Reductant composition for technetium-99m and method for making technetium-99m labelled ligands

Reductant compositions for reducing technetium to produce technetium labelled ligands comprise a substrate having attached thereto a reducing complex having sufficient redox potential to reduce technetium from the +7 oxidation state. Preferably the reducing complex comprises a reducing agent for technetium and a chelating ligand therefor. Technetium labelled ligands are prepared by mixing such ligands with pertechnetate in the presence of the reductant of this invention and separating the reductant from the resulting labelled ligand. Technetium labelled ligands that are substantially free from reducing agent used in their preparation may be made in this manner.

FIELD OF THE INVENTION 
This invention relates to materials and methods for the preparation of 
technetium-99m labelled compounds, particularly technetium-99m 
radiopharmaceuticals, that require the reduction of technetium from the +7 
oxidation state. More particularly, this invention relates to materials 
containing reducing agent for technetium immobilized on a separate or 
separable substrate, methods for using such materials to prepare 
technetium labelled compounds and the compounds prepared thereby. 
BACKGROUND OF THE INVENTION 
The use of tracer compounds, which emit radiation from within the body, as 
medical tools has long been known. Such materials have been used for 
testing liver function and biliary patency, for the analysis of the 
physiological structure and function of the kidneys, for imaging bone 
marrow, for scanning the skeletal bone structure of mammals, for blood 
pool imaging, for detecting tumors, and for analysis of the lungs, etc. 
Another development in radionuclide use is the detection, location and 
assessment of infarcts in various areas of the body. An infarct is a 
region of dead tissue caused by complete interference with the blood 
supply to that tissue usually as the result of occlusion of the supplying 
artery. Infarcts can occur in essentially any area of the body, the most 
serious including infarcts in the brain and infarcts in myocardium or 
heart muscle, caused by thrombi, embolisms, arterial sclerosis, etc. A 
number of attempts have been made to use radionuclides to confirm the 
presence of infarcts, and to give an assessment of their size and situs. 
Radioactively-labelled compounds which are selectively incorporated into 
infarcted tissue have been used for such purposes. Such agents include 
radioactive mercury derivatives of chlormerodrin and fluorescein, and 
technetium-labelled tetracycline, pyrophosphate and diphosphonates. See, 
Hubner, Cardiovascular Research 4:509 (1970) and Holman et al., J. of 
Nuclear Medicine 14:95 (1973). 
Technetium-99m (.sup.99m Tc) is a preferred radionuclide for radioactively 
scanning organs because of its short half-life and because it radiates 
gamma rays which can be easily measured, compared, for example, to beta 
rays. See Radiology, Vol. 99, April 1971, pages 192-196. 
The use of technetium-99m in radiopharmaceutical form has become an 
important non-invasive method for diagnosis with wide ranging medical 
application because of its ready availability from a generator source, 140 
KeV gamma radiation, and 6-hour half-life. 
Technetium-99m is obtained from either extraction from Molybdenum-99 with a 
solvent such as methyl ethyl ketone or elution from a column of alumina or 
other support on which is adsorbed the parent isotope Molybdenum-99 with 
an aqueous media. The most stable chemical form assumed under these 
conditions is pertechnetate (TcO.sub.4.sup.-) in a + 7 oxidation state. 
Most technetium-based radiopharmaceuticals, however, require a reduction 
to the +3, +4 or +5 oxidation state. Presently, these radiopharmaceuticals 
are frequently produced by combining an excess of the compound needed for 
labeling with a reducing agent, freeze-drying this mixture and adding 
pertechnetate. 
Suitable reducing agents have been known for some time. Examples of such 
include divalent stannous ion (Sn.sup.++) in the form of stannous 
chloride, tartrate, and phosphate, ferrous compounds (Fe.sup.++), 
ferric-ascorbate complexes and reduced zirconium. Such reducing agents are 
used to bind radioactive .sup.99m Tc to carriers, such as chelating 
agents, red blood cells, albumin and other proteins, and various other 
compounds which selectively seek out certain organs of the body, in order 
to carry the .sup.99m Tc with them to such organs of the body where it is 
concentrated, whereby such organ can be radioactively scanned or imaged 
for diagnostic or other purposes, e.g. radioactive treatment of a 
pathological condition. See Journal of Nuclear Medicine, Vol. 11, No. 12, 
1970, page 761; Journal of Nuclear Medicine, Vol. 12, No. 1, 1971, pages 
22-24; Journal of Nuclear Medicine, Vol. 13, No. 2, 1972, pages 180-181; 
Journal of Nuclear Medicine, Vol. 12, No. 5, May 1971, pages 204-211; 
Radiology, Vol. 102 , January 1972, pages 185-196; Journal of Nuclear 
Medicine, Vol. 13, No. 1, 1972, pages 58-65. 
Generally a radiopharmaceutical product containing a technetium-99mm 
labelled ligand (.sup.99m Tc-L) is made by mixing two components. A first 
component containing a reducing agent, such as stannous ions, and the 
ligand (L) to be labelled is mixed with radioactive pertechnetate solution 
from a generator to obtain the product. Thus, the radiopharmaceutical 
product contains technetium labelled ligand, stannous and stannic-ligand 
complexes, and excess ligand which is used to make sure that stannous or 
stannic salts do not precipitate out of solution and to reduce the 
quantity of free pertechnetate or reduced uncomplexed technetium in the 
solution, i.e. technetium that is not bound or complexed with the ligand. 
Certain disadvantages can be found in the above procedure. First, although 
the reducing agent is not necessary for the functioning of the resulting 
radiopharmaceutical product, it remains in the product and is injected 
into the patient. While the presence of tin or other reducing agents 
generally used to make these products has not been found harmful, it is 
not desirable to inject unnecessary chemicals into a patient. Thus, it 
would be desirable to separate or eliminate the reducing agent from the 
final product. 
Another disadvantage occurs when the ligand to be labelled is rare or 
difficult to obtain, or where the ligand to be labelled could be harmful 
to the patient and the amount injected must be minimized. Under such 
circumstances it is desirable to efficiently label small quantities of the 
ligand and not use any excess ligand in the labelling process. 
U.S. Pat. Nos. 4,001,387; 3,902,849 and 3,749,556 describe 
radiopharmaceutical generator kits in which particulate or sintered 
reducing agent is used to reduce the technetium-99m. As described therein, 
the reducing agent absorbs the technetium-99m and then reduced 
technetium-99m is eluted using a solution of the ligand to be labelled. 
The eluate thus contains technetium-99m labelled ligand. The eluant may be 
passed through a strongly acidic ion exchange column to eliminate any 
uncombined reducing agent. Thus, apparently reducing agent-ligand 
complexes formed during reduction and elution remain in the product. 
When stannous chloride is used conventionally as a reducing agent for 
labelling .sup.99m Tc radiopharmaceuticals, the excess tin is also 
chelated by the compound being tagged. Excess uncomplexed tin often forms 
a colloid which interferes with the use of the product. For most .sup.99m 
Tc labelled radiopharmaceuticals, tin is not an integral part of the 
Tc-complex but serves only as a reducing agent for pertechnetate. 
Therefore, it can be easily appreciated that a reduction/labeling system 
in which reducing agent is eliminated in the final labelled product would 
be highly desirable. 
SUMMARY OF THE INVENTION 
The present invention provides materials containing an immobilized reducing 
agent for technetium, methods for preparing radioactive ligands, 
particularly radiopharmaceuticals, using such materials, and 
radiopharmaceuticals produced thereby. Thus, one embodiment of this 
invention provides a reductant for reducing technetium comprising a 
substrate having attached thereto a reducing complex having sufficient 
redox potential to reduce technetium from the +7 oxidation state to an 
oxidation state at which the technetium forms a relatively stable complex 
with a ligand to be labelled. Preferably, the reducing complex comprises a 
reducing agent and a chelating ligand for binding the reducing agent to 
the substrate. 
In another embodiment, this invention provides a method for providing 
technetium-99m labelled radiopharmaceuticals that comprises mixing a 
solution containing technetium-99m and a ligand to be labelled with a 
reductant for sufficient time to reduce substantially all of the 
technetium-99m and label said ligand to form technetium-99m labelled 
ligand, and separating said reductant from said technetium-99m labelled 
ligand, said reductant comprising a material in a separable phase 
comprising a substrate having attached thereto a reducing complex having 
sufficient redox potential to reduce technetium-99m from the +7 oxidation 
state to an oxidation state at which said technetium-99m forms a 
relatively stable complex with said ligand to be labelled. By "relatively 
stable complex" is meant a complex which does not dissociate within the 
period oftimes necessary for the use of the product. As is well known in 
the art, this period of time can vary from a few seconds up to a day or 
more depending on the particular diagnostic test being used. 
In a preferred embodiment of this invention, technetium-99m labelled 
pharmaceuticals are provided that are substantially free from reducing 
agent. 
DETAILED DESCRIPTION OF THE INVENTION 
Technetium-99m labelled radiopharmaceuticals are generally prepared as 
needed by mixing a ligand (L) to be radioactively labelled and a reducing 
agent (R), such as, for instance, stannous chloride, with a solution of 
pertechnetate (.sup.99m TcO.sub.4 -) in saline. The pertechnetate is 
reduced and technetium-99m labelled ligand (.sup.99m TcL) is produced. 
This can be represented schematically as follows: 
EQU R.sub.(red) +L.fwdarw.R.sub.(red) L+L.sub.(excess) R.sub.(red) 
L+L.sub.(excess) +.sup.99m TcO.sub.4 -.fwdarw.R.sub.(red) L+R.sub.(ox) 
L+.sup.99m TcL+L.sub.(excess) 
where R.sub.(red) is the reducing agent in its lower oxidation state and 
R.sub.(ox) is the reducing agent in its higher oxidation state. In such a 
process the reducing agent competes with technetium for sites on the 
ligand to be labelled and a large excess of ligand is required to insure 
that all of the reducing agent and all of the technetium are complexed so 
that they do not precipitate out of solution in use. 
In accord with the present invention a reductant for reducing technetium 
and forming radiopharmaceuticals is provided that has the redox potential 
to reduce technetium but is not so readily available to compete with the 
technetium for sites to complex with the ligand to be labelled. Thus 
reductants of this invention are a separate or separable phase that can be 
easily separated from the technetium labelled pharmaceutical. 
Generally, reductants in accord with this invention comprise a substrate 
having a reducing complex attached thereto. The reducing complex may be 
any well known material having sufficient redox potential to reduce 
technetium from the +7 oxidation state. Suitable reducing complexes 
include oxidation reduction polymers such as described by Cassidy, et al., 
Oxidation-Reduction Polymers (Redox Polymers), Interscience Publishing 
(1965). Preferably, the reducing complex comprises a reducing agent for 
technetium and a chelating ligand for the reducing agent. Thus, a 
preferred reductant can be represented as 
##STR1## 
where Z is a chelating ligand for the reducing agent. 
Any known reducing agent for technetium can be used to make the reductants 
of this invention. Preferably, the reducing agent is a metal ion that can 
be immobilized on a substrate by a chelating ligand. After complexing with 
the chelating ligand the reducing agent complex must have sufficient redox 
potential to reduce technetium-99m from the +7 oxidation state to produce 
.sup.99m Tc ions capable of binding to the ligand being labelled. Suitable 
reducing agents include, for example, stannous ions, ferrous ions, cuprous 
ions, ferric-ascorbate complexes, and reduced zirconium. The stannous ion 
is a preferred reducing agent for technetium for many applications. 
Chelating ligands for the above reducing agents are well known. See, for 
example, Cotton and Wilkinson, Advanced Inorganic Chemistry, Interscience 
Publishers (1962). Chelating ligands are compounds with one or more 
appropriate functional groups for binding with the reducing metal (in both 
its reduced and oxidized forms). Chelating ligands useful in this 
invention are those that can be bound to a substrate, either directly or 
through a linking group, and can bind the reducing agent. As is well 
known, preferred chelating ligands are compounds that contain multiple 
functional groups such as, for example, --SH, --COOH, --NH.sub.2, and --OH 
phosphate and phosphonate groups. The number and configuration of such 
functional groups determine the ability of the compound to bind particular 
reducing agents. Preferably, the chelating ligand coordinates with the 
reducing agent forming a ligand-reducing agent complex that is more stable 
(either thermodynamically and kinetically) than a corresponding complex 
between the reducing agent and ligand to be labelled with technetium. 
Preferred chelating ligands include a polydentate ligand which forms a 1:1 
ligand:reducing metal ion complex in such a way that the metal ion is 
coordinately saturated; a macrocyclic ligand of appropriate ring size, 
preferably one where all coordinating atoms are in a planar configuration; 
and a bicyclic or polycyclic ligand that can encapsulate the reducing 
agent. 
Examples of chelating ligands for binding stannous ions include derivatives 
of ethylenediaminetetraacetic acid, 8-hydroxyquinoline, dihydrolipoamide, 
iminodiacetic acid, natural and synthetic macrocyclic complexes having 
multiple N, O, and/or S atoms, particularly those having 14-16 membered 
rings, such as cyclam, porphyrins, and corrins, etc., polycyclic ligands 
having N, O, and S atoms, e.g., cryptates such as [2,2,2] cryptate, 
sepulchrates, etc., and the like. Other suitable macrocyclic ligands are 
described in Lehn, "Cryptates: the chemistry of macropolycyclic inclusion 
complexes", Acc. Chem. Res., 11, 49 (1978) and Christensen et al., Chem 
Reviews, 74, 351 (1974) which are hereby incorporated by reference. 
Substrates useful in the practice of this invention include any material 
that is inert under conditions in which the reductant of this invention is 
used, that can be easily separated from the technetium labelled product 
and that can be bound substantially irreversibly to the chelating ligand 
or reducing complex either directly or through an intermediate group. By 
"substantially irreversibly" as used herein, we mean that the substrate 
and chelating ligand or reducing complex will maintain its bond under the 
conditions of use. Preferably, the bond is a covalent bond formed by 
reaction between the substrate and the chelating ligand or reducing 
complex. 
Preferably, substrates useful herein are materials that can be made sterile 
and pyrogen-free. In addition, preferred substrates for the practice of 
this invention also have a large surface area which allows for attachment 
of a large number of metal chelating ligands. Suitable substrates include, 
for example, glass, and natural and synthetic polymers such as 
styrene-co-divinylbenzene and polysaccharides. Preferably such substrates 
are used in the form of particles or beads. In a particularly preferred 
embodiment, the substrate is the inside of a vial, for example, a glass 
vial that will contain the radiopharmaceutical, preferably etched for 
maximum surface area and derivatized to provide appropriate sites for 
attachment of metal chelating ligands that will be used to bind the 
reducing agent. It will be readily apparent to those skilled in the art 
that a vast number of substrates can be used to practice this invention. 
All such substrates are contemplated to be within the scope of this 
invention. 
Substrates having chelating ligand already bound thereto are commercially 
available as functionalized glass beads, such as controlled porosity beads 
available from Corning Glass (such as CPG-550), and functionalized 
polysaccharide beads, such as those sold under the trademark 
Sepharose.RTM. available from Pharmacia Fine Chemicals Co. Presently 
preferred substrate-chelating ligand materials include Corning CPG-ED3A 
and CPG-8-hydroxyquinoline,. 
Reductants in accord with this invention are readily made. Some 
combinations of chelating ligand and substrate are available commercially 
and the reducing agent need only be chelated by mixing a solution of the 
reducing agent with the substrate-chelating ligand to bind the reducing 
agent thereto, and form the reductant of this invention. The reductant is 
then separated from the solution, rinsed to wash off any unbound reducing 
agent, and dried. 
If a substrate having the desired chelating ligand is not available 
commercially, then the desired chelating ligand is attached to the 
substrate by known chemical reactions. For example, many chelating ligands 
can be attached to substrates having free hydroxy groups by the well known 
cyanogen bromide reaction. See, for example, Axen et al., "Chemical 
coupling of peptides and proteins to polysaccharides by means of cyanogen 
halides", nature, 214, 1302-1304 (1967). Other well known reactions will 
be readily apparent to those skilled in the art for particular 
combinations of substrates and chelating ligands. See, for example, 
Weetall, "Enzymes immobilized on inorganic carriers", Res/Dev, pp. 18-22 
(Dec. '71); Bauman et al., "Coupled ligand chromatography applications to 
trace element collection and characterization", Analyt Chem, 39, 932-35 
(1967); Gozdzicka-Jozefiak, "Preparation of chelating exchangers with a 
polysaccharide network and low cross-linkage", J. of Chromatography, 131, 
91-97 (1977); Leyden et al, "Preconcentration of trace metals using 
chelating groups immobilized via silylation", Analyt Chem, 47, 9, pp. 
1612-1616 (Aug. 1975); and Schmuckler, "Chelating resins-their analytical 
properties and applications", Talanta, 12, pp. 281-290 (1965). 
Technetium-99m labelled ligands are prepared in accord with this invention 
by mixing the ligand to be labelled and pertechnetate (.sup.99m 
TcO.sub.4.sup.-) in the presence of the above reductant. A schematic of 
the reaction is as follows: 
##STR2## 
The reductant can then be separated from the technetium labelled product. 
Any ligand capable of being labelled with technetium-99m can be labelled in 
accord with this invention. Particularly useful ligands are polyhydroxy 
polycarboxylic acids, aminocarboxylic acids, phosphonates, phosphates and 
mercaptans, etc. Examples of such ligands include, for instance, plasma 
proteins such as human serum albumin (HSA), ethylhydroxydiphosphonate 
(EHDP), methylenediphosphonate (MDP), pyrophosphate, 
ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid 
(DTPA), dimercaptosuccinic acid (DSMA), gluconate, glucoheptonate, 
N-(2,6-dimethylphenylcarbamoylmethyl)iminodiacetic acid (HIDA), analogs of 
HIDA such as N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetic acid 
(PRIDA), N-(4-butylphenylcarbamoylmethyl)iminodiacetic acid (BIDA), 
clotting factors such as fibrinogen, gamma globulins, antibodies and their 
fractions, phytate, and the like. 
When the reducing agent is bound to a substrate in accord with this 
invention, it does not readily compete with the reduced technetium-99m for 
sites on the ligand being labelled. Therefore less ligand is necessary to 
insure that free (not bound to a ligand) technetium-99m is at a minimum 
acceptable level in the product radiopharmaceutical. Thus this invention 
makes it more practical to label biologically active materials that are 
available in small quantities only and to label such materials having no 
native binding site and in which such a binding site is added 
synthetically. 
As will be appreciated by those skilled in the art, the chelating ligand Z 
should be selected to provide a stable reductant under conditions of use. 
The chelating ligand should complex with the reducing agent to form a 
complex sufficiently stable (kinetically and/or thermodynamically) so that 
the reducing agent is not displaced by reduced technetium-99m and is not 
extracted by the ligand being labelled. Thus, it is readily apparent that 
the selection of the chelating ligand depends upon the particular ligand 
to be labelled and upon the particular reducing agent being used. 
Preferably, the chelating ligand should have a stronger affinity for the 
reducing agent than for technetium-99m and the chelating ligand should 
have a stronger affinity for the reducing agent than the ligand to be 
labelled has for the reducing agent. 
Chelating ligands that have been found useful, particularly when stannous 
ions are used for the reducing agent, include, for example, 
8-hydroxyquinoline, dihydrolipoamide, iminodiacetic acid, derivatives of 
ethylenediaminetetraacetic acid, and the like. 
Generally, radiopharmaceuticals can be prepared in accord with this 
invention so that there is less than 1.0 .mu.g per ml of reducing agent 
(calculated on the basis of the reducing metal ion salt) in the product. 
Preferably, radiopharmaceuticals are prepared having less than 0.1 .mu.g 
per ml of reducing agent on that basis, and most preferably less than 
0.001 .mu.g per ml. In accord with a particularly preferred embodiment of 
this invention, technetium-99m labelled pharmaceuticals are produced that 
are "substantially free" of reducing agent. By "substantially free" of 
reducing agent we mean that the reducing agent in the radiopharmaceutical 
product is less than 0.1 .mu.g per ml on the above basis. 
As will be readily appreciated by those skilled in the art, the quantity of 
reducing agent in the product can be minimized by proper selection of the 
chelating ligand for the particular reducing agent being used and ligand 
to be labelled, and by controlling the labelling conditions including the 
temperature and pH of the solution and the time that the solution is in 
contact with the reductant. 
Therefore, to minimize the amount of reducing agent in the labelled product 
one should select a chelating ligand so that the reducing agent-chelating 
ligand complex is considerably more stable than the reducing agent-ligand 
to be labelled complex and so that a non-labile reducing complex can be 
formed with the reducing agent. The reducing agent itself is preferably a 
non-labile reducing metal ion (i.e. slow in making and breaking bonds). 
Optimal labelling conditions for minimizing the amount of reducing agent 
in the product include minimizing the quantity of ligand being labelled, 
minimizing the contact time between the reductant and the labelling 
solution, and minimizing the quantity of reductant. 
It is also highly desirable to minimize the adsorption of technetium-99m by 
the reductant to obtain the technetium in the labelled product. This can 
be partially accomplished by following the criteria set forth above for 
minimizing reducing agent in the product. In addition, one should also 
select a chelating ligand so that the technetium labelled ligand complex 
is considerably more stable than a technetium-chelating ligand complex and 
should make sure that all possible binding sites on the chelating ligand 
are saturated with reducing agent. Furthermore, it has been found that 
adsorption of technetium by the reductant can be minimized by increasing 
the quantity of ligand being labelled. Proper selection of the chelating 
ligand and reducing agent for the particular ligand being labelled will 
enable both reducing agent in the product and technetium adsorption by the 
reductant to be minimized. 
It is readily apparent to those skilled in the art that various bond 
strengths and bond forming kinetics can be measured and/or calculated in 
order to select the appropriate chelating ligand and reducing agent for 
the particular ligand to be labelled. However, in practice it has been 
found simpler to conduct a series of tests using various combinations of 
chelating ligand and reducing agent for the reductant and to mix such 
reductant with the ligand to be labelled and pertechnetate for various 
lengths of time, from about 5 minutes to about 15 minutes being most 
suitable. At the end of such time the reductant and labelled ligand are 
separated and the labelled ligand is analyzed for the quantity of reducing 
agent and the reductant is analyzed for the quantity of adsorbed 
technetium.

The following examples are presented to further illustrate the practice of 
this invention. 
EXAMPLE 1 
A porous polyethylene frit of 0.25 cm thickness and average pore size of 70 
.mu.m was placed into a cylindrical glass column 7.0 cm high with an outer 
diameter of 1.0 cm and an inner diameter of 0.8 cm. Placed onto this was 
0.5 mg. of Corning ED3A-CPG-550 controlled pore glass beads of 550 A pore 
size with an ethylenediamine triacetic acid moiety convalently bonded onto 
the glass surface. Two rubber septa, 0.65 cm high and 0.85 cm in diameter, 
were pressed into the ends of the glass cylinder until their outer edges 
were flush with those of the cylinder. A hypodermic needle was inserted 
into the septum at the top of the column. A vacuum was induced in the 
column through this needle, and the process of evacuation was dynamically 
continued for several hours. After evacuation, the space within the column 
was filled to atmospheric pressure with nitrogen gas. 
An admixture of 60 mg sodium glucoheptonate and 600 .mu.g SnCl.sub.2 
.multidot.2H.sub.2 O (spiked with Sn-113) in 1.50 ml deoxygenated water, 
pH 5.0, 0.2 molar sodium acetate-acetic acid buffer, was added to the 
nitrogen gas-filled column. This stannous loaded column was then placed on 
a vertical rotary mixer for 15 minutes. 
After mixing, the stannous loading solution was removed via the bottom 
septum while a regulated 1 atmosphere of nitrogen gas simultaneously 
replaced the empty space. 
A similar procedure was used with deoxygenated water to wash off any 
residual glucoheptonate or unbound stannous compounds. 
An admixture of 1.0 mg of purified human serum albumin in 1.5 ml of 0.9% 
w/v agueous sodium chloride adjusted to pH 2 with dilute HCl, and 12.3 mCi 
of technetium-99m as .sup.99m TcO.sub.4.sup.- was loaded onto the above 
column and vertically mixed for 15 minutes. The mixture was withdrawn from 
the column and placed in an evacuated vial. The column was then washed 
with 1.5 ml of 0.9% w/v aqueous sodium chloride. This wash was combined 
with the first sample and yielded a product containing 10.7 mCi of 
technetium-99m-labelled human serum albumin and 0.4 .mu.g per ml of 
SnCl.sub.2 .multidot.2H.sub.2 O. 
Samples of the above solution were injected into the tail vein of rats for 
evaluation as a radiodiagnostic blood pool imaging agent. Other samples 
were spotted on Gelman ITLC (SG) chromatography strips developed in methyl 
ethyl ketone (MEK) for determination of free pertechnetate. 
Biodistribution results in rats 45 minutes after injection of 0.25 ml of 
the sample were as follows: 
______________________________________ 
% Injected Dose/Organ 
______________________________________ 
Blood* 35.0 
Liver 11.8 
Spleen 1.1 
Lungs and Heart 4.8 
Kidneys 10.7 
Gastrointestinal Tract 
5.3 
Stomach 0.4 
______________________________________ 
*based on 5% body weight. 
Free pertechnetate was 4.0% by ITLC (SG) in MEK. 
EXAMPLE 2 
A column was prepared and loaded with stannous as in Example 1, except that 
20 mg of ED3A-CPG-550 beads were placed on the frit. 
An admixture of 60 mg of glucoheptonic acid in 1.5 ml of 0.9% w/v aqueous 
sodium chloride adjusted to pH 8 with NaOH, and 94 mCi of technetium-99m 
as .sup.99m TcO.sub.4.sup.- was loaded onto the above column and 
vertically mixed for 15 minutes. 
Samples of the above were withdrawn from the column and injected into the 
tail vein of rats for evaluation as a radiodiagnostic kidney imaging 
agent. Other samples were spotted on Gelman ITLC (SG) chromatography 
strips developed in 0.9% w/v aqueous sodium chloride and methyl ethyl 
ketone (MEK) for the determination of radiocolloid and free pertechnetate 
respectively. 
Biodistribution results in rats 1 hour after injection of 0.25 ml of the 
sample were as follows: 
______________________________________ 
% Injected Dose/Organ 
______________________________________ 
Blood* 1.5 
Liver 0.7 
Kidneys 
21.8 
Intestines 
4.7 
Stomach 
0.1 
______________________________________ 
*based on 5% body weight? 
Radiocolloid was 0.9% by ITLC (SG) in saline. 
Free pertechnetate was 0.2% by ITLC (SG) in MEK. 
EXAMPLE 3 
A column was prepared and loaded with stannous as in Example 1, except that 
20 mg of the ED3A-CPG-550 beads were placed on the frit. 
An admixture of 1.0 mg tetrasodium pyrophosphate in 1.5 ml of 0.9% w/v 
aqueous sodium chloride, adjusted to pH 5 with dilute HCl, and 49.0 mCi of 
technetium-99m as .sup.99m TcO.sub.4.sup.- was loaded onto the above 
column and vertically mixed for 15 minutes. 
Samples of the above solution were withdrawn from the column and injected 
into the tail vein of mice for evaluation as radiodiagnostic bone imaging 
agents. Other samples were spotted on Gelman ITLC (SG) chromatography 
strips developed in 0.9% w/v aqueous sodium chloride and methyl ethyl 
ketone (MEK) for determination of radiocolloid and free pertechnetate, 
respectively. 
Biodistribution results in mice three hours after intravenous injection of 
0.05 ml of the sample were as follows: 
______________________________________ 
% Injected Dose/Organ 
______________________________________ 
Blood* 1.2 
Liver 1.2 
Kidneys 1.4 
Femur 1.9 
Gastrointestinal Tract and Stomach 
3.6 
______________________________________ 
*based on 5% body weight. 
Radiocolloid was 3.4% by ITLC (SG) in saline. 
Free pertechnetate was 6.2% by ITLC (SG) in MEK. 
EXAMPLE 4 
A column was prepared and loaded with stannous as in Example 1 except that 
(a) the immobilizing substrate-chelating ligand was BioRad chelating resin 
Chelex-100.RTM. which consists of a poly(styrene-co-divinylbenzene) with 
iminodiacetic acid convalently bonded thereto and (b) 20 mg (dry weight) 
of resin were placed on the frit. 
An admixture of 1.0 mg of methylene diphosphonic acid in 0.9% w/v aqueous 
sodium chloride adjusted to pH 5 with NaOH and 71.2 mCi of technetium-99m 
as .sup.99m TcO.sub.4.sup.- in 1.5 ml total volume was added to the 
column and vertically mixed for 15 minutes. 
Samples of the above solution were withdrawn from the column and injected 
into tail vein of mice for evaluation as radiodiagnostic bone agents. 
Other samples were spotted on Gelman ITLC8 SG) chromatography strips and 
developed in saline and methyl ethyl ketone (MEK) for determination of 
radiocolloid and free pertechnetate, respectively. 
Biodistribution results in mice 1 hour after injection of 0.05 ml of the 
sample were as follows: 
______________________________________ 
% Injected Dose/Organ 
______________________________________ 
Blood* 0.6 
Liver 1.0 
Kidneys 1.3 
Femur 2.1 
Gastrointestinal Tract and Stomach 
2.1 
______________________________________ 
*based on 5% body weight. 
Free pertechnetate was 1.2% by ITLC (SG) in MEK. 
Radiocolloid was 0.5% by ITLC (SG) in 0.9% w/v aqueous sodium chloride. 
EXAMPLE 5 
A column was prepared and loaded with stannous as in Example 1 except that 
(a) the immobilizing substrate-chelating ligand was BioRad chelating resin 
Chelex-100.RTM. and (b) 20 mg (dry weight) resin were placed on the frit. 
An admixture of 20.0 mg of 
N-(2,6-dimethylphenylcarbamoylmethyl)iminodiacetic acid (HIDA) in 1.5 ml 
of 0.9% w/v aqueous sodium chloride adjusted to pH 5 with NaOH and 20.6 
mCi of technetium-99m as .sup.99m TcO.sub.4 - was loaded onto the above 
column and mixed vertically for 15 minutes. 
Samples of the above solution were withdrawn from the column and injected 
into the tail vein of mice for evaluation as radiodiagnostic hepatobiliary 
imaging agents. Other samples were spotted in Gelman ITLC (SG) 
chromatography strips and developed in 0.9% w/v aqueous sodium chloride 
for determination of radiocolloid. 
Biodistribution results in mice 15 and 90 minutes after injection of 0.15 
ml of the sample were as follows: 
______________________________________ 
% Injected Dose Organ 
15 minutes 
90 minutes 
______________________________________ 
Blood* 1.8 0.8 
Stomach 0.4 0.4 
Intestines and Gall Bladder 
70.0 78.3 
Kidneys 1.3 0.7 
Liver 3.4 1.0 
______________________________________ 
*based on 5% body weight. 
Radiocolloid was 5.2% by ITLC (SG) in saline. 
EXAMPLE 6 
A porous polyethylene frit of 0.25 cm thickness and average pore size 70 
.mu.m was placed into a cylindrical glass column 7.0 cm high with an outer 
diameter of 1.0 cm and an inner diameter of 0.8 cm. Placed onto this was 
100 mg of Corning-ED3A-CPG-550 controlled pore glass beads of 550 A pore 
size with an ethylenediamine triacetic acid moiety covalently bonded onto 
the glass surface. Two rubber septa, 0.65 cm high and 0.85 cm in diameter 
were pressed into the ends of the glass cylinder until their outer edges 
were flush with those of the cylinder. A hypodermic needle was inserted 
into the septum at the top of the column. A vacuum was induced in the 
column through this needle. After evacuation, the space within the column 
was filled to atmospheric pressure with nitrogen gas. 
An admixture of 200 mg sodium glucoheptonate and 100 .mu.g SnCl.sub.2 
.multidot.2H.sub.2 O (spiked with Sn-113) in 1.50 ml deoxygenated acetate 
buffer, 0.1 M, pH 5.0 was added to the nitrogen gas filled column. This 
stannous loaded column was then placed on a vertical rotary mixer for 20 
minutes. 
After mixing, the stannous loading solution was removed via the bottom 
septum, while a regulated 1 atmosphere of nitrogen gas simultaneously 
replaced the empty space. 
A similar procedure was used with additional 1.5 ml aliquots of acetate 
buffer and 0.9% w/v aqueous sodium chloride adjusted to pH 3 to wash off 
any residual glucoheptonate or unbound stannous compounds. 
An admixture of 25 mg of purified human serum albumin in 1.5 ml of 0.9% w/v 
aqueous sodium chloride adjusted to pH 2 with dilute HCl, and 2.7 mCi of 
technetium-99m as .sup.99m TcO.sub.4 - was loaded onto the above column 
and vertically mixed for 15 minutes. The mixture was withdrawn from the 
column and placed in an evacuated vial. The column was then washed with 
1.5 ml of 0.9% w/v aqueous sodium chloride adjusted to pH 3. This wash was 
combined with the first sample and yielded a product containing 1.8 mCi of 
technetium 99m-labeled human serum albumin. 
Samples of the above solution were injected into the tail vein of rats for 
evaluation as radiodiagnostic blood pool imaging agents. Other samples 
were spotted on Gelman ITLC (SG) chromatography strips developed in methyl 
ethyl ketone (MEK) for determination of free pertechnetate. An additional 
sample was fractionated on a column of Pharmacia Sephadex G100, eluted 
with 0.9% sodium chloride. The results were as follows: 
% as .sup.99m TcO.sub.4.sup.31 :5.5% by chromatography on ITLC/MEK; 
% of Tc assoc. w HSA:100% by gel filtration on G100; 
Biodistribution in 2 rats, 45 min. after injection percent in 
blood:39.+-.0%. (based on 5% of body weight) 
EXAMPLE 7 
ED3A-CPG-550 beads were loaded with stannous ions by the procedure used in 
Example 1. Variable quantities of the stannous loaded ED3A-CPG-550 were 
mixed with variable quantities of human serum albumin in the presence of 
Tc-99m-pertechnetate (.about.10 mCi) in 1.5 ml of solution for 15 minutes 
to determine the amount of technetium adsorbed by the reductant. The 
results are given in the following table. 
______________________________________ 
Amount of .sup.99m Tc Adsorbed by Reductant 
Reductant 
Sn(II)-ED3A-CPG-550, mg 
HSA, mg %Tc on Reductant 
______________________________________ 
100 1 84 
20 1 70 
5 1 30 
1 1 14 
0.5 1 10 
20 20 8 
20 10 20 
20 5 30 
20 1 70 
______________________________________ 
EXAMPLE 8 
ED3A-CPG-550 beads were loaded with stannous ions by the procedure used in 
Example 1. Variable quantities of the stannous loaded ED3A-CPG-550 were 
mixed with variable quantities of 
N-(2,6-diisopropylphenylcarbamoylmethyl)-iminodiacetic acid (PRIDA) in the 
presence of Tc-99m-pertechnetate (.about.10 mCi) in 1.5 ml of a solution 
for 15 minutes to determine the amount of Sn in the product based on the 
amount of Sn originally on the reductant. The results are given in the 
following table. 
______________________________________ 
Sn in Product When Labelling PRIDA 
SnCl.sub.2 . 2H.sub.2 O 
Reductant PRIDA % Sn in in product, 
Sn(II)-ED3A-CPG-550, mg 
mg Product .mu.g/ml 
______________________________________ 
1 1 10 0.4 
20 55 2.1 
20 1 7 3.2 
20 57 21 
______________________________________ 
EXAMPLE 9 
Tests were run the same as in Example 8 except using human serum albumin as 
the ligand being labelled. The results are given in the following table. 
______________________________________ 
Sn in Product When Labelling HSA 
SnCl.sub.2 . 2H.sub.2 O 
Reductant % Sn in in Product 
Sn(II)-ED3A-CPG-550, mg 
HSA, mg product .mu.g/ml 
______________________________________ 
0.5 1.0 5 0.4 
20.0 1.0 2 2.7 
______________________________________ 
The above invention has been described in detail with particular reference 
to the preferred embodiments thereof, however, it will be appreciated that 
modifications within the spirit and scope of this invention may be 
effected by those skilled in the art.