CA1317066C - Ligand for forming radiolabeled technetium chelates for use in renal function determinations - Google Patents

Abstract

ABSTRACT

The present divisional application is directed to a ligand that may be reacted with a suitable Tc-99m-pertechnetate salt to form novel Tc-99m compounds (imaging agents incorporating Tc-99m as a radiolabel) having relatively high renal extraction efficiencies useful for conducting renal function imaging procedures. The ligand has the following general formula.

where X is S or N; and wherein Y is -H or wherein Y is -

Description

1 31 706h A LIGAND FOR FORMING RADIOLABELED TECHNETIUM CHEL~TES
FOR USE IN RENAL FIJNCTION DETERMINATIONS

BACKGROUND
1. (a) This application is a divisional application of patent application serial no. 485,2~0, filed 25 June, 1985, entitled "Radiolabeled Technetium Chelates For ~se In Renal Function Determinations".
1. (b) The Field of the Invention The present invention is related to methods and compounds for use in the field of determining renal function by means of scintigraphic urography. More particularly, the present invention is directed to renal system imaging in which technetium-99m radiolabeled chelates are used.
2. The Prior Art Regulation of the content and quantity of body fluids is critical to the basic physiology of bodily functions. For instance, it is necessary for the body to regulate such fluid-related variables as total body fluid volume, constituents of extracellular bod~ fluids, the acid-base balance in body fluids, and various factors that affect interchangè of extracellular and intracellular fluids (most notably, factors that affect the osmotic relationship between such fluids).
~5 The kidneys are the primary body organs that are responsible for regulation of the composition of body fluids. Thus, the kidneys maintain body fluids within a physiologically acceptable range by excreting most of the '~

13170~6 end products of metabolism and by regulatiny the concentra-tions of desirable body fluid constituents.
The human body contains two kidneys that function to form urine containing fluid constituents that are to be eliminated through the bladder. The basic biological unit that performs the work of the kidney is the "nephron." Each kidney is comprised of some one million nephrons, with each nephron being capable of regulating body fluid independently of other nephrons.
The kidneys function on body fluid by filtering a substantial volume of blood (about one-fifth of the total cardiae output is pumped directly to the kidneys~; this speeifie volume of blood is known as the "renal fraction."
Blood flow through the kidneys of a typical adult male aver-~ages about 1.2 liters per minute. As the blood passes through the kidneys, the nephrons "clear" the blood plasma of unwanted substances -- for example, the metabolie end produets (such as urea, creatinine, urie aeid, sulfates and phenols) and the nonmetabolie ionie substanees (sueh as excess sodium, potassium, and chloride).
The nephrons are basically comprised of a capillary bed termed the "glomerulus," a second capillary bed known as the "peritubular" capillaries, and a urine-forming component known as the "tubule." The tubule is separated from the glomerulus by a membrane known as the "glomerular membrane." As the renal fraction of blood flaws through the 1 31 706~
glomerulus, the glomerular membrane passes a small propor-tion (yenerally no more than 20%-25%) of the plasma comprising the renal fraction into the tubule. This filtered fluid then flows through the tubule, and towards the pelvis of the kidney, which in turn feeds into the bladder. As fluids flows through the tubule, most of the water and much of the electrolytes and other "wanted"
substances are reabsorbed and returned to the blood; the "unwanted" substances (such as metabolic end products and excess water and electrolytes) pass into the bladder for elimination as urine.
The remaining portion of the renal fraction that does not cross the glomerular membrane exits the glomerulus and then enters the peritubular capillaries; from there, a portion of the renal fraction is generally returned to the venous system. The large quantities of fluid components reabsorbed in the tubules are also transported to the peritubular capillaries by diffusion through the tubular membrane.
While large quantities of fluid diffuse from the tubule into the peritubular capillaries for return to the vascular system, diffusion of some plasma components occurs in the reverse direction -- from the peritubular capillaries to the tubules. For instance, sodium ions are actively transported across the tubular membrane and into the peritubular fluid, thereby conserving this important electrolyte. However, 1 31 70~h this creates a substantial negative charge within the tubule with respect to the peritubular fluid. In the proximal tubule, this electrical difference is approximately 20 millivolts, and can climb to as much as 120 millivolts in the distal tubule.
This difference in electrical potential potentiates diffusion o~ some positive ions, most notably potassium, from the peritubular fluid into the tubule. This flow of potassium into the tubules across the tubular membrane due to the electronegativity gradient is termed "passive secre-tion."
In addition to this passive secretion, some ionic materials are "actively" secreted into the tubules. For instance, para aminohippuric acid (generally referred to as lS - "PAH") .is actively secreted from the peritubular fluid into the tubules; although only about twenty percent (20%) of the renal fraction passes into the tubules as glomerular filtrate, nearly ninety percent (90%) of any PAH in the blood is removed by the kidneys. Thus, approximately seventy percent ~70~) of the P~H is removed from the plasma by active secretion into the tubules.
Occasionally, a kidney will become damaged and thus diminish or even cease its function of clearing the blood.
Various renal function tests have been devised to assist a physician to evaluate the extent and type of kidney damage that has occurred. Also, these renal function tests are --'I--1 31 706~, useful in evaluating whether a kidney is o~erating properly following a kidney transplant operation.
One such renal function testing procedure is known as intravenous scintigraphic urography (this procedure is also commonly known as a dynamic renal function imaging study).
This procedure has historically involved the intravenous administration of a radioactively labeled iodine substance, such as I-131 ortho-iodohippurate (often referred to as "I-131 OIH"). Like PAH, I-131 OIH is rapidly removed from the blood by active tubular secretion in addition to glomerular filtration, thereby causing significant ~uanti-ties of radiolabeled material to concentrate in the kidneys within a few minutes after administration. Images can be obtained using gamma scintillation cameras capable of showing the location of this radiolabeled material, and thereby giving a useful indication of the quality of renal unction in the kidneys.
Despite the fact that I-131 OIH is an important tool in evaluating renal function, it suffers from some significant drawbacks. First, because of the high-energy gamma radia-tion output (364 KeV) of iodine-131 ("I-131"), the use of I-131 OIH results in images having poor spatial resolution.
This makes it difficult to observe fine detail within the kidneys, and thereby limits the amount of useful information which is obtainable by this method.

.

1 31 70~h Further, the renal extraction efficiency (the ability to clear the radiopharmaceutical from blood passing through the kidneys) of I-131 OIH is only about 65%-80%, and, while this is quite good, a higher extraction efficiency would result in higher kidney-to-background ratios, which facili-tate detection of minimal renal function. In addition, I-131 emits a beta particle during radioactive decay which can cause damage to surrounding tissue. Further, because free radioiodine that accompanies I-131 OIH is readily taken up by the patient's thyroid gland, the maximum dose of I-131 OIH must generally be held to about 200 to 300 microcur-ies. This low dosage requires a significant exposure period when taking the radioactive image, which in turn decreases the temporal resolution of sequential images taken during renal function studies.
In order to improve upon the resolution obtainable in a radioactive renal function procedure, alternative radio-labeled materials have been earnestly sought. It is currently believed that the most desirable radioactive label is technetium-99m ("Tc-99m"), which has significantly improved resolution properties when compared to the I-131 label, because Tc-99m emits a lower energy (140KeV) radia-tion. This lower energy radiation is well-suited for use in connection with standard radiation-measuring instrumenta-tion. The radiation dose per millicurie is much less forTc-99m than is the case when using I-131; this is because 1 31 1~6~ `

Tc-99m has a half-life of only about six (6) hours (as opposed to a half-life of eight (8) days for I-131), and also because Tc-99m does not emit beta particles during its decay process.
The radioactive properties of Tc-99m result from the transition of the metastable excited nucleus to ground state Tc-99. The resulting Tc-99 has such a long half-life (200,000 years) as to be virtually innocuous. As a result, dosages of as much as 30,000 microcuries of Tc-99m may be administered without danger to the patient. The result is that much shorter exposure periods are re~uired than when I-131 OIH is used. This in turn makes it possible to take acceptable perfusion images during the first pass of radiopharmaceutical through the kidneys.
15 ; The foregoing properties o Tc-99m make it ideal as a tool in nuclear medicine, since it is well-suited for use with standard instrumentation, and because it subjects the patient with whom it is used to a relatively low dose of radiation.
Because of the demonstrated advantages of the Tc-99m label over the I-131 label, a great deal of effort has gone into developing a Tc-99m compound having a high renal extraction efficiency. A number of Tc-99m-labeled chelates have been reported in the literature.
One Tc-99m-labeled compound, Tc-99m diethylenetriamine-pentaacetic acid (generally referred to as "Tc-99m-DTPA") " 1 3 1 70~6 has sometimes been used in radioactive renal function evaluation procedures because of its excellent imaging characteristics. However, Tc-99m-DTPA is not actively secreted into the tubules of the kidney, and thus has a maximum extraction efficiency of only about 20-25%; this would be expected in connection with a substance entering the tubules only as a result of glomerular filtration. This lower extraction efficiency makes the use of Tc-99m-DTPA
less sensitive in detecting mild renal disease than is I-131 OIH. Even so, because of the ability of Tc-99m-DTPA to provide perfusion images of the renal blood supply to the kidneys during the first pass after injection, it is common to use Tc-99m-DTP~ together with I-131 OIH.
Another Tc-99m compound reported in the literature is Tc-99m-N,N'-bis (mercaptoacetyl)-ethylenediamine ("Tc-99m-DADS"). While this compound has been found to be secreted in the tubules, the tubular extraction efficiency of Tc-99M-DADS is only about 53~ in normal patients, and even less in patients having decreased renal function. This very low extraction efficiency makes this compound unsuitable as a replacement for I-131 OIH.
Although Tc-99m-DADS is itself deemed unsatisfactory as a replacement for I-131 OIH, the fact that it is actively secreted by the tubules led to experimentation with various analogs. For instance, various methyl, hydroxymethylene, .

benzo, carboxylate, dicarboxylate, and benzocarboxylate analogs have been synthesized and tested.
Of these, the most efficiently excreted analog has been Tc-99m-N,N'-bls (mercaptoacetyl)-2,3-diaminopropanoate (Tc-99mCO2-DADS). Unfortunately, this ligand exists as two stereoisomeric products upon chelation, referred to as Tc-99m-CO2-DADS-~ and Tc-99m-CO2-DADS-B. Further, the Tc-99m-CO2-DADS-B isomer was found to be far less effi-ciently removed by the kidneys than was the Tc-99m-CO2-~ADS-A isomer. Because of the inherent difficulty in separating these two isomers for clinical use, commercial development of Tc-99m-CO2-DADS-A has proven to be imprac-tical.
From the foregoing, it will be appreciated that it 15; would be a substantial improvement in the field of renal function imaging if a Tc-99m compound could be provided that has a relatively h.igh extraction efficiency, yet does not exhibit other adverse properties that would make it unsuit-able as a replacement for I-131 OIH. Because of the ~hort half-life of Tc-99m, it would also be a significant advance-ment if such Tc-g9m imaging compounds could be easily prepared immediatel~ prior to conducting a renal function diagnostic procedure. Such Tc-99m compounds and methods are disclosed and claimed herein.

_g_ BRIEF SUMMARY AND OBJECTS OF THE INVENTIOM

The invention of the applicant's parent and subject divisional applications is directed to novel radiopharmaceutical imaging agents incorporating Tc-99m as a radiolabel. In particular, the disclosed novel imaging agents have relatively high renal extraction efficiencies, and hence are useful for conducting renal function imaging procedures. The novel Tc-99m compounds of the invention are believed to have the following general formula:

/r \

1S N NJ~o O ~\

wherein X is S or N; and wherein Y is -H or wherein Y is IRl z ``~` 1 31 7066 and wherein Rl is -H, -CH3, or CH2CH3; R2 is -H, -CH2C02H, -CH2CONH2, -CH2CH2C02H, -CH2CH2CONH2, -CH3, -CH2CH3, or -CH20H; and Z is -H, -C02H, -CONH2, -S03H, -S02NH2, or -CONHCH2C02H; and wherein Tc is Tc-99m; and water-soluble salts thereof.
Of the foregoing, a preferred Tc-99m compound of the invention is Tc-99m-mercaptoacetylglycylglycylglycine (Tc-99m-MAGGG). Other preferred compounds are Tc-99m-MAGG-Alanine, Tc-99m-MAGG-Glutamine, and Tc-99m-MAGG-asparagine.
The invention also provides methods for preparing and using the novel Tc~99m compounds. The invention of the sub~ect divisional application is directed to a ligand capable of reacting with a suitable Tc-99m-pertechnetate salt to form a Tc-99m chelate. The ligand has the following genaral formula:
Y' /Y
X N`
N NJ ~0 O \\

where X and Y have the same definitions as above, and wherein y1 is -H2 when X is N, or wherein yl is -H, -COCH3, -COC6H5 or -CHzNHCOCH3, -COCF3, -COCH2OH, COCH2CO2H, or other s~itable protective group when X is S. `

BRIEF DESCRIPTION OF THE DRAWINGS
In the accompanying drawing:
Figure 1 is a diagram illustrating a pressently preferred process for synthesizing Tc-99m-MAGGG, a compound within the scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to novel Tc-99m compounds that are actively secreted into the tubules of the kidneys, and thus are excellent candidates for use in renal s function tests, such as radioactive urography procedures.
More particularly, the present invention is directed to Tc-99m-mercaptoacetyl-tri-amino acid compounds (an N3S
system), and especially Tc-99m-mercaptoacetylglycylglycyl-amino acid compounds having the general formula:

O

i ~ ~ o O

where Y is -H or where Y is:
7' z and where Rl is -H, -CH3, or -CH2CH3; R2 is -H, -CH2CQ2H, -CH2CONH2, -CH2cH2cO2H~ -CH2CH2CON~2' CH3, 2 3 -CH20H; and Z is -H, -C02H, -CONH2, -S03H, -S02NH2, or -coNHc~l2co2H .
The present invention also includes Tc-99m compounds similar to the general formula set forth above, except that the sulfur is replaced by nitrogen (an N~ system) as set forth below:

// i\ /
N / \ N
N N ~ O
O \\

lS where Y has the samè;definition as above.
In addition to the foregoing, the present invention is directed to Tc-99m compounds having the following general structure:

c \ N
N ~ N ~ ~f c d 1 ~il 7066 where X is S or N; where Y has the same definition as above;
and wherein each small letter a-f represents either 2 hydro-gens or a double-bonded oxygen: where X is S, then element a represents two hydrogens and at least two of elements b-f s are double-bonded oxygens, and the remaining elements are two hydrogens; where X is N, then at least two of elements a-f are double-bonded oxygens, and the remaining elements are two hydrogens.
In addition to each of the compounds illustrated above, the present invention includes the water-soluble salts thereof.
The present invention also includes novel chelating agents that may be reacted with Tc-99m to form the foregoing compounds. Such novel chelating agents have the following general formula: ;

X N

~ N N J O
Ot y where X and Y have the same definitions as above, and wherein Y' is -H2 when X is N, or wherein Y' is -H, -COCH3, -COC6H5, -CH2NHCOCH3, -COCF3, COCH2OH, -COCH2CO2H, or other suitable protective group when X is S. When X is S and Y' is one of ' `"` 1 3t 7066 the foregoing groups other than -H, the use of Y' serves to protect the sulfur from oYidation. No such protection is necessary when X is N.
The presently preferred process for synthesizing the 5 novel Tc-99m compounds of the present invention is set forth in Figure 1, wherein is specifically illustrated the process for synthesizing Tc-99m-mercaptoacetylglycylglycylglycine (Tc-99m-MAGGG).
. Thus, in Figure 1, it is seen that ~lycylglycylglycine (Compound I) is reacted with chloroacetyl chloride in order to produce chloroacetylglycylglycylglycine (Compound II).
Compound II is next reacted with sodium thiobenzoate in order to form benzoyl mercaptoacetylglycylglycylglycine (Compound III). Compound III is fin~lly reacted with sodium ~5 pertechnetate in t~e presence;of a suitable reducing agent so as to produce Tc-99m-MAGGG tCompound IV). It will be appreciated that the same general synthesis pathway may be used with other starting ligands in order to produce other Tc-99m compounds within the scope of the prese~t inven~
tion.
The novel Tc-99m compounds of the present invention are used in scintigraphic urography procedures by administration thereof to a patient by intravenous injection, followed by recording of images of the patient's kidneys by means of gamma scintillation cameras. As mentioned above, it is possible to administer dosages of as much as 30,000 `"` 1 31 7066 microcuries o~ Tc-99m; this is extremely beneficial in conducting dynamic tests of renal function because of the short e~posure periods that are possible with such a high dosage.
It has been found that the novel Tc-99m compounds of the present invention are actively secreted into the tubules of the kidneys, thereby providing significantly high extrac-tion efficiencies so that they are capable of serving as substitutes for I-131 OIH. Additionally, it will be appreciated from an examination of the structures set forth above that the novel compounds do not exist in stereo-isomeric forms that might make practical applications more difficult talthough diasteriomeric forms can exist dependent upon the choice of the Y group).
As mentioned above, Tc-99m has a half-life of only about six (6) hours. Because of this short half-life, it is not practical to package a Tc-99m-chelate ready for clinical use. An important feature of the presen~ invention is the ability to package the reagents in a kit form that permits easy preparation of the Tc-99m-chelate immediately prior to use as a radiopharmaceuticaI.
Thus, although in the laboratory it has been found practical to react mercaptoacetylglycylglycylglycine with Tc-99m pertechnetate in the presence of the reducing agent dithionite, both the Tc-99m and the dithionite must be freshly prepared.

"

A more convenient synthesis process has been discovered using stannous ion complexed with a suitable intermediate exchange ligand such as acetate, tartrate, malate, lactate, hydroxyisobutyrate, citrate, glucoheptonate, gluconate, pyrophosphate, N-methyl N,N'-bis (2-hydroxyethyl)ethylene-diamine, or glycine. It has been foun~ that the stannous ion is capable of inducing the reaction of a ligand such as that of Compound III of Figure l with sodium pertechnetate to form a Tc-99m compound such as Compound IV of Eigure l.
The stannous ion is not unstable in solution as is dithionite; hence, the process utilizing stannous ion is readily susceptible for packaging in a kit form, consisting of two parts, the stannous ion (and intermediate exchange ligand) and ligand to be attached to the Tc-99m being provided in one vial, capable of long-term storage, and sodium pertechnetate in another vial. Generally, the sodium pertechnetate will be locally prepared from readily available Mo-79/Tc-99m generators because of the short half-life of Tc-99m.
A few representative examples will assist in the under-; standing of the present invention. Examples I and II
describe a presently preferred process for synthesizing mercaptoacetylglycylglycylglycine and Tc-99m-MAGGG, respec-tively.

1 3~ 7066 EXAMPLE l Synthesis of a benzoyl mercaptoacetylglycylglycylgly-cine: The synthesis of benzoyl mercaptoacetylglycylglycyl-glycine was accomplished as a multi-step process beginning with dissolving 2.5 grams of glycylglycylglycine in 75 millimeters of 1.0 Normal sodium hydroxide in a 500 milli-liter flask at 0C and under a nitrogen atmosphere.
A solution of 13.0 grams of chloroacetyl ch]oride in 100 milliliters of ether was then added dropwise from one addition funnel, while 100 milliliters of 1.0 Normal sodium hydroxide was simultaneously added dropwise from a second addition funnel, at the same time continuously stirring the glycylglyc~lglycine solution. Following dropwise addition of the chloroacetyl chloride and sodium hydroxide, the reaction mixture was maintained at 0C while being stirred for an additional 1.5 hours.
Next, the reaction mixture was acidified to a pH of about 2 by addition of concentrated hydrochloric acid.
After stirring for yet an additional 30 minutes, the reac-tion mixture was warmed to 40C and concentrated to one-third of its volume under reduced pressure.
The concentrated mixture was then cooled in an ice bath in order to precipitate out chloroacetylglycylglycylglycine;
after two washings with water, it was found that 2.75 grams of chloroacetylglycylglycylglycine were obtained, a yield of :.
'.` ~. , .
.

1 3 1 70~)6 78.5% in the amount of glycylglycylqlycine dissolved in the starting mixture.
One gram of the crude chloroacetylglycylglycylglycine was next dissolved in 300 milliliters of anhydrous methanol s under a nitrogen atmosphere. Pifty (50) milliliters of a solution containing sodium thiobenzoate (prepared from 175 milligrams of sodium in methanol to which 1.1 gram of thiobenzoic acid was added) was then added to the flask, and the reaction mixture was re~luxed for 1.5 hours.
Next, the solvent was removed under reduced pressure.
The resultant solid was isolated by filtration and washed with chloroform. Crystallization from methanol resulted in recovery of 1.25 grams ~90%) of benzoyl mercaptoacetylgly-cylglycylglycine.
An elemental analysis was conducted to verify that benzoyl mercaptoacetylglycylglycylglycine was the product obtained from crystallization from methan~l. The calculated theoretical percentages of carbon, hydrogen, nitrogen, and sulfur comprising benzoyl mercaptoacetylglycylglycylglycine are 56.56, 4.92, and 5.74, and 13.11, respectively. The results of the elemental analysls were 56.50, 5.06, 5.67, and 13.27, respectively. The substantial agreement between the theoretical and experimental analyses clearly indicates that the product of this reaction sequence was benzoyl mercaptoacetylglycylglycylglycine.

1 3t 7066 Synthesis of Tc-99m merca~toacetyl~lycvlglycylgly-cine: As mentioned above, one important feature of the present invention is the ability to package precursors to the desired imaging agent in a kit form. This example des-cribes the preparation of such a "kit" in the context of Tc-99m-MAGGG as an imaging agent.
For instance, in preparing about 100 "kits," 80 milli-liters of 1.25 Molar intermediate exchange ligand (e.g., acetate, glycine, citrate, malonate, gluconate, glucohep-tonate, pyrophosphate, tartrate, malate, lactate, hydro-xyisobutyrate, or N-methyl N, N'-bis (2-hydroxyethyl) ethylened1amine) is ad~usted to a pH of about 5.5 and then deoxygenated by purging with nitrogen gas. One hundred milligrams of benzoyl mercaptoacetylglycylglycylglycine is then added with stirring until a clear solution is obtained.
Next, 0.20 milliliters of a freshly prepared 10.0 milligrams/milliliters soluti.on of SnC12 2H20 in 1 milli-liter of 0.1 Normal hydrochloric acid is added to themixture under a nitrogen atmosphere. Finally, the pH of the resulting mixture is adjusted to about 5 by addition of appropriate amounts o 0.1 Normal HCl or NaOH, diluted to 100 milliliters total volume, and the solution is sterilized by passage through a 0.2 micron filter, while still maintaining a nitrogen atmosphere. Finally, 1.0 milliliter .

.

~ 31 7~6h aliquots are dispensed into vials using a sterile technique, and the individual aliquots are frozen or freeze dried for storage. Optionally, a stannous ion stabilizer (such as gentisic acid or ascorbic acid) is also added.
S A volume o~ about l to 3 milliliters of Tc-99m pertechnetate in saline having the desired level of radio-activity ~as high as 50 mi]licuries per milliliter is acceptable~ is obtained from a Mo-99/Tc-99m generator and added to one of the vials containing reactants prepared as set forth above. Ater mixing, the vial is placed into a boiling water bath for five minutes in order to effect the reaction which results in formation of Tc-99m-MAGGG. Upon cooling, the preparation may be used with no further treat-ment.
``

Analysis procedures: Where desired, routine analysis of Tc-99m-MAGGG is advantageously conducted by use of thin layer chromatography on ITLC-SG silica gel impregnated glass fiber strips, such as those obtainable from Gelman, Inc., Ann Arbor, Michigan.
The amount of soluble, unbound Tc-99m pertechnetate is determined by the radioactivity at the solvent front of a strip developed in methylethyl ketone. The amount of insoluble Tc-99m is obtained by measuring the radioactivity from the raction at the origin on a strip developed in .

-- 131706~
saline. The percentage of bound Tc-99m is calculated according to the following formula:

~ bound = 100 - % unbound - ~ insoluble s An analysis of the chelated Tc-99m-MAGGG product may be conducted through use of high performance liquid chromato-graphy ("HPLC") using a 5 micron ODS column with a solvent system comprising 5% ethanol: 95~ 0.01 Molar phosphate at a pX of 6. Tc-99m-MAGGG is the major peak at about 4 minutes when using a 1.0 milliliter per minute flow rate. Other components that may be observed are Tc-99m pertechnetate at about 2.5 minutes.

~ EXAMPLE 4 Renal Uptake_of Tc-~9m-MAGGG in Normal Mice: Tc-99m-MAGGG was administered simultaneously with I-131 OIH (as a reference standard) to six mice in two groups. Each mouse was injected intravenously with 0.1 milliliters of a preparation containing 0.5 microcuries of Tc-99m-~AGGG and 0.2 microcuries o~ I-131 OIH, and then placed int~ a meta-bolic cage capable of collecting excreted urine.
Ten minutes after injection, each mouse's urethra was ligated and the mouse was sacrificed by chloroform vapor, various samples were then taken to determine biodistribution of radiolabeled material. The results of these samples are , ' ~ .
.

.

shown in Table I. Similar measurements, shown in Table II, were taken on the second group of mice 120 minutes after injection.

Table I
BIO~ISTRIBUTION IN NORMAL MICE AFTER 10 MINUTES
(Numbers expressed as percentage of radioactive agent initially injected) Ayent Blood Liver KidneYs Stomach Intestine Urine Tc-99m-MAGGG2.6 2.9 3.5 0.1 1.1 79.9 OIH 4.1 1.8 2.2 0.5 1.0 74.4 Table II

(Numbers expressed as percentage of 15 radioact`ive agent initially injected) Agent Blood Liver Kidneys Stomach Intestine Urine Tc--9 9m--MAGGG. 0.03 0.08 0.06 0.02 1.2 98.5 OIH0.14 0.11 0.06 0.98 . 0.16 96.0 Tables I and II clearly illustrate the rapid and selec-tive removal of Tc-99m-MAGGG by the kidneys, with only trace amounts be.ing taken up in other major organs. This is an important feature of an imaging agent so as to avoid damage to body tissues from radiation emitted by the radiolabel, and so as to minimize the amount of radiopharmaceutical required to be administered in order to obtain a suitable image.
Notably, these tables show that Tc-99m-MAGGG is even more rapidly excreted.by the kidneys than is I-131 OIH; the $ levels of Tc-99m MAGGG in the urine at 10 minutes and 120 minutes were equal to 107.3 percent and 102.6 percent of corresponding levels of I-131 OIH. Because Tc-99m causes less tissue damage than does I-131, these results indicate that Tc-99m-MAGGG is significantly safer to use than is I-131.

Renal Uptake of Tc-99m-MAGGG in Probenicid-Treated .
Mice: In order to test the biodistribution of Tc-99m-~AGGG
.- 15 in mice having inhibited renal tubular transport, six mice were injec.ted with a solution of probenicid at the rate of 50 milligrams of probenicid per kilogram of body weight.
Ten minutes later, each mouse was injected with 0.1 milliliters of a solution containing 0.5 microcuries of Tc 99m-MAGGG and 0.2 microcuries of I-131 OIH. After an additional ten minutes, each mouse was sacrificed and various samples taken to determine biodistribution of radiolabeled material. The results of these samples are set forth in Table III.

.
~ ' ', . ',. :
' .

1 31 70h6 Table II
BIODISTRIBUTION IN PROBENICID-TREATED MICE A~TER 10 MINUTES

(Numbers expressed as percentage of radioactive agent initially injected) Agent Blood Liver Kidneys Stomach Intestine Urine Tc-99m-MAGGG 6.0 5.9 5.4 0.24 1.6 64.7 OIH 7.0 3.7 3.6 0.75 2.0 59.2 Table III illustrates that kidneys having decreased function are still capable of removing Tc-99m-MAGG5 to a greater degree than they are capable of removing I-131 OIH. Since I-131 is the current standard against which other radiopharmaceuticals are measured, these results indicate that Tc-99m-MAGGG is an excellent compound for use in renal function diagnostic procedures.

., .
EXAMPLES 6 and 7 .
Renal Excretion of Tc-99m-MAGGG in Humans: The renal .. ... _ .
excretion of Tc-99m-MAGGG in humans was obtained based upon Z0 experiments on normal volunteers. For purposes of compari-son, tests were also conducted using I-131 OIH either immediately before or after the study using Tc-99m-MAGGG.
In Example 6, the Tc-99m-MAGGG used was prepared with stannous reduction similar to the procedure set forth in ~5 Example 2, with glucoheptonate being used as an intermediate exchange ligand. In Example 7, dithionite was used as a reducing agent instead of using stannous reduction. Both examples provided Tc-99m-MAGGG having the same ma~or peak on a HPLC column.
In both examples, the subject was injected with a dosage containing about 15 millicuries o Tc-99m-MAGGG. The comparative tests utilizing I-131 OIH involved administra-tion of about 300 microcuries of I-131 OIH. In both instances, imaging of the kidneys, ureters, and blood pool was conducted for a period of about 30 minute~, following which the count rate in the bladder was measured.
Thereafter, the bladder was voided, and an additional image was taken to detect the presence of residual radio-activity in order to allow determination of the percent of urine radioactivity at 30 minutes. Measurements were also ; 15 taken at 3 hours. The results of these tests are set forth in Table IV.

Table IV

(Numbers expressed as percentage of radioactive agent initially injected) ~ 30 minutes 3 hours Tc-99m-MAGGG 71.8 i 4.2 98.6 ~ 1.6 I-131 OIH 65.5 * 6.3 93.5 + 3.2 , Table IV clearly demonstrates that Tc-99m-MAGGG has a high renal extraction efficiency in humans, being even better than I-131 OIH. This makes Tc-99m-MAGGG an outstand-, .

t ~1 7066 ing imaging agent for use in scintigraphic urography. Incontrast, the most efficiently excreted Tc-99m compound prior to the present invention (Tc-99m-CO2-DADS-A) was excreted nearly 20 percent less effectively than I-131 5 OIHo Synthesis and_Renal Uptake of Tc-99m-MAGGGG: Following the general synthesis steps set forth in Figure 1 and Examples 1 and 2 with respect to the synthesis of Tc~99m-MAGGG, the compound Tc-99m-mercaptoacetylglycylglycylgly-cylglycine (Tc-99m-MAGGGG) was prepared. This was accom-plished utilizing glycylglycylglycylglycine in place of glycylglycylglycine ln Example 1.
Tc-99m-MAGGGG was then admi-nistered to mice using a procedure similar to that described in Example 4. Biodis-tribution measurements were taken 10 minutes and 120 minutes after injection. The results of these measurements are shown in Table V.

Table V
BIODISTRIBUTION OF Tc-99m-MAGGGG

(Numbers expressed as percentage of radioactive agent initially injected) Time_ Blood Liver ~ Stomach Intestine Urine (m1n) I0 2.7 3.9 3.3 .2 10.3 58.4 120 .04 1.1 .2 .05 16~0 82.5 1 3~ 7066 It was calculated that the levels of Tc-99m-MAGGGG in the urine at 10 minutes and 120 minutes were equal to 86.7 percent and 87.6 percent, respectively, of the corresponding levels of I-131 OIH, indicating that this compound is a possible substitute for I-131 OIH. Increases in hepatobiliary excretion as shown by lntestine radioactivity indicates decreased specificity, however.

Synthesis and Renal Uptake of Tc-99m-MAGG-Alanine:
Following the general synthesis steps set forth in Figure 1 and-Examples 1 and 2, the compound Tc-93m-mercaptoacetyl~ly-cylglycylalanine was prepared, utilizing glycylglycylalanine in place of glycylglycylglycine in Example lo Tc-99m-MAGG-Alanine was then administered to mice using a procedure similar to that described in Example 4. Biodis-tribution measurements were taken 10 minutes and 120 minutes after injection. The results of these measurements are shown in Table VI.
Table VI
BIODISTRIBUTION OF Tc-99m-MAGG-Alanine (Numbers expressed as percentage of radioactive agent initially injected) Time Blood Liver Kidneys Stomach Intestine Urine tmln 2.6 2.6 5.2 .2 1.6 75.1 120 .2 .1 .3 .2 2.2 96.0 2~

The levels of Tc-99m-MAGG-Alanine in the urine at 10 minutes and 120 minutes were equal to 106.4 percent and 102.2 percent, respectively, of the corresponding levels of I-131 OIH, indicating that this compound would be an excellent choice for use in scintigraphic urography procedures in place o~ I-131 OIH.

Synthesis and Re~ ptake of Tc-99m-MAGG-Aspartic Acid: Following the general synthesis steps set forth in Figure 1 and Examples 1 and 2, the compound Tc-99m-mercapto-acetylglycylglycylaspartic acid was prepared, utilizing glycylglycylaspartic acid in place of glycylglycylglycine in Example 1.
Tc-99m-MAGG-Aspartic Acid was~then administered to mice using a procedure similar to that described in Example 4.
Biodistribution measurements ar~ shown in Table VII.

Table VII
BIODISTRIBUTION OF Tc-99m-MAGG-Aspartic Acid (Numbers expressed as percentage of radioactive agent initially injected) Time Blood Liver Kidneys Stomach Intestines Urine (min?
6.6 6.51 4.7 .4 2.7 50.1 120 .3 1.5 .Z .3 6.2 87.

The levels of Tc-99m-MAGG-Aspartic Acid in the urine at 10 minutes and 120 minutes were equal to 64.2 percent and .

``` 1 31 70~
94.1 percent, respectively, of the corresponding levels of I-131 OIH. The low clearance at 10 minutes makes the suitability of this compound as a substitute for I-131 OIH
questionable.

Synthesis and Renal ~ptake of Tc-99m-MAGG-Glutamine:
The compound Tc-99m-MAGG-Glutamine was prepared following the general synthesis steps set forth in Figure 1 and Exam-ples 1 and 2, except that glycylglycylglutamine was used inplace of glycylglycylglycine in Example 1.
Tc-99m-MAGG-Glutamine was then administered to mice using a procedure similar to that described in Example 4.
Biodistribution measurements are shown in Table VIII.

Table VIII
BIODISTRIBUTION OF Tc-99m-MAGG-Glutamine ~Numbers expressed as percentage of radioactive agent initially injected) Time Blood Liver Kidne~ Stomach Intestines Urine 20 (min) 3.2 4.5 4.~ .2 .9 70.7 120 .04 .8 .1 .04 1.0 95.9 The levels of Tc-99M-MAGG-Glutamine in the urine at 10 minutes and 120 minutes were equal to 97.6 percent and 103.4 percent, respectively, of corresponding levels of I-131 OIH, indicating that this compound is an excellent substitute for I-131 OIH for use in scintigraphic urography procedures.

.. . .

1 31 7~66 Synthesis and ~enal Uptake__ of Tc-99m-MAGG--. Phenylalanine: The compound Tc-99m-MAGG-Phenylalanine was prepared following the general synthesis steps set forth in Figure 1 and in Examples ~ and 2, except that glycyl~lycyl-phenylalanine was used in place of glycylglycylglycine in Example 1.
Tc-99m-MAGG-phenylalanine exists in two separable diasteriomeric forms, ident1fied by the labels -A and -B.
The diasteriomeric forms Tc-99m-MAGG-Phenylalanine-A and Tc-99m-MAGG-Phenylalanine-B were separated and separately administered to mice using a procedure similar to that described in Example 4. Biodistribution measurements for ; 15 these two compounds are shown in Tables IX and X, respectively.

Table IX
BIODIST~IBUTION 0~ Tc-99~m-M~GG-Phenylalanine-A

- tNumbers expressed as percentage of radioactive a~en~ initially injected) Time Blood Liver ~ y~ Stomach Intestines Urine (min) ~ ~

9.7 220167.6 .4 7.7 32.3 120 .2 : 2.3 .2 .3 26.1 69.5 ~ 31 7066 ,, Table X

BIODISTRIB~TION OF Tc-99m-MAGG-Phenylalanine-B

(Numbers expressed as percentage of radioactive a~gent initially injected) 5 Time Blood Liver Kidneys Stomach Intestines Urine (min) .19.4 14.7 5.3 1.0 1~9 16.2 120 .8 3.0 .4 .9 41.7 48.6 The levels of Tc-99m-MAGG-Phenylalanine-A in the urine at 10 minutes and 120 minutes were equal to 43.9 percent and 73.5 percent, respectively, of the corresponding levels of I-131 OIHo The levels of Tc-99m-MAGG-Phenylalanine-B in the urine at 10 minutes and 120 minutes were equal to 22.0 percent and 52.3 percent, respectively, of corresponding levels of I-131 OIH~ These low percentages, taken together . 15 with the biodistribution measurements indicate that significant quantities of these compounds are taken up in various tissues, indicate that Tc-99m-MAGG-Phenylalanine is not well-suited for routine use in typical scintigraphic urography procedures.

EX~MPLE 13 SYnthesis and Renal Uptake cf Tc-99m-MAGG-Asparagine:
The compound Tc-99m-MAGG-Asparagine was prepared following the general synthesis steps step forth in Figure 1 and Examples 1 and 2, except that glycylglycylasparagine was used in place of glycylglycylglycine in Example 1.

... - - .

.
. . :

1 3~ 70S6 Tc-99m-MAGG-Asparagine exists in two separable diasteriomeric forms, identified by the labels -A and -B.
These diasteriomeric forms Tc-99m-MAGG-Asparagine-A and Tc-99m-MAGG-Asparagine-B were separated and each administered to mice using a procedure similar to that described in Example 4. Biodistribution measurements for these two compounds are shown in Tables XI and XII, respectively.

Table XI
BIODISTRIBUTION OF TC-99m-MAGG-Asparagine-A
(Numbers expressed as percentage of radioactive agent initially injected) Time Blood Liver Kidneys Stomach Intestines Urine (min) 2.6 5.3 4.1 .2 .9 7~.1 120 0.04 .6 0.04 .2 2.2 94.5 Table XII
BIODISTRIBUTION OF Tc-99m-MAGG-~sparagine-B
(Numbers expressed as percentage of radioactive agent initially injected) Time Blood Liver Kidneys Stomach Intestines Urine ~min) 2.6 6.3 4.2 .1 .9 73.6 120 0.03 .4 0.04 .02 1.8 96.7 The levels of Tc-99m-MAGG-Asparagine-A in the urine at 10 minutes and 120 minutes were equal to 98.9 percent and 102.2 percent, respectively, of corresponding levels of I-131 OIH. The levels of Tc-99m-MAGG-Asparagine-B in the .. . . . .

1 31 706h urine at 10 minutes and 120 minutes were equal to 97.1 percent and 103.5 percent, respectively, of corresponding levels of I-131 OIH.
These high percentages indicate that either diasteriomeric form of Tc-99m-MAGG-Asparagine would be suitable as a replacement for I-131 OIH in scintigraphic urography procedures. Further, since both diasteriomeric forms are suitable replacements for I-131 OIH, there is no need to separate the diasteriomeric forms from one another, making .he use of this compound in a kit form entirely practical.

Synthesis and Renal Uptake of Tc-99m-MAGG-Glutaric Acid: Following the general synthesis steps set forth in Figure 1 and Examples 1 and 2, the compound Tc-99m-MAGG-Glutaric ~cid was prepared, except that glycylglycylglutaric acid was used in place of glycylglycylglycine in Exam-ple 1.
Tc-g9m-MAGG-Glutaric Acid exists in two separable diasteriomeric forms, identified by the labels -A and -B.
These diasteriomeric forms were separated and each adminis-tered to mice using a procedure similar to that described in Example 4. Biodistribution measurements for these compounds are shown in Tables XIII and XIV, respectively.

. _ . . .. .

.. , ...................................................................... :

Table XIII
BIODISTRIBUTION OF Tc-99m-MAGG-Glutaric Acid-A
(Numbers expressed as percentage of radioactive agent initiall~ injected) Time Blood Liver Kidneys Stomach Intestines Urine (min) 107.5 4.8 5.2 .4 1~8 50.2 120.2 1.7 .1 .2 1.1 94.2 Table XIV
BIODISTRIBUTION OF Tc-99m-MAGG-Glutaric Acid-B
~Numbers expressed as percentage of radioactive agent initially injected~
Time Blood Liver Kidneys Stomach Intestines Urine (min?
104.2 3.6 6.5 .2 1.1 65.1 120.2 .9 .1 .4 ~.4 94.3 The levels of ~Tc-99m-MAGG-Glutaric Acid-A in the urine at 10 minutes and 120 minutes were equal to 73.0 percent and 99.8 percent, respectively, of corresponding levels of -131 OIH. The levels of Tc-99m-MAGG-Glutaric Acid-B in the urine at 10 minutes and 120 minutes were equal to 88.6 percent and 2098.8 percent, respectively, of corresponding levels of I-131 OIH. These findings indicate that this compound might be consi~ered as a substitute for I-131 OIH.

Synthesis of Other Tc-99m N3S System Compounds:
Following the general synthesis steps set forth in Figure 1 -` 1 31 7066 `
and in Examples 1 and 2 with respect to the synthesis of Tc-99m-MAGGG, other Tc-99m compounds incorporating the N3S
system within the scope of the present invention are synthe--sized. For instance, with respect to the following general formula: 1l S / Tc \ N

N N J
Il ~
O \\

one such Tc-99m compound is synthesized wherein Y is -CH2CH2CO2H. This compound is prepared by utilizing NH2CH2CONHCH2CONHCH2CH2CO2H in place of glycylglycylglycine in Example 1.
Based upon the results of tests with Tc-99m-MAGGG, it is to be expected that this compound will exhibit a signifi-cant extraction efficiency.

Another Tc-99m compound having the general formula set forth in Example 15, but where Y is -CH(CH2CH3)CO2H, is synthesized following the general synthesis steps set forth in Figure 1 and Examples 1 and 2, but where the starting ligand is NH2CH2CONHCH2COMHCH(CH2CH3)CO2H.

... . .

.

131706h Based upon the results of tests with Tc-99m-MAGGG, it is to be expected that this compound will exhibit a signifi-cant renal extraction efficiency.

Another Tc-99m compound having the general formula set forth in Example 15, but where Y is -CH2CONH2, is synthesized following the general synthesis steps set forth in Figure 1 and Examples 1 and 2, but where the starting ligand is NH2CH2CONHCH2CONHCH2CONH2.
Based upon the results of tests with Tc-99m-MAGGG, it is to be expected that this compound will exhibit a signifi-cant renal extraction efficiency.

: EXAMPLE 18 Synthesis of Tc- 9m Compounds Havin~ an N SYstem: In addition to those Tc-99m compounds synthesized above having an N3S ring systeml it is also possible to synthesize related Tc-99m compounds incorporating an N4 System. For example, with respect to the following general formula:

O
/ Tc \

~ N N J ~0 O ~\
o .
.

- 13170h6 a novel Tc-99m compound, where Y is -CH~CO2H, was synthe-sized utilizing the general synthesis steps set ~orth in Figure 1 and Example 2, but where the ligand NH2CH2CONHCH2--COHHCH2COHHCH2CO2H (glycylglycylglycylglycine) was reactedwith sodium pertechnetate~
The resulting compound, Tc-99m-GGGG, was administered to mice using a procedure similar to that described in Example 4. Biodistribution measurements taken 10 minutes and 120 minutes after injection are shown in Table XV~

Table XV
BIODISTRIBUTION OF Tc-99m-GGGG

(Numbers expressed as percentage of radioactive agent initially injected) ' ' ~ ` .
Time Blood Liver Kidneys Stomach Intestines Urine (Min) 3.8 3.4 4.2 1.1 2.2 69.3 120 .5 1.0 1.1 1.9 2.5 90.0 The levels of Tc-99m-GGGG in the urine at ten minutes and at 120 minutes were equal to 89.4 percent and 96.7 percent respectively, of corresponding levels o~ I-131 OIH, indicating that this compound is a possible substitute for I-131 OIH.

.
" , ~ ~ , .

1 3 t 7066 It is also possible to prepare other Tc-99m compounds involving some structural changes in the ring system. For example, it is expected that the following general class of compounds will also exhibit significant renal extraction efficiencies:

~ N N J `
/1 / \~
O O

For example, by starting with NH2CH2CH2NHCOCOMHCH2CO2H and lS following the general synthesis steps of Figure 1 and Examples 1 and 2, the compound having the general formula above is synthesized, where X is S, and Y is -CH2CO2H.

Another Tc-99m compound having the general formula set forth below:
o Tc \ /y J ~
2 5 N ~N O
I/ \~
O O

:
.

where X is S, and Y is -CHCH3CO2H, is synthesized following the general synthesis steps of Figure 1 and Example 2 by reacting C6H5COSCH2CH2NHCOCONHCH2CONHCHCH3CO2H with sodium pertechnetate.
Based upon the results of the tests done with Tc-99m-MAGGG, it is to be expected that this compound will exhibit a signi~icant renal extraction efficiency.

Another Tc-99m compound having the general formula:

/ Tc ~ N N J ~ ~

where X is S, and Y is ~C~2CH2CO2H, is synthesized following the general synthesis steps of Figure 1 an~ Example 2 by reacting CH3COSCH2CH2NHCH2CONHCH2CONHCH2CH2CO2H with sodium pertechnetate.
Based upon the results o the tests done with Tc-99m-MAGGG, it is to be expected that this compound will exhibit a significant renal extraction efficiency.

.

.
Another Tc-99m compound having the general formula:
o ~ N N J ~ O

where X is N, and Y is -CH2CO2H is synthesized following the general synthesis steps of Figure 1 and Example 2 by reacting H2NCH2CH2NHCH2CH2NHCOCONHCH2CO2H with sodlum per-technetate.

Based upon the results of the tests done with Tc-99m-MAGGG, it is to be expected that this compound will exhibit a significant renal extraction efficiency.

From the foregoin~, it will be appreciated that the novel Tc-99m compounds of the present invention will be useful as imaging agents in scintigraphic urography procedures because of their substantial renal extraction e~ficiencies and their substantial avoidance of adverse properties such as isomerism. Additionally, the ability to provide the precursors to these Tc-99m compounds in kit form :: .

-` 1 31 7066 requiring nothing more than a mixing and heating step makes the use of Tc-99m as a radiolabel extremely practical.
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

. , '' ' ~' ~

.

Claims

WHAT IS CLAIMED IS:

1. A ligand capable of reacting with a suitable Tc-99m-pertechnetate salt to form a Tc-99m chelate, said ligand having the general formula where X is S or N where X is S, Y' is -H or a suitable protective group which protects the sulfur from oxidation, where X is S, or where Y' is -H2 where X is N; and Y is -H2 or Y is:

and where R1 is -H, -CH3, or -CH2CH3; R2 is -H, CH2CO2H, -CH2CONH2, CH2CH2CO2H, -CH2CH2COHN2, -CH3, -CH2CH3, CH2C6H5, or -CH2OH; and Z
is -H, CO2H, -CONH2, -SO3H, -SO2NH2, or -CONHCH2CO2H.

2. A ligand as defined in claim 1 where X is S.

3. A ligand as defined in claim 1, wherein the suitable protective group is -COCH3, -COC6H5, -CH2NHCOCH3, -COCF3, or -COCH2OH.

4. A ligand as defined in claim 2, where Y' is -COC6H5, and Y is CH2CO2H.

5. A ligand as defined in claim 1, wherein Y is -CHCH3CO2H; -CH(CH2CONH2)CO2H; or -CH(CH2CH2CONH2) COOH.