Europ. J. Cancer Vol. 11, pp. 883-889. Pergamon Press 1975. Printed in Great Britain
Liposomes Containing H-Actinomycin D. Differential Tissue Distribution / Varying the Mode of Drug Incorporation* YUEH-ERH RAHMAN, WALTER E. KISIELESKI, EVELYN M. BUESS and ELIZABETH A. CERNY Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439 Abstract--Actinomycin D was encapsulated within liposomes in two different ways. When the drug was incorporated in the lipid phase, as p,~rt of the membrane bi-layers of the liposomes, they were called "lipid phase liposomes" (LPL) ; when the drug was incorporated in the aqueous solution in the center and between the lipid bi-layers of the liposomes, they were called "aqueous phase liposomes" (APL). Distributions of 3Hactinomycin D in tissues of mice were determined from 15 minutes to 48 hours after a single intravenous injection of either LPL, APL, or nonencapsulated actinomycin D. Marked differences in tissue distribution were shown. 3H-actinomycin D incorporated in LPL showed high concentrations in the lungs and low concentrations in the intestinal wall; whereas the reverse was found with APL. The spleen and bone marrow of mice receiving LPL showed an increase in 3H-rudioactivity between 3 and 24 hr after injection that was closely correlated with a concomitant decrease in activity in the liver and lungs. Mice receiving either APL or nonencapsulated actinomycin D had higher levels of the drug in the blood, kidneys and intestinal wall than did mice receiving LPL. In vitro studies showed that 3H-actinomycin D leaked out from APL significantly faster than that from LPL. We have demonstrated that the tissue distribution of a drug can be modified not only by liposome encapsulation, but also by varying the way of incorporating the drug within liposomes, and thereby altering the surface properties. The two forms of incorporating drugs within liposomes are potentially useful to direct antitumor agents to specific tumor bearing tissues.
INTRODUCTION
tion of antitumor agents by liposome encapsulation. Actinomycin D, an antibiotic with known inhibitory effects on certain tumors [3-6], has been encapsulated within liposomes. Liposomeencapsulated actinomycin D, when given either i.v. or i.p. to mice, was found to be much less toxic than the nonencapsulated, free form [7]. It was also shown that mice inoculated with Ehtlich ascites tumor cells survived significantly longer when given single or multiple injections ofliposome-encapsulated actinomycin D [7]. Since actinomycin D is soluble both in aqueous media and in lipid solvents, it can be incorporated in either the aqueous or the lipid phase of the liposomes. In the first case, in
MOST antitumor agents are highly toxic to animals. This toxicity could be alleviated if selected tissue distribution of the agent could be obtained. In earlier studies with chelating agents encapsulated within liposomes, i.e., multi-layered lipid spherules, we found that partial selective tissue uptake of the chelating agent can be obtained, depending on the lipid constituents of the liposomes [1, 2]. Therefore, we have attempted to direct the tissue deposiAccepted 29 August 1975. *Work supported by the U.S. Energy Research and Development Administration. 883
884
7ueh-Erh Rahman, Walter E. Kisieleski, Evelyn M. Buess and Elizabeth A. Cerny
which the actinomycin D is in the aqueous solution in the center and between the lipid bi-layers of the liposomes, the liposomes are designated "aqueous phase liposomes" (APL). When the actinomycin D is incorporated as part of the lipid bi-layers [8] which form the outer shells of the liposomes, they are called "lipid phase liposomes" (LPL). These two kinds of liposomes containing actinomycin D are different in their surface properties. The present report concerns a comparative study of APL, LPL and non-encapsulated actinomycin D, with emphasis on their pattern of distribution in various mouse tisses after i.v. injections.
MATERIAL AND METHODS
Phosphatidylcholine (PC) and cholesterol were respectively obtained from Sigma Chem. Co., St. Louis, Mo. and Applied Sciences Laboratories, Inc., State College, Pa. Actinomycin D (Cosmegen) was from Merck, Sharp and Dohme, West Point, Pa. Radioactive 3H-actinomycin D, with a specific activity of 3.0Ci/mmole, was from Amersham/Searle Corp., Arlington Hts., Ill.
Preparation of actinomycin D liposomes APL were prepared with a mixture of 3.0 mg of PC and 3.0 mg of cholesterol dissolved in chloroform and dried in a thin film inside of a round bottom flask. Actinomycin D, dissolved in 8 m M CaC12 at a concentration of 0.5 mg in 1 ml, was slowly added to the dried film of lecithin-cholesterol mixture with immediate and constant stirring. LPL were prepared by dissolving a mixture of 4-0 mg of PC, 2"0 mg of cholesterol and 0.5 mg of actinomycin D in chloroform. After being dried to a deep yellow film, an 8 m M CaCI2 solution was then slowly added, and liposomes were formed by immediate stirring. The subsequent steps in the preparation of both forms of liposomes are essentially the same as described previously [1, 9]. For both APL and LPL, a suitable amount of radioactive 3H-actinomycin D was added to the actinomycin D solution so that each mouse received about 2.0 x 10 s dis/rain of radioactivity together with a nonlethal dose of actinomycin D of approximately 350/tg/kg. We should emphasize that high purity of PC is essential for successful liposome preparations. Liposomes made of either partially degraded or oxidized PC tend to aggregate into small clumps that render the preparations unsuitable for injection into animals [1].
Injection of 3H-actinomycin D in liposome-encapsulated or nonencapsulatedforms Groups of 24 female Carworth Farm (CF 1) mice, 3 months of age, and weighing between 25-30g were used. 3H-actinomycin D was given in one of the following forms: (1) actinomycin D in nonencapsulated, free form; (2) actinomycin D in APL; (3) actinomycin D in LPL. Each mouse received a single intravenous injection of 3H-actinomycin D in a volume of 0.40 ml; and 8 m M CaC12 solution, was used as injection vehicle for all the three forms of actinomycin D. Three groups of 4 mice each were killed at the following time intervals after the injections: 15 rain, 0.5, 1, 3, 6, 24 and 48 hr. Mice were killed by an intravenous injection of an anesthetic dose of sodium pentobarbital (0.04 ml/mouse), followed by exsanguination. Samples of blood and of various tissues were removed immediately for 3Hradioactivity determinations.
Preparation of mouse tissues Blood samples, lungs, spleen, liver, kidneys, intestinal samples and a standard bone marrow sample from both tibias [10] were taken for 3H analysis. At 15 min after injection, the following additional tissues were also taken for analysis: thymus, brain, urinary bladder including the urine, and sample of muscle and skin. Blood from a jugular vein was collected with a capillary micropipette. Duplicate samples of 100 ~1 each were absorbed into a small ball of cotton and allowed to air dry before being wrapped in a filter paper (Whatman No. 1, 4.25 cm dia). After removal, the spleen, lungs, brain, thymus, urinary bladder, each kidney, samples of muscle and skin each weighing between 100 and 150mg were removed, weighed, wrapped in a filter paper, and air dried. And three 150-200 mg slices of liver were also wrapped separately for analysis. The total liver radioactivity was calculated as the mean counts per mg from the slices, times the total liver weight. The slices were always taken from the standard locations which preliminary determinations had shown to be representative of the whole. Three segments of the small intestine were excised and weighed after the contents had been extruded. Each segment was wrapped separately in a filter paper for radioactivity analysis. The bone marrow samples were washed out with 0.25 ml of water directly onto a small ball of cotton and then treated in the same manner as that described for the blood samples. The amount of 3H-radioactivity in the total marrow was estimated by multiplying the activity in the two tibial segments by a
Liposomes Containing 3H-Actinomycin D factor of 44 [11]. The absorbent paper used to collect urine samples was cut out, folded, and then wrapped in filter paper. The radioactivity in the total blood was calculated from two 100/~1 samples and the estimated mouse blood volume of 2 cm 3.
Analysis of 3H-radioactivity Each air dried biological sample, all wrapped in Whatman No. 1 filter paper, was pressed in a pellet press (Parr Instruments Co., Moline, Ill.). After automatic combustion in a Packard 305 sample oxidizer and addition of scintillation fluid, each sample was analysed for 3Hradioactivity in a Beckman liquid scintillation counter, Model LS-200B. The oxidation of each tissue sample was followed at least once by combustion of an empty filter paper to remove the residual activity in the system. In the case of tissues known to contain high levels of radioactivity, several successive empty filter papers were oxidized. The radioactivity obtained from these filter papers was added to that of the preceding tissue sample. Calculation of the amount of radioactivity injected was based on analysis of triplicate samples of 0.40 ml, measured with the same syringe that was used for injection. These samples were prepared for radioanalysis in the same manner as the blood and bone marrow. In vitro studies of release of 3H-actinomydn D from APL and LPL Preparations of APL or LPL containing 3H-actinomycin D, suspended either in 8 m M CaC12 or in culture medium (Eagle's medium containing 15% foetal calf serum), were incubated at 37°C in a water bath, with constant slow shaking. One ml aliquots from each preparation were then taken at hourly intervals. They were centrifuged in a Sorvall GLC-1 centrifuge at 2000rev/min for 5 min. The release of 3H-actinomycin D from the liposome was calculated from the 3H-radioactivity found in the supernatant compared to the total radioactivity in the sample before centrifugation. RESULTS
3H-actinomycin D distribution in mousetissues Figure 1 shows the distribution in blood, lungs, spleen, liver, kidneys, and bone marrow of 3H-actinomycin D, administered either in LPL, APL, or in nonencapsulated form. The disappearance of 3H-actinomycin D from the blood was very rapid (Fig. l(a)). Half of an hour after injection, the highest blood level
885
(almost 2% of the injected radioactivity) was found in APL-injected mice, and the lowest level (about 0.5%) was found in the LPLinjected mice. A small but significant rise in the blood level of 3H-actinomycin D in LPLinjected mice occurred between 24 and 48 hr after the injection (Fig. 1(a)). There was a striking difference in the lung uptake of the different forms of 3H-actinomycin D (Fig. l(b)). In mice receiving the LPL injection, about 40% of the total injected dose was found in the lungs half of an hour after the injection, and the high concentration was maintained at least up to 6 hr; 24 hr after the injection, 10% of the total injected radioactivity was still retained in the lungs. In contrast, mice injected either with APL or with nonencapsulated 3H-actinomycin D had very low concentrations of the drug in the lungs (Fig. l(b)), the mice receiving the nonencapsulated form consistently had the lowest levels. The initial uptake of 3H-actinomycin D in the spleen was low in all three groups of mice. However, the spleens of LPL-injected mice showed a significant increase in 3H-actinomycin D concentration at 3 hr and at 24 hr reached a peak concentration of about 20% of the total injected dose. The groups of mice receiving APL or free actinomycin D had very low concentrations of the drug in the spleen throughout the 48 hr after the injection. The LPL-injected mice also had a higher uptake of 3H-actinomycin D in the liver than mice injected either with APL or free actinomycin D (Fig. 1(d)). In LPL-injected mice, the level of the drug in the liver was maintained at least up to 6 hr, but then decreased. In the kidneys the highest concentration of 3H-actinomycin D occurred in mice injected with APL or free actinomycin D. The peak concentration was found at one half of an hour. The LPL-injected mice had very low concentrations of 3H-actinomycin D in the kidneys throughout the experimental period (Fig. 1(e)). Although the initial uptake of 3H-actinomycin D in bone marrow of all three groups of mice was similar, the uptake in the LPLinjected mice continued to increase for 24 hr reaching a peak concentration of about 10%. In free actinomycin D and LPL-injected mice, there was a significant decrease of radioactivity in the bone marrow between 24 and 48 hr; while a constant level was maintained in APLinjected mice (Fig. 1 (f)). In the intestinal wall, mice injected either with APL or with nonencapsulated actinomycin D have an initial concentration (one-
886
Yueh-Erh Rahman, Walter E. Kisieleski, Evelyn M. Buess and Elizabeth A. Cerny d Liver Liver
Blood
a
~
4O
I~. ~ "X
.. . . . .
30 I
o
F~E,~"r.
LPL
--- APL FREEACT,D
2c IC I
I '
I
I
I
II
J
II
I
C
'
'
'
I~ ......~ - - ~
e
E
i
~""~"~
Lungs - - LPL
-- 30
~.~
Kidneys
~ LPL . . . . APL ........ FREEACT.D
5
___ APL
[\~%,
4
r x,,...~.
3 2 I0
T
I
f LPL
--
/
- - - APL
0
I
6
?..... ] ~ : . L
~
i/
5
II
Bone Morrow
Spleen 20
I
NI
24
,:: Z 48
G 0
I
I
.I 3
N I
6
I~
24
~/" " - - - 48
TIME AFTER INJECTION, hours
Fig. 1. Percentinjecteddose of 3H-actinomycin D in : (a) blood, (b) lungs, (c) spleen, (d) liver, (e) kidneys and (f) bone marrow. A single intravenous injection of one of the following: LPL (lipid phase liposomes), APL (aqueousphase liposomes), or free act. D was given to each mouse. The radioactivity in the blood (total) was calculatedfrom two I00 #l samples and the estimated mouse blood volume of 2 cm3. The level in the bone marrow (total) was estimated by multiplying the radioactivity in the two tibial segments by a factor 0f44 [1 I]. Each point is the mean value from 4 mice. Vertical bars represent 2 standard errors of the mean.
half to one hour) about five-times that in LPL-
injected mice (Fig. 2). The radioactivity then decreases so that between 24 and 48 hr post injection, the levels of radioactivity in all three groups of mice are not significantly different (Fig. 2). .i
'l
t~
il--
I
I~
GUT WALL
Table 1. Tissue content of 3H-actinomycin D ( % injected dose, 15 min after injection) The data represent the averages obtained from four animals in each group
f] X " a =~
I{ f_;
k',,.
A?L
~.. ",,v .......
,-v
TISSUE
"l,
II
I~
o[
",
T
5. "-.~__
]
0 I
Table 1 shows the 3H-actinomycin D content of a few additional tissues, taken 15 rain after injection. In all three groups of mice, low concentrations of 3H-radioactivity were found in thymus, brain, urinary bladder, muscle and skin. However, the mice injected with nonencapsulated, free actinomycin D had higher concentrations of 3H-radioactivity in the
£
3
1
I~--
' ................ I, . . . . . . . . . .
6 24 TIME AFTER I.V, INJECTION, hours
'
FREE ACT. D.
APL
LPL
Thymu s
0.05
0.08
0.01
Brain
0.03
0.07
0.20
Urinary Bladder
0.05
0.31
0.03
12.75
5.02
5.37
8.38
3.35
3.94
48
Fig. 2. Concentration of 3H-actinomycin D (dis/rain per rag) in the gut wall. Each point is the mean valuefrom 4 mice. Vertical bars represent2 standard errors of the mean.
Muscle* Skin*
* For muscle and skin, the data represent dis/min per m g of tissue.
Liposomes Containing 3H-Actinomycin D
samples of muscle and skin compared to those of mice injected with either APL or LPL. In vitro release o f 3H-actinomycin D from liposomes The different rates of 3H-actinomycin D release from LPL and APL are shown in Fig. 3. The liposomes were incubated at 37°C, with slow shaking. When incubated in 8 triM CaC12, LPL released negligible radioactivity up to 4 hr, and no significant difference was found when incubated in culture medium (Eagle's medium containing 15% foetal calf serum). In contrast, APL released more than 60% of the total radioactivity within an hour. When APL were incubated in culture medium, there was slightly but significantly less release of radioactivity. The amount of radioactivity released from APL at 0 time (Fig. 3) was partially due to the additional centrifugation and manipulation inflected during the experimental procedure.
,-¢
lO0
LPL APL
....
CaCt2
hi --.I ~
6o
~
Culture medium
--
-O
,,;;,o"'"
. . . . . . . . . . . . . . . . .
4O ttO ~ zo °
iI
,,
~C~
0
y
I
"~
"~
cacle ,(
2 3 INCUBATION TIME, hours
4
Fig. 3. In vitro release of 3H-actinomycin D (% of total) from LPL and APL. The liposomes were incubated at 37°C
with slow shaking. • - - -, APL incubated in 8 m M CaClz; © - - - , APL incubated in culture medium (Eagle's medium containing 15% foetal calf serum); X - - , LPL incubated in 8 m M CaCl 2. DISCUSSION
The tissue distribution of a given drug can be modified by encapsulating it within liposomes. This modification has been demonstrated by studies on a liposome-encapsulated chelating agent, namely ethylenediaminetetraacetic acid [1]. The present report provides evidence that liposome encapsulation can be used to modify tissue distribution of other drugs, such as the anti-tumor agent actinomycin D. We have further demonstrated that by varying the way of incorporating the drug within liposomes, i.e., either in the aqueous or the lipid phase of the liposomes, marked differences in tissue distribution can be obtained. However, in comparison to results
887
obtained with nonencapsulated, free actinomycin D, the incorporation of this drug in the aqueous phase of the liposomes gave a quantitative difference only in their tissue distribution. Higher actinomycin D concentrations were generally found in tissues of mice receiving APL injections. Actinomycin D incorporated in the lipid phase shows high concentrations in the lungs, and low concentrations in the intestinal wall; whereas the reverse is true when it is in the aqueous phase. The different uptake in the lungs is not due to a size difference in the two forms of liposomes; the sizes are comparable when determined by a previously described method [1]. Studies on actinomycin D concentrations in mouse tissues by Schwartz, Sodergren and Ambaye [12] had shown a moderately high initial concentration in the small intestine, which was maintained only up to 3 hr after the injection of actinomycin D. Our present results are similar to theirs. The rapidly released 3H-actinomycin D from within the APL would presumably be distributed in a similar manner as the nonencapsulated, free form of actinomycin D. Therefore, it is not surprising to find relatively high concentrations of radioactivity in the intestinal wall of mice injected with APL. What seems surprising, though, are the very low concentrations of 3H-actinomycin D in the intestines of mice injected with LPL, and the very high concentrations in the lungs. In LPL, actinomycin D is incorporated within the lipid membranes which form the liposomes, so that one can assume that under such conditions, the surface properties of the actinomycin D molecule has been altered due to its bindings to the liposomal lipids. Consequently, the affinity between this drug and the tissues may also be modified. However, the chemical nature of the binding between actinomycin D and the liposomal lipids is not known. The tissue distribution is quite similar in mice injected either with nonencapsulated, free actinomycin D or APL, while the levels of 3H-actinomycin D concentration are generally higher in tissues of mice injected with APL (Fig. 1 and Table I). Again, the rapid release of actinomycin D from APL, as shown by in vitro studies (Fig. 3), would readily explain the similar distribution patterns in tissues of mice receiving either free actinomycin D or APL. The relatively high initial amounts of 3Hactinomycin D found in kidneys of mice receiving either free actinomycin D or APL are due to the rapid excretion of the drug in its free form.
888
Yueh-Erh Rahman, Walter E. Kisieleski, Evelyn M. Buess and Elizabeth A. Cerny
The spleen and bone marrow of mice receiving LPL show an increasing concentration of 3H-radioactivity between 3 and 24 hr after injection (Fig. l(c)). This increase is closely correlated with a concomitant decrease in actinomycin D concentration in the liver and lungs. Therefore, the increased 3H-radioactivity observed in the spleen and bone marrow, within this interval, seems to be the result of a translocation phenomenon between the liver and lungs on one hand and the spleen and marrow on the other. Actinomycin D is not being metabolized in animal tissues at least up to 2 4 h r after injection [12, 13], the 3H-radioactivity found between 3 and 24 hr in the spleen and bone marrow therefore could still represent intact molecules of actinomycin D. Liposome encapsulation of the drug could further protect it from being degraded within cells. In all three groups of mice, the liver has the highest 3H-actinomycin D activity among the tissues examined. In mice receiving either free actinomycin D or APL, the peak concentration in liver is at half an hour after injection; while the mice receiving LPL have the highest activity between one and three hours. Electron microscopic studies have shown that Kupffer cells as well as hepatocytes can rapidly take up liposomes containing chelating agents [14]. Preliminary results have indicated that both of these two cell types can also take up APL and LPL. The in vitro study of 3H-actinomycin D release from liposomes (Fig. 3) shows that the release from APL is very rapid compared to that from LPL. Even when APL were incubated in a culture medium more closely approximating the conditions in the blood, more than 40% of the total 3H-actinomycin D leaks out of the liposomes after only one-half hour of incuba-
tion (Fig. 3). The binding between actinomycin D and the liposomal lipids in LPL apparently retards the release of the drug from liposomes, thus preventing the drug from leaking out from liposomes at least during their passage in the blood stream. This phenomenon of slow drug release from LPL could be useful in future clinical applications. One of the most challenging problems in chemotherapy is to deliver a given drug specifically to a cell type within the target tissue. We have evidence that liposome uptake in tissues is not limited to phagocytic cells. As we have reported, parenchymal cells as well as Kupffer cells of the liver readily take up liposomes [14]. In addition, preliminary electron microscopic observations of mouse lung, after APL or LPL injections, indicate that several cell types incorporate liposomes. Therefore, higher tissue concentrations of a given drug obtained by liposome encapsulation, increase the potential for a higher drug concentration in any specific cell type, including tumor cells, within that tissue. Another aspect of the present study is that liposome encapsulation can be used as a tool to unravel problems related to tissue specificity, e.g., to learn the specific binding sites of a given tissue to liposomes. It should be emphasized, however, that before the problems of tissue specificity can be approached, the surface properties of APL and LPL, as well as the nature of the chemical binding between actinomycin D and the liposomal lipids, must be first understood. Furthermore, the quantitative uptake of liposomes by different cell types within a given tissue should be determined. We are currently investigating such problems. Acimowledgement--The authors wish to thank Ms. Gloria A. Kokaisl for her valuable assistance in the early phase of this work.
REFERENCES I. 2.
3. 4.
5.
Y.E. RAHMAN,M. W. ROSENTHAL,E. A. CERNYand E. S. MORETTI, Preparation and prolonged tissue retention of liposome-encapsulated chelating agents. J. lab. din. Med. 83, 640 (1974). M . M . JONAH, E. A. Cerny and Y. E. RAHMAN,Tissue distribution of EDTA encapsulated within liposomes of varying surface properties. Biochim. biophys. Acta401, 366 (1975). S. FARBER, Clinical and biological studies with actinomycins. Ciba Found. Syrup. Amino Acids Peptides Antimetab. Activity, pp. 138-148 (1959). G.T. Ross, L. L. STOLBACHand R. HERTZ, Actinomycin D in the treatment of methotrexate resistant trophoblastic disease in women. Cancer Res. 22, 1015 (1962). H.R. OETTGEN, P. CLIFFORDand D. BURKITT,Malignant lymphoma involving the jaw in African children: Treatment with alkylating agents and actinomycin D. CancerChemother. Rept. 28, 25 (1963).
Liposomes Containing 3H-Actinomycin D 6. J . H . COLEBATCH,R. HOWARD,A. L. WILLIAMS,G. R. KURRLE and A. C. L. Clark, Results of actinomycin D therapy for Wilm's tumor. Acta Un. int. Cancr. (N.Y.) 20,491 (1964). 7. Y.E. RAHMAN,E. A. CER~'¢, S. L. TOLLAKSEN,B. J. WRIGHT, S. L. NANCEand J. F. THOMSON, Liposome-encapsulated actinomycin D: Potential in cancer chemotherapy. Proc. Soc. Exp. Biol. Med. 146~ 1173 (1974). 8. G. GREOORtA91S,Drug entrapment in liposomes. FEBS Letters 36, 292 (1973). 9. Y.E. RAHMAN,M. W. ROSENTHALand E. A. CERN'¢, Intracellular plutonium: Removal by liposome-encapsulated chelating agent. Science 180~ 300 (1973). 10. M . W . ROSENTHAt,J. H. MARSHALLand A. LINDEm3AUM,Autoradiographic and radiochemical studies of the effect of colloidal state of intravenously injected plutonium on its distribution in bone and marrow, in Diagnosis and Treatment of Deposited Radionuclides, (Edited by H. A. KORt~BEROand W. D. NORWOOD), Excerpta Medica Foundation, Amsterdam, pp. 73-80 (1968). 11. M . W . ROSENTHAL,E. S. MORETTI and J. J. RUSSELL,Marrow deposition and distribution of monomeric and polymeric 239pu in the mouse, estimated by use of 59Fe. Health Phys. 22~ 743 (1972). 12. H . S . SCHWARTZ,J. E. SOD~RaRENand R. Y. A~BAYE, Actinomycin D: Drug concentrations and actions in mouse tissues and tumors. Cancer Res. 25~ 192 (1968). 13. H. WEISSBACH,B. R~DFmLD, T. O'CONNORand M. A. CHmIGOS, Studies on the disposition of actinomycin D-3H in virus-infected and tumor-bearing mice. Cancer Res. 26~ 1832 (1966). 14. Y. E. R.nHm~a'~ and B. J. WRIGHT, Liposomes containing chelating agents. Cellular penetration and a possible mechanism of metal removal. J. Cell Biol. 65, 112 (1975).
889
Liposomes Containing H-Actinomycin D. Differential Tissue Distribution / Varying the Mode of Drug Incorporation* YUEH-ERH RAHMAN, WALTER E. KISIELESKI, EVELYN M. BUESS and ELIZABETH A. CERNY Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439 Abstract--Actinomycin D was encapsulated within liposomes in two different ways. When the drug was incorporated in the lipid phase, as p,~rt of the membrane bi-layers of the liposomes, they were called "lipid phase liposomes" (LPL) ; when the drug was incorporated in the aqueous solution in the center and between the lipid bi-layers of the liposomes, they were called "aqueous phase liposomes" (APL). Distributions of 3Hactinomycin D in tissues of mice were determined from 15 minutes to 48 hours after a single intravenous injection of either LPL, APL, or nonencapsulated actinomycin D. Marked differences in tissue distribution were shown. 3H-actinomycin D incorporated in LPL showed high concentrations in the lungs and low concentrations in the intestinal wall; whereas the reverse was found with APL. The spleen and bone marrow of mice receiving LPL showed an increase in 3H-rudioactivity between 3 and 24 hr after injection that was closely correlated with a concomitant decrease in activity in the liver and lungs. Mice receiving either APL or nonencapsulated actinomycin D had higher levels of the drug in the blood, kidneys and intestinal wall than did mice receiving LPL. In vitro studies showed that 3H-actinomycin D leaked out from APL significantly faster than that from LPL. We have demonstrated that the tissue distribution of a drug can be modified not only by liposome encapsulation, but also by varying the way of incorporating the drug within liposomes, and thereby altering the surface properties. The two forms of incorporating drugs within liposomes are potentially useful to direct antitumor agents to specific tumor bearing tissues.
INTRODUCTION
tion of antitumor agents by liposome encapsulation. Actinomycin D, an antibiotic with known inhibitory effects on certain tumors [3-6], has been encapsulated within liposomes. Liposomeencapsulated actinomycin D, when given either i.v. or i.p. to mice, was found to be much less toxic than the nonencapsulated, free form [7]. It was also shown that mice inoculated with Ehtlich ascites tumor cells survived significantly longer when given single or multiple injections ofliposome-encapsulated actinomycin D [7]. Since actinomycin D is soluble both in aqueous media and in lipid solvents, it can be incorporated in either the aqueous or the lipid phase of the liposomes. In the first case, in
MOST antitumor agents are highly toxic to animals. This toxicity could be alleviated if selected tissue distribution of the agent could be obtained. In earlier studies with chelating agents encapsulated within liposomes, i.e., multi-layered lipid spherules, we found that partial selective tissue uptake of the chelating agent can be obtained, depending on the lipid constituents of the liposomes [1, 2]. Therefore, we have attempted to direct the tissue deposiAccepted 29 August 1975. *Work supported by the U.S. Energy Research and Development Administration. 883
884
7ueh-Erh Rahman, Walter E. Kisieleski, Evelyn M. Buess and Elizabeth A. Cerny
which the actinomycin D is in the aqueous solution in the center and between the lipid bi-layers of the liposomes, the liposomes are designated "aqueous phase liposomes" (APL). When the actinomycin D is incorporated as part of the lipid bi-layers [8] which form the outer shells of the liposomes, they are called "lipid phase liposomes" (LPL). These two kinds of liposomes containing actinomycin D are different in their surface properties. The present report concerns a comparative study of APL, LPL and non-encapsulated actinomycin D, with emphasis on their pattern of distribution in various mouse tisses after i.v. injections.
MATERIAL AND METHODS
Phosphatidylcholine (PC) and cholesterol were respectively obtained from Sigma Chem. Co., St. Louis, Mo. and Applied Sciences Laboratories, Inc., State College, Pa. Actinomycin D (Cosmegen) was from Merck, Sharp and Dohme, West Point, Pa. Radioactive 3H-actinomycin D, with a specific activity of 3.0Ci/mmole, was from Amersham/Searle Corp., Arlington Hts., Ill.
Preparation of actinomycin D liposomes APL were prepared with a mixture of 3.0 mg of PC and 3.0 mg of cholesterol dissolved in chloroform and dried in a thin film inside of a round bottom flask. Actinomycin D, dissolved in 8 m M CaC12 at a concentration of 0.5 mg in 1 ml, was slowly added to the dried film of lecithin-cholesterol mixture with immediate and constant stirring. LPL were prepared by dissolving a mixture of 4-0 mg of PC, 2"0 mg of cholesterol and 0.5 mg of actinomycin D in chloroform. After being dried to a deep yellow film, an 8 m M CaCI2 solution was then slowly added, and liposomes were formed by immediate stirring. The subsequent steps in the preparation of both forms of liposomes are essentially the same as described previously [1, 9]. For both APL and LPL, a suitable amount of radioactive 3H-actinomycin D was added to the actinomycin D solution so that each mouse received about 2.0 x 10 s dis/rain of radioactivity together with a nonlethal dose of actinomycin D of approximately 350/tg/kg. We should emphasize that high purity of PC is essential for successful liposome preparations. Liposomes made of either partially degraded or oxidized PC tend to aggregate into small clumps that render the preparations unsuitable for injection into animals [1].
Injection of 3H-actinomycin D in liposome-encapsulated or nonencapsulatedforms Groups of 24 female Carworth Farm (CF 1) mice, 3 months of age, and weighing between 25-30g were used. 3H-actinomycin D was given in one of the following forms: (1) actinomycin D in nonencapsulated, free form; (2) actinomycin D in APL; (3) actinomycin D in LPL. Each mouse received a single intravenous injection of 3H-actinomycin D in a volume of 0.40 ml; and 8 m M CaC12 solution, was used as injection vehicle for all the three forms of actinomycin D. Three groups of 4 mice each were killed at the following time intervals after the injections: 15 rain, 0.5, 1, 3, 6, 24 and 48 hr. Mice were killed by an intravenous injection of an anesthetic dose of sodium pentobarbital (0.04 ml/mouse), followed by exsanguination. Samples of blood and of various tissues were removed immediately for 3Hradioactivity determinations.
Preparation of mouse tissues Blood samples, lungs, spleen, liver, kidneys, intestinal samples and a standard bone marrow sample from both tibias [10] were taken for 3H analysis. At 15 min after injection, the following additional tissues were also taken for analysis: thymus, brain, urinary bladder including the urine, and sample of muscle and skin. Blood from a jugular vein was collected with a capillary micropipette. Duplicate samples of 100 ~1 each were absorbed into a small ball of cotton and allowed to air dry before being wrapped in a filter paper (Whatman No. 1, 4.25 cm dia). After removal, the spleen, lungs, brain, thymus, urinary bladder, each kidney, samples of muscle and skin each weighing between 100 and 150mg were removed, weighed, wrapped in a filter paper, and air dried. And three 150-200 mg slices of liver were also wrapped separately for analysis. The total liver radioactivity was calculated as the mean counts per mg from the slices, times the total liver weight. The slices were always taken from the standard locations which preliminary determinations had shown to be representative of the whole. Three segments of the small intestine were excised and weighed after the contents had been extruded. Each segment was wrapped separately in a filter paper for radioactivity analysis. The bone marrow samples were washed out with 0.25 ml of water directly onto a small ball of cotton and then treated in the same manner as that described for the blood samples. The amount of 3H-radioactivity in the total marrow was estimated by multiplying the activity in the two tibial segments by a
Liposomes Containing 3H-Actinomycin D factor of 44 [11]. The absorbent paper used to collect urine samples was cut out, folded, and then wrapped in filter paper. The radioactivity in the total blood was calculated from two 100/~1 samples and the estimated mouse blood volume of 2 cm 3.
Analysis of 3H-radioactivity Each air dried biological sample, all wrapped in Whatman No. 1 filter paper, was pressed in a pellet press (Parr Instruments Co., Moline, Ill.). After automatic combustion in a Packard 305 sample oxidizer and addition of scintillation fluid, each sample was analysed for 3Hradioactivity in a Beckman liquid scintillation counter, Model LS-200B. The oxidation of each tissue sample was followed at least once by combustion of an empty filter paper to remove the residual activity in the system. In the case of tissues known to contain high levels of radioactivity, several successive empty filter papers were oxidized. The radioactivity obtained from these filter papers was added to that of the preceding tissue sample. Calculation of the amount of radioactivity injected was based on analysis of triplicate samples of 0.40 ml, measured with the same syringe that was used for injection. These samples were prepared for radioanalysis in the same manner as the blood and bone marrow. In vitro studies of release of 3H-actinomydn D from APL and LPL Preparations of APL or LPL containing 3H-actinomycin D, suspended either in 8 m M CaC12 or in culture medium (Eagle's medium containing 15% foetal calf serum), were incubated at 37°C in a water bath, with constant slow shaking. One ml aliquots from each preparation were then taken at hourly intervals. They were centrifuged in a Sorvall GLC-1 centrifuge at 2000rev/min for 5 min. The release of 3H-actinomycin D from the liposome was calculated from the 3H-radioactivity found in the supernatant compared to the total radioactivity in the sample before centrifugation. RESULTS
3H-actinomycin D distribution in mousetissues Figure 1 shows the distribution in blood, lungs, spleen, liver, kidneys, and bone marrow of 3H-actinomycin D, administered either in LPL, APL, or in nonencapsulated form. The disappearance of 3H-actinomycin D from the blood was very rapid (Fig. l(a)). Half of an hour after injection, the highest blood level
885
(almost 2% of the injected radioactivity) was found in APL-injected mice, and the lowest level (about 0.5%) was found in the LPLinjected mice. A small but significant rise in the blood level of 3H-actinomycin D in LPLinjected mice occurred between 24 and 48 hr after the injection (Fig. 1(a)). There was a striking difference in the lung uptake of the different forms of 3H-actinomycin D (Fig. l(b)). In mice receiving the LPL injection, about 40% of the total injected dose was found in the lungs half of an hour after the injection, and the high concentration was maintained at least up to 6 hr; 24 hr after the injection, 10% of the total injected radioactivity was still retained in the lungs. In contrast, mice injected either with APL or with nonencapsulated 3H-actinomycin D had very low concentrations of the drug in the lungs (Fig. l(b)), the mice receiving the nonencapsulated form consistently had the lowest levels. The initial uptake of 3H-actinomycin D in the spleen was low in all three groups of mice. However, the spleens of LPL-injected mice showed a significant increase in 3H-actinomycin D concentration at 3 hr and at 24 hr reached a peak concentration of about 20% of the total injected dose. The groups of mice receiving APL or free actinomycin D had very low concentrations of the drug in the spleen throughout the 48 hr after the injection. The LPL-injected mice also had a higher uptake of 3H-actinomycin D in the liver than mice injected either with APL or free actinomycin D (Fig. 1(d)). In LPL-injected mice, the level of the drug in the liver was maintained at least up to 6 hr, but then decreased. In the kidneys the highest concentration of 3H-actinomycin D occurred in mice injected with APL or free actinomycin D. The peak concentration was found at one half of an hour. The LPL-injected mice had very low concentrations of 3H-actinomycin D in the kidneys throughout the experimental period (Fig. 1(e)). Although the initial uptake of 3H-actinomycin D in bone marrow of all three groups of mice was similar, the uptake in the LPLinjected mice continued to increase for 24 hr reaching a peak concentration of about 10%. In free actinomycin D and LPL-injected mice, there was a significant decrease of radioactivity in the bone marrow between 24 and 48 hr; while a constant level was maintained in APLinjected mice (Fig. 1 (f)). In the intestinal wall, mice injected either with APL or with nonencapsulated actinomycin D have an initial concentration (one-
886
Yueh-Erh Rahman, Walter E. Kisieleski, Evelyn M. Buess and Elizabeth A. Cerny d Liver Liver
Blood
a
~
4O
I~. ~ "X
.. . . . .
30 I
o
F~E,~"r.
LPL
--- APL FREEACT,D
2c IC I
I '
I
I
I
II
J
II
I
C
'
'
'
I~ ......~ - - ~
e
E
i
~""~"~
Lungs - - LPL
-- 30
~.~
Kidneys
~ LPL . . . . APL ........ FREEACT.D
5
___ APL
[\~%,
4
r x,,...~.
3 2 I0
T
I
f LPL
--
/
- - - APL
0
I
6
?..... ] ~ : . L
~
i/
5
II
Bone Morrow
Spleen 20
I
NI
24
,:: Z 48
G 0
I
I
.I 3
N I
6
I~
24
~/" " - - - 48
TIME AFTER INJECTION, hours
Fig. 1. Percentinjecteddose of 3H-actinomycin D in : (a) blood, (b) lungs, (c) spleen, (d) liver, (e) kidneys and (f) bone marrow. A single intravenous injection of one of the following: LPL (lipid phase liposomes), APL (aqueousphase liposomes), or free act. D was given to each mouse. The radioactivity in the blood (total) was calculatedfrom two I00 #l samples and the estimated mouse blood volume of 2 cm3. The level in the bone marrow (total) was estimated by multiplying the radioactivity in the two tibial segments by a factor 0f44 [1 I]. Each point is the mean value from 4 mice. Vertical bars represent 2 standard errors of the mean.
half to one hour) about five-times that in LPL-
injected mice (Fig. 2). The radioactivity then decreases so that between 24 and 48 hr post injection, the levels of radioactivity in all three groups of mice are not significantly different (Fig. 2). .i
'l
t~
il--
I
I~
GUT WALL
Table 1. Tissue content of 3H-actinomycin D ( % injected dose, 15 min after injection) The data represent the averages obtained from four animals in each group
f] X " a =~
I{ f_;
k',,.
A?L
~.. ",,v .......
,-v
TISSUE
"l,
II
I~
o[
",
T
5. "-.~__
]
0 I
Table 1 shows the 3H-actinomycin D content of a few additional tissues, taken 15 rain after injection. In all three groups of mice, low concentrations of 3H-radioactivity were found in thymus, brain, urinary bladder, muscle and skin. However, the mice injected with nonencapsulated, free actinomycin D had higher concentrations of 3H-radioactivity in the
£
3
1
I~--
' ................ I, . . . . . . . . . .
6 24 TIME AFTER I.V, INJECTION, hours
'
FREE ACT. D.
APL
LPL
Thymu s
0.05
0.08
0.01
Brain
0.03
0.07
0.20
Urinary Bladder
0.05
0.31
0.03
12.75
5.02
5.37
8.38
3.35
3.94
48
Fig. 2. Concentration of 3H-actinomycin D (dis/rain per rag) in the gut wall. Each point is the mean valuefrom 4 mice. Vertical bars represent2 standard errors of the mean.
Muscle* Skin*
* For muscle and skin, the data represent dis/min per m g of tissue.
Liposomes Containing 3H-Actinomycin D
samples of muscle and skin compared to those of mice injected with either APL or LPL. In vitro release o f 3H-actinomycin D from liposomes The different rates of 3H-actinomycin D release from LPL and APL are shown in Fig. 3. The liposomes were incubated at 37°C, with slow shaking. When incubated in 8 triM CaC12, LPL released negligible radioactivity up to 4 hr, and no significant difference was found when incubated in culture medium (Eagle's medium containing 15% foetal calf serum). In contrast, APL released more than 60% of the total radioactivity within an hour. When APL were incubated in culture medium, there was slightly but significantly less release of radioactivity. The amount of radioactivity released from APL at 0 time (Fig. 3) was partially due to the additional centrifugation and manipulation inflected during the experimental procedure.
,-¢
lO0
LPL APL
....
CaCt2
hi --.I ~
6o
~
Culture medium
--
-O
,,;;,o"'"
. . . . . . . . . . . . . . . . .
4O ttO ~ zo °
iI
,,
~C~
0
y
I
"~
"~
cacle ,(
2 3 INCUBATION TIME, hours
4
Fig. 3. In vitro release of 3H-actinomycin D (% of total) from LPL and APL. The liposomes were incubated at 37°C
with slow shaking. • - - -, APL incubated in 8 m M CaClz; © - - - , APL incubated in culture medium (Eagle's medium containing 15% foetal calf serum); X - - , LPL incubated in 8 m M CaCl 2. DISCUSSION
The tissue distribution of a given drug can be modified by encapsulating it within liposomes. This modification has been demonstrated by studies on a liposome-encapsulated chelating agent, namely ethylenediaminetetraacetic acid [1]. The present report provides evidence that liposome encapsulation can be used to modify tissue distribution of other drugs, such as the anti-tumor agent actinomycin D. We have further demonstrated that by varying the way of incorporating the drug within liposomes, i.e., either in the aqueous or the lipid phase of the liposomes, marked differences in tissue distribution can be obtained. However, in comparison to results
887
obtained with nonencapsulated, free actinomycin D, the incorporation of this drug in the aqueous phase of the liposomes gave a quantitative difference only in their tissue distribution. Higher actinomycin D concentrations were generally found in tissues of mice receiving APL injections. Actinomycin D incorporated in the lipid phase shows high concentrations in the lungs, and low concentrations in the intestinal wall; whereas the reverse is true when it is in the aqueous phase. The different uptake in the lungs is not due to a size difference in the two forms of liposomes; the sizes are comparable when determined by a previously described method [1]. Studies on actinomycin D concentrations in mouse tissues by Schwartz, Sodergren and Ambaye [12] had shown a moderately high initial concentration in the small intestine, which was maintained only up to 3 hr after the injection of actinomycin D. Our present results are similar to theirs. The rapidly released 3H-actinomycin D from within the APL would presumably be distributed in a similar manner as the nonencapsulated, free form of actinomycin D. Therefore, it is not surprising to find relatively high concentrations of radioactivity in the intestinal wall of mice injected with APL. What seems surprising, though, are the very low concentrations of 3H-actinomycin D in the intestines of mice injected with LPL, and the very high concentrations in the lungs. In LPL, actinomycin D is incorporated within the lipid membranes which form the liposomes, so that one can assume that under such conditions, the surface properties of the actinomycin D molecule has been altered due to its bindings to the liposomal lipids. Consequently, the affinity between this drug and the tissues may also be modified. However, the chemical nature of the binding between actinomycin D and the liposomal lipids is not known. The tissue distribution is quite similar in mice injected either with nonencapsulated, free actinomycin D or APL, while the levels of 3H-actinomycin D concentration are generally higher in tissues of mice injected with APL (Fig. 1 and Table I). Again, the rapid release of actinomycin D from APL, as shown by in vitro studies (Fig. 3), would readily explain the similar distribution patterns in tissues of mice receiving either free actinomycin D or APL. The relatively high initial amounts of 3Hactinomycin D found in kidneys of mice receiving either free actinomycin D or APL are due to the rapid excretion of the drug in its free form.
888
Yueh-Erh Rahman, Walter E. Kisieleski, Evelyn M. Buess and Elizabeth A. Cerny
The spleen and bone marrow of mice receiving LPL show an increasing concentration of 3H-radioactivity between 3 and 24 hr after injection (Fig. l(c)). This increase is closely correlated with a concomitant decrease in actinomycin D concentration in the liver and lungs. Therefore, the increased 3H-radioactivity observed in the spleen and bone marrow, within this interval, seems to be the result of a translocation phenomenon between the liver and lungs on one hand and the spleen and marrow on the other. Actinomycin D is not being metabolized in animal tissues at least up to 2 4 h r after injection [12, 13], the 3H-radioactivity found between 3 and 24 hr in the spleen and bone marrow therefore could still represent intact molecules of actinomycin D. Liposome encapsulation of the drug could further protect it from being degraded within cells. In all three groups of mice, the liver has the highest 3H-actinomycin D activity among the tissues examined. In mice receiving either free actinomycin D or APL, the peak concentration in liver is at half an hour after injection; while the mice receiving LPL have the highest activity between one and three hours. Electron microscopic studies have shown that Kupffer cells as well as hepatocytes can rapidly take up liposomes containing chelating agents [14]. Preliminary results have indicated that both of these two cell types can also take up APL and LPL. The in vitro study of 3H-actinomycin D release from liposomes (Fig. 3) shows that the release from APL is very rapid compared to that from LPL. Even when APL were incubated in a culture medium more closely approximating the conditions in the blood, more than 40% of the total 3H-actinomycin D leaks out of the liposomes after only one-half hour of incuba-
tion (Fig. 3). The binding between actinomycin D and the liposomal lipids in LPL apparently retards the release of the drug from liposomes, thus preventing the drug from leaking out from liposomes at least during their passage in the blood stream. This phenomenon of slow drug release from LPL could be useful in future clinical applications. One of the most challenging problems in chemotherapy is to deliver a given drug specifically to a cell type within the target tissue. We have evidence that liposome uptake in tissues is not limited to phagocytic cells. As we have reported, parenchymal cells as well as Kupffer cells of the liver readily take up liposomes [14]. In addition, preliminary electron microscopic observations of mouse lung, after APL or LPL injections, indicate that several cell types incorporate liposomes. Therefore, higher tissue concentrations of a given drug obtained by liposome encapsulation, increase the potential for a higher drug concentration in any specific cell type, including tumor cells, within that tissue. Another aspect of the present study is that liposome encapsulation can be used as a tool to unravel problems related to tissue specificity, e.g., to learn the specific binding sites of a given tissue to liposomes. It should be emphasized, however, that before the problems of tissue specificity can be approached, the surface properties of APL and LPL, as well as the nature of the chemical binding between actinomycin D and the liposomal lipids, must be first understood. Furthermore, the quantitative uptake of liposomes by different cell types within a given tissue should be determined. We are currently investigating such problems. Acimowledgement--The authors wish to thank Ms. Gloria A. Kokaisl for her valuable assistance in the early phase of this work.
REFERENCES I. 2.
3. 4.
5.
Y.E. RAHMAN,M. W. ROSENTHAL,E. A. CERNYand E. S. MORETTI, Preparation and prolonged tissue retention of liposome-encapsulated chelating agents. J. lab. din. Med. 83, 640 (1974). M . M . JONAH, E. A. Cerny and Y. E. RAHMAN,Tissue distribution of EDTA encapsulated within liposomes of varying surface properties. Biochim. biophys. Acta401, 366 (1975). S. FARBER, Clinical and biological studies with actinomycins. Ciba Found. Syrup. Amino Acids Peptides Antimetab. Activity, pp. 138-148 (1959). G.T. Ross, L. L. STOLBACHand R. HERTZ, Actinomycin D in the treatment of methotrexate resistant trophoblastic disease in women. Cancer Res. 22, 1015 (1962). H.R. OETTGEN, P. CLIFFORDand D. BURKITT,Malignant lymphoma involving the jaw in African children: Treatment with alkylating agents and actinomycin D. CancerChemother. Rept. 28, 25 (1963).
Liposomes Containing 3H-Actinomycin D 6. J . H . COLEBATCH,R. HOWARD,A. L. WILLIAMS,G. R. KURRLE and A. C. L. Clark, Results of actinomycin D therapy for Wilm's tumor. Acta Un. int. Cancr. (N.Y.) 20,491 (1964). 7. Y.E. RAHMAN,E. A. CER~'¢, S. L. TOLLAKSEN,B. J. WRIGHT, S. L. NANCEand J. F. THOMSON, Liposome-encapsulated actinomycin D: Potential in cancer chemotherapy. Proc. Soc. Exp. Biol. Med. 146~ 1173 (1974). 8. G. GREOORtA91S,Drug entrapment in liposomes. FEBS Letters 36, 292 (1973). 9. Y.E. RAHMAN,M. W. ROSENTHALand E. A. CERN'¢, Intracellular plutonium: Removal by liposome-encapsulated chelating agent. Science 180~ 300 (1973). 10. M . W . ROSENTHAt,J. H. MARSHALLand A. LINDEm3AUM,Autoradiographic and radiochemical studies of the effect of colloidal state of intravenously injected plutonium on its distribution in bone and marrow, in Diagnosis and Treatment of Deposited Radionuclides, (Edited by H. A. KORt~BEROand W. D. NORWOOD), Excerpta Medica Foundation, Amsterdam, pp. 73-80 (1968). 11. M . W . ROSENTHAL,E. S. MORETTI and J. J. RUSSELL,Marrow deposition and distribution of monomeric and polymeric 239pu in the mouse, estimated by use of 59Fe. Health Phys. 22~ 743 (1972). 12. H . S . SCHWARTZ,J. E. SOD~RaRENand R. Y. A~BAYE, Actinomycin D: Drug concentrations and actions in mouse tissues and tumors. Cancer Res. 25~ 192 (1968). 13. H. WEISSBACH,B. R~DFmLD, T. O'CONNORand M. A. CHmIGOS, Studies on the disposition of actinomycin D-3H in virus-infected and tumor-bearing mice. Cancer Res. 26~ 1832 (1966). 14. Y. E. R.nHm~a'~ and B. J. WRIGHT, Liposomes containing chelating agents. Cellular penetration and a possible mechanism of metal removal. J. Cell Biol. 65, 112 (1975).
889
