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Description  |
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FIELD OF THE INVENTION
The present invention relates to liposome therapeutic compositions, and
particularly to liposomal formulations which have enhanced circulation
time in the bloodstream, when administered intravenously.
REFERENCES
1. Allen, T. M. (1981) Biochem. Biophys. Acta 640, 385-397.
2. Allen, T. M., and Everest, J. (1983) J. Pharmacol. Exp. Therap. 226,
539-544.
3. Altura, B. M. (1980) Adv. Microcirc. 9, 252-294.
4. Alving, C. R. (1984) Biochem. Soc. Trans. 12, 342-344.
5. Ashwell, G., and Morell, A. G. (1974) Adv. Enzymology 41, 99-128.
6. Czop, J. K. (1978) Proc. Natl. Acad. Sci. USA 75: 3831.
7. Durocher, J. P., et al (1975) Blood 45: 11.
8. Ellens, H., et al. (1981) Biochim. Biophys. Acta 674, 10-18.
9. Gregoriadis, G., and Ryman, B. E. (1972) Eur. J. Biochem. 24, 485-491.
10. Gregoriadis, G., and Neerunjun, D. (1974) Eur. J. Biochem. 47, 179-185.
11. Gregoriadis, G., and Senior, J. (1980) FEBS Lett. 119, 43-46.
12. Greenberg, J. P., et al (1979) Blood 53: 916.
13. Hakomori, S. (1981) Ann. Rev. Biochem. 50, 733-764.
14. Hwang, K. J., et al (1980) Proc. Natl. Acad. Sci. USA 77: 4030.
15. Jonah, M. M., et al. (1975) Biochem. Biophys. Acta 401, 336-348.
16. Juliano, R. L., and Stamp, D. (1975) Biochem. Biophys. Res. Commun. 63,
651-658.
17. Karlsson, K. A. (1982) In: Biological Membranes, vol. 4, D. Chapman
(ed.) Academic Press, N.Y., pp. 1-74.
18. Kimelberg, H. K., et al. (1976) Cancer Res. 36, 2949-2957.
19. Lee, K. C., et al, J. Immunology 125: 86 (1980).
20. Lopez-Berestein, G., et al. (1984) Cancer Res. 44, 375-378.
21. Okada, N. (1982) Nature 299: 261.
22. Poznansky, M. J., and Juliano, R. L. (1984) Pharmacol. Rev. 36,
277-336.
23. Richardson, V. J., et al. (1979) Br. J. Cancer 40, 35-43.
24. Saba, T. M. (1970) Arch. Intern. Med. 126, 1031-1052.
25. Schaver, R. (1982) Adv. Carbohydrate Chem. Biochem. 40: 131.
26. Scherphof, T., et al. (1978) Biochim. Biophys. Acta 542, 296-307.
27. Senior, J., and Gregoriadis, G. (1982) FEBS Lett. 145, 109-114.
28. Senior, J., et al. (1985) Biochim. Biophys. Acta 839, 1-8.
29. Szoka, F., Jr., et al (1978) Proc. Natl. Acad. Sci. USA 75: 4194.
30. Szoka, F., Jr., et al (1980) Ann. Rev. Biophys. Bioeng. 9: 467.
31. Woodruff, J. J., et al (1969) J. Exp. Med. 129: 551.
BACKGROUND OF THE INVENTION
Liposome delivery systems have been proposed for a variety of drugs. For
use in drug delivery via the bloodstream, liposomes have the potential of
providing a controlled "depot" release of a liposome-entrapped drug over
an extended time period, and of reducing toxic side effects of the drug,
by limiting the concentration of free drug in the bloodstream.
Liposome/drug compositions can also increase the convenience of therapy by
allowing higher drug dosage and less frequent drug administration.
Liposome drug delivery systems are reviewed generally in Poznansky et al.
One limitation of intravenous liposome drug delivery which has been
recognized for many years is the rapid uptake of blood-circulating
liposomes by the mononuclear phagocytic system (MPS), also referred to as
the reticuloendothelial system (RES). This system, which consists of the
circulating macrophages and the fixed macrophages of the liver (Kupffer
cells), spleen, lungs, and bone marrow, removes foreign particulate
matter, including liposomes, from blood circulation with a half life on
the order of minutes (Saba). Liposomes, one of the most extensively
investigated particulate drug carriers, are removed from circulation
primarily by Kupffer cells of the liver and to a lesser extent by other
macrophage populations.
A variety of studies on factors which effect liposome uptake by the RES
have been reported. Early experiments, using heterogeneous preparations of
multilamellar liposomes (MLV) containing phosphatidylcholine (PC) and
cholesterol (CH) as their principal lipid constituents, demonstrated that
these liposomes are rapidly removed from circulation by uptake into liver
and spleen in a biphasic process with an initial rapid uptake followed by
a slow phase of uptake (Gregoriadis, 1974; Jonah; Gregoriadis, 1972;
Juliano). Half-time for removal of MLV from circulation was on the order
of 5-15 min. following intravenous (i.v.) injection. Negatively charged
liposomes are removed more rapidly from circulation than neutral or
positively charged liposomes. Small unilamellar liposomes (SUV) are
cleared with half-lives approximately three- to fourfold slower than MLV
(Juliano; Allen, 1983). Uptake of liposomes by liver and spleen occurs at
similar rates in several species, including mouse, rat, monkey, and human
(Gregoriadis, 1974; Jonah; Kimelberg, 1976; Juliano; Richardson;
Lopez-Berestein).
Liposomes which are capable of evading the RES would have two important
benefits. One is the increased liposome circulation time in the blood,
which would both increase the pharmacokinetic benefits of slow drug
release in the bloodstream, and also provide greater opportunity for
tissue targeting where the liver, spleen, and lungs are not involved. The
second benefit is decreased liposome loading of the RES. In addition to
the role of the RES in removing foreign particles, the RES is involved in
several other functions, including host defense against pathogenic
micro-organisms, parasites, and tumor cells, host responses to endotoxins
and hemorragic shock, drug response, and responses to circulating immune
complexes (Saba, Altura). It is important, therefore, in liposome
administration via the bloodstream, to avoid compromising the RES
seriously, by massive short-term or accumulated liposome uptake.
One approach which has been proposed is to increase liposome circulation
time by increasing liposome stability in serum. This approach is based on
studies by the inventor and others which have shown that factors which
decrease leakage of liposome contents in plasma also decrease the rate of
uptake of liposomes by the RES (Allen, 1983; Gregoriadis, 1980; Allen,
1981; Senior, 1982). The most important factor contributing to this effect
appears to be bilayer rigidity, which renders the liposomes more resistant
to the destabilizing effects of serum components, in particular high
density lipoproteins (Allen, 1981; Scherphof). Thus, inclusion of
cholesterol in the liposomal bilayer can reduce the rate of uptake by the
MPS (Gregoriadis, 1980; Hwang; Patel, 1983; Senior, 1985), and solid
liposomes such as those composed of distearoylphosphatidylcholine (DSPC)
or containing large amounts of sphingomyelin (SM) show decreased rate and
extent of uptake into liver (Allen, 1983; Ellens; Senior, 1982; Hwang).
However, this approach appears to have a very limited potential for
increasing liposome circulation times in the bloodstream. Studies carried
out in support of the present invention, and reported below, indicated
that 0.4 micron liposomes containing optimal membrane-rigidifying liposome
formulation are predominantly localized in the MPS two hours after
intravenous liposome administration. Although longer circulation times are
achieved with small unilamellar vesicles or SUVs (having a size range
between about 0.03-0.08 microns), SUVs are generally less useful in drug
delivery due to their smaller drug-carrying capacity and their tendency to
fuse to form large heterogeneous-size liposomes.
Several groups, including the inventor's, have also explored the
possibility of increasing liposome circulation times by designing the
liposome surface to mimic that of red blood cells. The role of cell
surface carbohydrates in cellular recognition phenomena is widely
appreciated (Ashwell, Hakomori, Karlsson). The chemistry, metabolism, and
biological functions of sialic acid have been reviewed (Schauer). Surface
sialic acid, which is carried by gangliosides, and glycoproteins such as
glycophorin, plays an important role in the survival of erthrocytes,
thrombocytes, and lymphocytes in circulation. Enzymatic removal of sialic
acid, which exposes terminal galactose residues, results in rapid removal
of erythrocytes from circulation, and uptake into Kupffer cells of the
liver (Durocher). Desialylation of thrombocytes (Greenberg) and
lymphocytes (Woodruff) also results in their rapid removal by the liver.
Although desialylated erythrocytes will bind to Kupffer cells or peritoneal
macrophages in vitro in the absence of serum, serum must be added in order
for significant phagocytosis to occur. The nature of the serum components
mediating endocytosis is speculative, but immunoglobin and complement
(C3b) are thought to be involved. Czop et al. (Czop) have shown that sheep
erythrocytes, which are not normally phagocytosed by by human monocytes,
will bind C3b and be phagocytosed upon desialylation. Okada et al. (Okada)
have demonstrated that sialyglycolipids on liposome membranes restrict
activation of the alternative complement pathway and that removal of the
terminal sialic acid from the glycolipids abolishes this restricting
capacity and results in activation of the alternative complement pathway.
Sialic acid, therefore, may be functioning as a non-recognition molecule
on cell membranes partly through its ability to prevent binding of C3b,
thus preventing phagocytosis via the alternative complement pathway. Other
immune factors may also be involved in liposome phagocytosis. Alving has
reported that 50% of the test sera from individual humans contain
naturally occurring "anti-liposome" antibodies which mediated
complement-dependent immune damage to liposomes.
The observations reported above suggest that surface sialic acid, and/or
other red-cell surface agents, incorporated into liposomes, for example,
in the form of ganglioside or glycophorin, may lead to increased
circulation half-lives of liposomes. This approach is described, for
example, in U.S. Pat. No. 4,501,728 for "Masking of Liposomes from RES
Recognition", although this patent does not disclose whether significant
RES masking is actually achieved by coating liposomes with sialic acid.
In fact, experiments conducted in support of the present applications
indicate that sialic acid, in the form of gangliosides, has a limited
ability to extend circulation half lives in vivo in liposomes which are
predominantly composed of conventional liposomes lipids, such as egg
phosphatidylcholine (egg PC) or egg PC:cholesterol mixtures. In vivo
uptake studies on PC:cholesterol:ganglioside liposomes (0.4 microns)
indicate that the injected liposomes are localized predominantly in the
MPS two hours post administration.
In summary, several approaches for achieving enhanced lipsome circulation
times in the bloodstream have been proposed. Heretofore, however, the
approaches have produced quite limited improvements in blood circulation
times, particularly in liposomes in the 0.1-0.4 micron size range which
are generally most desirable for therapeutic drug compositions.
SUMMARY OF THE INVENTION
It is therefore one general object of the present invention to provide an
improved liposome composition which gives significantly improved blood
circulation times.
A more specific object of the invention is to provide such a composition in
which liposomes are predominantly localized in the bloodstream, rather
than in the liver and spleen, several hours after liposome administration.
Yet another object of the invention is to provide such a composition
containing liposomes predominantly in the 0.1 to 0.4 micron size range.
Still another object of the invention is to provide an in vitro cell
culture method for evaluating lipid compositional factors which are
important to liposome uptake by the MPS in vivo.
According to one aspect of the invention, it has been discovered that
larger liposomes (0.08-0.4 microns) containing a high molar ratio of
particular membrane rigidifying agents and between about 5-15 mole percent
of a selected type of ganglioside give a much longer blood circulation
time than the sum of the circulation times of liposomes containing each
component alone. More precisely, the invention includes a composition of
liposomes which contain an entrapped pharmaceutical agent and which are
characterized by:
(a) liposome sizes predominantly having a selected size between about 0.08
and 0.5 microns,
(b) at least about 50 mole percent of a membrane-rigidifying component
including either sphingomyelin (SM) and/or neutral phospholipids with
predominantly saturated acyl chains,
(c) between 5-15 mole percent monosialylganglioside (G.sub.M), and
(d) an tissue distribution ratio, as measured by the amount of
intravenously administered liposomes in the blood divided by the combined
amount of administered liposomes in the liver and spleen, when measure 2
hours after intravenous administration of the composition to a subject,
which is substantially greater than the sum of the tissue distribution
ratios obtained with similarly constructed liposome compositions
containing in one case, at least about 50 mole percent of the membrane
rigidifying agent, but not G.sub.M, and in another case, between 5-15 mole
percent of G.sub.M, but not the membrane-rigidifying agent.
In a preferred embodiment of the composition, the liposomes are
oligolamellar vesicles sized to a selected size range between about
0.1-0.4 microns, the membrane rigidifying agent includes brain
sphingomyelin, at a molar ratio of between about 60-80 mole percent, and
the ganglioside is G.sub.M1. The preferred composition also contains
little or no cholesterol.
The liposome composition is used, in a liposome drug treatment, to achieve
a significantly extended lifetime of liposomes in the bloodstream.
In another aspect, the invention includes a method of assessing the effect
of a selected membrane component on the in vivo uptake of liposomes by the
reticuloendothelial system. The method is based on the discovery that
cultured macrophages, such as bone marrow macrophages, show a
discrimination in liposome uptake which is related to the same liposome
composition factors which effect liposome uptake by the MPS in vivo.
These and other objects and features of the invention will become more
fully apparent when the following detailed description of the invention is
read in conjunction with the accompany figures and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the time course of decrease of blood/MPS ratios in a test
subject injected i.v. with (1) liposomes containing ganglioside G.sub.M1,
but not SM (solid circles) and (2) with liposomes containing both G.sub.M1
and SM (open circles);
FIG. 2 shows the change in blood/MPS two hours post injection, in liposomes
containing SM:PC, 4:1, and increasing molar amounts of G.sub.M1 (solid
circles), and the blood/MPS values multiplied by the percent of total
liposome-associated radioactivity remaining two hours post-injection (open
circles), to correct for loss of liposome radiolabel in two hours;
FIG. 3 shows the uptake of PC liposomes by cultured macrophages in vitro,
where the solid squares represent non-specific uptake at 4.degree. C., the
solid circles, total liposome uptake at 37.degree. C., and the open
circles, the difference between total and non-specific uptake; (specific
uptake);
FIG. 4 shows the uptake of liposomes by cultured macrophages in vitro, at
increasing concentrations of added liposomes, where the open circles
represent non-specific uptake at 4.degree. C., and the the solid circles,
total liposome uptake at 37.degree. C.;
FIG. 5 shows the decline in uptake of PC liposomes, with increasing amounts
of G.sub.M1, by cultured macrophages in culture; and
FIG. 6 shows the decline in uptake of SM:PC PC liposomes, with increasing
amounts of G.sub.M1, by cultured macrophages in culture.
DETAILED DESCRIPTION OF THE INVENTION
The liposome composition of the invention is designed for delivering a drug
or other agent, such as nutritional supplements, vitamins, or chelated
metal, to a subject via the bloodstream, and for relatively slow uptake of
the lipsomes by the MPS, allowing the drug or agent to be released from
the liposomes into the bloodstream over an extended period of several
hours or more. Alternatively, the composition is designed, by appropriate
surface modification of the liposomes, for targeting via the bloodstream
to non-MPS target tissues, to allow the drug or agent to concentrate in
the immediate region of the target tissue.
Section IA below describes the general method used to evaluate liposome
uptake by the MPS in vivo, section IB, the combination of liposome
components which have been found, according to one aspect of the
invention, to give high blood circulation times for intravenously injected
liposomes, and section IC, methods for preparing, sizing, and sterilizing
drug-containing liposomes designed for intravenous administration. The
utility of the liposome composition, in drug delivery and drug targeting,
is discussed in Section II. A novel in vitro system for evaluating
liposomal factors which effect liposomal uptake by the MPS in vivo is
presented in Example III.
I. PREPARING THE LIPOSOMAL COMPOSITION
A. Measuring liposome uptake by the MPS in vivo
The method used for evaluating liposome circulation time in vivo measures
the distribution of intravenously injected liposomes in the bloodstream
and the primary organs of the MPS at selected times after injection. In
the standardized model which is used, MPS uptake is measured by the ratio
of total liposomes in the bloodstream to total liposomes in the liver and
spleen, the principal organs of the MPS. In practice, female ICR mice are
injected i.v. through the tail vein with a radiolabeled liposome
composition, and each time point is determined by measuring total blood
and combined liver and spleen radiolabel counts. Total blood counts are
calculated by assuming that the total blood volume makes up 7% of the
animal's body weight. The experimental methods are detailed in Example 2.
Since the liver and spleen account for nearly 100% of the initial uptake of
liposomes by the MPS, the blood/MPS ratio just described provides a good
approximation of the extent of uptake from the blood to the MPS in vivo.
For example, a ratio of about 1 or greater indicates a predominance of
injected liposomes remaining in the bloodstream, and a ratio below about
1, a predominance of liposomes in the MPS. For many of the lipid
compositions of interest, blood/MPS ratios were calculated at one-half
hour and at two hours, to identify lipid compositions which were effective
in both short-term evasion of the MPS, and to evaluate the rate of MPS
uptake shortly after injection. For some formulations of interest,
blood/MPS ratios during a 24 hour period post injection were also
measured, to demonstrate long-term MPS-evasion. As will be seen, only a
few prior art formulations gave blood/MPS ratios greater than 1 at
one-half hour, and none at two hours. By contrast, the formulations of the
invention typically gave blood/MPS ratios of 3-5 after 2 hours and greater
than 1 at 24 hours.
It is assumed that the data obtained with the model animal system can be
reasonably extrapolated to humans and veterinary animals of interest. This
is because, as mentioned above, uptake of liposomes by liver and spleen
has been found to occur at similar rates in several mammalian species,
including mouse, rat monkey, and human (Gregoriadis, 1974; Jonah;
Kimelberg, 1976; Juliano; Richardson; Lopez-Berestein). This result likely
reflects the fact that the biochemical factors which appear to be most
important in liposome uptake by the RES--including serum lipoproteins, and
cell shielding by carbohydrate moieties--are common features of all
mammalian species which have been examined.
B. Lipid Components
As indicated above, membrane-rigidifying components, such as SM,
cholesterol and saturated lipids, have been proposed heretofore in the
context of increased liposome circulation times in the bloodstream. The
effects of SM on egg PC liposomes was examined in studies which are
reported in part in Examples 3 and 4. With reference to Table 1 in Example
3, it is seen that addition of increasing amounts SM to 0.4 micron
oligolamellar vesicles (REVs) increased blood/MPS ratios at two hours
progressively from 0.01, in liposomes composed of egg PC alone, to 0.57,
in liposomes containing 80 mole percent SM. Brain and egg SM, which are
both composed of partially unsaturated hydrocarbon chains, gave higher
blood/MPS values than SM with saturated chains, such as stearoyl and
palmitoyl chains.
It is known that the polar head groups of SM are able to hydrogen bond with
each other and with the head groups of PC, and this feature may be
important to the ability of SM to rigidify lipid bilayer structures. If
so, the lower blood/MPS ratios seen with highly saturated SMs may be due
to the formation of SM domains which are in a non-fluid phase at
physiological temperature, and therefore either leakier and/or unable to
form polar-region hydrogen bonding
Liposomes containing egg PC and increasing mole ratios of distearoylPC
(DSPC) also gave increasing blood/MPS values at two hours, but not as high
as was seen with brain SM.
Addition of cholesterol (CH) to egg PC liposomes also increased blood/MPS
ratios at two hours, but only about one-fifth the increase seen with brain
SM, and addition of cholesterol to rigidified liposomes (containing SM and
PC) reduced the blood/MPS values observed with SM:PC alone. This result is
consistent with the proposed role of membrane rigidity in liposome uptake
by the MPS, since cholesterol produces a rigidifying effect on egg PC
liposomes, but a fluidizing effect on SM:PC liposomes. It is also noted,
from the data in Table 1, that negatively charged phospholipids, such as
phosphatidylserine (PS) and phosphatidic acid (PA) substantially negate
the increased blood/MPS ratios produced by brain SM.
Several gangliosides, including monosialylganglioside M.sub.1 (G.sub.M1),
asialylganglioside, produced by desialylation of G.sub.M1,
disialylganglioside (G.sub.D1a), and sulfatides were examined for their
ability to increase blood/MPS ratios in egg PC and egg PC:cholesterol
liposomes (0.4 micron REVs). The studies were designed to determine if one
or more gangliosides could increase liposome circulation time
significantly, as has been previously proposed, and if so, optimal
concentrations of gangliosides, with emphasis on 0.4 micron oligolamellar
vesicles. The results, some of which are reported in Examples 4 and 5
below, can be summarized as follows: (1) Addition of G.sub.M1 to egg PC or
egg PC:cholesterol increased blood/MPS ratios after two hours, but to a
lesser extent than did SM. (2) Optimal G.sub.M1 concentration was between
about 5-7 mole percent for egg PC:cholesterol liposomes. (3) The enhanced
blood/MPS value seen with G.sub.M1 is substantially abolished by
desialylating the liposomes with neuraminidase. (4) Monosialylganglioside
gave substantially higher blood/MPS values than did disialylgangliosides
and sulfatides.
The ability of GM.sub.1 to enhance liposome blood levels was studied during
a 24 hour period was also examined. A plot of blood/MPS ratios for
PC:CH:G.sub.M1 liposomes is shown in solid circles in FIG. 1. As seen, the
liposomes are largely removed from circulation after 2 hours, and
substantially completely removed after 6 hours.
The above data on membrane-rigidifying agents and gangliosides indicate a
rather limited ability of these lipid components by themselves to retard
liposome uptake by the MPS. The best membrane-rigidifying composition gave
blood/RES values of about 0.57, for 0.4 micron liposomes two hours post
injection, and the best ganglioside composition, blood/MPS gave values of
about 0.3 for the same conditions.
According to an important feature of the invention, it has been discovered
that a liposome composition formulated to include (a) at least about 50
mole percent membrane-rigidifying components which is either SM or a
saturated neutral phospholipid, and (b) 5-15 mole percent of
monosialylganglioside, gives much higher blood/MPS ratios than the sum of
the ratios which would be expected from either component alone. That is,
the blood/MPS ratio of the combined-component liposomes is substantially
higher than that of liposomes containing optimal membrane-rigidifying
agents alone plus that achieved with optimal ganglioside components alone.
Several studies on the blood/MPS ratios seen with SM and or DSPC and
G.sub.M1 are reported in Example 4. With particular reference to Table 3
in the example, which shows ratios for 0.4 micron REVs at two hours post
injection, it is seen that SM:PC:CH:G.sub.M1 liposomes give blood/MPS
ratios which are over 3 times the sum of the ratios seen with similarly
constructed liposomes containing either SM alone and G.sub.M1 alone. The
term "similarly constructed liposomes" is applied herein to denote
liposomes which are identical in all respects except for
membrane-rigidifying and/or ganglioside components.
Even more dramatic increases in liposome circulation time are observed in
liposomes containing SM and G.sub.M1, in the absence of cholesterol. Here
blood/MPS ratios of about 5 times the sum of the ratios seen with
similarly constructed liposomes containing either SM alone and G.sub.M1
alone were observed.
The data in Table 3 also shows the relatively poor increases in blood/MPS
ratios which can be achieved with SM in combination in G.sub.D1a or
sulfatide.
The data in Table 4 of Example 3 show blood/MPS ratios which are observed
when DSPC is substituted entirely or in part for SM. The data indicate
that DSPC is less effective than brain SM in enhancing liposome
circulation time, but that DSPC is also less sensitive to the "inhibitory"
effect of cholesterol on blood/MPS values.
The optimal concentration of G.sub.M1 was determined using the SM:PC, 4:1
formulation which appears to give the highest blood/MPS ratios when
combined with G.sub.M1. The results are plotted in FIG. 2, where blood/MPS
ratios determined on the basis of total radioactivity counts (solid
circles) and counts corrected for loss of liposomal counts over two hours
(open circles) are plotted. The plots show that G.sub.M1 concentrations
between about 7 and 15 mole percent are optimal. Details of the study are
provided in Example 5.
When an optimal liposome formulation containing SM:PC:G.sub.M1, 4:1:0.35 is
followed 24 hours post injection, the plot shown in open circles in FIG. 1
is obtained. The plot shows high blood/MPS ratios over 6 hours, and a
ratio of about 1 even at 24 hours. PC:CH:G.sub.M1 liposomes are indicated
by solid circles in the FIGURE. Thus it can be appreciated that the
improved liposome formulation gives unexpectedly high blood/MPS ratios
between 2-24 hours post injection.
In addition to the membrane-rigidifying agents and gangliosides required in
the liposome composition, the liposomes may be formulated to include other
neutral vesicle-forming lipids which do not significantly compromise the
MPS-evasion properties of the liposomes. An obvious example is egg PC
which is used in the optimal formulation described above. Although
cholesterol, or cholesterol derivatives may be used, the data above
indicates that the mole ratio of sterols should be kept well below about
30 mole percent and preferably less than 10 mole percent.
The liposomes may also include protective agents such alpha-tocopherol, or
other free-radical inhibitors, to minimize oxidative damage to the
liposomes and/or entrapped drug carried in the liposomes.
C. Preparing the Liposome Composition
The liposomes may be prepared by a variety of techniques, such as those
detailed in Szoka et al, 1980. One preferred method for preparing
drug-containing liposomes is the reverse phase evaporation method
described by Szoka et al and in U.S. Pat. No. 4,235,871. In this method, a
solution of liposome-forming lipids is mixed with a smaller volume of an
aqueous medium, and the mixture is dispersed to form a water-in-oil
emulsion. The drug or other pharmaceutical agent to be delivered is added
either to the lipid solution, in the case of a lipophilic drug, or to the
aqueous medium, in the case of a water-soluble drug. Here it is noted that
all lipid and aqueous components should preferably be sterile and pyrogen
free. After removing the lipid solvent by evaporation, the resulting gel
is converted to liposomes, with an encapsulation efficiency, for a
water-soluble drug, of up to 50%. The reverse phase evaporation vesicles
(REVs) have typical average sizes between about 2-4 microns and are
predominantly oligolamellar, that is, contain one or a few lipid bilayer
shells. The method is detailed in Example 1A.
The REVs are readily sized, as discussed below, by extrusion to give
oligolamellar vesicles having a maximum selected size preferably between
about 0.08 to 0.4 microns. Experiments conducted in support of the present
invention indicate that sized oligolamellar vesicles of this type show
substantially higher blood/MPS ratios than similar sized multilamellar
vesicles (MLVs), and that smaller REVs, e.g., 0.1 micron sizes, give
higher ratios than larger REVs, e.g., 0.4 microns. Another advantage of
REVs is the high ratio of encapsulated drug to lipid which is possible,
allowing greater drug doses to be administered in a given lipid dose.
MLVs, where desired, can be formed by simple lipid-film hydration
techniques. In this procedure, a mixture of liposome-forming lipids of the
type detailed above dissolved in a suitable solvent is evaporated in a
vessel to form a thin film, which is then covered by an aqueous medium.
The lipid film hydrates to form MLVs, typically with sizes between about
0.1 to 10 microns. These vesicles show relatively poor blood/MPS ratios,
compared with similar-composition REVs or SUVs, but do show the same
relationships between lipid composition and blood/MPS ratios. That is,
MLVs with the specified membrane-rigidifying and ganglioside components
give blood/MPS ratios which are substantially higher than the sum of the
ratios observed with MLVs containing one but not the other of the two
components. Like REVs, MLVs can be extruded to produce a suspension of
smaller, relatively homogeneous-size liposomes, in the 0.1-1.0, and
preferably 0.1-0.4 micron size range.
According to one important aspect of the invention, the circulation half
life of 0.4 micron REVs, having the above SM:PV:G.sub.M1 (4:1:0.35)
composition is substantially the same as that of SUVs having the same
composition. This contrasts with the findings from prior art studies on
ganglioside-containing liposomes, which show that smaller vessels,
especially SUVs, have substantially longer circulation times than larger
vesicles. In fact, experiments conducted in support of the present
invention indicate that liposomes containing G.sub.M1 but not SM show a
significant size effect, with SUVs being retained in circulation much
longer that 0.4 micron REVs. Thus, the lipid composition of the present
invention, comprising both G.sub.M1 and a membrane tightening component
such as SM, serves both to enhance blood circulation times severalfold,
and allow high circulation times to be achieved in larger vesicles, which
are generally preferred for therapeutic injection.
One effective sizing method for REVs and MLVs involves extruding an aqueous
suspension of the liposomes through a polycarbonate membrane having a
selected uniform pore size, typically 0.1, 0.2, or 0.4 microns (Szoka).
The pore size of the membrane corresponds roughly to the largest sizes of
liposomes produced by extrusion through that membrane, particularly where
the preparation is extruded two or more times through the same membrane.
This method of liposome sizing is used in preparing the 0.1 and 0.4 micron
REV compositions described in the examples below. As used herein, the
expressions "REVs sized to 0.1 micron" and "REVs sized to 0.4 microns"
refer to REV liposome compositions which have been sized by extrusion
through 0.1 and 0.4 micron pore size polycarbonate membranes,
respectively. A more recent method involves extrusion through an
asymmetric ceramic filter. The method is detailed in U.S. patent
application for Liposome Extrusion Method, Ser. No. 829,710, filed Feb.
13, 1986 and now U.S. Pat. No. 4,737,323.
Alternatively, the REV or MLV preparations can be treated to produce small
unilamellar vesicles (SUVs) which are characterized by sizes in the
0.04-0.08 micron range. However, as indicated above, SUVs have a
relatively small internal volume, for delivery of water-soluble drugs, and
they tend to fuse to form larger heterogeneous size liposomes with
heterodisperse drug leakage and MPS uptake characteristics, and are
leakier than REVs or MLVs. SUVs can be produced readily by homogenizing or
sonicating REVs or MLVs, as described in Example 1C.
After final sizing, the liposomes can be treated, if necessary, to remove
free (non-entrapped) drug. Conventional separation techniques, such as
centrifugation, diafiltration, and molecular-sieve chromatography are
suitable. The composition can be sterilized by filtration through a
conventional 0.45 micron depth filter.
II. UTILITY
The significantly increased circulation half life of liposomes constructed
as above can be exploited in two general types of therapeutic or
diagnostic liposome compositions. The first composition is designed for
sustained release of a liposome-associated agent into the bloodstream by
circulating liposomes. As seen above, liposomes constructed according to
the invention can be maintained predominantly in the bloodstream up to 24
hours, and therefore sustained released of the drug at physiologically
effective levels for up to about 1 day or more can be achieved. The second
composition is designed for concentrating a liposome-associated drug or
radiotracer material or the like via the bloodstream at a targeted non-MPS
tissue site, such as a tumor site. The extended lifetime of the liposomes
in the bloodstream makes it possible for a significant fraction of the
injected liposomes to reach the target site before being removed from the
bloodstream by the MPS.
A variety of drugs or other pharmacologically active agents are suitable
for delivery by the liposome composition. One general class of drugs
include water-soluble, liposome-permeable compounds which are
characterized by a tendency to partition preferentially into the aqueous
compartments of the liposome suspension, and to equilibrate, over time,
between the inner liposomal spaces and outer bulk phase of the suspension.
Representative drugs in this class include terbutaline, albuterol,
atropine methyl nitrate, cromolyn sodium, propranalol, flunoisolide,
ibuprofin, gentamycin, tobermycin, pentamidine, penicillin, theophylline,
bleomycin, etoposide, captoprel, n-acetyl cysteine, verapamil, vitamins,
and radio-opaque and particle-emitter agents, such as chelated metals.
Because of the tendency of these agents to equilibrate with the aqueous
composition of the medium, it is preferred to store the liposome
composition in lyophilized form, with rehydration shortly before
administration. Alternatively, the composition may be prepared in
concentrated form, and diluted shortly before administration. The latter
approach is detailed in U.S. patent application for "Liposome Concentrate
and Method", Ser. No. 860,528, filed May 7, 1986 and now abandoned.
A second general class of drugs are those which are water-soluble, but
liposome-impermeable. For the most part, these are peptide or protein
molecules, such as peptide hormones, enzymes, enzyme inhibitors,
apolipoproteins, and higher molecular weight carbohydrates are
characterized by long-term stability of encapsulation. Representative
compounds in this class include calcitonin, atriopeptin, .alpha.-1
antitrypsin (protease inhibitor), interferon, oxytocin, vasopressin,
insulin, interleukin-2, superoxide dismutase, tissue plasminogen activator
(TPA), plasma factor 8, epidermal growth factor, tumor necrosis factor,
lung surfactant protein, interferon, lipocortin, .alpha.-interferon and
erythropoetin.
A third class of drugs are lipophilic molecules which tend to partition
into the lipid bilayer phase of the liposomes, and which are therefore
associated with the liposomes predominantly in a membrane-entrapped form.
The drugs in this class are defined by an oil/water partition coefficient,
as measured in a standard oil/water mixture such as octanol/water, of
greater than 1 and preferably greater than about 5. Representative drugs
include prostaglandins, amphotericin B, progesterone, isosorbide
dinitrate, testosterone, nitroglycerin, estradiol, doxorubicin,
beclomethasone and esters, vitamin E, cortisone, dexamethasone and esters,
and betamethasone valerate.
For sustained drug-release via the bloodstream, the liposome composition is
administered intravenously in an amount which provides a suitable drug
dosage over the expected delivery time, typically 12-24 hours. The
injection may be given as a single bolus or slowly by i.v. drip, to allow
gradual dispersal of the liposomes from the site of injection.
Where it is desired to target the liposomes to a selected non-MPS tissue
site, the liposomes are preferably designed for surface recognition of
target-site molecules. For example, in the case of targeting to a solid
tumor, the liposomes may be prepared with surface-bound tumor recognition
molecules, such as antibodies directed against tumor-specific antigens.
Methods for coupling molecules of this type are well-known to those in the
field. These methods generally involve incorporation into the liposomes of
lipid components, such as phosphatidylethanolamine, which can be activated
for attachment of surface agents, or derivatized lipophilic compounds,
such as lipid-derivatized bleomycin.
In one particular liposome composition which is useful for radioimaging of
solid tumor regions, the liposomes are prepared with encapsulated
radio-opaque or particle-emission metal, typically in a chelated form
which substantially prevents permeation through the liposome bilayer, and
carrying surface-bound bleomycin molecules, for preferential liposome
attachment to tumor sites.
III. ASSESSING LIPID COMPOSITION FACTORS IN VITRO
The invention includes, in another aspect, a rapid in vitro method for
assessing the effect of various lipid composition factors on liposome
uptake by the MPS in vivo. This method is based on the discovery that that
liposome compositions which are most effective in increasing blood/MPS
values in vivo are also most effective in reducing uptake of the liposomes
by cultured MPS cells in vitro.
In practicing this aspect of the invention, MPS cells, and preferably
bone-marrow macrophages, such as murine bone-marrow macrophages, are
isolated and cultured according to standard cell-culture methods, such as
outlined in Example 7 below. Radiolabeled liposomes having the selected
lipid composition to be tested are added to the cells, and the uptake of
radioactivity by the cells measured. To date, three important parameters
of the system have been examined. These are liposome uptake
characteristics by the cultured cells (a) with increasing uptake times,
(b) with increasing amounts of liposome added, and (c) as a function of
liposome lipid composition.
FIG. 3 shows the uptake of liposomes by the cells at 37.degree. C. over a 4
hour culture period (solid circles). As seen, the extent of uptake, as
measured by nmol of liposome lipid/mg of cell protein, is substantially
linear over time. Non-specific uptake by the cells is measured at a cell
temperature, preferably about 4.degree. C., where endocytosis does not
occur. The solid squares in FIG. 3 shows non-specific uptake. Subtracting
the non-specific from the total uptake gives the specific uptake, shown in
open circles in the FIGURE.
FIG. 4 shows total (solid circles) and non-specific (open circles) uptake
levels with increasing amounts of added liposomes. The system is linearly
responsive to increasing liposome concentration, up to a maximum
concentration of about 0.8 nmol lipid/mg cell protein. The studies just
reported are detailed in Example 7.
The effect of brain SM on the in vitro uptake of egg PC liposomes was
examined, in a study reported in Example 8 below. The liposomes tested
contained increasing concentrations of SM, from 25 to 75 mole percent. As
seen from the data in Table 5 of the example, increasing amounts of SM
reduced cell uptake in vitro about 3 fold. The data is consistent with in
vivo data presented in Example 3, showing a severalfold increase in
blood/MPS ratios in vivo with addition of SM to egg PC liposomes.
The effect of increasing concentrations of G.sub.M1 on the in vitro uptake
of egg PC liposomes was similarly examined, in the study reported in
Example 9 below. The cell uptake data, which are plotted in FIG. 5, show
decreasing liposome uptake after 4 hours with increasing amounts of
G.sub.M1, up to a maximum G.sub.M1 concentration of about 10 mole percent.
It will be recalled from above that optimal G.sub.M1 concentrations in
PC:G.sub.M1 liposomes for in vivo uptake is between about 5-7 mole
percent.
A study of the combined effect of SM and G.sub.M1 on 0.1 oligolamellar PC
liposomes is also reported in Example 5. Here increasing amounts of
G.sub.M1 liposomes, up to about 15 mole percent, were added to SM:PC, 4:1
liposomes. The results of the study are shown in FIG. 6. As seen,
increasing G.sub.M1, up to 15 mole percent, decreased in vitro cellular
uptake, consistent with the in vivo study reported with respect to FIG. 2.
Studies conducted in support of the present invention also show a rapid
increase in cell uptake with increasing PS concentration in PC liposomes,
also consistent with the in vivo results presented above.
From the foregoing, it can be appreciated that the in vitro macrophage
culture system provides a simple, rapid system for testing lipid
composition factors which are important to liposome uptake by the MPS in
vivo. It is expected that the system can additionally be used to test the
effect of liposome size, lamellar structure | | |
