Antimetastatic effects of liposome entrapped indomethacin

Life Sciences, Vol. 48, pp. 149-154 Printed in the U.S.A.

Pergamon Press

ANTIMETASTATIC EFFECTS OF LIPOSOME F2q'IRAPPED INDOMETHACIN S. F. ALIlqO, A. IRUARRIZAGA, J. ALFARO, A. ALMENA, M. LEJARRETA and F. J. UNDA Departamento de Biologfa Celular y Ciencias Morfol6gieas, Faeultad de Medieina y Odontolo#a, Universidad del Pais Vaseo/Euskal Herriko Unibertsitatea 0.1PV/EHU), Leioa, 48940-Vizcaya, Spain. (Received in final form November 6, 1990)

~umrn~_ The "in vivo" administration of sized liposomes encapsulating indomethacin to mice bearing 31.,I, tumor, significantly reduced the incidence and/or number of superficial lung metastases. Also liposomes encapsulating indomethacin had significant inhibitory effects on the experimentally induced lung metastases. We conclude: i) indomethacin encapsulated in liposomes is more efficient than the free drug in mediating the antimetastatie effects and ii) liposomes are an valuable vehicle in evading the side metastatic effects of this drug during indomethacin treatment of tumor bearing mice. The most important property of malignant tumors is their ability to infiltrate normal tissue and to produce secondary growths in distant organs from primary tumor, named metastases. Primary tumor can be locally treated (e.g. surgically), but usually disseminated metastases result in the lethal event of most neoplastic diseases (1). Indomethacin (IND), a non-steroidal antiinflarnatory drug, has been reported to exhibit antitumoral activity against many human and experimental animal tumors (2), including 3LL tumor (3). However, it has been also described that direct indomethacin effect on some tumor/cells (including 3LL), increases the tumor cell ability to form metastatic loci (4, 5, 6). The metas/tatic effect of indomethacin on tumor cells detracts from its merits as an antitumoral drug ano could mask its beneficial effects in cancer therapy. In an attempt to prevent the metastatic effects of indomethaein after its systemic administration in tumor bearing mice, we have evaluated the effect of indomethaein encapsulated in sized liposomes on the spontaneous and experimental induced metastasis of 3LL carcinoma cells. Methods MATERIALS: egg phosphatidylcholine (PC) and sphingomyelin (SM) were purchased from Lipid Products (S.Nutfield, U.K.); cholesterol (CH) and indomethacin (IND) were from Sigma ChemicalCo. (St. Louis, USA). 5-6 carboxyfluorescein (CF) was obtained from Eastman K o d ~ , Co. and purified by a gel exclusion column. Sepharose CL-4B was from Pharmacia (Uppsala, Sweden) and polycarbonate filters (0.2 ~tm pore size) were from Nuclepore. The liposome encapsulated IND was quantified using a Waters Associated high-performance liquid chromatograph equipped with a variable wavelength UV detector (Waters, Lambda-Max 481). The column was a 30 cm x 3.9 mm I.D. tube packed with ~Bondapak C18 (Waters Associated). Indomethacin 20 mM was prepared by dilution in NaJ-ICO3 (100 raM) of a previously prepared ethanol solution of IND (100 raM).

Address for correslxmdence:Dr. S. F. Aliflo. Departamentode BiologfaCelular y CienciasMorfol6gicas.Facultadde Medicina y Odontologia.Universidaddel Pals Vasco(UPV/EHU).Leioa48940. Vizcaya.SPAIN. 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc

150

Liposome-lndomethacin and Metastasis

Vol. 48, No. 2, 1991

LIPOSOME PREPARATION: A lipid mixture of PC:CH:SM, dissolved in benzene-methanol (9:1, v:v), was distributed in glass tubes at 7:2:1 molar ratio, dried under N2 reduced pressure to form a thin film around the wall of the round-bottom tube and then lyophilized. Liposomes were prepared essentially as described previously (7). Briefly, lyophilized lipids were resuspended by mechanical dispersion in a 20 m M I N D solution and/or 50 mlVl CF to obtain multilamellar vesicles (MLV). Sized liposomes were prepared by several extrusions of MLV through policarbonate filters of 0.2 Ixm pore size, under N2 pressure. Liposomes were gel-filtrated on a Sepharose CL-4B column to remove free IND and/or CF and then sterilized by filtration through 0.45 Ixm pore size filters (lVIillilxre) and stored at 4°C. To evaluate the liposome encapsulation efficiency, the phospholipid concentration was determined by the ammonium ferrothiocyanate technique (8) and the liposome entrapped IND was quantified by HPLC (9). The elution solvent was 35% acetonitrile (CH3CH) in 0.7% NH+C1 (pH 7.75). The elution flow and detection conditions were 1 ml/min and 220 nm of wavelength, respectively. LIPOSOME STABILITY: Duplicate aliquots of 4°C stored liposomes entrapping CF (50 raM) or CF plus IND (50 mM and 20 mM, respectively) were collected at several interval days (from 1 to 21) and the fluorescence of CF was measured in absence (CFa) or presence (CFb) of Triton X100 (1% final concentration), using a spectrofluorimeter (Shimadzu RF-540) with excitation and emission wavelengths of 490 and 520 nm, respectively. Latent CF was determined from the following formula (CFb-CFa)/CFb. The values are expressed as a percentage of latency with respect to recently prepared liposomes. To evaluate the drug leakage of liposomes encapsulated IND, 100 gl of 4°C stored liposomes (from 1 to 21 days) or incubated at 37°C (between 1.5 and 24 h) in mice plasma (1:1, v/v) were separated from free drug on a Sepharose CL-4B column and liposome fraction was used to quantify IND associated to liposomes. The results are expressed as percentage of recovered IND. MICE: Male C57BL/6 mice 6-10 week old, were purchased from WFA-Credo laboratoires (France). Mice were maintained under standard laboratory conditions, at least one week before use. During the time the experiments took place, the mice were carefully controlled and the general state of health of the animals was satisfactory. CELL CULTURE: Lewis lung carcinoma cells (3LL) arc a continous lung tumor cell line of a poorly differentiated spontaneous carcinoma from C57BL/6 mouse. The cells were grown to confluence in 75 cm 2 tissue culture flasks using RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum, penicilin (100 U/ml) and streptomycin (100 ~tg/ml). The flasks were incubated in a humidified 37°C incubator with an atmosphere of 95% air-CO2 5%. The viability of the cells (> 90%) was monitored using a hemocytometer for counting by tripan blue exclusion. LUNG METASTASES: For studies of spontaneous metastases, subconfluent 3EL tumor cell cultures were released with 0.02% cold EDTA in PBS, then washed, counted and resuspcnded in PBS to appropriate concentrations (106 cells/ml) prior to i.m. injection of l0 s cells into the hind leg of syngeneic mice. After 48 h. of tumor transplant, separate groups of animals were subjected to intravenous treatment with free IND (F-IND) or liposome encapsulated IND (Lp-IND) for ten days, 0.5 mg 1ND/Kg]day. The respective control groups received an equivalent treatment with only the IND solvent or empty liposomes encapsulating IND solvent. Four weeks after tumor transplantation, the animals were sacrificed and the number of pulmonar surface metastases was counted, by means of a stereoscopic microscope. All groups were randomized and lung metastases were viewed without knowledge of their experimental group. For studies of experimental metastases, 105 3LL tumor cells in 0.1 ml Of PBS were injected into a lateral tail vein. The groups were performed and one week after the injection of tumor cells, animals were treated i.v. with FIND or Lp-IND for eight days, 1 or 0.2 mg IND/Kg/day, respectively. Three weeks later the animals were sacrificed and the number of pulmonar surface metastases was evaluated as mentioned before.

Vol. 48, No. 2, 1991

Liposome-lndomethacln and Metastasis

151

Results Yield of lND encapsulation: After the preparation of sized liposomes, the entrapped IND and the phospholipid concentration were quantified. In our experiments, the efficiency of liposome encapsulated IND was 9.8 + 3.0 pmol IND/~raol phospbolipid. Stability of stored liposomes : The liposome stability of entrapped material was carried out using CF as a model of entrapped solvent. The selection of CF is based on its capacity for self-quenching of fluorescence in relation to concentration (21). When liposomes containing quenched CF become leaky as a result of changes in their permeability, the encapsulated CF is released into the medium to attain the concentration of CF which provides an increase in the fluorescence. After liposome preparation, they were sterilized by filtration and stored at 4°C. The liposome stability was evaluated at intervals of several days (fig. 1).

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T I M E (DAYS) FIG. 1 Stability of liposomes stored at 4'~. Liposomes composed of PC:CH:SM (7:2:1, molar ratio) encapsulating CF (O) or CF plus IND (0) were prepared by extrusion through policarbonatefilters (0.2 pxn pore size) and stored at 4°C. At several days (from 1 m 21) aliquots of iiposomeswere taken and latent CF was evaluatedin absence(CFa) or presence(CFb) of Triton X-10O.Percentageof latent CF was determined as described in Methods. The values representthe mean of two measurementswhich had not differences higher than 5% and are expressed as % of latent CF respect to latency in recently prepared liposomes.

Our results show that sized liposomes entrapping CF (50 mM) are more stable than those encapsulating CF (50 raM) plus IND (20 mM). Thus, 14 and 21 days after liposome preparation the CF latency in liposomes entrapping CF was 81 and 63% respectively. Whereas in liposomes entrapping CF plus IND was 54 and 27%, respectively. However, in the first week after liposome preparation the CF latency in both cases was > 90%. The study of Lp-IND stability (Table 1) showed that the leakage of IND had a similiar behaviour than the stability of CF encapsulated in liposomes. In ~ddition, the results of Lp-IND stability in mice plasma showed that 80% of Lp-IND was stable after 6 h incubation. Since stability of IND was > 90% after 7 days of their storage at 4°C, we prepared the Lp-IND every week in the "in vivo" experiments.

152

L i p o s o m e - I n d o m e t h a e i n and M e t a s t a s i s

Vol. 48, No. 2, 1991

TABLE 1 Stability of lndomethacin Encapsulated in Lilx~omes

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Stability of indomethacin encapsulated in liposomes. Sized liposomes composed of PC:CH:SM (7:2:1, molar ratio) encapsulating IND were stored at 4°C (between 1 and 21 days) or incubated in mice plasma (1:1, v/v) at 37°(2 (between 1.5 and 24 h). Alliquots of 100 lal of Lp-IND were filtered through Sepharosa CL-4B column and I N was determined in the recovered liposome fraction. The values represent the mean~SE of three measurements and are expressed as % of IND recovered respect to recently prepared liposomes (time 0). Lung metastases: Spontaneous metastases were induced by previous implantation o f a primary tumor into the hind leg. The incidence rate of metastases (fig. 2, left) in two different control groups was 100% and a similar number of metastatic nodules was observed in both groups (11.8 + 2.2 and 9.7 + 2.7). A similar metastatic behaviour was observed in the group treated with empty liposomes. When mice were treated with Lp-IND (0.5 mg/Kg), the incidence rate of metastasis was reduced (55%) and the mean value of metastatic nodules (3.1 + 1.7) was significantly decreased. In contrast, treatment of tumor-bearing mice with free IND (0.5 mg/Kg) showed a 100% of metastatic incidence and the mean value of metastasis was increased (40%) but not significantly. Experimental metastases were induced by direct i.v. injection of 3LL tumor cells. Treatment of mice one week after tumor cell injection with free or liposome encapsulated IND (1 and 0.2 mg/Kg/day, respectively), during eight days, showed that only the latter had significant inhibitory effects (45% approx.) in the mean value of superficial lung metastases with respect to control group (fig. 2, right). Likewise, the treatment with doses 5 times higher of free IND had inhibitory effects but not significative. Discussion In the present study we have observed, in 3LL tumor bearing mice, that: i) encapsulated IND in sized liposomes, efficiently inhibits the incidence and number of spontaneous lung metastases whereas antimetastatic effects were not observed with equivalent doses of the free drug; ii) 0.2 mg/kg/day of IND encapsulated in liposomes reduced significantly the number o f experimental metastases in a similar way than free IND (1 mg/kg/day) although this last effect had not statistical significance. Liposomes have been considered as a potential and valuable drug delivery vehicle for "in vitro" and "in vivo" applications (10, 11). The phagocytic cells (e.g. macrophages) are those mainly involved in liposome clearance from circulation after i.v. injection (12, 13, 14). This property has been used as a strategy to avoid side effects of drugs (15-17) and/or enhance the therapeutic efficacy of drugs and immunomodulators in some experimental tumor systems (18-19). Our results on spontaneous metastases have shown us that free IND lightly increased (40%) the mean value of lung metastatic foci. In the last respect, it has been described that direct IND effects on these and

Vol. 48, No. 2, 1991

Llposome-lndomethacln

SPONTANEOUS METASTASES

153

EXPERIMENTAL METASTASES

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FIG. 2 Effect of free IND or liposome encapsulated IND on spontaneous (left) and experimental (right) lung metastases. For spontaneous metastases, C57BL/6 mice were inoculated i.m. into the hind leg with liP 3LL tumor cells in 0.1 ml of PBS and 48 h later, separate groups were treated with free IND (0.5 mg/Kg/day; F-IND) or fiposomeencapsulated IND (0.5 mg/Kg/day; Lp-IND) for tan days. Control groups received drug solvent (CON) or empty liposomes encapsulating IND solvent (LpO). The mice were sacrif'ar.ed28 days aftar tumor implantation and the number of superficial lung metastases was counted. For exoerimental metastases, C57BL/6 mice were injected in the tail vein with 105 3LL tumor cells. Seven days after injection of tumor ceils, mice were treated with free IND (1 mg/Kg/day; F-IND) or fiposome encapsulated IND (0.2 mg/Kg/day; Lp-IND) for eight days. Three weeks after 3LL cell inoculation, the mice were sacrificed and superficial lung metastases were counted. S ~ signifa~mce was evaluated respect to control group or empty liposomas (Lp • ) by a Wilcoxon test (p and p', respectively). other tumor cells, increase their metastatic potential (4, 5, 6). Because systemic administration of free-IND on tumor-bearing mice mediates effects on both normal host-cells and tumor ceils, we can not exclude a direct IND effect on the 3LL metastatic potential. In contrast, equivalent doses of encapsulated IND greatly reduced the metastatic event. Thus, in our experiments, more than 50% o f tumor-bearing mice were free of lung metastases. The antitumoral activity of IND (including 3LL) has been attributed to the ability o f this drug to increase the host immunoresistence mechanisms (3, 20-25), whereas the metastatic effects have been related to expression of receptors on tumor cell surface, which are involved in the cell attachment on extracellular matrix molecules (6), at least in the case of 3LL carcinoma. Since macrophage cells from tumor-bearing mice make an important contribution in the prostaglandin-mediated immunosupressive response (23-26), it would seem evident that antimetastatie effects of encapsulated-IND could involve drug delivery to phagocytic cells and inhibition o f their immunosupressive prostaglandins, but this question has still to be clarified. Because IN]) can have divergent effects on 3LL tumor progression, we have also evaluated the antitumoral activity o f free and liposorne encapsulated IND on the experimentally established lung metastases. It has been previously reported that 2 mg/kg/day of IND has antitumor activity (3); however in this work, we have not observed an inhibitor effect on primary tumor with 0.5 mg/kg/day of IND. This allows us to suppose that the antitumoral effect of Lp-IND on spontaneous lung metastases is not related to an IND inhibitor effect on primary tumor. In the present report, we have compared the effect o f free IND (lmg/kg/day) respect to a 5 fold lower dose of I N encapsulated in liposomes (0.2 mg/kg/day). The results showed that free-IND ( l m g / K g / d a y ) inhibited (20%) the mean value o f lung metastases but not significantly; whereas a significant 40% reduction was observed in the group treated with much lower doses o f encapsulated IND (0.2

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Liposome-lndomethacin and Metastasis

Vol. 48, No. 2, 1991

mg/Kg/day). These results are in agreement with previous observations that also lower doses of encapsulated IND with respect to the free drug were required in the "in vivo" induction of antiinflamatory effects (27). In summary, our results suggest that eneapsulated4ND in sized liposomes is several times more efficient than the free drug in inducing antimetastatic effects and may be an efficient way in evading the possible side effects of this drug after systemic administration. Acknowledgements The authors are grateful to Dr. Lee Leserman for his valuable suggestions and Mrs. M. Portuondo for her technical assistance. This work was supported by PGV-8903 grant from the Basque Goverment. References 1. I.J. FIDLER, Cytometry 10:673-680 (1989). 2. V.H. HO_NN, R.S. BOCKMAN and L.J. MARNET, Prostaglandins 21 : 833-864 (1981). 3. S.F. ALINO, F. UNDA, G. PEREZ-YARZA and M.L. CANAVATE, Biol. Cell. 66 : 225-261 (1989). 4. D.A. STRINGFELLOW and F.A. FITZPATRICK, Nature 2 ~ : 76-78 (1979). 5. F.A. FITZPATRICK and D.A. STRINGFELLOW, Proc. Natl. Acad. Sci. USA 76 : 17651769 (1979). 6. S.F. ALINO, F.J. UNDA and G. PEREZ-YARZA, Biochem. Biophys. Res. Commun. 167: 731-738 (1990). 7. S.F. ALINO, M. GARCIA, M. LEJARRETA, M. BOBADILLA, G. PEREZ-YARZA and F.J. UNDA, Biochem. Soc. Transac. 17 : 1000-1001 (1989). 8. J.C.M. STEWART, Anal. Biochem. 104:10-14 (1980). 9. L.J. DUSCI and L.P. HACKETT, J. Chromatogr. 172:516-519 (1979). 10. P. MACHY, B. ARNOLD, S.F. ALINO and L.D. LESERMAN, J. Immunol. 136:31103115 (1986). 11. L.D. LESERMAN, P. MACHY, D. ARAGNOLD and S.F. ALII~O, New Aven¢¢~ in Develooment Cancer Chemotherapy. pp. 299-331, Acad. Press, New York (1987). 12. I.R. MCDOUGAL, J.R. DOUNICK, M.C. MCNAMEE and J.B. KRISS, Proc. Natl. Acad. Sd. USA 71 : 348%3491 (1974). 13. G.L. SCHERPHOF, T. DAEMEN, H.H. SPANHER and F.H. ROERDINK, Lipids 2_.2_2: 891-896(1987). 14. S.F. ALINO, E. HILARIO, A. IRUARRIZAGA, J. ALFARO, G. PEREZ-YARZA and F.J. UNDA, Institute Physics Corfferenc¢ Series. pp 783-786, H.Y. Elder and P J Goodhew (eds.), Eastern Press. Ltd., London (1990). 15. LN. WEINSTEIN, LiDosomes. From Biophysics to Theraoeuties. pp. 277-338, M.J. Ostro (ed.), Marcel Decker, fnc, New York and Basel (1987). 16. G. STORM, P. A. STEERENBERG, F. EMMEN, M. VAN BORSSUM WALKES and D.J.A. CROMMELIN, Biochim. Biophys. Acta ~65 : 136-145 (1988). 17. J. SZEBEN, R. GARCIA, C.D. ESKELSON and M. CHVAPIL, Life Sci. 45 : 729-736 (1989). 18. I.L FIDLER, S. SONE, W.E. FOGLER and Z.L. BARNES, Proc. Nail. Acad. Sci. USA 78 : 1680-1684 (1981). 19. P.D. MONTE and F.C. SZOKA, J. Immunol. 142 : 1437-1443 (1989). 20. A.M. FULTON, J. Nail. Cancer Inst. 78 : 735-741 (1987). 21. O.L PLESCIA, A.H. SMITH and K. GRINWICH, Proc. Nail. Acad. Sci. USA 72 : 18481851 (1975). 22. N.R. LYNCH, M. CASTES, LC. ASTOIN and J.C. SALOMON, Br. J. Cancer 38 : 503-512 (1978). 23. R.S. PARHAR and K. LALA, Cell. ImmunoL 93 : 265-279 (1985). 24. A.B. TILDEN and C.M. BALCH, Surgery 90 : 77-84 (1981). 25. J.L. MURRAY, J. DOWD and E.M. HERSH, J. Biol. Response Modif. ~ : 12-19 (1986). 26. R.S. PARHAR and P.K. LALA, Proc. Am. Assoc. Cancer Res. 28 : 364 (1987). 27. A. GURSOY, J. AKBUGA, L. EROGLU and S. ULUTIN, J.'Phann. Pharmacol. 40 : 53-54 (1988).