Antitumor Efficacy of Taxane Liposomes on a Human Ovarian Tumor Xenograft in Nude Athymic Mice+ zyxwv AMARNATH SHARMA, ROBERT M. STRAUBINGERS, IWAO OJIMAS, AND RALPH J. BERNACKI~ Received June 29, 1 995, from the zyxwvutsrqp Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263, +Department of Pharmaceufics, University at Buffalo, State University of New York, Amherst, NY 14260, and §Department of Chemistty, State University of New York at Stony Brook, Stony Brook, NY 11 794. Accepted for publication September 1 1, 1995@. Abstract 0 Taxanes such as paclitaxel (Taxol) and docetaxel (Taxotere) are promising agents for use against ovarian cancer and other malignan- cies. Recently, SB-T-1011, a semisynthetic taxane, has been prepared from 14-hydroxy-l O-deacetylbaccatin 111. SB-T-1011 shows similar or greater zyxwvutsrqponm in vitro cytostatic activity than paclitaxel, depending on the tumor cell line. The administration of taxanes is problematic due to their low solubility in most pharmaceutically acceptable solvents; formulations used clinically contain Cremophor/ethanol (diluent 12) or polysorbate 80/ethanol, excipients which may cause serious adverse effects. To eliminate these vehicles, we have prepared paclitaxel liposome formulations. The objective of the present work was to evaluate the antitumor activity of paclitaxel and two semisynthetic analogs in Cremophor-based and liposomal formulations. Antitumor activity was evaluated against A121a, a taxane-sensitive human ovarian tumor, growing as subcutaneous xenografts in athymic nude mice. Free and liposomal formulations of each taxane showed similar antitumor effect. The antitumor activity of paclitaxel and SB-T-1011 was similar, and docetaxel was more potent than either paclitaxel or SB-T-1011. Overall, taxane liposomes were better tolerated and more easily administered iv than taxane formulated in Cremophorlethanol. Taxanes represent a new class of antineoplastic agents having a mechanism of antimicrotubule action. Paclitaxel, the first clinically used taxane, has shown significant anti- neoplastic activity in patients with advanced and platinum- resistant ovarian cancer, with response rates ranging from 20% to 50%.l Paclitaxel, originally known as taxol,2 is a naturally occur- ring taxane present in low abundance zyxwvuts (0.02% by weight) in the bark of the slow-growing Western Yew, zyxwvuts Taxus zyxwvuts brevifolia, and was obtained by a process that kills the trees. As a result, the limited supply of paclitaxel hindered widespread clinical trial. Therefore, major efforts have been directed toward the development of alternative sources of paclitaxel, as well as the synthesis of active analogs from more abundant precur- sors. Currently, paclitaxel can be produced by semisynthesis from 10-deacetylbaccatin zyxwvuts I11 (10-DAB 111), a taxane found in yew needles, a renewable resource. Docetaxel, an active analog also in clinical trial, is synthesized from 10-DAB Recently, the taxane SB-T-1011 has been synthesized from 14~-hydroxy-lO-DAB 111, also obtained from yew needle^.^ Docetaxel and SB-T-1011 are slightly more soluble in water than is paclitaxel. Given the low solubility of paclitaxel, the relatively small increase in solubility of these analogs is not sufficient to permit an aqueous formulation. Therefore, cosolvents and surfactants are used in the formulation, and these may cause adverse side effects. Presently, paclitaxel is formulated in Cremophor EL (poly- ethoxylated castor oil) containing 50% dehydrated ethanol, + Abbreviations: A121a, human ovarian carcinoma; diluent 12, Cre- mophor EL (polyethoxylatedcastor oil) containing 50% absolute ethanol; HPLC, high-performance liquid chromatography; MTD, maximum toler- ated dose; PC, phosphatidylcholine; PG, phosphatidylglycerol; ICm, drug concentration giving tumor 50% inhibition of cell growth zyxwvut in uitro. and docetaxel has been formulated in polysorbate 80 (Tween 80). Early docetaxel formulations contained 50% dehydrated ethanol and polysorbate 80. Cremophor has been shown to cause serious hypersensitivity reactions in animals5 and human@ to reduce the incidence and intensity of hypersen- sitivity, premedication with corticosteroids (dexamethasone) and antihistamines is being used. The premedication regimen has reduced the incidence of serious hypersensitivity reactions to less than 5%. However, milder reactions still occur in approximately 30% of patient^.^,^ Polysorbate 80 also has been observed to cause hemolysis in dogs.8 To minimize toxicity, docetaxel is given as a 1-2 h infusion in human clinical trials.s Polysorbate 80 may also contribute to the development of edema observed in some patients after treat- ment with a docetaxel-polysorbate 80 form~lation.~ There- fore, there is a strong rationale for reformulating taxanes in a safer, better-tolerated vehicle. The primary objectives of the present work are to evaluate and compare the preclinical antitumor efficacy of new taxanes in different vehicles. Most new taxanes have low solubility, thus raising the practical problem of preparing aqueous formulations in order to test their efficacy. Liposomes rep- resent a versatile drug carrier technology with considerable potential for improved solubilization of lipophilic drugs (re- viewed in refs 10-13), and various formulations are in clinical trials or under investigation for treatment of a number of neoplastic and infectious diseases. Previous work has identi- fied paclitaxel-liposome formulations that were well-tolerated at doses greater than or equal to the maximum tolerated dose (MTD) of free paclitaxel and which showed better in uivo antitumor activity against a paclitaxel-resistant murine tumor model, colon-26.14 In the present work, we have prepared taxane-liposome formulations and evaluated their antitumor activity against human ovarian tumor xenografts in athymic nude mice. Experimental Section Materials-Crystalline paclitaxel, diluent 12 (Cremophor EL (polyethoxylated castor oil containing 50% absolute ethanol), and paclitaxel dissolved in diluent 12 (30 mg/5 mL) were obtained from the National Cancer Institute, Bethesda, MD. Docetaxel and SB-T- 1011 were prepared as de~cribed.~J5 Cremophor EL was obtained as a gift from BASF Corp. Phospholipids were purchased from Avanti Polar Lipids (Birmingham, AL) or Princeton Lipids (Princeton, NJ) and stored in chloroform under argon at -70 "C. All organic solvents used were reagent or high performance liquid chromatography (HPLC) grade. Female athymic nude mice were obtained from HarladSprague-Dawley (Indianapolis, IN). Methods-Cell Growth Inhibition Experiments-Tumor cell growth inhibition was determined according to the method described by Skehan et a1.16 Briefly, human tumor cells (A121a ovarian carcinoma, HT-29 colon carcinoma, A549 non-small-cell lung carcinoma and MCF-7 breast carcinoma) were plated at a density of 400 cells/well in 96-well plates and allowed to attach overnight. These cell lines were maintained in RPMI-1640 medium (Roswell Park Memorial Institute growth medium) supplemented with 5% fetal bovine serum and 5% Nu Serum (Collaborative Biomedical Product, MA). Taxanes were solubilized in DMSO and further diluted with RPMI-1640 medium. Triplicate wells were exposed to various treatments. After 72 h incubation, 100pL of ice-cold 50% trichloroacetic acid (TCA) was 1400 / Journal of Pharmaceutical Sciences 0022-3549/953 184- 1400$09.00/0 Vol. 84, No. 12, December 1995 0 1995, American Chemical Society and American Pharmaceutical Association