presumably host tox protocols. Treatment failure may relate to the high tumor icity relates to valinomycin induced ion fluxes in key organ burden at later disease stages. Alternatively, it may indicate systems such as heart, liver, central nervous system, or kidney; that i.p. liposomal valinomycin is ineffective against tumor cells however, preliminary investigation of the blood chemistry of which have migrated out of the peritoneal cavity and are broadly mice treated with the MTD of free or liposomal valinomycin distributed in the host; this is known to occur between days 2 showed no abnormalities of ions or of markers indicative of and 4 for the P388 system (40). The data suggest that the latter heart, liver, or kidney damage (48). Thus we have no clear idea explanation is correct since i.p. administered MLV-VM has no of the mechanism of host toxicity. Finally, there is some indi effect on i.v. inoculated tumor indicating that the i.p. drug does cation that use of i.v. liposomal valinomycin may be quite not have access to the cells. By contrast, i.v. liposomal valino schedule dependent but we have not yet determined the opti mycin displays activity against i.v. P388 indicating that the mum scheduling. Despite these limitations, the present study drug reaches the tumor cells. Thus treatment of localized and seems quite promising in that it offers an approach for improv disseminated disease with liposomal valinomycin would prob ing the ease of pharmaceutical formulation of ionophoric drugs ably require both local and systemic administration of the drug. as well as the possibility of reducing their host toxicity, thus There has been a limited amount of previous work on the enhancing their possible utilization as chemotherapeutic agents. antitumor action of free valinomycin (28-30,41); most of these studies have been limited by the toxicities displayed by the drug. REFERENCES In the only other reported attempt to use a drug carrier system 1. Pratt, W. B., and Rudin, R. W. The Anti Cancer Drugs. Oxford, England: (Intralipid), (32), the drug carrier complex proved even more Oxford University Press, 1979. toxic than valinomycin itself and the attempt was abandoned. 2. Haskell, C. M. Drugs used in cancer chemotherapy. In: C. M. Haskeil (ed.). 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5523 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1986 American Association for Cancer Research. Reduced Toxicity and Enhanced Antitumor Effects in Mice of the Ionophoric Drug Valinomycin When Incorporated in Liposomes

Sayed S. Daoud and R. L. Juliano

Cancer Res 1986;46:5518-5523.

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