JOURNAL OF LIPOSOME RESEARCH, 7(2&3),24 1-260 (1997)

ORIGINAL ARTICLES

THE STUDY OF POLYETHYLENEGLYCOL-COATED LIPOSOMES

CONTAINING CPT-1 1

Yasuyuki Sadzuka*, Sachiyo Hirotsu, Atsuo Miyagishima,

Yasuo Nozawa and Sadao Hirota

Lab. of Pharmaceutical Engineering,

School of Pharmaceutical Sciences,

University of Shizuoka, 52-1 Yada, Shizuoka, 422, Japan

ABSTRACT

Polyethyleneglycol (PEG) -coated liposomal CPT-1 1 (PEG-LCPT( 1 1 ) )

was prepared and i t s pharmaceutical usefulness was examined. These
liposomes, plain liposomal CPT-1 1 (PLCPT( 1 1 )) and PEG-LCPT( 1 1 ),
were composed of dimyristoylphosphatidylcholine, cholesterol, and di-
myristoylphosphatidylglycerol (1 0 : 10 : 6, mol/mol) with or without
PEG. The mean particle diameters were both about 160 nm. The trapping
efficiencies were approximately 90%. In a distribution study, CDF1 mice
were injected with CPT-11 solution (CPT( 1 1 )sol), PLCPT( 1 1 ) and PEG-
LCPT( 1 1 ) a t a dose of 10 mg/kg (i.v.). Concentrations in each tissue of
CPT-1 1 and SN-38, the active metabolite of CPT-1 1 , were determined.
After the administration, CPT-1 1 and SN-38 concentrations in the blood
increased by liposomal encapsulation (liposomalization), and the
circulation time in the blood was prolonged further by PEG-modification of
the liposomes (PEGylation). In the liver, PLCPT( 1 1 ) was rapidly taken

24 1

Copyright 0 1997 by Marcel Dekker, Inc.

242 SADZUKA ET AL.

up by the reticuloendothelial system (RES), and the uptake was avoided by

PEGylation. Tumor accumulations of CPT-11 and SN-38 were accompanied
by an increase in antitumor activity of CPT-1 1 by liposomalization. Thus,
the prolongation of the circulation time in the blood by liposomalization and
the avoidance of the RES uptake by PEGylation caused passive targeting of
the tumor, with a resulting increase in the antitumor activity of CPT-1 1.

INTRODUCTION

Many reports have demonstrated that the encapsulation of antitumor

drugs such as adriamycin (ADR) in liposomes reduces its toxicity and

enhances its therapeutic index in animal system ( 1 -3). lrinotecan

hydrochloride (CPT-1 1 , Fig.1) was semisynthesized as a water-soluble

derivative of camptothecin, which is an alkaloid isolated from Camptotheca

acurninata ( 4 ) and has been known to have unique antitumor activity,

preventing DNA synthesis by inhibiting topoisomerase I ( 5 ) . CPT-1 1 i s

enzymatically hydrolyzed by carboxylesterase t o the active metabolite,

SN-38 (6). Although CPT-1 1 has a potent antitumor activity (7,8), i t

has lethal side effects such as myelosuppression and gastro-intestinal

disorders, mainly diarrhea (9). Although it has been on the market i n

Japan since 1994, it is used with severe limitations today. Therefore, we

considered CPT-1 1 to be a good example for improving the therapeutic

index of drug delivery system (DDS). We have reported that ADR

encapsulated in PEG-coated liposomes increases antitumor activity and

reduces cardiotoxicity (1 0). Encapsulation with liposome (liposomaliza-

tion) of CPT-I1 in the same manner is thus expected t o increase its

therapeutic index. In the present study, the tissue distribution and the
POLYETHYLENEGLYCOL-COATED LIPOSOMES 243

CH 2CH 3

CPT-1 1

H3C
carboxylesterase

CH 2CH 3
HO

(active metabolite of CPT-1 1 )

FIG. 1 Structural formulas of CPT-1 1 and SN-38

antitumor activity in mice of CPT-1 1 encapsulated in PEG-coated

liposomes (PEG-LCPT( 1 1 )) were investigated.

MATERIALS AND METHODS

Materials

CPT-1 1 solution (CPT( 1 1 )sol), Topotecin@ injection as a solution in

vials ( 1 00 mg/5 ml), was purchased from Daiichi Pharmaceutical Co.,

244 SADZUKA ET AL.

Ltd., Tokyo, Japan. CPT-11 for the preparation o f liposomal CPT-1 1 was

a gift from Yakult Honsha Co., Ltd., Tokyo, Japan. Dimyristoyl-

phosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG)

(COATSOME; MC-4040 and MGLS-4040, respectively), and 1-(mono-

methoxypolyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) were

gifts from Nippon Oil & Fats Co., Tokyo, Japan. The average molecular

weight of PEG in PEG-DMG was 2000. Cholesterol was purchased from

Wako Pure Chemical Industries Ltd., Osaka, Japan. All other chemicals

were commercial products of reagent grade. Polycarbonate membrane

filters were purchased from Nomura Micro Science Co., Tokyo, Japan.

Lactate buffer (1 0 mM) with 260 mM sucrose was filtered once through

membrane filters with a pore size of 0.1 ~ m .

Liposome oreparations

CPT-1 1 -containing liposornes (PLCPT(1 1)) were prepared as follows.

DMPC (1 00 cc rnol), cholesterol (1 00 cc mol), DMPG (60 M mol) and

CPT-1 1 (1 5 cc rnol) were dissolved in a chloroform/methanol mixture

(2:1, v/v). The chloroform and methanol were evaporated t o dryness

under a stream of nitrogen gas. The thin lipid film was evacuated in a

desiccator, and the lipid film was then hydrated with 8.0 ml of 1 0 mM

lactate buffer (pH 4.0) containing 260 mM sucrose in a water bath at 50-

60 "c. The suspension was sonicated for 20 min above the phase transition

temperature (Tc), 23"c, after nitrogen gas bubbling. The liposome

POLYETHYLENEGLYCOL-COATED LIPOSOMES 245

suspension was extruded through two stacked polycarbonate membrane

filters with 0.2 cLm pores, followed by extrusion five times through

0.1 LL m pores above the Tc; a homogeneously-sized liposome suspension

was thus obtained. PEG-LCPT( 1 1 ) and PLCPT( 1 1 ) were prepared by

adding 2.0 mi of 10 mM lactate buffer (pH 4.0), with or without 5.0 mo1%

PEG-DMG, respectively, t o 8.0 ml of the liposome suspension and

sonicating the mixture. The zeta potentials of the liposomes in lOmM

lactate buffer (pH 4.0) were measured by electrophoretic light scattering

and the particle size distributions by dynamic light scattering, both using

an ELS 800 apparatus (Otsuka Electronics Co., Ltd., Oseka, Japan).

Animal experiment

Male CDF1 mice (body weight 20-25 g, 5 weeks old) were obtained

from Japan SLC Ltd., Hamamatsu, Japan. Animals were housed under

conditions of 2 5 k 1 "C and 5 5 * 5 % humidity, and were allowed free

access to standard food and water throughout the study period.

In the distribution study, mice transplanted with Ehrlich ascites

carcinoma ( 5 x 1 05 cells/animal) subcutaneously on the back. Mice were

injected via the tail vein with CPT( 1 1 )sol, PLCPT( 1 1 ) or PEG-LCPT( 1 1 )

a t a dose of 10 mg/kg body weight (1 60 LL mot phospholipids/kg) on day

14 after tumor implantation. A t 0.5, 1, 2, 4 and 8 h after injection, the

mice were sacrificed by cervical dislocation and the blood was collected

from the heart. The liver, heart, lung, kidney, spleen and tumor were
246 SADZUKA ET AL.

immediately removed. The remaining blood in the tissue was carefully

removed so that the contribution of the blood to the data of each tissues was

within 5.0 %. Control mice were injected with sterile isotonic saline. The

tissues were homogenized in saline t o a 5% homogenate, using a Potter-

type Teflon homogenizer. Three ml of 1-butanol as extracting solvent was

added to 1 .O ml of homogenate, and this was then shaken using a vortex

mixer and centrifuged a t 1,200 g for 15 minutes. After centrifugation,

CPT-1 1 i n t h e upper layer were determined by fluorescence

spectrophotometry (CPT-11 Ex : 374 nm, Em : 435 nm and SN-38 Ex :

380 nm, Em : 556 nm). The extraction efficiency was more than 90% and

the detection limit was 1 .Ong/g tissue.

In the antitumor activity study, Ehrlich ascites carcinoma (5 X 1O5

cells/animal) was transplanted onto the backs of the mice on day 0. After

the tumor were grown until approximately 500 mm3, CPT( 1 1 )sol,

PLCPT( 1 1 ) or PEG-LCPT(1 1 ) was administered a t a dose of 1 0 mg/kg/day

(i.v.) on days 13, 16, and 19 after tumor inoculation. The animals were

killed by cervical dislocation on day 22 after tumor inoculation. The tumor

was rapidly removed and weighed.

Stability of the liposome in vitro

Liposomes containing CPT-11 were incubated in 50% fetal bovine

serum (FBS)/Hepes buffer (pH 7.0) for 8 h a t 20°C. After definite time,

reaction mixtures were centrifuged a t 40,000 g for 2 4 h and CPT-1 1

POLYETHYLENEGLYCOL-COATED LIPOSOMES 247

concentration in the supernatant was determined. Remaining percentage of

CPT-1 1 in liposomes calculated from the CPT-1 1 concentrations in the

supernatant of reaction mixture before and after the incubation.

Statistical analysis

Statistical analyses were performed by using Student's ttest.

RESULTS

Property of liposomes containinq CPT-1 1

The zeta potentials of PLCPT(1 1) and PEG-LCPT(1 1) were -22.2 mV

and -1 3.4 mV, and the mean particle size diameters were 165 _+ 27 nm and

1 6 0 f 25 nm, respectively. The trapping efficiencies were both

approximately 90%. The remaining % of CPT-1 1 in liposomes after 1 and

8 h-incubation with 50% FBS was 96.4% and 77.2%, respectively.

Tissue distribution of CPT(1 1 )sol and liposome-encapsulated CPT-1 1 in

mice

The time courses of CPT-1 1 and SN-38 in the plasma, liver, spleen and

tumor after i.v. injection of the 1 0 mg/kg dose of CPT( 1 1 )sol, PLCPT( 1 1 )

and PEG-LCPT(11) are shown in Figs. 2-5.

248 SADZUKA ET AL.

0.4
h
m
5
-
m
3 0.3
E
\
m
3
v

g
.-
0.2
c,
E
CI
C
W
0.1
U
m
T
5 0
Time (h) Time (h)

+CPT( 1 1 )sol --b PLCPT(11) PEG-LCPT( 1 1 )

FIG. 2 CPT-1 1 and SN-38 concentrations in the plasma. Mice were

injected with 1 0 mg/kg (i.v.) of CPT-1 1 in the form of (0)
CPT( 1 1 )sol; ( A ) PLCPT( 1 1 ); or (0) PEG-LCPT( 1 1 ). Each
point represents the mean f S.D. of three mice.

1.2

CPT-11 L
h SN-38
W
-2 0.9
\
m
a
v
c
.O
c,
0.6
e
+
C
W
u
g 0.3
V
m
2
* c
Time (h) Time (h)

4 CPT( 1 1 )sol 4~PLCPT(1 1) PEG-LCPT( 1 1 )

FIG. 3 CPT-1 1 and SN-38 concentrations in the liver. Mice were

injected with 1 0 mg/kg (i.v.) of CPT-11 in the form of (0)
CPT( 1 1 )sol; ( A ) PLCPT( 1 1 ); or (0)
PEG-LCPT( 1 1 ). Each
point represents the mean f S.D. of three mice.
POLYETHYLENEGLYCOL-COATED LIPOSOMES 249

h 0.4
CPT-11 5 SN-38

Time (h) Time (h)

+CPT( 1 1)sol 4PLCPT( 1 1 ) PEG-LCPT( 1 1 )

FIG. 4 CPT-1 1 and SN-38 concentrations in the spleen. Mice were

injected with 1 0 mg/kg (i.v.) of CPT-1 1 in the form of (0)
CPT( 1 1 )sol; ( A ) PLCPT( 1 1 ); or (0) PEG-LCPT( 1 1 ). Each
point represents the mean k S.D. of three mice.

-6
8 -z 0.10
E E
2m 5 a 0.08
\
\
m
?4 0
v
20.06
C
C
$3 0
U
e c! 0.04
c)
*
i2 C
W
C

6,
r W
5 0.02
c m
":
i
W 0 5 , O
Time (h) Time (h)

CPT(1l)sol PLCPT(1 1) PEG-LCPT(1 1 )

FIG. 5 CPT-11 and SN-38 concentrations in the tumor. Mice were

injected with 10 mg/kg (i.v.) of CPT-1 1 in the form of
CPT( 1 1 )sol, PLCPT( 1 1 ) or PEG-LCPT( 1 1 ). Each column
represents the means f S.D. of three mice.
250 SADZUKA ET AL.

1) Plasma (Fig.2)

After the administration of CPT-1 1 , the order of CPT-1 1

concentrations in the plasma was PEG-LCPT(1 1) > PLCPT(1 1 ) >

CPT(1 1)sol a t earlier time points. At 0.5 h post-injection, the CPT-1 1

levels of PEG-LCPT(1 1 ) and PLCPT(1 1 ) were 4.2-fold and 2.3-fold

higher than that of CPT(1 l)sol, and a t 1 h post-injection, the CPT-1 1

levels were 8.9-fold and 2.9-fold, respectively, higher than that of

CPT( 1 1 )sol. No difference was observed between PLCPT(1 1 ) and

CPT( 1 1 )sol, whereas the CPT-1 1 concentration of PEG-LCPT(1 1 ) was

14-fold higher than that of CPT(1 1 )sol after 2 agd 4 h post-injection.

The SN-38 concentration had a time course similar t o that of the CPT-1 1

concentration.

2 ) Liver (Fig.3)

In the liver, the order of CPT-1 concentrations was PLCPT(11) >

PEG-LCPT(11) = CPT(1 1)sol. The CPT-1 1 level of PLCPT(11 ) was

2.5-fold a t 0.5h post-injection and 2.1 -fold a t 1 h post-injection higher

than that of CPT(1 1 )sol. The active metabolite concentration had a time

course similar to that of the CPT-1 1 concentration. The SN-38 level of

PLCPT(1 1 ) was 2.3-fold and 1.6-fold a t 0.5 h and 1 h post-injection,

respectively, higher than that of CPT(1 1 )sol.

3) Spleen (Fig.4)

In the spleen, the CPT-1 1 level was increased slightly by PLCPT(1 1 )

and further increased by PEG-LCPT( 1 1 ), relative t o t hat with

POLYETHYLENEGLYCOL-COATED LIPOSOMES 25 1

CPT( 1 1 )sol, but the differences were not significant. The time course of

each group was similar. In addition, the active metabolite showed a peak a t

1 h a f t e r injection and had a time course similar to that of the CPT-11

concentration.

4) Heart, Kidney and Lung

In the heart, kidney and lung, no difference was observed in the CPT-1 1

concentrations among CPT( 1 1 )sol, PLCPT( 1 1 ) and PEG-LCPT( 1 1 ). The

CPT-1 1 level in all three forms decreased slightly with time. In the active

metabolite concentration, no difference was observed among the three

forms. SN-38 concentrations in the heart were lower than that in the

other tissues (data not shown).

5) Tumor (Fig.5)

In the tumor, the order of CPT-11 concentrations was PEG-LCPT( 1 1 ) >

PLCPT(1 1 ) > CPT(1 1 )sol . A t 8 h post-injection, the CPT-1 1 level of

PEG-LCPT( 1 1 ) was 4.9-fold higher than that of CPT( 1 1 )sol. The SN-38

concentration had a time course similar t o t h a t of the CPT-1 1

concentration.

Effect of CPT-11 liposomalization on the antitumor activity

Figure 6 shows the tumor weights after 30 mg/kg administration as

CPT-1 1 in the three forms. The tumor weight decreased slightly after
252 SADZUKA ET AL.

Control CPT( 1 1 )sol PLCPT( 1 1 ) PEG-LCPT( 1 1 )

FIG. 6 Effects of liposomalization on changes in tumor weight induced by

CPT-11. Mice were injected with 10 mg/kg/day (i.v.) x 3 days
of CPT-11 in the form of CPT(1 1 )sol, PLCPT( 1 1 ) or PEG-
*
LCPT( 1 1 ). Data represent mean S.D. of eight mice. Significant
differences from the level of the control group are indicated by a)
p < 0.02 and b) p < 0.05.

CPT( 1 1 )sol treatment. The decrease of tumor weight after PEG-LCPT( 1 1 )

treatment was significantly enhanced 1.9-fold, compared t o that after

CPT(1 1)sol treatment (p < 0.02 : significantly different from the level of

the control) and after PLCPT(1 1) treatment was enhanced 1.6-fold (p <

0.05 : significantly different from the level of the control).

DISCUSSION

CPT-1 1 is enzymatically hydrolyzed t o the active metabolite, SN-38

( 1 1). Concentrations of CPT-1 1 and SN-38 in the tissues have been

determined by HPLC (1 2). We developed fluorescence spectrophotometry

POLYETHYLENEGLYCOL-COATED LIPOSOMES 253

as a simpler method for the separate determinations of both compounds. In

the present study, we used l-butanol as an extracting solvent from mice

tissues, and determined CPT-1 1 by the detections of excitation a t 374 nm

and emission a t 435 nm, and SN-38 by the detections of excitation a t 380

nm and emission a t 556 nm (1 3). Using this method, the fluorescence of

CPT-1 1 did not prevent that of SN-38 in the SN-38 fluorescence

wavelength region, although SN-38 prevented 0.1 8% of the fluorescence of

the CPT-1 1 in the CPT-1 1 fluorescence wavelength region. However, in

the tissue the existence ratio of SN-38 t o CPT-1 1 after CPT-1 1

administration is approximately 1/ l o - 1 / l o 0 (1 4). Therefore, there is

no problem for accurate CPT-1 1 determination.

CPT-1 1 is quite hydrophilic like ADR and has positive charge. DMPG

which has negative charge was added to the liposome preparation four times

as much as CPT-1 1 in mol ratio. Therefore, CPT-1 1 combines with

liposome membrane electrostatically. Thus i t associates quite efficiently

with lipid bilayer. CPT-1 1, composed of lipophilic SN-38 and bulky

hydrophilic piperidino-piperidino moiety, is regarded as an amphiphile.

The lipophilic part i s incorporated in the membrane lipid, while

hydrophilic part exposed on the outer layer, protecting the SN-38 part

from the aqueous phase.

The DMPC used t o prepare these liposomes has the phase transition

temperature (Tc) 23"C, which is lower than body temperature.

Therefore, these liposomes will break easily in adequate time in the body.

CPT-1 1 entrapped in the liposomes will release from the liposomes, and
254 SADZUKA ET AL.

then CPT-11 will be enzymatically hydrolyzed t o the active metabolite SN-

38. Thus, it is expected that these liposomes can release CPT-11 more

effectively than liposomes consisting of phospholipid, which has a Tc that i s

higher than body temperature. We have previously reported the

distribution of ADR encapsulated in liposome included DMPC and i t s

usefulness (1 0). Therefore, DMPC was used for liposome preparation in

the present study.

We observed differences in zeta potentials between PEG-LCPT(1 1 ) and

PLCPT( 1 1 ). Addition of PEG-DMG t o PLCPT( 1 1 ) caused a decrease in the

zeta potential. We considered that this decrease depended on the formation

of fixed aqueous layer by PEG-modification (PEGylation), and it is possible

to assume that the fixed aqueous layer around the PEG-liposome inhibits

binding of opsonins (1 5).

In vitro, we examined the stability of liposome by incubating liposomes

with FBS. After 8 h-incubation, approxymately 20% of CPT-1 1

transferred to serum lipoproteins (or free-drug released from liposome).

Therefore, we thought that lipoprotein hardly contribute t o the tissue

distribution of liposomal CPT-1 1.

CPT-1 1 levels of PLCPT(11) and PEG-LCPT(11) in the plasma were

higher than that of CPT(1 l)sol, and the CPT-1 1 concentration of PEG-

LCPT( 1 1 ) was 14-fold higher than that of CPT( 1 1 )sol after 2 and 4 h

post-injections. AUCo.s-8h of PLCPT( 1 1 ) and PEG-LCPT(1 1 ) (7.42

-
b g hr/ml and 44.73 b g - hr/ml, respectively) were higher than that of

CPT(1 1)sol (4.28 w g - hr/ml) (Fig. 2). Drugs entrapped in liposomes

POLYETHYLENEGLYCOL-COATED LIPOSOMES 255

are generally more susceptible to uptake by the reticuloendothelial system

(RES) in the liver and spleen (16). Incorporation of PEG-derivatized

phospholipids or monosialoganglioside into liposomes has resulted in

altered pharmacokinetics of these liposomes, leading to increased blood

levels of liposomes and reduced uptake by RES (1 7, 1 8). In the present

study, PLCPT(11 ) also gave a CPT-1 1 concentration in the liver 2.5-fold

higher than that by CPT(1 1)sol. This result suggests that liposomes are

rapidly taken up by RES cells. However, PEG-LCPT(1 1 ) in the liver

showed a time course of the CPT-11 concentration similar t o the that of

CPT( 1 1 )sol, and the uptake by RES was certainly avoided by PEGylation.

Therefore, considering the CPT-11 concentration after PEG-LCPT( 1 1 )

administration compared t o this level in the PLCPT(1 1) group in the

liver, the avoidance of RES uptake by PEGylation resulted in prolonged

circulation time in the blood. The concentration of SN-38 was increased in

the blood, especially by the PEG-LCPT(11) group rather than the

CPT(1 1)sol group. The SN-38 concentration in the liver had a

distribution similar t o that of CPT-1 1.

In contrast, the distribution in the spleen did not show a distribution

like that in the liver. The time courses of CPT( 1 1 )sol, PLCPT( 1 1 ) and

PEG-LCPT( 1 1 ) were similar in the spleen. These results showed that the

liposomal CPT-1 1 was distributed differently in the liver and in the

spleen, although these tissues are both RES tissues.

In the tumors in the present study, the order of CPT-1 1 concentrations

was PEG-LCPT( 1 1 ) > PLCPT( 1 1 ) > CPT( 1 1)sol, and the CPT-11 level of
256 SADZUKA ET AL.

PEG-LCPT( 1 1) was 4.9-fold higher than that of CPT(11 )sol a t 8 h post-

injection. The concentration difference between PEG-LCPT( 1 1 ) and

CPT( 1 1)sol after 8 h post-injection was caused by the prolongation of the

circulation time in the blood by liposomalization. The SN-38 concentra-

tion had a time course similar t o that of CPT-11. The antitumor activity

after PLCPT( 1 1 ) treatment was enhanced, further after PEG-LCPT( 1 1 )

treatment. It is considered that the antitumor activity was enhanced as the

result of the prolongation of the circulation time in the blood by

liposomalization and the tumor accumulations of CPT-11 and SN-38. The

increased ratio of the concentration in the tumor of the PEG-LCPT( 1 1 )

group on that of the PLCPT(1 1 ) group is larger than the dfference of

antitumor activity in both group. For a human, the clinical dose of CPT-1 1

is 100-1 50 mg/m2/day. Surface area per unit weight for a mouse is

calculated to be about 20-fold greater than that for human. Therefore, the

therapeutic dose of CPT-1 1 for a mouse is approximately 50 mg/kg/day.

Thus, the CPT-11 1 0 mg/kg/day (i.v.) X 3days used in the present study

is less than the clinical dose. Therefore, it is considered that there is no

difference in therapeutic effect between the PEG and non-PEG liposomes.

In addition, regarding dosage and schedule, there were not any death of mice

or the decrease in weight due t o CPT-1 1 ; this result clearly showed only

the enhancement of antitumor activity. Thus, passive targeting t o tumor of

CPT-1 1 and SN-38 by liposomalization and by PEGylation suggests an

increase in the antitumor activity of CPT-1 1.

POLYETHYLENEGLYCOL-COATED LIPOSOMES 257

In the present study, total CPT-1 1 and total SN-38 were determined.

In a strict sense, the lactone forms of CPT-1 1 and SN-38, which have

antitumor activity, should be separated from the open ring forms (19).

However, the separation is diff;cult, and Sasaki e t al. reported that

monitoring of total CPT-1 1 and SN-38 has essentially the same clinical

significance as monitoring of lactone CPT-1 1 and SN-38 (20). Namely,

AUC of total CPT-1 1 and that of total SN-38 were significantly correlated

with the AUCs of the lactone CPT-11 and those of lactone SN-38,

respectively (20). We therefore examined the distribution of CPT-11 in

mice by determining the total drug concentration without separately

determining the open ring form and the lactone (closed ring) form. CPT-

1 1 is stabilized in an acid region (pH 3.0-4.0) and becomes the lactone

form with antitumor activity. These liposomes were prepared with a

lactose buffer (pH 4.0). Therefore, CPT-11 entrapped in the liposomes is

more stabilized than CPT-11 in solution in the body. If liposomal CPT-1 1

is taken up, as the intact liposome, metabolized and activated in the tumor,

liposomalization of CPT-1 1 may be advantageous for the stability of CPT-

1 1 and the maintenance of antitumor activity.

Thus, the prolongation of the circulation time in the blood by

liposomalization and the avoidance of the RES uptake by PEGylation were

clearly shown to cause passive targeting of the tumor and an increase in the

antitumor activity of CPT-1 1. CPT-11 is commonly used as a lung cancer

chemotherapeutic agent. We will n e x t investigate the effect of

liposomalization on survival time, using a lung cancer model.

258 SADZUKA ET AL.

REFERENCES

1. Gabizon, A., Goren, D. and Barenholz, Y. 1988. Investigations on the

antitumor efficacy of liposome-associated doxorubicin in murine
tumor models. Isr. J. Med. Sci. 24 : 5 1 2-51 7.

2. Mayhew, E., Cimino, M., Klemperer, J., Razo, R., Wiernikowski,

J. and Arbuck, S. 1990. Free and liposomal doxorubicin treatment
of intraperitoneal colon 26 tumor. Therapeutic and pharmacologic
studies. Select. Cancer Ther. 6(4) : 193-209.

3. Balazsovits, J. A. E., Mayer, L. D., Bally, M. B., Cullis, P. R.,

Ginsberg, R. S. and Falk, R. E. 1989. Analysis of the effect of
liposome encapsulation on the vesicant properties, acute and cardiac
toxicities, and antitumor efficacy of doxorubicin. Cancer Chemother.
Pharmacol. 23 : 81-86.

4. Sawada, S., Okajima, S., Aiyama, R., Nokota, K., Furuta, T.,
Yokokura, T., Sugini, E., Yamaguchi, K. and Miyasaka, K. 1991.
Synthesis and antitumor activity of 20(s)-camptotecin derivatives.
Carbomate-linked, water-soluble derivartives of 7 - e t h y l - 1 0 -
hydroxy-camptothecin. Chem. Pharm. Bull. 39(6) : 1446-1454.

5. Kawato, Y., Aonuma, M., Hirota, Y., Kuga, H. and Sato, K. 1991.
lntracellular roles of SN-38, a metabolite of the camptothecin
derivative CPT-1 1, in the antitumor effect of CPT-1 1. Cancer Res.
51(16) : 4187-4191.

6. Tsuji, T., Kaneda, N., Kado, K., Tokokura,.T., Yoshimoto, T. and

Tsuru, D. 1991. CPT-1 1 converting enzyme from r a t serum.
Purification and some properties. J. Pharmacobiodyn. 1 4 ( 6) :
341 - 3 4 9 .

7. Kawato, Y., Furuta, T., Aonuma, M., Yokokura, T. and Matsumoto,

K. 1991. Antitumor activity of a camptothecin derivative, CPT-1 1 ,
against human tumor xenografts in nude mice. Cancer Chemother.
Pharmacol. 28(3) : 192-1 98.

8. Kudoh, S., Takada, M., Masuda, N., Nakagawa, K., Itoh, K.,
Kusunoki, Y., Negri, S., Matsui, K., Takifuji, K., Morino, H. and
Fukuoka, M. 1993 Enhanced antitumor efficacy of a combination of
CPT-11 , a new derivative of camptothecin, and cisplatin against
human lung tumor xenografts. Jpn. J. Cancer. Res. 84 : 203-207.
POLYETHYLENEGLYCOL-COATED LIPOSOMES 259

9. Nishimura, G., Satou, T., Yoshimitsu, Y., Kurosaka, Y., Fujimura,

T., Sugiyama, K., Kanno, M., Yonemura, Y., Miwa, K. and Miyazaki,
I. 1995. Effect of chemotherapy using irinotecan (CPT-1 1) against
recurrent colorectal cancer. Jpn. J. Cancer Chemother. 22( 1 ) :
93-97.

10. Sadzuka, Y., Nakai, S., Miyagishima, A., Nozawa, Y. and Hirota, S.
1995. The effect of dose on the distribution of adriamycin
encapsulated in polyethyleneglycol-coated liposomes. J. Drug
Targeting 3 : 31-37.

11. Kono, A. and Hara, Y. 1991. Conversion of CPT-11 into SN-38 in

human tissues. Jpn. J. Cancer Chemother. 18(12) : 21 75-21 78.

12. Barilero, I., Gandia, D., Armand. J. P., Mathien-Bone, A., Re, M.,
Gouyette, A. and Chabot, G. G. 1992. Simultaneous determination of
the camptothecin analogue CPT-1 1 and its active metabolite SN-38
by high-performance liquid chromatography, application t o plasma
pharmacokinetics studies in cancer patients. J. Chromatogr. 575 :
275-280

13. Hirotsu, S., Sadzuka, Y., Miyagishima, A., Nozawa, Y. and Hirota, S.
1995. The preparation and the tissue distribution of liposomal CPT-
1 1. Drug Delivery System 1 O(4) : 283.

14. Taguchi, T., Wakui, A., Hasegawa, K., Niitani, H., Furue, H., Ohta,
K. and Hattori, T. 1990. Phase I clinical study of CPT-1 1.
Research group of CPT-1 1. Jpn. J. Cancer Chemother. 17(1) :
1 1 5-1 20.

15. Shimada, K., Miyagishima, A., Sadzuka, Y., Nozawa, Y. Mochizuki,

Y., Ohshima, H. and Hirota, S. 1995. Determination of the
thickness of the fixed aqueous layer around polyethyleneglycol-coated
liposomes. J. Drug Targeting 3 : 283-289.

16. Lopez-Berestein, G., Kasi, L., Rosenblum, M. G., Haynie, T., Jahns,
M., Glenn, H., Mehta, R., Mavlight, G. M. and Hersh, E. M. 1984.
Distribution and pharmacology of intravenous tecnetium-99m-
labeled multilamellar liposomes in rats and mice. Cancer Res. 44 :
3 75-3 78.

17. Klibanov, A. L., Maruyama, K., Torchilin, V. P. and Huang, I. 1990.

Amphipathic polyethyleneglycols effectively prolong circulation
time. FEBS Lett. 268 : 235-237.
260 SADZUKA ET AL.

18. Allen, T. M. and Chonn, A. 1987. Large unilameller liposomes with

low uptake into reticuloendothelial system. FEBS Lett. 223 : 42-46.

19. Mi, Z. and Burke, T. G. 1994. Differential interactions of campto-

thecin lactone and carboxylate forms with human blood components.
Biochemistry 33(34) : 10325-1 0336.

20. Sasaki, Y., Yoshida, Y., Sudoh, K., Hakusui, H., Fujii, H., Ohtsu,
T., Wakita, H., Igarashi, T. and Itoh, K. 1995. Pharmacological
correlation between total drug concentration and lactones of CPT-11
and SN-38 in patients treated with CPT-1 1. Jpn. J. Cancer Res. 86
: 111-116.