WO1996039622A1

WO1996039622A1 - Method for measuring intestinal integrity

Info

Publication number: WO1996039622A1
Application number: PCT/US1996/009467
Authority: WO
Inventors: Edward A. Deutsch; Mary Marmion Dyszlewski; Philip D. Thomson
Original assignee: Mallinckrodt Medical, Inc.
Priority date: 1995-06-05
Filing date: 1996-06-04
Publication date: 1996-12-12

Abstract

A method for measuring the intestinal integrity of an individual by orally administering a radiolabelled complex that is stable in the gastrointestinal environment is provided. The radiolabelled complex remains within the gastrointestinal tract of an individual with an uncompromised gastroinstestinal tract, but escapes from an individual with a compromised gastrointestinal tract. The individual is subsequently analyzed to determine the presence or absence of radioactivity outside of the gastrointestinal tract by either collecting a sample of blood or urine from the individual and determining the amount of radioactivity in that sample by a detecting device such as a gamma counter, or by scintigraphically evaluating the individual by imaging with a gamma camera. Preferably, a low oxidation state technetium 99m complex which is stable in the gastrointestinal tract is used.

Description

METHOD FOR MEASURING INTESTINAL INTEGRITY

Field of the Invention

This invention relates in general to a method for measuring intestinal integrity in an individual and, more particularly, to a method that utilizes a radionuclide labelled complex that is stable in the gastrointestinal environment.

Background of the Invention

It is well known that the gastrointestinal tract harbors many bacteria and endotoxins. Under normal conditions, these bacteria and endotoxins remain within the gastrointestinal tract and present no risk to systemic organs and tissues. In individuals exhibiting some clinically adverse conditions, however, intestinal bacterial escape from the gastrointestinal tract and invade extra-intestinal tissues causing systemic infections. These individuals have typically experienced a major stress event such as burns, trauma or hemorrhagic shock, are immunocompromised or malnourished, have developed multiple organ failure, or may have received therapeutic agents intended to alter the normal ecology of their gastrointestinal flora. It has been proposed that major stress events result in shunting blood away from the intestinal tract. This shunting results in intestinal ischemia which has been shown to allow gram negative bacteria or their endotoxins to enter the blood causing endotoxe ia or overt sepsis . It has been estimated that 300,000 patients per year suffer gastrointestinal tract complications, such as endotoxemia or overt sepsis, which result in approximately 100,000 deaths . It has also been suggested that cellular protein load in the circulation may also trigger the release of bacteria or endotoxins from the gastrointestinal tract. Thus, both mechanical and chemical components may contribute to bacterial translocation. Trauma of sufficient magnitude to destroy tissue may release tissue debris as well as skin flora endotoxin into the circulation. In concert with the local inflammatory response to injury, this may shunt blood away from the gut and release soluble mediators (cytokines) into the circulation which cause edema, vascular endothelial breakdown and separation. The accompanying gut ischemia would lead to the loss of gut mucosa and allow translocation. Mechanisms to detect circulating endotoxin in individuals exhibiting any of the conditions described have been well documented. A need exists, however, for a method to evaluate intestinal integrity of an individual to ascertain whether corrective action is necessary.

Summary of the Invention

The present invention is directed to a method for measuring the intestinal integrity of an individual by orally administering a radiolabelled complex that is stable in the gastrointestinal environment of the individual. The radiolabelled complex remains within the gastrointestinal tract of an individual with an uncompromised gastrointestinal tract, but escapes from an individual with a compromised gastrointestinal tract. An assay of a biological fluid of the individual, such as the blood or urine, for the presence or absence of radioactivity indicates whether the gastrointestinal tract is normal or compromised. According to the method, a diagnostically effective amount of a radiolabelled complex is orally administered to an individual and the individual is subsequently analyzed to determine the presence or absence of radioactivity outside of the gastrointestinal tract. The analysis step may be performed by either collecting a sample of blood or urine from the individual and then determining the amount of radioactivity in that sample by a detecting device such as a gamma counter, or by scintigraphically evaluating the individual by imaging with a gamma camera.

In a further embodiment of the present invention, the method comprises orally administering to an individual a diagnostically effective amount of a low oxidation state technetium 99m complex which is stable in the gastrointestinal tract and analyzing the individual to determine the presence or absence of the low oxidation state technetium 99m complex outside the gastrointestinal tract.

Among the several advantages of the present invention include the provision of a method that permits the evaluation of the intestinal integrity of an individual to determine the potential for bacterial or endotoxin translocation; a method that provides a convenient, orally administered means for determining intestinal integrity; a method that utilizes a radiolabelled complex that is stable in the gastrointestinal environment to permit a determination of intestinal integrity; and a method that utilizes a radiolabelled complex that passes into biological fluids from a compromised gastrointestinal tract while remaining in the gastrointestinal tract of an individual with an intact gastrointestinal tract. Other and further advantages of the present invention will become apparent from the following description and claims.

Detailed Description of the Preferred Embodiments It has been discovered that the intestinal integrity of an individual can be evaluated by the oral administration of a radiolabelled complex. Radiolabelled complexes suitable for use in the method of the present invention must be stable within the gastrointestinal environment and must be capable of passing through a compromised gastrointestinal tract. As used herein, the phrase "stable in the gastrointestinal environment" means that the complex is, at least, stable over a pH range from less than about 1 to about 8 or greater, stable to hydrolysis by resident gastrointestinal enzymes, and not susceptible to redox reactions; i.e. the stable complex must not decompose in vivo to produce a radioactive complex capable of passing through an uncompromised gastrointestinal tract. As used herein, the term "compromised gastrointestinal tract" means a set of clinical conditions existing within the gastrointestinal tract which permit resident bacteria or endotoxins to escape and invade extra-intestinal tissues or fluids. Preferably, the radiolabelled complex is also capable of excretion from the body. More specifically, the radiolabelled complexes will be low oxidation state technetium-99m complexes in the +1, +2, or +3 oxidation state coordinatively bonded to 6 atoms as shown in Formulas I (+1 oxidation state) and II (+2 or +3 oxidation state) below:

FORMULA I where Xi, X₂, X₃, X₄, X₅, and X₆ are coordinating groups and are the same or different and are each independently selected from π-acceptor ligands, such as monodentate and/or multidentate phosphines, phosphites, diphosphines, arsines, phosphonites, arenes, and isonitriles, and any ancillary ligands such as halogens; and

FORMULA II where X¹ _!, X^x ₂, X¹ ₃, X _{A l} X¹ ₅, X^x ₆ are coordinating groups and are the same or different and are each independently selected from monodentate and/or multidentate ligands such as phosphines, phosphites, arsines, diphosphines, phosphonites, isonitriles, isothiocyanides, thiols, thioethers, dithiocarbamates, halogens, oxygen chelates (such as acetylacetone and ethylenediaminetetraacetate) , nitrogen chelates (such as dioximeε, imines, pyridines, bipyridines, and terpyridines) , and Schiff base ligands (unsaturated N₂S₂ or N₂0₂ ligands) .

Although the majority of technetium (III) compounds are hexacoordinated complexes as in the case of technetium (I) , it should be noted that they may also be pentacoordinated or heptacoordinated complexes and thereby have one less or additional coordinating group. It is understood that the low oxidation state technetium compounds are stable in the gastrointestinal environment and are not oxidized to pertechnetate which transfers from the gastrointestinal tract to extra-intestinal fluids and tissues. Preferred technetium-99m (I) complexes include, but are not limited to, hexakis (trimethylphosphite) technetium 99m(I) , tris [ (dimethylphosphino)ethane] technetium 99m(I) ; and hexakis (2-methoxy-isobutylisocyanide) technetium 99m(I) . Suitable technetium-99m (II) or technetium- 9m (III) complexes include, but are not limited to, trans- dichloro-bis [1, 2-bis (dimethylphosphino)ethane] technetium 99m(III), trans- [N,N^'- ethylenebis (acetylacetoneiminato)bis (trimethylphosphine) ] technetium 99m(III) , trans [1,2-bis (dihydro-2, 2, 5, 5- tetramethyl-3 (2H) furanone-4-methylene- imino)ethane)bis (tris (3- methoxypropyDphosphine] technetium 99m(III) , chloro[bis (1, 2-cyclohexanedione dioximato (1-) -0(1,2- cyclohexane-dione dioximato(2-) -0) methyl borato (2-) -N^' ,

N^", N^'", N^"", N ] technetium 99m(III) , tris (acetylacetonato) technetium 99m(III) , bis (1,4, 7- trithiacyclononane) technetium 99m(II) , and trans- bis (methanethiolato)bis (1,2- bis (dimethylphosphino) ethane) technetium 99m(III) . A preferred low oxidation state technetium complex is trans [1,2-bis (dihydro-2,2,5, 5-tetramethyl-3 (2H) furanone- 4-methyleneimino)ethane)bis (tris (3- methoxypropyl)phosphine] technetium 99m(III) .

The radiolabelled complex useful in connection with the method of the present invention will preferably be provided in a kit form wherein the ligand capable of chelating a radionuclide is provided in one container and a solution containing the radionuclide is provided in another. The final radiolabelled complex is prepared by mixing the contents of the two containers prior to administration in a pharmaceutically acceptable carrier, such as saline, and heating the mixture to facilitate the labeling, if necessary. The provision of such radiolabelled complexes in kit form and the preparation of the final radiolabelled product are standard and routine in the field of nuclear medicine. The final radiopharmaceutical product should be of high radiochemical purity, preferably greater than 95%, and at least greater than 90%, as determined by standard protocols known in the art.

After the radiolabelled complex has been prepared, it is ready for oral administration to an individual . To prevent any significant absorption of the radiolabelled complex in the membranes of the oral cavity, it may be preferable to dilute the radiolabelled complex in a substance which solubilizes the radiolabelled complex prior to administration, such as milk, a soy product, enteral liquid, or the like. The radiolabelled complex is prepared to provide a radioactive dose of between about 0.5 mCi and about 40 mCi to the individual in accordance with standard radiopharmaceutical dosing determinations. As used herein, "a diagnostically effective amount" means an amount of the radiopharmaceutical sufficient to permit its detection by scintigraphic means.

After administration of the radiolabelled complex to an individual, samples of a biological fluid, such as blood or urine, are collected over a period not to exceed 24 hours, and samples will, preferably, be taken at 1-3 hours post ingestion for rapid diagnosis. The fluid samples are then evaluated by a means for detecting radioactivity in the sample to determine whether a significant amount of the radiolabelled complex has escaped the gastrointestinal tract. The individual may be evaluated by either collecting sample of blood or urine from the individual and then determining the amount of radioactivity in that sample by a detecting device such as a gamma counter, or by scintigraphically evaluating the individual by imaging with a gamma camera. If significant amounts of radioactivity are found in the extraintestinal biological fluid, it can be concluded that the individual has a compromised gastrointestinal tract and corrective action can and should be taken. Alternatively, after administration of the radiolabelled complex to an individual, the individual may be imaged by scintigraphic imaging to identify the location of the radiolabelled complex in the individual by known and routine methods such as described in Bernier, D.R. et al., eds. Nuclear Medicine Technology and Techniques, 2nd edition, 1989. The resulting images will identify whether the radiolabelled complex is within the gastrointestinal tract or whether it has escaped the gastrointestinal tract.

The following examples describe a preferred embodiment of the invention. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, taken together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims.

Example 1

This example is provided to illustrate the stability of a low oxidation state technetium 99m complex in vivo and that it does not pass through an uncompromised gastrointestinal tract.

A solution of Q12 (trans- (1,2-bis(dihydro-2,2,5,5- tetraethyl-3 (2H) furanose-4- methyleneimino)ethane)bis(tris(3- methoxypropyDphosphine) ) technetium(III) was prepared by reconstituting a lyophilized solution containing 50mg γ- cyclodextrin, 20mg Schiff base ligand, 1.5mg TMPP, 2.Omg Na ascorbate, and 1.5mg Na carbonate in a vial containing 2 ml of saline in which thirty ^*(30) mCi of additive-free, sterile, non-pyrogen sodium pertechnetate (Na99mTcO^~ ₄) had been dissolved. Proper lead shielding was used throughout and an inert atmosphere was maintained in the vial. The vial was placed upright in a boiling water bath for 15 to 30 minutes and allowed to cool to room temperature. The cooled solution was visibly inspected and found to be clear to slightly opalescent and free of particulate matter and, therefore, acceptable for use. The resulting technetium 99m Q-12 solution was assayed by HPLC and a Sep-Pak® procedure to determine radiochemical purity. The HPLC was performed on a PRP-1 column with a mobile phase consisting of 80% methanol and 20% 0.01M ammonium acetate at a flow rate of 1.0 ml/minute. The Sep-Pak® procedure was performed on a Waters Sep-Pak® Alumina A cartridge. Using a 1 ml syringe with a needle, 0.1 ml of the Tc-99m Q-12 solution was applied to the cartridge and eluted stepwise with 10 ml of 200 proof ethanol and 10 ml of 0.9% NaCl injection, USP. The ethanol eluent contains the Tc-99m Q-12 complex. The radiochemical purity was determined to be 92% by HPLC and 90.5% by the Sep-Pak® protocol. From the original preparation, 0.7 ml was dissolved in 10 ml saline. The solution was taken orally by a male volunteer. The dose received was 8.2 mCi. Blood samples were taken at 1, 3.5 and 6 hours post injection(p.i.) . Urine was collected from 0-6 hours p.i. (total volume = 750 ml) and 6-21 hours p.i. (total volume = 630 ml). Sample volumes of 1 ml were taken and counted with 1%, 0.1%, and 0.01% standards in a gamma counter (LKB 1282 Universal Gamma Counter) in order to calculate the percent dose contained in both the urine and blood samples. The results are tabulated below:

Table I Q-12 GASTROINTESTINAL STUDY RESULTS % injected dose [(counts x total volume) /standard]

Blood 1% 0.1% 0.01% AVG STD

1 hour 1.57 2.15 1.77 1.83 0.24

3.5 hours 1.51 2.07 1.70 1.76 0.23

6 hours 1.39 1.90 1.57 1.62 0.21

Urine

0-6 hours 0.87 1.19 0.98 1.01 0.13

6-21 hours 1.03 1.40 1.15 1.19 0.15

Standards n=2 6586210 482723 58553

By use of the equations below, the calculated total blood volume of the volunteer was 6.1 liters.

8.2 x [height (cm)] + 17.3 x [weight (kg) ] -693 = red blood cells

23.7 x [height (cm)] + 9 x [weight (kg) ] -1709 = plasma volume red blood cells + plasma volume = total blood volume (ml)

As shown in Table 1, approximately 2.2% of the ingested dose excreted in the urine over 0-21 hours and approximately 1.7% of the injected dose was present in the blood over 0-6 hours. Thus, the maximum of activity that "leaked" from the gastrointestinal tract over 21 hours is less than about 4% of the ingested dose which is less than the 8% of impurities (all more hydrophilic than Q-12 and more likely to decompose to pertechnetate) present in the ingested preparation. Therefore, 21 hours after ingestion greater than 96% of the dose remained within the gastrointestinal tract and scintigraphic images taken of the individual confirm this result.

Example 2 This example is provided to illustrate the in vivo fate of an orally administered dose of Q-12 (as prepared in Example 1) in conscious, normal rats.

99mTc labeled Q-12 was diluted with non-radioactive Q-12 solution reconstituted with saline and with saline to provide 10X an anticipated clinical dose with an activity of approximately 250μCi in 1ml. The purity of the radiopharmaceutical was 92.9% by HPLC analysis immediately prior to injection into the rats, [retention time = 12.9 minutes; conditions = reverse phase HPLC using a Hamilton PRP-1 column (250 x 4.1 mm, lOμ) and an acetonitrile:0.005M KH₂P0₄ linear gradient] .

Four normal rats were fasted overnight. Three conscious rats were gavaged with 1ml of the test mixture and returned to shoebox cages. The fourth rat was gavaged with 1ml of the test mixture and placed in a metabolism cage. At 2, 4, and 6 hours post dosing, a single rat was removed from the shoebox cage, anesthetized, and an image taken on the Picker Gamma Camera. The animal was then sacrificed and the organs, tissues and fluid listed in Table II were removed or collected for the determination of radioactivity. The skeletal muscle was obtained as a lg sample from the quadriceps femoris muscle, the urine was from the bladder (if available), and the blood was collected as two 0.5ml samples by cardiac puncture using a heparinized syringe. At 24 hours post dosing the final animal was anesthetized and an image obtained on the gamma camera. The animal was sacrificed and all organs plus accumulated urine and feces were taken for a determination of radioactivity. Dosing solution standards were prepared in duplicate and each sample or standard was assayed in a calibrated gamma scintillation counter (Packard Model 500C Auto- Gamma scintillation spectrometer) with windows set to detect photoelectric energies of 40-240 keV. Raw data were reported in counts per minute (CPM) . Blank tubes were assayed together prior to and immediately following the samples and then averaged together as a measure of background. Background CPM was subtracted from all standards and samples prior to calculation of the percent administered dose (%ID) .

The %ID was calculated as the product of the net CPM in the injection solution standards, dilution factor of the standards, and the dose volume administered. %ID/g and %ID/organ recovered for each rat was determined by adding the %ID values for all organs or tissues and excreta. The total %ID excreted by the 24-hour rat was determined by adding the %ID in urine and feces at that time. For the purposes of the calculation, the blood was assumed to represent 5.0% of the total body weight and the muscle 45.5% of the body weight as according to Caster, et al. (1956) Proc. Soc. Exp. Biol. Med. 91:122- 126. The results from each rat are shown in Table II below.

Table II BIODISTRIBUTION OF ORALLY- ADMINISTERED Q-12

% Administered Dose per Organ

ORGAN 2 HR 4 HR 6HR24HR

Blood . 030 . 023 . 024 . 011 Liver .091 .099 .114

.038 Kidney .023 .019 .230

.020 Spleen .001 .006 .005

.001 Muscle .103 .421 .230

.652 Heart .003 .003 .004 .002 Stomach 6.492 2.356 1.768

.150 Lung .003 .004

.001 .001 Esophagus .184 .023 .011 .001 Bladder .001 .001 .009

.001 Urine .20/ml .013/ml .017/ml

.001 Stomach Contents 1.530 7.552 5.993

3.980 Small Intestine 26.860 18.560 5.712

.047 Small Intestine Contents 48.567 29.050 21.080 .586 Cecum .064 1.651 10.506

.019 Cecum Contents.014 24.006 30.730

2.918 Large Intestine .017 .076

.064 .015

Large Intestine Contents .001 .088 None 2.300 Feces —97.4

The results in Table II illustrate that orally administered Q-12 to a rat having an uncompromised gastrointestinal tract remains within the gastrointestinal tract and only trace amounts of radioactivity are found in the blood or urine.

Claims

What is claimed is:

1. A method for measuring gastrointestinal tract integrity in an individual comprising:

administering orally to an individual a diagnostically effective amount of a radiolabelled complex stable in the gastrointestinal tract; and analyzing the individual to determine the presence or absence of the radiolabelled complex outside of the gastrointestinal tract.

2. The method of claim 1 wherein the analyzing step is performed by collecting a sample of blood or urine from the individual and assaying the blood or urine for the presence or absence of the radiolabelled complex.

3. The method of claim 2 wherein the assay comprises evaluating the collected blood or urine for the presence or absence of radioactivity.

4. The method of claim 1 wherein the analyzing step is performed by scintigraphic imaging of the individual to locate radioactivity both within and without the gastrointestinal tract.

5. The method of claim 1 wherein the radiolabelled complex is stable in an environment having a pH less than about 5.5.

6. The method of claim 1 wherein the radiolabelled complex is resistant to decomposition by gastrointestinal enzymes.

7. A method for measuring gastrointestinal tract integrity in an individual comprising:

administering orally to an individual a diagnostically effective amount of a low oxidation state technetium 99m complex stable in the gastrointestinal tract; and analyzing the individual to determine the presence or absence of the low oxidation state technetium 99m complex outside of the gastrointestinal tract.

8. The method of claim 7 wherein the analyzing step is performed by collecting a sample of blood or urine from the individual and assaying the blood or urine for the presence or absence of the low oxidation state technetium 99m complex.

9. The method of claim 8 wherein the assay comprises evaluating the collected blood or urine for the presence or absence of radioactivity.

10. The method of claim 7 wherein the analyzing step is performed by scintigraphic imaging of the individual to locate radioactivity both within and without the gastrointestinal tract.

11. The method of claim 7 wherein the low oxidation state technetium 99m complex is stable in an environment having a pH less than about 5.5.

12. The method of claim 7 wherein the low oxidation state technetium 99m complex is resistant to decomposition by gastrointestinal enzymes.

13. The method of claim 7 wherein the low oxidation state technetium 99m complex is selected from the group consisting of stable technetium 99m(I) complexes, stable technetium 99m(II) complexes and stable technetium 99m(III) complexes.

14. The method of claim 13 wherein the stable technetium 99m(I) complexes have the formula:

Xi

Xe. .X₂

Tc⁺¹ ₅" \ ^ ₃ χ₄

where X_lf X₂, X₃, X₄, X₅, and X₆ are coordinating groups and are the same or different and are each independently selected from π-acceptor ligands, such as monodentate and/or multidentate phosphines, phosphites, diphosphines, arsines, phosphonites, arenes, and isonitriles, and any ancillary ligands such as halogens and Tc is in its +1 oxidation state.

15. The method of claim 14 wherein the stable technetium 99m(I) complexes are selected from the group consisting of hexakis (trimethylphosphite) technetium

99m(I) , tris [ (dimethylphosphino)ethane] technetium 99m(I) ; and hexakis (2-methoxy-isobutylisocyanide) technetium 99m(I) .

16. The method of claim 13 wherein the stable technetium 99m(II) or stable technetium 99 (III) complexes have the formula:

where X¹!, X^x _2/ X¹ ₃, x X¹ ₅, X^x ₆ are coordinating groups and are the same or different and are each independently selected from monodentate and/or multidentate ligands such as phosphines, phosphites, arsines, diphosphines, phosphonites, isonitriles, isothiocyanides, thiols, thioethers, dithiocarbamates, halogens, oxygen chelates, nitrogen chelates, and Schiff base ligands and Tc is in its +2 or +3 oxidation state.

17. The method of claim 16 wherein the stable technetium 99m(II) and stable technetium 99 (III) complexes are selected from the group consisting of trans-dichloro-bis[1,2- bis(dimethylphosphino)ethane]technetium 99m(III) , trans- [N,N^'- ethylenebis(acetylacetoneiminato)bis(trimethylphosphine) ] technetium 99m(III) , trans[1,2-bis(dihydro-2,2,5,5- tetramethyl-3 (2H) furanone-4-methylene- imino)ethane)bis(tris(3- methoxypropyl)phosphine]technetium 99m(III) , chlorofbis(1,2-cyclohexanedione dioximato(1-)-0(1,2- cyclohexane-dione dioximato(2-)-0) methyl borato(2-)-N^', N^", N^'", N^"", N ] technetium 99m(III) , tris(acetylacetonato)technetium 99m(III) , bis(l,4,7- trithiacyclononane)technetium 99m(II) , and trans- bis(methanethiolato)bis(1,2- bis(dimethylphosphino)ethane)technetium 99m(III) .

18. The method of claim 17 wherein the stable technetium 99m(III) complex is trans[1,2-bis(dihydro- 2,2,5,5-tetramethyl-3 (2H)furanone-4-methylene- i ino)ethane)bis(tris(3- methoxypropyDphosphine]technetium 99m(III) .