728 Vol. 10, 728–738, January 15, 2004 Clinical Cancer Research In Vitro and in Vivo Characterization of Doxorubicin and Vincristine Coencapsulated within Liposomes through Use of Transition Metal Ion Complexation and pH Gradient Loading Sheela A. Abraham,1,2 Cheryl McKenzie,1 studies evaluating doxorubicin and vincristine combinations Dana Masin,1 Rebecca Ng,1 Troy O. Harasym,4 analyzed using the Chou and Talalay median effect princi- Lawrence D. Mayer,1,3,4 and Marcel B. Bally1,2,4 ple. These data clearly indicated that simultaneous addition 1 of vincristine and doxorubicin resulted in pronounced an- Division of Medical Oncology, Department of Advanced tagonism. Therapeutics, BC Cancer Agency; 2Department of Pathology and Laboratory Medicine, Faculty of Medicine, and 3Faculty of Conclusion: These results emphasize that in vitro drug Pharmaceutical Sciences, University of British Columbia; and combination screens can be used to predict whether a co- 4Celator Technologies Incorporated, Vancouver, British Columbia, formulated drug combination will act in an antagonistic or Canada synergistic manner. ABSTRACT INTRODUCTION Purpose: There is an opportunity to augment the ther- Combination treatment regimes must take into consider- apeutic potential of drug combinations through use of drug ation drug resistance mechanisms (1) and tumor heterogeneity delivery technology. This report summarizes data obtained (2). In practice, however, drug combinations usually take ad- using a novel liposomal formulation with coencapsulated vantage of nonoverlapping toxicities and unique mechanisms of doxorubicin and vincristine. The rationale for selecting these action. We believe that there is an opportunity to capture the drugs is due in part to the fact that liposomal formulations therapeutic potential of drug combinations through the use of of doxorubicin and vincristine are being separately evalu- drug delivery technology. This hypothesis is supported by three ated as components of drug combinations. lines of evidence: (a) appropriately designed drug delivery Experimental Design: Doxorubicin and vincristine were systems can improve drug delivery and efficacy for single coencapsulated into liposomes using two distinct methods of agents (Refs. 3–5; b) polymer, protein, and lipid-based drug drug loading. A manganese-based drug loading procedure, carriers have the capacity to coformulate two or more therapeu- which relies on drug complexation with a transition metal, tic agents (6–8); and (c) the pharmacokinetic behavior of the was used to encapsulate doxorubicin. Subsequently the iono- coformulated drugs will be dictated by the pharmacokinetic phore A23187 was added to induce formation of a pH gra- behavior of the drug carrier system used, thus offering the dient, which promoted vincristine encapsulation. potential to coordinate the plasma elimination and tissue distri- Results: Plasma elimination studies in mice indicated bution of the combined agents. that the drug:drug ratio before injection [4:1 doxorubicin: There are a multitude of approved chemotherapy combi- vincristine (wt:wt ratio)] changed to 20:1 at the 24-h time nations that could be used to test this principle, and this report point, indicative of more rapid release of vincristine from summarizes data obtained using a novel liposomal formulation the liposomes than doxorubicin. Efficacy studies completed with coencapsulated doxorubicin and vincristine. The rationale in MDA MB-435/LCC6 tumor-bearing mice suggested that for selecting these drugs is due in part to the fact that liposomal at the maximum tolerated dose, the coencapsulated formu- formulations of the individual agents have already proven to be lation was therapeutically no better than liposomal vincris- of clinical value in the treatment of patients with HIV-associated tine. This result was explained in part by in vitro cytotoxicity Kaposi sarcoma (9), ovarian cancer (10), breast cancer (11), and various hematological malignancies (12, 13), including relapsed non-Hodgkin’s lymphoma. Furthermore, these liposomal for- mulations of doxorubicin and vincristine are now being evalu- Received 7/31/03; revised 9/24/03; accepted 9/29/03. ated as components of drug combinations used routinely in the Grant support: Canadian Institutes for Health Research and Celator management of patients with cancer. Liposomal formulations of Technologies Incorporated. S. A. A. is a recipient of a Science Council doxorubicin, for example, have been evaluated as part of the of British Columbia GREAT scholarship and the William Armstrong vincristine, doxorubicin, and dexamethasone regimens used to Memorial Award. The costs of publication of this article were defrayed in part by the treat patients with multiple myeloma (14), and in the cyclophos- payment of page charges. This article must therefore be hereby marked phamide, doxorubicin, vincristine, and prednisone regimen for advertisement in accordance with 18 U.S.C. Section 1734 solely to treatment of aggressive non-Hodgkin’s lymphoma (15). Alter- indicate this fact. natively, this regimen has been modified by incorporation of a Requests for reprints: Marcel B. Bally, Department of Advanced Therapeutics, BC Cancer Agency, 601 West 10th Avenue, Vancouver, liposomal vincristine formulation for use in the treatment of a British Columbia V5Z 1L3, Canada. Phone: (604) 877-6098, extension similar patient population (16). A liposomal formulation of 3191; Fax: (604) 877-6011; E-mail: [email protected]. doxorubicin has been used in combination with cyclophospha- Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 729 mide for treatment of patients with metastatic breast cancer (17), Technologies, Vancouver, British Columbia, Canada) supple- and with cyclophosphamide and fluorouracil for first-line treat- mented with 10% fetal bovine serum from Hyclone Laboratories ment of patients with metastatic breast cancer (18). More re- (Logan, UT), 1% L-glutamine, and 1% penicillin and strepto- cently, liposomal formulations of doxorubicin have been tested mycin solution (Stem Cell Technologies, Vancouver, British in combination with novel agents targeting the multidrug resist- Columbia, Canada). ance protein Pgp as well as with trastuzumab (Herceptin) for All of the animal studies were done according to proce- treatment of breast cancer patients with tumors overexpressing dures approved by the University of British Columbia Animal Her-2/neu (19, 20). Care Committee. These studies meet the requirements outlined These examples highlight two points. First, liposomal for- in the current guidelines for animal use established by the mulations of anticancer drugs are gaining wider acceptance by Canadian Council of Animal Care. the clinical oncology community, which is evaluating how these Preparation of Liposomes. Lipids (DSPC/Chol; 55/45; 3 14 formulations function when used as part of combination regi- mol%) were dissolved in chloroform, and [ H]CHE or [ C]- mens. Second, vincristine and doxorubicin (or other anthracy- cholesterol hexadecyl ether was added to achieve ϳ5 ␮Ci/100 clines) are still in use together as part of many combination mg lipid. The chloroform was removed under a gentle stream of regimens. This is likely a consequence of many decades of nitrogen gas, and subsequently the lipid samples were placed research providing data highlighting the different modes of under a high vacuum for at least3htoremove residual solvent. action and nonoverlapping toxicities of these agents. Dried lipid films were hydrated with 300 mM MnSO4 (adjusted Previous studies from our laboratory have attempted to to pH 3.5 with dilute HCl; 0.1 N) to achieve a final lipid coformulate these two drugs into a single liposomal formulation concentration of 100 mg/ml. After hydration the multilamellar by an established transmembrane pH gradient; however, this vesicles were subjected to 5 freeze-thaw cycles (freezing in effort proved unsuccessful because of the instability of the liquid nitrogen and thawing at 60°C; Ref. 22). Samples were resulting formulation (21). More recently, a novel doxorubicin extruded 10 times through stacked polycarbonate filters with 0.1 encapsulation method, which relies on drug binding to an en- and 0.08 ␮m pore size at 60°C using a water-jacketed Extruder capsulated transition metal, manganese, has been characterized. (Northern Lipids Inc., Vancouver, British Columbia, Canada). It is proposed here that a manganese metal gradient across The mean size distribution of the resulting liposome prepara- liposomes could be used to encapsulate doxorubicin and, in a tions ranged between 100 and 120 nm as determined by a second step, to form a pH gradient across the doxorubicin- NICOMP Submicron Particle Sizer Model 270 (Pacific Sci- loaded liposomes by addition of the electroneutral ionophore, entific, Santa Barbara, CA) with an argon laser operating at A23187 (divalent cation/proton exchanger). It is demonstrated 632.8 nm. here that the resulting pH gradient can facilitate the rapid In Vitro Drug Loading. Before the addition of drug to accumulation of vincristine without loss of encapsulated doxo- liposomes, a pH gradient (ϳ3 units) and a transition metal rubicin. The coencapsulated formulation exhibited plasma elim- gradient was established across liposome bilayers. To generate ination and drug release rates comparable with those observed the gradients, DSPC/Chol liposomes in MnSO4 buffer (pH 3.5) with liposomes containing