Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1994-05-03
2002-02-19
Lovering, Richard D. (Department: 1712)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C128SDIG003, C428S402200, C436S829000, C514S078000, C514S824000, C514S878000, C514S879000
Reexamination Certificate
active
06348213
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for reversing age-related changes in the lipid composition of heart muscle tissue and other age-related physiological characteristics.
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BACKGROUND OF THE INVENTION
One of the biochemical changes which occur with aging is a change in membrane lipid composition. In mammalian plasma membranes, the main variation occurs in the relative composition of phosphatidylcholine (PC). which decreases with age, and sphingomyelin (SM) and cholesterol, which increase with age (Barenholz). The changes in the relative amounts of PC and SM is especially great in tissues which have a low phospholipid turnover. For example, plasma membranes associated with the aorta and arterial wall show a 6-fold decrease in PC/SM ratio with aging. SM also increases in several diseases, including atherosclerosis. The SM content can be as high as 70-80% of the total phospholipids in advanced aortic lesion (Barenholz 1982, 1984).
The most striking differences between PC and SM derived from biological membranes are (a) the phase transition temperature of the lipids, and (b) the hydrogen-bonding character of the two lipids in a lipid-bilayer. Most sphingomyelins have transition temperatures in the physiological temperature range between 30° and 40° C., whereas most naturally occurring phosphatidylcholines are well above their transition temperature at 37° C. (Barenholz 1980, 1982, 1984). In terms of hydrogen bonding, the difference in the polar regions of these two lipids enables SM to be both a donor and acceptor of hydrogen in hydrogen bonding, while PC can only serve as a hydrogen donor.
Whether or not related to these differences, the relative content of PC to SM in mammalian plasma membranes appears to affect cell functioning significantly. The inventors and colleagues have recently reported on changes in the lipid composition and activity of primary rat myocytes in culture over time. Measurements of PC and SM content in the cells showed a decline of PC/SM ratio from 5 to about 2 in the first three days in culture, and from 2 to about 1 over the next 14 days in culture. The lipid composition changes were accompanied by a dramatic change in heart cell activity, as measured by the beating rate of the cultured cells. Between days 7 and 12 in culture, the beats/minute fell from about 160 to about 20, and significant increases in the activities of at least seven enzymes, expressed as Vmax/DNA, were also observed. One of these enzymes was creatine phosphokinase (CPK), which plays a major role in intracellular energy transport from mitochondria to myofibrils, and in the regulation of energy production coupled to energy utilization (Yechiel 1985a, 1985b).
The ratio of cholesterol to phospholipid also appears to be an important determinant in regulating the properties of biological membranes (Cooper 1977). Several studies have shown that certain properties of biological membranes can be altered by enrichment with or depletion of cholesterol (Borochov, Cooper 1978, Hasin). In general, there is a strong positive correlation between changes in SM and cholesterol levels in mammalian plasma membranes. That is, changes in the content of one are followed by changes in the other (Wallach, Barenholz 1982, 1984).
It is not clear how cells maintain the various lipid compositions in their different membranes, or why lipid composition changes with aging. In theory, lipid compositional changes could result from changes in the rates of synthesis or degradation of specific lipid components, or changes in the rate of lipid exchange or transfer between serum and the cell membrane. The latter mechanism has received considerable attention with regard to in vitro studies on lipid exchange in cultured biological cells. A number of studies have shown lipid exchange between biological membranes and artificial lipid bilayer vesicles or liposomes (Pagano, Martin, Cooper 1977, Frank). In general, phospholipid exchange between cells and liposomes is accelerated by the presence of a variety of phospholipid transfer proteins, including high and low density serum apolipoproteins (Wirtz, Kader). Cholesterol exchange between biological membranes and liposomes, and/or serum lipoprotein particles is also well known (Hasin, Grunze, Pal).
The ability to alter the lipid composition of biological cells by lipid exchange provides a means for studying the effect of lipid variation on cell function. For example, in the above-discussed myocyte culture system in which a decline in PC/SM ratio over time is accompanied by a drop in beating frequency, it can be asked whether (a) the original lipid composition of the cells can be restored by lipid exchange; and (b) if so, if original cell functioning, i.e., initial beating rate, is also restored. The inventors and coworkers have investigated this question, using small unilamellar PC liposomes as a vehicle for lipid exchange with the cells (Yechiel 1985a, 1985b). The study showed that lipid exchange increased both PC/SM and PC/cholesterol ratios, and thus reversed the normal lipid compositional changes which occur in the cultured cells over time. Interestingly, lipid exchange also restored cell beating frequency to its original levels, with the beating frequency showing a jump from about 20 to 160 within one day of cell exposure to the PC liposomes. Lipid exchange also led to a reduction in cellular enzymes, such as CPK, which were normally increase with time in culture.
SUMMARY OF THE INVENTION
According to an important aspect of the present invention, it has been discovered that PC-rich liposomes are able to reverse age-related changes in the lipid composition of heart muscle cells in animals which have received the liposomes by parenteral administration. One significant benefit of the liposome treatment is that the ability to withstand respiratory stress, which normally shows a gradual loss with increasing age (starting above the age of about 15 months in rats), is significantly improved. Furthermore, lipid exchange and concomitant improvement in respiratory hardiness are produ
Barenholz Yechezkel
Yechiel Elishalom
Foley & Lardner
Lovering Richard D.
Yissum Research and Development Co. of The Hebrew University of
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