WO2000043004A1

WO2000043004A1 - 1,4,8,11-tetraazacyclotetradecane derivatives as radiodiganostic agents and their use in determining hypoxia and radioresistance of tumors

Info

Publication number: WO2000043004A1
Application number: PCT/US2000/001754
Authority: WO
Inventors: J. Donald Chapman; Richard F. Schneider; Edward L. Engelhardt
Original assignee: Fox Chase Cancer Center
Current assignee: Fox Chase Cancer Center
Priority date: 1999-01-26
Filing date: 2000-01-26
Publication date: 2000-07-27
Anticipated expiration: 2001-07-26
Also published as: EP1148878A1; EP1148878A4; CA2361115A1

Abstract

1,4,8,11-Tetraazacyclotetradecane which is chemically linked to from one to four nitroimidazol groups provides an effective radiodiagnostic agent for determining tissue hypoxia and radioresistance of tumor tissue.

Description

1,4,8,11-TETRAAZACYCLOTETRADECANE DERIVATIVES AS

RADIODIAGNOSTIC AGENTS AND THEIR USE

IN DETERMINING HYPOXIA AND RADIORESISTANCE OF TUMORS

Pursuant to 35 U.S.C. §202(c), it is hereby acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Institutes of Health (Grant No. CA 06927)

BACKGROUND OF THE INVENTION

This invention relates to derivatives of 1,4,8,11- tetraazacyclotetradecane (cyclam) which demonstrate hypoxic cell selectivity and retention and which are useful as radiodiagnostic agents for assessing tissue oxygenation status non-invasively, as well as to methods for preparing such derivatives and radionuclide metal-containing complexes thereof and their use for imaging and non-invasively determining tissue hypoxia and radioresi stance of tumors.

Hypoxic cells are known to exist in both animal and human tumors and this cell population has been shown to be 2.5 to 3 times more resistant than normally oxygenated cells to therapeutic radiation. The presence of these radioresistant populations in tumors presents a serious obstacle to the curative potential of clinical radiotherapy. Radiobiologic hypoxic fraction (HF), the fraction of clonogenic tumor cells that exhibit maximum radioresistance, has been shown to be an important parameter for predicting tumor treatment resistance and for the selection of aggressive and metastatic cell phenotypes. If this tumor property were known by clinicians at the time of diagnosis, it would be a useful predictor of tumor treatment response and would define subsets of patients for whom targeted therapies would produce improved cure rates. Thus, radiodiagnostic agents that can accurately assess the presence and extent of hypoxic tissues in tumors would be of invaluable assistance in designing the appropriate therapeutic regimen and in following the response of tumor tissue to therapy. Although various agents and methods have been proposed for the detection and measurement of hypoxic cells in tumors, and direct pO₂ measurements have been obtained from accessible tumors using microelectrodes, there is no practical, clinically useful diagnostic agent or method currently available. Some experimental procedures to assess tumor hypoxia are invasive, requiring multiple biopsy specimens that cannot always be obtained.

A promising approach to the assessment of hypoxic tissue is suggested by the ability of certain classes of compounds to selectively localize in these tissues after intravenous administration. The radiosensitizing drug, misonidazole (MISO), was shown to become selectively bound to the macromolecular fraction of EMT-6 murine tumor and V-79 hamster lung cells in hypoxic in vitro incubation studies (J.D. Chapman et al., Cancer Res., 43: 1523- 1528 (1983)) and to EMT-6 tumors in BALB/C mice (B.M. Garrecht et al., Brit. J. Radiol., 56: 745-753 (1983)). Selective binding of a γ-emitting analogue of a suitable compound would allow imaging and measurement of hypoxic tissue by conventional nuclear medicine techniques. Based on this approach, a number of analogues of 2-nitroimidazole have been investigated as potential non-invasive, hypoxic tissue specific, nuclear medicine imaging agents. The selective toxicity to hypoxic cells by 2-nitroimidazole has been shown to correlate with the accumulation of cellular reduction products of these compounds (J.D. Chapman, Cancer, 54: 2441-2449 (1984)).

The exact mechanism of the binding to the macromolecular fraction of cells remains under investigation, but is believed to rely on reduction of the nitroheterocycle through a series of one-electron transfers that produce nitroso, hydroxylamino and amino products (J.E. Biaglow et al., Biochem. Pharmacol, 35: 77-90 (1986)). This process is reliant on flavoproteins known as nitroreductases. The enzymes are functional only in viable cells and the progression of the reduction past the one-electron stage (nitro radical anion) is strongly inhibited by oxygen, since this species can accept the electron from the nitro radical anion, thereby regenerating the nitroheterocycle. Further reductive metabolism and subsequent binding will, therefore, only be expected to take place in poorly oxygenated yet viable (hence hypoxic) cells.

A number of experimental hypoxic tissue imaging agents incorporating γ-emitting radionuclides have been investigated. These include 4- bromomisonidazole (D.C. Jette et al., Int. J. Nucl. Med. Biol, 10: 205-210 (1983); J.S. Rasey et al., Radiat. Rse., 91: 542-554 (1982)), l-(2-iodophenoxy)- ethyl)-2-nitroimidazole (L.I. Wiebe, Nucelarmedizine, 23: 63-67 (1984)), a series of iodinated acetophenone derivatives of 2-nitroimidazole (J.R. Mercer et al., J. Lab. Comp. Radiopharm., 25: 107-108 (1988)), fluoromisonidazole (F-MISO) (P.A. Jerabek et al., Appl. Radiat. hot, 37: 599-605 (1986)) the 2-nitroimidazole nucleoside analogue, iodoazomycin riboside (IAZR) (D.C. Jette et al., Radiat. Res., 105: 169-179 (1986); L.I. Wiebe et al., In Nuclear Medicine in Clinical

Oncology, Heidelberg, Springer- Verlag, pp. 402-407 (1986)) and certain azomycin nucleosides, namely iodoazomycin arabinoside (IAZA), iodoazomycin galactopyranoside (IAZGP) and iodoazomycin xylopyranoside (IAZXP), which are the subject of U.S. Patent 5,401,490. At millimolar concentrations, IAZGP and IAZA have been found to be selectively toxic to hypoxic EMT-6 tumor cells and also sensitize these cells to the lethal effects of ionizing radiation. At micromolar concentrations, IAZA is taken up preferentially in EMT-6 tumor tissue at a level useful for non-invasive imaging. An imaging study using ¹²⁵I-IAZA showed EMT-6 tumor tissue to be clearly delineated from surrounding tissue. More recently, pilot clinical studies using ¹²³I-IAZA with single photon emission computed tomography (SPECT) and ¹⁸F-FMISO with positron emission tomography (PET) have been reported. These data were recently reviewed and it appears unlikely that either marker is optimal for routinely determining the HF of individual human tumors (J.D. Chapman et al., Radiother. Oncol., 46: 229-37 (1998)). Among the azomycin-nucleoside markers, IAZXP and IAZGP have been shown to exhibit the most favorable properties for marking the HF of solid rodent tumors in vivo (J.D. Chapman et al. (1988), supra).

Although the azomycin nucleosides, and particularly IAZGP, have been found to have useful hypoxic marking properties, an extensive research effort is ongoing to identify hypoxic markers which exhibit good bioavailability to all tissues, low lipophilicity to promote rapid renal excretion, are amenable to detection at optimal times after administration and can be convenientily labelled with the preferred nuclear medicine isotopes.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, cyclam derivatives are provided having the formula:

, wherein at least one of said R,, R₂, R₃ and R₄ contains 2-nitro-imidazol- 1-yl moiety of the formula:

which is chemically linked to the cyclam nitrogen through any of various divalent linking groups. The remaining Rs are hydrogen. Preferably, the linking group comprises a chain of 2 to 7 atoms selected from the group of carbon or nitrogen, wherein the moiety connecting the cyclam nitrogen and the 1-imidazole nitrogen to the remainder of the linking group is a -CH₂- moiety, the carbons in the remainder of the linking moiety being optionally substituted with a hydroxyl group, and two adjacent carbons in the linking groups being optionally replaceable by an amide group. Stereoisomers, including both enantiomers, meso forms and diastereomers of the cyclam derivatives of the above general formula, as well as the pharmaceutically acceptable salts of such cyclam derivatives and their isomers, are included within the present invention.

The present invention also provides radiodiagnostic complexes comprising a cyclam derivative, as described above, and various complex- forming radionuclide metals.

According to a further aspect of the invention, a process is provided for preparation of the cyclam derivatives of the invention. Briefly, this method involves the nucleophilic displacement by cyclam of a suitable leaving group on a moiety containing azomycin.

According to yet another aspect, the present invention provides radiodiagnostic compositions comprising the above-mentioned radionuclide metal-containing complexes and a biologically compatible carrier medium for use in determining tumor hypoxia and radioresistance, as well as in imaging body tissue of a living test subject.

There is also provided, in accordance with this invention, a method of preparing a radionuclide metal-containing complex, as described above, by causing a salt or chelate of a radionuclide metal to undergo a complex- forming reaction with a cyclam derivative of the above formula, or a metal adduct thereof. A kit is also provided for carrying out such method. The kit contains

(i) a cyclam derivative, or a metal adduct thereof, and (ii) instructions for reacting the cyclam derivative or metal adduct thereof, with a radionuclide metal selected from the group of the radioactive isotopes of Tc, Cu, Ru, Co, Pt, Fe, Os, Ir, W, Re, Cr, Mo, Mn, Ni, Rh, Pd, Nb, Pb, Ga, As, In and Ta to produce the complex. According to yet a further aspect of the invention, a novel non- invasive method is provided for the detection and measurement of tissue hypoxia in a mammal comprising the steps of:

(a) administering a radiodiagnostic complex, as described above, such that that complex is taken up selectively and retained by the hypoxic tissue; and (b) quantifying the extent of retained complex by the γ- emission from said complex.

According to still a further aspect of this invention, there is provided a method for imaging body tissue of a living test subject by administering to the test subject a radionuclide metal-containing complex, as described herein, and detecting the localization of the complex in the body tissue using a suitable radiation detector. Of course, different detectors may be employed depending on the nature of the emission produced by the radioactive metal present in the complex. Suitable detectors include, without limitation, SPECT and PET cameras. The cyclam derivatives of the present invention are considered superior to previously reported radiodiagnostic agents for measuring hypoxia and predicting tumor radioresistance because of their high specific activity and the large differential labelling of experimental tumors having a an HF of 15-20%, as compared to tumor tissue that exhibits no detectable HF. Furthermore, in that radiation resistance and some chemotherapeutic drug resistance correlate with tumor hypoxia, the ability to define one type of tumor resistance in advance of therapy is important, since various modalities of targeted cancer treatment, such as chemical radiosensitizers, chemopotentiation agents and bioreductive drugs, can now be directed towards treatment-resistant hypoxic cells.

In addition, a radiodiagnostic agent for oxygenation status will be useful in detecting and defining other disease states, including myocardial infarct and cerebrovascular hemorrhage, in which ischemia and/or infarct play a role and in infections which involve anaerobic foci. BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon payment of the necessary fee. FIGURE 1 is a graphical representation of typical marker biodistribution data obtained by measuring percentage of injected dose of marker/gram of assessed tissue (% ID/G) as a function of time for eight different tissues from five EMT6 tumor-bearing scid mice, up to 10 hours after intravenous

(i.v.) administration of a Tc-99m containing mono-[2-hydroxy-3-(2- nitroimidazol- l-yl)propyl-substituted cyclam complex. The eight tissues evaluated are represented as follows: • = blood; ■ = liver; Δ = kidney; v = spleen; ♦ = lungs; = muscle; O = brain; D = tumor.

FIGURES 2A-D show collimated, gamma-camera planar images of two pair of R 3327-AT (left; hypoxic) and R 3327-H (right; non-hypoxic) tumor-bearing rats six hours after i.v. administration of Tc-99m-labelled

Ceretec® (Figures 2A and 2B) and a Cu-67-containing tetra-[2-hydroxy-3-(2- nitroimidazol-l-yl)-propyl] -substituted cyclam (Figures 2C and 2D). The tumor site is indicated by an arrow in each image.

FIGURE 3 is a graphical representation of data showing the binding rate (dpm/10⁶ cells/hr) of a Cu-64 containing tetra-[2-hydroxy-3-(2- nitroimidazol-l-yl)propyl]-substituted cyclam to tumor cells in vitro, as a function of oxygen concentration.

FIGURES 4A and B show collimated, gamma camera planar images of a pair of R3327-AT (left; hypoxic) and R3327-H (right; non-hypoxic) tumor bearing rats 5-7 hours after administration of a Cu-64 containing tetra-[2- hydroxy-3-(2-nitroimidazol-l-yl)propyl]-substituted cyclam.

DETAILED DESCRIPTION OF THE INVENTION

The cyclam derivatives of formula I, above, can be prepared from known starting materials and the syntheses of specific embodiments of such derivatives that are within the scope of the invention are exemplified below. Certain of these derivatives have been used for radiodiagnostic imaging of animal tumor models and for evaluating their ability to determine tissue hypoxia and assess their hypoxic marking properties, as reported below.

Suitable linking groups for linking the 2-nitro-imidazolyl moiety to the cyclam nucleus are those of the formula

-(CHR_a)_n-X-(CHR_b)_m-Y-(CHR_c)_p-, wherein R_a and R_c represent hydrogen or alkyl (C_rC₄) and R_b represents hydrogen, alkyl ( -C , hydroxyl, X and Y may be the same or different and represent a structural group, such as an amide (-C(=O)NH-), urea (-NH-C(=O)-NH-), thiourea (-NH-C(=S)-NH-), carbamic (-NH-C(=O)-O-), amine (-NH-), carbonyl (-C(=O)-), and amidine

(-NH-C=(NHe)-); disulfide (-S-S-), ether (-O-), thioether (-S-) or sulfonamide (-S(=O)₂-NH-) group, n is an integer from 1 to 6, m is an integer from 1 to 6 and p is an integer from 1 to 6.

Preferably, at least one of the cyclam ring nitrogens is linked to a nitroimidazolyl moiety of the above formula through an unsubstituted or hydroxyl-substituted divalent alkylene linking moiety of 2 to 7 carbon atoms, which may optionally be interrupted by at least one group containing the amide moiety. Representative examples of suitable linking moieties include -CH₂- CH(OH)-CH₂-; -(CH₂)₂-NH-C(=O)-CH₂-; -CH₂-C(=O)-NH-(CH₂)₂-; (CH₂)₂-C(=O)-NH-(CH₂)₃-; -(CH₂)₃-.

Particularly preferred embodiments of the present invention are the mono-, di- and tetra-2-nitroimidazol-l-yl-substituted cyclam derivatives in which the nitroimidazolyl moiety is linked to the ring nitrogen of cyclam via a linkage having the structure -CH₂-CH(OH)-CH₂-. These particular derivatives have an asymmetric center in the linking moiety. This makes the mono- substituted compound resolvable into a pair of optical isomers. On the other hand, the di- and tetra-substituted derivatives are separable into d, 1 (racemic) and meso forms, which contain a plane of symmetry and, therefore, are resolvable into optical isomers. The cyclam derivatives of this invention can form soluble salts with pharmaceutically acceptable acids, such as hydrochloric, phosphoric, tartaric and citric acids, and these salts are also within the scope of the present invention. The pharmaceutically acceptable salts of the cyclam derivatives described herein can be prepared following procedures that are familiar to those skilled in the art. The cyclam derivatives described herein form a complex with various radionuclide metals selected from the group of the radioactive isotopes of

Tc, Cu, Ru, Co, Pt, Fe, Os, Ir, W, Re, Cr, Mo, Mn, Ni, Rh, Pd, Nb, Pb, Ga, As, In and Ta, for example, Tc-99m, Cu-67, Cu-64, Cu-62, Pb-203, Ga-67, Ga-68, As- 72, In-I l l, In-113m, Ru-97, Fe-52, Mn-52m, Cn-51 and Co-57. Particularly preferred complex -forming radionuclide metals for use in the present invention are Tc-99m, Cu-67, Cu-64 and In-111. The complexes have a net positive charge and are associated with counter anions, which may be BF₄-, ClO₄-, Br-, CF₃COO-, C1-, or the like, depending on the manner in which they are prepared.

The radionuclide metal-containing complexes, as described above, are prepared by causing a salt or chelate of a suitable radionuclide metal to undergo a complex-forming reaction with a cyclam derivative of the invention.

Complex formation is effected by contacting the cyclam derivative with the desired metal in the form of a salt or in the form of a chelate wherein the metal is bound to a relatively weak chelator. In using a metal chelate as starting material for the complex formation, the desired complex is formed via the principle of ligand exchange.

A reducing agent may be required for the above reaction, as in cases where technetium metal is used. Suitable reducing agents for this purpose include Sn(LI) compounds, dithionites, sodium borohydride, and the like.

Given the relatively short half-life of the radionuclide metal used in the practice of this invention, it is frequently impossible to put the ready-for- use complexes at the disposal of those wanting to use them. In such cases, the user will carry out the labeling reaction with the radionuclide in the clinical or laboratory setting. To that end, the various reaction components may be conveniently provided in kit form. Because the radiodiagnostic composition of the present invention can be prepared in a sample manner, this process may be readily carried out by experienced researches or clinicians. Such a kit will typically contain (i) a cyclam derivative of the general formula I, shown above, to which an inert biologically acceptable carrier medium and/or formulating agent(s), e.g., reducing agent(s), and/or auxiliary substance(s) may optionally be added, and (ii) instructions for carrying out a complex-forming reaction using the components present in the kit. As mentioned above, for such complex-forming reaction the desired radionuclide may be contacted with cyclam derivative in the form of a chelate, bound to a relatively weak chelator, such as a glucoheptonate, pyrophosphate, a polyphosphate, a phosphonate or polyphosphonate, an oxinate, a carboxylate, a hydroxycarboxylate, an aminocarboxylate, an enolate or a mixture thereof, such reaction being carried out under moderate conditions.

The kit embodiment of this invention may optionally comprise a solution of the radionuclide metal. Because such solutions have a limited shelf life, however, they may be made available separately.

The components of the above-described kit may be supplied as a solution, for example, in the form of a physiological saline solution, or in a buffer solution. If desired, the above-mentioned components may be stabilized in a usual way with suitable stabilizers such as ascorbic acid, gentisic acid or salts of these acids.

Radiodiagnostic compositions comprising a radionuclide metal- containing complex, as described above, may be conveniently formulated for administration with a biologically acceptable carrier medium. According to a preferred embodiment, the carrier medium is sterile, pyrogen-free phosphate buffered sale (PBS). The radiodiagnostic agents of the invention should be delivered to tissue in trace amounts, on the order of 10^"12M. Nuclear medicine markers are usually administered according to their biodistribution, with isotope dose up to 40 mCi in the case of Tc-99m, for example. In all cases, any substance used in formulating a radiodiagnostic composition in accordance with this invention should be virus-free, pharmaceutically pure and substantially non- toxic in the amount used. If necessary or desirable, the risk of contaminating microorganisms may be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, surbic acid, thimerosal or the like. It may also be beneficial to include in the formulation isotonic agents such as glucose.

As used herein, the term "biologically acceptable carrier medium" is intended to include any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the composition.

The use of such media for hypoxic markers is known in the art. Except insofar as any conventional carrier medium is incompatible with the particular radiodiagnostic agents described herein, its use in the compositions of the invention is contemplated. The radiodiagnostic agents of the invention are administered parenterally, intravenous administration being the preferred route.

After administration of the radiodiagnostic composition to a test subject, an image is obtained to detect accumulated radioactivity and thus to determine the location thereof in the body tissue of the test subject. The radionuclide metal-containing complexes of the invention may be utilized in a number of different clinical and research imaging techniques, including without limitation, SPECT and PET.

PET has been gaining increasing acceptance in the field of oncology. One of the principal advantages of positron imaging currently is its high detection efficiency and higher resolution, as compared to prior gamma- detectors. This technique is particularly well suited to the detection of tumor oxygen levels, providing results that are consistent with those obtained through invasive polarographic electrode measurements.

Cu-64 is the preferred radionuclide for PET applications. It can be obtained from the Mallinckrodt Institute of Radiology, or may be prepared as described in U.S. Patent 6,001,825. PET imaging systems are available from commercial sources. See also, U.S. Patent 5,451,789 to Wong et al. ("High Performance Positron Camera").

The following examples are provided to describe the invention in futher detail. These examples, which set forth the best mode presently contemplated for carrying out the invention, are intended to illustrate and to not limit the invention. Examples 1-4 describe the synthesis and purification of representative cyclam derivatives of the invention, as well as the preparation of complexes of such derivatives with suitable γ-emitting radionuclides. In the examples, all temperatures are given in degrees Centigrade, unless otherwise indicated.

EXAMPLE 1

Preparation of mono-[2-hydroxy-3-(2-nitroimidazoI-l-yI) propylj-substituted cyclam 1,4,8,11-Tetraazacyclotetradecane (98%), 1.88 g (9.4 mmol) was dissolved in 12 ml of methanol. A solution of l-(2,3-epoxypropyl)-2- nitroimidazole, 366.8 mg (2.17 mmol) in 3.0 ml of methanol, was added dropwise to the cyclam solution with stirring. After 20 minutes at 25°, a trace of white solid appeared. The flask was wrapped in aluminum foil and allowed to stand five days.

Thin-layer chromatography (TLC) on an E. Merck silica gel 60F plate, developed in chloroform 3: abs. ethanol 6: cone. NH₄OH 1, showed no evidence of the starting epoxide. A 5.00 ml aliquot of the reaction mixture was evaporated on a film evaporator and the residue dried over CaCl₂ in vacuo. The solid was refluxed with 10 ml of methylene chloride for 35 minutes and the insoluble material separated by filtration. The clear yellow solution was evaporated to give 0.4393 g of residue that was dissolved in 1.0 ml of abs ethanol and applied to a 30 x 153 mm column of E. Merck silica gel 60 (230-400 mesh). The column was developed with a mixture of chloroform 400 ml; abs. ethanol 500 ml; and cone. NH₄OH 100 ml; 20 ml fractions collected.

Fractions 7-14 gave a clear yellow oil that was dissolved in 4.0 ml of 1 N HCl. The flask was placed in an evacuated desiccator over CaCl₂. After 4 days, some small white floes were removed from the yellow solution by filtration through sintered glass and the filtrate concentrated in a stream of N₂ at 60°. The flask was placed in a beaker with 25 ml of abs. ethanol; the whole placed in a dessicator. After 7 days, the product consisted of a conical mass of pale yellow crystal at the bottom of the flask and large clusters of fine white crystals over the surface of the liquid and on the upper walls of the flask. After removing the supernatant and drying overnight over CaCl₂ in vacuo, the types of crystals were separated mechanically. The yellow crystals weighed 190.1 mg. A mixture of white and yellow crystals that could not be readily separated weighed 86.1 mg.

This material was dissolved in 300 μl of 1 N HCl. The flask was placed in a beaker containing 25 ml of abs. ethanol in a dessicator. After 2 days, a large portion of the white crystals was removed and the remaining crystals combined with the 190 mg fraction. The combined products were dissolved in 2 ml of water, the solution rendered basic with 3.0 ml of 1 N NaOH and extracted with 5 x 2 ml of methylene chloride. The solvent was evaporated from the combined extracts and the residue dried to give 184.6 mg of partially crystalline yellow material. This product was dissolved in 300 μL of abs. ethanol and applied to a 20 x 153 mm column of E. Merck silica gel 60 (230-400 mesh). The column was developed with: chloroform 175 ml; abs. ethanol 275 ml; cone. NH₄OH 50 ml.

Fractions (10 ml) 7-19 were collected, concentrated and the oily yellow residue dissolved in methanol. This solution was filtered through a 0.45 μ PTFE syringe filter and the methanol evaporated. The residue was dried over CaCl₂ in vacuo. This product weighed 114 mg. TLC showed two slow moving components. This material was rechromatographed in the same flash chromatography system.

Fractious 8-11 gave 60.5 mg of a yellow syrup from which a few needle-like crystals separated. This product was dissolved in 820 μL of 1 N HCl. The flask was placed in a beaker of abs. ethanol in a dessicator at atmospheric pressure for 4 days. At this time, there was a small area of crystaline material in the neck. There were numerous small globules of clear yellow liquid on the walls. The slightly cloudy mother liquor was transferred to another flask that was placed in a beaker of alcohol for 4 days. Cream colored crystals were deposited. After drying over CaCl₂ followed by P₂O₅ in vacuo, this product, the tetrahydrochloride of l-[2-hydroxy-3-(2-nitroimidazol-l-yl)propyl]-,l,4,8,l 1- tetraazacyclotetradecane weighed 44.1 mg.

Electrospray mass spectrometry gave (M+l) = 370.2; calculated value 370.

EXAMPLE 2 Di-[2-hydroxy-3-(2-nitroimadazol-l-yl)- propylj-substituted cyclam

Fractions 3 and 4 from the first column chromatography in the preparation of the mono-substituted compound (Example 1) gave 16.6 mg of material that was dissolved in 200 μL of 1 N HCl. The flask was placed in a beaker with abs. ethanol and kept in a dessicator at atmospheric pressure for 21 days. The yield of small bright yellow crystals was 12.5 mg.

A small portion was converted to the base, extracted into chloroform and subjected to TLC in the system described in Example 1. A major component of Rf 0.36 was by far the largest and most intense. Smaller spots were seen at Rf 0.44, 0.54 and 0.68. In another experiment, a very small amount of a bright yellow crystalline salt was obtained that on electrospray mass spectrometry gave (M+l) = 539.4. Calculated value 539.

EXAMPLE 3 Tetra-[2-hydroxy-3-(2-nitroimidazoI-l-yl)- propylj-substituted cyclam l-(2,3-Epoxypropyl)-2-nitroimidazole, 152.3 mg (0.900 mmol) was dissolved in 500 μL of acetonitrile. 1,4,8,11-Tetraazacyclotetradecane, 41.0 mg (0.205 mmol) was added, followed by 100 μL of acetonitrile and 10 μL of water. The cyclam did not dissolve. Methanol, 300 μL, was added and the mixture stirred at 32°. All but a trace of the cyclam dissolved. After standing 2 days, the thick slurry, containing a light yellow, very finely divided solid, was heated to 56-61° with stirring under reflux for 2-1/4 hours. Methanol, 500 μL, was added after the first hour. After standing 3 weeks, the solvent was removed in a stream of N₂ while heating in a bath at 55°. The residue was broken up under 3.0 ml of tetrahydrofuran. Di-tert-Butyl dicarbonate (99%) 301.5 mg, dissolved in 1.0 ml of tetrahydrofuran was added and the mixture stirred at room temperature for 4 days. The finely divided pale yellow solid was collected and dried overnight. This solid weighed 92.9 mg. It was stirred with 1.5 ml of 1.0 N HCl. The material that did not dissolve was stirred with 1.5 ml of cold trifluoroacetic acid.

On warming to 25°, a clear solution resulted. After filtration to remove a few white crystals, the bulk of the trifluoroacetic acid was evaporated in a stream of N₂ and the residue dried over NaOH pellets in vacuo. The clear light yellow glass was dissolved in 1.00 ml of water and passed over a cation exchange column (9 mm dia x 11 mm long) containing 3.0 ml of IR-120 (plus) resin (capacity 1.9 meq/ml). Water, 4.0 mL was used as chaser. The effluent turned cloudy and a fine white precipitate separated. The solution was stirred while 6.0 N HCl was added dropwise until it was clear. After standing overnight, small pale yellow crystals of the tetrahydrochloride salt had deposited on the walls of the flask. These were collected and dried over CaCl₂ in a vacuum dessicator.

Electrospray mass spectrometry gave (M+l)= 877. Calculated value 877.

EXAMPLE 4 Radiolabelling of Cyclam Derivatives a) N-[2-hydroxy)-3-(2-nitroimidazole-l-yl)propyl]-l,4,8,l 1- tetraazacyclotetradecanato-technetium V.

Into an 4 ml screw-cap vial were placed: 3.0 mg (5.8157 x 10^"6 moles) cyclam derivative prepared in Example 1, above; 3.0 ml deareated distilled water; 0.332 ml deareated 0.1 N sodium hydroxide solution; 0.2 ml

(13.55 mCl) NH₄TcO₄ generator eluate; and 20 μl saturated stannous tartarate solution prepared fresh using deareated distilled water. The mixture was incubated at room temperature for 0.5 hr.

The solution was passed through a 0.8 X 4 cm BioRad Poly-Prep Chromatography Column (cat #731-1550) containing 0.31 g BioRad Ag 1-X8 ion exchange resin, chloride form, 200-400 mesh (cat # 140-1451). The column was rinsed with 1.0 ml deareated distilled water. At this point, the radiolabeled ligand in the eluant was ready as a radiodiagnostic agent.

The relative levels of Tc-99m delivered using the complex of this example in animal tissues, as shown in Figure 1, did not change significantly over the first ten hours after administration. [Add explanation of symbols in Fig. 1]. b) N, N', N", N'"-Tetra[(2-hydroxy)-3-(2-nitroimidazole-l- yl) propyl]- 1,4,8,11-tetraazacyclotetradecanato-copper U.

To the 10 ml serum vials (each of which contained 5.1 m Cu ⁶⁷CuCl₂ in 0.240 ml 0.1 N HCl) were added: 3.00 ml distilled water; 3.8 mg (3.7155 x 10^"6 moles) of the cyclam derivative prepared in Example 3, above; and

0.240 ml 0.1 N sodium hydroxide solution. The pH was determined and adjusted to 7.52 with additional IN or 0.1 N sodium hydroxide solution as required.

The solution was allowed to incubate at room temperature for one- half hour. The solution was filtered through a 0.22 μ filter, which renders it ready for use as a radiodiagnostic agent.

Figure 2 shows two pairs of Fischer X Copenhagen rats in which R3327-AT and R3327-H tumors of approximately equal volume were growing and to which Ceretec®, radiolabeled with Tc-99m, and the complex of this example were administered i.v.. Ceretec® is a commercial tumor perfusion marker. Planar images using a collimated gamma camera were acquired six to seven hours after marker administration. After the imaging procedure, animals were sacrificed and the specific activity of hypoxic marker in blood, muscle and tumor tissue was measured. The tumor specific activity of these markers is presented in Example 5, below. c.) A Cu-64-containing N, N', N", N'"-tetra(2-hydroxy)-3-(2- nitroimidazole-l-yl)propyl]-substituted cyclam was prepared following the same general procedure described in Example 4b, above.

The absolute binding rate of this radiodiagnostic complex to DU- 145 prostate cancer cells (ATCC) in vitro, as a function of oxygen concentration is shown in Figure 3. In this experiment, background binding to aerobic cells (21% O₂) was observed, which was probably due to ionic trapping of isotope on the filters.

Figure 4 shows the uptake of the radiodiagnostic complex of this example in a pair of Fischer X Copenhagen rats in which R3327-AT (Fig. 4A) and R3327-H (Fig. 4B) tumors of approximately equal volume were growing. The tumors, of 8-10 gm. weight, are indicated by a black circle in each figure. Tumor specific activity was determined to be about 2.2.

EXAMPLE 5

The following table sets forth data concerning hypoxic marking properties of radiolabelled cyclam derivatives of Examples 1 and 3, above.

HSF - hypoxia-specific factor refers to the amount of marker bound to (or retained in) hypoxic cells relative to aerobic cells in vitro. See Chapman et al., Radiotherapy and Oncology, 46: 229-37 (1998) Km-concentration of O2 at which binding rate of marker is at 50% of maximum (normally defined in vitro)

T/B-maximal tumor/blood ratio of tissue-specific radioactivity T/M-maximal tumor/muscle ratio of tissue-specific radioactivity %ID/g AT - percentage of injected dose of marker/g in anaplastic tumor tissue assessed 6 hours following injection. Specific activities were corrected to a standard rat weight of 400 g. Anaplastic tumors formed following injection of cells from the Dunning rat prostate carcinoma, R3327-AT.

%ID/g H - percentage of injected dose of marker/g in well-differentiated tumor tissue assessed 6 hours following injection. Specific activities were corrected to a standard rat weight of 400 g. Well-differentiated tumors formed following injection of cells from the Dunning rat prostate carcinoma, R3327-H. ^e AT/H - ratio of specific activities in anaplastic (hypoxic) versus well- differentiated (non-hypoxic) tumor tissues.

ND = Not determined

As the data show in the above table, the radiodiagnostic agent of Example 4b, above, was recovered from the hypoxic tumors at a level of about three times higher than the non-hypoxic, well oxygenated tumors. It is also noteworthy that up to six-fold higher levels were observed in individual pairs of animals. By comparison, Tc-99m labelled Ceretec® distributed to the well oxygenated tumors at a level of about 1.7 times higher than hypoxia tumors. In this connection, see Moore et al., Brit. J. Cancer, 65: 491-97 (1992). The following example describes PET imaging, using Cu-64- labelled cyclam derivatives of the present invention to detect and quantify tumor hypoxic microenvironments in prostate carcinomas of laboratory animal test subjects.

EXAMPLE 6

A micro-PET imaging system using luetium oxyorthosilicate detector elements (Concord Microsystems, Knoxville, TN) with an 8-cm field of view, a spatial resolution of 2 mm at the center of the field of view and a volumetric resolution of about 8 μl may be used to image and quantitate rat tumor metabolism and viable hypoxic cells. The animals, bearing implanted hypoxic and non-hypoxic prostate carcinomas, are anesthetized with Avertin (Aldridge, Milwaukee, WI) before the imaging procedure. The long access of the rats is parallel to the long access of the scanner. On the day of the procedure, each rat is injected i.v. with 2 mCi of labelled fluorodeoxyglucose (¹⁸FDG) (1.0-1.5 ml) followed by a line flush with 500 μl of physiological saline. Three sequential whole body studies are performed on each rat using 5 minute acquisitions for each bed position, between 3-8, depending on the rat length. The following day, each rat is reanesthesized and repositioned so as to acquire PET images from the tumor region. The rat is then injected with 2 mCi of optimal Cu-64-labelled cyclam derivative. Sequential scans are obtained from each rat using 5 minute aquisitions at 6 different times up to 8 hours after marker administration.

Attenuation correction is determined with a 1 mCi Ge-68 ring transmission source and rat images are reconstructed using the 3-dimensional filtered back projection algorithm with the appropriate ramp filtered cutoff. Time activity curves are established for each organ in the field, including tumor, and the relative distribution of FDG and labelled cyclam derivative are determined. The standardized unit value (SUV) of metabolic activity and HF are calculated for each tumor at various times and statistical analysis of data are performed to assess differences in metabolism and hypoxic fraction of the two tumor types. Although the present invention has been described and exemplified in terms of certain preferred embodiments, other embodiments will be apparent to those skilled in the art. The invention is, therefore, not limited to the particular embodiments described and exemplified, but is capable of modification or variation without departing from the spirit of the invention, the full scope of which is delineated by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A derivative of 1,4,8,11-tetraazacyclotetradecane (cyclam), said derivative having the formula:

, wherein at least one of said R,, R₂, R₃ and R₄ contains a 2-nitroimidazol- 1-yl moiety of the formula:

each said 2-nitroimidazolyl moiety being linked to a cyclam ring nitrogen through a linking group, any remaining R,, R₂, R₃ and R₄ radicals representing hydrogen, and the stereoisomers and pharmaceutically acceptable salts of said derivative.

2. A cyclam derivative as claimed in claim 1, wherein said linking group comprises a chain of 2 to 7 atoms selected from the group of carbon or nitrogen, wherein the moiety connecting the cyclam nitrogen and the 1- imidazole nitrogen to the remainder of said linking group is a -CH₂- moiety, the carbons in the remainder of said linking moiety being optionally substituted with a hydroxyl group, and two adjacent carbons in said linking groups being optionally replaceable by an amide group.

3. A cyclam derivative as claimed in claim 1, wherein said linking group is selected from those consisting of

-CH₂- CH(OH)-CH₂-; -(CH₂)₂-NH-C(=O)-CH₂-; -CH₂-C(=O)-NH-(CH₂)₂-; - (CH₂)₂-C(=O)-NH-(CH₂)₃; -(CH₂)₃-.

4. A cyclam derivative as claimed in claim 3, wherein R_[ is a

2-nitroimidazolyl group and R₂, R₃ and R₄ represent hydrogen.

5. A cyclam derivative as claimed in claim 3, wherein two of said R,, R₂, R₃ and R₄ represent 2-nitroimidazolyl, and the other two of said R,, R₂, R₃ and R₄ represent hydrogen and said linking moiety is -CH₂-CH(OH)-CH₂-.

6. A cyclam derivative as claimed in claim 3, wherein each of said R,, R₂, R₃ and R₄ groups represents 2-nitroimidazolyl and said linking moiety is -CH₂-CH(OH)-CH₂-.

7. A radionuclide metal complex comprising a cyclam derivative, as claimed in claim 1, and a γ ray-emitting radionuclide.

8. A complex as claimed in claim 7, wherein said γ ray- emitting radionuclide is selected from the group consisting of Tc-99m, Cu-67 and

In-111.

9. A radionuclide metal-containing complex comprising a cyclam derivative as claimed in claim 1 and a radionuclide metal selected from the group of the radioactive isotopes of Tc, Cu, Ru, Co, Pt, Fe, Os, Ir, W, Re, Cr,

Mo, Mn, Ni, Rh, Pd, Nb, Pb, Ga, As, In and Ta.

10. A method of identifying hypoxic tissue in a living test subject, said method comprising the steps of

(a) administering to said test subject a complex, as claimed in claim 9, such that said complex is taken up selectively and retained by said hypoxic tissue; and

(b) quantifying the radiation emitted from said complex.

11. A method as claimed in claim 10, wherein said complex is formed with a γ ray-emitting radionuclide selected from the group consisting of

Tc-99m, Cu-67 and In-111

12. A method as claimed in claim 10, wherein said complex is administered intravenously.

13. A method as claimed in claim 10, wherein said hypoxic tissue is a component of tumor tissue.

14. A method as claimed in claim 10, wherein said hypoxic tissue is ischaemic heart tissue.

15. A method as claimed in claim 10, wherein said hypoxic tissue is ischaemic brain tissue.

16. A radiodiagnostic composition comprising a complex as claimed in claim 9 and a biologically acceptable carrier medium.

17. A method of preparing a radionuclide metal-containing complex as claimed in claim 9, wherein a salt or chelate of said radionuclide metal is caused to undergo a complex-forming reaction with said cyclam derivative.

18. A kit for preparing a radionuclide metal-containing complex containing (i) a cyclam derivative as claimed in claim 1, or a metal adduct thereof, and (ii) instructions for reacting said cyclam derivative with a radionuclide metal selected from the group of the radioactive isotopes of Tc, Cu,

I Ru, Co, Pt, Fe, Os, Ir, W, Re, Cr, Mo, Mn, Ni, Rh, Pd, Nb, Pb, Ga, As, In and Ta to produce said complex.

19. A kit as claimed in claim 18, which further contains a solution of a salt or chelate of said radionuclide metal.

20. A method for imaging body tissue of a living test subject comprising administering to said test subject a radionuclide metal-containing complex as claimed in claim 9, and detecting the localization of said complex in said body tissue by a radiation detector.

21. A method for imaging body tissue as claimed in claim 20, wherein said complex contains a radionuclide metal selected from the group of Tc-99m and Cu-67, and said detector is a gamma detector.

22. A method for imaging body tissue as claimed in claim 20, wherein said complex contains Cu-64 as said radionuclide metal, and said detector is a positron detector.