(CANCER RESEARCH 52. 2431-2439. May 1. I992|

Stealth Liposomes: An Improved Sustained Release System for1-/?-D-ArabinofuranosyIcytosine '

Theresa M. Allen,2 Tarun Mehra, Christian Hansen, and Yeen Cheel Chin

Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7

ABSTRACT

Newly developed liposomes with prolonged circulation half-lives anddose-independent pharmacokinetics (Stealth" liposomes) have been

tested for their efficacy as a slow release system for the rapidly degraded,schedule-dependent, antineoplastic drug 1-0-D-arabinofuranosylcytosine(ara-C) in the treatment of murine L1210/C2 leukemia. Mice were giveninjections of either 10* cells or 10' cells by either the i.v. or the i.p.routes. Leukemia-bearing mice were treated with either i.v. or i.p. injections of free drug, i.v. or i.p. injections of liposome-entrapped drug, or24-h i.v. infusions of free drug. Long-circulating liposomes contained, asthe stealth component, either monosialoganglioside or polyethylene gly-col-distearoylphosphatidylethanolamine. Liposomes lacking the stealthcomponents (non-stealth liposomes) were also injected for comparison.At lower dose ranges, stealth liposomes were superior to non-stealthliposomes in prolonging mean survival times of the mice, and all liposomepreparations were superior to injections of the free drug. Drug entrappedin stealth liposomes, when administered at or near the maximum tolerateddose of 100 mg/kg ara-C were considerably superior to 24-h free druginfusions given at the same total drug dose. Therapeutic effect was relatedto the half-life of leakage of ara-C from the liposome formulations, aswell as to circulation half-life, with maximum therapeutic effect achievedwith long circulation half-lives and more rapid leakage rates. The therapeutic efficacy of non-stealth liposomes increased with increasing liposome (and drug) dose as a result of saturation of liposome uptake by themononuclear phagocyte system, which resulted in longer circulation half-lives for these liposomes at higher doses (Michaelis-Menten pharmacokinetics). Liposome entrapment can protect rapidly degraded drugs frombreakdown in n'TO,with release of the drugs in a therapeutically active

form over periods of up to several days. The dose-independent pharmacokinetics and reduced mononuclear phagocyte system uptake of stealthliposomes gives them distinct advantages over non-stealth liposomes.

INTRODUCTION

There have been at least 2 major drawbacks to the use ofliposomes as sustained release systems for drugs in vivo, (a)The high affinity of conventional liposome formulations(termed here "non-stealth" liposomes) for the MPS' (reticulo-

endothelial system) leads to their rapid removal from circulation resulting in adverse effects on this important host defensesystem (reviewed in Refs. 1 and 2) and reduced availability ofliposome-entrapped drug to other tissues, (b) Avid uptake into

Received 7/31/91; accepted 2/19/92.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by a grant from the Medical Research Council ofCanada (MA-9127) and by Liposome Technology. Inc., Menlo Park, CA. whoprovided the PEG-DSPE and HSPC for the experiments. T. M. was supportedby a graduate studentship, and Y. C. C. by a summer studentship, from theAlberta Heritage Foundation for Medical Research.

2To whom requests for reprints should be addressed.3The abbreviations used are: MPS, mononuclear phagocyte system (reticulo-

endothelial system); ara-C, 1-ff-D-arabinofuranosylcj tosine; PC. egg phosphati-dylcholine; HSPC, hydrogenated soy phosphatidylcholine; SM, bovine brainsphingomyelin; CH, cholesterol; PC, egg phosphatidylglycerol; GMi. monosialoganglioside; PEG, polyethylene glycol; PEG-DSPE. polyethylene glycol (M, 2,average) derivative of dislcaroylphosphatidylethanolamine; REV, large unilamellar liposomes prepared by the reverse-phase evaporation technique; USP. UnitedStales Pharmacopoeia; IIS, percent increase in the mean survival times of testmice as compared to control mice; q3h. every 3 h; LD«.SO^c lethal dose; T,/2,half-life.

the MPS of non-stealth liposomes leads to saturable (nonlinear)pharmacokinetics for the carrier, which complicates calculations of the amount of liposome-entrapped drug necessary toachieve a given therapeutic drug dose (3-6). In addition, manyconventional liposome formulations are susceptible to the action of plasma proteins and other biological fluids in vivo,leading to rapid loss of their drug contents (7-9).

New liposomal formulations have now been developed thathave considerably reduced MPS uptake, remain in circulationfor extended periods of time (9-17) with dose-independentpharmacokinetics (6), and have reduced susceptibility to protein-induced leakage (13, 18). These improved liposome formulations, termed Stealth4 liposomes for their ability to avoid

detection by the MPS (12), would be expected to be of utilityas drug-sustained release systems for drugs that normally experience rapid degradation in vivo.

The model system chosen to test this proposed therapeuticapplication was one in which the drug ara-C, free or liposome-entrapped, was tested for its ability to prolong survival times ofmice bearing L1210/C2 leukemia. ara-C was chosen because itis a schedule-dependent antineoplastic drug, used clinically inthe treatment of acute leukemia, that is rapidly inactivated invivo by cytidine deaminases with an initial half-life of 16 to 20min in mice, close to the value found in humans (19, 20). It isan inexpensive model for other drugs, e.g., the immune modulators, which are candidates for delivery by drug-sustainedrelease systems. In addition, several investigators have previously tested non-stealth liposomal preparations of ara-C andhave found good therapeutic efficacy for their formulations(21-28). These prior experiments provide data against which

the efficacy of stealth liposomes can be compared, although thecomparison will be complicated by the nonlinear pharmacokinetics of the previous preparations. Measurement of meansurvival times of tumor-bearing mice in response to varioustreatment regimens is a simple means of comparing the therapeutic efficacy of the different therapeutic groups.

The objective of our initial experiments was to compare thetherapeutic efficacy of the various treatments, not to producelong-term survivors, although this is achievable at high dosesof ara-C at low tumor burdens. The presence of long-termsurvivors prevents statistical comparisons. Therefore, initially,low drug doses of ara-C were chosen, so that single injectionsof free drug had marginal effects on increasing mean survivaltimes of the mice. The same total dose given by infusionprolonged the mean survival times, but did not result in cures.In the final experiments, the objective was to evaluate thetherapeutic efficacy, and the maximum tolerated dose, of anewly developed, inexpensive stealth liposome formulation consisting of HSPC, CH, and PEG-DSPE. Single or multipleinjections of liposome-entrapped drug were given in order todetermine the conditions that resulted in long term survivors.

In addition, drug leakage, pharmacokinetic, and tissue distribution studies were carried out in order to provide data that

4Stealth is a registered trademark of Liposome Technology Inc.. Menlo Park.

CA.

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ARA-C IN STEALTH LIPOSOMES

would aid in our understanding of the mechanism by whichliposome-entrapped drug was having its therapeutic effect.

MATERIALS AND METHODS

Materials. PC, SM, and PC were purchased from Avanti Polar Lipids(Birmingham. AL). CH and ara-C were purchased from Sigma Chemical Co. (St. Louis, MO) and [5-'H]ara-C (0.55-1.1 TBq/mmol) was

purchased from Amersham (Oakville, Ontario, Canada). HSPC andPEG-DSPE were generous gifts of Liposome Technology, Inc. (MenloPark, CA). The PEG used in these experiments was an average of A/r2. The synthesis of PEG-DSPE has recently been described (17). GMrwas purchased from Makor Chemicals (Jerusalem, Israel). Pyrogen-free saline (0.9% USP) was obtained from Travenol Canada, Inc.(Mississauga, Ontario, Canada).

Liposome Preparation. Liposomes were prepared by the REV method(29) with an aqueous solution of 247 ¿IMara-C (60 mg/ml) in distilledwater (290 mOsm) containing [5-'H]ara-C such that the specific activityof the final ara-C solution was 37 kBq/ml. For some experimentsinvolving the effect of osmolarity on leakage of ara-C from liposomes,the concentration of ara-C was 150 mg/ml (850 mOsm). The liposomeswere then extruded (Extruder; Lipex Biomembranes, Vancouver, British Columbia, Canada) 10 times through 2 stacked 0.4-^m Nucleporepolycarbonate filters (Nuclepore Corp., Pleasanton, CA) either at roomtemperature, or at 5 to 10°Cabove the phase transition temperature in

the case of high phase transition phospholipids (30, 31). Concentrationsof lipids are expressed as molar ratios of phospholipids.

Liposome-entrapped ara-C was separated from free ara-C in one of3 ways depending on the desired concentration of liposomal ara-C. Forexperiments involving low doses of liposomal ara-C, liposomes werechromatographed over Sephadex G-50 in sterile, pyrogen-free NaCI(0.9%, USP) containing 10 mM /V-tris(hydroxymethyl)methyl-2-ami-noethanesulfonic acid, pH 7.4 (buffer). For higher doses of liposomalara-C, where dilution of the liposomes was undesirable, liposomes weredialyzed through Spectrapore dialysis tubing (cutoff, M, 12) againstseveral changes of buffer at 4°C.For the highest doses of liposomal

ara-C, liposomes were concentrated by centrifugation at 100,000 x gfor 1 h, and free ara-C was removed by washing the liposomes 3 timesin buffer. Following the final washing step, the liposome pellet wasmade up to the desired concentration by the addition of buffer. In someliposome preparations, liposomes were pelleted by centrifugation, thesupernatant containing free ara-C was removed, but the pellet was notwashed. The pellet was resuspended in buffer to the desired drugconcentration. In those preparations, the total amount of free ara-Cwas equivalent to the total amount of liposomal ara-C, as determinedby chromatography of the samples over Sephadex G-50. Samples ofthe liposome preparations were taken before and after separation of thefree drug, and the concentration of ara-C in the preparations wascalculated from the specific activity of [5-'H]ara-C.

Extruded vesicles were sized by quasielastic light scattering using aBI-90 particle sizer (Brookhaven Instruments, Holtsville, NJ). Phos-pholipid concentration was determined using the method of Bartlett(32), and trapped volumes of the liposomes were determined from thespecific activity of [3H]ara-C. The average mean diameter of the lipo

somes was approximately 190 to 220 nm and the trapped volume wasin the range of 4 to 6 liters/mol phospholipid. These results could bereliably reproduced from sample to sample.

Leakage of ara-C from Liposomes. ara-C and [5-3H]ara-C were en

trapped in liposomes, as above, at concentrations of 60 or 150 mg/ml,corresponding to osmolarities of 290 and 850 mOsm (Westcor VapourPressure Osmometer, model 5500; Westcor, Logan, UT). The effect ofosmolarity on leakage was determined for liposomes composed ofSM:PC:CH:GMi, 1:1:1:0.14 or PC:CH, 2:1 (molar ratios of phospholipid). The rate of leakage was determined at 37°Cin either buffer or

25% pooled human plasma in buffer, by measuring the free and entrapped ara-C peaks following chromatography of samples over Sephadex G-50 columns. Half-times for leakage were calculated usingGraphpad software (ISI, Philadelphia. PA). Because liposomes containing hyperosmolar concentrations of ara-C had increased contents leak

age in the presence of plasma, all therapeutic experiments and allsubsequent leakage experiments using other liposome compositionswere done with isoosmolar ara-C.

Pharmacokinetic Experiments. The time course for the eliminationof liposomes from the circulation of C57BL/6J x DBA/2J (B6D2F,)mice was determined by either i.p. or i.v. injection of liposomes containing entrapped ['"Ijtyraminylinulin (33), a marker of intact lipo

somes (10, 13, 33). Free label was separated from entrapped label, priorto injection, by gel filtration over Ultragel AcA-34 columns (IBFBiotechnics, Columbia, VtD). Mice were sacrificed at various timepoints postinjection, and blood, liver, spleen, and remaining carcasswere sampled. These tissues were corrected for blood volume usingcorrection factors determined previously (12). Bone marrow levels(cpm/mg) were determined as described previously (13). Results areexpressed as percent of in vivo cpm per organ, which gives total organuptake, corrected for leakage of drug from the liposomes (6). Becausetotal bone marrow weight could not be determined, percent of in vivocpm could not provided for bone marrow. Results are also expressedas cpm/mg tissue, normalized to IO6 injected cpm, which is propor

tional to the concentration of liposomes in each tissue. Similar experiments were performed in mice that had received IO6L1210/C2 leuke

mia cells 24 h previous in order to determine whether the presence ofthe tumor cells affected the pharmacokinetics of the liposomes.

Survival Experiments. L1210/C2 leukemia cells (34) were passagedin vivo by weekly i.p. transplants in either male or female B6D2F,hybrid mice. After 25 weeks of in vivo passage, the passage line wasrestarted from frozen stock to preclude the possibility of genetic drift.Groups of 5-10 mice of either sex (2-4 months, 18-30 g) were giveninjections by either the i.p. or the i.v. (tail vein) route with either 10sor 10" L1210/C2 cells. Twenty-four h after implantation of cells,

treatment began. Treatment consisted of either single or multipleinjections of free drug or liposome-entrapped drug by either the i.v. orthe i.p. route (Table 1). Control mice received injections of sterile saline(0.9%, USP). Other groups of control mice received empty (i.e., containing no drug) liposomes. Some groups of mice received free ara-Cover 24 h by tail-vein infusion according to described procedures (35,36). Infused drug was administered in a total volume of 3 ml at a flowrate of 0.0021 ml/min using a Harvard Infusion Pump (model 940).Survival times were noted for all mice. Survival times of mice in thevarious treatment groups were compared by analysis of variance. Sometimes the results were expressed as %ILS.

RESULTS

The rates of leakage from liposomes of ara-C at isoosmolarand hyperosmolar concentrations were determined for liposomes composed of PC:CH 2:1, or SM:PC:CH:GM, 1:1:1:0.14.This was to determine whether the presence of hyperosmolarconcentrations of ara-C, which was desirable in order to reducethe total amount of lipid administered to the mice at the higherara-C doses, would affect the rate of ara-C leakage from theliposomes in the presence of buffer and plasma. In buffer, at37°C,the rates of leakage of ara-C from liposomes of both

compositions were low and independent of the concentrationof the entrapped ara-C, with terminal half-lives averaging 442h for POCH liposomes and 475 h for SM:PC:CH:GMi liposomes (data not shown). In the presence of 25% plasma in

Table 1 Experimental protocols for treatment ofLI2IO/C2 leukemia-bearingmice with ara-C

Route of injection of cells(10'or 10'cells/mouse)

Route of drug treatment( 10-100 mg/kg ara-C)

.V..V..V..p.•p..p.i.v.Lp.Infusioni.p.I.V.Infusion

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buffer, the rates of leakage of hyperosmolar ara-C were rapidcompared to isoosmolar ara-C (Fig. 1), and leakage of ara-C

from PC:CH liposomes was more rapid than fromSM:PC:CH:GMi liposomes under both conditions of osmolar-ity. The leakage of hyperosmotic ara-C from SM:PC:CH:GMiliposomes (Fig. 1, solid squares) was characterized by an initialrapid loss of contents, followed by a slow leakage, with a half-life similar to that seen with isoosmotic conditions. All therapeutic experiments were performed under isoosmotic conditions because of the susceptibility of liposomes containinghyperosmotic ara-C to plasma-induced loss of contents.

We used large liposomes, formed by the REV technique, inthese experiments in order to increase the trapped volume anddecrease the administered lipid dose, particularly at the higherdoses of ara-C. The in vivo behavior of the larger stealthliposomes used in these experiments was characterized in orderto aid our understanding of how liposome-entrapped drug wasexerting its therapeutic effect in vivo. Blood, liver, spleen, andremaining carcass levels, as a function of time postinjec-

tion, were determined for PC:CH, SM:PC:CH:GM,, andHSPC:CH:PEG-DSPE liposomes (REV, 0.4 Mmextruded) following either i.v. or i.p. injection. The results for blood, following i.v. injection of a phospholipid dose of 0.5 /umol/mouse, areshown in Fig. 2. Liposomes containing PEG-DSPE or G\nwere removed from circulation with similar pharmacokineticsat a rate that was significantly slower than that seen for liposomes lacking stealth components (Fig. 2). PC:CH liposomesreached undetectable levels in blood by 48 h postinjection.Following i.p. injection, intact liposomes of all compositionsbegan to move from the peritoneal cavity into blood approximately 2 h postinjection. Following their appearance in blood,liposomes were removed with a time course very similar to thatseen following i.v. injection (data not shown), as has beenpreviously seen for smaller liposomes (17).

For liposomes composed of HSPC:CH:PEG-DSPE, bloodand liver levels were not significantly different for mice receiving either 0.5 or 4 ¿imolphospholipid/mouse, the latter dosebeing the average phospholipid dose injected into mice receiving50 mg/kg ara-C for liposomes of this composition (Table 2).For non-stealth liposomes composed of HSPC:CH:PG, at thehigher dose, liver levels were significantly lower (P > 0.001)and the blood levels were significantly higher (P> 0.001) thanat the lower dose, due to saturation of MPS uptake of the

Fig. I. Leakage of [3H)ara-C from liposomes, as a function of time, at 37°Cin

25% human plasma. Liposomes were composed of SM:PC:CH:GMi 1:1:1:0.14(D. •¿�)or PC:CH 2:1 (O. •¿�)and were REV-extruded through 0.4-^m Nucleporefilters (0.5 ¿imolphospholipid/ml 25'V plasma). ara-C was entrapped in liposomesal isoosmolar (•,G) or hyperosmolar (O. •¿�)conditions.

Time post-injection (hours)

Fig. 2. Blood levels in B6D2F, mice as a function time postinjection forliposomes (0.4 firn extruded REV) of different compositions. Mice (3 per group)were given 0.5 ^mol/mouse injections of liposomes composed of PC:CH 2:1 (O),SM:PC:CH:GMi 1:1:1:0.14 (d). or HSPC:CH:PEG DSPE 2:1:0.1 (A). Resultsare given as percent of in rivo cpm of liposome-entrapped [';'l|lyraminylinulin.

Data are mean ±SD.

liposomes (6). Bone marrow uptake, expressed as cpm/mg bonemarrow, for both compositions at both doses was not significantly different.

Although these larger stealth liposomes did not have half-lives in circulation as long as those seen for smaller stealthliposomes of the same composition (13, 17), their half-life incirculation of approximately 12 h was significantly longer thanthe 15-min initial half-life seen for PC:CH liposomes (Fig. 2).Liposomes containing GM1 (data not shown) or PEG-DSPE(Table 2) had decreased uptake into liver and spleen comparedto PC:CH (data not shown) or HSPC:CH:PG liposomes (Table2) as has been reported previously (10-13), although liveruptake, and particularly spleen uptake, were higher for thelarger liposomes than had previously been seen for smallerliposomes, accounting for the decrease in circulation half-lives.Leukemic mice, given injections of liposomes 24 h after receiving 10" LI 210 cells, did not show any significant differences in

liposome pharmacokinetics as compared to nonleukemic mice(data not shown).

Fig. 3 shows the survival data for mice given either i.v. or i.p.injections of IO5 L1210/C2 leukemia cells and treated eitheri.v. or i.p. with 10 mg/kg ara-C as single injections of free drug,as 24-h infusions of free drug, or as single injections of each of2 liposome compositions (PC:CH or SM:PC:CH:GMi). It canbe readily seen that the stealth liposomes are superior to thenon-stealth liposomes, which are in turn superior to singleinjections of free drug, at the same drug dose, in prolonging thesurvival time of the mice. All of these treatment groups aresignificantly different from each other (P > 0.001).

The optimum dosage schedules for free ara-C are reported tobe either by infusion or by frequent injection5 (37). We havecompared our results to either 24-h infusions or to literaturevalues for frequent injections. Free drug given by 24-h infusion(10 mg/kg total dose) was significantly better than 10 mg/kgara-C entrapped in stealth liposomes (P > 0.001) against i.v.tumor (Fig. 3O), but not significantly better than stealth liposomes (P < 0.05) against i.p. tumor (Fig. 3fi). The injection of

'The single i.v. dose LD,„for ara-C is 2500 mg/kg and (his provides acytotoxic body fluid level of ara-C for approximately 6 h. Optimum dosageschedules for injection have been determined to be every 3 h. The LD,0 for a q3h(x8) schedule was 244 mg/kg. The best dosing regimen for free ara-C was q.lh(X8); q4d (X4) (15 mg/kg/dose) which resulted in 5'i cures when the cell burdenwas i.v. 10*cells (37).

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Table 2 Tissue distribution in B6D2F¡mice of'^l-laoeled liposomes of 2 compositions (0.4 ¡imextruded REV) as a function of liposome dose, 24 h postinjection via

the tait veinResults are expressed either as percent of m vivo cpm (mean ±SD, n = 6) for liver, spleen, and blood, or as cpm/mg tissue (mean ±SD, n = 6, except n = 3 for

bone marrow), normalized to 10* cpm of injected counts.

% of m vivo cpm remaining in body 24 h postinjection (cpm/mg)

Liposome composition (Mratio)HSPC:CH:PG(2:1:0.1)

HSPC:CH:PEG-DSPE (2:1:0.1)Dose

(/¿mol)0.5

4

0.54Liver67.3

±4.6(370.1 ±41.2)

55.9 ±4.0(247.7 ±37.5)

40.0 ±3.0(220.3 ±45.8)

43.2 ±1.9(205.2 ±68.0)Spleen15.9

±3.1(1068.5 ±247.1)

22.4 ±3.9(1101. 1 ±198.7)

29.3 ±3.2(2121.8 ±509.2)

18.1 ±2.3(1219.1 ±275.7)Blood0.5

±0.3(1.7 ±1.3)2.2 ±0.9

(7.1 ±2.4)12.4 ±3.6

(48.1 ±17.3)17.3 ±6.4

(78.0 ±42.5)Bone

marrowND(107.1

±16.2)ND

(88.5 ±11.4)ND

(7 1.4 ±6.4)ND

(115.5± 20.7)

IDO

75

50

25

B10 15

Days survived

20 25

100 r 100 r

25

10 15 20 10 15 20 25

Days survivedDays survived

Fig. 3. Percentage of surviving B6D2F, mice (5 to 10 per group), as a function of days postinoculation of tumor, for mice receiving either i.p. (A and B) or i.v. (Cand D) injections of 10* L12IO/C2 leukemia cells. Twenty-four h later the mice were treated either i.p. (A and C) or i.v. (B and D) with single injections of sterileisotonic saline (A). 10 mg/kg free ara-C (O), or 10 mg/kg of ara-C entrapped in liposomes composed of SM:PC:CH:GM, 1:1:1:0.14 (D) or PC:CH 2:1 (V). Micereceived approximately 1 ¿imolof lipid. Some mice received 24-h tail-vein infusions of a total of 10 mg/kg ara-C (O).

empty liposomes had no therapeutic effect (data not shown).Liposomes administered by one route of injection were effectiveagainst tumors given by the other route route of injection, i.e.,i.v. liposomal ara-C was effective against i.p. tumor and viceversa (Fig. 3, B and C). A single i.v. injection of ara-C at a doseof 1000 mg/kg against IO5 LI210 tumor cells is reported to

result in a %ILS of 30% (37). This was superior to 10 mg/kgara-C administered in PC:CH liposomes, which resulted in a%ILS of 11%, but inferior to SM:PC:CH:GM, liposomes(%ILS = 52%) (Fig 3fi).

Very similar relationships to those seen in Fig. 3 were ob

served between the various therapeutic groups at higher tumorburdens (IO6 cells/mouse) and at higher drug doses (data not

shown). For subsequent experiments, even though experimentswere done for all 4 injection groups (Table 1), the results fori.v. tumor and i.v. treatment will primarily be presented sincethese data best mimic the clinical situation. As the drug doseincreased, the liposomes became superior in their therapeuticeffect to the free drug given by 24-h infusion. These results areshown in Fig. 4 for i.v. treatment against i.v. leukemia. Formice receiving IO6 L1210/C2 cells and stealth liposomes, bothby the i.v route, treatment with 10 mg/kg ara-C was signifi-

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100

25

of 80 mg/kg ara-C. Here, stealth and non-stealth liposomeswere equivalent (data not shown). At i.v. doses of 80 mg/kgliposome-entrapped ara-C, PC:CH liposomes were therapeuti-cally superior (%ILS = 141%) to 24-h free drug infusions (P >0.001 ) against i.v. tumor, but still significantly inferior to stealthliposomes (P> 0.001).

The maximum tolerated dose of liposomal ara-C was foundto be on the order of 100 to 120 mg/kg ara-C, and did notappear to vary in any significant way between stealth and non-stealth liposomes (data not shown). This value can be comparedto a 10% lethal dose of 95 mg/kg for an optimized i.v. injectionschedule of q3h (xl6) (37) or a LD<<,(which is approximately1.5 times the 10% lethal dose) of 135 mg/kg for a 2-day druginfusion (38).

Stealth liposomes composed of inexpensive starting materials, i.e., HSPC, cholesterol, and PEG-DSPE (17), were ex-

100

25

Days survived

Fig. 4. Percentage of surviving B6D2F, mice (5 to 10 per group), as a functionof days postinoculation of tumor and ara-C dose, for mice receiving IO* LI210/C2 cells by the i.v. route and treated 24 h later with a single i.v. injection of ara-C entrapped in liposomes (0.4 um extruded REV) composed of SM:PC:CH:GMi1:1:1:0.14 or 24-h infusions of ara-C. Mice received either 10 mg/kg (D. •¿�),20mg/kg (O, •¿�),or 80 mg/kg (O. *) liposome-entrapped (A, O. O. O) or infused (B,•¿�.•¿�,*) ara-C. Control mice (A) received sterile saline. Phospholipid dosesaveraged I. 2. and 10 ^mol/mouse. respectively.

cantly inferior to infusion (P > 0.001), treatment with 20 mg/kg was equivalent to infusion (P < 0.05), and treatment with80 mg/kg was significantly superior to infusion (P > 0.001),resulting in a %ILS of 192%, compared to 122% for infusionat the same total dose (Fig. 4, A versus B). The results at 80mg/kg for stealth liposomes can be compared against those formice, bearing a tumor burden of IO6i.v. leukemia cells, injectedwith free ara-C at a multiple injection schedule of i.v. drugevery 3 h for 3 days (24 injections), at a slightly higher totalara-C dose of 105 mg/kg, resulting in a %ILS of 142% (37). Inother words, at the highest drug dose used in this series ofexperiments, the liposomal preparations were superior to anoptimized multiple dose schedule of free ara-C administeredover 3 days, and to 24-h infusions of free drug, suggesting thatthe liposomes were able to maintain therapeutic drug levels forperiods longer than 24 h.

As the dose of liposome-entrapped drug increased, the efficacy of non-stealth liposomes, for all 4 injection groups, wassignificantly inferior to stealth liposomes (range = P > 0.05 to0.001) with the exception of i.p. drug versus i.p. tumor at adose

100

75

50

25

10 15

Dayssurvived

20 25

100

80

40

20

B0 10 20 30 40 50 60 70 80

Days survived

Fig. 5. Percentage of surviving B6D2F, mice (5 per group), as a function ofdays postinoculation of tumor and ara-C dose, for mice receiving 10* L1210/C2leukemia cells by the i.v. route or the i.p. route and treated 24 h later with single(A) or multiple (B) injections of ara-C entrapped in liposomes composed ofHSPC:CH:PEG-DSPE, 2:1:0.1 (0.4 ^m extruded REV). A. mice received i.v.tumor and single i.v. injections of sterile saline (A), 25 mg/kg (O), 50 mg/kg (+),or 100 mg/kg (D) liposome-entrapped ara-C. Some mice received 50 mg/kg (O)or 100 mg/kg (V) 24-h infusions of free ara-C. Phospholipid doses averagedapproximately 2. 4. and 10 jjmol/mouse. B. mice received tumor by cither the i.v.(O, D) or the i.p. (O, V) route and 3 injections of 50 mg/kg liposome-entrappedara-C (<0.V) (4 /jmol phospholipid/mouse) on days 1, 8. and 15 postinoculationof tumor. Two treatment groups received liposome-entrapped ara-C containingan additional 50 mg/kg of free ara-C (O. D). Control mice received sterile saline(A).

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Table 3 Relationship between leakage rate for ara-C in plasma and increased life spanB6D2F, mice (5 per group) received injections by the i.v. or the i.p. route of 10" L1210 leukemia cells, and 24 h later treatment was begun with single injections

of 50 mg/kg ara-C entrapped in various liposome compositions (0.4 ^m extruded REV). Mice received an average of 4 ¿jmollipid/mouse. Leakage of ara-C wasdetermined in 25'< human plasma at 37°C.

Liposome composition (Mratio)HSPC:CH:PEG-DSPE

(2:1:0.1)HSPC:CH:PG

(2:1:0.1)SM:PC:CH:PEG-DSPE

(1:1:1:0.1)24-h

infusion of free ara-CRoute

ofinjection ofcellsi.v.i.p.I.V.I.p.I.V.I.p.I.V.I.p.Route

ofinjection ofara-Ci.v.Lp.I.V.Lp.I.V.Lp.I.V.I.V.T,/,

for leakage of[5-3Hlara-C (h) %ILS786

138133184

16014241

197163907770-day

survivors0/50/50/50/50/50/50/50/5

amined, as a function of dose, for their ability to prolongsurvival times of mice given either i.v. or i.p. injections of IO6

L1210/C2 leukemia cells. The results for i.v. tumor with singleinjection i.v. treatment, or 24-h i.v. infusion, are given in Fig.5A. Treatment of the mice with 50 mg/kg liposome-entrappedara-C was equivalent to 24-h infusions of 100 mg/kg ara-C, andtreatment of the mice with 100 mg/kg liposomal ara-C resultedin a significant prolongation of survival times (P > 0.001compared to infusion) with %ILS increasing to 229%. Theseresults can be compared to a q3h (x24) injection schedule (totaldose, 105 mg/kg), which resulted in a %ILS of 142% (37). Inorder to achieve a %ILS similar to that achieved with a singleinjection of 100 mg/kg of ara-C entrapped in stealth liposomes,free ara-C would have to be given at a dosage schedule of q3h(x24) at 10 mg/kg/dose for a total dose of 240 mg/kg (%ILS= 257%) (37).

The half-life of leakage of ['H]ara-C from liposomes composed of HSPC:CH:PEG was determined at 37°Cin 25%

human plasma. The results, given in Table 3, show that theliposomes are extremely stable in plasma with a T./, of 786 h.We were interested in comparing the results obtained withHSPC:CH:PEG-DSPE liposomes with results for liposomes ofother compositions that had more rapid leakage rates in plasma,as it was believed that leakage rates that were very slow mighttend to decrease the therapeutic effect of the liposomes. Acomparison of the therapeutic results for i.v. tumor and drug,and for i.p. tumor and drug, for 3 liposome compositions withwidely differing ara-C leakage rates, is given in Table 3. It isapparent that, as the rate of ara-C release from liposomesincreases, the therapeutic effect increases. The liposomecomposition with the most rapid release for ara-C(SM:PC:CH:PEG-DSPE, T,/; = 41 h) resulted in a %ILS of197% for a single injection of 50 mg/kg entrapped ara-C, whichis superior to the %ILS of 178% (similar tumor burden androute of injection) seen for q3h (x24) at a dose of 6.7 mg/kg/dose of free ara-C (3-fold higher total dose of 160 mg/kg).

In a final series of experiments, mice bearing IO6 L1210/C2

leukemia cells, given by either the i.p. or the i.v. route, weretreated with multiple injections of ara-C entrapped inHSPC:CH:PEG-DSPE liposomes at doses of 25 or 50 mg/kgby the i.v. or the i.p. routes of injection. Doses of ara-C (25mg/kg) in stealth liposomes given on days 1 through 4, beginning 24 h after tumor implantation (total dose, 100 mg/kg),resulted in no therapeutic advantage over a single liposome-entrapped dose of 100 mg/kg given on day 1 (data not shown).Doses of 25 mg/kg liposomal ara-C given on days 1, 5, 9, and13 (total dose, 100 mg/kg) resulted in a %ILS of 348% for i.v.tumor with i.v. treatment, while i.p. tumor and i.p. treatmentresulted in a %ILS of 242% with 40% long-term survivors.These results can be compared to the results for IO6i.v. LI210

cells treated with 15 mg/kg/dose free ara-C (total dose, 480mg/kg) with a multiple administration schedule of q3h (x8);every 4 days (x4), which resulted in a %ILS of 300% with 5%long-term survivors (37). Treatment of l O6i.p. L1210 cells with5 mg/kg/dose free ara-C (total dose, 160 mg/kg) using the samemultiple dose schedule resulted in a %ILS of 194% with 55%long-term survivors (37), similar to the liposome results for i.p.tumor at a slightly lower total dose.

Administration, to mice bearing IO6L1210/C2, of 50 mg/kgliposomal ara-C on days 1 and 3 gave decreased life span dueto toxicity (data not shown). Administration of 3 doses, atweekly intervals, of 50 mg/kg of ara-C entrapped inHSPC:CH:PEG-DSPE liposomes, i.e., days 1, 8, and 15, resulted in high %ILS (Fig. 5B). Treatment and tumor given bythe i.v. route resulted in %ILS of 465%, while treatment andtumor given by the i.p. route resulted in %ILS of 493% with40% long-term survivors (Fig. 5B). Mice treated with free drug(50 mg/kg) died before the second injection could be given.

Because free ara-C is degraded rapidly in vivo, it has a veryhigh LOâ„¢of approximately 4 g/kg in mice with single i.v.injections (38). Therefore, it was believed that leaving some freedrug in the liposome preparations would not significantly increase the toxicity, would greatly simplify liposome preparation, and may be of some therapeutic advantage. Mice that hadreceived injections of IO6 L1210/C2 leukemia cells, by eitherthe i.v. or the i.p. route, were treated with 3 doses of ara-Centrapped in liposomes composed of HSPC:CH:PEG-DSPE atweekly intervals (days 1, 8, and 15), similar to the previousexperiment. However, free ara-C at the same dose of 50 mg/kgwas also included in the preparation. The results are shown inFig. 5B. Treatment and tumor given by the i.v. route, as well astreatment and tumor given by the i.p. route, resulted in 100%long-term survivors, demonstrating the utility of this approach.While 100% cures could be obtained at initial tumor burdensof less than IO6 L1210 cells/mouse against i.p. tumor usingoptimal free ara-C dosing schedules of q3h (x8), every 4 days(x4) (15 mg/kg/dose), it was not possible to obtain 100% cureswith IO6cells or higher tumor burdens at the start of treatmentusing free ara-C (37). Using the same optimal dosing scheduleand ara-C dose against i.v. tumor, 100% survivors could not beobtained with initial tumor burdens as low as IO4cells (37), so

it would appear that the liposomal treatment is more effectivethan optimal free drug schedules.

DISCUSSION

The mechanism of the increased efficacy of liposomal ara-C,compared to free drug, is 2-fold: (a) The liposomes protect ara-

C from rapid degradation by deaminases; and (b) the liposomesact as a slow release depot, altering the pharmacokinetics of

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ARA-C IN STEALTH LIPOSOMES

ara-C in vivo by increasing the area under the concentrationversus time curve (26,27,39). The ability of liposome-entrappedara-C to increase survival times in mice bearing LI210 leukemia, as compared to bolus injections of free drug, can beexplained solely on the basis of these 2 factors without invokingthe uptake of liposome-entrapped drug by tumor cells in vivo(26, 27). However, the ability of the liposomes to localize inbone marrow, liver, and spleen may also be contributing totheir therapeutic effect. It has been reported that high numbersof tumor cells can be found in bone marrow and spleen within3 h following i.v. injection of leukemia cells (37). Following i.p.injection of cells, spleen levels of cells begin to rise on day 2and bone marrow levels of cells on day 6 postinjection (37).Because liposome-entrapped drug accumulates in significantconcentrations in the mononuclear phagocyte system, includingliver, spleen, bone marrow, and lymph nodes (Table 2) (13, 17),leukemic cells localized in these tissues may be more readilydestroyed.

An estimate can be made of the number of days over whichcytotoxic levels of ara-C are maintained in vivo. Administrationof a single dose of 50 mg/kg ara-C entrapped in stealth liposomes, containing PEG-DSPE, was significantly more effectiveat prolonging survival of the mice than 24-h infusions of 50mg/kg ara-C (Fig. 5A; Table 3). This could happen only if theara-C were being released from the liposomes in the therapeuticconcentration range of 0.01 to 3.0 fig/ml (40) over a periodconsiderably longer than 24 h. At a dose of 20 mg/kg, liposomalara-C and 24-h infusions of free ara-C were not statisticallydifferent (Fig. 4), suggesting that at this dose the liposomeswere maintaining suprathreshold levels of drug for approximately 24 h.

The LD50 of free ara-C given by infusion over 1, 2, and 4days has been reported to be 500, 135, and 80 mg/kg, respectively (38) and for 5-day infusion, 50 mg/kg (41 ). The maximumtolerated dose (100% survivors) for ara-C in stealth liposomesis approximately 100 mg/kg, and all mice die at a dose of 150mg/kg. Therefore, the LD50 for the stealth liposome formulations is between 100 and 150 mg/kg. Comparison with theinfusion toxicity data suggests that the stealth liposomes havea toxicity level approximately equivalent to that seen for a 2-

day infusion of free drug.From the pharmacokinetic standpoint, a primary difference

between administration of a rapidly degraded drug by bolusinjection, by infusion, and via a liposomal slow release systemare the profiles of blood drug levels with time. Predicted druglevels for an i.v. administration of 50 mg/kg are given in Fig. 6for each type of drug treatment. In the case of bolus injections,therapeutic drug levels will be rapidly achieved, but will bemaintained for only a short period of time relative to thedoubling time of the L1210 cells (0.4 day) (40). When drug isadministered by infusion, constant drug levels will be maintained within the therapeutic range over the infusion period(after the initial period required to reach steady state of approximately 5 times the half-life, or about 2 h for ara-C) andthey will fall rapidly to subtherapeutic levels upon terminationof the infusion (Fig. 6). Free ara-C is assumed to distributethroughout the fluid volume of the mouse, which is taken to be77% of body weight, and ara-C is assumed to have an initialhalf-life of 16 min (20).

When the drug is administered in a liposomal slow releasesystem, the situation becomes more complex, and will dependnot only on the factors governing blood and tissue levels of freedrug but also on a number of other factors. These include the

100

0 01

0.1 1

16 24 32

Time post-injection (hours)

48

Fig. 6. Predicted levels of free ara-C in plasma following administration of 50mg/kg ara-C as a bolus injection ( ), as a 24-h infusion (••¿�•¿�•¿�).or entrappedin liposomes composed of SM:PC:CH:PEG-DSPE 1:1:1:0.1 ( ).

blood and tissue levels of liposome-entrapped drug, the rate ofremoval of liposomes to compartments where the drug is degraded or otherwise unavailable, the rate of leakage of drugfrom the liposomes, and the ability of the liposome-entrappeddrug to move from one compartment to another.

Analytical procedures that can distinguish liposome-associ-ated drug from free or protein-bound drug have only recentlybeen developed and have been used to measure plasma clearanceof total and liposome-associated doxorubicin (42). We canspeculate that the pharmacokinetics of ara-C entrapped inliposomes are similar to those seen for doxorubicin. Therefore,approximate predications of blood levels, as a function of time,of available drug released from liposomes containing 50 mg/kgliposome-entrapped ara-C have been made based on the rate ofremoval of liposomes from circulation by MPS uptake (Fig. 2),the rate of leakage of drug from liposomes in the presence ofplasma (Fig. 1; Table 3), the volumes of distribution of theliposomal and the free drug (6, 42, 43), and the half-life of thefree drug (19. 20) (Fig. 6).

These predictions are based on the assumptions that drugremaining entrapped in the liposomes experiences no degradation: that the half-life for ara-C, once released from liposomes,is 16 min; that the volume of distribution of the liposomal ara-C averages 2.2 ml (6); and that the volume of distribution ofthe free ara-C is equal to the mouse fluid volume (19.4 ml fora 25-g mouse). Free ara-C will be slowly released from liposomes, but rapidly degraded once released. This would resultin free ara-C levels that decreased slowly with increasing time,with drug levels above the minimum cytotoxic concentrationmaintained for more than 24 h for liposomal ara-C doses of 50mg/kg (Fig. 6). Lower doses of liposomal ara-C would shift thecurve downward, resulting in drug levels remaining in thetherapeutic concentration range for shorter periods of time.More rapid leakage of ara-C from liposomes would shift theliposomal curve towards the free drug curve. Less rapid leakagerates could result in free drug levels below the minimal effectiveconcentration. Longer half-lives of the liposomes in circulationwould extend the period of time over which therapeutic druglevels could be maintained, while shorter half-lives would decrease that time.

A comparison of leakage and pharmacokinetic data for onestealth and one non-stealth formulation (Figs. 1 and 2) suggeststhat the inferior performance of PC:CH liposomes was a resultof 2 main factors: rapid leakage of ara-C (Fig. 1) and a shortcirculation half-life (Fig. 2). The most effective liposomal drug

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slow release system would be one with a long (several h)circulation half-life balanced with an appropriate drug releaserate, with most of the drug being released while the liposomeswere still in circulation. A test of this hypothesis resulted in thedata in Table 3, which showed that therapeutic efficacy increased as leakage rates of ara-C increased. There is obviouslyan optimum leakage rate above which therapeutic efficacywould decrease because of rapid release and degradation of ara-C, and this is probably playing a role in the decreased efficacyof PC:CH liposomes (see above). This points out the importance of assessing the rates of release of drugs, peptides, etc.,from liposomal carriers in experiments to optimize therapeuticeffects.

The inclusion of free ara-C in a formulation that had a veryslow leakage rate for liposome-entrapped ara-C(HSPC:CH:PEG-DSPE) resulted in a significant improvementin therapeutic effect (Fig. 5B), probably because high doses ofthe free drug, even if present at therapeutic levels for only a fewh. resulted in a substantial reduction in tumor burden by killingcells going through the S-phase of the cell cycle during thosefew h. Subsequently, the slow release form of the drug acted tokill residual cells as they went through DNA replication. Aformulation such as this would be of interest from the manufacturing point of view because of the relatively inexpensivestarting materials, the stability of the lipids, and the simplification of product production by eliminating the need to removefree drug.

From previous research, ara-C in conventional liposome formulations, given at high lipid doses (up to 20 ¿/mol/mouse),resulted in increases in mean survival times that were almostas high as those seen when ara-C was entrapped in stealthliposomes (21-28). Such high doses of lipid would be expectedto markedly increase circulation half-lives of liposomes because

of MPS saturation (6), leading to increased therapeutic effects.What, then, are the advantages of stealth liposomes? Theadvantages lie primarily in the dose-independent pharmacoki-netics of stealth liposomes, and their decreased MPS uptake(6). The dose-independent pharmacokinetics ensures that theamount of entrapped drug that can gain access to tumor cellswill increase in proportion to lipid dose. For non-stealth liposomes, which have dose-dependent pharmacokinetics, entrapped drug will be sequestered almost completely within theMPS at very low lipid doses, and at high lipid doses the amountof entrapped drug available to non-MPS tissues will approachthat achieved with stealth liposomes (6). This observation willassume paramount importance when highly active drugs e.g.,peptides are entrapped in liposomes, as these drugs will beadministered at very low drug and lipid doses.

Delivery of large amounts of cytotoxic drugs in non-stealthliposomes to the MPS will increase MPS impairment andincrease the chances of adverse effects to the host (1,2). Becausestealth liposomes appear to accumulate in the MPS at ratesthat do not exceed the ability of the MPS to recycle or regenerate (6), they will cause less MPS impairment, and decreasethe incidence of adverse effects such as increased susceptibilityto infections, increased chance of systemic endotoxemia, andincreased rates of tumor growth and métastases(1,2).

The ideal controls in these experiments would have been freedrug infusions given over 2- to 3-day periods. Because of thedifficulties in maintaining free drug infusions over this periodof time, we have compared our results to 24-h infusions, andto data from the literature in which exhaustive experimentshave been conducted to find the optimum doses, and multiple

dose schedules, for both i.v. and i.p. LI210 tumor given at avariety of tumor burdens (37, 40). Doses of free ara-C given byinjection every 3 h were found to be optimal in these studies,and the resulting %ILS compared favorably with %ILS foundwhen similar doses of free ara-C, at similar tumor burdens,were given by infusion over the same period of time (this studyand Refs. 38 and 41).

In summary, we have shown that stealth liposomes are aneffective drug slow release system, with particular applicationsfor rapidly degraded drugs. Single injections of ara-C entrappedin stealth liposomes were superior to non-stealth liposomes,and both were superior to single injections of free drug. Singleinjections of stealth liposome-entrapped ara-C resulted in therapeutic effects that were equivalent to effects seen in the pastfor higher doses of free drug given at optimized multiple doseschedules of free ara-C. Multiple injections of liposome-entrapped ara-C resulted in %ILS, or cure rates, higher than thebest survival data, at similar tumor burdens, which could beobtained in the past from multiple, frequent injections of freedrug using a variety of dosage schedules. Combination of liposome-entrapped drug with an equivalent amount of free drug

further increased the efficacy of the preparations. The availability of an inexpensive, nontoxic, easily synthesized componentsuch as PEG-DSPE, which will impart long circulation half-lives to liposomes, greatly improves the commercial acceptability of stealth formulations for therapeutic applications. Theirdose-independent pharmacokinetics and lack of MPS saturation give them important advantages over previous liposomeformulations.

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-d-Arabinofuranosylcytosineβ1-Stealth Liposomes: An Improved Sustained Release System for

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