Patent Application: US-40490203-A

Abstract:
the present invention provides methods , preparations , and uses of a variety of liposomal - digitalis glycoside compositions . the present invention also provides protein - stabilized nanoparticle formulations containing liposomal - digitalis glycosides such as oleandrin , digitoxin , and digoxin with reduced toxicity , high drug to lipid ratio , long circulating time in the bloodstream and the ability to deliver the drug to tumor sites . in another aspect , the present invention provides an effective method to reduce the growth of cancers or reduce the incidence of metastases .

Description:
it is understood as “ digitalis activity ” the ability to inhibit na + , k + - atpase through acting onto the digitalis receptor , along with the ability to display a positive inotropic effect . such an action is performed by several natural , semisynthetic and synthetic compounds ( thomas 1992 ). among the natural compounds , there are three groups : steroidal butenolides and pentadienolides , known as “ cardiotonic steroids ” or “ digitalic compounds ” and erythrophleum alkaloids . the word “ digitalis ” is often used as a generic word for all cardiotonic steroids ; similarly , the receptor for these compounds is generally known as “ digitalis receptor ”. digitalis glycosides or also called as digitalis - type glycosides or also called as cardiac glycosides are compounds bearing a steroidal genin or aglycone with one or several sugar molecules attached to position c - 3 . in the case of toad venom , sugar is replaced by suberylarginine . as used herein , the term “ micron ” refers to a unit of measure of one one - thousandth of a millimeter . as used herein , the term “ nm ” or the term “ nanometers ” refers to a unit of measure of one one - billionth of a meter . as used herein , the term “ biocompatible ” describes a substance that does not appreciably alter or affect in any adverse way , the biological system into which it is introduced . as used herein , the term “ substantially water insoluble pharmaceutical agent ” means biologically active chemical compounds which are poorly soluble or almost insoluble in water . examples of such compounds are paclitaxel , oleandrin , cyclosporine , digitoxin and the like . as used herein , the term “ cell - proliferative diseases ” is meant here to denote malignant as well as non - malignant cell populations which often appear morphologically to differ from the surrounding tissue . as discussed above , the present invention provides liposomal and psl nanoparticle formulations of digitalis glycosides and methods of preparing and employing such formulations . the advantages of these psl nanoparticle formulations are that a drug is entrapped in either dissolved or precipitated form . these compositions have been observed to provide a very low toxicity form of the pharmacologically active agent that can be delivered in the form of nanoparticles or suspensions by slow infusions or by bolus injection or by other parenteral or oral delivery routes . these psl nanoparticles have sizes below 400 nm , preferably below 200 nm , and more preferably below 120 nm having hydrophilic proteins coated onto the surface of the nanoparticles . the vesicle forming lipids such as , egg phosphatidylcholine ( epc ), hydrogenated soy phosphatidylcholine ( hspc ), phosphatidylethanolamine ( pe ), phosphatidylglycerol ( pg ), phosphatidylinsitol ( pi ), monosialogangolioside and spingomyelin ( spm ); the derivatized vesicle forming lipids such as poly ( ethylene glycol )- derivatized distearoylphosphatidylethanolamine ( peg - dspe ) and poly ( ethylene glycol )- derivatized cerarmides ( peg - cer ); and cholesterol are dissolved in organic solvents along with one or more digitalis glycoside . the phospholipids can be either synthetic or derived from natural sources such as egg or soy . the phospholipids can be distearoylphosphatidylcholine ( dspc ), dimyristoylphosphatidylcholine ( dmpc ), dimyristoylphosphatidylglycerol ( dmpg ), and dipalmitoylphosphatidylcholine ( dppc ). these lipids are dissolved in the organic solvent along with the digitalis glycoside , and the protein is dissolved in the aqueous phase . the organic phase is added to the aqueous phase and subjected to high shear stress . this results in a fine oil - in - water emulsion . evaporation of the solvent from the emulsion leads to the formation psl nanoparticles with a high digitalis glycoside to lipid - protein ratio ( wt / wt ). the drug to lipid - protein weight ratio varies between 0 . 01 and 1 , preferably between 0 . 05 and 1 . in order to make the protein stabilized liposomal nanoparticles , digitalis glycoside , lipid and other agents are dissolved in a suitable solvent ( e . g ., chloroform , methylene chloride , ethyl acetate , ethanol , tetrahydrofuran , dioxane , acetonitrile , acetone , dimethyl sulfoxide , dimethyl formamide , methyl pyrrolidinone , or the like , as well as mixtures of any two or more thereof ). additional solvents contemplated for use in the practice of the present invention include soybean oil , coconut oil , olive oil , safflower oil , cotton seed oil , sesame oil , orange oil , limonene oil , c1 - c20 alcohols , c2 - c20 esters , c3 - c20 ketones , polyethylene glycols , aliphatic hydrocarbons , aromatic hydrocarbons , halogenated hydrocarbons and combinations thereof . in the next stage , in order to make the protein stabilized liposomal nanoparticles , a protein ( e . g ., human serum albumin ) is added ( into the aqueous phase ) to act as a stabilizing agent for the formation of stable nanodroplets . protein is added at a concentration in the range of about 0 . 05 to 25 % ( w / v ), more preferably in the range of about 0 . 5 %- 5 % ( w / v ). in the next stage , in order to make the protein stabilized liposomal nanoparticles , an emulsion is formed by homogenization under high pressure and high shear forces . such homogenization is conveniently carried out in a high pressure homogenizer , typically operated at pressures in the range of about 3 , 000 up to 30 , 000 psi . preferably , such processes are carried out at pressures in the range of about 6 , 000 up to 25 , 000 psi . the resulting emulsion comprises very small nanodroplets of the nonaqueous solvent containing the digitalis glycoside , lipid and other agents . acceptable methods of homogenization include processes imparting high shear and cavitation such as high pressure homogenization , high shear mixers , sonication , high shear impellers , and the like . finally , in order to make the protein stabilized liposomal nanoparticles , the solvent is evaporated under reduced pressure to yield a colloidal system composed of protein stabilized liposomal nanoparticles of digitalis glycoside in liposome and protein . acceptable methods of evaporation include the use of rotary evaporators , falling film evaporators , spray driers , freeze driers , and the like . following evaporation of solvent , the liquid suspension may be dried to obtain a powder containing the pharmacologically active agent and protein . the resulting powder can be redispersed at any convenient time into a suitable aqueous medium such as saline , buffered saline , water , buffered aqueous media , solutions of amino acids , solutions of vitamins , solutions of carbohydrates , or the like , as well as combinations of any two or more thereof , to obtain a suspension that can be administered to mammals . methods contemplated for obtaining this powder include freeze - drying , spray drying , and the like . in accordance with a specific embodiment of the present invention , there is provided a method for the formation of unusually small submicron liposomal particles containing digitalis gfycoside , i . e ., particles which are less than 200 nanometers in diameter . such particles are capable of being sterile - filtered before use in the form of a liquid suspension . the ability to sterile - filter the end product of the invention formulation process ( i . e ., the drug particles ) is of great importance since it is impossible to sterilize dispersions which contain high concentrations of protein ( e . g ., serum albumin ) by conventional means such as autoclaving . in order to obtain sterile - filterable protein stabilized liposomal particles of digitalis glycosides ( i . e ., particles & lt ; 200 nm ), the digitalis glycoside , lipids and other agents are initially dissolved in a substantially water immiscible organic solvent ( e . g ., a solvent having less than about 5 % solubility in water , such as , for example , chloroform ) at high concentration , thereby forming an oil phase containing the digitalis glycoside , lipids and other agents . suitable solvents are set forth above . next , a water miscible organic solvent ( e . g ., a solvent having greater than about 10 % solubility in water , such as , for example , ethanol ) is added to the oil phase at a final concentration in the range of about 1 %- 99 % v / v , more preferably in the range of about 5 %- 25 % v / v of the total organic phase . the water miscible organic solvent can be selected from such solvents as ethyl acetate , ethanol , tetrahydrofuran , dioxane , acetonitrile , acetone , dimethyl sulfoxide , dimethyl formamide , methyl pyrrolidinone , and the like . alternatively , the mixture of water immiscible solvent with the water miscible solvent is prepared first , followed by dissolution of the digitalis glycoside , lipids and other agents in the mixture . in the next stage , in order to make the protein stabilized liposomal nanoparticles of digitalis glycosides , human serum albumin or any other suitable stabilizing agent as described above is dissolved in aqueous media . this component acts as a stabilizing agent for the formation of stable nanodroplets . optionally , a sufficient amount of the first organic solvent ( e . g . chloroform ) is dissolved in the aqueous phase to bring it close to the saturation concentration . a separate , measured amount of the organic phase ( which now contains the digitalis glycosides , the first organic solvent and the second organic solvent ) is added to the saturated aqueous phase , so that the phase fraction of the organic phase is between about 0 . 5 %- 015 % v / v , and more preferably between 1 % and 8 % v / v . next , a mixture composed of micro and nanodroplets is formed by homogenization at low shear forces . this can be accomplished in a variety of ways , as can readily be identified by those of skill in the art , employing , for example , a conventional laboratory homogenizer operated in the range of about 2 , 000 up to about 15 , 000 rpm . this is followed by homogenization under high pressure ( i . e ., in the range of about 3 , 000 up to 30 , 000 psi ). the resulting mixture comprises an aqueous protein solution ( e . g ., human serum albumin ), the digitalis glycoside , lipids , other agents , the first solvent and the second solvent . finally , solvent is rapidly evaporated under vacuum to yield a colloidal dispersion system ( liposomal digitalis glycoside and protein ) in the form of extremely small nanoparticles ( i . e ., particles in the range of about 50 nm - 200 nm diameter ), and thus can be sterile - filtered . the preferred size range of the particles is between about 50 nm - 170 nm , depending on the formulation and operational parameters . the protein stabilized liposomal nanoparticles prepared in accordance with the present invention may be further converted into powder form by removal of the water therefrom , e . g ., by lyophilization at a suitable temperature - time profile . the protein ( e . g ., human serum albumin ) itself acts as a cryoprotectant , and the powder is easily reconstituted by addition of water , saline or buffer , without the need to use such conventional cryoprotectants as mannitol , sucrose , glycine , and the like . while not required , it is of course understood that conventional cryoprotectants may be added to invention formulations if so desired . the liposomal shell containing digitalis glycoside allows for the delivery of high doses of the pharmacologically active agent in relatively small volumes . according to this embodiment of the present invention , the liposome containing digitalis glycoside has a cross - sectional diameter of no greater than about 10 microns . a cross - sectional diameter of less than 5 microns is more preferred , while a cross - sectional diameter of less than 1 micron is presently the most preferred for the intravenous route of administration . proteins contemplated for use as stabilizing agents in accordance with the present invention include albumins ( which contain 35 cysteine residues ), immunoglobulins , caseins , insulins ( which contain 6 cysteines ), hemoglobins ( which contain 6 cysteine residues per α2 β2 unit ), lysozymes ( which contain 8 cysteine residues ), immunoglobulins , α - 2 - macroglobulin , fibronectins , vitronectins , fibrinogens , lipases , and the like . proteins , peptides , enzymes , antibodies and combinations thereof , are general classes of stabilizers contemplated for use in the present invention . a presently preferred protein for use is albumin . specific antibodies may also be utilized to target the nanoparticles to specific locations . in the preparation of invention compositions , a wide variety of organic media can be employed to suspend or dissolve the substantially water insoluble digitalis glycosides . organic media contemplated for use in the practice of the present invention include any nonaqueous liquid that is capable of suspending or dissolving the pharmacologically active agent , but does not chemically react with either the polymer employed to produce the shell , or the pharmacologically active agent itself . examples include vegetable oils ( e . g ., soybean oil , olive oil , and the like ), coconut oil , safflower oil , cotton seed oil , sesame oil , orange oil , limonene oil , aliphatic , cycloaliphatic , or aromatic hydrocarbons having 4 - 30 carbon atoms ( e . g ., n - dodecane , n - decane , n - hexane , cyclohexane , toluene , benzene , and the like ), aliphatic or aromatic alcohols having 2 - 30 carbon atoms ( e . g ., octanol , and the like ), aliphatic or aromatic esters having 2 - 30 carbon atoms ( e . g ., ethyl caprylate ( octanoate ), and the like ), alkyl , aryl , or cyclic ethers having 2 - 30 carbon atoms ( e . g ., diethyl ether , tetrahydrofuran , and the like ), alkyl or aryl halides having 1 - 30 carbon atoms ( and optionally more than one halogen substituent , e . g ., ch 3 cl , ch 2 cl 2 , ch 2 cl — ch 2 cl , and the like ), ketones having 3 - 30 carbon atoms ( e . g ., acetone , methyl ethyl ketone , and the like ), polyalkylene glycols ( e . g ., polyethylene glycol , and the like ), or combinations of any two or more thereof . especially preferred combinations of organic media contemplated for use in the practice of the present invention typically have a boiling point of no greater than about 200 ° c ., and include volatile liquids such as dichloromethane , chloroform , ethyl acetate , benzene , and the like ( i . e ., solvents that have a high degree of solubility for the pharmacologically active agent , and are soluble in the other organic medium employed ), along with a higher molecular weight ( less volatile ) organic medium . when added to the other organic medium , these volatile additives help to drive the solubility of the pharmacologically active agent into the organic medium . this is desirable since this step is usually time consuming . following dissolution , the volatile component may be removed by evaporation ( optionally under vacuum ). the liposomes containing digitalis glycoside stabilized with protein , prepared as described above , are delivered as a suspension in a biocompatible aqueous liquid . this liquid may be selected from water , saline , a solution containing appropriate buffers , a solution containing nutritional agents such as amino acids , sugars , proteins , carbohydrates , vitamins or fat , and the like . for increasing the long - term storage stability , the psl nanoparticle formulations may be frozen and lyophilized in the presence of one or more protective agents such as sucrose , mannitol , trehalose or the like . upon rehydration of the lyophilized psl nanoparticle formulations , the suspension retains essentially all the drug previously loaded and the particle size . the rehydration is accomplished by simply adding purified or sterile water or 0 . 9 % sodium chloride injection or 5 % dextrose solution followed by gentle swirling of the suspension . the potency of the drug in a psl nanoparticle formulation is not lost after lyophilization and reconstitution . the psl nanoparticle formulation of the present invention is shown to be less toxic than the drug administered in its free form . determination of toxicity in mice has shown about 1 - to 20 - fold decrease in acute ld 50 values for psl oleandrin nanoparticle formulations as compared to the free oleandrin . the ld 50 values are dependent on the lipid and protein compositions . furthermore , psl nanoparticle formulations containing oleandrin exhibit 1 to 100 - fold decrease in toxicity as compared to the drug in its free form . psl nanoparticle formulations with low ld 50 values show low drug accumulation levels in heart , lung and kidney tissues . although administration psl nanoparticle formulations lead to their uptake by liver , acute liver damage is not observed . in order to make the protein stabilized nanoparticles without the lipids , digitalis glycoside is dissolved in a suitable solvent ( e . g ., chloroform , methylene chloride , ethyl acetate , ethanol , tetrahydrofuran , dioxane , acetonitrile , acetone , dimethyl sulfoxide , dimethyl formamide , methyl pyrrolidinone , or the like , as well as mixtures of any two or more thereof ). additional solvents contemplated for use in the practice of the present invention include soybean oil , coconut oil , olive oil , safflower oil , cotton seed oil , sesame oil , orange oil , limonene oil , c1 - c20 alcohols , c2 - c20 esters , c3 - c20 ketones , polyethylene glycols , aliphatic hydrocarbons , aromatic hydrocarbons , halogenated hydrocarbons and combinations thereof . unlike conventional methods for nanoparticle formation , a polymer ( e . g . polylactic acid ) is not dissolved in the solvent . the oil phase employed in the preparation of invention compositions contains only the digitalis like molecules dissolved in solvent . next , in order to make the protein stabilized nanoparticles , a protein ( e . g ., human serum albumin ) is added ( into the aqueous phase ) to act as a stabilizing agent for the formation of stable nanodroplets . protein is added at a concentration in the range of about 0 . 05 to 25 % ( w / v ), more preferably in the range of about 0 . 5 %- 5 % ( w / v ). unlike conventional methods for nanoparticle formation , no surfactant ( e . g . sodium lauryl sulfate , lecithin , tween 80 , pluronic f - 68 and the like ) is added to the mixture . next , in order to make the protein stabilized nanoparticles , an emulsion is formed by homogenization under high pressure and high shear forces . such homogenization is conveniently carried out in a high pressure homogenizer , typically operated at pressures in the range of about 3 , 000 up to 30 , 000 psi . preferably , such processes are carried out at pressures in the range of about 6 , 000 up to 25 , 000 psi . the resulting emulsion comprises very small nanodroplets of the nonaqueous solvent ( containing the dissolved pharmacologically active agent ) and very small nanodroplets of the protein stabilizing agent . acceptable methods of homogenization include processes imparting high shear and cavitation such as high pressure homogenization , high shear mixers , sonication , high shear impellers , and the like . finally , in order to make the protein stabilized nanoparticles , the solvent is evaporated under reduced pressure to yield a colloidal system composed of protein stabilized nanoparticles of pharmacologically active agent and protein . acceptable methods of evaporation include the use of rotary evaporators , falling film evaporators , spray driers , freeze driers , and the like . following evaporation of solvent , the liquid suspension may be dried to obtain a powder containing the pharmacologically active agent and protein . the resulting powder can be redispersed at any convenient time into a suitable aqueous medium such as saline , buffered saline , water , buffered aqueous media , solutions of amino acids , solutions of vitamins , solutions of carbohydrates , or the like , as well as combinations of any two or more thereof , to obtain a suspension that can be administered to mammals . methods contemplated for obtaining this powder include freeze - drying , spray drying , and the like . in accordance with a specific embodiment of the present invention , there is provided a method for the formation of unusually small submicron particles of digitalis glycosides ( nanoparticles ), i . e ., particles which are less than 200 nanometers in diameter . such particles are capable of being sterile - filtered before use in the form of a liquid suspension . the ability to sterile - filter the end product of the invention formulation process ( i . e ., the drug particles ) is of great importance since it is impossible to sterilize dispersions which contain high concentrations of protein ( e . g ., serum albumin ) by conventional means such as autoclaving . in order to obtain sterile - filterable particles of digitalis glycosides ( i . e ., particles & lt ; 200 nm ), the pharmacologically active agent is initially dissolved in a substantially water immiscible organic solvent ( e . g ., a solvent having less than about 5 % solubility in water , such as , for example , chloroform ) at high concentration , thereby forming an oil phase containing the pharmacologically active agent . suitable solvents are set forth above . unlike conventional methods for nanoparticle formation , a polymer ( e . g . polylactic acid ) is not dissolved in the solvent . the oil phase employed in the process of the present invention contains only the pharmacologically active agent dissolved in solvent . next , a water miscible organic solvent ( e . g ., a solvent having greater than about 10 % solubility in water , such as , for example , ethanol ) is added to the oil phase at a final concentration in the range of about 1 %- 99 % v / v , more preferably in the range of about 5 %- 25 % v / v of the total organic phase . the water miscible organic solvent can be selected from such solvents as ethyl acetate , ethanol , tetrahydrofuran , dioxane , acetonitrile , acetone , dimethyl sulfoxide , dimethyl formamide , methyl pyrrolidinone , and the like . alternatively , the mixture of water immiscible solvent with the water miscible solvent is prepared first , followed by dissolution of the pharmaceutically active agent in the mixture . next , in order to make the nanoparticles of digitalis glycosides , human serum albumin or any other suitable stabilizing agent as described above is dissolved in aqueous media . this component acts as a stabilizing agent for the formation of stable nanodroplets . optionally , a sufficient amount of the first organic solvent ( e . g . chloroform ) is dissolved in the aqueous phase to bring it close to the saturation concentration . a separate , measured amount of the organic phase ( which now contains the digitalis glycosides , the first organic solvent and the second organic solvent ) is added to the saturated aqueous phase , so that the phase fraction of the organic phase is between about 0 . 5 %- 015 % v / v , and more preferably between 1 % and 8 % v / v . next , a mixture composed of micro and nanodroplets is formed by homogenization at low shear forces . this can be accomplished in a variety of ways , as can readily be identified by those of skill in the art , employing , for example , a conventional laboratory homogenizer operated in the range of about 2 , 000 up to about 15 , 000 rpm . this is followed by homogenization under high pressure ( i . e ., in the range of about 3 , 000 up to 30 , 000 psi ). the resulting mixture comprises an aqueous protein solution ( e . g ., human serum albumin ), the water insoluble digitalis glycosides , the first solvent and the second solvent . finally , solvent is rapidly evaporated under vacuum to yield a colloidal dispersion system ( digitalis glycosides and protein ) in the form of extremely small nanoparticles ( i . e ., particles in the range of about 10 nm - 200 nm diameter ), and thus can be sterile - filtered . the preferred size range of the particles is between about 50 nm - 170 nm , depending on the formulation and operational parameters . colloidal systems prepared in accordance with the present invention may be further converted into powder form by removal of the water therefrom , e . g ., by lyophilization at a suitable temperature - time profile . the protein ( e . g ., human serum albumin ) itself acts as a cryoprotectant , and the powder is easily reconstituted by addition of water , saline or buffer , without the need to use such conventional cryoprotectants as mannitol , sucrose , glycine , and the like . while not required , it is of course understood that conventional cryoprotectants may be added to invention formulations if so desired . the polymeric shell containing solid or liquid cores of digitalis glycosides allows for the delivery of high doses of the pharmacologically active agent in relatively small volumes . in addition , the walls of the polymeric shell or coating are generally completely degradable in vivo by proteolytic enzymes ( e . g ., when the polymer is a protein ), resulting in no side effects from the delivery system as is the case with current formulations . according to this embodiment of the present invention , particles of substantially water insoluble digitalis glycosides have a cross - sectional diameter of no greater than about 10 microns . a cross - sectional diameter of less than 5 microns is more preferred , while a cross - sectional diameter of less than 1 micron is presently the most preferred for the intravenous route of administration . proteins contemplated for use as stabilizing agents in accordance with the present invention include albumins ( which contain 35 cysteine residues ), immunoglobulins , caseins , insulins ( which contain 6 cysteines ), hemoglobins ( which contain 6 cysteine residues per α2 β2 unit ), lysozymes ( which contain 8 cysteine residues ), immunoglobulins , α - 2 - macroglobulin , fibronectins , vitronectins , fibrinogens , lipases , and the like . proteins , peptides , enzymes , antibodies and combinations thereof , are general classes of stabilizers contemplated for use in the present invention . a presently preferred protein for use in the formation of a polymeric shell is albumin . optionally , proteins such as α - 2 - macroglobulin , a known opsonin , could be used to enhance uptake of the shell encased particles of substantially water insoluble pharmacologically active agents by macrophage - like cells , or to enhance the uptake of the shell encased particles into the liver and spleen . specific antibodies may also be utilized to target the nanoparticles to specific locations . in the preparation of invention compositions , a wide variety of organic media can be employed to suspend or dissolve the substantially water insoluble digitalis glycosides . organic media contemplated for use in the practice of the present invention include any nonaqueous liquid that is capable of suspending or dissolving the pharmacologically active agent , but does not chemically react with either the polymer employed to produce the shell , or the pharmacologically active agent itself . examples include vegetable oils ( e . g ., soybean oil , olive oil , and the like ), coconut oil , safflower oil , cotton seed oil , sesame oil , orange oil , limonene oil , aliphatic , cycloaliphatic , or aromatic hydrocarbons having 4 - 30 carbon atoms ( e . g ., n - dodecane , n - decane , n - hexane , cyclohexane , toluene , benzene , and the like ), aliphatic or aromatic alcohols having 2 - 30 carbon atoms ( e . g ., octanol , and the like ), aliphatic or aromatic esters having 2 - 30 carbon atoms ( e . g ., ethyl caprylate ( octanoate ), and the like ), alkyl , aryl , or cyclic ethers having 2 - 30 carbon atoms ( e . g ., diethyl ether , tetrahydrofuran , and the like ), alkyl or aryl halides having 1 - 30 carbon atoms ( and optionally more than one halogen substituent , e . g ., ch 3 cl , ch 2 cl 2 , ch 2 cl — ch 2 cl , and the like ), ketones having 3 - 30 carbon atoms ( e . g ., acetone , methyl ethyl ketone , and the like ), polyalkylene glycols ( e . g ., polyethylene glycol , and the like ), or combinations of any two or more thereof . especially preferred combinations of organic media contemplated for use in the practice of the present invention typically have a boiling point of no greater than about 200 ° c ., and include volatile liquids such as dichloromethane , chloroform , ethyl acetate , benzene , and the like ( i . e ., solvents that have a high degree of solubility for the pharmacologically active agent , and are soluble in the other organic medium employed ), along with a higher molecular weight ( less volatile ) organic medium . when added to the other organic medium , these volatile additives help to drive the solubility of the pharmacologically active agent into the organic medium . this is desirable since this step is usually time consuming . following dissolution , the volatile component may be removed by evaporation ( optionally under vacuum ). particles of pharmacologically active agent associated with a polymeric shell , prepared as described above , are delivered as a suspension in a biocompatible aqueous liquid . this liquid may be selected from water , saline , a solution containing appropriate buffers , a solution containing nutritional agents such as amino acids , sugars , proteins , carbohydrates , vitamins or fat , and the like . in the present invention , efficacy of psl nanoparticle formulations of the present invention with varying lipid compositions , particle size , and drug to lipid - protein ratio have been investigated on various systems such as human cell lines and animal models for cell proliferative activities . furthermore , effects of psl nanoparticle formulations and various drugs in their free form on the body weight of mice with different sarcomas and healthy mice without tumor have been investigated . effects of psl nanoparticle formulations and various drugs in their free form on the dna fragmentation in different normal and tumor cells are investigated . these examples are not intended , however , to limit or restrict the scope of the present invention in any way and should not be construed as providing conditions , parameters , reagents , or starting materials which must be utilized exclusively in order to practice the art of the present invention . it is known that certain anionic polysaccharides ( baba , 1988 ), such as dextran sulphate , pustulan sulphate stimulate cell - mediated t - cell dependent immune responses without stimulating anti - body mediated immune responses that are b - cell dependent . on the other hand , unmodified polysaccharides stimulate only b - cells and certain other polysaccharides are known to stimulate both t - cell and b - cell responses under certain conditions . the polysaccharides present in water extract of the plant nerium oleander has been shown to contain galacturonic acids similar to pectin . these polysaccharides are claimed to be immune stimulants . thus the formulations of the present inventions can contain suitable polysaccharides such as pectin to provide the stimulant effect . compositions employing the novel compounds will contain a biologically effective amount of the compounds . as used herein a biologically effective amount of a compound or composition refers to an amount effective to alter , modulate or reduce tumor growth or related conditions . for intravenous administration , a satisfactory result may be obtained employing the compounds in an amount within the range of from about 0 . 001 mg / kg to about 5 mg / kg , preferably from about 0 . 002 mg / kg to about 2 mg / kg and more preferably from about 0 . 004 mg / kg to about 0 . 5 mg / kg alone or in combination with one or more additional anti - tumor compounds in an amount within the range from about 0 . 01 mg / kg to about 50 mg / kg , preferably from about 0 . 05 mg / kg to about 20 mg / kg and more preferably from about 0 . 1 mg / kg to about 10 mg / kg both being employed together in the same intravenous dosage form or in separate oral or intramuscular or intravenous dosage forms taken at the same time . the percentage of the compositions and preparations may , of course , be varied and may conveniently be between about 0 . 1 to about 50 % of the weight of the unit . the amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained . the pharmaceutical formulations of oleandrin according to the present invention offer several advantages over the existing formulation of nerium oleander extract administered parenterally . they can be intravenously administered and relatively high concentrations of oleandrin can be loaded into patients . thus the frequency of dosage can be reduced . thus within the spirit , the invention is related to improved formulations and methods of using the same when administering such formulations to patients . as mentioned herein above a number of excipients may be appropriate for use in the formulation which comprise the composition according to the present invention . the inclusion of excipients and the optimization of their concentration for their characteristics such as for example ease of handling or carrier agents will be understood by those ordinarily skilled in the art not to depart from the spirit of the invention as described herein and claimed herein below . the invention will now be further described with reference to the following examples . these examples are intended to be merely illustrative of the invention and are not intended to be limiting . a lipid mixture containing hspc : cholesterol : peg2000 - dspe in a molar ratio of 55 : 40 : 5 was dissolved in a chloroform : ethanol ( 8 . 5 . : 1 . 5 vol / vol ) mixture . for example , 3 . 55 g of hspc , 1 . 39 g cholesterol and 1 . 33 g peg2000 - dspe were dissolved in 30 ml of chloroform : ethanol ( 8 . 5 : 1 . 5 vol / vol ) mixture . a chloroform - ethanol solution of oleandrin , in the range of 50 - 150 mg / ml was added to the above solution , resulting in a drug to lipid ratio of 1 : 10 ( wt / wt ). the above organic solution was added to the aqueous phase , with a ph of 8 . 0 - 8 . 5 , while mixing from 3000 to 10000 rpm . the mixture was subjected to either high - pressure microfluidization or homogenization . the pressure was varied between 20 , 000 and 30 , 000 psi . this resulted in a homogeneous and extremely fine oil - in - water emulsion . the emulsion was rapidly evaporated in an evaporator to a nanoparticle suspension . the evaporator pressure and the bath temperature during evaporation were 10 - 50 mm hg and 30 - 70 ° c ., respectively . the particle size of the suspension was determined by photon correlation spectroscopy with the malvern zetasizer . the suspension was sterile - filtered through a 0 . 22 μm filter . the particle size of the suspension was between 30 and 220 nm . the suspension was frozen below − 40 ° c . and lyophilized . the lyophilized cake was reconstituted prior to further use . the particle size did not change appreciably following lyophilization and reconstitution . in a similar manner , the liposomal formulations of neriifolin , odoroside a , odoroside h and proscillaridin a were prepared . a lipid mixture containing hspc : cholesterol : peg2000 - dspe in a molar ratio of 55 : 40 : 5 was dissolved in a chloroform : ethanol ( 8 . 5 . : 1 . 5 vol / vol ) mixture . for example , 3 . 55 g of hspc , 1 . 39 g cholesterol and 1 . 33 g peg2000 - dspe were dissolved in 30 ml of chloroform : ethanol ( 8 . 5 : 1 . 5 vol / vol ) mixture . a chloroform - ethanol solution of oleandrin , in the range of 100 - 200 mg / ml was added to the above solution , resulting in a drug to lipid - protein ratio of 1 : 10 ( wt / wt ). a 1 - 10 % human albumin solution was prepared . the ph of the solution was adjusted to 7 . 4 . the above organic solution was added to the albumin phase while mixing from 3000 to 10000 rpm . the mixture was subjected to either high - pressure microfluidization or homogenization . the pressure was varied between 20 , 000 and 30 , 000 psi . this resulted in a homogeneous and extremely fine oil - in - water emulsion . the emulsion was rapidly evaporated in an evaporator to a nanoparticle suspension . the evaporator pressure and the bath temperature during evaporation were 10 - 50 mm hg and 30 - 70 ° c ., respectively . the particle size of the suspension was determined by photon correlation spectroscopy with the malvern zetasizer . the suspension was sterile - filtered through a 0 . 22 μm filter . the particle size of the suspension was between 30 and 220 nm . the suspension was frozen below − 40 ° c . and lyophilized . the lyophilized cake was reconstituted prior to further use . the particle size did not change appreciably following lyophilization and reconstitution . in a similar manner , the psl formulations ofneriifolin , odoroside a , odoroside h and proscillaridin a were prepared . lipid mixtures ( egg sphingomyelin : phosphatidylcholine : cholesterol : peg2000 - dspe = 1 : 1 : 1 : 0 . 02 molar ratio ) were dissolved in a chloroform : ethanol ( 9 . 5 : 0 . 05 vol / vol ) mixture . a chloroform - ethanol solution of oleandrin , in the range of 100 - 200 mg / ml was added to the above solution , resulting in a drug to lipid ratio of 1 : 10 ( wt / wt ). the above procedure described in example 2 was employed to prepare psl - oleandrin . the particle size of the suspension before lyophilization and after reconstitution was between 50 and 220 nm . the above procedure described in example 2 was employed to prepare psl - oleandrin formulation . however , instead of the lipid , peg2000 - dspe , peg2000 - ceramide was used . the particle size of the suspension before lyophilization and after reconstitution was between 50 and 220 nm . the above procedure described in example 2 was employed to prepare psl - oleandrin . lipid mixtures ( distearylphosphatidylcholine : cholesterol : peg2000 - ceramide = 1 . 5 : 1 : 0 . 02 molar ratio ) were dissolved in a chloroform : ethanol ( 8 : 2 vol / vol ) mixture . the particle size of the suspension before lyophilization and after reconstitution was between 50 and 220 nm . the above procedure described in example 2 was employed to prepare psl - oleandrin . lipid mixtures ( egg phosphatidylcholine : cholesterol = 55 : 45 molar ratio ) were dissolved in a chloroform : ethanol ( 8 : 2 vol / vol ) mixture or in chloroform or dicloromethane . the particle size of the suspension before lyophilization and after reconstitution was between 50 and 220 nm . the above procedure described in example 2 was employed to prepare psl - oleandrin formulation . lipid mixtures ( 1 , 2 - di ( 2 , 4 - tetradecadienoyl )- 3 - phosphatidylcholine : cholesterol = 2 : 1 molar ratio ) were dissolved in a chloroform : ethanol ( 8 : 2 vol / vol ) mixture or in chloroform or dicloromethane . the particle size of the suspension before lyophilization and after reconstitution was between 50 and 220 nm . 100 mg oleandrin is dissolved in 10 ml methylene chloride . the solution was added to 81 ml of human serum abumin solution ( 1 % w / v ). the mixture was homogenized for 5 minutes at low rpm ( vitris homogenizer ) in order to form a crude emulsion , and then transferred into a high pressure homogenizer ( avestin ). the emulsification was performed at 9000 - 18 , 000 psi while recycling the emulsion for at least 5 cycles . the resulting system was transferred into a rotary evaporator , and methylene chloride was rapidly removed at 40 ° c ., at reduced pressure ( 30 mm hg ), for 20 - 30 minutes . the resulting dispersion was translucent , and the typical diameter of the resulting oleandrin particles was 160 - 220 ( z - average , malvern zetasizer ). the dispersion was further lyophilized for 48 hrs . without adding any cryoprotectant . the resulting cake could be easily reconstituted to the original dispersion by addition of sterile water or saline . the particle size after reconstitution was the same as before lyophilization . 20 mg of oleandrin is dissolved in 1 . 0 ml methylene chloride . the solution is added to 4 . 0 ml of human serum abumin solution ( 5 % w / v ). the mixture is homogenized for 5 minutes at low rpm ( vitris homogenizer , model : tempest i . q .) in order to form a crude emulsion , and then transferred into a 40 khz sonicator cell . the sonicator is performed at 60 - 90 % power at 0 degree for 1 min ( 550 sonic dismembrator ). the mixture is transferred into a rotary evaporator , and methylene chloride is rapidly removed at 40 ° c ., at reduced pressure ( 30 mm hg ), for 20 - 30 minutes . the typical diameter of the resulting oleandrin particles was 350 - 420 nm ( z - average , malvern zetasizer ). the dispersion was further lyophilized for 48 hrs . without adding any cryoprotectant . the resulting cake could be easily reconstituted to the original dispersion by addition of sterile water or saline . the particle size after reconstitution was the same as before lyophilization . 10 mg of oleandrin is dissolved in 0 . 55 ml chloroform and 0 . 05 ml ethanol . the solution is added to 29 . 4 ml of human serum abumin solution ( 1 % w / v ), which is presaturated with 1 % chloroform . the mixture is homogenized for 5 minutes at low rpm in order to form a crude emulsion , and then transferred into a high pressure homogenizer ( avestin ). the emulsification is performed at 9000 - 18 , 000 psi while recycling the emulsion for at least 6 cycles . the resulting system is transferred into a rotary evaporator , and the chloroform is rapidly removed at 40 ° c ., at reduced pressure ( 30 mm hg ), for 15 - 30 minutes . the resulting dispersion is translucent , and the typical diameter of the resulting oleandrin particles is 140 - 160 nm ( z - average , malvern zeta sizer ). the dispersion is filtered through a 0 . 22 micron filter ( millipore ), without any significant change in turbidity , or particle size . hplc analysis of the oleandrin content revealed that more than 97 % of the oleandrin was recovered after filtration , thus providing a sterile oleandrin dispersion . the sterile dispersion was further lyophilized for 48 hrs . without adding any cryoprotectant . the resulting cake could be easily reconstituted to the original dispersion by addition of sterile water or saline . the particle size after reconstitution was the same as before lyophilization . 225 mg oleandrin is dissolved in 2 . 7 ml chloroform and 0 . 3 ml ethanol . the solution is added to 97 ml of human serum abumin solution ( 3 % w / v ). the mixture is homogenized for 5 minutes at low rpm ( vitris homogenizer ) in order to form a crude emulsion , and then transferred into a high pressure homogenizer ( avestin ). the emulsification is performed at 9000 - 18 , 000 psi while recycling the emulsion for at least 6 cycles . the resulting system is transferred into a rotary evaporator , and the chloroform is rapidly removed at 40 ° c ., at reduced pressure ( 30 mm hg ), for 15 - 30 minutes . the resulting dispersion is translucent , and the typical diameter of the resulting oleandrin particles is 140 - 160 nm ( z - average , malvern zeta sizer ). the dispersion is filtered through a 0 . 22 micron filter ( sartorius , sartobran 300 ), without any significant change in turbidity , or particle size . hplc analysis of the oleandrin content typically revealed that 70 - 100 % of the oleandrin could be recovered after filtration , depending on the conditions employed . thus , a sterile oleandrin dispersion was obtained . the sterile dispersion was aseptically filled into sterile glass vials and lyophilized without adding any cryoprotectant . the resulting cake could be easily reconstituted to the original dispersion by addition of sterile water or saline . the particle size after reconstitution was the same as before lyophilization . allen t m , hansen c , martin f j , redemann c , and yau - 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