Patent Application: US-81231604-A

Abstract:
the present invention provides method , preparation and use of a variety of pharmaceutical composition containing at least one digitalis glycosides such as oleandrin , odoroside - a , neriifolin , proscillaridin - a , methyl - proscillaridin - a , digitoxin , digoxin and amorphous cyclodextrins . in another aspect , the present invention provides an effective method to reduce the growth of cancers or reducing the incidence of metastases . in yet another aspect , the present invention provides an effective method for treating diseases in a warm - blooded animal .

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 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 “ nanometer ” refers to a unit of measure of one one - billionth of a meter . as used herein , the term “ ng ” or the term “ nanogram ” refers to a unit of measure of one one - billionth of a gram . as used herein , the term “ ml ” refers to a unit of measure of one one - thousandth of a liter . as used herein , the term “ mm ” refers to a unit of measure of one one - thousandth of a mole . 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 . by cyclodextrin is meant α -, β -, or γ - cyclodextrin . cyclodextrins are described in detail in pitha et al ., u . s . pat . no . 4 , 727 , 064 which is incorporated herein by reference . cyclodextrins are cyclic oligomers of glucose ; these compounds form inclusion complexes with any drug whose molecule can fit into the lipophile - seeking cavities of the cyclodextrin molecule . 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 . by amorphous cyclodextrin is meant non - crystalline mixtures of cyclodextrins wherein the mixture is prepared from α -, β -, or γ - cyclodextrin . in general the amorphous cyclodextrin is prepared by non - selective additions , especially alkylation of the desired cyclodextrin species . reactions are carried out to yield mixtures containing a plurality of components thereby preventing crystallization of the cyclodextrin . various alkylated and hydroxyalkyl - cyclodextrins can be made and of course will vary , depending upon the starting species of cyclodextrin and the addition agent used . among the amorphous cyclodextrins suitable for compositions according to the invention are hydroxypropyl , hydroxyethyl , glucosyl , maltosyl and maltotriosyl derivatives of β - cyclodextrin , carboxyamidomethyl - β - cyclodextrin , carboxymethyl - β - cyclodextrin , hydroxypropyl - β - cyclodextrin and diethylamino - β - cyclodextrin . in the compositions according to the invention hydroxy - β - cyclodextrin is preferred . the substituted γ - cyclodextrins may also be suitable , including hydroxypropyl , hydroxyethyl , glucosyl , maltosyl and maltotriosyl derivatives of γ - cyclodextrin . the cyclodextrin of the compositions according to the invention may be α -, β -, or γ - cyclodextrin . α - cyclodextrin contains six glucopyranose units ; β - cyclodextrin contains seven glucopyranose units ; and γ - cyclodextrin contains eight glucopyranose units . the molecule is believed to form a truncated cone having a core opening of 4 . 7 - 5 . 3 å , 6 . 0 - 6 . 5 å and 7 . 5 - 8 . 3 å in α -, β -, or γ - cyclodextrin respectively . the composition according to the invention may comprise a mixture of two or more of the α -, β -, or γ - cyclodextrins . usually , however the composition according to the invention will comprise only one of the α -, β -, or γ - cyclodextrins . the particular α -, β -, or γ - cyclodextrin to be used with the particular digitalis type of cardiac glycosides such as oleandrin , digitoxin , digoxin to form the compositions according to the invention may be selected based on the known size of the molecule of the digitalis type of cardiac glycosides such as oleandrin , digitoxin , digoxin and the relative size of the cavity of the cyclodextrin compound . generally if the molecule of the digitalis type of cardiac glycosides such as oleandrin , digitoxin , digoxin is relatively large , a cyclodextrin having a larger cavity is used to make the composition according to the invention . furthermore , if the molecule selected from the digitalis type of cardiac glycosides such as oleandrin , digitoxin , digoxin is administered with an excipient it may be desirable to use a cyclodextrin compound having a larger cavity in the composition according to the invention . the unmodified α -, β -, or γ - cyclodextrins are less preferred in the compositions according to the invention because the unmodified forms tend to crystallize and are relatively less soluble in aqueous solutions . more preferred for the compositions according to the invention are the α -, β -, and γ - cyclodextrins that are chemically modified or substituted . chemical substitution at the 2 , 3 and 6 hydroxyl groups of the glucopyranose units of the cyclodextrin rings yields increases in solubility of the cyclodextrin compound . most preferred cyclodextrins in the compositions according to the invention are amorphous cyclodextrin compounds . by amorphous cyclodextrin is meant non - crystalline mixtures of cyclodextrins wherein the mixture is prepared from α -, β -, or γ - cyclodextrin . in general , the amorphous cyclodextrin is prepared by non - selective alkylation of the desired cyclodextrin species . suitable alkylation agents for this purpose include but are not limited to propylene oxide , glycidol , iodoacetarnide , chloroacetate , and 2 - diethylaminoethlychloride . reactions are carried out to yield mixtures containing a plurality of components thereby preventing crystallization of the cyclodextrin . various alkylated cyclodextrins can be made and of course will vary , depending upon the starting species of cyclodextrin and the alkylating agent used . among the amorphous cyclodextrins suitable for compositions according to the invention are hydroxypropyl , hydroxyethyl , glucosyl , maltosyl and maltotriosyl derivatives of β - cyclodextrin , carboxyanidomethyl - β - cyclodextrin , carboxymethyl - β - cyclodextrin , hydroxypropyl - β - cyclodextrin and diethylamino -, β - cyclodextrin . in the compositions according to the invention hydroxypropyl - β - cyclodextrin is preferred although the α - or γ - analogs may also be suitable . the particular alkylated α -, β -, or γ - cyclodextrin to be used with the particular compound of digitalis glycosides such as oleandrin , digitoxin , digoxin and proscillaridin - a to form the compositions according to the invention will be selected based on the size of the molecule of the compound and the relative size of the cavity of the cyclodextrin compound . as with the unsubstituted cyclodextrins mentioned above , it may be advantageous to use alkylated cyclodextrin having a larger cavity when the composition according to the invention also includes an excipient . the use of a particular α -, β -, or γ - cyclodextrin with a particular digitalis type of cardiac glycosides such as oleandrin , digitoxin , digoxin and proscillaridin - a compound or the compound selected from the digitalis type of cardiac glycosides such as oleandrin , digitoxin , digoxin and proscillaridin - a and excipient in the compositions according to the invention may of course be optimized based on the effectiveness in of maintaining the compound of the digitalis type of cardiac glycosides such as oleandrin , digitoxin , digoxin and proscillaridin - a or mixture there of in solution . as mentioned above , the compositions of matter of the invention comprise an aqueous preparation of preferably substituted amorphous cyclodextrin and one or more digitalis glycosides . the relative amounts of digitalis glycosides and cyclodextrin will vary depending upon the relative amount of each of the digitalis glycosides and the effect of the cyclodextrin on the compound . in general , the ratio of the weight of compound of the digitalis glycosides to the weight of cyclodextrin compound will be in a range between 1 : 1 and 1 : 100 . a weight to weight ratio in a range of 1 : 5 to 1 : 50 and more preferably in a range of 1 : 10 to 1 : 20 of the compound selected from digitalis glycosides to cyclodextrin are believed to be the most effective for increased circulating availability of the digitalis glycoside . for example , oleandrin or proscillaridin - a in a ratio of between 1 : 10 and 1 : 50 ( drug : amorphous cyclodextrin , wt : wt ), and a final concentration of the injection solution of 0 . 3 mg / ml of oleandrin is expected to significantly reduce the toxicity as compared to free oleandrin or proscillaridin - a due to the complexation with amorphous cyclodextrin . importantly , if the aqueous solution comprising the digitalis glycosides and amorphous cyclodextrin is to be administered parenterally , especially via the intravenous route , the amorphous cyclodextrin will be substantially free of pyrogenic contaminants . amorphous hydroxypropyl - β - cyclodextrin may be purchased from a number of vendors including sigma - aldrich , inc . ( st . louis , mo ., usa ). in addition , other forms of amorphous cyclodextrin having different degrees of substitution or glucose residue number are available commercially . a method for the production of hydroxypropyl - β - cyclodextrin is disclosed in pitha et al ., u . s . pat . no . 4 , 727 , 064 which is incorporated herein by reference . to produce the formulations according to the invention , a pre - weighed amount of hydroxypropyl - β - cyclodextrin compound , which is substantially pyrogen free is placed in a suitable depyrogenated sterile container . methods for depyrogenation of containers and closure components are well known to those skilled in the art and are fully described in the united states pharmacopeia 23 ( united states pharmacopeial convention , rockville , md . usa ). generally , depyrogenation is accomplished by exposing the objects to be depyrogenated to temperatures above 400 ° c . for a period of time sufficient to fully incinerate any organic matter . as measured in u . s . p . bacterial endotoxin units , the formulation will contain no more than 10 bacterial endotoxin units per gram of amorphous cyclodextrin . by substantially pyrogen free is meant that the hydroxypropyl - β - cyclodextrin contains less than 10 u . s . p . bacterial endotoxin units per gram using the u . s . p . method . preferably , the hydroxypropyl - β - cyclodextrin will contain between 0 . 1 and 5 u . s . p . bacterial endotoxin units per mg , under conditions specified in the united states pharmacopeia 23 . sufficient sterile water for injection is added to the substantially pyrogen free amorphous cyclodextrin until the desired concentration of hydroxypropyl - β - cyclodextrin is in solution . to this solution a pre - weighed amount of the compound selected from the digitalis type of cardiac glycosides such as oleandrin , digitoxin , digoxin is added with agitation and with additional standing if necessary until it dissolves . the solution is then filtered through a sterile 0 . 22 micron filter into a sterile holding vessel and is subsequently filled in sterile depyrogenated vials and is capped . for products that will be stored for long periods of time , a pharmaceutically acceptable preservative may be added to the solution of oleandrin and hydroxypropyl - β - cyclodextrin prior to filtration , filling and capping or alternatively , may be added sterilely after filtration . as discussed above , the present invention provides improved water soluble formulations of digitalis glycosides and methods of preparing and employing such formulations . the advantages of these water soluble formulations are that a drug is entrapped in cyclodextrin in dissolved 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 by slow infusions or by bolus injection or by other parenteral or oral delivery routes . for increasing the long - term storage stability , these water soluble 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 formulations , the solution retains essentially all the drug previously loaded . 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 water soluble formulation is not lost after lyophilization and reconstitution . the digitalis glycosides in the cyclodextrin complex may be in the form of pharmaceutically acceptable salts , esters , amides or prodrugs or combinations thereof . however , conversion of inactive ester , amide or prodrug forms to an active form must occur prior to or upon reaching the target tissue or cell . salts , esters , amides and prodrugs of the active agents may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described , for example , by j . march , advanced organic chemistry : reactions , mechanisms and structure , 4th ed . ( new york : wiley - interscience , 1992 ). for example , acid addition salts are prepared from the free base ( typically wherein the neutral form of the drug has a neutral — nh2 group ) using conventional means , involving reaction with a suitable acid . generally , the base form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto . the resulting salt either precipitates or may be brought out of solution by addition of a less polar solvent . suitable acids for preparing acid addition salts include both organic acids , e . g ., acetic acid , propionic acid , glycolic acid , pyruvic acid , oxalic acid , malic acid , malonic acid , succinic acid , maleic acid , fumaric acid , tartaric acid , citric acid , benzoic acid , cinnamic acid , mandelic acid , methanesulfonic acid , ethanesulfonic acid , p - toluenesulfonic acid , salicylic acid , and the like , as well as inorganic acids , e . g ., hydrochloric acid , hydrobromic acid , sulfuric acid , nitric acid , phosphoric acid , and the like . an acid addition salt may be reconverted to the free base by treatment with a suitable base . conversely , preparation of basic salts of acid moieties which may be present on a drug are prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide , potassium hydroxide , ammonium hydroxide , calcium hydroxide , trimethylamine , or the like . preparation of esters involves functionalization of hydroxyl and / or carboxyl groups which may be present within the molecular structure of the drug . the esters are typically acyl - substituted derivatives of free alcohol groups , i . e ., moieties which are derived from carboxylic acids of the formula rcooh where r is alkyl , and preferably is lower alkyl . esters can be reconverted to the free acids , if desired , by using conventional hydrogenolysis or hydrolysis procedures . preparation of amides and prodrugs can be carried out in an analogous manner . other derivatives and analogs of the active agents may be prepared using standard techniques known to those skilled in the art of synthetic organic chemistry , or may be deduced by reference to the pertinent literature . in addition , chiral active agents may be in enantiomerically pure form , or they may be administered as an enantiomeric mixture . in the present invention , the efficacy of water soluble formulations of oleandrin and proscillaridin - a of the present invention have been investigated on various systems such as human cell lines for cell proliferative activities and found to be active against tumors . 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 , preferably , modified citrus pectin to provide the stimulant effect . further , it has been previously shown ( glycan stimuation of macrophages in vitro , r . seljelid , g . bogwald and a . lundwall , experimental cell research 131 ( 1981 ) 121 ), that certain glucans , particularly such glucans containing 1 , 3 - bound β - d - glucose entities , activate macrophages in vitro making same cytotoxic . thus the formulations of the present inventions can contain suitable 1 , 3 - β - d glucans and their derivatives such as phosphorylated 1 , 3 - β - d glucan , aminated 1 , 3 - β - d glucan , sulfated 1 , 3 - β - d glucan and carboxymethylated 1 , 3 - β - d glucan to provide the desired immune stimulant effect . previously , the effect of citrus pectin ( cp ), a complex polysaccharide rich in galactosyl residues , and its ph - modified derivative , modified citrus pectin ( mcp ) on the experimental metastasis of b16 melanoma and prostate was analyzed as described in the articles ( platt 1992 ; inohara 1994 ; pienta 1995 and raloff 1995 ). u . s . pat . no . 5 , 834 , 442 and u . s . pat . no . 5 , 895 , 784 claims the oral administration of modified citrus pectin to treat prostate and melanoma cancer . it was found that co - injection of mcp with the b16 - f1 cells intravenously resulted in a marked inhibition of their ability to colonize the lungs of the injected mice . the ph modification of cp results in the generation of smaller sized non - branched carbohydrate chains of similar sugar composition of the unmodified cp . mcp appears to be non - toxic , in vitro and in vivo and is sold as nutritional supplement by herbalists and natural medicine vendors . compositions employing the water soluble formulations of digitalis glycosides such as proscillaridin - a , digitoxin and oleandrin , will contain a biologically effective amount of digitalis glycosides . 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 . 1 microgram / kg to about 100 microgram / kg , preferably from about 0 . 2 microgram / kg to about 50 microgram / kg and more preferably from about 0 . 2 microgram / kg to about 10 microgram / 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 amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained . the composition of matter according to the invention may be supplied as a dry powder or as a solution . if the composition of matter is to be injected into a subject it will be rendered sterile prior to injection . accordingly , the composition of matter according to the invention may be supplied as a sterile cake , plug or powder or as a sterile lyophilized preparation in a sterile vial suitable for the addition of a sterile diluent , or as a sterile liquid solution in a sterile container . the compositions of matter according to the invention may be supplied as a powder comprising the active pharmaceutical digitalis glycoside and amorphous cyclodextrin compound . if the composition is to be administered parenterally , for example intravenous , the composition of matter will be rendered sterile prior to such administration . any of the several known means for rendering such pharmaceutical preparations sterile may be used so long as the active pharmaceutical compound is not inactivated and the complex with the amorphous cyclodextrin is not degraded . if the active pharmaceutical compound is heat stable , the composition of matter according to the invention may be heat sterilized . if the digitalis glycoside is not heat - stable but is not photo degraded the composition may be sterilized by exposure to ultraviolet light or by ionizing radiation . alternatively , the composition of matter if in a powder form may be gas sterilized using for example ethylene oxide gas . in another alternative , the composition of matter according to the invention may be filter - sterilized using a 0 . 22 micron filter . if the composition of matter is an aqueous liquid , it may be filled in a sterile container and supplied as a sterile liquid ready for further dilution or injection neat . alternatively such sterile liquids may be freeze - dried or lyophilized in a sterile container and capped . in general the compositions of matter according to the invention will be made by dissolving the cyclodextrin in water and adding digitalis glycoside compound to the aqueous cyclodextrin solution . excipients , if any are desired , may be added with or subsequent to adding the oleandrin or other digitalis glycoside compound . the resulting solution may be sterilized using any of the known methods appropriate to preserving the compound without significant degradation . preferably the solution will be sterile filtered , although other means such as terminal heat sterilization or irradiation may be employed as is known in the art , provided that the cyclodextrin compound is not significantly degraded . alternatively , the components may be sterilized by any of the known methods appropriate to preserving the compound prior to mixing in water and may be mixed using sterile equipment and technique . the solution may be lyophilized in sterile containers and capped . prior to use the lyophilized composition of matter may be reconstituted using sterile water for injection . the container closure system used for containing the formulation according to the invention will also be treated to remove or destroy pyrogenic substances by means known in the art prior to filling and further processing . thus the preferred compositions of matter according to the invention for parenteral administration , especially by the intravenous route will be nonpyrogenic . nonpyrogenic preparations according to the invention , when administered to a subject , does not cause a febrile ( basal body temperature raising ) reaction . although some bacterial endotoxin may be present , the amount is insufficient to elicit a febrile reaction . in general , such non - pyrogenic compositions will contain less than 10 u . s . p . bacterial endotoxin units per gram of product . the formulation according to the invention may be supplied as a dry lyophilized powder as mentioned above or as a sterile non pyrogenic aqueous solution in a sterile container closure system such as a stoppered vial suitable for puncturing with a sterile syringe and needle . alternatively the formulation according to the invention may be supplied as a sterile non - pyrogenic aqueous solution in a sterile syringe or syringe and needle . as a sterile solution or powder it may also include a pharmaceutically acceptable preservative . the formulation according to the invention may also be included in other dosage forms in addition to those appropriate for parenteral administration . preferably , such other dosage forms will include one or more of the digitalis glycosides . such dosage forms may be in the form of aqueous suspensions , elixirs , or syrups suitable for oral administration , or compounded as a cream or ointment in a pharmaceutically acceptable topical base allowing the digitalis glycoside compounds to be absorbed across the skin . in addition the formulation according to the invention may be compounded in a lozenge or suppository suitable for trans - mucosal absorption . for the intended oral mode of administration , the pharmaceutical compositions containing cyclodextrin - digitalis glycoside complex may be in the form of solid , semi - solid or liquid dosage forms , such as , for example , tablets , suppositories , pills , capsules , powders , liquids , suspensions , or the like , preferably in unit dosage form suitable for single administration of a precise dosage . the cyclodextrin - digitalis glycoside complex can be lyophilized and the lyophilized powder can be used for preparing solid dosage forms . the compositions will include an effective amount of the selected cyclodextrin - digitalis glycoside complex in combination with a pharmaceutically acceptable carrier and , in addition , may include other pharmaceutical agents , adjuvants , diluents , buffers , etc . the compounds may thus be administered orally , in dosage formulations containing conventional non - toxic pharmaceutically acceptable carriers , adjuvants and vehicles . the equivalent amount of active digitalis glycoside compound administered as cyclodextrin - digitalis glycoside complex will , of course , be dependent on the subject being treated , the subject &# 39 ; s weight , the manner of administration and the judgment of the prescribing physician . for solid compositions , conventional nontoxic solid carriers include , for example , pharmaceutical grades of mannitol , lactose , starch , magnesium stearate , sodium saccharin , talc , cellulose , glucose , sucrose , magnesium carbonate , and the like . liquid pharmaceutically administrable compositions can , for example , be prepared by dissolving , dispersing , etc ., an active compound as described herein and optional pharmaceutical adjuvants in an excipient , such as , for example , water , saline , aqueous dextrose , glycerol , ethanol , and the like , to thereby form a solution or suspension . if desired , the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents , ph buffering agents and the like , for example , sodium acetate , sorbitan mono - laurate , triethanolamine sodium acetate , triethanolamine oleate , etc . actual methods of preparing such dosage forms are known , or will be apparent , to those skilled in this art ; for example , see remington &# 39 ; s pharmaceutical sciences , referenced above . for oral administration , the composition will generally take the form of a tablet or capsule , or may be an aqueous or nonaqueous solution , suspension or syrup . tablets and capsules are preferred oral administration forms . tablets and capsules for oral use will generally include one or more commonly used carriers such as lactose and corn starch . lubricating agents , such as magnesium stearate , are also typically added . when liquid suspensions are used , the active agent may be combined with emulsifying and suspending agents . if desired , flavoring , coloring and / or sweetening agents may be added as well . other optional components for incorporation into an oral formulation herein include , but are not limited to , preservatives , suspending agents , thickening agents , and the like . oral dosage units preferably contain equivalent of digitalis glycoside such as oleandrin , in the cyclodextrin - digitalis glycoside complex , in the range of about 50 to not more than 1000 micrograms ( μg ), preferably in the range of about 100 and about 400 μg so long as the dose received by the patient is accompanied by minimal or substantially no undesirable side effects . a particularly preferred oral dosage unit contains about 250 μg equivalent oleandrin , more preferably about 150 μg equivalent oleandrin . the pharmaceutical formulations of digitalis glycoside according to the present invention offer several advantages over the existing formulation of oleander extract administered parenterally . they can be intravenously administered and relatively high concentrations of oleandrin or other digitalis glycoside 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 . 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 . 10 milligrams ( mg ) of oleandrin was stirred and shaken with 10 ml of water in a test tube . appreciable quantities of compound remained out of solution after 20 minutes accumulating as white crystals at the bottom of the test tube . 100 milligrams of oleandrin was weighed and placed in a 5 ml scintillation tube . 1 . 5 ml of absolute ethanol was added to the tube and shaken until the oleandrin was completely dissolved . 5 grams of pyrogen free hydroxypropyl - β - cyclodextrin ( sold by , sigma - aldrich , inc ., st . louis , mo ., usa ) was weighed on an analytical scale and placed in a graduated cylinder . water was added with shaking until the volume reached 90 ml . the above ethanolic solution of oleandrin was added to the aqueous solution containing hydroxypropyl - β - cyclodextrin with stirring . a clear solution was obtained . water was added to the clear solution to make the total volume to 100 ml . thus , 1 mg oleandrin was effectively solubilized in 1 ml of 5 % solution of hydroxypropyl - β - cyclodextrin . the solution was sterile - filtered through a 0 . 22 μm filter . the suspension was frozen below − 40 ° c . and lyophilized . the lyophilized cake was reconstituted with sterile water for injection prior to further use . the previous experiment was repeated using a 2 % solution of hydroxypropyl - β - cyclodextrin prepared as in example 1 . 100 mg of oleandrin was dissolved in 100 ml of water containing 2 . 5 grams of hydroxypropyl - β - cyclodextrin . the solution was sterile - filtered through a 0 . 22 μm filter . the solution was frozen below − 40 ° c . and lyophilized . the lyophilized cake was reconstituted with sterile water for injection prior to further use . the experiment in example 1 was repeated using a 100 mg of odoroside - a instead of oleandrin . 100 mg of odoroside - a was dissolved in 5 grams of hydroxypropyl - β - cyclodextrin in 100 ml of water . the solution was sterile - filtered through a 0 . 22 μm filter . the solution was frozen below − 40 ° c . and lyophilized . the lyophilized cake was reconstituted with sterile water for injection prior to further use . 100 milligrams of oleandrin was weighed and placed in a 5ml scintillation tube . 1 - 2 ml of absolute ethanol was added to the tube and shaken until the oleandrin was completely dissolved . 2 . 5 grams of pyrogen free hydroxypropyl - γ - cyclodextrin ( sold by , sigma - aldrich , inc ., st . louis , mo ., usa ) was weighed on an analytical scale and placed in a graduated cylinder . water was added with shaking until the volume reached 90 ml . the above ethanolic solution of oleandrin was added to the aqueous solution containing hydroxypropyl - γ - cyclodextrin with stirring . a clear solution was obtained . water was added to the clear solution to make the total volume to 100 ml . thus , 1 mg oleandrin was effectively solubilized in 1 ml of 2 . 5 % solution of hydroxypropyl - γ - cyclodextrin . the solution was sterile - filtered through a 0 . 22 μm filter . the suspension was frozen below − 40 ° c . and lyophilized . the lyophilized cake was reconstituted with sterile water for injection prior to further use . 100 milligrams of proscillaridin - a was weighed and placed in a 5 ml scintillation tube . 1 - 2 ml of absolute ethanol was added to the tube and shaken until the proscillaridin - a was completely dissolved . 2 grams of pyrogen free hydroxypropyl - β - cyclodextrin ( sold by , sigma - aldrich , inc ., st . louis , mo ., usa ) was weighed on an analytical scale and placed in a graduated cylinder . water was added with shaking until the volume reached 90 ml . the above ethanolic solution of proscillaridin - a was added to the aqueous solution containing hydroxypropyl - β - cyclodextrin with stirring . a clear solution was obtained . water was added to the clear solution to make the total volume to 100 ml . thus , 1 mg proscillaridin - a was effectively solubilized in 1 ml of 2 % solution of hydroxypropyl - β - cyclodextrin . the solution was sterile - filtered through a 0 . 22 μm filter . the suspension was frozen below − 40 ° c . and lyophilized . the lyophilized cake was reconstituted with sterile water for injection prior to further use . the previous experiment was repeated using a 5 % solution of hydroxypropyl - β - cyclodextrin prepared as in example 5 . 100 mg of proscillaridin - a was dissolved in 100 ml of water containing 5 grams of hydroxypropyl - β - cyclodextrin . the solution was sterile - filtered through a 0 . 22 μm filter . the solution was frozen below − 40 ° c . and lyophilized . the lyophilized cake was reconstituted with sterile water for injection prior to further use . 100 milligrams of proscillaridin - a was weighed and placed in a 5 ml scintillation tube . 1 . 5 ml of absolute ethanol was added to the tube and shaken until the proscillaridin - a was completely dissolved . 2 . 5 grams of pyrogen free hydroxypropyl - γ - cyclodextrin ( sold by , sigma - aldrich , inc ., st . louis , mo ., usa ) was weighed on an analytical scale and placed in a graduated cylinder . water was added with shaking until the volume reached 90 ml . the above ethanolic solution of proscillaridin - a was added to the aqueous solution containing hydroxypropyl - γ - cyclodextrin with stirring . a clear solution was obtained . water was added to the clear solution to make the total volume to 100 ml . thus , 1 mg proscillaridin - a was effectively solubilized in 1 ml of 2 . 5 % solution of hydroxypropyl - γ - cyclodextrin . the solution was sterile - filtered through a 0 . 22 μm filter . the suspension was frozen below − 40 ° c . and lyophilized . the lyophilized cake was reconstituted with sterile water for injection prior to further use . a formulation with 0 . 1 % of oleandrin , 2 . 5 % hydroxypropyl beta - cyclodextrin , 0 . 5 % sodium ascorbate and 0 . 04 % ascorbic acid as antioxidants , 0 . 1 % methylparaben sodium and 0 . 01 % propylparaben sodium as preservatives ( table 2 ) was prepared under aseptic conditions following the method given below . the ingredients sodium ascorbate , ascorbic acid , methylparaben sodium and propylparaben were purchased as usp grade materials from spectrum chemical and safety products . the ingredients hydroxypropyl beta - cyclodextrin and trehalose dihydrate were purchased from sigma chemicals co . a 150 ml sterile beaker was weighed and tarred , and the ingredients , except the oleandrin and trehalose dihydrate , listed in table 2 were weighed and transferred directly into the beaker . type i water was added and the volume was adjusted to 100 ml . the solution was heated to 70 - 80 ° c . in a pre - heated circulating water - bath . the amount of oleandrin listed in table 2 was weighed and dissolved in 1 - 2 ml of purified ethanol in a sterile test tube . the ingredients were stirred and the ethanol solution of oleandrin was slowly added over a period of 5 - 10 minutes to the aqueous solution while stirring . the trehalose dihydrate was added to the solution and the resulting solution was stirred for additional 10 - 15 minutes to form a clear solution . the ph of the solution measured using a orion ph meter was approximately 6 . 50 . when required , the ph of the solution was adjusted using either 1n naoh or 1n hcl to 6 . 5 ± 0 . 2 . the hot solution was filtered using a 0 . 22 μm sterile cellulose acetate bottle - top filter with a glass pre - filter attached to a sterile media receiver bottle in the laminar flow hood . a diaphragm pump ( laboport ) was connected to the bottle - top filter to filter - sterilize the solution into the media bottle . immediately after filtering the solution , the bottle - top filter was removed and a bottle - top dispenser ( dispensette ii , brinkmann ) was attached to fill 5 ml of the sterile liquid at a time in 10 ml sterile glass vials in the laminar flow hood . to each one of the 10 ml vials filled , a 3 - leg gray butyl rubber stopper ( wheaton ) was placed in such a way that the stopper openings were exposed outside the vial &# 39 ; s mouth . these vials were arranged in two sterile stainless steel trays , and these trays were then placed onto the freeze - dryer stoppering trays pre - cooled to − 40 ° c . after 7 hours , the sample was freeze - dried using following the following temperature cycle : − 35 ° c . for 6 hours ; 0 ° c . for 72 hours ; 30 ° c . for 24 hours . the vials with freeze - dried oleandrin - cyclodextrin complex were stoppered under a vacuum level of about 250 × 10 − 3 mbar in the stoppering tray . the vacuum pump was turned off , vacuum in the stoppering tray compartment was released , and the stainless steel trays with the vials were removed from the freeze - dryer . each one of the vials was sealed with a flip - cap aluminum seal ( wheaton ) employing a hand operated e - z crimper . the pharmaceutically formulated freeze - dried oleandrin - cyclodextrin complex , when stored at room temperature in the vacuum sealed vials is stable at least for about 3 to 5 years . reconstitution testing : the reconstitution test of the formulated oleandrin - cyclodextrin complex powder in the vacuum sealed vials was performed following the procedure given below : 1 . five ml of sterile water for injection was withdrawn using a sterile 10 ml syringe with a 20 g × 1 ″ needle attached to it . 2 . the flip - cap was detached from the aluminum seal in the vial , and the exposed surface of the rubber stopper was cleaned with 70 % isopropanol . 3 . the water for injection from the syringe was administered into the vial . because the powder in the vial was under vacuum , as soon as the syringe needle was inserted , the vacuum automatically withdrew the water into the vial without having to syringe &# 39 ; s plunger . 4 . after adding the water for injection , the powder was reconstituted into a clear solution within a minute . the reconstituted oleandrin - cyclodextrin complex solution was used for the further studies . stability testing : the testing was performed by storing the vials containing the reconstituted solution at 4 ° c . and room temperatures . the formulated oleandrin - cyclodextrin complex solution was stable for at least one month when stored at these temperatures . there was no visible precipitation of particles from the solution in this period . steriility testing : the freeze - dried formulated oleandrin - cyclodextrin complex powder was reconstituted with 5 ml sterile water for injection in a laminar flow hood under aseptic conditions at the southwest bioscience laboratories , san antonio and tested in accordance with the procedure recommended by us pharmacopeia xxiii . the formulated solution was inoculated in a culture bottle ( bbl septi - check ) containing either 70 ml casein digest broth with sps and co 2 or thioglycollate broth with sps and co 2 . the casein digest broth was aerated using a 0 . 2 μm filter for aerobic growth . the casein digest and thioglycollate broths were incubated at 25 ° c . and 35 ° c . for 7 days , respectively . these samples were examined each day for growth and retained for 14 days before discarding . for 14 days the cultures were also observed for microbial growth after incubating under the same conditions as the samples . no microbial growth was observed in the cultures with or without formulated oleandrin - cyclodextrin complex solution , indicating that the freeze - dried formulated oleandrin - cyclodextrin complex powder was sterile . a formulation with 0 . 05 % of oleandrin , 1 . 5 % hydroxypropyl cyclodextrin , 0 . 5 % sodium ascorbate and 0 . 02 % ascorbic acid as antioxidants , 0 . 1 % methylparaben sodium and 0 . 01 % propylparaben sodium as preservatives ( table 3 ) was prepared under aseptic conditions following the method given in example 8 . a formulation with 0 . 1 % of proscillaridin - a , 2 . 5 % hydroxypropyl cyclodextrin , 0 . 5 % sodium ascorbate and 0 . 05 % ascorbic acid as antioxidants , 0 . 1 % methylparaben sodium and 0 . 01 % propylparaben sodium as preservatives ( table 4 ) was prepared under aseptic conditions following the method given in example 8 . a formulation with 0 . 1 % of oleandrin , 2 . 5 % hydroxypropyl cyclodextrin , 0 . 5 % sodium ascorbate and 0 . 05 % ascorbic acid as antioxidants , 0 . 1 % methylparaben sodium and 0 . 01 % propylparaben sodium as preservatives and 1 . 5 % modified citrus pectin which is derived from pectin by controlled hydrolysis ( table 5 ) was prepared under aseptic conditions following the method given in example 8 . preparation of nerium oleander polysaccharide : the branches of nerium oleander plant grown under quarantine conditions were washed thoroughly two times with tap water , one time each with di water and sterile type i water ( purity water system , san antonio ) and then cut into pieces of about one inch . the cut stems and leaves were weighed and transferred into a 50 l glass round bottom flask , which was placed onto a mantle . to approximately 7 kg of leaves and stems , 30 l of sterile type i water was added to the flask . a ground joint with a condenser and a thermometer was then attached to the flask , and it was heated for 4 - 6 hours after the mixture started boiling . the boiled oleander extract was cooled then to between 60 ° and 70 ° c . the solution was transferred , employing a peristaltic pump , into a sterile corning 0 . 22 μm cellulose acetate bottle - top filter with a glass - fiber pre - filter , attached to a sterile 2 l media bottle ( corning ) in a laminar flow hood ( labgrad ). a diaphragm pump ( laboport ) was connected to the bottle - top filter to filter - sterilize the solution into the media bottle . immediately after filtering about 2 l of the solution , the bottle - top filter was removed from the media bottle and the bottle was closed tightly with a cap , inside the hood . thirteen such 2 l bottles with about 26 . 5 l of the filter - sterilized solution were heated to about 100 ° c . for 1 hour by placing these bottles in a water - bath ( precision scientific ) pre - heated to 100 ° c . these media bottles with the hot sterile solution were cooled to room temperature , and then stored at 4 ° c . in a refrigerator . the above water extract of the oleander plant was mixed with ethanol ( 96 %) in a 1 : 1 ratio . this solution was allowed to set for 12 hours , the resulting gel suspension was filtered off . the gel suspension filtrate was dissolved in distilled water . this solution was again mixed with ethanol ( 96 %) in a 1 : 1 ratio . after allowing the solution to set for at least 12 hours , the gel suspension was filtered off . again the gel suspension filtrate was dissolved in distilled water , frozen and lyophilized . in this way , the freeze dried polysaccharide extract from the oleander plant was obtained . a formulation with 0 . 1 % of oleandrin , 2 . 5 % hydroxypropyl cyclodextrin , 0 . 5 % sodium ascorbate and 0 . 05 % ascorbic acid as antioxidants , 0 . 1 % methylparaben sodium and 0 . 01 % propylparaben sodium as preservatives and 1 . 0 % freeze dried polysaccharide from the oleander plant ( table 6 ) was prepared under aseptic conditions following the method given in example 8 . a formulation with 0 . 1 % of proscillaridin - a , 2 . 5 % hydroxypropyl cyclodextrin , 0 . 5 % sodium ascorbate and 0 . 05 % ascorbic acid as antioxidants , 0 . 1 % methylparaben sodium and 0 . 01 % propylparaben sodium as preservatives an 1 . 5 modified citrus pectin ( table 7 ) was prepared under aseptic conditions following the method given in example 8 . 100 mg of oleandrin was weighed and placed in a sterile test tube . the oleandrin was dissolved in 2 - 3 ml of purified absolute ethanol . 50 ml of 9 . 8 % solution of hydroxypropyl - β - cyclodextrin was prepared in a 150 ml sterile beaker and the solution was heated to 70 - 80 degree centigrade while stirring on a hot plate . the ethanolic solution of oleandrin was slowly added to the beaker with stirring . within 10 - 30 minutes , the oleandrin dissolved , leaving a clear solution with no accumulation of crystals . thus , 100 mg was effectively solubilized in 50 ml of 9 . 8 % solution of hydroxypropyl - β - cyclodextrin . the solution was sterile - filtered through a 0 . 22 μm filter . the solution was frozen below − 40 ° c . and lyophilized . the lyophilized cake was powdered and used for the tablets , capsule and coated pills formulations and the lyophilized powder is denoted as oleandrin - cyclodextrin complex . the tablet composition is compounded from the following ingredients given in table 8 . preparation : the oleandrin - cyclodextrin complex is intensively milled with ten times its weight of lactose , the milled mixture is admixed with the remaining amount of the lactose and the potato starch , the resulting . mixture is moistened with an aqueous 10 % solution of the gelatin , the moist mass is formed through a 1 . 5 mm - mesh screen , and the resulting granulate is dried at 40 degree c . the dry granulate is again passed through a 1 mm - mesh screen , admixed with the magnesium stearate , and the composition is compressed into 120 mg - tablets in a conventional tablet making machine . each tablet contains 0 . 125 mg of oleandrin and is an oral dosage unit composition with effective therapeutic action . the pill core composition is compounded from the ingredients given in table 9 . preparation : the oleandrin - cyclodextrin complex is intensively milled with the lactose , the milled mixture is admixed with the corn starch , the mixture is moistened with an aqueous 15 % solution of the polyvinylpyrrolidone , the moist mass is forced through a 1 mm - mesh screen , and the resulting granulate is dried at 40 degree c . and again passed through the screen . the dry granulate is admixed with the magnesium stearte , and the resulting composition is compressed into 50 mg - pill cores which are subsequently coated in conventional manner with a thin shell consisting essentially of a mixture of sugar and talcum and finally polished with beeswax . each coated pill contains 0 . 125 mg of oleandrin complexed with hydroxypropyl - cyclodextrin and is an oral dosage unit composition with effective therapeutic action . the solution is compounded from the ingredients given in table 10 . preparation : the oleandrin - cyclodextrin complex and the flavoring are dissolved in the ethanol , and the sorbic acid and the saccharin sodium are dissolved in the distilled water . the two solutions are uniformly admixed with each other , and the mixed solution is filtered until free from suspended matter . 1 ml of the filtrate contains 0 . 125 mg of the oleandrin and is an oral dosage unit composition with effective therapeutic action . the suppository composition is compounded from the ingredients given in table 11 . preparation : the oleandrin - cyclodextrin complex and the lactose are admixed , and the mixture is milled . the milled mixture is uniformly stirred with the aid of an immersion homogenizer into the suppository base , which had previously been melted and cooled to 40 degree c . the resulting composition is cooled at 37 degree c ., and 1700 mg - portions thereof are poured into cooled suppository molds and allowed to harden therein . each suppository contains 0 . 125 mg of the oleandrin and is rectal dosage unit composition with effective therapeutic action . the capsule composition is compounded from the following ingredients given in table 12 . preparation : the oleandrin - cyclodextrin complex is intensively milled with ten times its weight of lactose , the milled mixture is admixed with the remaining amount of the lactose , the micronized beta - glucan and the r - alpha lipoic acid . the mixed powder is again milled and the composition is filled into 400 mg - capsule in a conventional capsule making machine . each capsule contains 0 . 125 mg of oleandrin and is an oral dosage unit composition with effective therapeutic action . 100 mg of proscillaridin - a was weighed and placed in a sterile test tube . the proscillaridin - a was dissolved in 2 - 3 ml of purified absolute ethanol . 50 ml of 9 . 8 % solution of hydroxypropyl - β - cyclodextrin was prepared in a 150 ml sterile beaker and the solution was heated to 70 - 80 degree centigrade while stirring on a hot plate . the ethanolic solution of proscillaridin - a was slowly added to the beaker with stirring . within 10 - 30 minutes , the proscillaridin - a dissolved , leaving a clear solution with no accumulation of crystals . thus , 100 mg was effectively solubilized in 50 ml of 9 . 8 % solution of hydroxypropyl - β - cyclodextrin . the solution was sterile - filtered through a 0 . 22 μm filter . the solution was frozen below − 40 ° c . and lyophilized . the lyophilized cake was powdered and used for the tablets , capsule and coated pills formulations and the lyophilized powder is denoted as proscillaridin - a - cyclodextrin complex . the tablet composition is compounded from the following ingredients given in table 13 . preparation : the proscillaridin - a - cyclodextrin complex is intensively milled with five times its weight of lactose , the milled mixture is admixed with the remaining amount of the lactose and the potato starch , the resulting mixture is moistened with an aqueous 10 % solution of the gelatin , the moist mass is formed through a 1 . 5 mm - mesh screen , and the resulting granulate is dried at 40 degree c . the dry granulate is again passed through a 1 mm - mesh screen , admixed with the magnesium stearate , and the composition is compressed into 120 mg - tablets in a conventional tablet making machine . each tablet contains 0 . 250 mg of proscillaridin - a and is an oral dosage unit composition with effective therapeutic action . the pill core composition is compounded from the ingredients given in table 14 . preparation : the proscillaridin - a - cyclodextrin complex is intensively milled with the lactose , the milled mixture is admixed with the corn starch , the mixture is moistened with an aqueous 15 % solution of the polyvinylpyrrolidone , the moist mass is forced through a 1 mm - mesh screen , and the resulting granulate is dried at 40 degree c . and again passed through the screen . the dry granulate is admixed with the magnesium stearte , and the resulting composition is compressed into 50 mg - pill cores which are subsequently coated in conventional manner with a thin shell consisting essentially of a mixture of sugar and talcum and finally polished with beeswax . each coated pill contains 0 . 250 mg of proscillaridin - a complexed with hydroxypropyl - cyclodextrin and is an oral dosage unit composition with effective therapeutic action . the solution is compounded from the ingredients given in table 15 . preparation : the proscillaridin - a - cyclodextrin complex and the flavoring are dissolved in the ethanol , and the sorbic acid and the saccharin sodium are dissolved in the distilled water . the two solutions are uniformly admixed with each other , and the mixed solution is filtered until free from suspended matter . 1 ml of the filtrate contains 0 . 250 mg of the proscillaridin - a and is an oral dosage unit composition with effective therapeutic action . the suppository composition is compounded from the ingredients given in table 16 . preparation : the proscillaridin - a - cyclodextrin complex and the lactose are admixed , and the mixture is milled . the milled mixture is uniformly stirred with the aid of an immersion homogenizer into the suppository base , which had previously been melted and cooled to 40 degree c . the resulting composition is cooled at 37 degree c ., and 1700 mg - portions thereof are poured into cooled suppository molds and allowed to harden therein . each suppository contains 0 . 250 mg of the proscillaridin - a and is rectal dosage unit composition with effective therapeutic action . the capsule composition is compounded from the following ingredients given in table 17 . preparation : the proscillaridin - a - cyclodextrin complex is intensively milled with five times its weight of lactose , the milled mixture is admixed with the remaining amount of the lactose , the micronized beta - glucan and the r - alpha lipoic acid . the mixed powder is again milled and the composition is filled into 400 mg - capsule in a conventional capsule making machine . each capsule contains 0 . 250 mg of proscillaridin - a and is an oral dosage unit composition with effective therapeutic action . likewise , the amount of active ingredient in these illustrative examples may be varied to achieve the dosage unit range set forth above , and the amounts and nature of the inert pharmaceutical carrier ingredients may be varied to meet particular requirements . while the present invention has been illustrated with the aid of certain specific embodiments thereof , it will be readily apparent to others skilled in the art that the invention is not limited to these particular embodiments , and that various changes and modifications may be made without departing from the spirit of the invention or the scope of the appended claims . müller b w , waaler t , helm h and anderson a : complex formation of β - 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