CA1070241A - Radioactive diagnostic composition - Google Patents

Abstract

DIAGNOSTIC AGENTS

Abstract of the Disclosure Radionuclide diagnostic agents, comprising technetium-99m, a reducing agent, preferably a stannous reducing agent, and certain polyhydroxycarboxylic acids and derivatives there-of, i.e. glucoheptonic acid, lactobionic acid, galacturonic acid and glucuronic acid and salts thereof, are highly useful, both in the study and diagnosis of kidney morphology and function, and in the diagnosis of ischemic, infarcted or diseased tissue.

Description

~70~

This invention pertains to agents which are useful in medicine as aids in detecting and diagnos.ing disease, in the examina-tion and evaluation of body organs, and/or for other purposes, and to diagnostic and evaluatory processes using such agents. More particularly, it is concerned with certain radionucli.de tracer agents which a,id in radiological visualization of various types of tissues, including body organs, particularly the kidneys, and cysts, tumors or other diseased tissue, and/or dead or dying tissue.
The use of tracer compounds, which emit radiation from within the body, as medical tools has long been known. Early work included the use of such materials for testing liver function and biliary patency, and for the analysis of the physiological structure and function o~ the kidneys. A parti-cularly important use of such agents is in the analysis of kidney ` , morphology function and disorders, where radionuclides are ~
used in a number of ways in order to get a number of different ~ ' types of informatlon. ` ~ :
In order to appreciate the various ways such agents ,' ' can be used in connection with the kidneys, a brief and ,, , necessarily oversimplified description of kidney function is : ' , ' , ': .
' ' -' ~

., ~

: .
~,~ '' .

~ 7~
t ¦I necessary. 1`he bulk of the kid~ley consists of an outer portioncalled t~e cortex ancl an inner portion called the rnedulla; The cort:ex is reddish brown and contains approxlmately one million .~
nephrons, the primary workin~ unit of the kidney. The nephron .
consists of vessels and capillaries which carry the.:blood supply, a'renal ~lomerulus, and a ~enerally convoluted renal tubule. The renal ~lomerulus consists of capillary tuft having a few loops of capillary, which are almost completely encapsu-lated by an expansion of the renal tubule called Bowman's Capsule. The only parts of the ~lomerulus which are not covered by that capsule are the points at which a blood-supplyln~
~afferent) vessel, and a blood withdrawin~ (efferent) vessel '.
enter and leave the capillary tuft. The renal tubule~extends 1'~
.. from a hi~hly convoluted route proximal the capsule in the ~: : -cortex, into the medulla, where it reverses ltself in Henle's '' ~ :
'loop. The tubule returns to the cortex, where its distal portion .~ . .' . follows a convoluted course until it empties into a collectin~
duct for the waste products. The collectin~ ducts lead to' .
further kidney structures from which the urine.leaves the body throu~h the ureter, the bladder, and thence-the urethra. The . :
renal tubules are closely associated throu~hout their length ' ' "
with a close meshwork of capillaries, which carry an abundant : I .
blood supply. - ; ' Essentially three processes are involved in urine forma- ~. ;
tion: filtration by the ~lomerulus 7 reabsorption of materials ~
from the tubular fluid, primarily back into the blood stream, .
and secretion of materials into the tubular fluid.
The ~lomerulus. functions as àn ultrafilter, perm'ittin~
particles smaller than the size of the pores in its walls to ~:
escape into the tubular fluid, thus filterin~ small colloidal :~
and noncolloidal components of plasma. There is-no.sel.ectivity ¦~ t this filtration other than that of par=icle size, so the , ' ,'; ~' l -2- . - :~
I
... ...

'70Z~l chemical composition and concentrations of materials in the filtrate are otherwise the same as those of plasma. The filter-ing process is directly related to the blood pressure. The effective filtering pressure is equal to the hydrostatic pressure in the glomerulus (normally about 75 to 80 mm Hg), minus the pro-tein osmotic pressure (normally about 25 mm Hg), and minus the backpressure caused by resistance to movement to fluid in the tubule ~normally about 7 mm Hg). Backpressure in the tubules is increased by increased intraureteral pressure, which is transmitted upward through the renal pelvis and inhibits the onward movement of tubular fluid. Pressure in the ureter rises if urine flow is impeded by obstruction of the ureteral openinys into the bladder, or of the urethra.
While the total glomerulus filtration rate is very high, normally about 120 ml per minute (170 - 180 liters per day), the urine output is low, about 1 ml per minute (1 1/2 liters per day).
Thus as the filtrate passes through the tubules, it is changed ~ ~
in both volume and composition by the reabsorption of about 119 ~ ~;
ml/minute of water and certain of its constituents. The cells of the tubule walls are responsible for the reabsorbing of materials to their approximate normal blood vessels. Much of the reabsorption is an "active" proc0ss, which involves so-called "carriers" within the tubular cell to aid in transporting the -substance across the membrane. There is a limit to the rate at which such active transport systems canoperate, and when this limit is reached, this material, e.g. glucose, which is present in excessive amounts in the filtrate, then fails to be reabsorbed and is excreted in the urine. In addition to active transport, passive transport occurs across the membrane through the processes ~ -of diffusion and osmosis. Secretion of materials by some of thecells in the tubule walls also plays an important part in renal function, some secretions marely being eliminated from the ~07~Z~
body, and other secretions being compounds which have specific functions in the renal system, such as controlling reabsorption of various materials.
More complex descriptions of the functions of the kidney are readily available, e.g. Smith, The Kidney, Scientific American 188:40 (1953).
. .
The use of radionuclides in the diagnosis of kidney problems has been known for some time, the bulk of the work in this area having been done since the early or middle fifties.
Such techniques can generally be divided into dynamic tests and static tests. The dynamic tests follow the course of radionuclides which have been injected into the blood as they are taken up and eliminated by the kidney. Such tests may ; merely monitor the radioactivity in the kidney over a period of time, or may consist of the taking of anumber of rapid, sequential, short exposure radiophotographic images of the ~ ~
radioactivity in the kidney over a period time, or may ; ;;
use one of a number of other similar techniques. Static tests comprise a num~er of techniques for obtaining imagee of the kidney containing the radionuclides, usually taken with longer exposure times, in order to optimize the visualization of the kidney morphology or structure.
For many years both types of renal investigation with radionuclides have enjoyed limited acceptance, largely because of the poor properties of the radionuclides used. Some, such as 131I-iodopyracet, did not allow adequate visualization of the kidneys, because of simultaneous accumulation in surrounding tissue,~especially the liver. Some techniques utilized to avoid this problem produced undesirable side effects. Others, such as 131I-iodohippuran, were more kidney specific, and, being predominantly eliminated through active secretion by tubular cells as composed to elimination by glo-~CI713;~

merular filtration, emphasized a dif~erent renal function than agents eliminated by filtration alone. However these agents were eliminated from the kidney so fast that rectilinear scanner images could only be obtained by giving a constant infusion of the agent, rather than a single dose. Alternate, longer lasting agents included the radioactive mercurials, but these are partially retained in the kidney for weeks or months after administration, resulting in continuing radiation ex-posure long after the scan is completed. Further, mercury scans were not as detailed in some respects as those using other agents, ~nd mercurial agents were unable to attain high kidney/background contrast ratios rapidly. An e~cellent review of developments in this art is given in Blaufox et al., Evaluation of Renal Function and Disease with Radionuclides , (S. Kanger AG, 1972). ~
The development of technetium-labelled agents has `
led to better visualization of the kidney morphology and more ;~
functional information, yet with far less radiation exposure.
Such agents include combinations of technetium-99m with chelating agents, such as diethylenetetraamine pentaacetic acid (DTPA), or nitrilotriacetic acid (NTA), or more complicated combinations such as that offered under the trademark Renotec, by E.R. Squibb & Sons, Inc., which is a combination of ferric chloride, ascorbic acid and DTPA at higher pH. ~ more recent development has been the suggestion of agents consisting o radioactive technetium-gluconate complexes. See Boyd et al., Tc Gluconate Complexes for Renal Scintigraphy, Brit. J.
Radioloqy, 46:604 (1973). While these agents were in many ways an improvement over those previously known, still some problems persisted, more particularly with regard to the uptake contrast ratios ~hich enable the kidney to be distinguished from ;

the surrounding tissues or organs, e.g. the liver, as well as the blood which fills the background.
'~
_ 5 _ ", ".

3~0'7~
Ano-ther development in radionuclicle use, which is ~ :
more recent ~han the use of the above a~ents in connection with the kidneyJ has been the attempt to find radionuclides which ¦ ~Jould enable detection, location and assessment of infarcts ~ `
i 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 supply-ing ~artery. Infarcts can occur in essentially any area of the I bodyt 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 whlch are selectively ;15 incorporated into infarcted tissue have ~een used for such purposes. Such agents include chlormerodrin, radioactive mercury derivatives of fluorescein, and technetium-labelled tetracycline.
See, Hubner, Cardiovascular Research L~:509 (1970) and Holman et al., J. of Nuclear Medicine 1~: 95 (1973). The high uptake ¦ and long half life of the mercurial compounds, the di~ficulties i ¦ encountered in their preparation, and interference by agent up-take in surroundin~ organs, e.~. the liver made the mercurial compounds unacceptable. Nei~hboring organ uptake was also a l problem with tetracycline agent~
¦ Accordingly, it is an object of the present invention ¦ to provide radionuclide agents that are highly suited as ; I dia~nostic agents, and as agents for the observation or study ¦ of the function of human or animal tissue. It is a further object to provide radionuclide agents whlch are suited for use -~
in vivo, giving maximal information while at the same time ~ exposi g the body to minimal radlati~n dosage and disc~mfort.

Il . ' . ~ .' 6- .

It is a further object to provide radionuclide agents for use in renal studies, which agents are highly kidney specific and are transferred to the kidney quickly with minimal accumu-lation in the surrounding organs, tissues or fluids. It is a further object to provide radionuclide agents for renal study which distinctly emphasize renal function differences.
It is a further object to provide radionuclide agents for renal study which are highly suitable -for both dynamic testing and static testing, being suitable to provide information concerning renal function, patency of excretory pathways, and renal morphology, in a single study. It is a further object to provide radionuclide agents for renal study which are eliminated by both glomerular filtration and tubular secretion, and give high kidney/background contrast ratios and permit high quality visualization of kidney morphology. It is a further object of the invention to provide radionuclide agents which are useful in detectln~, locating and visualizing infarcted areas. It is a further object to provide radionuclide ; agents which are~highly s-electively incorporated or accumulated in ischemic, infarcted or diseased tissue. It is still a ;,.~
further object of this invention to provide radionuclide agents which are usable to advantage both in renal studies and in diagnosis of ischemic or diseased tissue and to methods of renaI study and diagnosis which utilize such radionuclide agents.
Other objects and advantages will be apparent to those skilled in the art upon consideration of this disclosure or upon practice of the invention disclosed herein.

., .
Briefly, it has now been discovered that combinations ;
of radioactive materials with certain polyhydroxycarboxylic acids and derivatives thereof (hereafter referred to simply ~
as "P~C compounds") have highly advantageous properties as ~ -.. ' ~, .

. .

- ...... . .. .

compared with pre~iollsly known radionuclide a~ents. More ¦ specifically, it ~las been found tllat combinations of technetium I
¦ 99 with salts of glucoheptonic acid, lactobionic acid, galac-I turonic acid and glucuronic acid, give extremely effective ! radionuclide a~en-ts. Moreover, it has $urprisingly been found that such a~en-ts are useful not only in renal study but also in diagnosis of ischemic, infarcted or diseased areas.
The radionuclide agents of the present invention are kidney specific diagnostic agents which make it possible to ¦ identify a variety of renal disorders and to obtain in a single ¦ investigation information which had previously required a ¦ plurality of different investigations. Unlike the mercurial ~
agents, the short effective half life of these technetium agents ¦ minimize any danger from overexposure to radiation. On the l otherhand, the period of retention of usable levels of the ¦ technetlum agents of this invention in the kidney i9 larger than the period of retention for example of radioactive iodohippuran, and thus allows ~reatly improved static images l of the kidney to be made lon~ after administration which would ¦ be practically impossible with iodohippuran. The agents Or the instant invention are more kidney specific and give higher kidney/background contrast ratios than those agents previously l known, even the known technetium stannous ~luconate complexes.
¦ Further, the same agents are useful not only in renal ~tudies, ¦ but also in the location and identification of ischemic, infarcted or diseased areas in the body, by radionuclide tracing or imaging techniques, applied for example to the heart I or the brain. Suitable techniques are known to the art, and ¦ are disclosed for example in Hubner, supr_, in Holman, supra, in Ph sician's Desk ReferenF~ for RadloloEy ~nl ~u le~r Me'lclne I ~ ~ .
I
! ~ ,: -, )Z4~

tMedical Economics Co., 1973) and in HanVboc~ Rad~ tive Nuclides (Chemucal Rubber Co., 1969)~ -The agents of the present invention are produced by binding technetium-99m to the above P~C compounds either by using chemical reducing agents, or electrolytically, both methods of binding being known per se in the art.
According to the invention there is provided a method of preparing a radioactive diagnostic agent, comprising (a) admixing a reducing agent and a polyhydroxycarboxylic acid or a salt thereof, said acid being selected from the group consisting of glucohe~tonic acid, lactobionic acid, galacturonic acid and glucuronic acid, and combining technetium-99m with the admixture, or (b) forming a mixture of technetium-99m and a polyhydroxycarboxylic acid selected from the group of glucoheptonic acid, lactobionic acid, galacturonic acid and glucuronic acid, and subjecting said mixture to electrolysis in the presence of an oxidizable electrode.
In another aspect of the invention there is provided a radioactive diagnostic composition comprising technetium-99m bound to a polyhydr~xycarboxylic acid or a salt thereof, the acid being selected from the group consisting of glucoheptonic acid, lactobionic acid, galacturonic acid and glucuronic acid.
Where the binding is by chemical reducing agent, the PHC compound i9 preferably mixed with a reducing agent, e~g., stannous ions, and pertechnetate is thereafter added to that mixture. While not wishing to be bound by theory, it is believed that the pertechnetate is reduced by the stannous ions and reacted with the PHC compound to form the radionuclide agents of the present invention, which is believed to be some type of technetium-stannous-carbohydrate acid complex. The PHC
~ompound may be any salt of glucoheptonic acid, lactobionic acid, galacturoni~ acid or glucuronic acid which i5 soluble ..

_ g _ `` 1(~70Z41 '~
in the muxing medium. Preferably the components are mixed in aqueous medium and the PHC compound is a soluble alkali or alkaline earth metal salt. However, other pharmacologically acceptable media can be used. Preferably the reducing agent is also soluble in the mixing medium, with water soluble sources of stannous ions, such as stannous chloride, or of ferrous ions, such as ferrous ascorbate being preferred.
Stannous ions constitute the most preferred reducing agent.
Other suitable reducing agents will be a~parent to those skilled in the art.
The source of technetium should also preferably be water soluble, with preferred sources being alkali and alkaline earth metal pertechnetates. The technetium is preferably obtained in the form of fresh sodium pertechnetate from a ~terile ~EN 9mTc GeneratorO Any other source of pharmacologically acceptable 99mTc can be used, and a numher of 99TC gene ators are available.

- 9a -7~
The maximum amount of reducing agent which can be used is t}le arnount beyond which preci~itation of the reducing agen~ occurs, and the minimurn amount required i-s that amount necessary to bind a sufficient amount of 99mTc to the carbo-'I hydrate acid to achieve si~nificant selective tissue or -or~an uptake. These amounts can be readily determined for particular technetium-reducing agent - carbohydrate acid mixtures -by routine experimentation. Very small amounts of reducing a~ent are effecti~e for this purpose, but because such agents are usually easily oxidized, compositions using extremely small ;
amounts are likely to loose this effectiveness over a period of time after handling or during use. Thus as a minimum, the amount of reducing agent used should be sufficient to supply 0.1 micrograms of reducing agent per milliliter of the diagnostic agent to be injected. As the amount of reduclng agent is increased~ there appears to be a point for any given combination of particular reducing agent and PHC compound, beyond which blnding effectiveness no longer increases, and in fact may decrease, upon further additive of reducing agent. Some level of binding effectiveness appears to be achieved for even very high levels of reducing agent. Advantage can sometimes be taken of the natural attrition of reducing through oxidation during handling or storage, by providing ~;
compositions containing more than the optimum amount of reducing agent, which in effect will be reduced to the optimum ¦
amount by that attrition pri-or to use. In the presently prefer-red technetium-99m-stannous-glucoheptonate system, the amount of stannous reducing agent is between about 1 and 100 micrograms, preferably between 5 or lO and 40 micrograms per milliliter 3 ~ of t diagnostic agent ~o be injocteo.
. ' .
I . ' , . ''; ' ~ ! 10 ~ .
._ . . ...... . . .
,_ . . .

:

11D70Z4~
.

It also appears important that the PHC compound be in large excess of the reducing agent. Preferably the reducing agent should be present in an amount of about -0.0005% to 0.5%, more preferably 0.005% to 0.1% of the PHC
compound.
Sufficient technetium-99m should be present to give easy detection in the body. The amount necessary appears to depend essentially completely on the level of radioactivity --desired, since if the proper amounts and ratios of reducing agent and PHC compound are present, as much as 9~/O to 95%
or greater of the technetium-99m is bound to the PHC compound.
In the presently preferred system, a sterile, non pyrogenic lyophilized mixture of about O~l mg. of stannous chloride dihydrate reducing agent and about 200 mg. of sodium glucoheptonate is provided in a sterile vial, which is preferably mixed w1th 3 to 7 ml of the output of a NEN 9mTc Generator, shortly before use.
While binding of the technetium-99m through use of a chemical reducing agent is presently preferred, it may also be done electrolytically, using at least one electrode which comprises an oxidizable material such as iron, tin, zirconium, or the like. Such binding methods are known in the art, as disclosed, for example, by ~oyd et al., supra.

.
The diagnostic compositions of the invention may also contain additional pharmacologically acceptable ingredients - which do not interfere with their diagnostic functions. For example, the eluate obtained from standard 9mTc generators may contain saline salts, or saline so1utions may be used to dilute highly radioactive diagnostic~ compositions to the proper level for administration.
~ ' ~

.: -- 1 1 -- . ~

~ ~L07~Z~

The pH of the agents should be adjusted if necessary ;
by pharmacologically acceptable acids or bases, so that the pH
of the agent is from about 3 to 10, preferably about 5 to 8.
Aseptic techniques and sterile non-pyrogenic ingredients should be used at all steps, such procedures being standard to those skilled in the art~
In order to prevent oxidation of the stannous ions other than in formation of the complex, care should be taken to exclude all oxidizing agents from the radionuclide agent and ~ ~-the starting materials. For this reason, sources of technetium-99m containing significant amounts of oxidants should not be used. Oxygen should also be excluded, as by purging the various containers used in preparation and storage with an inert gas, e.g. nitrogen, for a sufficient length of time. However, it is not essential (but highly preferred) to use a nitrogen flushed system.
After mixing, the solution containing the reducing agent and the PHC compound can be sterilized if necessary by ~- .
standard procedures, as by passing them through a Millipore biological filter of about 0.22 micron pore size under a nitrogen atmosphere. Thereafter milliliter portions of the ;~
sterile solutions are poured into individual sterile and non-pyrogenic storage glass vials under a nitrogen atmosp~ere. ~;~
As indicated above, the individual portions are preferably lyophilized by conventional freeze drying techniques under aseptic conditions to remove water. This provides a solid stannous-PHC complex or mixture of some sort, which ;~
aids in shipping and storage and is more stable than the complex in solution. The vials can be sealed and stored until needed to form the fresh 99mTc-stannous-PHC agent at the use situs.
,: .

107~24~L

While it is preferred that the stannous chloride and the PHC compound be mixed together prior to admixtu~e with the technetium, the order of admixture c~n also be technetium plus PHC compound followed by admixture of stannous chloride, or even stannous chloride plus technetium followed by addition of PHC compound. -~
The technetium-99m-stannous-PHC (e.g. glucoheptonate) complex or mix~ure is aseptically intravenously injected into the blood stream. The preferred dosages are between about 1 and 20 or 25 millicuries, preferably between about 10 and 15 mCi of 99mTc-Stannous Glucoheptonate for the normal 70 kg patient. Eigher and lower amounts may be used in certain circumstances although greater dosages increase patient radiation exposure. Lower amounts may be desirable for example when simpler tests such as blood clearance rate tests are run. The study may be commenced immediately after administration either by sequential visuaIization devices such as scintilla~
tion cameras, or by the probes conventionally used in producing a renogram. Static imagery is optionally performed one hour or more after injection. Distribution data in rats concern-ing the glucoheptonate agent indicates that about 15 - 25%
of the injected dose is taken up by the kidneys within one hour post injection, with the total blood activity rapidly clearing to less than 2% within the same time period. Data for humans indicates that about 25% of the injected dose is excreted in the urine within the first hour after injection.
The study may be conducted with a conventional gamma-ray-excited scintillation device or camera or rectilinear scanners, or external probes having the capabiLity of detecting gamma rays of the energy released by ~ c.

' ~0'7~Z4~

Standard p~ecautions for use of radioactive materials should be observed in handling those solutions containing 99mTc.
The acute toxicity level in mice (~50) for stannous glucoheptonate adminstered intravenously in about 1500 mg/kg.
Sub-acute toxicity studies in mice at levels of 800, 400 and 80 mg/k~ per day for 15 days have shown no ill effects.
Similarly no untoward effects were noted in sub-acute toxicity studies in dogs at a level of 19 mg/kg per day for 14 days.
The invention will be further clarified with reference -~
to the following illustrative embodiments, which are intended to be purely exemplary and not to be construed in any limiting -~
sense.
Examples 1 - 8 Technetium-99m-stannous-PHC agents are prepared as follows. First, a mixture containing a~out 0~1 mg. of stannous chloride dihydrate with 200 mg. of the sodium salt of various ;
PMC compounds per milliliter of water is prepared. In Example 1, the salt is sodium glucoheptonate. In Example 2, that salt is sodium lactobionate. In Example 3, that salt is sodium galacturonate. In Example 4, that salt is sodium glucuronate.
In Example 5, the salt is sodium gluconate. The resulting mixtures are lyophilized using standard freeze drying equipment, and in each case the resulting solid is mixed with 3 - 7 ml of the eluate of a sterile NEN 99mTc Generator, which eluate also contains 0.~/0 saline salt. The amount of eluate added to the solid is determined by the dosage required and the activity of the eluate at the time of addition. After thoroughly dispersing and dissolving the lyophilized solid, the radio-nuclide agents are ready for injection.

; ~ ~ 7~'~ 4~
i Example 6 concerns the use Or a commercially available renal ima~in~ a~ent, Renotec, obtained as a kit from E.R.
Squibb & Sons, Inc., of Princeton, Ne~ Jersey. The kit comprises a vial and two disposable syringes. The vial con~ains about

2 ml of an aqueous solution reportedly consisting of about 10 m~. ferric chloride, 5-10 m~. of acorbic acid and sodium hydroxide to adjust pH to 2.0-~Ø One syringe contains 2 ml of a sterile 0.07 N sodium hydroxide solution, and the other syringe contains 2 ml of a sterile diethylenetriamine ¦ pentaacetic acid (DTPA) solution, containing about 5 mg. of DTPA. The radionuclide agent was prepared in accordance with instructions supplied with the kit. Briefly, 1 to 5 ml of 99mTC eluate, a~ain depending on the dosages and activity at time of preparation, were mixed with the contents of the vial.
T~e contents of the sodium hydroxide syringe were then injected and mixed in the vial, and then the contents of the DTPA
syringe were injected and mixed with the vial contents. The Renotec scanning agent was then ready for injection.
Example 7 concerns the use ;of a technetium-99m-stannous-I DTPA agent similar to those known to the art. A mixture contain-in~ about 0.25 m~. of stannous chloride dihydrate and about 5 mg. of the calcium trisodium salt of DTPA per milliliter of water is prepared, and one ml amounts are dispersed in indi-vidual vials and lyophilized using standard freeze-drying procedures. The agent results from dissolving the lyophilized mixture in 3-7 ml of the 99mTc eluate.
Example B concerns the use of a nitrilotriacetic acid radionuclide agent similar to those previously known.~ A
mixture containing about 50 micrograms of stannous chloride dihydrate is prepared. One milliliter portions are dispersed ln vials, and lyophilized using standard freeze-drying techniques -The lyophilized mixture is dissolved in 3-7 ml Or the standard ¦ 99mTc eluate, and lS ready for use.

!

~ ~15~

7~)Z~

Each of the foregoing renal scanning agents is aseptical-ly intravenously injected (0.25 ml containing about 3-5 mCi) into adult rats. One hour later the rats are sacrificed, and the blood collected, weighed and counted. The organs of interest, e.g. the kidney, the liver, and the gastrointestinal tract, are individually weighed and the radioactivity contained therein counted. The amount of radioactivity injected is determined by counting the radioactivity in the syringe before and after injection. The organ distribution values are reported ; ~;
as the percent of the activity injec~ed which is observed in the organ, corrected for the radioactive decay of technetium.

~ . . .
The organ uptake contrast ratios are determined by determining ~ ~-the relative activitycontained per gram of the organs compared.
Thus for example the kidney/liver organ uptake contrast ratio is equal to the radioactivity found in the kidney divided by the welght of the kidney, all dividedby the radioactivity per gram in the liver. The results are reported in Table I
.
below. Each value represents the average of observations on at least two animals, and while there is some variation between individual animals the results reported are representa-tive for the radlonuclide agent tested.
As can be seen the kidney/blood organ uptake contrast ratios for the compositions of this invention vary between 82 and 105, while those for the corresponding gluconic .; .
acid complex (40), the commercial agent Renotec* (22) and the chelating agent complexes DTPA (22) and NTA (6) are quite low, all being less than half of that obtained by the agents of the invention. The ratio lS a measure of the ability to * trademark .,' ~

- 16 - ~ ~
.
:.

- : :. .

~7~Z4~ :
____ _ ~ , . -. :
~D ~ ,~ ~ ~ ~
_ ~_ ' :' d~, O ~ ~ O , u~ ~1 0~ ~i C~ D
` ~, .

~1 P; o D __ _ ~ .. ~

U~ O~ ~ ~ ~ O U~ '~
t~
__________ ~ ~ ~_. _ D __ _ U :

~: d' O D O 11~ 0 ~i ~ ~ :
~ : S~ , ,1 ~ ~ _ _ o _ u o ~: : . ~ . ' ~ ~ ~rl 0 ~ ~1 H O ~ I¢ ~-J L~l 0 r-l 0 0 ~
: ' a~ 1~: ~ : ~ C : : ~ ~ , ~ ~ _ _ ; -~ ~ ~
rl ~ ~ ~ 0 O ~c~l ~0 ~ ~ : ~ U ~ .
~ .1-l `~ 0 o u ~ 1~

~~ u ~ o r~ ~1 P~
U O ,~ . : 0 ~ ~ ~ o LO d~ ~ .~
~ o ~ 3 ~_ - E~ U ~
: ~ ~ . ~ O ~ H ~_1 o ~
~1) ~ ~ ~ 1 ~
~d H ~ O ~ C u~ 3 .~ . .,, ~ .,, .,, .,, ~ ~ m x x x ~
. :

- 17 - ~:~

1~711D2~

accurately distinguish radioactivity emanating from the kidney as against a blood filled background. Striking improvements are also observed in the kidney/liver contrast ratio, which is a measure of the ability to radiographically distinguish the kidney from the liver, a well known problem with previous agents. Generally the kidney/gastrointestinal contrast rations are of about the same order of magnitude as the better of the prior agents.
This difference in ability to accurately define the kidney against the neighboring organs is quite surprising, especially in view of the closeness in structure between, ~`
for example, the glucoheptonic acid agent and previously known gluconic acid agents. No solid reasons for these observed differences are presently known.
Examples 9 a_d 10 The technetium-99m-stannous-glucoheptonate agent of Example 1, supra is compared with a technetium-99m-stannous~
tetracycline agent, see Holman et al, supra, for effectiveness in preferentially accumulating in infarcted, rather than normal, myocardial tissue. In each case a myocardial infarct is produced in adult rats by surgery. In Example 9,doses of ?
about 3-5 mCi each of the glucoheptona~e and tetracycline agents respectively are lnjected three hours after infarct was induced. The animals are sacrificed one hour after injection and the heart removed. The infarcted tissue, which is visibly distinguishable from normal myocardial tissue, was excised, weighed and counted. The normal myocardial tissue was also weighed and its activity counted and the results are reported as the ratio of the activity per gram of the infarcted 7~%4L~ r tissue to the ac~ivity per gram of the normal tissue (A), and the ratio of the activity per gram o~ the infarcted tissue to activity per gram in the blood (B). Exarnple 10 reports similar .
. I results, the difference being that in that example the radionu-clides were injected into the live rats eighteen hours after the infarct was induced, rather than three hours. Examples 9 and 10 are reported in Table II below, , ' : ~ .,.
, Table II
. Infarction Tests A~ent _ Example 9 ~ ~:
Glucoheptonate A 10.3 6.7 complex - ~
:, B 4-5 3.2 Tet,racycline A 5,9 3.7 . .
complex ' B 2.5 2.0 . . .' . , ' .
The results of Examples 9 and 10 both indicate that :
.. , the glucoheptonate agent of the present invention was more -~ effective in preferentially accumulating in infarcted, rather ¦ than normal, tissue than the .tetracycline infarct agent.
l ' ' ' '' ' " .
While particular embodiments of the present invention have been described herein, they are intended to be exemplary only, with the true'scope and spirit of the invention being indicated in the spec}-ication and t~e follo~i-g cl~:ms:

:
Il . , . , Il . :

~l ~19-. ' .

Claims (38)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A method of preparing a radioactive diagnostic agent, comprising (a) admixing a reducing agent and a polyhydroxy-carboxylic acid or a salt thereof, said acid being selected from the group consisting of glucoheptonic acid, lactobionic acid, galacturonic acid and glucuronic acid, and combining technetium-99m with the admixture, or (b) forming a mixture of technetium-99m and a poly-hydroxycarboxylic acid selected from the group of gluco-heptonic acid, lactobionic acid, galacturonic acid and glucuronic acid, and subjecting said mixture to electrolysis in the presence of an oxidizable electrode.

2. A method according to claim 1(a), comprising admixing a reducing agent and a salt of a polyhydroxycarboxylic acid selected from the group consisting of glucoheptonic acid, lactobionic acid, galacturonic acid and glucuronic acid, and combining technetium-99m with the admixture.

3. A method according to claim 1(b), comprising forming a mixture of technetium-99m and a polyhydroxycarboxylic acid selected from the group of glucoheptonic acid, lactobionic acid, galacturonic acid and glucuronic acid, and subjecting said mixture to electrolysis in the presence of an oxidizable electrode.

4. A method according to claim 2, wherein said mixture is lyophilized prior to being combined with technetium-99m.

5. A method according to claim 2, wherein said reducing agent comprises a stannous reducing agent.

6. A method according to claim 5, wherein the stannous reducing agent is present in an amount of at least 0.1 micro-gram per milliliter of composition.

7. A method according to claim 6, wherein the stannous reducing agent is present in an amount of from about 1 to 100 micrograms per milliliter of composition.

8. A method according to claim 6, wherein the stannous reducing agent is present in an amount of from 10 to 40 micrograms per milliliter of composition.

9. A method according to claim 6, wherein the stannous reducing agent is present in an amount of from 0.0005% to 0.5% by weight of the polyhydroxycarboxylic acid salt.

10. A method according to claim 6, wherein the stannous reducing agent is present in an amount of from 0.005% to 0.1%
by weight of the polyhydroxycarboxylic acid salt.

11. A method according to claim 10, wherein the poly-hydroxycarboxylic acid salt is an alkali or alkaline earth metal salt of glucoheptonic acid.

12. A method according to claim 10, further including a pharmacologically acceptable carrier in said admixture.

13. A method according to claim 1, wherein said technetium-99m is present in an amount to give radioactivity in the amount of from about 1 to 25mCi per 75 kilograms of body weight.

14. A method according to claim 1, wherein said technetium-99m is present in an amount to give radioactivity in the amount of from about 10 to 15 mCi per 75 kilograms of body weight.

15. A method according to claim 2, wherein said reducing agent comprises a stannous reducing agent, and is present in an amount of at least 0.1 micrograms per milliliter of said composition.

16. A method according to claim 2, wherein said reducing agent is a stannous reducing agent, and is present in an amount of from about 0.0005% to 0.5% by weight of the poly-hydroxycarboxylic acid.

17. A method according to claim 16, wherein the poly-hydroxycarboxylic acid is glucoheptonic acid.

18. A method according to claim 2, wherein said reducing agent is a stannous reducing agent present in an amount of from .01 to .1% by weight of said polyhydroxycarboxylic acid,

19. A method according to claim 3, wherein the oxidizable electrode comprises a metal selected from the group of iron, tin and zirconium.

20. A method of preparing a radioactive diagnostic agent comprising admixing a reducing agent and a salt of a poly-hydroxycarboxylic acid selected from the group consisting of glucoheptonic acid, lactobionic acid, galacturonic acid and glucuronic acid, said reducing agent being present in an amount of from about 1 to 100 micrograms per milliliter of composition, the amount of said reducing agent being from about 0.0005 to 0.5% of said salt, and combining technetium-99m with the admixture in an amount sufficient to provide radio-activity in the amount of from about 1 to 25 mCi.

21. A radioactive diagnostic composition comprising technetium-99m bound to a polyhydroxycarboxylic acid or a salt thereof, said acid being selected from the group consisting of glucoheptonic acid, lactobionic acid, galacturonic acid and glucuronic acid whenever prepared by the method of claim 1 or by an obvious chemical equivalent thereof.

22. A radioactive diagnostic composition, comprising a product of admixture of technetium-99m, a reducing agent, a salt of a polyhydroxycarboxylic acid selected from the group consisting of glucoheptonic acid, lactobionic acid, galacturonic acid and glucuronic acid, whenever prepared by the method of claim 2, or by an obvious chemical equivalent thereof.

23. A composition as defined in claim 22, wherein said reducing agent comprises a stannous reducing agent whenever prepared by the method of claim 5, or by an obvious chemical equivalent thereof.

24. A composition as defined in claim 22, wherein the reducing agent is a stannous reducing agent present in an amount of at least 0.1 microgram per milliliter of composition whenever prepared by the method of claim 6, or by an obvious chemical equivalent thereof.

25. A composition as defined in claim 22, wherein the reducing agent is a stannous reducing agent present in an amount of from about 1 to 100 micrograms per milliliter of composition whenever prepared by the method of claim 7, or by an obvious chemical equivalent thereof.

26. A composition as defined in claim 22, wherein the reducing agent is a stannous reducing agent present in an amount of from 10 to 40 micrograms per milliliter of com-position, whenever prepared by the method of claim 8, or by an obvious chemical equivalent thereof.

27. A composition as defined in claim 22, wherein the reducing agent is a stannous reducing agent present in an amount of from 0.0005% to 0.5% by weight of the polyhydroxy-carboxylic acid salt, whenever prepared by the method of claim 9, or by an obvious chemical equivalent thereof.

28. A composition as defined in claim 22, wherein the reducing agent is a stannous reducing agent present in an amount of from 0.005% to 0.1% by weight of the polyhydroxy-carboxylic acid salt, whenever prepared by the method of claim 10, or by an obvious chemical equivalent thereof.

29. A composition as defined in claim 22, wherein the polyhydroxycarboxylic acid salt is an alkali or alkaline earth metal salt of glucoheptonic acid and the reducing agent is a stannous reducing agent present in an amount of from 0.005% to 0.1% by weight of said salt, whenever prepared by the method of claim 11, or by an obvious chemical equivalent thereof.

30. A composition as defined in claim 22, further comprising a pharmacologically acceptable carrier, whenever prepared by the method of claim 12, or by an obvious chemical equivalent thereof.

31. A composition as defined in claim 22, wherein said reducing agent comprises a stannous reducing agent, and is present in an amount of at least 0.1 micrograms per milli-liter of said composition, whenever prepared by the method of claim 15, or by an obvious chemical equivalent thereof.

32. A composition as defined in claim 2 2, wherein said reducing agent is a stannous reducing agent, and is present in an amount of from about 0.0005% to 0.5% by weight of the polyhydroxycarboxylic acid, whenever prepared by the method of claim 16, or by an obvious chemical equivalent thereof.

33. A composition as defined in claim 22, wherein said reducing agent is a stannous reducing agent, and is present in an amount of from about 0.0005% to 0.5% by weight of the polyhydroxycarboxylic acid, and said polyhydroxycarboxylic acid is glucoheptonic acid, whenever prepared by the method of claim 17, or by an obvious chemical equivalent thereof.

34. A composition as defined in claim 22, wherein said reducing agent is a stannous reducing agent present in an amount of from .01 to .1% by weight of said polyhydroxy-carboxylic acid, whenever prepared by the method of claim 18, or by an obvious chemical equivalent thereof.

35. A composition as defined in claim 20, whenever prepared by the method of claim 20, or by an obvious chemical equivalent thereof.

36. A method of preparing a radioactive diagnostic agent comprising binding technetium-99m to a polyhydroxy carboxylic acid selected from the group of glucoheptonic acid, lactobionic acid, galacturonic acid and glucoronic acid or a salt thereof.

37. A method according to claim 36, wherein the poly-hydroxy carboxylic acid or salt is an alkali or alkaline earth metal salt of glucoheptonic acid.

38. A radioactive diagnostic agent whenever prepared by the method of claim 36 or 37, or by an obvious chemical equivalent thereof.