the above discussed needs are filled by a conjugate for treating infected cells , especially cells stressed by a protozoan infection , that , in one embodiment includes a targeting agent that is attracted to a receptor that is expressed in higher numbers or more frequently by cells infected by a protozoa than by normal uninfected cells , and an anti - protozoan drug . the targeting agent can be any material that is attracted to receptors on cells that present in higher numbers or more frequently when a cell is stressed from a protozoan infection . preferably , the targeting agent is transferrin . attachment of the drug to the targeting agent may be by any mechanism that prevents their separation , at least until after the targeting agent has been positioned in the corresponding receptor . presently , the best known mechanism for attachment for a transferrin - doxorubicin conjugate is a gluteraldehyde linker , but the linker can be any material useful for the targeting agent / drug combination in question . technical details of the conjugation procedure can vary , but the requirement of any procedure is to prepare defined conjugates that are ( a ) active in binding and killing experiments with protozoan infected cells , and that ( b ) do not bind or kill significant numbers of normal cells . in light of these requirements , when transferrin is used as the targeting agent and doxorubicin is used as the anti - protozoan drug , the preferred method for preparing the conjugates according to the present invention is the following process . the synthesis of large amounts of homogeneous transferrin - doxorubicin conjugates with predetermined molecular ratios was done stoichiometrically by employing the only amino group of doxorubicin ( dox ), which is at the 3 ′ amino position , to react with one of the two reactive groups on glutaraldehyde ( glu ). thus , the first step was drop - wise addition of a saline solution of dox into a saline solution of glu containing a solvent such as dmso or another suitable cryopreservative , to a final concentration of a 1 : 1 molar ratio of dox - to - glu . the resulting solution of dox - glu was stirred three hours at room temperature in the dark . the molarities of dox and glu were the same in the above reaction in order to produce a final solution of dox - glu that contains neither free dox nor free glu . however , there is the possibility of free glu in solution if one glu reacts with two dox to produce dox - glu - dox , but this possibility is minimized by the mass action kinetics generated by drop - wise addition of monovalent dox into the solution of bivalent glu . the volumes of these reactants are not restricted , so large amounts of homogeneous dox - glu can be prepared . the second step in the conjugation reaction was drop - wise addition of dox - glu into a saline solution of transferrin ( trf ). the trf can be either iron - free ( apo - transferrin ) or iron - saturated ( holo - transferrin ). the desired molar ratio of dox to trf was obtained by appropriately adjusting the volume of trf . the resulting solution of trf - glu - dox was stirred for 20 hours at room temperature in the dark . unlike the reaction of dox with glu , the reaction of dox - glu with trf is not restricted to one binding site , for the glu component of dox - glu can react with any one of several epsilon - amino lysine groups in the trf molecule . the number of dox molecules bound to trf was determined in the second step . for example , if the starting ratio of dox - glu to trf was 7 . 2 : 1 . 0 , the final solution of trf - glu - dox would have contained 2 . 5 molecules of dox per molecule of trf . however , if the starting ratio of dox - glu to trf was 4 . 0 : 1 . 0 , the final solution of trf - glu - dox would have contained 1 . 4 molecules of dox per molecule of trf . similarly , if the starting ratio of dox - glu to trf was 2 . 5 : 1 . 0 , the final solution of trf - glu - dox would have contained 0 . 9 molecules of dox per molecule of trf . in this way , large amounts of trf - glu - dox with predetermined ratios of dox - to - trf can be provided according to the need . one skilled in the art will appreciate that there may be unreacted linker and a small amount of unintended constructions , such as dox - glu - dox in the reaction , product and that it will be desirable to optimize the reaction product by removing them . ethanolamine or another substance suitable for scavenging any excess linker may be added to the reaction product , followed by centrifugation and dialysis , may be used to remove excess glu and such unintended constructions . although reactions with dox and trf theoretically consume all of the glu , ethanolamine was added to the final reaction mixture to bind any available glu . this reaction was allowed to continue for 30 minutes in the dark . the final solution was centrifuged at 2000 rpm for 10 minutes , dialyzed twice for 6 hours in a 100 - fold excess of saline and three times in the same excess of hepes buffered saline , and the resulting trf - glu - dox conjugates were ready for use . by using hplc and polyacrylamide gel electrophoresis , the homogeneity of trf - glu - dox conjugates can be determined . also , by using spectrophotometry , the molecular ratio of dox - to - trf can be determined . these techniques repeatedly have revealed a consistent homogeneity of the trf - glu - dox conjugates . in addition , chromatography is not required in the preparation of these conjugates , because there are no aggregates or fragments . this allows for the preparation of large volumes of homogeneous transferrin - drug conjugates , which increases yields and decreases costs . the expenses caused by losses of trf and dox in other types of transferrin - drug conjugates have been an impediment to their use . for example , yields of dox and trf are decreased by using procedures such as thiolation that alter the drug and / or protein . yields also - are decreased by using solvent systems and by chromatography used to prepare acid - stable and acid - labile linkages . the glu bond between dox and trf is acid - stable , and yields of useful conjugates prepared according to this invention are high indeed , compared to other procedures , the yield for useful conjugate is increased 5 - fold . none of the previously known approaches to the preparation of transferrin - doxorubicin conjugates are capable of producing large amounts of homogeneous conjugates with predetermined ratios of the number of drug molecules per molecule of transferrin . in addition , the known approaches employ chromatography to eliminate aggregates and to harvest fractions that are enriched in homogeneous conjugates . these procedures decrease yields , increase costs , and lack the ability to predetermine molecular ratios . after the conjugates are isolated , they can optionally be characterized by polyacrylamide gel electrophoresis to determine their molecular weight , and the number of drug molecules per protein molecule can be determined . experience with drug - protein conjugates in other systems has shown that a functional drug : protein ratio is 0 . 1 – 4 . 0 molecules of drug per molecule of protein ( berczi et al ., arch biochem biophy 1993 ; 300 : 356 ), recent unpublished data - suggest that lower conjugation numbers are still significantly cytotoxic , while higher conjugation numbers ( e . g ., & gt ; 4 . 0 ) tend to be associated with unstable conjugates . other steps in the characterization of the conjugates are to ( a ) determine if the conjugates bind to transferrin receptors on the surface of infected cells and not uninfected cells , and ( b ) determine if the conjugates kill protozoan infected cells and not uninfected cells . the binding studies can be done by using flow cytometry , and the killing studies can be done by using microculture techniques to determine the concentration of free drug required to kill 50 % of a culture of infected cells compared to the concentration of drug in the drug - protein conjugate required to kill the same number of infected cells . experience with drug - protein conjugates in other systems indicates that approximately 10 - fold more free drug compared to the drug in drug - protein conjugates should be required to kill the same number of infected cells . for example , the dosage of a conjugate of transferrin - doxorubicin is expected to be between 0 . 5 – 50 mg per 28 day period for a 150 pound ( 68 kg ) person the dosage can be administered as smaller doses at varying intervals during the 28 day period . for a conjugate to be efficacious , preferably it should kill none or only a minimum of uninfected cells . since drug resistance ( world health org ., technical report series no 692 , 2000 ) and drug toxicity ( winstanley , j roy col phy london 1998 ; 32 : 203 ) are major problems in the treatment of malaria , the aim of the present invention is to provide a ligand - receptor method for the targeted delivery of anti - malarial drugs designed to utilize the pathways employed by plasmodia to acquire iron in one embodiment , the ligand is human transferrin , the receptor is plasmodial transferrin receptor , and the drug is either the cytotoxic drug doxorubicin which also is an iron chelator ( myers , seminars oncol 1998 ; 25 : 10 ); or the iron - chelating siderophore deferoxamine ( also known as desferrioxamine or desferal ). deferoxarnine is a hydroxamate - based hydrophilic chelator of iron ( tsafack et al ., j lab clin med 1996 ; 127 : 574 ). the molecule has a terminal nh 2 that has been derivatized with molecules such as nitrobezyl - diazole and n - methylanthranile without reducing its property of iron chelation ( loyevsky et al ., j clin invest 1993 ; 91 : 218 ). a carrier is needed that could transport trypanocidal compounds across the blood - brain - barrier . the normal plasma protein transferrin has been shown to accomplish this task by means of interacting with transferrin receptors on endothelial cells that compose the microcirculation of the blood - brain - barrier ( broadwell et al ., exp neurol 1996 ; 142 : 47 ). for example , a conjugate of transferrin with nerve growth factor has been shown to be transported from blood into the brain ( li et al ., j natural tox 2000 ; 9 : 73 ), and the object of the present invention is to provide conjugates of trypanocidal drugs with transferrin that can be transported from blood across the blood - brain - barrier into the central nervous system , thereby providing effective therapy for both early and late stages of trypanosomiasis . in addition to being an effective transporter of trypanocidal drugs across the blood - brain - barrier , transferrin can be targeted to transferrin receptors present on trypanosomal plasma membranes ( borst et al ., science 1994 ; 264 : 1872 ). like human transferrin receptors , trypanosomal receptors are regulated post - transcriptionally by iron ( fast et al ., biochem j 1999 ; 342 : 691 ). each trypanosome contains about 3000 receptors , which are heterodimers linked by a glycosylphosphatidylinositol anchor to the plasma membrane where they concentrate in flagellar pockets , among a sea of variant surface glycoprotein ( borst & amp ; fairlamb , ann rev microbiol 1998 ; 52 : 745 ). trypanosomes require iron , which they obtain from the transferrin of their host ( schell et al ., embo j 1991 ; 10 : 1061 ). since they can thrive in many different mammalian hosts , and since transferrins differ in different mammals , trypanosomes have about 20 gene copies of transferrin receptors , which allows them to produce a high - affinity receptor to bind and internalize host transferrins , whether they be in animals or human patients ( bitter et al ., nature 1998 ; 391 : 499 ). thus , intravenously administered trypanocidal drug conjugates of transferrin circulate throughout the body , including the central nervous system , where they are bound by trypanosomal transferrin receptors and exercise their trypanocidal properties . the present invention is a drug - protein conjugate which can be used for the targeted delivery of a cytotoxic drug to trypanosomes in infected patients , whether they are in early or late stages of disease , and regardless of which trypanosoma species with which they are infected . targeted delivery of drugs is possible in this invention because the preferred protein in the drug - protein conjugate is transferrin , which is relevant because trypanosomes have transferrin receptors on their surfaces ( bitter et al ., nature 1998 ; 391 : 499 ). in addition , the drug in the drug - transferrin corrugate can be a known trypanocidal agent , or cytotoxic drug such as doxorubicin . while being present on the surfaces of cancer cells ( yeh et al ., vox sang 1984 ; 46 : 217 ), transferrin receptors usually are not present on the surface of normal , adult , resting cells ( berczi et al ., arch biochem biophy 1993 ; 300 : 356 ). thus , most normal cells in trypanosomiasis patients will not be affected , and the only cells to be eliminated by cytotoxic transferrin conjugates will be the trypanosomes , whether they are in blood , lymph or the central nervous system . one way to illustrate targeted drug delivery to trypanosomes in patients is to focus on the use of transferrin , which carries iron in the blood . transferrin can be obtained by isolation from blood plasma , from commercial suppliers , or from recombinant technology ( ali et al ., j biol chem 1999 ; 274 ; 2406 6 ). to form the drug - protein conjugate , transferrin molecules must be modified in such a way as to prepare them to be coupled with a trypanocidal or cytostatic drug . the drug can be an arsenical such as melarsopral , a cytotoxic antibiotic such as doxorubicin or an inhibitor of polyarnine synthesis such as eflomithine , but any compound can be used , including plant toxins such as ricin , and bacterial mutant toxins such as modified diphtheriatoxin ( laske et al ., nature med 1997 ; 41 : 1039 ). several coupling processes such as glutaraldehyde coupling ( yeh & amp ; faulk , clin immunol immunopathol 1984 ; 32 : 1 ), disulfide coupling ( sasaki et al ., jap j can res 1993 ; 84 : 191 ) or benzyl hydrazine coupling ( kratz et al ., j pharm sci 1998 ; 87 : 338 ) have been used to couple transferrin with other molecules . the wide variety of coupling procedures allows the conjugation of a broad range of drugs to transferrin , resulting in either permanent or dissociable bonding of the drugs with the transferrin molecule ( barabas et al ., j biol chem 1992 ; 267 : 9437 ). following the coupling reaction , drug - protein conjugates can be separated from uncoupled drug and free protein , if necessary by using chromatographic procedures or selective dialysis . while the present invention has been described in relation to transferrin being the delivery protein , it is known that other proteins exist in the body which are capable of binding to receptor sites on infected cells . if the receptor site is activated in infected cells , and is inactive in uninfected cells , then any protein or other compound which binds to such a receptor site can be used to deliver the drugs used in the present invention . one example of such a binding protein is transcobalamin , which delivers vitamins , especially vitamin b12 , to transcobalamin receptors on cells in the human body ( seetheram , ann rev nutr 1999 ; 19 : 173 ). other examples include but are not limited to ceruloplasmin , vitamin binding proteins , hormones , cytokines , low density lipoproteins , and growth factors . the conjugates according to the present invention are administered to an animal in an effective amount in treating protozoan infections , an effective amount includes an amount effective to reduce the amount of protozoa the dosage for the conjugates can be determined taking into account the age , weight and condition of the patient and the pharmacokinetics of the anti - protozoan agent . the amount of the conjugate required for effective treatment will be less than the amount required using the anti - protozoan agent alone and depends upon the anti - protozoan agent used . for example , the dosage of a conjugate of transferrin - doxorubicin is expected to be between 0 . 5 – 50 mg for a 150 pound ( 68 kg ) person . the dosage can be administered as smaller doses at varying intervals and repeated if necessary . the pharmaceutical compositions of the invention can be administered by a number of routes , including but not limited to orally , topically , rectally , ocularly , vaginally , by pulmonary route , for instance , by use of an aerosol , or parenterally , including but not limited to intramuscularly , subcutaneously , intraperitoneally , intra - arterially or intravenously . the compositions can be administered alone , or can be combined with a pharmaceutically - acceptable carrier or excipient according to standard pharmaceutical practice . for the oral mode of administration , the compositions can be used in the form of tablets , capsules , lozenges , troches , powders , syrups , elixirs , aqueous solutions and suspensions , and the like . for parenteral administration , sterile solutions of the conjugate are usually prepared , and the phs of the solutions are suitably adjusted and buffered . for intravenous use , the total concentration of solutes should be controlled to render the preparation isotonic . for ocular administration , ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers . for pulmonary administration , diluents and / or carriers will be selected to be appropriate to allow the formation of an aerosol . it is preferred that the conjugate of the present invention be administered parenterally , i . e . intravenously or intraperitoneally , by infusion or injection . as used in the present document , the term “ substantially homogeneous conjugates ” means that the conjugates can be used without further purification to remove protein dimers , polymers or aggregates . in other words , little or no protein dimers , polymers or aggregates are present . preferred embodiments of the present invention are described below . it will be apparent to those of ordinary skill in the art after reading the following description that modifications and variations are possible , all of which are intended to fall within the scope of the claims . the synthesis of large amounts of homogeneous transferrin - doxorubicin conjugates with predetermined molecular ratios was done stoichiometrically by employing the only amino group of doxorubicin ( dox ), which is at the 3 ′ amino position , to react with one of the two reactive groups on glutaraldehyde ( glu ). the first step was to add glu drop - wise to dmso in an ice cold water bath . next was the drop - wise addition of a saline solution of dox into a saline solution of glu + dmso to a final concentration of a 1 : 1 molar ratio of dox - to - glu . the resulting solution of dox - glu was stirred three hours at room temperature in the dark . the molarities of dox and glu were the same in the above reaction in order to produce a final solution of dox - glu that contains neither free dox nor free glu . however , there is the possibility of free glu in solution if one glu reacts with two dox to produce dox - glu - dox , but this possibility is minimized by the mass action kinetics generated by drop - wise addition of monovalent dox into the solution of bivalent glu . the volumes of these reactants are not restricted , so large amounts of homogeneous dox - glu can be prepared . the second step in the conjugation reaction was drop - wise addition of dox - glu into a saline solution of transferrin ( trf ). the trf can be either iron - free ( apo - transferrin ) or iron - saturated ( holo - transferrin ). the desired molar ratio of dox to trf was obtained by appropriately adjusting the volume of trf . the resulting solution of trf - glu - dox was stirred for 20 hours at room temperature in the dark . unlike the reaction of dox with glu , the reaction of dox - glu with trf is not restricted to one binding site , for the glu component of dox - glu can react with any one of several epsilon - amino lysine groups in the trf molecule . the number of dox molecules bound to trf was determined in the second step . for example , if the starting ratio of dox - glu to trf was 7 . 2 : 1 . 0 , the final solution of trf - glu - dox would have contained 2 . 5 molecules of dox per molecule of trf . however , if the starting ratio of dox - glu to trf was 4 . 0 : 1 . 0 , the final solution of trf - glu - dox would have contained 1 . 4 molecules of dox per molecule of trf . similarly , if the starting ratio of dox - glu to trf was 2 . 5 : 1 . 0 , the final solution of trf - glu - dox would have contained 0 . 9 molecules of dox per molecule of trf . in this way , large amounts of trf - glu - dox with predetermined ratios of dox - to - trf can be provided according to the need . in an optimization of the production of the conjugate , ethanolamnine is added , followed by centrifugation and dialysis . although reactions with dox and trf theoretically consume all of the glu , ethanolamine was added to the final reaction mixture to bind any available glu . this reaction was allowed to continue for 30 minutes in the dark . the final solution was centrifuged at 2000 rpm for 10 minutes , dialyzed twice for 6 hours in a 100 - fold excess of saline and three times in the same excess of hepes buffered saline , and the resulting trf - glu - dox conjugates were ready for use .
targeting agents such as transferrin and transcobalamin can be conjugated with anti - protozoan drugs for the treatment of protozoan infections . any suitable anti - protozoan drug can be used , preferably the drug is selected from the group consisting of apoptosis inducing compounds , cytotoxic antibiotics , alkalating agents , plant toxins , and bacterial mutant toxins . the targeting agent is preferably coupled to the antiprotozoan drug by means of glutaraldehyde .