A new series of technetium chelating agents based on amide and mercaptide donor groups is described, exemplified by 2,3-bis(mercaptoacetamido)propanoic acid. Chelation of Tc-99 m with 2,3-bis(mercaptoacetamido)propanoic acid resulted in two components that were separable by high-performance liquid chromatography. The component that eluted first demonstrated high specificity for renal excretion with over 90% in the urine in rabbits at 35 minutes and 87% in the urine of mice at 2 hours and 1.6% or less in the intestines of mice. Excretion was rapid with the first component equal to or greater than I-131 Hippuran in the urine of rabbits at all times. The second or latter component demonstrated comparable specificity, but slower renal excretion kinetics. 2,3-Bis-(mercaptoacetamido)succinic acid forms similar chelates with technetium which are also useful as radiopharmaceuticals for the renal systems of mammals.

This invention relates to novel chelating agents and chelates thereof with 
technetium. More particularly this invention relates to 
bis(mercaptoalkanoamido)alkanoic acids of the general formula 
##STR1## 
wherein X represents hydrogen or carboxyl and R and R' represent hydrogen 
or lower alkyl, and water-soluble salts thereof. The invention also 
relates to chelates of the compounds of the foregoing general formula with 
radioactive technetium, the method of producing such chelates, and the 
method of producing compounds of the foregoing general formula. 
BACKGROUND OF THE INVENTION 
1,2-Bis(mercaptoacetamido)ethane (commonly referred to as DADS) is a 
tetradentate chelating agent which has been evaluated as a technetium-99m 
renal function radiopharmaceutical. See A. R. Fritzberg et al, J. Nucl. 
Med. 22: 258-263, 1981. Biological studies have indicated that this agent 
is cleared by the kidneys significantly faster than Tc-99m 
diethylenetriaminepentaacetic acid (Tc-99m DTPA, p&lt;0.01) and slightly 
slower than I-131 o-iodohippurate (p&gt;0.05), with no evidence of 
significant renal retention. While this agent has shown promise as an 
imaging agent for the renal system there is need for such agents with 
higher extraction efficiency. 
Ideally, renal function should be evaluated with a single 
radiopharmaceutical which possesses high extraction efficiency, such as 
I-131 o-iodohippurate (OIH), and also is labeled with a radionuclide 
having good physical properties such as technetium-99m. Currently two 
agents are commonly used in the evaluation of renal function, OIH and 
Tc-99m DTPA. Renal perfusion is evaluated by rapid serial imaging during 
the first circulation after bolus injection of Tc-99m DTPA. OIH cannot be 
used for this purpose because the iodine-131 label limits the amount of 
radioactivity that can be injected. Renal clearance can be evaluated with 
either Tc-99m DTPA or OIH, but since Tc-99m DTPA is limited to clearance 
by glomerular filtration, the maximum extraction efficiency is 20%. 
Secretion of OIH by the renal tubular cells, in addition to some 
filtration, results in extraction efficiency of about 67%. The higher 
extraction efficiency of OIH increases the kidney-to-background image 
ratio, thus increasing the sensitivity of OIH for detection and evaluation 
of reduced renal function. Davison and coworkers J. Nucl. Med. 20: 641, 
1979; Inorg. Chem. 20: 1629-1632, 1981, have described the 
benzoyl-protected dimercaptodiamides (PhCOS(CH.sub.2).sub.n CONH).sub.2 X 
(n=1, X=(CH.sub.2).sub.2, (CH.sub.2).sub.3, and o-C.sub.6 H.sub.4 ; n=2, 
X=(CH.sub.2).sub.2 and (CH.sub.2).sub. 3). From these, via the sodium 
dithionite reduction of TcO.sub.4.sup.- in base, the technetium complexes 
[TcO(S(CH.sub.2).sub.n CONXNCO(CH.sub.2).sub.n S)].sup.- (n=1, 
X=(CH.sub.2).sub.2, (CH.sub.2).sub.3, and o-C.sub.6 H.sub.4 ; n=2, 
X=(CH.sub.2).sub.2) were prepared. The synthesis and characterization of 
the complexes, and their precursors, were reported, and their 
radiopharmaceutical applicability was discussed. 
OBJECTS OF THE INVENTION 
It is an object to provide radio-pharmaceutical or radiolabeled contrast 
agents for evaluation of renal function which do not contain radioactive 
iodine. It is another object of this invention to provide technetium 
chelating agents which exhibit high specificity for renal tubular 
excretion which is equal to or greater than I-131 o-iodohippurate renal 
excretion. It is a further object to provide technetium chelating agents 
for renal excretion which are cleared rapidly by the kidneys as I-131 
o-iodohippurate. It is another object to provide a method of producing 
such chelating agents. These and other objects are apparent from and are 
achieved in accordance with the following disclosure. 
GENERAL DESCRIPTION OF THE INVENTION 
This invention relates to 2,3-bis(mercaptoalkanoamido)alkanoic acids of the 
formula 
##STR2## 
and water-soluble salts thereof, wherein X is hydrogen or carboxyl and R 
and R' are hydrogen or lower alkyl (e.g., methyl or ethyl). Among the 
water-soluble salts of the compounds represented by the foregoing formula 
are the alkali metal and ammonium salts, which are readily formed in 
aqueous solution. 
The 2,3-bis(mercaptoalkanoamido)alkanoic acid compounds of the foregoing 
general formula can be produced from the corresponding 2,3-diaminoalkanoic 
acids of the general formula 
##STR3## 
wherein R' and X have the meanings given above, by esterification with a 
lower alcohol containing dry hydrogen chloride, followed by treatment with 
a chloroalkanoyl chloride to form the bis(chloroalkanoamido) derivative of 
the ester, and then by treatment of the latter with sodium thiobenzoate to 
form the 2,3-bis(benzoylmercaptoalkanoamido)alkanoic acid ester, which on 
alkaline hydrolysis produces the 2,3-bis(mercaptoalkanoamido)alkanoic acid 
(as its alkali metal salt). 
DESCRIPTION OF THE DRAWINGS 
In the attached drawings, FIG. 1 is a diagram illustrating the synthesis of 
2,3-bis(mercaptoacetamido)propanoic acid and the formation of the chelate 
of that acid with technetium-99m. 
FIG. 1a illustrates another modification of the invention, namely the 
Tc-99m chelate of 2,3-bis(mercaptoacetamido)succinic acid. 
FIG. 2 is a high-performance liquid chromatogram of 
Tc-99m-2,3-bis(mercaptoacetamido)propanoate chelate, showing two forms A 
and B with differing solubility characteristics. 
FIG. 3 comprises blood disappearance curves of 
Tc-99m-2,3-bis(mercaptoacetamido)propanoate [Tc-CO.sub.2 -DADS] components 
A and B and reference radiopharmaceuticals OIH and Tc-DTPA in rats. Data 
are plotted as mean for 5 or more animals at each sampling time. The 
disappearance slope of component A is comparable to or slightly greater 
than that of OIH, reflecting similar clearance values. The disappearance 
of component B is also significantly faster than Tc-DTPA, an indicator of 
glomerular filtration rate. 
FIG. 4 illustrates renal excretion of 
Tc-99-m-2,3-bis(mercaptoacetamido)propanoate [Tc-CO.sub.2 -DADS] 
components A and B and reference radiopharmaceuticals OIH and Tc-DTPA for 
comparison. Data are mean and range for 3 studies at each point. Component 
A appears slightly faster than OIH from 10 minutes onward. Component B is 
always faster than Tc-DTPA, but significantly slower than OIH or component 
A at early times. By 35 minutes both components and OIH are found in 
similar amounts.

DETAILED DESCRIPTION OF THE INVENTION 
Referring to FIG. 1 of the drawings, the synthesis of 
2,3-bis(mercaptoacetamido)propanoic acid is outlined. The starting 
material, 2,3-diaminopropanoic acid (I), in the form of its hydrochloride, 
is reacted with absolute ethanol and dry HCl gas. The mixture is refluxed, 
then evaporated to a dry solid (ethyl 2,3-diaminopropanoate (II) as the 
hydrochloride) which is then dissolved in a mixture of toluene and 
saturated sodium bicarbonate solution and reacted with chloroacetyl 
chloride in toluene to form ethyl 2,3-bis(chloroacetamido)propanoate(III). 
From this reaction mixture the organic layer is separated and evaporated, 
isolating compound III. This product is treated with a solution of sodium 
thiobenzoate in dry ethanol and ethyl 
2,3-bis(benzoylmercaptoacetamido)propanoate(IV) is produced. The latter, 
dissolved in warm ethanol, is treated with aqueous alkali and heated, 
hydrolyzing the ethyl ester and the benzoyl groups to form V. Then V, 
treated with pertechnetate solution in the presence of a reducing agent, 
forms the chelate VI of technetium with 
2,3-bis(mercaptoacetamido)propanoic acid, which is a renal imaging agent 
when the technetium is the gamma radiation-emitting isotope, Tc-99m. 
The same series of reactions can be carried out to produce the chelate 
shown in FIG. 1a. This series starts with 2,3-diaminosuccinic acid (in 
lieu of 2,3-diaminopropanoic acid) and all the steps are the same, carried 
out with the same reagents under the same conditions. The resulting 
chelate with Tc-99m, illustrated in FIG. 1a, is also a useful renal 
imaging agent. 
The invention is described in further detail in the following examples 
which illustrate specific modes of the invention and their physiological 
properties. 
EXAMPLE 1 
Synthesis of ethyl 2,3-bis(benzoylmercaptoacetamido)-propanoate 
Into a dry flask under nitrogen were placed 1.40 g (0.010 mol) of 
2,3-diaminopropionic acid hydrochloride and 250 ml of absolute ethanol. 
Then dry HCl gas was bubbled into the solution. The mixture was refluxed 
for one to two days or until proton magnetic reasonance spectra (pmr) 
analysis of aliquots with solvent removed indicated complete formation of 
the ethyl ester. The product was then concentrated to a dry solid. The 
hydrochloride ester thus obtained was dissolved by rapid stirring at ice 
bath temperature in a mixture of 50 ml of toluene and 50 ml of saturated 
sodium bicarbonate. Then 5.0 (0.444 mol) of chloracetyl chloride in 10 ml 
of toluene was added dropwise. After addition was complete the mixture was 
allowed to come to room temperature and stirred for an additional 30 
minutes. Layers were separated and the aqueous portion extracted twice 
with ethyl acetate. The organic layers were combined, washed with water 
and with brine, and dried (M.sub.g SO.sub.4). Removal of solvent left 2.28 
g of ethyl 2,3-bis(chloroacetamido)propanoate as a white solid (87% 
yield). Pmr (CDCl.sub.3): .delta.1.31 (t, 3.0IH,CH.sub.3), 3.74 (t, 
1.9H,--HNCH.sub.2 --CH), 4.07 (s,4.0, ClCH.sub.2 NH--), 4.21 (q, 2.0 H, 
OCH.sub.2 CH.sub.3), 4.67 (broad.sub.Q, 1.0 H, --CH.sub.2 
--CH--(--NH--)--CO.sub.2 CH.sub.2 CH.sub.3), and 7.12 and 7.58 (broad, 1.0 
H each, --CONH--). The material was used without further purification. 
A solution of 953 mg (4.45 mmol) of the ethyl 
2,3-bis(chloroacetamido)propanoate was prepared in 10 ml of dry ethanol 
under nitrogen. To this was added a solution of sodium thiobenzoate in dry 
ethanol (prepared from 204 mg of sodium (8.87 mmol) in ethanol) to form 
sodium ethoxide which was reacted with 1.23 g (8.90 mmol) of thiobenzoic 
acid). After a few minutes at room temperature precipitation occurred. The 
reaction was heated to reflux for 30 minutes. It was then allowed to cool, 
diluted with ethyl acetate, washed with water, brine, and dried (M.sub.g 
SO.sub.4). Removal of solvent left 2.68 g of a cream colored solid. 
Recrystallization in toluene gave 1.30 g of ethyl 
2,3-bis(benzoylmercaptoacetamido)propanoate: mp 129.5.degree.-131.degree. 
C.; pmr (CDCl.sub.3,--DMSOd.sub.6), .delta.1.20 (t, 3.0 H,OCH.sub.2 
CH.sub.3), 3.72 and 3.81 (singlets superimposed on triplet, --COCH.sub.2 
S--) and 3.70 (triplet with singlets, --NHCH.sub.2 CH(--NH--)--CO--, 6.0 H 
combined), 4.10 (q, 2.0 H, --OCH.sub.2 CH.sub.3), 4.60 (broad quartet, 1.0 
H, --CH.sub.2 --CH--(--NH--)--CO.sub.2), and 6.75-8.15 (complex aromatic 
and amide H, 12.3 H), Anal: calcd. for C.sub.23 H.sub.24 N.sub.2 O.sub.6 
S.sub.2 : C 56.56, H 4.92, N 5.74, S 13.11; found: C 56.64, H 5.09, N 
5.76, S 13.26. 
EXAMPLE 2 
Synthesis of dimethyl 2,3-bis(benzoylmercaptoacetamido)succinate 
2,3-Diaminosuccinic acid dihydrochloride (400 mg, 2.7 mmol) was converted 
to the methyl ester by refluxing in dry methanol saturated with dry HCl 
gas. Removal of solvent left 420 mg of solid ester. 
A mixture of 40 ml of toluene and 15 ml of an aqueous suspension of 2 g of 
sodium bicarbonate was added to the ester while cooling. During rapid 
stirring 0.83 ml (1.18 g, 0.4 mmol) of chloroacetyl chloride diluted in 5 
ml of toluene was added dropwise. The mixture was stirred with cooling for 
45 min. Separation of layers and extraction of the aqueous residue with 
ethyl acetate gave 350 mg of dichloroacetamidosuccinic acid which was 
reacted with sodium thiobenzoate, prepared from 346 mg (2.5 mmol) of 
thiobenzoic acid and 57 mg of sodium metal (2.5 mmol) in methanol. After 
refluxing under nitrogen for 2 hours the reaction mixture was reduced to 
dryness and the residue washed with hexane. Extraction of the product with 
hot methyl ethyl ketone gave 420 mg. Recrystallization from ethanol and 
methyl ethyl ketone provided white crystalline dimethyl 
2,3-bis(benzoylmercaptoacetamido)succinate of mp 165.degree.-167.5.degree. 
C.; nmr (CDCl.sub.3): 3.77-3.80 (superimposed singlets, 10H), 5.12 (broad 
t, 2H), 7.2-8.1 (complex, 12H). 
EXAMPLE 3 
Radiolabeling with Tc-99m 
Approximately 100 mCi of Tc-99m pertechnetate in generator eluate saline 
was made basic by the addition of 5 N NaOH (4 parts saline solution to 1 
part NaOH). The radioactivity was extracted twice with methyl ethyl ketone 
and the extracts combined and reduced to dryness in a nitrogen stream with 
warming. Then a solution of the hydrolyzed chelating agent 0.5 mg ethyl 
2,3-bis(benzoylmercaptoacetamido)propanoate dissolved in 0.3 ml of 
ethanol, treated with 30 ul of 5 N NaOH and 0.30 ml H.sub.2 O, and heated 
15 minutes at 95.degree. C. in about 0.25 ml volume was added to the 
Tc-99m pertechnetate residue. Reducing agent (1.0 mg sodium dithionite in 
30 ul) was added and the mixture heated at 95.degree. C. for 15 min. The 
mixture was neutralized with 20 ul of 6 N HCl and injected onto an 
octadecylsilyl HPLC column (250.times.4.6 mm, Altex Ultrasphere, 5u) and 
eluted with 0.01 M sodium phosphate, pH 6 (96%) and ethanol (4%). The 
column effluent was collected as the A component came out and then 
sterilized by passage through a 0.22 micron filter. The 
Tc-99m-2,3-bis(mercaptoacetamido)propanoate preparations were analyzed for 
reduced hydrolyzed technetium on silica gel thin-layer strips. Both main 
components had Rf values of 1 in 0.9% NaCl. 
Studies of protein binding were carried out in plasma using an 
ultrafiltration method described at J.Nucl.Med. 22, 258-263, 1981. 
ANIMAL STUDIES--GENERAL 
Organ biodistribution and acute toxicity studies were carried out in mice, 
blood disappearance and bile appearance rates were determined by sampling 
of bood or bile in rats, and renal excretion rates of the two 
Tc-2,3-bis(mercaptoacetamido)propanoic acid components, Tc-DTPA, and OIH 
were determined in rabbits because of greater ease of urine sampling. The 
HPLC-purified Tc-2,3-bis(mercaptoacetamido)propanoate components were used 
directly except for dilution as necessary. In general HPLC collected 
volumes were 1 to 1.5 ml and were not diluted for rabbit studies, were 
diluted 2- to 10-fold for rat studies and were diluted about 20-fold for 
mouse studies. In most cases determinations were made with simultaneous 
administration of OIH. 
Biodistribution 
The time course of organ distribution was determined in groups of six Hal 
Cr female albino mice. They were injected with 0.10 ml (0.5 uCi) of the 
preparation. For comparison purposes, 0.2 uCi OIH was added to each 
injection. The mice were placed in metabolic cages for the collection of 
excreted urine. At indicated intervals after injection, the urethra was 
ligated and the mice killed with chloroform vapor. The organs were removed 
and counted in a dual channel counter with correction for I-131 crossover 
into the Tc-99m channel. 
Blood Disappearance Rates and Biliary Excretion 
These determinations were made in male Sprague-Dawley rats. For blood 
studies the animals were anesthetized with sodium pentobarbital, a line 
was placed in a femoral vein for tracer injections and hydration, and 
another was placed in a carotid artery for blood sampling. About 20.mu.Ci 
(0.25 ml) each of OIH and Tc-99m complex under study were injected and 0.1 
to 0.2 ml samples of blood were taken at 1, 2, 3, 4, 5, 6, 9, 12, 15, 20, 
25, 30, 45, 60, 75, and 90 minutes after injection. Biliary excretion in 
the absence of renal function was determined by cannulating the common 
bile duct, ligating the renal pedicles, and collecting bile in 4-minute 
fractions for 90 minutes. 
Renal Excretion 
The rate of renal excretion was measured in New Zealand albino male 
rabbits. They were anesthetized with ketamine and xylazine and placed on a 
gamma camera provided with digital storage. After injection of 0.5 to 1 
mCi of the Tc-99m complex containing 0.5.mu.Ci of OIH, images were 
collected on tape for 45 minutes. At 35 minutes after injection, urine was 
expelled from the bladder and the percentage of injected dose contained in 
the expelled urine was determined. Accumulation of bladder radioactivity 
was monitored by placing a region of interest over the bladder. The drop 
in the radioactivity in the bladder after expelling the urine sample 
(50-80% of the bladder radioactivity) allowed calibration of the bladder 
time-activity curve in terms of percent injected dose. OIH excretion was 
based on the change in bladder Tc-99m radioactivity and the amount of 
I-131 in the urine sample. These values were compared with studies made 
with OIH alone, in which 250.mu.Ci were injected and the bladder 
radioactivity monitored for I-131. 
Tubular Transport Inhibition Studies 
The effect of probenicid as an inhibitor of renal tubular transport on the 
excretion and organ distribution of 
Tc-2,3-bis(mercaptoacetamido)propanoate-A was studied in mice as 
described. A dose of 50 mg/kg of probenicid was given 10 minutes prior to 
injection of radiochemicals. OIH was injected simultaneously. The values 
at 10 minutes were determined because they have been found to be 
kinetically representative. 
Toxicity 
Acute toxicity studies were performed in 52 Hal Cr female mice. The 
formulation was carried out with added ethanol, NaOH, and HCl and with the 
heating steps described. The doses administered ranged from 200 to 500 
mg/kg. No deaths were observed in the 48-hour period following injection. 
In vitro studies 
Analysis of a typical Tc-2,3-bis(mercaptoacetamido)propanoate preparation 
by high-performance liquid chromatography is shown in FIG. 2. Two major 
components have been present in variable ratios in every preparation. The 
first peak, Tc-2,3-bis(mercaptoacetamido)propanoate A, represents the 
component with superior biological properties. Heating at 95.degree. C. 
for 30 minutes without added reducing agent resulted in less than 10% of 
both components in a ratio similar to that shown. The use of stannous ion 
at room temperature as reducing agent gave the two components shown in 
about 65% yield with a small early component (5%) and a larger one (30%) 
with a longer retention time. However, the ratio of the main components 
was about 2:3 (A:B). The use of dithionite at room temperature as reducing 
agent resulted in over 90% A and B but in the 2:3 ratio. Formamidine 
sulfinate as reducing agent at levels of 20 .mu.g and heating for 15 
minutes at 95.degree. C. gave over 95% A and B, but in about a 7:3 (A:B) 
ratio. This result was shown to be independent of reducing agent since the 
same result was observed with dithionite when the preparation was heated 
after reducing agent addition, but before neutralization. 
For animal studies the radioactivity corresponding to HPLC peaks A and B 
was collected. The chemical stability of the isolated material is high 
since no change was observed with time, dilution, or treatment with NaOH 
and heating at 95.degree. C. for 30 minutes. 
At 10 minutes duplicate determinations of binding to plasma proteins 
resulted in 93% (range 93 to 94) of 
Tc-2,3-bis(mercaptoacetamido)propanoate-A bound and at 30 minutes 94% (94 
to 94) bound. Simultaneously determined OIH was 64% (61 to 67) bound at 10 
minutes and at 30 minutes 56% (55 to 57) in agreement with previously 
reported values. The fraction bound of 
Tc-2,3-bis(mercaptoacetamido)propanoate-B was 87% (82 to 91) at 10 minutes 
and 90% (89 and 92) at 30 minutes. These values are similar to the high 
protein binding value found for Tc-DADS of 95%. 
In vivo studies 
The results in different species showed good qualitative and, in the case 
of renal excretion, quantitative agreement. No reaction was noted to the 
small doses of acetonitrile resulting from HPLC elution of the 
radiochemicals at about 15% acetonitrile, and the OIH values from 
simultaneous administration were in good agreement with earlier values 
(J.Nucl.Med. 22: 258-263, 1981). 
Organ Biodistribution 
Table 1 shows the organ distribution in mice of 
Tc-2,3-bis(mercaptoacetamido)propanoate-A from 5 minutes to 2 hours post 
injection and Tc-2,3-bis(mercaptoacetamido)propanoate-B at 10 minutes and 
2 hours for comparison. Over the first 15 minutes component A was slightly 
higher than or comparable to OIH in renal excretion. Blood disappearance 
was rapid with 0.6% remaining in the blood at 30 minutes. The initial 
liver radioactivity of about 8% at 5 minutes appeared to return to the 
blood since 1.6% or less was seen in the intestine at any time interval 
and less than 1% was in the liver at 2 hours. The kidneys with 5.6% of the 
dose at 5 minutes contained less than the 12.6% found for Tc-DADS at 5 
minutes. Since 45% of Tc-2,3-bis(mercaptoacetmido)propanoate-A was already 
in the urine compared to 28% of Tc-DADS at 5 minutes, it appears that the 
peak kidney radioactivity level is at a time less than 5 minutes after 
injection. Retention in the kidneys was low with less than 1% of the dose 
remaining in them at 30 minutes. That 
Tc-2,3-bis(mercaptoacetamido)proponoate-B was more slowly cleared is shown 
by lower levels in the urine at 10 minutes. The higher liver radioactivity 
seems to account for this. At 2 hours the liver had cleared and renal 
excretion was only slightly lower than OIH determined simultaneously. 
Negligible biliary excretion is seen with 
Tc-2,3-bis-(mercaptoacetamido)propanoate-B as well. 
Blood Disappearance 
Comparative blood disappearance curves in rats for both components A and B, 
OIH, and Tc-DTPA, are shown in FIG. 3. OIH showed the most rapid 
disappearance through 75 minutes. However, 
Tc-2,3-bis(mercaptoacetamido)propanoate-A with a higher initial value 
resulted in comparable curve slopes. The high initial values in the 1- to 
5-minute period following injection may be due to a smaller initial volume 
of distribution due to the high degree of protein binding. For comparison 
purposes the radioactivity remaining in the blood of 
Tc-2,3-bis(mercaptoacetamido)propanoate-A was 5% at 16.5 minutes and 2% at 
36 minutes while comparative values for Tc-DADS were 5% at 19 minutes and 
2% at 68 minutes. The disappearance of 
Tc-2,3-bis(mercaptoacetamido)propanoate-B (0.05), while faster than 
Tc-DTPA, was significantly slower than 
Tc-2,3-bis(mercaptoacetamido)propanoate-A. 
Renal Extraction 
Comparative rates of renal appearance are shown in FIG. 4. From 10 minutes 
to 35 minutes Tc-2,3-bis(mercaptoacetamido)propanoate-A was found in the 
urine to a slightly greater extent than OIH although differences were only 
significant at 35 minutes (p&lt;0.05). In contrast, 
Tc-2,3-bis(mercaptoacetamido)propanoate-B was significantly lower at early 
times, but similar to component A and OIH at 35 minutes. These results are 
consistent with a similar degree of overall specificity, but different 
renal handling kinetics among these compounds. All were found in the urine 
in amounts exceeding Tc-DTPA over the 35-minute period measured. 
Tubular Transport Inhibition 
Clinical studies with Tc-DADS indicated that decreased renal function 
reduced renal excretion of Tc-DADS to a greater extent than OIH. 
Experiments with probenicid as an inhibitor of tubular secretion 
demonstrated a decrease in renal excretion of Tc-DADS to a much greater 
extent than OIH. Since these results appear to be consistent and suggest 
that the effect of probenicid may be an indicator of renal excretion 
efficiency in patients with reduced renal function 
Tc-2,3-bis(mercaptoacetamido)propanoate was studied under these 
conditions. The results indicate that the decrease in renal excretion for 
Tc-99m-2,3-bis(mercaptoacetamido)propanoate-A is much less than for 
Tc-DADS, but still greater than for OIH (Table 2). Biliary excretion was 
not increased significantly for component A, but liver radioactivity was. 
Biliary Excretion 
Biliary excretion of radioactivity in the absence of renal function was 
slow and amounted to 3.1% (range 2.4 and 3.6) in 90 minutes which compares 
to 19% for Tc-DADS in the same amount of time. At 90 minute measurement of 
Tc-99m-2,3-bis(mercaptoacetamido)propanoate-A radioactivity in other 
organs was 1% in the kidneys, 21% in the blood, 4% in the liver, 0.1% in 
the spleen, 0.3% in the stomach, and 22% in muscle. 
Acute Toxicity 
Acute toxicity studies resulted in no deaths over 48 hours in doses up to 
500 mg/kg. Little or no reaction was observed on injection as well. 
BIOLOGICAL EVALUATION 
Clinical evaluation of Tc-DADS in a limited number of renal transplant 
patients demonstrated high kidney to background ratios (similar to OIH) 
and thus high extraction efficiencies in patients with good renal 
function, but poor ratios (much lower than OIH) in patients with moderate 
to severe decreases in renal function. Moreover, in patients with 
decreased levels of renal function hepatobiliary excretion became 
significant. An unanticipated finding was that the high degree of protein 
binding of Tc-DADS confined radioactivity to the vascular system rather 
than the extracellular space as with Tc-DTPA and increased the ease of 
placing well defined regions of interest on major vessels. This feature 
facilitated quantitative analysis of renal function. 
The biological results of the studies with 
Tc-2,3-bis(mercaptoacetamido)propanoate-A indicate that the early or "A" 
component has significantly improved parameters for renal function 
evaluation compared to Tc-DADS. In animals with normal renal function the 
rate of renal excretion is equivalent to OIH and the specificity for renal 
excretion is nearly complete. The only indication of biological 
inferiority to OIH is lower renal excretion than OIH in mice pretreated 
with probenicid as a renal tubular transport inhibitor. However, the 
depression of renal excretion by probenicid treatment was from 68 to 39% 
at 10 minutes, a decrease of 43%, while Tc-DADS shows a depression of 61 
to 11%, a decrease of 82%, under the same conditions. The depression of 
OIH was from 65 to 52% or a 20% decrease. Biliary excretion in the absence 
of renal function amounted to less than 3% in 90 minutes in contrast to 
19% for Tc-DADS. In addition, these improvements have been demonstrated 
without the loss of a high degree of protein binding. 
The availability of Tc-2,3-bis(mercaptoacetamido)propanoate lies in the 
HPLC purification step. It appears that the two major components in the 
Tc-2,3-bis(mercaptoacetamido)propanoate preparations are a result in 
chelate ring isomers as indicated by VI in FIG. 1. In support of this 
conclusion are the observations that heating for one minute or less after 
reducing agent addition results in only 
Tc-2,3-bis(mercaptoacetamido)propanoate-B, reduction with dithionite and 
no heating favors Tc-2,3-bis(mercaptoacetamido)propanoate-B while heating 
favors Tc-2,3-bis(mercaptoacetamido)propanoate-A, and the analog prepared 
from 3,4-diaminobenzoic acid in which the carboxylate group must lie in 
the plane of the chelate ring carbon atoms gives only one component. 
TABLE 1 
__________________________________________________________________________ 
MICE BIODISTRIBUTION DATA OF TC--CO.sub.2 --DADS--A and 
Tc--CO.sub.2 --DADS--B WITH COMISON 
OIH URINE PERCENTAGES* 
OIH 
Time Blood 
Liver 
Kidneys 
Stomach 
Intestines 
Urine 
Urine 
__________________________________________________________________________ 
Tc--CO.sub.2 --DADS--A 
5 min 
8.19 
7.81 
5.61 0.24 1.61 45.52 
42.02 
.+-.0.51 
.+-.0.43 
.+-.0.67 
.+-.0.02 
.+-.0.18 
.+-.2.16 
.+-.6.45 
10 min 
3.10 
4.26 
2.23 0.11 0.82 67.97 
64.94 
.+-.0.19 
.+-.0.32 
.+-.0.31 
.+-.0.004 
.+-.0.05 
.+-.0.97 
.+-.0.89 
15 min 
1.95 
3.79 
1.30 0.09 0.86 75.08 
78.24 
.+-.0.21 
.+-.0.46 
.+-.0.10 
.+-.0.02 
.+-.0.16 
.+-.2.06 
.+-.0.46 
30 min 
0.59 
1.34 
0.51 0.07 1.05 80.82 
.+-.0.05 
.+-.0.13 
.+-.0.05 
.+-.0.01 
.+-.0.12 
.+-.1.31 
120 min 
0.21 
0.78 
0.25 0.04 1.46 86.57 
82.43 
.+-.0.04 
.+-.0.20 
.+-.0.05 
.+-.0.01 
.+-.0.19 
.+-.0.92 
.+-.0.95 
Tc--CO.sub.2 DADS--B 
10 min 
3.64 
14.83 
2.87 0.13 1.00 51.94 
.+-.0.32 
.+-.1.33 
.+-.0.33 
.+-.0.02 
.+-.0.10 
.+-.2.31 
120 min 
0.14 
3.71 
0.14 0.02 0.21 79.16 
.+-.0.01 
.+-.0.43 
.+-.0.03 
.+-.0.002 
.+-.0.01 
.+-.1.09 
__________________________________________________________________________ 
*Values are mean and S.E.M. percent injected dose for 6 mice at each time 
post injection. 
TABLE 2 
______________________________________ 
EFFECT OF PROBENICID ON BIODISTRIBUTION 
AMETERS OF RENAL AGENTS* - 
Tc--CO.sub.2-- 
Tc--DADS 
DADS--A OIH 
Con- Con- Con- 
Organ trol Treated trol Treated 
trol Treated 
______________________________________ 
Blood 1.85 19.53 3.37 10.20 4.58 7.49 
.+-.0.37 
.+-.0.69 .+-.0.16 
.+-.0.81 
.+-.0.24 
.+-.0.41 
Kid- 4.25 2.69 2.53 3.93 2.83 3.79 
neys .+-.1.16 
.+-.0.05 .+-.0.24 
.+-.0.19 
.+-.0.38 
.+-.0.18 
Liver 2.24 17.13 4.21 14.36 1.31 2.68 
.+-.0.26 
.+-.1.69 .+-.0.24 
.+-.0.59 
.+-.0.10 
.+-.0.19 
Intes- 
4.69 12.78 0.77 1.83 1.12 2.18 
tines .+-.0.35 
.+-.0.91 .+-.0.03 
.+-.0.09 
.+-.0.11 
.+-.0.13 
Urine 60.90 10.69 67.14 38.68 64.94 52.62 
.+-.2.34 
.+-.1.18 .+-.0.73 
.+-.2.47 
.+-.0.89 
.+-.2.53 
______________________________________ 
*Values are mean and S.E.M. percent injected dose at 10 min. post 
injection for 6 or more mice with each agent. Probencid at a dose of 50 
mg/kg was given 10 min. prior to injection of radiopharmaceuticals. 
TABLE 3 
______________________________________ 
ORGAN DISTRIBUTION OF 
Tc--99m--2,3--(MERCAPTOACETAMIDO) SUCCINATE* 
Time 
(min) 
Blood Kidneys Liver Stomach 
Intestines 
Urine 
______________________________________ 
Component A 
10 10.13 3.41 5.80 0.40 2.16 32.32 
.+-.0.92 
.+-.0.50 .+-.0.93 
.+-.0.04 
.+-.0.23 
.+-.5.33 
120 0.44 7.44 3.17 0.03 0.30 77.73 
.+-.0.13 
.+-.1.26 .+-.1.00 
.+-.0.01 
.+-.0.13 
.+-.3.99 
Component B 
10 16.21 3.43 7.06 0.53 2.88 19.32 
.+-.0.90 
.+-.0.31 .+-.1.01 
.+-.0.10 
.+-.0.34 
.+-.2.86 
120 0.87 0.65 2.33 0.49 8.85 66.92 
.+-.0.28 
.+-.0.11 .+-.1.20 
.+-.0.57 
.+-.1.18 
.+-.7.72 
______________________________________ 
*Values are mean .+-. S.D. for 6 mice at each time period post injection.