Patent Application: US-56946600-A

Abstract:
new macromolecular carriers for drugs and diagnostic agents are described that make use of the chemical attachment of new leashes to oligomeric backbone structures . the synthesis of these leashes and their facile creation , reaction and conjugation with chelators and ligands makes them ideal candidates for use in medicine , and especially diagnostics .

Description:
the invention pertains to the targeting of drugs and diagnostic agents to specific tissues using a macromolecular delivery vehicle . before now , there has been only limited clinical success for such vehicles due to lack of inexpensive and nontoxic molecular backbones to which drugs and target substrates can be attached . the invention remedies this by providing new macromolecules produced by novel processes that suppress unwanted cross - linking while preserving nontoxicity , and optimizing leash and load capacity per backbone size . although useful when combined or conjugated with therapeutic agents , the invention is especially useful for cardiovascular and / or tumor imaging . a successful cardiovascular and tumor contrast agent for magnetic resonance imaging ( mri ) or computed tomography ( ct ) should possess an inexpensive and nontoxic molecular backbone capable of carrying a high number of contrast substrates . prior to the invention , this had not been the case . one embodiment of the invention features a chemical scheme that produces an inexpensive macromolecule with a large number of attachment sites called “ leashes ”. a high number ( hundreds ) of leashes per backbone molecule permits the attachment of a high density of substrates , drugs , and / or other molecular entities . an additional useful feature can be found in a preferred embodiment in which the backbone of the macromolecule is dextran . dextran has an excellent safety record as a cardiovascular volume expander . the extensive human - use of dextran increases the probability that the delivered drug or diagnostic agent will also be nontoxic . there are many potential uses for the invention , e . g ., as pharmaceuticals ( i . e ., therapeutics ) and as diagnostics ( i . e ., probes ). in theory , although most any drug can be used with the invention , the following uses are especially foreseen : attachment of radioprotectors , such as wr2721 , ( rasey j s et al . specific protection of different normal tissues . pharmac ther 39 : 33 - 43 , 1988 ), chemoprotectors , such as amifostine ( ethyol ) ( capizzi r l . protection of normal tissues from the cytotoxic effects of chemotherapy by amifostine ( ethyol ): clincial experiences . semin oncol 21 ( s11 ): 8 - 15 ( 1994 )), and radiosenisitizer molecules , such as misonidazole ( phillips t l . sensitizers and protectors in clinical oncology . semin oncol 8 : 67 - 82 , 1881 ) and appropriate receptor substrates for tissue specific protection or sensitization ; attachment of antiviral drugs and appropriate receptor substrates for tissue - specific antiviral therapy , e . g ., to treat hepatitis ; attachment of immunomodulators and appropriate receptor substrates or ligands for tissue - specific immunotherapy ; attachment of dna and appropriate receptor substrates for tissue - specific gene therapy , e . g ., targeting p52 or suicide genes into cancer cells to induce apoptosis , immunotargeting , etc . ; and attachment of peptides and mono - or polysaccharides for targeting to specific receptors . see , e . g ., molteni l ( 1979 ), dextrans as drug carriers . in : gregoriadis g , ed . drug carriers in biology and medicine . san diego , academic press ; 107 - 125 . in terms of diagnostics , the following uses are especially contemplated : attachment of gadolinium complexes for magnetic resonance imaging ( mri ) of the cardiovasculature and / or tumors ; attachment of iodinated molecules for computer tomographic ( ct ) imaging of the cardiovasculature and / or tumors ; attachment of ytterbium , dysprosium , or other heavy metal complexes for computer tomographic ( ct ) imaging of the cardiovasculature and / or tumors ; attachment of technetium - 99m complexes for scintigraphic imaging of the cardiovasculature and / or tumors ; attachment of gadolinium complexes and appropriate receptor substrates for tissue - specific imaging of the liver , lymph nodes , bone marrow , and / or other tissues ; attachment of iodinated molecules and appropriate receptor substrates for tissue - specific ct imaging ; attachment of ytterbium , dysprosium , or other heavy metal complexes and appropriate receptor substrates for tissue - specific ct imaging ; attachment of technetium - 99m complexes and appropriate receptor substrates for tissue - specific scintigraphy ; and attachment of dyes and fluorescent agents for optical imaging . again , the preceding uses are only illustrative of the many possible applications for which the invention can be used . specific examples for some of these applications follow ; other applications will be readily apparent and can be practiced by one of ordinary skill in the art viewing the application . dextran , a preferred embodiment for the molecular backbone of the invention , possesses , as do many polysaccharides and branched polysaccharides , a plethora of reactive hydroxyl groups to which chemical attachments can be made . dextran is a natural product derived from bacteria . it is isolated in high molecular weight form and can be hydrolyzed and purified in controlled fashion to various smaller molecular weight commodities , e . g ., molecular weight (“ mw ”) 1 , 000 , 10 , 000 , 40 , 000 , 70 , 000 , 110 , 000 , 150 , 000 and 500 , 000 . ( amersham pharmacia biotech , usa ). each of the listed mw species can be used with the invention and each may have more or less suitable effect for a given application . for example , for tumor imaging one would select a dextran with a size that after conjugation of leashes and reporter groups would yield a final molecular weight within the 50 - 70 kilodalton ( kd ) range . this would permit the selective diffusion into tumors , which have greater permeability than normal tissue ( see seymour l w . passive tumor targeting of soluble macromolecules and drug conjugates . critical reviews of therapeutic drug carrier systems 9 : 135 - 187 ( 1992 )). imaging of cardiovascular structures will require conjugates with molecular weights in the 70 - 100 kd range . the lower limit is set by the desire to minimize renal filtration . the upper limit is set by the increased viscosity of high molecular weight macromolecules , which produces administration problems . as stated previously , much pharmacological data is available for dextran based on its wide use as a plasma volume expander , in which it has been demonstrated to persist for several weeks after infusion into patients and during which time it is gradually oxidized into smaller forms and cleared via the kidneys . see goodman and gilman &# 39 ; s the pharmalogical basis of therapeutics , eighth edition , pp . 690 - 91 , pergamon press , new york , ( 1990 ). the leash attachment was a two - step process , as shown in fig1 . the first step is the activation of the dextran hydroxyl units with allylbromine ( holmberg , 1993 ; gedda , 1996 ). this reaction uses 10 grams of pm10 in 75 ml deionized water is carried out at 50 ° c . and ph 11 ( maintained by dropwise addition of 2 . 5n naoh ) in the presence of sodium hydroxide ( 2 . 5 grams ) and sodium borohydride ( 0 . 2 grams ). after 3 hours the solution is neutralized with acetic acid ( 2 . 5 m ) the reaction is placed in a 5 ° c . refrigerator for 2 hours , and the top organic layer is decanted . after addition of 100 ml deionized water , the resulting solution is then filtered ( 5 mm ) into an ultrafiltration cell ( mwco = 3 , 000 ), and dialyzed with 10 exchange volumes of deionized water . the product , allyl - dextran , is concentrated , lyophilized , and stored at − 80 ° c . the mean molecular diameter is measured by laser light scattering ( honeywell microtrac upa 150 ). the average number of allyl groups per dextran is calculated in the following manner . the lyophilized allyl - dextran is dissolved in saline and the glucose concentration of the sample is measured by the sulfuric acid method ( dubios , 1956 ) using lyophilized dextran as a standard . the allyl density is calculated by subtracting the amount of glucose in the sample from the total weight of the sample . the result is assumed to be the amount of allyl hydrocarbon in the sample . the allyl concentration is then divided by the glucose concentration followed by multiplication by the average number of glucose units per dextran . in the second step , the allyl - groups were reacted with 7 . 5 grams aminoethanethiol ( cysteamine ) in dmso ( 30 ) to produce the amino - terminated leashes . this reaction is initiated with ammonium persulfate ( 99 . 99 %, 1 . 0 g ) and carried out at under a nitrogen atmosphere . after 3 hours the reaction volume is doubled with deionized water and the solution is adjusted to ph 4 with sodium hydroxide ( 2 . 5 n ). after addition of 140 ml sodium acetate buffer ( 0 . 02 m , ph 4 ), the product is filtered ( 5 mm ) into an ultrafiltration cell and dialyzed with five exchange volumes of acetate buffer ( 0 . 02 m , ph 4 ) followed by five exchange volumes of deionized water . after concentration , the amino - terminated - dextran is then lyophilized . a sample is then assayed for the average number of amino groups per dextran , which is defined as the amino density . this lyophilized product is stored at − 80 ° c . the mean molecular diameter is measured by laser light scattering . using these methods , substitution levels of 516 amino groups per molecule were determined for a amino - dextran t70 , which contains an average of 388 glucose residues linked by α - 1 , 6 and 1 , 4 glucosidic bonds . the mean diameter of this preparation of amino - dextran - t70 in 0 . 9 % saline was 10 . 8 nanometers ; the mean diameter of dextran t70 in saline was 10 . 6 nanometers . using t500 , which contains an average of 2881 glucose units , an average of 2900 amino leashes were found substituted . the average number of amino groups per dextran is calculated in the following manner . the lyophilized dextran conjugate is dissolved in saline and the amine concentration is measured by the tnbs assay ( fields , 1972 ) using hexylamine as a standard . the glucose concentration of the same sample is also measured by the sulfuric acid method ( dubios , 1956 ). the amino density is calculated by dividing the amine concentration by the glucose concentration followed by multiplication by the average number of glucose units per dextran . following are examples of how amino - dextran as formulated above can be conjugated with other compounds to synthesize various diagnostic agents . attachment of a chelator ( dtpa ) for blood pool imaging via magnetic resonance or computed tomography attachment of diethylenetriamine pentaacetic acid ( dtpa ) to amino - dextran yields dtpa - amino - dextran which can in turn be labeled with gadolinium or dysprosium ( see fig2 a ) to produce a blood pool contrast agent for magnetic resonance imaging or computed tomography . the mixed anhydride method of krejcarek et al . ( 1977 ), biochem . biophys . res . commun . 77 : 581 - 583 , is employed . this is in addition to the novel chemistry used to first establish the leashes . this process will not promote cross - linking of the dextran if an excess of dtpa ( compared to activating reagent , ibcf ) is employed . the mixed anhydride method ( krejcarek , 1977 ) was used to conjugate dtpa to the dextran backbone . the synthesis starts with the activation of dtpa ( diethylenetriamine pentaacetic acid ) ( 20 g ) with isobutylchloroformate ( ibcf ) ( 3 . 1 ml ). this is carried out in acetonitrile ( 83 ml ) at − 30 ° c . the activated dtpa is slowly added to the amino - terminated dextran ( 2 g ) in bicarbonate buffer ( 0 . 1 m , ph 9 ) at 4 ° c . ( see fig3 .) this solution is stirred overnight at room temperature . after extensive diafiltration the product with five exchange volumes of bicarbonate buffer ( 0 . 1 m , ph 9 ), followed by five exchange volumes of deionized water , the retentate is concentrated , and freeze dried . a sample is then assayed for dtpa and amino densities . this lyophilized product is stored at − 80 ° c . the mean molecular diameter is measured by laser light scattering . the average number of dtpa units per dextran is calculated in the following manner . lyophilized dtpa - dextran is dissolved in 0 . 1 mmol / l triethanolamine hcl ( ph = 6 . 0 ). we added a 1 . 5 ( mol / mol with respect to dtpa ) excess of gadolinium in a 66 mm solution ( filtered , 0 . 2 micron ) of 0 . 1 n hcl and then adjusted the ph to 6 with 2 . 5 n naoh . after stirring 24 hours at 37 ° c . the solution is transferred to an amicon ultrafiltration system for diafiltration at a molecular weight cutoff of 3 , 000 da . after 10 exchange volumes with deionized water followed by another 10 exchange volumes with acetate buffer ( 0 . 2 m , ph 4 ), the retentate is concentrated and assayed for gadolinium concentration by icp spectrometry ( vera , 1995 ). the glucose concentration of the same sample is also measured by the sulfuric acid method ( dubios , 1956 ). the dtpa density is calculated by dividing the gadolinium concentration by the glucose concentration followed by multiplication by the average number of glucose units per dextran . procedures for coupling of other chelators , including dota and mag3 , to attachment sites are shown in fig4 and 5 , respectively . the presence of mannose appended to the dextran backbone will target the compound , depending on the mode of administration , to a receptor ( mannose binding protein ) that resides on liver , spleen , lung , bone marrow and lymph nodes . steer c j , ashwell g ( 1990 ). receptor - mediated endocytosis : mechanisms , biologic function , and molecular properties . in : hepatology . a textbook of liver disease . ( 2nd ed .) zakim d , boyer t d , eds . w . b . saunders , philadelphia . if the compound is administered subcutaneously , the mannose - terminated dextran will enter the lymphatic system and bind to receptors within lymph nodes ; if administered intravenously , binding will occur to the liver , spleen , lung and bone marrow . it is further known that mannose - binding protein exhibits increased affinity to cluster glycosides , e . g ., mannose and mannose - derivatized compounds . if the dextran backbone is also made to carry a chelating agent such as dtpa or dota ( sieving et al . ( 1990 ), bioconj . chem . 1 : 65 - 71 ), the resulting combination can further be labeled with gadolinium , technetium - 99m , or ytterbium . gadolinium ( see , e . g ., fig6 ) permits the detection of liver or lymph node tumors via magnetic resonance imaging . the radiolabel technetium - 99m ( see , e . g ., fig7 ) permits liver or lymph node imaging by scintigraphy . ytterbium , like gadolinium and dysprosium , permits imaging via computed tomography . the chemical attachment of mannose to amino - dextran can be accomplished by a number of methods , e . g ., that described for attachment to human serum albumin ( vera et al . ( 1985 ) j . nucl . med . 26 : 1157 - 1167 ) and that described for attachment to polylysine ( vera et al . ( 1995 ) acad . radiol . 2 : 497 - 596 ). see , e . g ., fig8 . this attachment can be carried out as described above for galactose . conjugation of dtpa - dextran with mannose is accomplished by reductive alkylation ( vera , 1997 ). the mannosyl coupling reagent , cyanomethyl 2 , 3 , 4 , 6 - tetra - o - acetyl - 1 - thio - β - d - mannoside ( cnm - thiomannose ), is synthesized by the following route . tetra - o - acetyl - α - d - mannopyranosyl bromide ( this carbohydrate is not commercially available ) is produced by a two - step reaction in chloroform ( lee , 1994 ). after rotary evaporation to a syrup , the product is immediately reacted with thiourea to produce 2 - s -( 2 , 3 , 4 , 6 - tetra - o - acetyl - β - d - mannopyranosyl )- 2 - peusdothiourea - hydrobromide ( chipowsky , 1973 ), which is recrystallized in acetone . immediately prior to mannosyl conjugation , this product is deacetylated with sodium methoxide ( 0 . 05 mg / ml ) in dry freshly distilled methanol ( 250 ml ) using a cnm - thiomannose concentration of 0 . 04 gram per ml of methanol . after removal of the methanol by rotary evaporation , the coupling reaction is carried out by the addition of an appropriate amount of dtpa - dextran ( 0 . 5 gram ) in a 20 mg / ml solution of bicarbonate buffer ( 0 . 2 m , ph 9 . 0 ). the reaction is conducted at room temperature 2 hours ( fig8 ). after diafiltration the product with five exchange volumes of bicarbonate buffer ( 0 . 1 m , ph 9 ), followed by another five exchange volumes of deionized water , the retentate is concentrated , and freeze dried . a sample is then assayed for mannose , dtpa , and amine densities . this lyophilized product is stored at − 80 ° c . the mean molecular diameter as measured by laser light scattering is 7 . 6 nm . the average number of mannose groups per dextran is calculated in the following manner . the lyophilized dextran conjugate is dissolved in triethanolamine and labeled with gadolinium as described above . after diafiltration and measurement of dtpa concentration , the mannose concentration of the same sample is also measured by the sulfuric acid method ( dubios , 1956 ). the mannose density is calculated by dividing the mannose concentration by the dextran concentration , which is calculated based on the known dtpa density of the dtpa - dextran conjugate . a sentinel node imaging agent dtpa - mannosyl - dextran was synthesized using pharmaceutical grade dextran , pm10 . see , e . g ., fig7 . this preparation had a mean diameter of 7 . 6 nanometers , mannose density of 44 units per dextran , an amine density of 23 units per dextran , and a dtpa density of 8 units per dextran . the molecular weight was 36 , 288 grams per mole . the presence of the substrate galactose can deliver the dextran to a receptor that resides exclusively in the liver . if the dextran is also carrying dtpa , it can be labeled with gadolinium or technetium - 99m . the use of gadolinium permits the detection of liver cancer via magnetic resonance imaging . the radioactive label technetium - 99m permits liver imaging by scintigraphy . the chemical attachment of galactose to amino - dextran can be accomplished by a number of methods , e . g ., as described for attachment to human serum albumin ( vera et al . ( 1985 ) j . nucl . med . 26 : 1157 - 1167 ) and polylysine ( vera et al . ( 1995 ) acad . radiol . 2 : 497 - 596 ). the coupling reaction for the attachment of galactose to amino - terminated dextran is the same as the attachment ( see below ) of mannose . fig1 a and 13b are examples of an mr liver imaging agent and a nuclear medicine imaging agent , both of which use an embodiment of the invention . conjugation of amino - terminated dextran starts with the activation of mercaptoacetylglycylglycyl - glycine ( mag3 ) ( fritzberg , 1986 ) with tetraflorophenol ( tfp ). this is carried out with an equivalent amount of 1 , 3 dicyclohexylcarbodiimide ( dcc ) in n , n - dimethylformamide ( dmf ) at room temperature . this solution is rotary evaporated , dissolved in chloroform , and filtered . this resulting solution is rotary evaporated and the solution product , tfp - mag3 stored in the freezer . finally , the aminoterminated - dextran in a dmso / water solution ( 1 : 10 ) is combined with tfp - mag3 in a similar solution . this solution is stirred overnight at room temperature . after extensive diafiltration with water the product is concentrated and freeze dried . sentinel node imaging : prospects for the improved diagnosis and treatment of cancers , i . e ., breast cancer , melanoma , and colorectal cancer in a effort to reduce the morbidity and costs of detection for lymph node metastases , surgical oncologists have developed a method by which the sentinel lymph node ( the first node in a draining basin ) is identified intraoperatively and removed ( morton et al . ( 1992 ) arch . surg . 127 : 292 - 299 .). this technique , called sentinel node biopsy , has extremely high negative predictive values for melanoma and breast cancer metastases ( giuliano et al . ( 1994 ) arch . surg . 220 : 391 - 401 ); the false - negative rates range from zero to 9 % for both cancers . there is also growing evidence that sentinel node biopsy will have a significant impact on the management of colorectal cancer ( saha et al . ( 1998 ) j . surg . oncol . 69 : 183 .). as stated , sentinel node imaging is a nuclear medicine examination which identifies for the surgeon the first lymph node to receive lymphatic flow from the primary tumor site . this node is removed and sent for frozen section analysis for detection of malignant cells . by identifying the sentinel node prior to surgery , a small incision can be use to remove the node and a smaller dissection can be employed . the extremely high negative predictive value of the technique seems to provide an accurate staging procedure and may spare patients who are sentinel node negative the morbidity of a complete lymph node dissection . consequently , staging of the cancer by sentinel node imaging may be equivalent to axillary node dissection without the attendant post - surgical morbidity . when performing sentinel node biopsy , the imaging agent is injected around the tumor site and a hand held gamma probe is used to find and remove the sentinel node . often , however , the nuclear activity either spills over into the nodal basin making differentiation between node and tumor difficult , or it disseminates into multiple nodes causing more than the necessary number of nodes to be removed . an ideal sentinel node imaging agent would exhibit rapid clearance from the injection site , high retention within the lymph channel , rapid , complete , and sustained uptake by the sentinel lymph node , and low uptake by the remaining lymph nodes . in breast cancer cases the sentinel node is sometimes not found because the tumor site is so close to the sentinel node , with radioactivity between the two sites being indiscernible . the standard features of low radiation absorption , high biological safety , convenient , rapid , and stable technetium - 99m labeling , and biochemical purity also apply . filtered colloids exhibit poor clearance from the injection site . the half times for filtered tc - albumin colloid and tc - sulfur colloid are 5 . 5 hr and 10 . 5 hr , respectively ( glass , 1995 ). this translates to approximately 65 % and 85 % of the dose at the injection site at 3 hours postinjection , the optimal time for the intraoperative search for the sentinel node ( uren , 1995 ). conversely the labeled macromolecules , such as tc - dextran and tc - hsa ( t ½ = 2 . 8 hr ), offer the fastest clearance ( henze , 1982 ; glass , 1995 ). the procedures described for the present invention improve the combination of technetium - 99m - labeled mag3 - mannosyl - dextran . this agent has already been demonstrated effective , even when prepared using the sub - optimal , cross - link - prone procedures of the prior art . technetium - 99m - labeled mag3 - mannosyl - dextran , as previously described , is a 10 - kilodalton molecule of dextran to which multiple units of mannose and mag3 are attached . this molecule has a mean diameter of 5 . 5 nanometers ( nm ), which is substantially smaller than tc - 99m antimony trisulfide ( 10 nm ), filtered tc - 99m sulfur colloid ( 50 nm ), and unfiltered tc - 99m sulfur colloid ( 650 nm ), which had previously been the prevailing standards . the employment of mannose acts as a substrate for the mannose - binding protein receptor and the mag3 serves as a chelating agent for labeling with technetium - 99m ( fig9 ). the resulting radiolabeled conjugate rapidly clears the injection site and binds to the sentinel lymph node . that prior study is reproduced below ; use of the improved chemistry described herein promises to only enhance the success already obtained . after anesthesia with an intramuscular injection of ketamine and xylazine , 0 . 5 ml of tcmag3 - mannosyl - dextran ( 19 , 000 g / mole ) was injected to the right front ( 0 . 42 mci ) and right rear ( 0 . 43 mci ) foot pads and 0 . 5 ml of filtered tc - sulfur colloid to the left front ( 0 . 41 mci ) and left rear ( 0 . 42 mci ) foot pads . the rabbit was hydrated with 10 cc of saline infused to the neck every 60 minutes . after injection of each radiopharmaceutical , the foot pads were massaged for 5 minutes . periodic static images ( 1 , 000 kcts , 256 × 256 × 16 ) were acquired on a large field - of - view gamma camera ( high resolution , low energy collimator ) at 15 , 45 , 100 , and 135 minutes with the front limbs and axillary nodes in view , and at 21 , 53 , 109 , and 154 minutes with the rear limbs and popliteal limbs in view . the limbs were exercised for 15 minutes before each image . imaging standards ( 10 × dilution ) of both tcmag3 - mannosyl - dextran and the tcsc were also positioned within the field - of - view . approximately 150 minutes post - injection the rabbit was euthanized . each sentinel node was excised and assayed for radioactivity with diluted ( 2 - fold ) samples of the tcmag3 - mannosyl - dextran and tcsc doses . these values were normalized by each imaging standard to yield the sentinel node % id , which were then plotted as a function of time ( fig1 ). for each serial image , regions - of - interest ( rois ) were drawn around the injection site , sentinel node , and imaging standards . the total counts within each roi were then calculated using standard nuclear medicine software . the absolute counts in the injection site roi &# 39 ; s were then divided by the counts of the standard and the fraction - of - injected dose was calculated . these values were then plotted on a logarithmic scale , shown in fig1 . the data obtained demonstrates that the imaging properties of tc99m - mag3 - mannosyl - dextran are superior to the filtered tc99m sulfur colloid . the percent - of injected dose , plotted in fig1 , demonstrated greater lymph node uptake of tcmag3 - mannosyl - dextran in both axillary and popliteal sentinel nodes over a 150 - minute period . additionally , tcmag3 - mannosyl - dextran exhibited significantly faster clearance from the injection site , indicated by the lower curve of fig1 . axillary lymph nodes imaged 15 minutes after administration of tcmag3 - mannosyl - dextran and tcsc appeared as shown in fig1 . the injection sites are at the top of the image ( tcmag3 - mannosyl - dextran on the left and tcsc filtered on the right side ). there is a pair of standards at either side of the rabbit . in addition to the bilateral sentinel nodes , a focus of activity adjacent to the left sentinel node medially may represent tcsc activity in a distal node , corresponding to the frequently observed behavior of tcsc where distal lymph nodes are commonly observed in delayed images . this activity , however , may represent a mediastinal lymph node shared by both sides of the axillary lymphatic chain , in which case this finding will not be of much significance . the rapid injection site clearance of tcmag3 - mannosyl - dextran may be explained by the small size of the particle ( 5 . 5 nanometers ), which allows it to also diffuse into the blood capillaries . lymph node uptake of tcmag3 - mannosyl - dextran for the three rabbit studies ranged from 1 . 2 to 2 . 5 percent of the injected dose . the range for filtered tcsulfur colloid was 1 . 5 - 4 . 9 percent . magnetic resonance imaging with gddtpa - dextran was performed using both one healthy and one tumor - bearing rabbit weighing 3 . 0 and 2 . 7 kg , respectively . each rabbit was anesthetized with a mixture of 50 mg / kg ketamine and 8 . 8 mg / kg xylazine . following anesthesia administration each rabbit was then intubated with a 3 - mm tracheal tube and positioned within the scan field . each rabbit was ventilated at 60 bpm with a stroke volume of 25 cc , which permitted respiratory gating . both rabbits received a 0 . 17 mmole gadolinium - per - kilogram dose ( 0 . 13 grams gddtpa - dx / kg ) of gddtpa - dextran ( mw = 398 , 000 g / mole , 515 gd per dextran ). images were acquired using a ge signa ™ 1 . 5 t magnetic resonance imaging unit ( version 4 . 83 software ) ( ge medical systems , milwaukee , wis .) with each rabbit placed in a knee coil . we explored a number of acquisition parameters ; the most successful was a 3d time of flight ( tof ) fast spoiled gradient echo ( fspgr ) sequence : tr = 15 . 1 msec , te = 4 . 2 msec , flip angle = 30 °, 256 × 192 matrix , 16 cm fov , 80 slices of 1 . 5 mm thickness . images of the healthy rabbit ( 3 kg ) were acquired prior to injection ( 2 . 0 ml ), and at 15 , 28 , 33 , 46 minutes , and 3 hours post injection . with the exception of the 28 - minute scan , which was in the coronal plane , all scans were acquired in the axial projection . the uncorrected signal intensities within the aorta at the level of the right kidney were : pre - injection , 193 ; 28 minutes , 552 ; 33 minutes , 516 ; and 46 minutes , 472 . fig1 a is a maximum intensity projection image of the healthy rabbit acquired 15 minutes post - injection . fig1 b is a similar image of the same animal acquired three hours post - injection . fig1 a and 16 b are magnetic resonance images of the tumor - bearing rabbit ( 2 . 7 kg ). the tumor is avx2 carcinoma in the right hind leg . the tumor vessels are not visible in fig1 a , which was acquired before the gddtpa - dextran injection ( 1 . 8 ml , 0 . 17 mmole gd / kg ). one hour after administration of gddtpa - dextran the tumor vessels are easily visualized ( fig1 b ). based on axial 3d fspgr images ( tr = 15 . 1 msec , te = 4 . 2 msec ) we calculated the contrast - to - noise ratio cnr by measuring the mean signal intensity within the tumor region - of - interest ( roi ), subtracting the mean signal intensity from muscle , and dividing the result by the standard deviation of an roi taken from a region outside the body . the cnr for a region within the center of the tumor was 0 . 63 before injection and 9 . 6 fifty - four minutes after gddtpa - dextran administration . the cnr for blood vessel within the tumor was − 21 . 4 before injection and 120 after administration . the negative number is the result of a higher signal for muscle than the unenhanced tumor vessel . our 54 - minute cnr of 120 is approximately three - fold higher then the typical values ( cnr = 42 ) reported ( grist tm et al . radiology 207 : 539 - 544 .) in the human aorta using ms - 325 , the albumin - targeted agent at a similar time postinjection . two rabbits were scanned on a g . e . 9800 quick ™ ct scanner . one rabbit ( 2 . 6 kg ) bearing a large vx2 tumor was imaged with dydtpa - t40 ( mw = 101 , 537 g / mole , 95 dy per t40 ) and a second healthy rabbit ( 3 kg ) was imaged with optiray - 320 ( mw = 807 g / mole ). each rabbit was anesthetized with a mixture of 50 mg / kg ketamine and 8 . 8 mg / kg xylazine . following anesthesia administration each rabbit was intubated with a 3 - mm tracheal tube and positioned within the scan field . each rabbit was ventilated at 60 bpm with a stroke volume of 25 cc to allow for breath holding during each image . a localization image was then obtained to discern the location of the heart , tumor , right lobe of the liver , kidney and spleen . all subsequent images were obtained using 120 kv , 200 ma , 3 mm slice thickness , 2 sec scan time , 512 matrix , and fov of 16 cm . serial images , 3 mm apart , were taken from the middle of the heart to the middle of the left kidney . from this image series , axial positions were selected for the heart , liver , tumor , kidney and spleen . after three images at each position we injected [ dy ] dtpa - t40 ( 5 ml , 190 mg dy / kg , 37 mmole dy / kg ) over a 120 - second period and acquired images at 2 , 5 , 7 , 15 , 30 and 37 minutes post - injection . images for the tumor - bearing rabbit taken before injection and at 2 , 5 , 30 and 37 minutes post - injection are shown in fig1 a - e , respectively . we injected the optiray - 320 ( 640 mg l / kg , 5 . 0 mmole l / kg ) over a 60 - sec period and imaged at 2 through 10 minutes at one - minute intervals and at 20 minutes after injection . fig1 a , 18 b , 18 c and 18 d are a time series of liver cross - sections before and 2 , 5 , and 10 minutes after an injection of optiray - 320 into a healthy rabbit . only the five - minute image shows the aorta , ivc and portal vein at a greater intensity than the liver . the two and five minutes images ( fig1 b and 18 c , respectively ) in the dydtpa - t40 time series show these vessels , as well as , intra - hepatic vessels with a greater intensity then the surrounding liver . the ivc and liver baseline - corrected curves ( fig1 a - d ) for both agents are consistent with the images . most important , is that the ivc curve for dydpta - t40 persists for at least ten minutes , while the optiray - 320 ivc curve has rapidly declined with a half - time of approximately two minutes . the y - axis of the curves in fig1 a - d were calculated by subtracting the ct # of the ivc roi from the liver ct #. our purpose was to remove the baseline and preserve the difference between vessel and liver signals . also significant is the lack of uptake of dydtpa - dx by the spleen and renal cortex , as indicated in fig1 b . the renal medulla intensity indicates filtration of either intact macromolecule , dy dissociated from the chelate , or and dy - metabolic product . to test for evidence of dextran crosslinking resulting from our leash attachment chemistry we chromatographed a amino - terminated - t10 conjugate ( nt10 ) using sephacryl s - 300hr ( 27 × 1 cm ) and 0 . 9 % saline as the mobile phase ( 30 ml / hr ). we monitored the elution at 226 nm . pharmacia lists the useful fractionation range for dextran as 2 × 10 3 - 1 × 10 5 da . fig1 a is a chromatogram of human serum albumin ; the void volume is at vo and the total volume is at vt . fig1 b is a chromatogram of the amino - terminated - t10 conjugate , which has 105 leashes ; this is a leash density of 1 . 8 amino groups per glucose unit . evidence of crosslinking would occur at the void volume , where the absorbance profile would immediately rise in response to the presence of high molecular weight dextran . the mean diameter of this conjugate , as measured by dynamic laser light scattering ( honeywell microtrac ™ upa150 ), was 0 . 043 micron , with a standard deviation of 0 . 0010 microns . the albumin was measured at 0 . 0092 −/+ 0 . 0011 micron , which is slightly higher than the published diameter of 0 . 0072 micron . the advantages and useful contributions of the two - step leash attachment scheme described herein are : high attachment yield , low cross - linking , and the lack of charge at the attachment site . the dextran backbone further offers low - cost and extensive human - use experience . the existence of a large number of leashes ( i . e ., amino groups ) per dextran molecule also permits the attachment of a high density of substrates and / or drugs to each dextran backbone . methods prior to the invention produced unwanted cross - linking of the molecular backbone . see , e . g ., rebizak et al . ( 1997 ), bioconj . chem . 8 : 605 - 610 ; rebizak et al . ( 1998 ), bioconj . chem . 9 : 94 - 99 ( describing reaction of ethylenediamine with dextran carboxylic acid in the presence of 2 - ethoxy - 1 -( ethoxycarbonyl )- 1 , 2 - dihydroquinoline ( eedq )). cross - linking is a serious problem because it increases the molecular weight of the backbone , consequently decreasing the solubility of the product . furthermore , it may induce toxicity by destabilizing the complexation constant between the highly toxic radiotracer ( e . g ., gd 3 + ) and chelator group in such macromolecular structures . as a result , these conventional reaction schemes can only be used to attach a low density of leashes per molecule of dextran ; accordingly , conventional procedures permit the dextran molecule to carry only a few substrates and / or drugs , thereby requiring higher doses of dextran to achieve the desired diagnostic or drug effect . this increases cost and diminishes safety , as well as presenting solubility problems ( increased cross - linking decreases solubility ). the amount of tissue receptor also limits the number of dextran molecules that can bind to the receptor and enter the cells . the resulting low density of drug or diagnostic agent cannot deliver an adequate amount to produce the desired effect . the invention solves these problems by providing a carrier - molecule with a sufficiently high density of attachment site “ leashes ”. the invention also overcomes the shortcoming of low attachment flexibility provided by other schemes , e . g ., gedda et al . ( 1996 ), bioconj . chem . 7 : 584 - 591 , and holmberg et al . 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