Treatment of gamma radiation-induced transplanted leukemia in ICRC mice by liposomally encapsulated 5-fluoro uracil

Vol. 17, No. 7, pp. 601--607. 1993. Printed in Great Britain.

0145-2126/93 $6.00 + .00 © 1993 PergamonPress Ltd

Leukemia Research

T R E A T M E N T OF GAMMA RADIATION-INDUCED TRANSPLANTED LEUKEMIA IN ICRC MICE BY LIPOSOMALLY ENCAPSULATED 5-FLUORO URACIL* S I N D H U V . JOSHI, S. G . V A I D Y A , V . R . N E R U R K A R a n d C H I T R A L E K H A S O M A N ~

Chemotherapy Division, Cancer Research Institute and tPathology Department, Tata Memorial Hospital, Tata Memorial Centre, Pard, Bombay 400 012, India (Received 14 June 1992. Revision accepted 31 January 1993) Abstract--The present investigation reports on the efficacy of 5-FU encapsulated in liposomes for the treatment of leukemia using a murine model of gamma radiation-induced transplantable leukemia in ICRC strain of mice. Multi-lamellar vesicles (MLVs) or large unilamellar vesicles (REVs) prepared using phosphatidyl choline and cholesterol in a molar ratio of 8 : 2 for neutral and 7 : 2 : 1 for charged vesicles were administered as a single i.p. dose in mice, 5-FU encapsulated in MLVs at a concentration of 0.6-2.5 mg/kg had no effect, whereas REVs at a single i.p. dose of 9 mg/kg increased survival of leukemic mice with T/C = 138%, decreased peripheral blood count and considerably reduced infiltration of leukemic cells in different tissues (supported by histopathology) as compared to 60 mg/kg of free drug (LD10 = 70 mg/kg). Key words: Gamma radiation-induced, murine leukemia model, liposomally encapsulated 5-FU, treatment of transplanted leukemia, leukemic infiltration.

seem to limit the use of this versatile drug carrier system when administered intravenously [11]. Treatment of cancer patients in clinics using liposomally encapsulated drugs for fungal infections, ovarian, bladder and breast carcinoma etc. has been reported [12-151 . The use of drug delivery by liposomes for effective treatment of leukemia seems to be an area of interest wherein the possibility of direct interaction of drugloaded liposomes with diseased cells in circulation, and release of drugs to the cell interior bringing about cell kill with minimal toxicity to other tissues, could be visualized. The adequate concentration for effective cell kill could be maintained by selecting liposomes of suitable type, and extensive uptake of liposomes in R.E.S. could be beneficially utilized further, adding to cell kill. Based on this rationale, a study was undertaken in our laboratory using liposomally encapsulated anti-cancer drugs for the treatment of leukemia. A murine model of transplantable lymphocytic leukemia induced by whole body yirradiation in albino mice of ICRC strain, developed by us, has been used for treatment. Our murine model of leukemia simulates a clinical picture of human disease with high peripheral blood count, presence of large numbers of blasts in circulation and also infiltrating different tissues including testes and brain [16]. One of the most effective drugs used for treatment

INTRODUCTION LIPOSOMES are lipid vesicles readily prepared from natural and synthetic phospholipids with diversity of size, charge and composition [1]. The possibility of using liposomes as carriers for the direct delivery of drugs to tumor cells or as reservoirs for the sustained release of drugs for phase-specific drugs is based on their intrinsic properties [2, 3]. Liposomally encapsulated anti-neoplastic drugs show altered pharmacokinetic behavior [4], and when used for treatment on experimental tumor models were found to be therapeutically more effective than the free drugs in terms of increased survival, reduced toxicity and tumor load, and overcome drug resistance due to membrane impermeability [5-10]. Extensive uptake of liposomes by cells of the reticulo endothelial system (R. E.S.) and micro-circulation barrier Abbreviations: 5-FU, 5-fluoro-uracil; MLVs, multilamellar vesicles; REVs, large-sized single walled liposomes prepared by reversed phase evaporation method; PC, phosphatidyl choline; Chol, cholesterol; 5-FU REVs, 5-fluoro-uracil encapsulated in liposomes prepared by reversed phase evaporation method. Correspondence to: Dr Sindhu V. Joshi, 63, Pranav, Green Garden Apartments, Waman Patil Marg, Govandi, Bombay 400 088, India. * Partly presented at the II International Conference of Anticancer Research, held at Saronis, Greece, 11-15 October 1988. 601

s.w. JOSHIet al.

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of solid tumors in clinics, either alone or in combination [17] is 5-fluoro-uracil. The growth inhibitory activity of this S-phase-specific anti-metabolite was reported earlier by Heidelberger et al. [18] and Liebling [19] against different transplanted tumors in rats and mice. M a z u m d e r observed increased survival of Swiss mice bearing Ehrlich ascites carcinoma on treatment with 5-FU encapsulated in negatively charged M L V s [20]. We report on the increased efficacy of 5-FU encapsulated in liposomes for the treatment of murine transplanted leukemia as c o m p a r e d to free drug.

of lipid concentration on encapsulation of 5-FU in liposomes was studied by preparing MLVs using different amounts of lipid (25-200 pm) for 16 mg of the drug. The amount of drug encapsulated was determined by dissolving a known volume of liposomes in methanol (5 ml) with a spectral absorption at 266 mtx.

Stability of liposomes Liposomes diluted to 10 ml in saline (0.85%) were stored at 4°C. The samples were centrifuged at different time intervals. The amount of 5-FU in the pellet or in the supernatant was determined spectrophotometrically. The stability of liposomal 5-FU was calculated as a percentage of the amount of 5-FU present/amount initially present.

Toxicity of 5-FU MATERIALS AND METHODS

Chemicals Phosphatidyl choline (PC) was prepared from fresh chicken egg yolks using the method of Singleton et al. [21]. Purity of PC was tested by thin layer chromatography using CHCI3: CH3OH : HzO (65 : 25 : 4 v/v) as a solvent system [22]. A single spot was exhibited at an applied amount of 10 pg of PC. Cholesterol (CHOL), spectroscopic grade, was obtained from SISCO Laboratories, Bombay. Stearyl amine (SA) and dicetyl phosphate (DCP) were Sigma products. Biochem Pharmaceutical Industries India supplied the 5-FU. ]4C-5-FU (specific activity 4.65 ~tCi/mMol) was synthesized and supplied by the isotope division of Bhabha Atomic Research Centre (BARC), Bombay and 7 (n) 3HCHOL (specific activity: 5 Curies/mMol) by the Radiochemical Centre, Amersham, U.S.A. Solvents chloroform and methanol were of high purity grade and redistilled prior to use. Diethyl ether (AR grade) was used as such (SDS Fine Chemicals).

Preparation of liposomes Liposomes were prepared using PC and CHOL in a molar ratio of 8 : 2 for neutral, and 7 : 2 : 1 for charged vesicles, positive with stearyl amine and negative with dicetyl phosphate. Multilamellar vesicles (MLV) were prepared by the method of Bangham [23] by rotary evaporation of phospholipid solution in chloroform, followed by hydration of lipid film with aqueous solution of 5-FU (16 mg/ml in 0.85% saline/50 pM of lipid). Large-sized single walled liposomes were prepared by the reversed phase evaporation method as described by Szoka et al. [24]. In short, phospholipids dissolved in 3 ml of diethyl ether with 1 ml of aqueous solution of 5-FU in a round-bottom flask held in an ice-bath were sonicated using a probe-type (1 cm diameter) sonicator (Soniprep, Ralsonics) for 10 spurs (30 s on and 30 s off) at 80% input efficiency. Ether was removed carefully under reduced pressure. Unentrapped 5-FU was removed by ultracentrifugation (Kontran refrigerated centrifuge, Model T 1065) at 100,000 g for 35 min at 4°C three times.

Encapsulated efficiency The amount of 5-FU encapsulated in MLVs and REVs was determined by using 14C-5-FU as an aqueous marker. The degree of encapsulation was determined by the percent radioactivity present/total radioactivity added. The effect

ICRC mice of either sex, 6-8 weeks old, 28-30 g average weight, were used. The animals were evenly grouped by weight. Ten animals were used for each concentration of drug 5-FU (25-200 mg/kg) administered as a single i.p. dose. The animals were kept under observation for 10 days. The plot of % mortality vs concentration (mg/kg) was used to calculate LDs0and LD10(concentration of drug causing death of 50% and 10%, respectively) values for 5-FU. In vivo activity of free and liposomally encapsulated 5-FU Mice of either sex, 6-8 weeks old, 6 in each group, were used. Leukemic cells, 10 7, were implanted by i.p. route in each animal. Twenty-four hours after treatment with free drug (concentration lower than LD10 dose), empty liposomes, empty liposomes + free drug and drug-laden liposomes were administered by i.p. route (0.1 ml/10 g wt of mouse). Animals without any treatment were taken as untreated controls. Peripheral leukocyte count and leukemic infiltration in different tissues was studied. The antitumor activity was assessed from the increase in survival, i.e. T/C=survival of treated/survival of untreated × 100 (T/C _>--125% considered as significant using NIH protocol).

Peripheral leukocyte and differential blood count Blood from a cut tail vein was removed on day 7 and used for W.B.C. and differential counts. Student's t-test was applied to evaluate the statistical significance of the treatment.

DOSE R E S P O N S E C U R V E OF 5 F U IN ICRC MICE. 100 90

70

50 50

10

/ ..... ~...... _ - - - ~ - ~,,. /

to

so

so

7o

,o

,o

t~o

tso

tTo

~Jo 20o

DOSE OF 5 FU I m g / k ( J )

FIG. 1. Dose-response curve of 5-FU in ICRC mice.

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Murine transplanted leukemia treated by liposomally encapsulated 5-FU TABLE 1. ENCAPSULATION OF 5-FU IN LIPOSOMES

Concentration of 5-FU (mg/ml)

Type of liposomes

Composition with molar ratio

Entrapment efficiency

50.0 40.0 30.0 20.0 16.0 16.0 16.0 20.0 16.0 16.0 16.0

MLV neutral MLV neutral MLV neutral MLV neutral MLV neutral MLV positive MLV negative REVs neutral REVs neutral REVs positive REVs negative

PC : CHOL [8 : 2] PC: CHOL [8: 2] PC:CHOL [8:2] PC : CHOL [8:2] PC:CHOL [8:2] PC:CHOL: SA [7:2: 1] PC: CHOL : DCP [7 : 2 : 1] PC : CHOL [8 : 2] PC'CHOL [8: 2] PC: CHOL : SA [7:2: 1] PC: CHOL : DCP [7:2: 1]

Not formed Not formed 0.50% 0.50% 0.75% 1.30% 1.40% 3.00% 6.20% 6.70% 5.60%

Histopathology of different tissuesfor leukemic infiltration The animals were sacrificed on day 7. The tissues were fixed and sections of 6 ~t thickness were stained with Haematoxylin & Eosin and observed for leukemic infiltration. RESULTS

Toxicity of 5-FU The effect of an increase in concentration of 5-FU on survival of normal animals was studied in the range 25-200 mg/kg. The drug was found to be toxic at a dose of 125 mg/kg and above. From the doseresponse curve (Fig. 1) (concentration of drug vs % mortality), the LD10, i.e. concentration of drug at which 10% of treated animals died, was found to be 70 mg/kg and the drug was used at a concentration lower than the LD10 dose in the present study.

Encapsulation of 5-FU in liposomes After preparation of both MLVs and REVs, the unentrapped free drug was removed from the drug encapsulated in liposomes by ultracentrifugation. It was not possible to separate free 5-FU by column chromatography on Sepharose 4B or Sephadex Gs0 (column size 1.5 x 32 cm, eluted with 0.85% saline). The liposome preparation of REVs travelled down the column to a distance of 1-2cm initially, was retarded and remained adsorbed to the column material in the shape of a wedge. The free drug, however, was eluted in the bed volume. The encapsulation efficacy of liposome using 14C5-FU as an aqueous marker and calculated from the percentage of total radioactivity added is shown in Table 1. At 16 mg/ml of 5-FU used, the degree of encapsulation for neutral MLVs is 0.5%, whereas a two-fold increase is seen in the amount of drug present in liposomes bearing positive and negative charges. In REVs, single bilayered liposomes with a

high capacity to entrap, a many-fold increase in the degree of encapsulation is observed and is not affected, as expected, by the presence of charge. No liposomes could be formed when the concentration of 5-FU used was 40 and 50 mg/ml for 50 ~tM of phospholipid used. At a constant amount of drug used (16mg), the quantity of drug encapsulated increased with increase in lipid concentration of MLV from 0.5% at 50 ~xM to 0.7% at 100 ~tM and 1% at 200 ~tM.

Stability of liposomes Liposomes stored at 4°C released 10% of encapsulated drug after 48 h. Liposomes were used within 1-2 h.

Treatment of murine leukemia with 5-FU encapsulated in liposomes On treatment of leukemic mice (i.p., single dose) with 5-FU encapsulated in MLVs (concentration 0.62.5 mg/kg) and with empty liposomes, free drug, free drug + empty MLVs as control groups, no effect on survival was seen (T/C = 100%). With 5-FU encapsulated in REVs, an increase in survival was observed with T/C = 138% at a concentration of 9 mg/kg, but not at a dose of 3 and 6 mg/kg (Tables 2 and 3). However, at all three doses used, a decrease in peripheral leukocyte count was seen which was statistically significant, with p values < 0.05. Empty REVs alone or free drug at 20 mg/kg had no effect on survival; however, empty REVs + free drug (20 mg/ kg) is found to be effective with T/C = 133%. The effectiveness of treatment as seen from the increase in survival and decrease in peripheral blood count was also supported by histopathological observations on leukemic infiltration in different tissues. The number of blasts present in the bone marrow was reduced and the presence of normal granulocytic elements

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S. V. JOSHI et al. TABLE 2. EFFECT OF TREATMENT ON MURINE LEUKEMIA

Survival Expt

Type of treatment

WBC mean+ SEM*

p significance

Range (days)

Average (days)

T/C %

Untreated Empty REVs Empty REVs + Free 5-FU (20 mg/kg) Free 5-FU (20 mg/kg)

194,800 ± 19,673 147,200 ± 17,488

N.S.

14-17 13-17

15 15

100

20,000 ± 894 36,800 ± 11,230

p < 0.001 p<0.001

19-21 14-17

20 15

133 100

Untreated REVs entrapped 5-FU (3 mg/kg) REVs entrapped 5-FU (6 mg/kg) REVs entrapped 5-FU (9 mg/kg)

200,366 ± 15,000

13-14

13

38,000 ± 13,905

p < 0.01

13-14

13

100

34,000 ± 7304

p < 0.01

13-18

14

104

29,083 ± 11,005

p<0.01

16-18

17

138

* Blood counts were taken on day 7, after treatment.

TABLE 3. TREATMENT OF MURINE LEUKEMIA BY FREE 5-FLUOROURACIL

Survival Type of treatment free drug (single I.P.) Untreated 5-FUFree

Dose (mg/kg)

Range (days)

Average (days)

T/C %*

12 20 40 60

14-16 14-18 14-17 19-22 20-23

15 15 15 20 21

-105 100 133 140

median survival of treated * T/C = median survival of control significantT/C %/> 125.

with neutrophils and megakaryocytes was seen. The extensive infiltration of leukemic cells observed in tissues of untreated animals was considerably reduced on treatment with 5-FU encapsulated REVs (concentration 9 mg/kg). The leukemic cells appeared as scanty loci of infiltrates with normal parenchymatous structures in liver and normal lymphoid structures in spleen, mesenteric mass and thymus. Treatment with free drug (5-FU, 60 mg/kg) seemed to bring about a massive reduction in the number of infiltrating cells in these tissues. DISCUSSION 5-FU is a polar drug with limited water solubility (partition coefficient 6.9 x 10-4) [25]. It does not associate with lipid bilayers [26] and distributes in aqueous compartments of liposomes [27]. Such hydrophilic compounds are poorly entrapped in liposomes as compared to non-polar solutes. In general,

the degree of encapsulation in liposomes depends on size, phospholipid composition, nature and molar ratio of components, the degree of saturation of acyl chains present in phospholipid, and the presence of charge on lipid membrane and cations in aqueous compartment [4, 28, 29]. From our own data it is seen that with high capacity to encapsulate in aqueous compartment, REVs, show higher degree of encapsulation for 5-FU as compared to MLVs but with a single lipid bilayer no further increase is seen in the amount of drug entrapped in REVs as observed with charged MLVs (Table 1). Encapsulation of 5-FU in liposomes of different types has been reported by other workers [25, 26, 27, 29, 30, 31]. Kirby and Gregoriadis [32] developed a novel method using the rehydration-dehydration procedure and for 16.5 ixmol of phospholipid as SUV, reported 46% entrapment at concentration of 10 mg/ ml for 5-FU. Using this procedure for MLVs, we obtained a value of 12.6%. The encapsulation

Murine transplante-I leukemia treated by liposomally encapsulated 5-FU

efficiency of liposomes prepared by us from time to time was reproducible and considered satisfactory for work. Removal of unentrapped drug from liposome dispersions is often carried out by dialysis, column chromatography or ultracentrifugation; of these methods, dialysis is time consuming. As observed by us and reported by others, MLVs show a tendency to remain adsorbed to the bed material at the top of the column, whereas REVs are readily eluted. Compounds with ring structures are known to be retarded during elution on Sephadex column. Our observation that 5-FU-REVs remain adsorbed to the Sepharose 4B column material in the shape of a wedge, while free 5-FU gets eluted in bed volume, prompted us to use ultracentrifugation method for separation of free 5-FU from 5-FU-REVs. Ozer and Talsma [30] reported that their negatively charged preparation of 5-FU-REVs could not be separated from free drug by column chromatography using ion exchangers such as Dowex 50, WX4, SP Sephadex Gs0 or Amberlite X A D 2 due to chemical interaction and adsorption, and also observed that Silica Gel 60H was not effective, and therefore used ultracentrifugation. From our data it was seen that at a dose of 0.62.24 mg/kg, MLVs neutral and charged, single i.p. or 1-5-9 schedule, had no effect on survival of animals or peripheral blood count. 5-FU-REVs at doses of 3 and 6 mg/kg have no effect on survival; however, they reduce peripheral blood count to a value which is statistically significant with a p value of < 0.01.5FU-REVs, at a dose of 9 mg/kg, seem to be most effective in terms of decrease in peripheral leukocyte count, increase in survival with T/C = 138% and reduction in leukemic infiltration in different tissues. This is comparable to the effect shown by free drug at a concentration of 40-60 mg/kg. The therapeutic effect of the drug thus seems to depend on concentration of the drug available. The mechanism of action of 5-FU is well understood. The drug is eliminated fast through excretion. Use of drug delivery would protect the drug from excretion, making it available for inhibition of DNA synthesis by blocking thymidylate synthetase activity for cells in S-phase. Thus the possibility of increasing the effectiveness of the drug at a lower concentration would be realised. That liposomally encapsulated 5-FU administered by i.p. route enters circulation and remains there for a longer time is supported by our own data on the clearance of i.p. injected free 5-FU and 5-FU-REVs (data not shown) as well as by other workers. Ellens et al. [33], Senior et al. [34], and Kirby and Gregoriadis [35] observed that i.p. injected drug-laden liposomes enter circulation intact within 90 min and

605

remain so for up to 4 h. The i.p. injected 5-FU loaded REVs seem to enter the circulation and bring about the observed therapeutic effect by interacting with cells in circulation and possibly entering different tissues to decrease peripheral blood count and reduce leukemic infiltration in different tissues including bone marrow. We observed delayed take up of leukemia when cells from the bone marrow of animals treated with free or encapsulated 5-FU were used for transplantation. With our observations that both empty liposomes or free drug alone (concentration 20 mg/kg) have no effect on survival of leukemic mice whereas empty liposomes + free drug at same concentration increase the survival, further experiments were carried out using liposomes of different size (MLV, REVs and SUVs) as well as of different cholesterol content + free drug. Empty MLVs prepared with PC alone were found to be toxic while other groups had no effect on the survival of leukemic mice. It is observed that the peripheral leukocyte count may decrease with or without an increase in survival, whereas increase in survival is always associated with a reduction in peripheral blood count. In experimental studies using 5-FU encapsulated in positively charged MLVs, injected in testicles of rats, Segal et al. observed delayed liberation of labeled drug in surrounding tissues [36]. Mazumder observed an increase in survival and regression of Ehrlich ascites carcinoma in Swiss mice on treatment with negatively charged MLVs (dipalmitoyl PC : CHOL: PA, molar ratio of 7 : 2 : 1) when injected both by i.p. and i.v. route and administered 48 h after implantation of tumor cells [20]. Chemical modification of 5-FU to lipophilic prodrug increases its incorporation in lipid bilayer and could be effectively used for treatment at a dose much higher than polar 5-FU. Sasaki et al. investigated the effect of alkyl promoieties and carbomoyl linkage encapsulated in liposomes on L1210 leukemia in BDF1 mice [26]. Hashida et al. reported on in v i v o antitumor activity of cholesterol promoiety of 5-FU as a prodrug on B6D2F 1 mice bearing P388 leukemia [25]. Tsurao et al. [37] studied lymph node metastasis and the effect of Ara-C and 5-FU and their lipophilic derivatives in an experimental model system using P388 leukemia.

Iigo et al. studied the effect of anti-tumor activity of 1-hexyl-carbamoyl 5-FU in Lewis lung carcinoma and B16 melanoma [38]. Thus, 5-FU encapsulated in REVs, administered as a single i.p. dose at a concentration of 9 m g / kg, shows a beneficial effect with increased survival, reduction in peripheral blood count and partial eradication of leukemic cells infiltrating different tissues

606

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in our murine model. The liposomally encapsulated 5-FU seems to be 4-6-fold more effective as compared to free drug at concentrations of 40-60 mg/kg. The usefulness of this observation in terms of reduction in toxicity and cost is considerable. The increase in survival observed, however, was not more than T / C = 140%. This could be a limitation of the murine model developed by us or related to the type of anti-cancer drug used. Further work is in progress.

Acknowledgements--We are thankful to Dr S. V. Gokhale, formerly In-Charge, Pharmacokinetics and Drug Targeting Unit and Dr Lalitha Rao, In-Charge, Neurooncology Unit, Cancer Research Institute, Parel, Bombay for useful comments and discussions during the preparation of this manuscript.

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