Patent Application: US-19050494-A

Abstract:
the present invention relates to a method of delivering antioxidants to cells and tissues and to compositions suitable for use therein . the invention also relates to methods of disease treatment involving the use of such compositions .

Description:
reactive oxygen species have been implicated in both the pathogenesis and altered physiologic responses of atherosclerosis . oxidation of ldl , a critical event in atheroma formation , is associated with enhanced cellular production of o 2 - ( 37 , 38 ). probucol &# 39 ; s efficacy in the prevention of intimal lesion development in watanabe hyperlipidemic rabbits is related to its antioxidant properties ( 39 ). in addition , the presence of oxidized ldl in atheromatous plaque correlates with progression of atherosclerotic cartoid disease ( 40 ). oxidized ldl has also been shown to directly inhibit ec - dependent vasorelaxation ( 41 , 42 ). generation of o 2 - in situ reduces endothelial - dependent relaxation in normal vessels ( 16 , 43 ) and may be involved in the abnormal ec - dependent relaxation in atherosclerosis ( 22 ). indirect measures of excess production of reactive oxygen species in hypercholesterolemia have been reported , including elevated levels of cholesterol oxides and oxidant - modified proteins ( 44 , 45 ). however , the precise role of oxidants and their critical reaction sites in the initiation of liproprotein oxidation and impairment of vascular function in atherosclerotic processes have not been previously defined . the data presented in the example below indicate that a cholesterol - enriched environment enhances vascular production of o 2 - . this excess o 2 - can then react with . no , reducting the vasoactive levels of . no , and diminishing the response to ec - dependent vasodilators . in this case , normal or even elevated levels of sod may be insufficient to effectively scavenge the excess o 2 - . this is suggested by the slightly higher endogenous levels of sod measured in chol - fed animals . the hypercholesterolemic state may lead to increased expression of vessel sod ( 45 , 46 ), but in the studies described below it is not significantly different from controls . impaired ec - dependent relaxation may then be due to enhanced reaction of . no with o 2 - , yielding onoo - ( 23 ). this reaction is rapid , with a rate constant of k = 6 . 7 × 10 9 m - 1 s - 1 , faster than both the sod - catalyzed and spontaneous dismutation of o 2 - to h 2 o 2 ( 47 ). at physiological ph , onoo - is protonated to peroxynitrous acid ( onooh ), yielding nitrogen dioxide (. no 2 ) and a molecule with - oh - like reactivity ( 23 , 48 ). onoo - has been observed to be a less potent activator of vsmc guanylate cyclase than . no . ec - dependent relaxation thus appears to be impaired in the present model due to diminished rates of cgmp formation . alternatively , the impaired relaxation may be the result of onoo - -- derived - oh , which stimulates guanylate cyclase activity to a lesser extent than . no ( 49 ). peroxynitrite exhibited potent oxidative effects of β - vldl , the principal carrier of cholesterol in this model . when compared to 5 μm cu 2 + , a commonly employed in vitro lipoprotein oxidant ( 50 ), onoo - rapidly generated greater quantities of lipid peroxidation products . similar results have been observed using the syndominine , sin - 1 , a compound yielding . no and o 2 - , which react to form onoo - ( 51 ). thus , the product of . no and o 2 - reaction appear to play a critical role in the initiation and extension of atherosclerotic lesions as well as the altered vascular reactivity associated with the disease . the studies described in the example also demonstrate that ph - sensitive liposomes are effective vectors for the delivery of antioxidant enzymes to the wall of both normal and atherosclerotic vessels . pharmacologic efficiency of lip - sod , as indicated by enzymatic analysis and changes in vascular function , contrasts with the minimal effect of native sod . it is not unexpected that native sod did little to restore ec - dependent relaxation . the 32k da sod is electrostatically repelled from cell surfaces at ph 7 . 4 , and is thus excluded from intracellular compartments , where significant extents of both the production and reactions of o 2 - and . no will occur . in the studies described below ph - sensitive liposomes facilitated delivery of sod to internal sites of o 2 - production , thus lowering steady state o 2 - concentration , and limiting o 2 - reaction with . no to yield onoo - . once injected intravenously , liposomes gain direct contact with the blood vessel wall and become incorporated via endocytosis . in the acidic environment of the endocome ( ph 5 . 0 ), the liposomal membrane undergoes a phase transition ( 52 ), promoting liposome - endosome fusion and release of liposomal contents to the cytosol . an increase in sod content was demonstrated in both control and chol - fed liposome - injected rabbits . parallel immunocytochemical quantitation of sod distribution reveals that sod gained access not only to ec , but vsmc and the interstitium as well . these locales are all critical sites of excess production of oxidants associated with the development and maintenance of atherosclerosis . thus , lip - sod delivery is particularly effective in raising tissue levels of the enzyme . use of polyethylene glycol - derivatized sod to enhance vascular endothelial antioxidant enzyme levels has been previously employed ( 53 ). however , higher doses of peg - sod ( 41 , 000 u / kg / d vs . 1 , 500 u / k / d ) were used to achieve similar results in an animal model of atherosclerosis ( 54 ). certain aspects of the invention are described in greater detail in the non - limiting example that follows . bovine cuzn sod was from diagnostic data inc . ( mountain view , calif .). dioleoyl - phosphatidylethanolamine and dioleoyl - glycero - 3 - succinate were obtained from avanti polar lipids ( birmingham , al .). l - arginine , ach , indomethacin , papaverine , phenylephrine and tetraethylammoniumhydroxide were from signa chemical co . ( st . louis , mo .). new zealand white rabbits ( 2 . 5 - 3 . 0 kg ) were maintained on rabbit chow containing 1 % cholesterol ( ralston purina , inc .) for 6 months prior to study ( chol - fed group ). age - and weight - matched controls were fed a standard diet . after exsanguination under ketamine / rompun anesthesia , vessels were isolated and changes in tension were measured in fermoral artery ring segments , as previously described ( 28 ). following maximal contraction with 70 mm kcl and recovery , phenylephrine was added to achieve 30 % of maximal tone . rings were then exposed to increasing doses of ach ; relaxation is reported as the percent decrease in pre - existing tone . after the generation of cumulative ach dose - response curves , rings were exposed to 3 × 10 - 5 m papaverine . in some experiments , rings from control and chol - fed rabbits were incubated with 3 mm l - arginine for 30 minutes prior to the administration of ach . in other studies , vessels were treated with native bovine sod ( 200 u / ml ) before measuring ach - induced relaxation . all studies were performed in the presence of 5 μm indomethacin . liposomes were composed of dioleoyl - phosphatidylethanolamine and dioleoyl - glycero - 3 - succinate ( 1 : 1 ). lipids were dried under n 2 nd hydrated 36 hr in 210 mm sucrose , 7 mm hepes . during hydration ph 8 . 5 was maintained with tetraethylammoniumhydroxide . lipids were added to sod , dissolved in sucrose - hepes buffer , and the mixture was extruded through a 600 nm filter under n 2 pressure ( extruder , lipex biomembranes ); mean liposome diameter was 217 nm . final sod concentration was 3 , 000 u / ml . liposomal - entrapped sod ( lip - sod ) was injected daily ( 1 , 500 u / kg ) via the marginal ear vein for 5 days before sacrifice . plasma cholesterol levels were determined using an enzymatic method ( 29 ) modified for 96 well plates . aortic sod activity was assayed in a 10 % homogenate in 50 mm kpi , 0 . 1 mm edta , 0 . 1 % chaps , ph 7 . 8 . after centrifugation at 10 , 000 × g × 10 min at 4 ° c ., supernatant sod activity was measured by inhibition of xanthine oxidase - mediated reduction of cytochrome c ( 30 ). tissue dna was measured by fluorescence ( 31 ). femoral ring segments were prepared for quantitative electron microscopic immunocytochemistry as previously ( 32 ). cryo - ultrathin sections were incubated with rabbit antibovine cuzn sod ( 1 : 100 dilution ) and 10 nm gold colloid conjugated to protein a . distribution of sod labeling was measured by counting gold granules and tissue points of randomly selected photographic fields . because of variable loss of endothelium during cryosectioning , ec sod density was quantitated in selected segments with intact endothelium . peroxynitrite was synthesized as previously ( 23 ). β - vldl , isolated as described ( 33 ), was exposed to onoo - or 5 μm cuso4 . lipid oxidation was assessed by measurement of thiobarbiturnc acid - reactive substances ( tbars ) and formation of conjugated dienes ( 25 ). plasma cholesterol was markedly elevated in rabbits fed a 1 % cholesterol diet for six months , from 43 ± 6 to 2696 ± 292 mg / dl . intimal thickening was apparent , with extensive plaque deposition , about 50 % of the luminal surface of the femoral artery . control and chol - fed groups exhibited no differences in the maximal tone generated with 70 mm kcl nor in the phenylephrine concentrations required to achieve submaximal contraction of vessels . chol - fed rabbits manifested a significant shift in the ec 50 to ach - induced relaxation : 1 . 73 × 10 - 6 m vs . 5 . 21 × 10 - 8 m ( p & lt ; 0 . 01 ) as well as a 33 % reduction in maximal response ( fig1 a ). incubation of ring segments with l - arginine failed to augment ach - induced relaxation in chol - fed rabbits ( fig1 b ). pretreatment with native cuzn sod had a minor effect on the ach dose - response profile in control , but not chol - fed rabbits ( fig2 a ). due to the limited cellular uptake of native sod , we delivered sod in vivo via ph - sensitive liposomes . vessels isolated from both control and chol - fed rabbits treated for 5 days with lip - sod demonstrated enhanced ach - induced relaxation ( fig2 b ). liposomal - sod restored ach - induced relaxation in chol - fed rabbits close to control values ( ec 50 ; 1 . 65 × 10 - 7 m ). in addition , the δec 50 for ach - induced relaxation was greater ( p & lt ; 0 . 01 ) in the chol - fed group than in the control group . further , lip - sod treatment enhanced the maximum relaxation of segments to ach in chol - fed rabbits . treatment of both control and chol - fed rabbits with empty ph - sensitive liposomes had no effect on ach responses . there was no difference between groups in the response to papaverine , an endothelial - independent vasodilator ; vessels relaxed below the initial vessel tone measured prior to phenylephrine administration : 114 . 52 %± 3 . 13 ( control ) vs . 116 . 35 %± 6 . 51 ( chol - fed ). lip - sod had no effect on responses to papaverine in either group . quantitative electron microscopic immunocytocheimistry assessed the distribution of sod in the blood vessel wall . lip - sod localized not only in the endothelium , but also in the subendothelial space and in underlying vsmc ( table 1 , fig3 ). a small degree of crossreactivity was evident between rabbit anti - bovine sod and endogenous rabbit sod . however , multivariate analysis of labeling densities showed significant increases in labeling in sections from lip - sod injected rabbit vessels compared with controls ( table 1 ). sod activity was similar in vessel homogenates from control and chol - fed rabbits ( table 1 ). liposomal - sod led to an almost 2 - fold increase in vessel sod activity in both groups . exposure of β - vldl to onoo - resulted in rapid generation of lipid oxidation products as indicated by both tbars and conjugated diene formation ( fig4 a and 4b ). control experiments demonstrated that up to 100 μm . no alone did not stimulate lipid peroxidation . table 1__________________________________________________________________________vascular enzymatic activity and tissue distribution of cuzn soddelivered via ph - sensitive liposomes . - sod liposomes + sod liposomesmeasurement control chol - fed control chol - fed__________________________________________________________________________enzymatic activity 0 . 92 ± 0 . 13 1 . 06 ± 0 . 43 1 . 73 ± 0 . 46 * 1 . 73 ± 0 . 55 * u sod / μg dnatissue distributionpoint density of colloidal gold particles (* 10 . sup .- 2 )( relative ratio of # immunogold particles / tissue point ) endothelium 0 . 70 ± 0 . 02 1 . 53 ± 0 . 27 ** smooth muscle 0 . 99 ± 0 . 08 1 . 88 ± 0 . 11 ** extracellular matrix 0 . 40 ± 0 . 12 1 . 18 ± 0 . 14 ** collagen 0 . 31 ± 0 . 06 1 . 16 ± 0 . 32 ** elastin 1 . 95 ± 0 . 16 2 . 43 ± 0 . 33__________________________________________________________________________ sod activity : mean ± sd of aortae from 3 animals from each group . morphometric data : mean ± sd from analysis of 4 sites from each treatment group . because there was no difference in sod distribution between control and cholfed groups , the groups were combined and analyzed for treatment with and without lipsod . 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( 1988 ) j . biol . chem . 263 , 6884 - 6892 . 54 . mugge , a ., elwell , j . e ., peterson , t . e ., hofmeyer , t . g ., heistad , d . d . & amp ; harrison , d . g . ( 1991 ) circ . res . 69 , 1293 - 1300 . ach , acetyichofine ; sod , superoxide dismutase ; β - vldl , β - very low density lipoprotein ; lip - sod , liposomal - entrapped sod ; edrf , endothelium - derived relaxing factor ; vsmc , vascular smooth muscle cell ; ec , endothelial cell ; ldl , low density lipoprotein ; chol - fed , rabbits fed a 1 % cholesterol diet ; tbars , thiobarbituric acid reactive substances .