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Application of lipid microspheres for the treatment of cancer
advanced
drug deliiry reviews ELSEVIER
Advanced Drug Delivery Reviews 20 (1996) 209-219
Application
of lipid microspheres
for the treatment
of cancer
Mitsuko Takenaga’” Institute
of Medical Science, St. Marianna University School of Medicine, 2-16-i Sugao, Miyamae-ku, Kawasaki 216, Japan
Abstract Lipid microspheres can act as a carrier for antitumor agents. We incorporated a lipophilic antitumor agent, 1,3-bis(2-chloroethyI)-l-nitrosourea (BCNU) into microspheres by homogenizing a soybean oil solution of BCNU with egg yolk lecithin. Lipid microsphere-encapsulated BCNU showed a significantly enhanced antitumor activity
with reduced toxicity in mice with L1210 leukemia when compared to the corresponding dose of free BCNU. Lipid nanospheres, smaller particles containing BCNU with an average size of 50 nm, also showed a similar level of in vivo antitumor activity. An in vitro study showed that [“Cltriolein uptake by tumor cells was increased by incorporation into microspheres. The in vitro uptake of small microspheres was lower than that of standard microspheres. However, the in vivo half-life of small microspheres was longer, they avoided capture by the reticuloendothelial system and showed higher accumulation at tumor sites. Thus, lipid microspheres may be useful for delivering various lipophilic chemotherapy agents. Keywords:
nanospheres;
Chemotherapy; Drug delivery system; 1,3-bis(2-chloroethyl)-l-nitrosourea; L1210 leukemia
Lipid microspheres;
Lipid
Contents 1. Introduction .......................................................................................................................................................................... 2. Stability of lipo-BCNU ......................................................................................................................................................... 3. In viva antitumor activity of lipo-BCNU in L1210-bearing mice .......................................................................................... 3.1, Consecutive administration study .................................................................................................................................. 3.2. Single-dose study ........................................................................................................................................................... 3.3. Intermittent administration study .................................................................................................................................. 4. Toxicity of lipo-BCNU .......................................................................................................................................................... 5. In vivo antitumor activity of BCNU in lipid nanospheres ..................................................................................................... 6. In vitro uptake of LM by tumor cells.. .................................................................................................................................. 7. In vivo distribution of LM.. ................................................................................................................................................... 7.1. [ ‘“C]Triolein-containing LM.. ........................................................................................................................................ 7.2. CDDP-D-containing LM ............................................................................................................................................... 8. Conclusions.. ......................................................................................................................................................................... References.. ...............................................................................................................................................................................
*Corresponding
author.
0169-409X/961$32.00 @ SSDI 0169-409X(95)00124-7
1996 Elsevier
Science
B.V. Al1 rights reserved
210 210 211 211 211 212 213 214 214 215 215 216 217 218
1. Introduction Much attention has been focused on drug delivery system (DDS) for cancer chemotherapy which aim at the specific targeting of tumor cells or tumor tissues. thus enhancing the efficacy and reducing the toxicity of antitumor agents. For example, microcapsules coated with a monoclonal antibody directed against a tumor-specific antigen have been suggested as an extremely effective targeting vehicle for antitumor agents. In fact, numerous studies have demonstrated that microcapsules containing chemotherapy agents show excellent activity against certain types of experimental tumors [l-S]. However. most of these approaches have been clinically unsuccessful because the microcapsules are trapped by the reticuloendothelial system before reaching the target tumor cells [6-S]. Lipid microspheres provide a suitable carrier for lipophilic antitumor agents which are easy to dissolve in soybean oil however most chemotherapy agents are hydrophilic and not hydrophobic. Among them, the nitrosoureas are one of the few lipophilic drug classes [Y-13]. They can cross the blood-brain barrier and show excellent activity against brain tumors originating from neuronal tissues. Nitrosoureas are also highly effective for malignant lymphoma. Their adverse effects are bone marrow suppression. renal toxicity and pulmonary toxicity which arise as a of drug consequence accumulation during chronic treatment [Y.lO]. Among the several nitrosourea agents used clinically. we selected BCNU (Fig. l), since it is the most lipophilic compound [ 111. In this study. BCNU in lipid microspheres (lipo-BCNU) was administered intravenously to mice bearing L1210 tumors and the antitumor activity was compared with that of BCNU dissolved in saline (free BCNU). The antitumor activity of BCNU encapsulated in smaller lipid particles (lipid nanospheres) was also examined.
y” CICH2CHzNHCONCH
Fig. 1. Structure (BCNLJ).
of
zCH2CI
I .3-his-( 2-chloroethyl)-
I-nitrosourea
1
1 ‘/
/
,
,
,
0
1
2
3
4 Weeks
I 0
3
5
I
6
9
,
,
6
7
I 12
Days
Fig. 2. Stability of BCNU. Lipo-BCNU (4 q/ml) (0) or a B<‘NI 1aqueoussolution (A) was prepared and stored at 4°C (a) or room temperature (b). Then the BCNU content was determined by HPLC at the specified times. Results are the mean of 2-j aSSa”b.
since smaller liposomes have been reported to accumulate better in tumors [6.14]. In addition, several kinds of lipid micropheres (LM) were prepared and their distribution and uptake were assessed by in vitro and in vivo studies.
2. Stability of lipo-BCNU Lipid microspheres (LM) containing BCNU were prepared as described previously [15] and suspended at a final concentration of 4 mg/ml. The average diameter of the microspheres thus prepared was 198 ? 34 nm as measured by a Coulter N4 particle analyzer (Coulter, FL, USA). When lipo-BCNU was stored at 4°C the BCNU content was still almost 100% after 2 weeks (Fig. 2a). Even after 3 months. 80% of the BCNU was
M. Takenaga I Advanced Table Effect
Drug Delivery Reviews 20 (1996) 209-219
211
I of consecutive
administrations
of free BCNU
and Lipo-BCNU
to L1210 tumor-bearing
Preparation
Drug dose (mgi kg)
Survival
Saline LM Free BCNU
0 0 0 3 10 3 10
777777777778 7 1 > ,, 3 3 3 2 >, 777771777711 > 7 ,. 7 > > 3. 3 7777777778.8 >, >. 1>>>1 7778888889 >33 > 337 , , 12,13,13,13,13,13,14,14,14,14 78888999910 33, 32, 311 16,17,17,18,18,18,18,19,19.19
Lipo-BCNU
time (day)
mice
Mean ? SD (day)
% Increase
7.08 t 0.27 7.36 -t 1.20 7.18 + 0.38 7.8 ? 0.60 13.3 IT 0.64 8.5 2 0.81 17.9 + 0.94
4 1 10 88 20 152
1 *
CDF, mice were inoculated intraperitoneally with L1210 cells on day 0 and were subsequently treated intravenously on days 2-6. The percent increase lifespan of survival time (% increase) was determined as a median survival time of treated animals divided by the median survival time of control animals. * P < 0.01 by Student’s t-test.
retained by the LM. On the other hand, the BCNU content of a solution in physiological saline decreased over time to 38.9%, after 4 weeks. With storage at room temperature, the BCNU content of lipo-BCNU was still about 80% after 5 days (Fig. 2b), while that of an aqueous solution was only about 3.5% even after 3 days. At 37°C however, the BCNU content of both preparations decreased rapidly, with the residual BCNU content in lipo-BCNU, incubated for 3 and 24 h, being 18.5% and 3.8%) respectively. Also, when lipo-BCNU was incubated for 5 min at 37°C with human serum, more than 90% of the BCNU was released from the LM, as determined after Sepharose 4B column chromatography (data not shown). The particle size of lipo-BCNU remained at 206 5 41 nm after 2 months of storage at 4°C. At 37°C however, the emulsion was destroyed and the oil and water phases showed separation.
The results are shown in Table 1. The average survival time of the saline controls was 7.08 t 0.27 days (mean + SD) and the LM controls showed a similar survival time of 7.36 2 0.27 days. Therefore, LM seemed to have no effect on the survival of the mice. When the average survival time was compared between the two groups of mice given BCNU at 3 mg I kg per day, the lipo-BCNU group showed a longer survival than the free BCNU group. With a BCNU dose of 10 mg/kg per day, the survival time of the lipo-BCNU group was significantly longer than that of the free BCNU group (P < 0.01). The highest BCNU dose tested (30 mg/kg) was very toxic to the mice. In the free BCNU group, the mice rapidly lost weight and began to die on day 12. Survival time was not prolonged when compared with that at 10 mg/kg per day of BCNU. Treatment with lipo-BCNU at this high dose was also toxic and the mice began to die on day 16. 3.2. Single-dose study
3. In vivo antitumor LUlO-bearing
activity of lipo-BCNU
in
mice
3.1. Consecutive administration study
CDF, mice were randomly allocated to 12 groups (n = 10-11) and L1210 ascites cells (1 X 105) were injected intraperitoneally. Treatment was initiated on the second day, after tumor cell injection was given once daily for 5 days. The agent or vehicle was injected via the tail vein in a volume of 0.2 ml per 25 g.
The second experiment employed the same conditions as described above, except that 20 mg/kg of either BCNU or lipo-BCNU was given once. on day 2 after tumor cell injection to avoid the toxicity seen in the first experiment. As shown in Table 2, the survival time was 8.3 -+ 0.46 days in saline controls while BCNU at a single dose of 20 mg/kg prolonged survival by 49.4%. Treatment with lipo-BCNU produced a significantly longer survival time by 74.7% than treatment with BCNU in saline (P < 0.01).
M. Takenuga
212
Table 2 Effect of a single dose of free BCNU
I Advunced
Drlrg Delivery
Reviews
20 (1996) 20%219
or Lipo-BCNU
Preparation
Drug dose (mgikg)
Survival
time (days)
Free BCNU Lipo-BCNU Saline
20 700
11.12.12,12.12.12,13.13,13,14 14.14.14.14.14.14.15,15,15.16 8,8,8.8.8,8,X.Y,Y.Y
Mean + SD (day)
12.4 t 0.8 1* * I45 8.3 f2 0.67 0.46
1 *
% increase
4Y.4 74.7
CDF, mice (10 animals per group) wcrc inoculated intrapcritoneally with 10’ L1210 cells on day 0 and were subsequently treated intravenously on day 2. The percent increase of survival (% increase) was determined as the median survival time of treated animals divided bv the median survival time of control animals. * P
3.3. Intermittent udministration study
Intermittent treatment with either BCNU or lipo-BCNU on days 2, 9 and 16 after tumor injection was attempted to increase the efficacy. Treatment with BCNU alone significantly prolonged the survival by 36.5% at a dose of 10 mgikg, by 97.2% at 20 mglkg and by 400.6% at 30 mg/kg, indicating that intermittent treatment was superior to consecutive treatment (Table 3). In the high-dose group, 4 out of 10 mice survived longer than 60 days after tumor cell injection and were regarded as cured. Treatment with lipo-BCNU resulted in a significantly longer survival time than the corresponding dose of BCNU alone. Treatment with lipo-BCNU at 10 mg/kg prolonged the survival time by 65.6% as compared with the LM controls, while 20 mglkg achieved prolongation by 412% and 30 mg/ kg did so by 626.9%. In both the medium- and high-dose lipo-BCNU groups, 4 of 10 and 9 of 10 mice, respectively, survived for longer than 60 days. These survival rates indi-
Table 3 Effect ot intermittent
treatment
with free BCNU
or Lipo-BCNU
Preparation
Drug dose (mgikg)
Survival
Saline Free BCNLJ
0 IO 20 30 0 10 20 30
7.8,8.8,8,8.8.8,8,8.8 9,10.10,10.10,10,10.10.11.14,15 13,13.13,15,16,16.l6,lh,lX.20 21,25,26.27,28,29 8,8,8,8,8,8.8,8,8.X,9 12.12.12,13,13,13.14.14,14.l4 27,27.27.28,28,28 35
LM Lipo KNlJ
time (day)
cated that intermittent lipo-BCNU treatment had a significantly better antitumor activity aginst Ll210 leukemia. Besides increasing the survival time, the number of 60-day survivors was significantly greater in the lipo-BCNU-treated groups of mice. The 60-day survivors were killed and the body and spleen weights were measured along with determination of the white blood cell (WBC) count and hemoglobin (Hb) concentration which were determined by using a PC-607 particle counter (Erma, Tokyo, Japan). The body weight of the survivors recovered faster in the lipo-BCNUtreated groups than in the free BCNU groups. When BCNU was delivered in the saline vehicles, weight gain was retarded and the minimum was reached on day 20. The mean body weight of survivors on day 60 was 24.8 g (Table 4). while that of survivors treated with lipoBCNU was 27.8 g. In addition, the ratio of spleen weight to body weight and the WBC count of survivors from the free BCNU groups were higher than those of animals treated with lipo-
on the lifespan
of L1210 tumor-bearing
Mean -t SD (day) 7.Yl I? 0.29 10.x + I.80 IS.6+ 2.15 I .>39.6 -r 16.7 8.09 t 0.29 13.1 kO.83 >40.5 + 1S.Y BS7.7 i- 7.Y
% ILS
mice
Survivors/tested
on day 60
0111 5:
~400.6 6.5.6 36.5 97.2 2
0110 Oil1 4110 o/11 oi 10
>412.0 >62h.Y
4110 9/ 10
CDF, mice were inoculated intraperitoneally with L1210 cells on day 0 were subsequently treated i.v. on days 2.9 and 16. The percent increase of survival time was determined as the median survival time of treated animals/the median survival time of control animals. * P
M. Takenaga Table 4 Body weight,
spleen
weight,
WBC count
Group
Dose
I Advanced Drug Delivery Reviews 20 (1996) 209-219
and Hb in L1210-bearing n
(mg/kg) Day 0 Ll210-bearing mice Survivors on day 60 Free BCNU Lipo-BCNU i4-week-old Values
normal
_
123
30 20 30 -
mice
4 4 9 5
mice on days 0 and 60
Body weight
Spleen weight
(9)
(mg)
22.6 ? 0.47
_
24.8 28.1 27.8 28.4
124.2 74.2 78.3 71.2
5 * -’ 2
213
0.34 1.22 1.15 0.23
WBC count ( x 1021~1)
69.4 t 9.12 2 ? ? 2
55 12.6 10.2 3.96
227.5 82.8 71.4 57 t
? 187.2 2 17.6 2 11.8 17.5
Hb concentration (g/d])
16.5 2 1.21 14.4 16.4 16.4 17.1
t 2.3 t 0.1 ? 0.69 -+ 0.74
are mean f SD.
BCNU. Furthermore, the free BCNU survivors showed an increased WBC count and a decreased Hb concentration. The mean body weight of normal 14-week-old mice was 28.4 _’ 0.23 g, while the spleen weight was 71.2 2 3.96 mg, the WBC count was 57 2 17.5/1_~1and the Hb was 17.1 ? 0.74 g/d1 (n = 5, mean 2 SD). These values were similar to those found in lipoBCNU-treated survivors.
4. Toxicity of lipo-BCNU The similar normal mittent
toxicity of lipo-BCNU appeared to be or more severe than that of BCNU in male CDF, mice (Table 5). When interintravenous injection was performed, the
Table 5 Toxicity of free BCNU Preparation
and Lipo-BCNU
in normal
LD,, values of lipo-BCNU and BCNU calculated by Behrens’ method were 71 mg/kg and 73.5 mg/kg, respectively. Three doses of 10 or 30 mg/kg of lipo-BCNU had little effect over 60 days despite transient weight loss after drug treatment. However, the mice given 60 mg/kg all died within 14 days after showing marked weight loss. After treatment at 90 mg/kg, 7 mice died within 7 days and the last one died on day 8. As compared to lipo-BCNU, free BCNU appeared to be somewhat less toxic. Three doses of 10 or 30 mg/kg of free BCNU also had little effect over 60 days. However, with treatment at 60 mg/kg, 4 out of 8 mice died within 14 days and the others died by day 17. After treatment at 90 mglkg, 4 mice died by day 7 but the others remained alive until days 9-10. After a single
mice
Drug dose
Total dose
Survival
(mgikg)
(mg/kg)
60 days
Mean t SD
Saline Free BCNIJ
0 10 30 60 90
_ 30 90 a)* b)*
8/B 8/B O/8 O/8
60> 60> 14.6 t 1.32 7.63 ? 2.0
Lipo BCNU
10 30 60 90
30 90 c)* d)*
8/B 818 018 O/8
60> 60> 13.1 i 1.27 6.6 + 0.99
Normal CDF, mice (n = 8) were treated intravenously (BWD) data are expressed as percentages. * a) 180 mgikg (n = 4). others 120 mg/kg. b) 90 mgikg mgikg (n = 1).
time (day)
with drugs
BWD(%) Day 3
Day 14
Day 26
Day 60
1.7 0.9 4.3 7.2 8.1
+ 6.5 + 4.0 - 2.6 -21.1 ~27.3
+9.5 +8.5 -6.0 -40.8 -
+15.2 t13.7 - 5.6 -.
+29.0 +27.0 + 15.8 _ _
+ 0.9 - 3.1 -11.4 - 13.4
+ 5.1 ~ 2.2 -26.5 -26.8
i7.3 -3.5 ~27.7 -
113.4 --3.5
+24.7 + 19.6 _ _
+ ~ ~ -
three
(n = 4), others
Day 7
times
180 mglkg.
(days
0,7 and 14). The body
c) 120 mglkg.
d) 90 mgikg
weight
differences
t’n = 7). others
180
214
M. Takenaga
i
Advanced
Drug
dose of BCNU, mice given 90 mgikg died on days 5-14 (7.83 2 3.24 days, mean ? SD).
Delivers
Reviews
20 (1996)
209-219
treated survivors. as was the case with lipoBCNU-treated survivors.
6. In vitro uptake of LM by tumor cells 5. In vivo antitumor nanospheres
activity of BCNU in lipid
The antitumor activity of BCNU incorporated into lipid nanospheres (LN), a finer lipid emulsion with an average size of 50 nm. was also examined since smaller liposomes have been reported to accumulate better in tumors [6.14]. The size of LN varied according to the homogenizing conditions and the lecithin content while the stability was similar to that of lipoBCNU. Consecutive treatments with BCNU in LN (slipo-BCNU) also resulted in a significantly longer survival time than the corresponding dose of BCNU in saline. Treatment at both 3 mg/kg and 10 mg/kg produced a significant extension of survival time by 28% and 163%. respectively, as compared with LN controls. In particular, even 3 mg/kg of s-lipo-BCNU prolonged the survival time significantly compared to free BCNU while lipo-BCNU did not, although the difference between the two emulsions was not statistically significant. Also, a single dose of s-lipo-BCNU (20 mg/kg) prolonged the survival time by 84.3%, which was slightly superior to the effect of lipo-BCNU. Intermittent treatment with s-lipo-BCNU also resulted in a significantly longer survival time than that of the corresponding free BCNU group. Administration of s-lipo-BCNU at 10 mgi kg prolonged the survival time by 61.8%, while the increase was 327.3% at 20 mglkg and 626.9% at 30 mgikg. 2 and 9 mice out of the 10 animals. each in the medium- and high-dose groups receiving s-lipo-BCNU. survived for longer than 60 days. The respective mean body weight of survivors treated with medium and high doses of s-lipo-BCNU was 28.9 g and 28.1 g. The ratio of spleen weight to body weight and the WBC count of the s-lipo-BCNU-treated survivors were lower than those of animals given free BCNU. while the Hb was higher. All these parameters were within the normal range in s-lipo-BCNU-
In vitro uptake of LM by tumor cells was studied. [ “C]Triolein was used as a tracer of LM, since it is a lipophilic triglyceride. LM preparations labeled with [ “C]triolein (NEN Research Products. Beverly, MA, USA) were incubated with several kinds of tumor cells (lO’/ml) including L1210 cells. After 4 h of incubation at 37°C the LM uptake by the tumor cells was determined from the radioactivity incorporated. The results are shown in Table 6. Compared to free [ “C]triolein, a 3-fold larger amount of [ “C]triolein incorporated into LM consisting of phosphatidylinositol, phosphatidylserine or phosphatidylcholine (lip0 [‘4C]triolein was taken up by L1210 cells. Data in the table are for standard lipid emulsions with an average particle diameter of 200 nm. In contrast, smaller LM of about 140 nm showed only one tenth of the uptake obtained with the standared LM preparations. The other tumor cells also showed a higher uptake of lipo [r4C]triolein than of free [‘“Cltriolein. Although smaller LM composed of phosphatidylcholine were taken up by MM46 -1‘ahle
h
Influence of phosphohpid composition and size on the uptake of lipid microspheres into tumor cells Phospholipid
PI PS PC S-PC” Free
Tumor
cells
L 1210
MM46
Sarcoma
1.118 0.897 1.102 0.136 0.33 1
2.766 1.864 1.127 1.021 0.958
1.045 0.713 1.030 0.856 0.487
180
P388 0.663 0.399 1.005 0.167 0.304
PI. phosphatidylinositol: P.S. phosphatidylserine; PC, phosphatidylcholine, Lipid microspheres, incorporating [“Cltriolein were incubated with tumor cells (IO’ cells/ml) for J h at 37°C and washed, after which the radioactivity was measured. Values arc the mean percentage of tumor cell radioactivity uptake (n = 2). ” The average diameter of the small microspheres composed of PC (s-PC) was 140 nm. while that of the other microspheres was 200 nm.
M. Takenaga
I Advanced Drug Delivery Reviews 20 (1996) 209-219
cells at much the same rate as the standard LM, uptake of small LM by the other tumor cells was reduced.
7. In vivo distribution
of LM
7.1. [‘JC]Triolein-containing
LM
The effects of vesicle size and lipid composition on the in vivo distribution of LM were studied. LM preparations incorporating [ “C]triolein were injected into C,H/He mice (pretreated with 10” MM46 tumor cells subcutaneously into the flank), via the tail vein in a volume of 0.2 ml (about 2 x lo6 dpm). At appropriate times, blood was slowly collected from the heart into a heparized syringe and used for the determination of radioactivity. After each mouse was killed by
0
0.250.5
Hours
after
1
2
215
cervical dislocation, the animal was perfused with saline. Subsequently, the tumor, the liver and other tissues were removed and a sample of each tissue (100 mg) was solubilized with 1 ml of tissue solubilizer (Soluene-350, Packard Company Meriden, CT, USA) and isopropyl alcohol, followed by the addition of hydrogen peroxide. The radioactivity of the tissue solution was then determined in a liquid scintillation counter. Fig. 3a shows the tumor uptake of LM. Standard LM with a particle size of 200 nm were delivered to the tumors up to 0.5 h after injection. All LM preparations were retained by the tumors. Smaller LM (phosphatidylcholine with a particle size of 140 nm) accumulated at tumor sites in a similar manner to standard LM up to 4 h after injection. At 24 h after injection, however, 0.969% of the injected dose of smaller LM was retained compared to 0.632% of the standard LM dose (data not shown). Therefore,
0
4
0.250.5
Hours
injection
1
after
2
4
injection
:
01s /
0
0.250.5
Hours
after
1
2
0
4
Hours
injection
Fig. 3. Pharmacodynamics of several kinds of LM. Male C,H/He mice were after tumor inoculation, several kinds of LM incorporating [‘4C]triolein (2 times, tumor (a), blood (b), liver (c) and spleen (d) samples were harvested are expressed as percent injected dose per gram or milliliter of the specimen (0). phosphatidylserine (A) and phosphatidylcholine (0). The average phosphatidylcholine (0) was 140 nm. while that of the other microspheres
I
0.250.5 after
I
1
I
2
4
injection
subcutanously injected with 10” MM46 cells. 14 days 10’ dpm) were given intravenously. At appropriate and used for the determination of radioactivity. Data (n = 3). LM were prepared using phosphatidylinositol diameter of small lipid microspheres composed of was 200 nm.
X
216
M. Takenaga I Advanced
Drug Delivery Rewews 20 (1996) 209-219
smaller LM seems to be retained better at the tumor site. LM, prepared from soybean phosphatidylinositol showed the maximum tumor localization at 2 h after injection (1.871% of the injected dose per gram of tumor). LM composed of phosphatidylserine showed less than one half of the tumor uptake obtained with LM composed of phosphatidylinositol. As shown in Fig. 3b. elimination from the blood seemed to be influenced by LM size and smaller particles disappeared more slowly compared to standard LM with an average size of 200 nm. LM showed marked accumulation in the liver (Fig. 3~). In particular, LM composed of phosphatidylinositol showed 2-fold more accumulation than LM composed of phosphatidylcholine. LM with a smaller particle size showed a tendency not to be trapped in the liver. Smaller LM showed much less uptake by the spleen (Fig. 3d). In contrast to small sized LM. the standard LM were predominantly localized to the spleen. 7.2. CDDP-D-contuining
LM
Another LM distribution study was carried out using an antitumor agent, cisplatin derivative (CDDP-D) which is much more lipophilic than cisplatin itself. Intravenous injection of 4 X 10” L5178Y-ML cells (kindly donated by Dr. Okura, Banyu Pharmaceutical, Tsukuba, Japan) induced liver metastasis [16]. Eight days after tumor inoculation, an LM preparation incorporating CDDP-D (4 mg/ kg, as cisplatin) was given intravenously to tumor-bearing or tumor-free mice. At 10 min and 30 min after treatment. the amount of cisplatin in liver and plasma was determined and compared with the values after treatment with free cisplatin (4 mg/kg). The results are illustrated in Fig. 4. In normal mice given free cisplatin. the amount of cisplatin in the liver was 0.394 _’ 0.042 pug/g (mean ? SD) at 10 min and 0.490 ? 0.001 @g/g at 30 min (Fig. 4a), while the respective amount of cisplatin in lipo CDDP-D-treated mice was 0.879 2 0.084 ,ug/g and 0.965 ? 0.13 ,uglg. LM are well known
(b)
0
10 20 30 MIII after b”,ectlon
40
II
IO 20 30 Mln after m,ect,on
40
Fig. 4. Distribution of LM-encapsulated antitumor agents. Female CDF, mice were intravenously injected with 4 X 10’ L5 17XY-ML cells. Eight days after tumor maculation. an LM preparation incorporating a cisplatin CDDP derivative (CDDP-D) (0) or free cisplatin (0) were given intravenously. At 10 or 30 mitt after treatment. the cisplatin level in liver or plasma was determined by inductively coupled plasma (ICP) method. Lipo CDDP-D (0) and free cisplatin (0) were also given to tumor-free mice. The cisplatin content is expressed as pgip liver (a) or fig/ml plasma (b). Mean k SD (n = 3).
to be extensively taken up by the reticuloendothelial system in the liver. Therefore, lipo CDDP-D was preferentially delivered to the liver and retained there, with the hepatic CDDP concentration being twice that after treatment with free cisplatin. Tumor-bearing mice had metastatic nodules in the liver. as confirmed by hematoxylin-eosin staining. After free cisplatin treatment, hepatic uptake was about twice that observed in normal mice. Treatment with lipo CDDP-D achieved the highest hepatic cisplatin levels which were 0.965 + 0.131 pg/g at 10 min and 1.173 -t 0.117 ,ug/g at 30 min. These results indicated that the delivery of cisplatin to the liver including the metastases was increased by encapsulation in LM. The pharmacodynamics of free cisplatin in plasma were quite different from those of LMencapsulated CDDP-D (Fig. 4b). Free cisplatin disappeared very rapidly from the blood in both normal and tumor-bearing mice. However, treatment with LM-encapsulated CDDP-D led to much higher plasma levels of cisplatin, indicating that the LM-encapsulated agent was retained in the plasma.
M. Takenaga I Advanced
Drug Delivery Reviews 20 (1996) 209-219
8. Conclusions
These studies showed that lipo-BCNU not only had increased activity against L1210 leukemia in mice but also had reduced toxicity, and the potency of lipo-BCNU was much greater than that of the equivalent dose of free BCNU. The best response was achieved by intermittent treatment with lipo-BCNU. Besides increasing the survival time, the number of 60-day survivors was significantly greater in the lipoBCNU-treated mice. Also, the body weight of the survivors recovered faster in lipo-BCNUtreated groups than in the free BCNU-treated groups which showed splenomegaly, an increased WBC count, and a decreased Hb concentration. Splenomegaly might have reflected the recurrence of leukemia in the free BCNU-treated survivors. In contrast, the weight gain and spleen:body weight ratio were both within the normal range in lipo-BCNU-treated survivors. All of these findings indicate that intermittent administration of lipo-BCNU was superior for the treatment of L1210 leukemia in mice. This improvement may be partly explained by the modification of BCNU distribution induced by the LM drug carrier. Previously, we have shown that LM preferentially accumulates in inflamed tissues and vascular lesions in vivo [15,17,18]. Tumors always require a high energy supply and would actively take up LM particles. Fusion of LM with lipoprotein particles may also take place in vivo [19-211. We have already demonstrated that LM are readily taken up by tumor cells in vitro [22]. The present studies also showed that larger amounts of [14C]trioleinlabeled LM were taken up by tumor cells including L1210 cells when compared to free [‘4C]triolein. It 1s . well known that oil such as lipiodol is retained selectively in tumor tissues [23] and our in vivo study also demonstrated that an LM-encapsulated cisplatin derivative (CDDPD) was preferentially localized at the liver including the metastases. Accordingly, the improved therapeutic efficacy of lipo-BCNU would be related to increased uptake and longer retention of BCNU in tumor sites. The toxicity of lipo-BCNU appeared to be the
211
same as that of free BCNU in normal mice. This may have been because lipo-BCNU is retained in the tissues and has a slow rate of clearance. In tumor-bearing mice, however, lipo-BCNU appeared to be retained preferentially in the tumor compartment. The tissue distribution of liposomes is considered to be influenced by both vesicle size and lipid composition [6,14]. Although there was no significant improvement, the lipid emulsion with an average diameter of 50 nm (s-lipo-BCNU) tended to show a higher antitumor activity in vivo. Reducing the size of the LM decreased in vitro incorporation by tumor cells although in vivo tumor accumulation was satisfactory. In addition, smaller LM were retained longer in plasma so antitumor agents would exert more activity against solid tumors. The balance between these factors that variously increase and decrease antitumor activity would account for the lack of a significant improvement when slipo-BCNU was used. A number of reports have shown that the lipid components of LM can also influence antitumor activity or can exihibit such activity themselves [24-291. We found that LM composed of plant phosphatidylinositol showed the highest uptake by tumor cells in vitro and the in vivo study also indicated that such LM had the highest uptake by MM46 tumors. However, the antitumor activity of lipo-BCNU prepared using plant phosphatidylinositol may be almost the same as that of standard lipo-BCNU prepared with egg lecithin (data not shown). Thus, plant phosphatidylinositol may only be active against certain types of tumors. However, LM prepared from other glyceryl ether phospholipids or from eicosapentadecanoic acid, which has been reported to have antitumor activity [28], may be worth investigating for their targeting ability and/or antitumor activity. BCNU has been reported to decompose at relatively rapid rates under physiological conditions without the need for enzymatic attack [30] and a solution in which BCNU is no longer detectable by HPLC, exhibited no antitumor activity (data not shown). When lipo-BCNU was stored at 4°C however, the BCNU content
218
M.
Takrnaga
I Advancrd
Drug
remained at almost 100% for 2 weeks, indicating that lipo-BCNU is stable at 4°C. This may be partly because lecithin surrounds and protects the oil phase containing BCNU, since it was confirmed that BCNU remained intact in soybean oil stored at more than 37°C as determined by HPLC (data not shown). In contrast, lipo-BCNU seemed to leak out from LM and be destroyed in the aqueous phase when incubated in serum at 37°C and the residual content of BCNU was less than 10%. We have previously found [31] that the amount of prostaglandin E, retained in LM after incubation with human serum was only 0.5%. Despite this. LM containing prostaglandin E, have a potent clinical activity [32]. In the case of lipo-BCNU, the LM may be taken up by tumor sites before breakdown and the release of BCNU can occur. Liposome encapsulated BCNU has already been reported to exhibit a high antitumor activity against L1210 leukemia and Ehrlich ascites [33]. Also, Inaba et al. [34] have already demonstrated that liposomal MeCCNU, a nitrosourea antitumor agent, has a greater efficacy compared with free MeCCNU. However. compared to liposomes, LM are superior in terms of stability and safety. Therefore. LM (and LN) may be able to serve as useful carriers for other lipophilic agents besides BCNU [35].
Delivrr_v
20 (IYYh)
209%21Y
rinc MC’A-38 hepatic 2132.
IhI
metastasis.
Cancer
Res. 51, 21277
Mayer, L.D., Tai, L.C.L.. Ko. D.S.CJ.. Masin, D., Ginsberg. R.S., Cullis. P.R. and Bailey, M.B. (1989) Infuence of vesicle size, lipid composition and drug-tolipid ratio on the biological activity of liposomal doxorubicin in mice. Cancer Res. 49. 5922-5930.
I71 Maruyama,
K., Kennel, S.J. and Huang, L. (1990) Lipid composition is important for highly efficient target binding and retention of immunoliposomes. Proc. Nat]. Acad. Sci. USA X7. 574445748.
1x1 Dinney.
C.P.N.. Utsugi. T.. Fidler, I.J., von Eschenhach. A.(‘. and Killion. J.J. (1992) Immunotherapy of murine renal adenocarcinoma by systemic administration of liposomes containing the synthetic macrophage activators GCP 31362 or CGP lY853A in combination with intcrleukin-2 or gammma-interferon. Cancer Res. 52. 1155~1161.
101Bono. studies 707.
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Papahadjopoulos, 0. (1978) Liposomes and theit- uses in biology and medicine. Ann. N.Y. Acad. Sci. 308. l-462. drugs in targeted cancci121Kato. T.(1983) Encapsulated therapy. In: S.R. Bruck (Ed). Controlled Drug Delivery. CRC Press, Boca Raton. FL. pp. 189-105. T.. Kumai. K.. 131Konno, H.. Suzuki, H.. Tadakuma. Yasuda, T., Kuhota, T., Ohta S.. Nagaike, K., Hosokawa. S.. Ishihiki, K.. Abe, 0. and Saito, K. (1987) Antitumor effect of adriamycin-entrapped liposomes conjugated with antihuman cu-fetoprotcin monoclonal antibody. Cancer Res. 47. 4471-4477. K.K.. Ahai, A.M.. Cobb. S.. Hong. K.. 141 Matthay, Papahadjopoulos. D. and Straubinger. R. (1980) Role 01 ligand and antibody-directed endocytosis of liposomes by human T-leukemia cells. Cancer Res. 49, 487Y-4886. P., Katsanis. E.. 151 Loeffler, CM.. Platt, J.L.. Manderson, Ochoa. J.B., Urba. W.J.. Longo, D.L.. Lenard, A.S. and Ochoa. A.C. ( 1YYl ) Antitumor effect of interleukin-2 liposomes and anti-CD%stimulated T cells against mu
Hrvrew3
] IX]
VH. (1976) Review of mechanism of action ot the nitrosoureas. Cancer Treat. Rep. 60, 6YY-
Carter. S.K. (lY81) Design of clinical trials with nitrosoureas. In: A.W. Prestayko, ST. Crooke, L.H. Baker, SK. Caster and P.S. Schein (Eds), Nitrosoureas, Current Status and New Developments. Academic Press. New York. NY. pp. 41 I-416. Wasserman, T.H. (1976) The nitrosoureas: an outline of clinical schedules and toxic effects. Cancer Treat. Rep. 60. 70997 1I. Kanamaru. R., Asamura, M., Sato. H.. Saito, S.. Wakui. A. and Saito. T. (1980) Studies on the mechanism of action of ACNIJ. I-(4.amino-2-methylpyrimidine-S-yl) methyl-3-(2.chloroethyl)-3nitrosourea hydrochloride: effect on cultured HeLa S3 cells. Tohoku J. Exp. Med. 132.431I441. Sekido, S.. Ninomiya, K. and Iwasaki. M. (197’)) Biologic activity of MCNIJ: a new antitumor agent. Cancer Treat. Rep. 63. 961-070. Gahizon. A., Price. D.C.. Huherty. J.B., Resalier, R.S. and Papahadjopoulos, D. (1990) Effect of liposome composition and other factors on the targeting of liposomes to experimental tumors: Biodistrihution and imaging studies. Cancer Res. SO. 6731-6378. Mizushima. Y., Hammano. T. and Yokoyama. K. (1082) I’issuc distribution and anti-inflammatory activity of corticoids incorporated in lipid emulsion. Ann. Rheum. Dis. 41. 263-267. Watanabe, Y.. Okura, A.. Naito, K. and Kobayashi, M. (1988) Murine liver metastasis model using LS178Y-ML lymphoma and the effect of antitumor agents on the metastasis. Jpn. J. Cancer Res. 7Y, 1208-1216. Mizushima, Y., Shiokawa, Y.. Homma, M.. Kashiwazaki. S.. Ichikawa, Y., Hashimoto. H. and Sakuma. A. (1987) A multicenter double blind controlled study of lipoPGE, incorporated in lipid microspheres in peripheral vascular disease secondary to connective tissue disorders. J. Rheumatol. 14, 97-101. Mizushima. Y. (IYYI ) Lipo-prostaglandin preparations
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(review) Prostaglandins Leukotrienes Essent. Fatty Acids 42, l-6. Karaiovic. R.C., Andrew, M., Zepp, E. and Cenedella. R.J. (1977) Studies of the mechanism of carcass fat depletion in experimental cancer. Eur. J. Cancer 13. 1071-1079. Chernick, S.S., Spooner, P.M., Garrison, M.M. and Scow, R.O. (1986) Effect of epinephrine and other lipolytic agents on intracellular lipolysis and lipoprotein activity in 3T3Ll adipocyte. J. Lipid Res. 27, 286-294. Noguchi, Y., Vydelingum, N.A., Youners, R.N., Fried, S.K. and Brenan, M.F. (1991) Tumor-induced alterations in tissue lipoprotein lipase and mRNA levels. Cancer Res. 51, 863-869. Shoji. Y., Mizushima, Y. and Kametani, T. (1986) Affinity of lipid microspheres for MM-46 tumor cells. J. Pharm. Sot. Jpn. 106, 605-608 (in Japanese). Konno. T.. Maeda. H. and Iwai, K.(1983) Effect of arterial administration of high-molecular weight anticancer agent SMANCS with lipid lymphographic agent on hepatoma: a preliminary report. Eur. J. Cancer Clin. Oncol. 19. 105331065. Hayashi, Y.. Fukushima, S., Kishimoto, S., Kawaguchi. T., Numata, M.. Isoda. Y., Hirano, J. and Nakano, M. (1992) Antitumor effects of free polyunsaturated fatty acids in an oily lymphographic agent following intrahepatic arterial administration to a rabbit bearing VX-2 tumor. Cancer Res. 52, 400-405. Noseda, A., Berens, M.E., White. J.G. and Modest, E.J. (1988) In vitro antiproliferative activity of combination of ether lipid analogues and DNA-interactive agents against human tumor cells. Cancer Res. 48, 1788-1791. Chabot, M.C., Wykle, R.L., Modest, E.J. and Daniel, L.W. (1989) Correlation of ether lipid content of human leukemia cell lines and their susceptibility to l-o-octadecyl-2-o-methyl-rat. glycero-3-phosphocholine. Cancer Res. 49. 4441-4445.
Delivery
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20 (1996)
209-219
21’)
[27] Figiwara, K., Modest, E.J., Welander, C.E. and Wallen, C.A. (1989) Cytotoxic interactions of heat and ether lipid analogues in human ovarian carcinoma cells. Cancer Res. 49. 6285-6289. [28] Beck, S.A., Smith, K.L. and Tisdale. M.J. ( 1991) Anticachectic and antitumor effect of eicosapentadecanoic acid and its effect on protem turnover. Cancer Res. 51. 6089-6093. [29] Jett. M., Chudzik, J., Alving. CR. and Stanacev, N.Z. (1985) Metabolic fate of liposomal phosphatidylinositol in murine tumor cells: implications for the mechanism of tumor cell cytotoxicity. Cancer Res. 45, 4810-481.5. [30] Carter. S.K., Shabel, F.M., Broder, L.E. and Johnston. T.P. (1972) 1,3-Bis(2-chloroethyI)-I-nitrosourea (BCNU) and other nitrosoureas in cancer treatment: a review. Adv. Cancer Res. 16. 273-332. [31] Igarashi, R., Mizushima, Y., Takenaga. M., Matsumoto, K.. Morizawa, Y. and Yasuda. A. (1992) A stable PGE, prodrug for targeting therapy. J. Control. Rel. 20,37-46. [32] Hoshi. K.. Mizushima. Y., Kiyokawa. S. and Yanagawa. A. (1986) Prostaglandin E, incorporated in lipid microspheres in the treatment of peripheral vascular diseases and diabetic neuropathy. Drug Exp. Clin. Res. 12. 68l685. [33] Rutman, R.J., Ritter. CA., Avadhani. N.G. and Hansel, J. (1976) Liposomal potentiation of the antitumor activity of alkylating drugs. Cancer Treat. Rep. 60, 617-618. [34] Inaba, M., Yoshida, N. and Tsukagoshi, S. (1981) Preferential action of liposome-entrapped I-(2chloroethyl).3. (4.methylcyclohexyl)-I-nitrosourea on lung metastasis of Lewis lung carcinoma as compared with the free drug. Gann 72, 341-345. [35] Takenaga, M., Igarashi, R., Tsuji, H. and Mizushima. Y. (1993) Enhanced antitumor activity and reduced toxicity of 1,3-bis-2-chloroethyl-1-nitrosourea administered in lipid microspheres to tumor-bearing mice. Jpn. J. Cancer Res. 84, 1078-1085.
drug deliiry reviews ELSEVIER
Advanced Drug Delivery Reviews 20 (1996) 209-219
Application
of lipid microspheres
for the treatment
of cancer
Mitsuko Takenaga’” Institute
of Medical Science, St. Marianna University School of Medicine, 2-16-i Sugao, Miyamae-ku, Kawasaki 216, Japan
Abstract Lipid microspheres can act as a carrier for antitumor agents. We incorporated a lipophilic antitumor agent, 1,3-bis(2-chloroethyI)-l-nitrosourea (BCNU) into microspheres by homogenizing a soybean oil solution of BCNU with egg yolk lecithin. Lipid microsphere-encapsulated BCNU showed a significantly enhanced antitumor activity
with reduced toxicity in mice with L1210 leukemia when compared to the corresponding dose of free BCNU. Lipid nanospheres, smaller particles containing BCNU with an average size of 50 nm, also showed a similar level of in vivo antitumor activity. An in vitro study showed that [“Cltriolein uptake by tumor cells was increased by incorporation into microspheres. The in vitro uptake of small microspheres was lower than that of standard microspheres. However, the in vivo half-life of small microspheres was longer, they avoided capture by the reticuloendothelial system and showed higher accumulation at tumor sites. Thus, lipid microspheres may be useful for delivering various lipophilic chemotherapy agents. Keywords:
nanospheres;
Chemotherapy; Drug delivery system; 1,3-bis(2-chloroethyl)-l-nitrosourea; L1210 leukemia
Lipid microspheres;
Lipid
Contents 1. Introduction .......................................................................................................................................................................... 2. Stability of lipo-BCNU ......................................................................................................................................................... 3. In viva antitumor activity of lipo-BCNU in L1210-bearing mice .......................................................................................... 3.1, Consecutive administration study .................................................................................................................................. 3.2. Single-dose study ........................................................................................................................................................... 3.3. Intermittent administration study .................................................................................................................................. 4. Toxicity of lipo-BCNU .......................................................................................................................................................... 5. In vivo antitumor activity of BCNU in lipid nanospheres ..................................................................................................... 6. In vitro uptake of LM by tumor cells.. .................................................................................................................................. 7. In vivo distribution of LM.. ................................................................................................................................................... 7.1. [ ‘“C]Triolein-containing LM.. ........................................................................................................................................ 7.2. CDDP-D-containing LM ............................................................................................................................................... 8. Conclusions.. ......................................................................................................................................................................... References.. ...............................................................................................................................................................................
*Corresponding
author.
0169-409X/961$32.00 @ SSDI 0169-409X(95)00124-7
1996 Elsevier
Science
B.V. Al1 rights reserved
210 210 211 211 211 212 213 214 214 215 215 216 217 218
1. Introduction Much attention has been focused on drug delivery system (DDS) for cancer chemotherapy which aim at the specific targeting of tumor cells or tumor tissues. thus enhancing the efficacy and reducing the toxicity of antitumor agents. For example, microcapsules coated with a monoclonal antibody directed against a tumor-specific antigen have been suggested as an extremely effective targeting vehicle for antitumor agents. In fact, numerous studies have demonstrated that microcapsules containing chemotherapy agents show excellent activity against certain types of experimental tumors [l-S]. However. most of these approaches have been clinically unsuccessful because the microcapsules are trapped by the reticuloendothelial system before reaching the target tumor cells [6-S]. Lipid microspheres provide a suitable carrier for lipophilic antitumor agents which are easy to dissolve in soybean oil however most chemotherapy agents are hydrophilic and not hydrophobic. Among them, the nitrosoureas are one of the few lipophilic drug classes [Y-13]. They can cross the blood-brain barrier and show excellent activity against brain tumors originating from neuronal tissues. Nitrosoureas are also highly effective for malignant lymphoma. Their adverse effects are bone marrow suppression. renal toxicity and pulmonary toxicity which arise as a of drug consequence accumulation during chronic treatment [Y.lO]. Among the several nitrosourea agents used clinically. we selected BCNU (Fig. l), since it is the most lipophilic compound [ 111. In this study. BCNU in lipid microspheres (lipo-BCNU) was administered intravenously to mice bearing L1210 tumors and the antitumor activity was compared with that of BCNU dissolved in saline (free BCNU). The antitumor activity of BCNU encapsulated in smaller lipid particles (lipid nanospheres) was also examined.
y” CICH2CHzNHCONCH
Fig. 1. Structure (BCNLJ).
of
zCH2CI
I .3-his-( 2-chloroethyl)-
I-nitrosourea
1
1 ‘/
/
,
,
,
0
1
2
3
4 Weeks
I 0
3
5
I
6
9
,
,
6
7
I 12
Days
Fig. 2. Stability of BCNU. Lipo-BCNU (4 q/ml) (0) or a B<‘NI 1aqueoussolution (A) was prepared and stored at 4°C (a) or room temperature (b). Then the BCNU content was determined by HPLC at the specified times. Results are the mean of 2-j aSSa”b.
since smaller liposomes have been reported to accumulate better in tumors [6.14]. In addition, several kinds of lipid micropheres (LM) were prepared and their distribution and uptake were assessed by in vitro and in vivo studies.
2. Stability of lipo-BCNU Lipid microspheres (LM) containing BCNU were prepared as described previously [15] and suspended at a final concentration of 4 mg/ml. The average diameter of the microspheres thus prepared was 198 ? 34 nm as measured by a Coulter N4 particle analyzer (Coulter, FL, USA). When lipo-BCNU was stored at 4°C the BCNU content was still almost 100% after 2 weeks (Fig. 2a). Even after 3 months. 80% of the BCNU was
M. Takenaga I Advanced Table Effect
Drug Delivery Reviews 20 (1996) 209-219
211
I of consecutive
administrations
of free BCNU
and Lipo-BCNU
to L1210 tumor-bearing
Preparation
Drug dose (mgi kg)
Survival
Saline LM Free BCNU
0 0 0 3 10 3 10
777777777778 7 1 > ,, 3 3 3 2 >, 777771777711 > 7 ,. 7 > > 3. 3 7777777778.8 >, >. 1>>>1 7778888889 >33 > 337 , , 12,13,13,13,13,13,14,14,14,14 78888999910 33, 32, 311 16,17,17,18,18,18,18,19,19.19
Lipo-BCNU
time (day)
mice
Mean ? SD (day)
% Increase
7.08 t 0.27 7.36 -t 1.20 7.18 + 0.38 7.8 ? 0.60 13.3 IT 0.64 8.5 2 0.81 17.9 + 0.94
4 1 10 88 20 152
1 *
CDF, mice were inoculated intraperitoneally with L1210 cells on day 0 and were subsequently treated intravenously on days 2-6. The percent increase lifespan of survival time (% increase) was determined as a median survival time of treated animals divided by the median survival time of control animals. * P < 0.01 by Student’s t-test.
retained by the LM. On the other hand, the BCNU content of a solution in physiological saline decreased over time to 38.9%, after 4 weeks. With storage at room temperature, the BCNU content of lipo-BCNU was still about 80% after 5 days (Fig. 2b), while that of an aqueous solution was only about 3.5% even after 3 days. At 37°C however, the BCNU content of both preparations decreased rapidly, with the residual BCNU content in lipo-BCNU, incubated for 3 and 24 h, being 18.5% and 3.8%) respectively. Also, when lipo-BCNU was incubated for 5 min at 37°C with human serum, more than 90% of the BCNU was released from the LM, as determined after Sepharose 4B column chromatography (data not shown). The particle size of lipo-BCNU remained at 206 5 41 nm after 2 months of storage at 4°C. At 37°C however, the emulsion was destroyed and the oil and water phases showed separation.
The results are shown in Table 1. The average survival time of the saline controls was 7.08 t 0.27 days (mean + SD) and the LM controls showed a similar survival time of 7.36 2 0.27 days. Therefore, LM seemed to have no effect on the survival of the mice. When the average survival time was compared between the two groups of mice given BCNU at 3 mg I kg per day, the lipo-BCNU group showed a longer survival than the free BCNU group. With a BCNU dose of 10 mg/kg per day, the survival time of the lipo-BCNU group was significantly longer than that of the free BCNU group (P < 0.01). The highest BCNU dose tested (30 mg/kg) was very toxic to the mice. In the free BCNU group, the mice rapidly lost weight and began to die on day 12. Survival time was not prolonged when compared with that at 10 mg/kg per day of BCNU. Treatment with lipo-BCNU at this high dose was also toxic and the mice began to die on day 16. 3.2. Single-dose study
3. In vivo antitumor LUlO-bearing
activity of lipo-BCNU
in
mice
3.1. Consecutive administration study
CDF, mice were randomly allocated to 12 groups (n = 10-11) and L1210 ascites cells (1 X 105) were injected intraperitoneally. Treatment was initiated on the second day, after tumor cell injection was given once daily for 5 days. The agent or vehicle was injected via the tail vein in a volume of 0.2 ml per 25 g.
The second experiment employed the same conditions as described above, except that 20 mg/kg of either BCNU or lipo-BCNU was given once. on day 2 after tumor cell injection to avoid the toxicity seen in the first experiment. As shown in Table 2, the survival time was 8.3 -+ 0.46 days in saline controls while BCNU at a single dose of 20 mg/kg prolonged survival by 49.4%. Treatment with lipo-BCNU produced a significantly longer survival time by 74.7% than treatment with BCNU in saline (P < 0.01).
M. Takenuga
212
Table 2 Effect of a single dose of free BCNU
I Advunced
Drlrg Delivery
Reviews
20 (1996) 20%219
or Lipo-BCNU
Preparation
Drug dose (mgikg)
Survival
time (days)
Free BCNU Lipo-BCNU Saline
20 700
11.12.12,12.12.12,13.13,13,14 14.14.14.14.14.14.15,15,15.16 8,8,8.8.8,8,X.Y,Y.Y
Mean + SD (day)
12.4 t 0.8 1* * I45 8.3 f2 0.67 0.46
1 *
% increase
4Y.4 74.7
CDF, mice (10 animals per group) wcrc inoculated intrapcritoneally with 10’ L1210 cells on day 0 and were subsequently treated intravenously on day 2. The percent increase of survival (% increase) was determined as the median survival time of treated animals divided bv the median survival time of control animals. * P
3.3. Intermittent udministration study
Intermittent treatment with either BCNU or lipo-BCNU on days 2, 9 and 16 after tumor injection was attempted to increase the efficacy. Treatment with BCNU alone significantly prolonged the survival by 36.5% at a dose of 10 mgikg, by 97.2% at 20 mglkg and by 400.6% at 30 mg/kg, indicating that intermittent treatment was superior to consecutive treatment (Table 3). In the high-dose group, 4 out of 10 mice survived longer than 60 days after tumor cell injection and were regarded as cured. Treatment with lipo-BCNU resulted in a significantly longer survival time than the corresponding dose of BCNU alone. Treatment with lipo-BCNU at 10 mg/kg prolonged the survival time by 65.6% as compared with the LM controls, while 20 mglkg achieved prolongation by 412% and 30 mg/ kg did so by 626.9%. In both the medium- and high-dose lipo-BCNU groups, 4 of 10 and 9 of 10 mice, respectively, survived for longer than 60 days. These survival rates indi-
Table 3 Effect ot intermittent
treatment
with free BCNU
or Lipo-BCNU
Preparation
Drug dose (mgikg)
Survival
Saline Free BCNLJ
0 IO 20 30 0 10 20 30
7.8,8.8,8,8.8.8,8,8.8 9,10.10,10.10,10,10.10.11.14,15 13,13.13,15,16,16.l6,lh,lX.20 21,25,26.27,28,29 8,8,8,8,8,8.8,8,8.X,9 12.12.12,13,13,13.14.14,14.l4 27,27.27.28,28,28 35
LM Lipo KNlJ
time (day)
cated that intermittent lipo-BCNU treatment had a significantly better antitumor activity aginst Ll210 leukemia. Besides increasing the survival time, the number of 60-day survivors was significantly greater in the lipo-BCNU-treated groups of mice. The 60-day survivors were killed and the body and spleen weights were measured along with determination of the white blood cell (WBC) count and hemoglobin (Hb) concentration which were determined by using a PC-607 particle counter (Erma, Tokyo, Japan). The body weight of the survivors recovered faster in the lipo-BCNUtreated groups than in the free BCNU groups. When BCNU was delivered in the saline vehicles, weight gain was retarded and the minimum was reached on day 20. The mean body weight of survivors on day 60 was 24.8 g (Table 4). while that of survivors treated with lipoBCNU was 27.8 g. In addition, the ratio of spleen weight to body weight and the WBC count of survivors from the free BCNU groups were higher than those of animals treated with lipo-
on the lifespan
of L1210 tumor-bearing
Mean -t SD (day) 7.Yl I? 0.29 10.x + I.80 IS.6+ 2.15 I .>39.6 -r 16.7 8.09 t 0.29 13.1 kO.83 >40.5 + 1S.Y BS7.7 i- 7.Y
% ILS
mice
Survivors/tested
on day 60
0111 5:
~400.6 6.5.6 36.5 97.2 2
0110 Oil1 4110 o/11 oi 10
>412.0 >62h.Y
4110 9/ 10
CDF, mice were inoculated intraperitoneally with L1210 cells on day 0 were subsequently treated i.v. on days 2.9 and 16. The percent increase of survival time was determined as the median survival time of treated animals/the median survival time of control animals. * P
M. Takenaga Table 4 Body weight,
spleen
weight,
WBC count
Group
Dose
I Advanced Drug Delivery Reviews 20 (1996) 209-219
and Hb in L1210-bearing n
(mg/kg) Day 0 Ll210-bearing mice Survivors on day 60 Free BCNU Lipo-BCNU i4-week-old Values
normal
_
123
30 20 30 -
mice
4 4 9 5
mice on days 0 and 60
Body weight
Spleen weight
(9)
(mg)
22.6 ? 0.47
_
24.8 28.1 27.8 28.4
124.2 74.2 78.3 71.2
5 * -’ 2
213
0.34 1.22 1.15 0.23
WBC count ( x 1021~1)
69.4 t 9.12 2 ? ? 2
55 12.6 10.2 3.96
227.5 82.8 71.4 57 t
? 187.2 2 17.6 2 11.8 17.5
Hb concentration (g/d])
16.5 2 1.21 14.4 16.4 16.4 17.1
t 2.3 t 0.1 ? 0.69 -+ 0.74
are mean f SD.
BCNU. Furthermore, the free BCNU survivors showed an increased WBC count and a decreased Hb concentration. The mean body weight of normal 14-week-old mice was 28.4 _’ 0.23 g, while the spleen weight was 71.2 2 3.96 mg, the WBC count was 57 2 17.5/1_~1and the Hb was 17.1 ? 0.74 g/d1 (n = 5, mean 2 SD). These values were similar to those found in lipoBCNU-treated survivors.
4. Toxicity of lipo-BCNU The similar normal mittent
toxicity of lipo-BCNU appeared to be or more severe than that of BCNU in male CDF, mice (Table 5). When interintravenous injection was performed, the
Table 5 Toxicity of free BCNU Preparation
and Lipo-BCNU
in normal
LD,, values of lipo-BCNU and BCNU calculated by Behrens’ method were 71 mg/kg and 73.5 mg/kg, respectively. Three doses of 10 or 30 mg/kg of lipo-BCNU had little effect over 60 days despite transient weight loss after drug treatment. However, the mice given 60 mg/kg all died within 14 days after showing marked weight loss. After treatment at 90 mg/kg, 7 mice died within 7 days and the last one died on day 8. As compared to lipo-BCNU, free BCNU appeared to be somewhat less toxic. Three doses of 10 or 30 mg/kg of free BCNU also had little effect over 60 days. However, with treatment at 60 mg/kg, 4 out of 8 mice died within 14 days and the others died by day 17. After treatment at 90 mglkg, 4 mice died by day 7 but the others remained alive until days 9-10. After a single
mice
Drug dose
Total dose
Survival
(mgikg)
(mg/kg)
60 days
Mean t SD
Saline Free BCNIJ
0 10 30 60 90
_ 30 90 a)* b)*
8/B 8/B O/8 O/8
60> 60> 14.6 t 1.32 7.63 ? 2.0
Lipo BCNU
10 30 60 90
30 90 c)* d)*
8/B 818 018 O/8
60> 60> 13.1 i 1.27 6.6 + 0.99
Normal CDF, mice (n = 8) were treated intravenously (BWD) data are expressed as percentages. * a) 180 mgikg (n = 4). others 120 mg/kg. b) 90 mgikg mgikg (n = 1).
time (day)
with drugs
BWD(%) Day 3
Day 14
Day 26
Day 60
1.7 0.9 4.3 7.2 8.1
+ 6.5 + 4.0 - 2.6 -21.1 ~27.3
+9.5 +8.5 -6.0 -40.8 -
+15.2 t13.7 - 5.6 -.
+29.0 +27.0 + 15.8 _ _
+ 0.9 - 3.1 -11.4 - 13.4
+ 5.1 ~ 2.2 -26.5 -26.8
i7.3 -3.5 ~27.7 -
113.4 --3.5
+24.7 + 19.6 _ _
+ ~ ~ -
three
(n = 4), others
Day 7
times
180 mglkg.
(days
0,7 and 14). The body
c) 120 mglkg.
d) 90 mgikg
weight
differences
t’n = 7). others
180
214
M. Takenaga
i
Advanced
Drug
dose of BCNU, mice given 90 mgikg died on days 5-14 (7.83 2 3.24 days, mean ? SD).
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20 (1996)
209-219
treated survivors. as was the case with lipoBCNU-treated survivors.
6. In vitro uptake of LM by tumor cells 5. In vivo antitumor nanospheres
activity of BCNU in lipid
The antitumor activity of BCNU incorporated into lipid nanospheres (LN), a finer lipid emulsion with an average size of 50 nm. was also examined since smaller liposomes have been reported to accumulate better in tumors [6.14]. The size of LN varied according to the homogenizing conditions and the lecithin content while the stability was similar to that of lipoBCNU. Consecutive treatments with BCNU in LN (slipo-BCNU) also resulted in a significantly longer survival time than the corresponding dose of BCNU in saline. Treatment at both 3 mg/kg and 10 mg/kg produced a significant extension of survival time by 28% and 163%. respectively, as compared with LN controls. In particular, even 3 mg/kg of s-lipo-BCNU prolonged the survival time significantly compared to free BCNU while lipo-BCNU did not, although the difference between the two emulsions was not statistically significant. Also, a single dose of s-lipo-BCNU (20 mg/kg) prolonged the survival time by 84.3%, which was slightly superior to the effect of lipo-BCNU. Intermittent treatment with s-lipo-BCNU also resulted in a significantly longer survival time than that of the corresponding free BCNU group. Administration of s-lipo-BCNU at 10 mgi kg prolonged the survival time by 61.8%, while the increase was 327.3% at 20 mglkg and 626.9% at 30 mgikg. 2 and 9 mice out of the 10 animals. each in the medium- and high-dose groups receiving s-lipo-BCNU. survived for longer than 60 days. The respective mean body weight of survivors treated with medium and high doses of s-lipo-BCNU was 28.9 g and 28.1 g. The ratio of spleen weight to body weight and the WBC count of the s-lipo-BCNU-treated survivors were lower than those of animals given free BCNU. while the Hb was higher. All these parameters were within the normal range in s-lipo-BCNU-
In vitro uptake of LM by tumor cells was studied. [ “C]Triolein was used as a tracer of LM, since it is a lipophilic triglyceride. LM preparations labeled with [ “C]triolein (NEN Research Products. Beverly, MA, USA) were incubated with several kinds of tumor cells (lO’/ml) including L1210 cells. After 4 h of incubation at 37°C the LM uptake by the tumor cells was determined from the radioactivity incorporated. The results are shown in Table 6. Compared to free [ “C]triolein, a 3-fold larger amount of [ “C]triolein incorporated into LM consisting of phosphatidylinositol, phosphatidylserine or phosphatidylcholine (lip0 [‘4C]triolein was taken up by L1210 cells. Data in the table are for standard lipid emulsions with an average particle diameter of 200 nm. In contrast, smaller LM of about 140 nm showed only one tenth of the uptake obtained with the standared LM preparations. The other tumor cells also showed a higher uptake of lipo [r4C]triolein than of free [‘“Cltriolein. Although smaller LM composed of phosphatidylcholine were taken up by MM46 -1‘ahle
h
Influence of phosphohpid composition and size on the uptake of lipid microspheres into tumor cells Phospholipid
PI PS PC S-PC” Free
Tumor
cells
L 1210
MM46
Sarcoma
1.118 0.897 1.102 0.136 0.33 1
2.766 1.864 1.127 1.021 0.958
1.045 0.713 1.030 0.856 0.487
180
P388 0.663 0.399 1.005 0.167 0.304
PI. phosphatidylinositol: P.S. phosphatidylserine; PC, phosphatidylcholine, Lipid microspheres, incorporating [“Cltriolein were incubated with tumor cells (IO’ cells/ml) for J h at 37°C and washed, after which the radioactivity was measured. Values arc the mean percentage of tumor cell radioactivity uptake (n = 2). ” The average diameter of the small microspheres composed of PC (s-PC) was 140 nm. while that of the other microspheres was 200 nm.
M. Takenaga
I Advanced Drug Delivery Reviews 20 (1996) 209-219
cells at much the same rate as the standard LM, uptake of small LM by the other tumor cells was reduced.
7. In vivo distribution
of LM
7.1. [‘JC]Triolein-containing
LM
The effects of vesicle size and lipid composition on the in vivo distribution of LM were studied. LM preparations incorporating [ “C]triolein were injected into C,H/He mice (pretreated with 10” MM46 tumor cells subcutaneously into the flank), via the tail vein in a volume of 0.2 ml (about 2 x lo6 dpm). At appropriate times, blood was slowly collected from the heart into a heparized syringe and used for the determination of radioactivity. After each mouse was killed by
0
0.250.5
Hours
after
1
2
215
cervical dislocation, the animal was perfused with saline. Subsequently, the tumor, the liver and other tissues were removed and a sample of each tissue (100 mg) was solubilized with 1 ml of tissue solubilizer (Soluene-350, Packard Company Meriden, CT, USA) and isopropyl alcohol, followed by the addition of hydrogen peroxide. The radioactivity of the tissue solution was then determined in a liquid scintillation counter. Fig. 3a shows the tumor uptake of LM. Standard LM with a particle size of 200 nm were delivered to the tumors up to 0.5 h after injection. All LM preparations were retained by the tumors. Smaller LM (phosphatidylcholine with a particle size of 140 nm) accumulated at tumor sites in a similar manner to standard LM up to 4 h after injection. At 24 h after injection, however, 0.969% of the injected dose of smaller LM was retained compared to 0.632% of the standard LM dose (data not shown). Therefore,
0
4
0.250.5
Hours
injection
1
after
2
4
injection
:
01s /
0
0.250.5
Hours
after
1
2
0
4
Hours
injection
Fig. 3. Pharmacodynamics of several kinds of LM. Male C,H/He mice were after tumor inoculation, several kinds of LM incorporating [‘4C]triolein (2 times, tumor (a), blood (b), liver (c) and spleen (d) samples were harvested are expressed as percent injected dose per gram or milliliter of the specimen (0). phosphatidylserine (A) and phosphatidylcholine (0). The average phosphatidylcholine (0) was 140 nm. while that of the other microspheres
I
0.250.5 after
I
1
I
2
4
injection
subcutanously injected with 10” MM46 cells. 14 days 10’ dpm) were given intravenously. At appropriate and used for the determination of radioactivity. Data (n = 3). LM were prepared using phosphatidylinositol diameter of small lipid microspheres composed of was 200 nm.
X
216
M. Takenaga I Advanced
Drug Delivery Rewews 20 (1996) 209-219
smaller LM seems to be retained better at the tumor site. LM, prepared from soybean phosphatidylinositol showed the maximum tumor localization at 2 h after injection (1.871% of the injected dose per gram of tumor). LM composed of phosphatidylserine showed less than one half of the tumor uptake obtained with LM composed of phosphatidylinositol. As shown in Fig. 3b. elimination from the blood seemed to be influenced by LM size and smaller particles disappeared more slowly compared to standard LM with an average size of 200 nm. LM showed marked accumulation in the liver (Fig. 3~). In particular, LM composed of phosphatidylinositol showed 2-fold more accumulation than LM composed of phosphatidylcholine. LM with a smaller particle size showed a tendency not to be trapped in the liver. Smaller LM showed much less uptake by the spleen (Fig. 3d). In contrast to small sized LM. the standard LM were predominantly localized to the spleen. 7.2. CDDP-D-contuining
LM
Another LM distribution study was carried out using an antitumor agent, cisplatin derivative (CDDP-D) which is much more lipophilic than cisplatin itself. Intravenous injection of 4 X 10” L5178Y-ML cells (kindly donated by Dr. Okura, Banyu Pharmaceutical, Tsukuba, Japan) induced liver metastasis [16]. Eight days after tumor inoculation, an LM preparation incorporating CDDP-D (4 mg/ kg, as cisplatin) was given intravenously to tumor-bearing or tumor-free mice. At 10 min and 30 min after treatment. the amount of cisplatin in liver and plasma was determined and compared with the values after treatment with free cisplatin (4 mg/kg). The results are illustrated in Fig. 4. In normal mice given free cisplatin. the amount of cisplatin in the liver was 0.394 _’ 0.042 pug/g (mean ? SD) at 10 min and 0.490 ? 0.001 @g/g at 30 min (Fig. 4a), while the respective amount of cisplatin in lipo CDDP-D-treated mice was 0.879 2 0.084 ,ug/g and 0.965 ? 0.13 ,uglg. LM are well known
(b)
0
10 20 30 MIII after b”,ectlon
40
II
IO 20 30 Mln after m,ect,on
40
Fig. 4. Distribution of LM-encapsulated antitumor agents. Female CDF, mice were intravenously injected with 4 X 10’ L5 17XY-ML cells. Eight days after tumor maculation. an LM preparation incorporating a cisplatin CDDP derivative (CDDP-D) (0) or free cisplatin (0) were given intravenously. At 10 or 30 mitt after treatment. the cisplatin level in liver or plasma was determined by inductively coupled plasma (ICP) method. Lipo CDDP-D (0) and free cisplatin (0) were also given to tumor-free mice. The cisplatin content is expressed as pgip liver (a) or fig/ml plasma (b). Mean k SD (n = 3).
to be extensively taken up by the reticuloendothelial system in the liver. Therefore, lipo CDDP-D was preferentially delivered to the liver and retained there, with the hepatic CDDP concentration being twice that after treatment with free cisplatin. Tumor-bearing mice had metastatic nodules in the liver. as confirmed by hematoxylin-eosin staining. After free cisplatin treatment, hepatic uptake was about twice that observed in normal mice. Treatment with lipo CDDP-D achieved the highest hepatic cisplatin levels which were 0.965 + 0.131 pg/g at 10 min and 1.173 -t 0.117 ,ug/g at 30 min. These results indicated that the delivery of cisplatin to the liver including the metastases was increased by encapsulation in LM. The pharmacodynamics of free cisplatin in plasma were quite different from those of LMencapsulated CDDP-D (Fig. 4b). Free cisplatin disappeared very rapidly from the blood in both normal and tumor-bearing mice. However, treatment with LM-encapsulated CDDP-D led to much higher plasma levels of cisplatin, indicating that the LM-encapsulated agent was retained in the plasma.
M. Takenaga I Advanced
Drug Delivery Reviews 20 (1996) 209-219
8. Conclusions
These studies showed that lipo-BCNU not only had increased activity against L1210 leukemia in mice but also had reduced toxicity, and the potency of lipo-BCNU was much greater than that of the equivalent dose of free BCNU. The best response was achieved by intermittent treatment with lipo-BCNU. Besides increasing the survival time, the number of 60-day survivors was significantly greater in the lipoBCNU-treated mice. Also, the body weight of the survivors recovered faster in lipo-BCNUtreated groups than in the free BCNU-treated groups which showed splenomegaly, an increased WBC count, and a decreased Hb concentration. Splenomegaly might have reflected the recurrence of leukemia in the free BCNU-treated survivors. In contrast, the weight gain and spleen:body weight ratio were both within the normal range in lipo-BCNU-treated survivors. All of these findings indicate that intermittent administration of lipo-BCNU was superior for the treatment of L1210 leukemia in mice. This improvement may be partly explained by the modification of BCNU distribution induced by the LM drug carrier. Previously, we have shown that LM preferentially accumulates in inflamed tissues and vascular lesions in vivo [15,17,18]. Tumors always require a high energy supply and would actively take up LM particles. Fusion of LM with lipoprotein particles may also take place in vivo [19-211. We have already demonstrated that LM are readily taken up by tumor cells in vitro [22]. The present studies also showed that larger amounts of [14C]trioleinlabeled LM were taken up by tumor cells including L1210 cells when compared to free [‘4C]triolein. It 1s . well known that oil such as lipiodol is retained selectively in tumor tissues [23] and our in vivo study also demonstrated that an LM-encapsulated cisplatin derivative (CDDPD) was preferentially localized at the liver including the metastases. Accordingly, the improved therapeutic efficacy of lipo-BCNU would be related to increased uptake and longer retention of BCNU in tumor sites. The toxicity of lipo-BCNU appeared to be the
211
same as that of free BCNU in normal mice. This may have been because lipo-BCNU is retained in the tissues and has a slow rate of clearance. In tumor-bearing mice, however, lipo-BCNU appeared to be retained preferentially in the tumor compartment. The tissue distribution of liposomes is considered to be influenced by both vesicle size and lipid composition [6,14]. Although there was no significant improvement, the lipid emulsion with an average diameter of 50 nm (s-lipo-BCNU) tended to show a higher antitumor activity in vivo. Reducing the size of the LM decreased in vitro incorporation by tumor cells although in vivo tumor accumulation was satisfactory. In addition, smaller LM were retained longer in plasma so antitumor agents would exert more activity against solid tumors. The balance between these factors that variously increase and decrease antitumor activity would account for the lack of a significant improvement when slipo-BCNU was used. A number of reports have shown that the lipid components of LM can also influence antitumor activity or can exihibit such activity themselves [24-291. We found that LM composed of plant phosphatidylinositol showed the highest uptake by tumor cells in vitro and the in vivo study also indicated that such LM had the highest uptake by MM46 tumors. However, the antitumor activity of lipo-BCNU prepared using plant phosphatidylinositol may be almost the same as that of standard lipo-BCNU prepared with egg lecithin (data not shown). Thus, plant phosphatidylinositol may only be active against certain types of tumors. However, LM prepared from other glyceryl ether phospholipids or from eicosapentadecanoic acid, which has been reported to have antitumor activity [28], may be worth investigating for their targeting ability and/or antitumor activity. BCNU has been reported to decompose at relatively rapid rates under physiological conditions without the need for enzymatic attack [30] and a solution in which BCNU is no longer detectable by HPLC, exhibited no antitumor activity (data not shown). When lipo-BCNU was stored at 4°C however, the BCNU content
218
M.
Takrnaga
I Advancrd
Drug
remained at almost 100% for 2 weeks, indicating that lipo-BCNU is stable at 4°C. This may be partly because lecithin surrounds and protects the oil phase containing BCNU, since it was confirmed that BCNU remained intact in soybean oil stored at more than 37°C as determined by HPLC (data not shown). In contrast, lipo-BCNU seemed to leak out from LM and be destroyed in the aqueous phase when incubated in serum at 37°C and the residual content of BCNU was less than 10%. We have previously found [31] that the amount of prostaglandin E, retained in LM after incubation with human serum was only 0.5%. Despite this. LM containing prostaglandin E, have a potent clinical activity [32]. In the case of lipo-BCNU, the LM may be taken up by tumor sites before breakdown and the release of BCNU can occur. Liposome encapsulated BCNU has already been reported to exhibit a high antitumor activity against L1210 leukemia and Ehrlich ascites [33]. Also, Inaba et al. [34] have already demonstrated that liposomal MeCCNU, a nitrosourea antitumor agent, has a greater efficacy compared with free MeCCNU. However. compared to liposomes, LM are superior in terms of stability and safety. Therefore. LM (and LN) may be able to serve as useful carriers for other lipophilic agents besides BCNU [35].
Delivrr_v
20 (IYYh)
209%21Y
rinc MC’A-38 hepatic 2132.
IhI
metastasis.
Cancer
Res. 51, 21277
Mayer, L.D., Tai, L.C.L.. Ko. D.S.CJ.. Masin, D., Ginsberg. R.S., Cullis. P.R. and Bailey, M.B. (1989) Infuence of vesicle size, lipid composition and drug-tolipid ratio on the biological activity of liposomal doxorubicin in mice. Cancer Res. 49. 5922-5930.
I71 Maruyama,
K., Kennel, S.J. and Huang, L. (1990) Lipid composition is important for highly efficient target binding and retention of immunoliposomes. Proc. Nat]. Acad. Sci. USA X7. 574445748.
1x1 Dinney.
C.P.N.. Utsugi. T.. Fidler, I.J., von Eschenhach. A.(‘. and Killion. J.J. (1992) Immunotherapy of murine renal adenocarcinoma by systemic administration of liposomes containing the synthetic macrophage activators GCP 31362 or CGP lY853A in combination with intcrleukin-2 or gammma-interferon. Cancer Res. 52. 1155~1161.
101Bono. studies 707.
Il’Jl
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[27] Figiwara, K., Modest, E.J., Welander, C.E. and Wallen, C.A. (1989) Cytotoxic interactions of heat and ether lipid analogues in human ovarian carcinoma cells. Cancer Res. 49. 6285-6289. [28] Beck, S.A., Smith, K.L. and Tisdale. M.J. ( 1991) Anticachectic and antitumor effect of eicosapentadecanoic acid and its effect on protem turnover. Cancer Res. 51. 6089-6093. [29] Jett. M., Chudzik, J., Alving. CR. and Stanacev, N.Z. (1985) Metabolic fate of liposomal phosphatidylinositol in murine tumor cells: implications for the mechanism of tumor cell cytotoxicity. Cancer Res. 45, 4810-481.5. [30] Carter. S.K., Shabel, F.M., Broder, L.E. and Johnston. T.P. (1972) 1,3-Bis(2-chloroethyI)-I-nitrosourea (BCNU) and other nitrosoureas in cancer treatment: a review. Adv. Cancer Res. 16. 273-332. [31] Igarashi, R., Mizushima, Y., Takenaga. M., Matsumoto, K.. Morizawa, Y. and Yasuda. A. (1992) A stable PGE, prodrug for targeting therapy. J. Control. Rel. 20,37-46. [32] Hoshi. K.. Mizushima. Y., Kiyokawa. S. and Yanagawa. A. (1986) Prostaglandin E, incorporated in lipid microspheres in the treatment of peripheral vascular diseases and diabetic neuropathy. Drug Exp. Clin. Res. 12. 68l685. [33] Rutman, R.J., Ritter. CA., Avadhani. N.G. and Hansel, J. (1976) Liposomal potentiation of the antitumor activity of alkylating drugs. Cancer Treat. Rep. 60, 617-618. [34] Inaba, M., Yoshida, N. and Tsukagoshi, S. (1981) Preferential action of liposome-entrapped I-(2chloroethyl).3. (4.methylcyclohexyl)-I-nitrosourea on lung metastasis of Lewis lung carcinoma as compared with the free drug. Gann 72, 341-345. [35] Takenaga, M., Igarashi, R., Tsuji, H. and Mizushima. Y. (1993) Enhanced antitumor activity and reduced toxicity of 1,3-bis-2-chloroethyl-1-nitrosourea administered in lipid microspheres to tumor-bearing mice. Jpn. J. Cancer Res. 84, 1078-1085.