Studies of the Organ Distribution in Mice of Teniposide Liposomes Designed for Treatment of Diseases in the Mononuclear Phagocytic System
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0031-3998/95/3801-0007$03.00/0 PEDIATRIC RESEARCH Vol. 38, No. 1, 1995 Copyright O 1995 International Pediatric Research Foundation, Inc. Printed in U.S.A. Studies of the Organ Distribution in Mice of Teniposide Liposomes Designed for Treatment of Diseases in the Mononuclear Phagocytic System EVA LILIEMARK, JAN LILIEMARK, NILS K~LBERG,MAGNUS BJORKHOLM, BRITA SJOSTROM,AND CURT PETERSON Departments of Clinical Pharmacology [E.L, J.L., C.P.] and Medicine, Section of Hematology and Medical Immunology [M.B.], and Karolinska Pharmacy [N.K.], Karolinska Hospital, Stockholm, and Institute for Sulface Chemistry [B.S.], Stockholm, Sweden Liposomes can be used for the delivery of drugs in cancer ing radiolabeled teniposide or phospholipid were given i.v. to chemotherapy. After i.v. injection liposomes are to a large extent mice. By increasing the size of the vesicles, the MPS uptake taken up by the mononuclear phagocytic system (MPS). When could be modulated. When vesicles of 200 nm and 1 and 3 pm treating diseases in the MPS, such as the histiocytic syndromes, were injected, the drug levels in the spleen were increased 2.6-, this property is of potential value for drug targeting and may lead 6.8-, and 21-fold 40 min after injection, compared with levels to a more efficient therapy with less systemic toxicity. Teniposide after injection of the commercial teniposide formulation. It was (VM-26) is a potent anti-tumor drug. Its lipophilicity makes it concluded that organ distribution of teniposide in mice could be suitable for liposomal formulation. Teniposide liposomes were modified by administering the drug in liposomal form with the prepared by dissolving egg phosphatidylcholine and dioleoyl potential of improved treatment of diseases engaging the MPS. phosphatidic acid (19:l molar ratio) in methylene chloride to- (Pediatr Res 38: 7-10, 1995) gether with teniposide. After solvent evaporation, the dry lipid film was dispersed in a glucose solution (50 mg/mL), and size Abbreviations calibration was obtained by filtration through polycarbonate FHL, familial hemophagocytic lymphohistiocytosis filters. The amount of teniposide incorporated was 2.5 mol %. To MPS, mononuclear phagocytic system investigate the organ distribution, teniposide liposomes contain- PC, phosphatidyl choline Most anti-tumor agents have severe side effects limiting the is dose-limiting, and liposomal formulations could improve the therapeutic possibilities when given systemically. Selective therapeutic index of teniposide. Liposomal teniposide therapy localization of anti-tumor drugs may improve treatment results might also be of value for other malignant and autoimmune of chemotherapy. After i.v. injection most liposomes are rap- disorders involving phagocytic cells. idly cleared from the circulation by phagocytosis by the MPS The purpose of this work was to prepare and evaluate in liver, spleen, and bone marrow (1). Liposome-mediated drug teniposide liposomes in an attempt to achieve enhanced drug delivery is therefore an alternative if the target is tissues of the uptake in the MPS. Mice were used for drug distribution MPS. studies. The podophyllotoxin derivative teniposide is active against solid tumors such as small cell lung cancer and germinal METHODS tumors as well as hematologic malignancies (2, 3), and lipid- soluble drugs such as teniposide can easily be encapsulated Chemicals. [14~]~eniposide,specific activity 0.77 GBq/ within the membrane of liposomes (4). mmol, was kindly provided by Dr Kathleen Deardorff, Bristol The podophyllotoxins are the drugs of choice in the treat- Myers-Squibb (Syracuse, NY). Teniposide in the commercially ment of FHL, a rare childhood disease which affects the available formulation, Vumon, was received as a gift from macrophages in liver and spleen (5, 6). Bone marrow toxicity Bristol-Myers Squibb (Bromma, Sweden). Phosphatidylcholine, ~-(~-[2-~almito~l-l-~~~]di~alrnito~l, Received May 26, 1994; accepted January 26, 1995. specific activity 2.12 GBq/mmol, radiochemical purity 99%, Correspondence and reprint requests: Eva Liliemark, Department of Clinical Pharma- and phosphatidylcholine, L-a-[choline-methyl-3~]dipalmitoyl, cology, Karolinska Hospital, S-171 76 Stockholm, Sweden. Supported by grants from the Swedish Cancer Society, the Swedish Children Cancer specific activity 1480 GBqImmol, radiochemical purity 99% Society, and Bristol Myers-Squibb in Sweden. was purchased from NEN Research Products, Du Pont Scan- 8 LILIEMARK ET AL. dinavia A.B. (Stockholm, Sweden). Phosphatidylcholine, L-a- warming of the animal at 45°C to obtain vasodilatation, 150- [2-palmitoyl-l-14~]dipalmitoylwith specific activity 4.2 GBql 200 pL of liposomal or free teniposide were injected in a tail mmol, radiochemical purity 99%, from Amersham vein. At different time intervals, the mice were anesthetized by International (Amersham, UK) was also used. Egg PC and diethyl ether. Blood obtained by neck incision was collected in dioleoyl phosphatidic acid were purchased from Avanti Polar heparin tubes and kept on ice until plasma was separated by Lipids, Inc. (Pelham, Ala). All solvents used were of analytical centrifugation. Organs were washed in ice-cooled PBS, pH 7.4, or HPLC grade. weighed, and homogenized. Radioactivity in plasma, liver, Preparation and characterization of liposomes. Liposomes spleen, lung, and heart was determined by liquid scintillation were produced in small batches the day before use in animal counting. experiments. The phospholipids, PC, 138 mg, and phosphatidic One or two experiments using one or two animals per time acid, 6.2 mg (19:l molar ratio), were dissolved in 10 mL point was performed with free teniposide and liposomes la- methylene chloride together with teniposide, 2.9 mg. The beled with [14~]teniposide,liposomes labeled with [14c]pc, radiolabeled teniposide or PC was added to the solvent. After and liposomes labeled with both [14~]teniposideand [3~]~~. evaporation, the dry lipid film on the inside walls of the flask With double labeling the fate of both drug and lipid could be was dispersed in 8.8 mL glucose solution (50 mg/mL) by followed in the same animal. By dividing radioactivity in the manual shaking. Large unilamellar vesicles were produced by samples with the injected amount of radioactivity per gram repeated filtration through Nucleopore polycarbonate filters, body weight of the mice, the results from the different exper- Costar Corp. (Cambridge, MA), with different pore sizes (200 iments could be compared. nm and 1 and 3 pm) (7). The Extruder Lipex Biomembranes Inc. (Vancouver, Canada), was used to produce vesicles by RESULTS extrusion two to three times through double 200-nm filters. Larger vesicles were filtered manually two to four times In the attempts to optimize the amount of teniposide possible through membranes with 1- and 3-pm pores. The teniposide to incorporate into our liposomes, druglipid molar ratios of concentration of the preparations was 365 pg/mL, and the 1-15% were studied. The recovery of teniposide was >90% phospholipid concentration 16.4 mg/mL. when the teniposide molar ratio was up to 7%. Stability tests Teniposide concentrations in the preparations were deter- were performed on liposomes extruded twice through double mined by reversed phase high performance liquid chromatog- 200-nm polycarbonate filters. Drug crystals occurred if the raphy using a Spherisorb Phenomenex mini-bore column teniposide content was 3% or higher. With 2 mol % teniposide eluted with methanol/water/acetic acid (30:69:1). Detection the liposomes could be stored at +8OC for 3 mo without was performed fluorometrically, hexcitation/emission = 2301 formation of drug crystals as judged from the results of filtra- 330. The 14c or 3~ radioactivity in the preparations was tion through 1-pm polycarbonate filters without significant loss determined by P-counting using a Packard Tri-Carb 460C of teniposide and from microscopic examination in polarized liquid scintillation system. The mean diameter of the 1- and light. The mean liposome diameter was 200-300 nm and the 3-pm liposomes was measured by laser diffraction with a size distribution was homogenous, reproducible, and stable. Mastersizer, Malvern Instruments (Malvern, UK), and the size The liposomes were stable for at least 3 mo in PBS, pH 7.4, of the 200-nm liposomes was measured by dynamic light and in glucose, 50 mg/mL. scattering at a fixed angle of 90" using an Autosizer IIc, During the filtration procedure for liposome production the Malvern Instruments (Malvern, UK). The unilamellar structure size distribution approached the size of the pores with number of the vesicles has been verified with cryo-transmission elec- of passages through the filter. Figure 1 illustrates how the tron microscopy as previously described (8). number of passages affect mean liposome size using a 3-pm Preparation of radiolabeled teniposide. Radiolabeled teni- polycarbonate membrane. After four filtrations the size is poside was obtained by dissolving [14~]teniposidein methanol rather homogenous with a mean diameter of 3.5 pm, and 95% and evaporating to dryness under nitrogen in a glass tube. The of the liposomes are below 7 pm (volume distribution), which commercially available teniposide solution, Vumon, contain- means smaller than the red blood cells. ing cremophore, was used to solubilize the radiolabeled teni- To evaluate the in vivo distribution of teniposide liposomes poside. Vumon, 290 pL (teniposide concentration 10 mg/mL), we used mice and injected the commercial formulation