Patent Abstract:
the use of ph - sensitive liposomes enhances the delivery of drugs that target the endoplasmic reticulum , reducing the required dosage compared to direct administration of the drug without such liposomes . in particular , antiviral compounds such as n - butyldeoxynojirnmycin can be used in lower amounts when administered in a ph - sensitive liposome .

Detailed Description:
unless otherwise specified , the words “ a ” or “ an ” mean “ one or more ”. the term “ treating ” means preventing , reducing or ameliorating the symptoms of a disease or a condition that could lead to a disease . one embodiment is a ph - sensitive liposome comprising a biologically active compound that acts upon or within the endoplasmic reticulum (“ er ”). a biologically active compound may be included in the liposome as a single active agent or in combination with other biologically active compounds . the biologically active compound is preferably an anti - viral compound or a combination of biologically active compounds useful for treating a disease , preferably a viral disease , and more preferably hepatitis . a preferred type of anti - viral compound is a compound that inhibits er n - glycosylation processing . a particularly preferred biologically active compound is n - butyldeoxynojirimycin (“ nb - dnj ”). the ph - sensitive liposome preferably comprises dioleoylphosphatidylethanolamine (“ dope ”) and cholesteryl hemisuccinate (“ chems ”). another embodiment is a method of sing a ph - sensitive liposome comprising a biologically active compound to treat a viral infection . preferably , the biologically active compound is a compound that acts upon or within the endoplasmic reticulum . more preferably , the biologically active compound is an anti - viral compound . the method preferably uses a ph - sensitive liposome which reduces the amount of anti - viral compound needed to achieve the same effect as a method which does not use such liposomes by a factor of at least 100 , more preferably at least 500 and most preferably at least 1000 . another embodiment is a method of using a ph - sensitive liposome comprising a biologically active compound that acts upon or within the endoplasmic reticulum to treat pigmentation diseases . another embodiment is a method of preparing a ph - sensitive liposome comprising a compound that modifies skin color , which can be included in a cosmetic composition . “ m +” refers to b16 - f1 mouse melanoma cells incubated in the presence of 0 . 5 mm free nb - dnj ; “ m + l ” refers to b16 - p1 mouse melanoma cells incubated in the presence of 0 . 5 mm nb - dnj included in ph - sensitive liposomes ; “ m + l 0 ” refers to b16 - f1 mouse melanoma cells incubated in the presence of “ empty ” ph - sensitive liposomes ; the present invention is further illustrated by , though in no way limited to , the following examples . to monitor the efficiency of encapsulated nb - dnj delivery in the cytoplasm of mammalian cells , b16 mouse melanoma cells were cultivated in the presence of various lipid formulations and the modifications in the activity of tyrosinase synthesized in these cells was assayed . encapsulation of nb - dnj in ph - sensitive liposomes reduces the required dose of nb - dnj by a factor of 1000 . lipids for liposome preparation — dioleoylphosphatidylethanolamine ( dope ), cholesterylhemisuccinate ( chems ), phosphatidylethanolamine ( pe ), phosphatidylcholine ( pc ), phosphatidylserine ( ps ) and cholesterol ( chol ) were purchased from sigma chemical co . ( st . louis , mo ., and u . s . a .). all other chemicals were analytical grade or the best grade available . b16 - f1 mouse melanoma cells ( european collection of animal cell culture , porton down , u . k .) were cultured in rpmi 1640 medium ( life technologies , paisley , scotland , and u . k .) as described in ref . [ 8 ]. cells were incubated with liposomes for 72 h at 37 ° c . and their viability was determined by using the trypan blue staining test . following the incubation time , cells were harvested , washed three times with pbs and lysed in 10 mm phosphate buffer , ph 6 . 8 containing 1 % nonidet p40 and proteinase inhibitors . liposomes were made according to the general method for small unilamellar vesicles ( suv ) preparation from multi - lamellar vesicles ( mlv ) ( ref . [ 15 ]). in particular , a mixture of the appropriate amounts of phospholipids from stock solutions in chloroform / methanol ( 95 : 5 ) was dried in a rotary evaporator under reduced pressure . dried lipids were hydrated with pbs containing different concentration of the inhibitor to be encapsulated . resulting mlv were sonicated in a bath - type sonicator ( at least 1 h , at room temperature ) followed by a probe sonicator ( about 10 min , with intervals , cooled in ice / water ) to form a stable emulsion . after sonication , metal particles from the probe and large mlv were removed by centrifugation ( 15 min , 12 000 rpm , 4 ° c .). the diameter of suv liposomes , as shown by transmission electron microscopy varied between 50 and 200 nm . liposomal formulations were filtered under sterile conditions through a 0 . 22 μm filter ( milipore ). in some experiments , liposomes were separated from free nb - dnj by ultracentrifugation at 159 , 000 g for 4 h . the amount of encapsulated nb - dnj was monitored with 14 c labelled nb - dnj . the inclusion yield was 1 - 5 % as calculated by experiments using labelled nb - dnj ( ref . [ 16 ]). tyrosinase activity was measured by the dopa oxidase assay that measures the conversion of dopa to dopachrome via dopa quinone . briefly , 50 μl of cell lysate was mixed with 950 μl of 1 mm dopa in 0 . 1 m sodium phosphate buffer , ph 7 . 2 at 37 ° c . and the reaction was followed spectrophotometrically at 475 nm by the chromogenic appearance of dopachroine from 1 mm dopa ( ref . [ 8 ]). to investigate the uptake of liposome encapsulated nb - dnj in living cells ,. the activity of tyrosinase was monitored , a constitutively expressed enzyme of melanoma cells , highly sensitive to small variations of the nb - dnj concentration in the culture medium ( ref . [ 8 ]). inhibition of n - glycosylation processing in the presence of nb - dnj yields a tyrosinase polypeptide caring glc3man7 - 9glcnac glycans . as a result , the interaction with the er chaperone calnexin is abolished and tyrosinase folding escapes the er quality control resulting in an enzyme devoided of its biological activity ( ref . [ 7 ]). a dramatic decrease of tyrosinase activity was observed when b16 mouse melanoma cells were cultivated in the presence of concentrations varying fom 0 . 5 mm - 5 mm nb - dnj ( ref . [ 6 ]), recommending tyrosinase as a sensitive probe to monitor the intracellular delivery of liposome encapsulated nb - dnj . five lipid compositions were tested generating two types of liposomes , ph sensitive ( s ) and ph insensitive ( i ): s 1 , consisting of phosphatidylethanolamine ( pe ) and cholesterylhemisuccinate ( chems ); s 2 , made of dioleoylphosphatidylethanolamirie ( dope ) and chems ; i 1 , consisting of phosphatidylcholine ( pc ) and cholesterol ( chol ); i 2 , prepared from pc , chol and phosphatidylserine ( ps ); i 3 , containing pc and chems ( table 1 ). liposomes prepared in 0 . 5 mm nb - dnj were added to the culture medium and b16 - f1 mouse melanoma cells were cultivated at 37 ° c . in 5 % co 2 for 72 h . as shown in fig1 when cells were cultured in the presence of nb - dnj included in ph - insensitive liposomes , tyrosinase activity was decreased to 62 % for pc / chol liposomes ( i 1 ) and to 51 % in the case of pc / chol / ps liposomes - entrapped inhibitor ( i 2 ). although the level of tyrosinase inhibition went down to 25 % for nb - dnj included in pc / chems liposomes ( i 3 ), any of the three ph insensitive liposomes formulations tested was able to produce the inhibition level obtained in the presence of free nb - dnj ( fig1 ). similar experiments were performed with different formulations of ph - sensitive liposomes . tyrosinase activity decreased by 85 % in cells incubated in the presence of ph - sensitive pe / chems liposomes including nb - dnj ( s 1 ). when cells were cultivated in the presence of ph - sensitive liposomes composed of dope / chems ( s 2 ) tyrosinase activity decreased by 92 %, recommending this liposomal formulation as the most efficient in the delivery of the glycosylation inhibitor . similar results have been obtained with liposomes purified by ultracentrifugation . usually , ph - insensitive liposomes are taken up by endocytosis , which limits the efficient delivery of intact encapsulated material to the cytosol . a partial delivery of nb - dnj into the cytoplasm could explain the ˜ 50 % inhibition of tyrosinase obtained with these liposomes as compared with the 98 % inhibition resulted following the treatment of the b16 - f1 cells with the drug included in ph - sensitive liposomes . chems included in the composition of either ph sensitive or ph insensitive liposomes confers a remarkable property of efficient cytoplasmic delivery , possibly due to its negative charge that facilitates their penetration through the plasma membrane . the higher efficiency observed when chems was used in conjunction with dope , in ph - sensitive liposomes , could be explained by the destabilization of ph sensitive liposomes due to the protonation of chems in the acidic endosomes , resulting in the release of the - imino sugars in the cytoplasm . total reversibility of tyrosinase activity was observed when nb - dnj containing liposomes were washed out of the culture ( fig2 ), confirming that liposome included nb - dnj induces reversible modifications in the cell , hence reinforcing the potential use of this iminosugar as a potential therapeutic drugs . recovery of tyrosinase activity is slower in liposome treated cells as compared to free drug treated cells ( 50 % recovery in 24 h time as compared to 75 % recovery for free nb - dnj ). to ensure that liposomes do not induce side effects to the cultured mammalian cells , their cytotoxicity was evaluated . the effect of a preferred lipid formulation ( ph - sensitive lihposomes dope / chems ) was evaluated using various lipid concentrations ( 25 - 1000 μm ). the results in fig3 show that cell viability was affected at high liposomal lipid concentration . the cytotoxicity gradually - appeared following the incubation in the presence of 250 μm lipid concentration and became significant after a 500 μm lipid concentration . it was established that a preferred lipid concentration in the culture medium that allows a cell viability of more than 90 %/ p was 100 μm . this lipid concentration was used in all liposomes formulations . as a second test , tyrosinase activity was monitored in b16 - f1 cells incubated with “ empty ” ph - sensitive liposomes in identical conditions . as shown in fig4 tyrosinase enzymatic activity in b16 - f1 cells is not affected by empty ph - sensitive liposomes . to evaluate the ph - sensitive liposomes ( dope / chems 6 : 4 ) efficiency in delivering reduced amounts of inhibitor that inactivate tyrosinase , several concentrations of nb - dnj were tested . b16 - f1 cells were incubated in the presence of different amounts of nb - dnj included in ph - sensitive liposomes and tyrosinase activity was assayed . the amount of the inhibitor included in ph - sensitive liposomes required for the er inactivation of tyrosinase was about 100 - 1000 times lower than the amount of free nb - dnj added in the culture medium ( fig5 ). higher dilutions of ab - dnj included in liposomes had no more effect on tyrosinase enzymatic activity ( fig5 ). similar results were obtained when two other iminosugars , deoxynojirimycin ( dnj ) and nonyl - deoxynojirimycin ( nonyl - dnj ) have been entrapped in dope / chems liposomes . as shown in fig6 tyrosinase activity was dramatically decreased when cells were treated with liposome included drugs as compared to the free compounds . these results show that the liposome formulations used in this study could be used for inclusion of various glycosylation inhibitors , with highly efficient intracellular delivery and displaying high stability in serum containing media . tyrosinase is the regulatory enzyme of melanogenesis in melanocytes . therefore , inactivation of tyrosinase is associated with lack of pigmentation due to the absence of melanin synthesis in specialized cells . to correlate the results on tyrosinase activity , with the melanin content , the pigmentation of the melanoma cells treated with liposomes was observed . visual inspection of untreated melanoma cells showed a normal level of pigmentation , whereas cells treated with 0 . 5 mm free nb - dnj and 0 . 5 μm liposome entrapped nb - dnj , respectively , showed a similar dramatic decrease in their pigmentation ( fig7 ). the lack of pigmentation in melanoma cells treated with minute amounts of liposomal nb - dnj correlates well with the abolishment of tyrosinase activity as a result of n - glycosylation processing inhibition , reinforcing the efficiency of the cytoplasmic delivery of the drug encapsulated in liposomes . thus , the present invention provides a liposomal system that offers a direct , effective and selective delivery of biologically active materials into cells . while the invention is not bound by its theory of operation , it is probable that liposomes adsorb to the cell surface by a mechanism that depends on liposome negative charge and are internalized into the cell , through the endocytic pathway . nb - dnj has been shown to be effective in retaining the hepatitis b virus inside the hepatic cells , thus preventing the multiplication of the virus . taking into account the potential use of nb - dnj in treating the hepatitis b , the encapsulation of the drug in liposomes could be used to target small and less toxic nb - dnj concentration to the cytoplasm and into the er of the hepatic cells . 3 . metha , a ., lu , x ., block , t ., blumberg , b . and dwek , r . a . 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( 2000 ) chem . rev . 100 , 4863 - 4696 10 . branza - nichita , n ., petrescu , a . j ., negroiu , g ., dwek , r . a ., petrescu , s . m . ( 2000 ), chem . rev . 100 , 4697 - 4711 . 11 . karlsson , g ., butters , t ., dwek , r . a . and platt , f . m . ( 1993 ) j . biol . chem . 268 , 570 - 576 . 13 . alving , c . r ., velinova , m ., regts , j . and scherpmof , g . l . ( 1997 ) hepatology 26 , 416 - 422 . 14 . lee , k .- d ., oh , y .- k ., portnoy , d . a . and swanson , j . a .- ( 1996 ) j . biol . chem . 271 , 7249 - 7252 . 15 . gregoriadis , g ., liposome preparation and related techniques in liposome technology , 2nd edition , crc press , boca raton , fla . 1993 it will be apparent to those skilled in the art that various modifications and variations can be made to the compositions , methods , and processes of this invention . thus , it is intended that the present invention cover such modifications and variations , provided they come within the scope of the appended claims and their equivalents . the disclosure of all publications cited above are expressly incorporated herein by reference in their entireties to the same extent as if each were incorporated by reference individually .