WO1996000085A1

WO1996000085A1 - Difference imaging method for the identification of multidrug resistant tumor cells

Info

Publication number: WO1996000085A1
Application number: PCT/US1995/008089
Authority: WO
Inventors: Mary Marmion Dyszlewski; Bart J. Doedens; B. Daniel Burleigh
Original assignee: Mallinckrodt Medical, Inc.
Priority date: 1994-06-27
Filing date: 1995-06-27
Publication date: 1996-01-04

Abstract

A method for identifying tumor cells exhibiting the Multidrug Resistance (MDR) characteristic utilizing a pair of radiolabeled compounds to form a difference image of the cells is provided. One of the radiolabeled compounds is a substrate for P-glycoprotein and the other is not. The radiolabeled compounds may be labeled with the same or different radioisotope, and the compounds may be introduced or administered sequentially or simultaneously to obtain the difference image in one or two imaging sessions, respectively.

Description

DIFFERENCE IMAGING METHOD FOR THE IDENTIFICATION OF MULTIDRUG RESISTANT TUMOR CELLS

Background of the Invention

1. Field of the Invention

This invention relates to a method for imaging tumor cells to identify cells exhibiting the multidrug resistance (MDR) characteristic, and more particularly to a method that utilizes a pair of radiolabeled imaging agents, only one of which accumulates sufficiently in cells exhibiting the MDR characteristic and thereby capable of producing a radioimage of the cell in response to scintigraphic imaging, such that cells exhibiting the MDR characteristic are identified by a difference imaging method.

2. Description of Background Art

The phenomenon of broad spectrum resistance to a range of chemotherapeutic drugs in the treatment of human cancer has been called multidrug resistance (MDR). Patients exhibiting this phenomenon can be classified in two general groups: those that do not respond to chemotherapy, and those that respond initially to chemotherapy but later have cancerous tumors recur that do not respond to chemotherapy. In the latter group, the patient is often diagnosed as having overcome the cancer only later to suffer a relapse or develop another cancer. For either group of patients the treating physician does not know before prescribing chemotherapy whether the patients' cancer exhibits the MDR characteristic and, therefore, whether chemotherapy will be an effective treatment. Moreover, if the cancer has metastasized or if secondary cancerous foci have developed in the patient which cannot yet be detected by existing metastasized or if secondary cancerous foci have developed in the patient which cannot yet be detected by existing diagnostic methods, and these metastases or foci exhibit the MDR characteristic (MDR positive cells), no diagnostic method exists to identify their existence prior to surgery or chemotherapy. Because of the considerable adverse side effects associated with chemotherapy, and the fact that many chemotherapeutic agents are inherently toxic, it would be desirable to be able to identify patients whose tumors exhibit the MDR characteristic prior to prescribing chemotherapy. Likewise, it would be desirable to be able to identify specific metastases and /or secondary foci that are MDR positive in patients so that the MDR positive metastases or foci can be removed at the same time as the primary tumor or delineated and specifically removed by surgery and/or focused radiotherapy.

Considerable research on the multidrug resistance phenomenon has been conducted and definitive evidence has been obtained that cells exhibit the MDR characteristic due to increased expression of a single protein identified as P-glycoprotein, which is also referred to as PGP or PI 70. This type of drug resistance is referred to as P-glycoprotein mediated multidrug resistance, or MDR. Endicott, J.A. & Ling, V. (1989) "The Biochemistry of P-Glycoprotein- Mediated Multidrug Resistance" Annu. Rev. Biochem. 58:137-71. It has been shown that P-glycoprotein acts by increasing efflux of drugs from the cell through the cell membrane. Although the mechanism of action of P- glycoprotein mediated multidrug resistance has not been completely elucidated, it is known that P-glycoprotein functions as an energy dependent, ATP-driven pump to remove substances within its substrate range from the cell. It is believed that P-glycoprotein binds drugs as they enter the plasma membrane from the intracellular, cytosolic side and transports them outside the cell. Furthermore, it is believed that this transport occurs through a single channel transporter mechanism. Gottesman, M.M. & Pastan, I. (1993) "Biochemistry of Multidrug Resistance Mediated By The Multidrug Transporter" Annu. Rev. Biochem. 62:385-427.

One approach to diagnosing MDR positive cells by an imaging method has been described in European Patent Application No. WO93/00064. Briefly, this method involves making a primary image of a tumor after systemic administration of an imaging agent that is a substrate for P-glycoprotein, followed by making a second image after co-administration of an imaging agent with an inhibitor of P-glycoprotein activity, such as Verapamil or Cyclosporin A. MDR positive cells will be weakly, if at all, imaged in the first imaging session, and more strongly imaged in the second imaging session. The difference between the two images is asserted to form an image of MDR positive cells. A potential impediment to the practice of this proposed method may be the toxiάty of the inhibitor compounds, or reversal agents, at the high systemic doses which would be needed for their effectiveness. It is known that the toxicity of some of the proposed reversal agents, e.g. Cyclosporin A and Verapamil, is significant when provided at systemic doses and that a significant quantity of the agent must be administered to block at least a substantial percentage of the P-glycoproteins to produce a worthwhile image. The side effects of some of the proposed reversal agents are also known to be problematic and this may limit the amount of the dose that could be used.

A need exists, therefore, for a diagnostic method for identifying, MDR positive cells in an individual that overcomes the foregoing problems.

Summary of the Invention

The present invention is directed to a method for identifying cells exhibiting the multidrug resistance characteristic by a difference imaging method. According to the method, a pair of radiolabeled compounds are introduced in trace amounts into an individual, one of the radiolabeled compounds being substantially transported out of cells exhibiting the MDR characteristic such that a radioimage from this radiolabeled compound is not obtained after scintigraphic imaging of MDR positive cells, and the other of the radiolabeled compounds being capable of accumulating in cells exhibiting the MDR characteristic such that a radioimage can be obtained after scintigraphic imaging of these cells or tumor masses. The radioimages are digitally or otherwise compared and a difference image obtained which identifies tumor masses or cells that generate an enhanced image only in response to the radiolabeled compound that accumulates in the cell. These are cells that exhibit the MDR characteristic. Cells that produce an image in response to both radiolabeled compounds do not exhibit the MDR characteristic.

Therefore, in one significant aspect of the invention, a method for identifying cells which exhibit the multidrug resistance (MDR) characteristic is provided which involves the steps of introducing a first radiolabeled compound into an individual wherein the first .radiolabeled compound is a substrate for P-glycoprotein and is substantially removed from cells exhibiting the MDR characteristic such that the first radiolabeled compound does not accumulate in cells exhibiting the MDR characteristic; introducing a second radiolabeled compound into an individual wherein the second radiolabeled compound accumulates in cells exhibiting the MDR characteristic; performing scintigraphic imaging on the individual to obtain a radioimage of both radiolabeled compounds in the individual; and producing a difference image identifying cells which produce an enhanced image in response to the second radiolabeled compound and not the first radiolabeled compound.

In another significant aspect of the present invention, a method for determining the presence or absence of cells exhibiting the multidrug resistance (MDR) characteristic in an individual in a single imaging session is provided which comprises introducing a first radiolabeled compound into an individual, the first radiolabeled compound being labelled with a first gamma emitting radioisotope and the compound being a substrate for P-glycoprotein such that it is substantially exported out of cells exhibiting the MDR characteristic and does not accumulate therein; introducing a second radiolabeled compotmd into the individual, the second radiolabeled compound being labelled with a second radioisotope having gamma emissions at a different energy than the gamma emissions of the first radioisotope and wherein the second radiolabeled compound accumulates in cells exhibiting the MDR characteristic; performing scintigraphic imaging on the individual and simultaneously obtaining the radioimages from both radioisotopes; and separating the gamma emissions from each radioisotope to form a difference image identifying cells which produce an enhanced image only in response to the second radiolabeled compotmd. m a further embodiment of the present invention, the radiolabeled compound that is not the substrate for P-glycoprotein in the pair of . radiolabeled compounds is a lipophilic cationic compound containing a labile sidechain, the compound is capable of diffusing into a cell but having the labile sidechain cleaved by the intracellular or membranous environment such that an acidic functional group is formed resulting in a lipophilic polyanion having a negative net charge which will remain within the cell because of its negative net charge.

Among the several advantages found to be achieved by the present invention include the provision of a method for identifying cells exhibiting the MDR characteristic by a difference imaging approach using two different radiolabeled compounds, only one of which accumulates in MDR positive cells; the provision of a sensitive and selective method for identifying tumor cells exhibiting the MDR characteristic in an individual that avoids the need to introduce an inhibitor of P-glycoprotein into the individual at toxic, systemic dose levels; the provision of a method that is capable of identifying metastases and /or secondary foci exhibiting the MDR characteristic in an individual that would not be identified by existing imaging techniques; the provision of a method that utilizes a pair of radiolabeled compounds that are both used in trace amounts and that are rapidly cleared from the body; and the provision of a method for identifying cells exhibiting the MDR characteristic using a pair of radiolabeled compounds utilizing radioisotopes having different gamma emission energies which permits the identification of MDR positive cells by difference imaging in a single scintigraphic imaging session.

Detailed Description of the Preferred Embodiments

In accordance with the present invention, it has been discovered that the use of a pair of radiolabeled compounds, one of which is a substrate for P- glycoprotein and does not accumulate in MDR positive cells and the other of which is a radiolabeled compound that does accumulate in MDR positive cells, provides a means for identifying MDR positive cells when both radiolabeled compounds are introduced into an individual and the difference image based on the individual images produced by each of the radiolabeled compounds is obtained. The difference image so obtained identifies those cells or tumor masses that are MDR positive. The method is performed by introducing the two radiolabeled compounds into the individual either sequentially or simultaneously depending upon whether the same radioisotope is used in the radiolabeled compounds. When radioisotopes having different gamma emission energies are used in the method, the compounds can be introduced simultaneously and digital scintigraphic imaging cameras used to capture the biodistributions of both isotopes. Thus, the difference image can be obtained in a single imaging session. When the same radioisotope is used with the radiolabeled compound that is a substrate for P-glycoprotein and the radiolabeled compotmd that is not a substrate for P-glycoprotein, the compounds are introduced sequentially and an image obtained after each administration. These images are then digitally or otherwise compared to produce an enhanced image identifying those cells or tumors that are MDR positive.

The radiolabeled compounds useful in the methods of this invention are compounds that can readily enter the cell by diffusion through the cell membrane. Preferably, the compounds are lipophilic cationic compounds. One of the radiolabeled compounds, however, does not accumulate in cells that are MDR positive because it is a substrate for P-glycoprotein. A compound is a "substrate ior P-glycoprotein" if it is actively transported out of a MDR positive cell such that it does not substantially accumulate in the cell. The compounds are radiolabeled with known gamma emitting radioisotopes useful in diagnostic imaging such as ^WmTc, ¹⁸F, ²⁰¹T1 and ^mIn. Compounds that are good substrates for P-glycoprotein preferably have an octonal: buffer ratio between about 5 and about 30 and have 60 minute heart/liver clearance values of greater than 3 in the guinea pig. Compounds that are good substrates for P-glycoprotein include, but are not limited to, "^mTc-sestamibi, ^WmTc- tetrofosmin, 99mTc-Q12 (trans-(l,2-bis(dihydro-2,2^^-tetramethyl- 3(2H)furanone-4-memyleneiιιιino)ethane)bis(tris(3-methoxy-l- propyljphosphinejtechnetium^j^θm), "^mTc-Q51 (trans-[5^'- (l,2,ethanediyldiimino)bis(2-ethoxy-2-methyl-4-penten-3- one)]bis(tiimethylphosphine)technetiιιm(iπ)), trans-(l,2-bis(dihydro-2,2,5,5- tetiame yl-3(2H)furanone-4-memyleneinr no)ethane)bis(tiis(3,3-dimethyl-3- methoxy-l-propyl)phosphine)technetium(πi)-99m, hereinafter referred to as To-QiS, and "^mTc-Q3 (trans-[N,N'- ethylenebis(acetylacetoneiminato)]bis(tris(3- memoxypropyl)phosphine)technetium(iπ)). The synthesis of these compounds is known to those skilled in the art. The preparation of "^mTc-Q12 is described in U.S. Patent No. 5,112,595, the entirety of which is incorporated herein by reference hereto. The general structure of ^99mTc-Q12 is shown below:

The general structure of "^mTc-Q3 is shown below:

The general structure of "^mTC Q-51 is shown below:

Preferably, "^mTc Q-51 or "^mTc Q-3 is used as the radiolabeled compound that does not accumulate in MDR positive cells.

The second member of the pair of radiolabeled compounds is a compound that is not a substrate for P-glycoprotein and therefore accumulates in cells that are MDR positive such that an image of the cell can be produced from the radiolabeled compotmd. This compotmd is also radiolabeled by standard labelling procedures known to those skilled in the art with radioisotopes useful in diagnostic imaging such as "^mTc, ¹⁸F, ²⁰¹T1 and ^mIn. In general, compounds that are not substrates for P-glycoprotein are highly hydrophobic compounds that are sparingly soluble in water or compounds having structural features that sterically inhibit its transport through the single channel transporter of the P-glycoprotein mechanism. In addition, Tl-201 and F-18FDG are examples of agents that are not substrates for P-glycoprotein. Furthermore, various of the "Q" complexes as described herein are examples of compounds that are not substrates for P-glycoprotein and for these compounds it is preferred that at least one of the radionuclide coordinating atoms be a sulfur atom, preferably two or more sulfur atoms as coordinating atoms. Preferred chemical groups that sterically hinder the transport of this radiolabeled compotmd include bulky, rigid, lipophilic groups. Suitable compounds for use as the radiolabeled compotmd that is not a substrate for MDR positive cells and therefore accumulates in such cells includes, but is not limited to, "^mTc-Q17 (tians-{l,2-bis[dihydro-2,2,5,5-tetiamethyl-3(2H)- furanthione-4-methylene-imino] ethane}bis(tris(3-methoxypropyl)phosphine)technetium(πi)), the general structure of which is shown below:

^luIn sal₃TAME(saHcylaldiminomethylethane), an ^mIn DTPA complex which includes a covalently bonded lipophilic group, ²⁰¹T1, and ¹⁸F- FDG(fluorodeoxyglucose). Preferably, the radiolabeled compound that accumulates in MDR positive cells is "^mTc-Q17. Thus, a pair of compounds can be selected, one which is a good substrate for p-glycoprotein and a second which is not a good substrate for p-glycoprotein by identifying a pair of compounds that have similar uptakes in drug-sensitive tumors or foci, but have substantially different exclusion properties from drug-resistant tumors or foci so that a difference image may be formed.

In a further preferred embodiment of the invention, the radiolabeled compound that is not a substrate for P-glycoprotein is a "^mTc or In labelled compound that is unstable in the intracellular environment such that it degrades upon entry into the cell or is altered by the properties of the intracellular environment and the released radionuclide remains in the cell to permit a radioimage to be formed of the cell. This member of the difference imaging substrate pair is a lipophilic cation having the above properties which contains, as a part of its ligand structure, labile groups, e.g. esters, whose lysis is activated by properties of the intracellular environment, e.g. pH decrease or esterase activity. These groups can be specifically designed to have the desired reactivity rates by specific neighboring groups. Upon entering the cell by diffusion through the plasma membrane, these labile groups react, exposing acidic functional groups, e.g. carboxylic or sulfonic acids, which convert the complex from a lipophilic cation having a positive net charge to a lipophilic polyanion having a negative net charge. This complex remains within the cell, because it will not be a substrate for p-glycoprotein and will not diffuse through the membrane, being repelled by the negative charge of the phospholipid inner surface of the bilayer. The radiolabeled compound may include an ^mIn DTPA complex which includes a covalently bonded lipophilic group, which is known to degrade intracellularly, or any of the compounds shown below:

where for the ester, R is an appropriate leaving group having the desired reactivity such as methyl, ethyl, t-butyl, and Rj-Rg is selected from hydrogen, methyl, or an ether of the formula COR, and where for the amide, R is hydrogen or methyl, and Rr- β is selected from hydrogen, methyl, or an ether of the formula COR. It being understood that these groups are representative examples and are not intended to be limiting as it is well known in the art that various other groups may be introduced without affecting the reactive properties of the compounds described.

One method for identifying radiolabeled compounds that are useful in the method of this invention, either as a substrate for P-glycoprotein or as a non-substrate for P-glycoprotein, involves deteixniriing the differential selective retention of the radiolabeled compounds by colchicine-sensitive (parent) and colchicine-resistant mutant strains of Chinese Hamster Ovary (CHO) cells in culture. The following general protocol would be followed: Chinese Hamster Ovary (CHO) cells are obtained as parent strain stock. These cells are established by suspension culture at 37°C in minimal medium (e.g., Eagle's MEM, 10%(v/v) fetal bovine serum, supplemented with antibiotics). The general procedure for these suspension cultures is the use of spinner flasks and methods in general use (e.g., R.L.P. Adams, Cell Culture for Biochemists. Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 8, Elsevier, 1990, pp. 47-50). The CHO cells are established by these means as the parent, colchicine-sensitive (CHO-S) strain. This CHO-S strain is maintained by suspension culture in minimal medium. A Chinese Hamster Ovary, high colchicine resistant strain (CHO-R) is selected by stepwise, 3-step, serial suspension culture of these parent cells in the minimal medium with addition of increasing concentrations of colchicine (e.g., 0J0 μM, 0.50 μM, and 1.0 μM). This CHO-R strain is maintained by suspension culture in minimal medium plus 1.0 μM colchicine.

Small volume (e.g., 10 ml) suspension cultures of both CHO-S and CHO-R cells, established as above, are subsequently incubated with the addition of doses of the selected radiolabeled compounds having a known specific activity. At different time points, aliquots of the incubation suspension are removed and the cells pelleted by centrifugation. The cell pellets are counted in a gamma counter and uptake /retention of the radiolabeled compound is calculated, and typically expressed as fmole/cell or fmole/mg cellular protein. Differences in uptake/retention for different radiolabeled compounds in CHO-S and CHO-R cells are used to select appropriate pairs of radiolabeled compounds for use in the practice of MDR difference imaging in accordance with the method of this invention. In the practice of the claimed invention, the pair of radiolabeled compounds are prepared as injectable doses and injected, either by systemic i.v. injection or by close arterial infusion, and scintigraphic imaging is performed after an appropriate period of uptake as is well known in the art. The dose amount is an amount of each of the radiolabeled compounds that can be effectively imaged. Standard determinations for optimal dosage amounts are routine to those skilled in the art. Typically, "" c radiolabeled compounds are introduced as dose levels in a range of about lOmCi to about 40mCi, ^U1ln radiolabeled compounds are introduced at dose levels in a range of about 3mCi to about 6mCi, and ²⁰¹T1 radiolabeled compounds are introduced at dose levels in a range of about 2mCi to about 4mCi. If the radiolabeled compounds are being introduced sequentially, the compound that is a substrate for P- glycoprotein is preferably introduced first and a scintigraphic image made. After an appropriate period of time, the radiolabeled compound that is not a substrate for P-glycoprotein is introduced and the scintigraphic imaging process repeated. The two images so obtained can then be digitally balanced, enhanced, and subtracted using standard scintigraphic imaging apparatus to obtain a difference image identifying cells that are MDR positive. When the radiolabeled compounds are introduced simultaneously, a single imaging session utilizing a digital scintigraphic camera capable of capturing the biodistributions of both isotopes provides the difference image identifying MDR positive cells.

The method of the present invention may be used for detection or imaging of internal organ tumors, such as kidney, pancreatic acinar, colorectal, and other digestive tract tumors as well as liver hepatomas, or any other tumors, foci within tumors, or metastatic sites that are, or include, MDR positive cells.

The following examples are offered to further illustrate the present invention. These examples are intended to be purely exemplary and should not be viewed as a limitation on any claimed embodiment. Example 1

"^mTc-Q45 was prepared based on the standard two-step procedure as described in U.S. Patent No. 5,112,595. In the first step, "^mTc0₄- from a commercial generator (20, 0.74GBq, 1ml) was added to a vial containing 15mg of 1,2-bis (dihydro-2,2^^-tetiamethyl-3(2H)ftu:anone-4-methylenamino)ethane in OJml of ethanol. The solution was deaerated for 15 minutes with a vigorous stream of argon; 15μg of SnCl₂ (in degassed ethanol) and 0.03ml of 1M NaOH were then added. The preparation was subsequently incubated for 5 minutes at 100°C to yield the "^mTc(V) intermediate. Radiochemical purity was determined by reverse phase HPLC to be >95% (PRP-1 column: 150 X 4J mm, lOμ; 45% acetonitrile/OJM NH₄OAc, 2.0ml/min, t,= 3.0 minutes). In the second step, 10 mg of a 3,3-dimethyl-3-methoxy-l-propyl)- phosphine (lg in 10 ml ethanol) was added to the ^{9 m}Tc(V) preparation and the solution heated for 15 minutes at 100°C to yield the desired "^∞Tc-Q/iS complex. Separation of the radiolabeled complex from reagents was performed by diluting the preparation to 20ml with water, loading it onto a pre-wet C₁₈ Sep- Pak and washing the loaded Sep-Pak with water (20ml) and ethanolcwater (80:20, 4ml); the product was then eluted in about 70% yield with 2ml of 80:20 ethanoksaline. Quality control effected by reverse phase HPLC as described above (t,. = 24 minutes) showed > 95% radiochemical purity.

Example 2

"Tc Q-3 was prepared by a two-step procedure as follows: In the first step, ""TcO^ (20-200 mCi, 0.74-7.4 GBq, 1 mL) was added to a vial containing 15 mg of N,N'-ethylene(acetylacetoneimine) (acac₂en) in 0J mL of ethanol. The solution was deaerated for 15 minutes with a vigorous stream of argon; 15 μg of SnCl₂ (in degassed ethanol) and 0.03 mL of 1 M NaOH were then added to this solution of Schiff base and 99m-pertechnetate. The preparation was then incubated for 7 minutes at 75° C to yield the [^99mTc(V)(0)(acac₂en)]⁺ intermediate.

In the second step, OJ mL of a TMPP -HCl solution (1 g TMPP -HCl in 10 mL ethanol) was added to the [^99mTC(V)(0)(acac₂en)]⁺ preparation and the solution heated for 8 minutes at 75° C to yield the desired "^∞Tc Q-3 complex. Separation of the radiolabeled complex from reagents was performed by reverse phase HPLC (PRP-1 column: 250 X 4J mm, lOμ; linear gradient containing acetonitrile/0.005M KH₂P0₄, 2.0 mL/minutes, t,. = 14.7 minutes). Quality control effected by reverse phase HPLC (same conditions as above, showed >95% radiochemical purity.

Example 3

"^∞Tc Q-51 was prepared by a two-step procedure as follows:

In the first step, "^mTc0₄- (20-200 mCi, 0.74-7.4 GBq, 1 mL) was added to a vial containing 15 mg of 5^'-(l,2-^thanediyldiimino)bis(2-ethoxy-2-methyl-4- penten-3-one) in 0J mL of ethanol. The solution was deaerated for 15 minutes with a vigorous stream of argon; 15 μg of SnCl₂ (in degassed ethanol) and 0.03 mL of 1 M NaOH were then added to this solution of Schiff base and 99m- pertechnetate. The preparation was then incubated for 7 minutes at 75° C to yield the "^mTc(V) intermediate.

In the second step, 0J mL of a PMe₃ -HCl solution (1 g in 10 mL ethanol) was added to the preparation and the solution heated for 8 minutes at 75° C to yield the desired "^mTc Q-51 complex. Separation of the radiolabeled complex from reagents was performed by reverse phase HPLC (PRP-1 column: 250 X 4J mm, lOμ; linear gradient containing acetonitrile/0.005M KH-JPO^ 2.0 mL/minutes, t₄ = 13.4 minutes). Quality control effected by reverse phase HPLC (same conditions as above, showed >95% radiochemical purity. Example 4

^WmTc Q-17 was prepared by a two-step procedure as follows: In the first step, "^mTcO₄- (20-200 mCi, 0.74-7.4 GBq, 1 mL) was added to a vial containing 2 mg of l,2-bis[dihydro-2,2_/5^,-tetramethyl-3(2H)-furanthione- 4-methylene-imino]ethane in 0J mL of ethanol. The solution was dearated for 15 minutes with a vigorous stream of argon; 15 μg of SnCl₂ (in degassed ethanol) and 0.03 mL of 1 M NaOH were then added to this solution of Schiff base and 99m-pertechnetate. The preparation was then incubated for 7 minutes at 75° C to yield the Tc(V) intermediate. In the second step, 0J mL of a TMPP -HCl solution (lg TMPP.HC1 in 10 mL ethanol) was added to the Tc(V) preparation and the solution heated for 8 minutes at 75° C to yield the desired "^mTc Q-17 complex. ^" Separation of the radiolabeled complex from reagents was performed by reverse phase HPLC (PRP-1 column: 250 X 4J mm, lOμ; linear gradient containing acetonitrile/0.005M KE O^ 2.0 mL/minutes, t. =. 17.9 minutes). Quality control effected by reverse phase HPLC (same renditions as above, showed >95% radiochemical purity.

Example 5 This example illustrates the in vitro differential effects of various compounds regarding their p-glycoprotein substrate affinity and provides a means for selecting a pair of compounds for use in the difference imaging method of the invention.

Human renal carcinoma cell lines KB-3-1 (drug sensitive) or KB-8-5 (drug resistant, expressing p-glycoprotein) which serve as models for drug sensitive and resistant tumors were grown to confluence at 37° C on prepared coverslips in DMEM medium (Dulbecco's Modified Eagle's medium, GIBCO) supplemented with L-glutamine (1%, v/v), penicillin/ streptomycin (0.1%, v/v), and fetal bovine serum (10%, v/v).

The coverslips with confluent cells were removed from the medium, rinsed in buffer, and pre-equilibrated 60 seconds in control buffer (Modified Earle's Balanced Salt Solution (MEBSS, GIBCO): 145 mM Na⁺, 5.4 mM K⁺, 1.2 mM Ca²⁺, 0.8 mM Mg²*, 152 mM Cl^", 0.8 mM H₂P0₄ ^", 0.8 mM S0₄ ^2", 5.6 mM - dextrose, 4.0 mM HEPES, plus 1% (v/v) bovine calf serum, pH 7.4 +/- 0.05. Following pre-equilibration, the coverslips with cells (0.3mg cellular protein) were immersed in buffer at 37° C containing 0J - 0.6 nM of the "Tc-complex (5-9 pmole/mCi; 5-20 uCi/ml) to be tested for 30 minutes, rinsed 3 times with ice cold buffer, placed in 35 mm plastic Petri dishes, and counted in a well- type sodium iodide gamma counter. Cell protein was determined on control coverslips with confluent cells by extraction into 10 mM Na borate, 1% (v/v) sodium dodecylsulfate and assay by the method of Lowry versus a standard of bovine serum albumin. Data were calculated and reported as fmoles "" c- complex retained, per nM concentration in the incubation solution, per mg cell protein (fmole/nM-mg). For example, in the experiment to determine whether Q3 was or was not a substrate for p-glycoprotein, the cellular protein amount was determined to be ).3mg, the amount of Q3 was 0.5nM and the S.A. was 0J43mCi/pmole. From this the values as shown in Table 1 were calculated The results for the various compounds tested are shown in Table 1 below.

Table 1

KB-8-5

30 min. uptake

(fmole/nM-mg)

5.9

46.4

5.7

2.2

184.0

71.0

2.9

64.0

370.0

223.0 5.8 2.3

7.3

It can be seen that complexes Q3 and Q17 form a suitable pair of compounds for use in the method of this invention because of their similar uptake values in drug-sensitive cells and the difference in their exclusion from drug resistant cells. Q51 also demonstrates significant exclusion from drug sensitive cells.

*Q5: tians-[N,N'-ethylenebis(acetylacetoneiminato)[bis(tris({2-2[-l(2 - dioxyanyl)]}ethyl)phosphine)technetium(πi); Qll: trar^-[2,2'-(l,2-ethanediyldiimino)bis(l^-dimethoxy-5-methyl-2-hexen- - one)]bis(tris(3-methoxypropyl)phosphine)technetium(πi);

Q15: trans-[N,N'-ethylenebis(acetylacetoneiminato)]bis(tris(3-methoxy-3- methylbutyl)phosphine)technetium(πi);

Q19: trans-{l-[dihydro-2,2^^_/-tetramethyl-3(2H)-furanthione-4-methylene- imino]-2-[dihydro-2,2^,-tetramethyl-3(2H)-furanone-4-methylene- immoemane}bis(tris(3-memoxypropyl)phosphine)technetium(iπ);

Q25: trans-[l,2-bis(dihydro-2,2^^-tetramethyl-3(2H)-furanone-4- memyleneimino)ethane]bis(tris{2-2[-(l^-dioxanyl)]}ethyl) phosphine)technetium(iπ);

Q40: trans-[l,2-bis(dihydro-2,2^^-tetramethyl-3(2H)-ftιranone-4- memyleneimino)ethane]bis(triethylphosphine)technetitιm(πi);

Q41: trans-[l,2-bis(dihydro-2,2,4,4-tetramethyl-3(2H)-cyclohexene- -methylene- immo)ethane]bis(tris(3-me oxypropyl)phosphine)te metium(IlI).

Claims

What is claimed is:

1. A method for identifying cells which exhibit the multidrug resistance (MDR) characteristic, the method comprising the steps of:

introducing a first radiolabeled compound into an individual wherein the first radiolabeled compound is a substrate for P-glycoprotein and is substantially removed from cells exhibiting the MDR characteristic such that the first radiolabeled compound does not accumulate in cells exhibiting the MDR characteristic;

introducing -a second radiolabeled compound into an individual wherein the second radiolabeled compotmd accumulates in cells exhibiting the MDR characteristic;

performing scintigraphic imaging to obtain separate radioimages from each radiolabeled compound; and

identifying cells which produce an image in response to the second radiolabeled compound and not the first radiolabeled compotmd.

2. The method of claim 1 wherein the second radiolabeled compound is not a substrate for P-glycoprotein.

3. The method of claim 2 wherein the first and second radiolabeled compounds are lipophilic cationic compounds.

4. The method of claim 3 wherein the second radiolabeled compound is trapped intracellularly or in the membrane of the cells.

5. The method of claim 4 wherein the second radiolabeled compound is an ln labelled cationic compound.

6. The method of claim 2 wherein the second radiolabeled compound is selected from the group consisting of an ^mln DTPA complex having a lipophilic group covalently bonded thereto, ln sal₃TAME, ²⁰¹T1, ¹⁸F-

7. The method of claim 6 wherein the second radiolabeled compotmd is ^99mTc-Q17.

8. The method of claim 3 wherein the first radiolabeled compotmd is selected from the group consisting of "^mTc-sestamibi, "^mTc-Q3, ^Tc-Qδl and ^99mTc-Q12.

9. The method of claim 8 wherein the first radiolabeled compound is "^mTc-Q51.

10. The method of claim 3 wherein the first and second radiolabeled compounds are radiolabeled with different diagnostic isotopes.

11. The method of claim 10 wherein at least one of the first and second radiolabeled compounds is radiolabeled with "^mTc.

12. A method for determining the presence or absence of cells exhibiting the multidrug resistance (MDR) characteristic in an individual in a single imaging session, the method comprising the steps of:

introducing a first radiolabeled compound into the individual, the first radiolabeled compotmd being labelled with a first gamma emitting radioisotope and the compotmd being a substrate for P-glycoprotein such that it is substantially exported out of cells exhibiting the MDR characteristic and does not accumulate therein;

introducing a second radiolabeled compound into the individual, the second radiolabeled compound being labelled with a second radioisotope having gamma emissions at a different energy than the gamma emissions of the first radioisotope and- wherein the second radiolabeled compound accumulates in cells exhibiting the MDR characteristic;

performing scintigraphic imaging on the individual and simultaneously obtaining the radioimages from both radioisotopes; and

separating the gamma emissions from each radioisotope to form a difference image identifying cells which produce an image only in response to the second radiolabeled compound.

13. The method of claim 12 wherein the second radiolabeled compound is not a substrate for P-glycoprotein.

14. The method of claim 13 wherein the first and second radiolabeled compounds are lipophilic cationic compounds.

15. The method of claim 14 wherein the second radiolabeled compound is trapped intracellularly or in the membrane of the cells.

16. The method of claim 13 wherein the second radiolabeled compound is selected from the group consisting of an ^mIn DTPA complex having a lipophilic group covalently bonded thereto, ^luIn sal₃TAME, ^Tl, ¹⁸F- FDG, and "^mTc-Q17.

17. The method of claim 16 wherein the second radiolabeled compound is ⁹⁹ Tc-Q17.

18. The method of claim 14 wherein the first radiolabeled compotmd is selected from the group consisting of "Tc-sestamibi, "^∞Tc-QS, "^mTc-Q51 and ^99mTc-Q12.

19. The method of claim 18 wherein the first radiolabeled compound is " Tc-QSl.

20. The method of claim 12 wherein at least one of the first and second radiolabeled compounds is radiolabeled with Tc.