WO1999049900A1

WO1999049900A1 - Tumour imaging and therapeutic agents

Info

Publication number: WO1999049900A1
Application number: PCT/CA1999/000261
Authority: WO
Inventors: John Robert Scott; Alummoottil Varghese Joshua; Alexander James Baird Mcewan
Original assignee: Alberta Cancer Board
Priority date: 1998-03-31
Filing date: 1999-03-26
Publication date: 1999-10-07
Also published as: AU2918199A; CA2233736A1

Abstract

New methods and compositions are provided for detecting tumours by scintigraphic imaging and for treating tumours, employing radiolabelled diamines.

Description

TUMOUR IMAGING AND THERAPEUTIC AGENTS

Field of Invention This invention relates to nuclear medicine techniques for imaging and treating tumours. In particular, the invention relates to compounds and methods for detecting and localising tumours and for treating tumours.

Background of the Invention

The early detection of cancers is of great importance in their management and treatment. Early detection significantly increases patient median survival times. In addition, the determination of whether or not a particular tumour has metastasised influences the type and extent of therapy administered to the patient . A radiolabelled agent that is preferentially localised and retained by tumours would have widespread application in cancer diagnosis and treatment .

One example of a cancer whose incidencs has been increasing alarmingly is malignant melanoma. Occurrence of this tumour is now reaching epidemic proportions in countries like Australia, New Zealand and the United States, (Elwood M, (1993) , Melanoma Res., v. 3, pp. 149 - 156), presumably due to increased levels of exposure to ultraviolet radiation, either as a consequence of thinning of the ozone layer or failure of the public to heed to the advice of experts concerning minimizing solar exposure. In the United States, it has been estimated that approximately 38,000 individuals developed melanoma in 1996, a 12% increase from 1995, of which there were approximately 7,300 deaths. Similarly in Canada, the incidence of melanoma has been increasing steadily at an annual rate of 6.8% in men and 4.8% in women .

Despite improvements in survival rates over the past five decades and the detection of earlier, thinner melanomas, the mortality rate from melanoma continues to increase, albeit more slowly than the incidence rate (2.5%/yr. vs 4.6%/yr., respectively) . The mortality rate from this devastating cancer can be reduced in two ways; either by reducing the number of new - 2 -

cases by eliminating or minimizing those factors responsible for its pathogenesis (namely ultraviolet light exposure) , or by the early detection of melanoma when the likelihood of cure is highest . Diagnosis of melanoma by direct inspection of suspicious skin lesions is of limited accuracy and various non-invasive diagnostic techniques have been investigated as possible aids in differentiating benign from malignant pigmented skin lesions. These techniques include surface microscopy (dermoscopy or epiluminescence microscopy), computer image analysis, confocal scanning laser microscopy, and high-frequency ultrasonography. Imaging techniques have also been used, including magnetic resonance imaging (Takahashi M, et al . , 1992; Zemstov A., et al . , 1989) and scintigraphy (Alonso O, et al . , 1996; Ryu YH, et al. , 1995) .

Positron Emission Tomography (PET) imaging has been employed for melanoma detection, using F-fluorodeoxyglucose ( FDG) as imaging agent (Gritters et al . (1993), J. Nucl . Med. , v.34, pp. 1420 - 1427) . The data derived from PET ¹⁸FDG imaging clearly show the potential of a radiopharmaceutical which can effectively image the whole body to detect recurrent or mets.static melanoma. PET is, however, an expensive technology which is not routinely available in most clinical environments. Single Photon Emission Computed Tomography (SPECT) imaging is a recent advance in nuclear imaging that results in improved three dimensional resolution compared with previous two dimensional techniques. This technology offers the potential advantage of early diagnosis of cancer and requires equipment which is less costly and more generally available than PET.

There is, however, a scarcity of imaging agents for detecting tumours by SPECT.

Once a diagnosis of melanoma has been confirmed histologically, the stage of the tumour must be determined, both by pathology and clinical assessment. It has recently been shown that radioimmunoscintigraphy can increase the accuracy of staging by 20% over clinical and radiologic examinations (Blend MJ, et al., (1996), J. Nucl . Med. , v. 37, pp. 252 - 257). Due to the significant risk of recurrence, even after treatment there is a need for follow up of melanoma patients.

With an increased interest in the role of biological response modifiers (immunotherapy, melanoma vaccines, angiosuppressive agents, etc.) it is imperative that patients with metastasis be detected when the tumour burden is least, for these agents to have a beneficial effect. Towards this end, newer melanoma- specific imaging techniques such as radioimmunoscintigraphy and PET scan may prove to be of value, both to the initial staging process (Blend MJ, et al . , (1996), supra ; Wagner JD, et al . , (1997), J. Surg. Oncol., v. 64, pp. 181 - 189; Sergieva S. et al., (1994), Nucl. Med. Commun. , v. 15, pp. 168 - 172), and in follow up.

Although conventional wisdom has it that melanoma is radioresistant, advances over the past 10-15 years in radiotherapy suggest that this is not the case. Recent developments include the recognition that larger doses per fraction are required, or that radiotherapy should be combined with hyperthermia or chemotherapy. Various groups have been assessing the role of "targeted" radiotherapeutic agents. These agents are either selectively taken-up by melanoma cells (e.g., phenol and catechol-based chemicals) or are tagged with monoclonal antibodies reactive against melanoma cells (Ji bow K, et al., (1996), Elsevier, Amsterdam, pp. 257 - 268; Link EM, et al., (1996), Acta Oncologica, v. 35, pp. 331- 41). In the last few years, a number of radioiodinated benzamides and related compounds have been suggested as potential agents for the imaging of tumours. Some clinical studies, involving the use of [1-123] - (S) -IBZM, and [¹²³I] -N- (2-diethylaminoethyl-4- iodobenzamide for the scintigraphic detection of malignant - 4 -

melanoma and metastases have been reported (Maffioli et al . (1994), J. Nucl. Med., v. 35, p.1741; Michelot et al . , (1993), J.Nucl. Med., v.34, p.1260).

These compounds have not, however, proved very successful for clinical use due to poor tumour uptake and/or poor tumour retention.

Multiple myeloma is a malignancy of plasma cells, occurring usually in the bone marrow, but sometimes at other sites in the body. Multiple myeloma comprises about 1% of all malignant disease. Multiple myeloma is not curable and survival ranges from a few months to a few years after diagnosis.

The proliferating malignant plasma cells produce monoclonal immunoglobulins but the presence of these monoclonal immunoglobulins may not be diagnostic as they may be seen in a variety of other diseases. Diagnosis is often difficult as it is usually based on clinical presentations that may mimic other diseases .

A closely related disease, plasmacytoma, is not widely spread throughout the bone marrow as is multiple myeloma but is localized to a specific site, either in the bone marrow or in other areas of the body.

An imaging agent that localizes in multiple myeloma and plasmacytoma would be of considerable value in the diagnosis and localization of these diseases. There remains a need for improved imaging agents to assist in the early detection of tumours, to allow early therapeutic intervention and to facilitate the monitoring of possible tumour progression and the need for further treatment. There is also a need for compounds which inhibit tumour growth and which accumulate with sufficient specificity in tumour tissue that they can be used for tumour ablation with minimal damage to surrounding tissue. - 5 -

Summary of the Invention

The inventors have identified a group of diamine compounds which are taken up by tumour tissue to a level sufficiently greater than their uptake into normal tissues to permit detection of tumours by whole body scintigraphic imaging after administration of radioactive derivatives of these compounds.

Radioactive derivatives of the compounds of higher specific activity can be used as therapeutic agents to inhibit tumour growth.

Summary of the Drawings

Certain embodiments of the invention are described, reference being made to the accompanying drawings, wherein:

Figure 1 shows a whole body image 3 days after the administration of 1-131 ERC 9 to a patient with malignant melanoma.

Figure 2 shows SPECT images of the thorax, at 24 hours after injection of 1-131 ERC 9, of the same patient as seen in Figure 1. Figure 3 shows a whole body image 4.5 hours after the administration of 1-123 ERC 9 to a patient with multiple myeloma.

Detailed Description of the Invention The inventors have found that certain diamines show sufficiently selective accumulation in tumour tissue to make these compounds useful either as diagnostic imaging agents, for detection and localisation of tumours, or as therapeutic agents, for inhibition of tumour growth. These diamines are of the formula :

R 2 _/ j I (CH₂)„-

I 1 R

wherein n is 1 to 10; X is a halogen atom; R₁ and R₂ are the same or different and are hydrogen, hydroxyl or lower alkyl; R₃ is lower alkyl; and N' is a nitrogen atom having two lower alkyl substituents or a nitrogen atom forming part of a 4- to 8- membered heterocyclic ring containing one or two hetero atoms, one of which is the nitrogen, the heterocyclic ring being unsubstituted or substituted with one or more lower alkyl groups, or a pharmaceutically acceptable acid addition salt thereof .

As used herein, "lower alkyl" denotes a straight or branched chain alkyl radical having 1 to 6 carbon atoms. Preferred lower alkyl radicals are methyl and ethyl.

In the substituted benzene ring of formula I, R_x and R₂ are the same or different and are hydrogen, hydroxyl or lower alkyl. The preferred configurations for the benzene ring are 2- hydroxy-3-lower alkyl-5-iodo or 2-hydroxy-3-iodo-5-lower alkyl. X may be any radioactive halogen isotope, including At, F, Br and I. F, Br and I are preferred and I is especially preferred.

In a preferred embodiment, the compound of formula I, as shown above, has n = 1 to 10, R and R₂ are the same or different and are hydroxyl or lower alkyl; R₃ is lower alkyl and N¹ is a nitrogen atom having two lower alkyl substituents having 1 to 3 carbon atoms or a nitrogen atom forming part of a morpholino, pyrrolidino or piperidino ring which is unsubstituted or substituted with one or more lower alkyl groups.

Preferred diamines for use in the methods and compositions of the present invention include N [3 (4-morpholino) ropyl] -N-methyl- 2-hydroxy-3-methyl-5-halobenzylamine; N[3 (1-pyrrolidino) propyl] - N-methyl-2 -hydroxy-3 -methyl-5-halobenzylamine; N[3 (1- piperidino) propyl] -N-methyl-2-hydroxy-3 -methyl-5-halobenzylamine and N[3 (diethylamino) propyl] -N-methyl-2 -hydroxy-3 -methyl-5- halobenzylamine .

The iodo derivatives of these preferred diamines, N[3(4- morpholino) propyl] -N-methyl-2-hydroxy-3 -methyl-5-iodobenzylamine (ERC 9) ; N [3 (1-pyrrolidino) propyl] -N-methyl-2 -hydroxy-3 -methyl- 5-iodobenzylamine (ERC 46); N [3 (1-piperidino) propyl] -N-methyl-2- hydroxy-3-methyl-5-iodobenzylamine (ERC 90) and

N[3 (diethylamino) propyl] -N-methyl-2-hydroxy-3-methyl-5- iodobenzylamine (ERC 94) , are especially preferred.

The compounds employed in the methods and compositions of the invention may, for example, be synthesised as described in United States Patent No. 5,349,063, the contents of which are incorporated herein by reference, or by minor modifications of the described method.

For example, iodine radiolabelled compounds may be synthesised by preparing the non-radioactive iodinated compounds as described in USP 5,349,063 and radiolabelling by exchange, as also described therein. Similarly, bromine radiolabelled compounds may be made by exchange, by reacting the iodocompound with ammonium radiobromide in the presence of a cuprous catalyst and an excess of a reducing agent (such as gentisic acid, ascorbic acid or citric acid) by the method of Mertens et al . (J. Lab. Com. Radiopharm. v. 22, p. 89, 1985) .

Fluorine radiolabelling of the compounds may be done by direct radiofluorination of the deiodo-compounds . The deiodo- compounds may be synthesized by the process described in USP 5,349,063 with omission of the iodination step and condensation of the phenolic aldehyde with the appropriate diamine, reduction and subsequent reductive alkylation. The deiodo-compounds may then be radiofluorinated with radio-acetylhypofluorite (CH₃COOF ) as described in Murali et al . (Internat. J. Radiation Applications & Instrumentation - Part A, Applied Radiation & Isotopes, (1992), v. 43, pp. 969 - 77).

As will be understood by those of skill in the art, the choice of radioisotope to be incorporated into a compound of formula I, and the specific activity of the compound to be prepared will depend on whether the compound is to be used for diagnostic imaging or for tumour treatment.

For diagnostic imaging, the radiolabel should emit gamma radiation or positrons with sufficient energy to penetrate tissue and to be detected by scintigraphic imaging devices. Most radionuclides used in single photon clinical imaging studies have gamma energies in the range between 50 and 400 keV ("Single Photon Imaging", M.V. Green and J. Seidel in "Nuclear Medicine Diagnosis and Therapy", Ed: J.C. Harbert, W.C. Eckelman and R.D. Neumann, Thieme Medical Publishers, Inc., New York,

1996, p. 87) . Positron emitting radionuclides used for positron emission tomography emit 512 keV gamma rays. The selection of a suitable label is within the knowledge of one of ordinary skill in the art. For imaging, gamma or positron emitting radionuclides such as 1-121, 1-123, 1-124, 1-125, 1-131, F-18, Br-76 and Br-77 are preferred. 1-123, 1-131 and F-18 are especially preferred.

For tumour therapy, the radiolabel should preferably be a beta or alpha emitter which will produce a local cell killing effect. Potential radionuclides for therapeutic use have been reviewed by Ackery ("Principles of Radionuclide Therapy", D. Ackery, in "Nuclear medicine in Clinical Diagnosis and Treatment", Ed: I.P.C. Murray and P.J. Ell, Churchill Livingstone, New York, 1998, pp. 1040 - 1041) . Preferred radiolabels are 1-131, At-211, Br-76 and Br-77. 1-131 is especially preferred.

In order to be suitable for tumour imaging, a radiolabelled diamine in accordance with formula I should be taken up by the tumour tissue and retained for a sufficient time to allow the - 9 -

desired imaging techniques to be carried out. The compound should also be bound to the target tumour at a higher level than to the surrounding tissues. A target tissue/non-target tissue ratio of about 1.5 is generally sufficient for satisfactory imaging. It is within the expertise of one of ordinary skill in the art to determine the suitability of a particular diamine of formula I for tumour imaging and to determine a diagnostic imaging amount of the compound. Preliminary assessment of a compound can be carried out in an animal model, as described in the Examples herein.

The particular dose of a compound to be used for imaging can be determined by the physician attending the patient, but based on the studies described herein both with a mouse model and human studies, for example, a dose of about 0.1 to about 10 mg of diamine labelled, for diagnostic imaging, with about 50 MBq to about 800 MBq of radioisotope, should be administered to a human patient.

The radiolabelled diamines described herein, as exemplified by compounds ERC 9, ERC 46, ERC 90 and ERC 94, show high levels of tumour uptake and slow clearance from tumour tissue, making these compounds valuable agents for imaging tumours or for radiotherapy of tumours .

For use in tumour imaging or in tumour therapy, the radiolabelled compounds of the invention are prepared as sterile, pyrogen-free preparations in pharmaceutically acceptable vehicles by conventional techniques known to those skilled in the art . The radiolabelled agent is preferably administered by intravenous injection, formulated, for example, as a solution in 0.9% sodium chloride, following generally accepted criteria such as those outlined in the U.S.

Pharmacopeia (U.S. Phamacopeia 23, 1995, Injections pp. 1650 - 1652) . The amount of radiolabelled diamine, as outlined above, may be formulated in about 1 to 3 millilitres of vehicle.

Imaging for tumour detection and location may be done by - 10 -

conventional scintigraphic or gamma camera imaging methods, with or without single photon emission computed tomography (SPECT) capability, such methods being well known to those skilled in the art. Scintillation imaging of the radiolabelled compound should be done at such time periods after administration as to provide the optimum tumour uptake and tumour/normal tissue ratios. For example, for SPECT imaging, it has been found that the optimum time using 1-123 ERC 9 is around 3-4 hours after administration. For whole body imaging, around 3 to 4 hours after administration was found to be optimum for tissues in which the compound administered had a short retention time. For tissues such as liver with a longer retention time, whole body imaging could be delayed to around 24 hours, giving better definition due to clearance of the compound from non-tumour liver tissue.

These imaging times may vary depending upon the compound and upon the radioisotope being used, as will be understood by those skilled in the art.

For tumour therapy, a radiolabelled diamine should concentrate in, and be retained by, tumour tissue to a sufficient extent so as to be effective as a tumour inhibiting agent. At the same time, the concentration and retention of the radiolabelled diamine in normal tissue should be low, so as to give a high tumour/normal tissue radiation absorbed dose and minimal damage to normal tissue. Those skilled in the art are well aware of how to estimate appropriate ratios and doses, aided by information available in the literature, for example in Wegst (1987), Nucl. Med. Biol., v. 14, pp. 269-271. For example, for radio-sensitive tissue such as bone marrow and lungs, this tumour : normal tissue radiation absorbed dose ratio should be 10:1 or higher.

It is within the expertise of one skilled in the art to determine a tumour inhibiting amount of a radiolabelled diamine for use in the method of the invention. For example, a dose of - 11 -

about 0.1 mg to about 10 mg of diamine, labelled with about 40 MBq to about 30,000 MBq of radioisotope, may be administered. Radioisotope in a range of about 400 MBq to about 20,000 MBq is preferred. Repeat administrations of the compound in these quantities, at intervals varying from a few weeks to a few months, may be necessary to achieve a full therapeutic effect.

The diagnostic methods and compositions of the invention may be employed to detect and locate any tumour which accumulates a compound of formula I with sufficient specificity to permit satisfactory imaging, as described above.

The therapeutic methods and compositions of the invention may be employed to treat any tumour which accumulates a compound of formula I with sufficient specificity to provide tumour inhibition.

Tumours which may be detected or treated by the methods and compositions of the invention include malignant melanoma, breast cancer, non-small cell lung carcinoma, prostate tumours, multiple myeloma and plasmacytoma. Imaging studies in malignant melanoma patients, for example, using the methods and compositions of the invention, gave a sensitivity and specificity of 93% on a per patient basis which is superior to the performance of currently available tumour- diagnostic imaging techniques.

EXAMPLES

Example 1

Biodistribution studies were performed with 1-125 ERC 9 in C57BL/6CrlR mice bearing B16F10 melanoma tumours to assess tumour uptake and retention.

C57BL/6CrlR mice, weighing 20 to 25 grams, were injected subcutaneously in the flank with 2 x 10⁵ B16F10 melanoma cells suspended in 0.1 ml of 0.9% saline. The tumours were allowed to - 12 -

grow for approximately seven days at which point the tumours were from 20 to 150 mg in weight.

A preparation of 1-125 labelled ERC 9 (10 kBq 1-125, 1 _μg ERC 9, 0.1 ml) was injected into the tail vein of each animal. Three samples (0.1 ml) of the 1-125 ERC 9 were retained as standards.

At various times, from 30 minutes to 24 hours after injection of the 1-125 ERC 9, groups of 5 mice were killed by C0₂ asphyxiation. Blood samples were taken by cardiac puncture and, noting the volumes, placed in gamma counting tubes. The animals were dissected and the tumours and various organs were removed, weighed and placed in gamma counting tubes.

The blood samples, tumours, organs and standards were counted for 1-125 radioactivity in a Canberra Packard automatic gamma counter. The percent of injected dose per gram was determined by comparing the radioactivity in the organ with that in the standards and dividing by the weight of the organ.

The tumour to blood ratios were calculated by dividing the mean percent of injected dose per gram of tumour by the mean percent injected dose per gram of blood at each time period. The biological half-time of a radiolabelled compound in a particular organ or tissue is the clearance half-time of the compound ignoring the physical half-time due to decay of the radioisotope. This was calculated using a computer based plotting and curve stripping program (Plot, Oak Ridge National Laboratories) . For most of the organs in this study, 1-125 ERC 9 had a two component biological clearance .

The biodistribution of 1-125 ERC 9 in B16F10 melanoma bearing C57BL/6CrlBR mice is shown in Table 1 as percent injected dose per gram. Initial uptake (30 minutes after injection) is high in lungs, liver, spleen, kidneys, brain, pancreas and tumour.

Clearance of this material is primarily via the hepatobiliary system into the intestine. Thus the amount of material in the intestine is high up to 4 hours after injection but then decreases significantly by 24 hours. - 13 -

By 4 hours after injection, the tumour issue contained more radioactivity, on a per gram basis, than any of the organs with the exception of pancreas.

With the exception of the pancreas and tumour tissue, this material was cleared biphasically (i.e. with two components) from the organs. The short lived component varied from 1 to 3 hours in the organs studied. The long lived component is shown in Table 1, as is the single clearance component for the pancreas and tumour issue. The biological half times of the long lived component varied from 7 to 14 hours. In contrast, the biological clearance half times for the pancreas and tumour tissue was greater than 30 hours.

The high uptake and slow clearance of 1-125 ERC 9 from tumour tissue as compared to normal tissue (with the exception of pancreas) indicates its value for tumour detection and therapy.

Example 2

Biodistribution studies were performed with 1-125 ERC 46 in C57BL/6CrlBR mice bearing B16F10 melanoma tumours to assess tumour uptake and retention.

Tumour-bearing mice were prepared as in Example 1 and a preparation of 1-125 labelled ERC 46 (10 kBq 1-125, 1 _μg ERC

46, 0.1 ml) was injected into the tail vein of each animal.

Three samples (0.1 ml) of the 1-125 ERC 46 were retained as standards.

At 2 hours and 24 hours after injection of the 1-125 ERC 46, mice were sacrificed and examined as in Example 1.

The biodistribution of 1-125 ERC 46 in B16F10 melanoma bearing C57BL/6CrlBR mice at 2 hours and 24 hours after administration is shown in Table 2 as percent injected dose per gram.

At 2 hours after injection, levels of the material are high in the lungs, spleen, kidneys, brain, pancreas and tumour.

Clearance of this material is primarily via the - 14 -

hepatobiliary system into the intestine. Thus the amount of material in the intestine is high at 2 hours after injection but then decreases significantly by 24 hours.

Between 2 hours and 24 hours after injection, the amount of radioactivity, as percent of injected dose per gram, decreased significantly in all organs and tissues with the exception of the pancreas and tumour, both of which increased. The tumour tissue had considerably higher levels than any other tissue, with the exception of the pancreas. The high uptake and slow clearance of 1-125 ERC 46 from tumour tissue as compared to normal tissue (with the exception of pancreas) indicate its suitability for tumour detection and therapy.

Example 3

Biodistribution studies were performed with 1-125 ERC 90 in C57BL/6CrlBR mice bearing B16F10 melanoma tumours to assess tumour uptake and retention.

Tumour-bearing mice were prepared as in Example 1, and a preparation of 1-125 labelled ERC 90 (10 kBq 1-125, 1 _μg ERC

90, 0.1 ml) was injected into the tail vein of each animal.

Three samples (0.1 ml) of the 1-125 ERC 90 were retained as standards .

At 2 hours and 24 hours after injection of the 1-125 ERC 90, mice were sacrificed and examined as in Example 1.

The biodistribution of 1-125 ERC 90 in B16F10 melanoma bearing C57BL/6CrlBR mice at 2 hours and 24 hours after administration is shown in Table 3 as percent injected dose per gram. At 2 hours after injection levels of the material is high in the lungs, spleen, kidneys, brain, pancreas and tumour.

Between 2 hours and 24 hours after injection, the amount of radioactivity, as percent of injected dose per gram, decreased significantly in all organs and tissues with the exception of - 15 -

the pancreas and tumour. The tumour tissue had considerably higher levels than any other tissue, with the exception of the pancreas .

1-125 ERC 90 shows high uptake and slow clearance from tumour tissue as compared to normal tissue (with the exception of pancreas) .

Example 4

Biodistribution studies were performed with 1-125 ERC 94 in C57BL/6CrlBR mice bearing B16F10 melanoma tumours to assess tumour uptake and retention.

Tumour-bearing mice were prepared as in Example 1 and a preparation of 1-125 labelled ERC 94 (10 kBq 1-125, 1 _μg ERC

94, 0.1 ml) was injected into the tail vein of each animal. Three samples (0.1 ml) of the 1-125 ERC 94 were retained as standards .

At 2 hours and 24 hours after injection of the 1-125 ERC 94, mice were sacrificed and examined as in Example 1.

The biodistribution of 1-125 ERC 94 in B16F10 melanoma bearing C57BL/6CrlBR mice at 2 hours and 24 hours after administration is shown in Table 4 as percent injected dose per gram.

At 2 hours after injection levels of the material is high in the lungs, spleen, kidneys, brain, pancreas and tumour. Between 2 hours and 24 hours after injection, the amount of radioactivity, as percent of injected dose per gram, decreased significantly in all organs and tissues with the exception of the pancreas and tumour. The tumour tissue had considerably higher levels than any other tissue, with the exception of the pancreas.

1-125 ERC 94 shows high uptake and slow clearance from tumour tissue as compared to normal tissue (with the exception of pancreas) . - 16 -

Example 5

Fifty one patients with either current or previously treated malignant melanoma, were imaged with either 1-123 ERC 9 or 1-131 ERC 9.

Fifty imaging studies were done in 44 patients with 1-123

ERC 9 (6 patients had two imaging studies each) . An average of

185 MBq of 1-123 ERC 9 (range 123 MBq - 327 MBq, containing approximately 2 mg of ERC 9 compound) , was administered by slow intravenous injection.

Simultaneous anterior and posterior whole body imaging (scan time 30 minutes) was done immediately after injection and at approximately 1.5 hours, 3-4 hours and 18-24 hours. SPECT imaging was done at areas of interest at about 3-4 hours after injection. All images were acquired with a Picker Prism 2000 gamma camera interfaced to an Odyssey 750 computer.

To evaluate the longer term retention of the radiopharmaceutical in normal and tumour tissue, seven imaging studies were done using 1-131 ERC 9. 1-131 ERC 9, average 370 MBq (range 254 MBq - 396 MBq, containing approximately 2 mg of ERC 9 compound) , was administered by slow intravenous injection. The imaging procedure used for 1-131 ERC 9 was similar to that used for I- 123 ERC 9 except that daily whole body imaging was done for four to five days following administration.

Results of the radioiodinated ERC 9 studies were compared with histological, clinical, and other diagnostic imaging studies .

Of the 57 I-ERC 9 studies, 26 were true positive as confirmed by biopsy, other imaging techniques or clinical follow-up, 27 true negative, two were false positive and two were false negative, which gave a sensitivity and specificity of 93% on a per patient basis. Lesions visualized ranged from 0.8 to 8 cm in size. In several patients, previously unknown - 17 -

sites of metastatic disease were detected.

Typical imaging results are shown in Figures 1 and 2.

Figure 1 is a whole body image of a patient 3 days after administration of 1-131 ERC 9 and shows the long retention of the imaging agent in multiple tumour sites in the thigh, abdomen, lungs, shoulder and head.

Figure 2 shows sequential images as one centimetre slices through the thorax from back to front. Smaller tumours are seen in individual images but larger tumours can be seen on several consecutive images. The figure indicates the ability of the method of the invention to determine the exact location of metastases .

Example 6 Four patients with terminal malignant melanoma have been treated with between 3700 and 7400 MBq of 1-131 ERC 9 (2mg of compound) under the Health Canada Emergency Drug Release Programme .

One of these patients received two doses of 1-131 ERC 9 (total administered dose 7700 MBq) . Multiple whole body images were obtained post therapy to evaluate biodistribution and to calculate radiation dosimetry. Toxicity was assessed by serial complete blood count determinations. Response to the therapy was evaluated by clinical assessment . Short lived (<4 weeks) symptomatic relief (lessening of pain) was seen in two patients.

In two patients, thrombocytopenia (a drop in platelet count) was seen. No other adverse affects of the therapy was observed. In patients with cutaneous (skin) lesions these lesions showed erythema (reddening) followed by pallour after therapy. In one patient with a scalp cutaneous lesion, there was hair loss only at the lesion site following therapy. Both of these indicate that a significant radiation absorbed dose was delivered to the tumours. - 18 -

The calculated radiation absorbed dose delivered to the tumours ranged from 4.0 to 6.5 mGy per MBq administered. The red marrow, lung and whole body radiation absorbed doses were calculated to be 0.1-0.3, 0.3-0.5 and 0.2-0.3 mGy per MBg administered, respectively.

The relatively high radiation dose to tumours as compared to the total body indicates a good therapeutic ratio i.e. a good potential of effective killing of tumour cells as compared to potential side effects.

Example 7

Three patients with proven multiple myeloma received 1-123 ERC 9 (180 MBq, 152 MBq and 165 MBq, respectively) by slow intravenous injection. Anterior and posterior whole body images were obtained immediately after injection and at 3 - 4.5 hours after injection and, for patients 1 and 2, also at approximately 24 hours after injection.

All three patients had increased bone marrow uptake of the 1-123 ERC 9 at sites of known and suspected disease. In patient 1, there was abnormal non-homogenous uptake of the radiotracer in the spine, ribs, pelvis and proximal humeri and femora, all sites of known or suspected multiple myeloma involvement. There was also abnormal uptake in the spleen. Disease at this site has not been confirmed.

Figure 3 shows an anterior and posterior whole body image of patient 1 obtained 4.5 hours after the intravenous administration of 180 MBq of 1-123 ERC 9. The abnormal uptake of the radiotracer in the sites mentioned above can be readily seen.

Patient 2 had abnormal uptake in the sternum, lumbar spine, sacrum and sacro- iliac joints and in each femur and humerus .

Patient 3 had abnormal uptake in the spine and sacrum. In each case these were sites of known or suspected multiple - 19 -

myeloma involvement .

The present invention is not limited to the features of the embodiments described herein, but includes all variations and modifications within the scope of the claims.

20

Table 1

Biodistribution of 1-125 ERC 9 in C57BL/6CrlBR Black Mice Bearing B16F10 Melanoma Tumour Mean Percent of Injected Dose Per Gram (Standard Deviation) . n=5

Time After 30 Min 1 Hour 2 Hour 4 Hour 24 Hours Biol.Tl/2 *

Injection (Hours)

Lung IS.31 17.54 9.17 4.59 1.61 13.23

(1.99) (10.27) (1.82) (0.34) (0.27)

Heart 1.98 2.52 1.50 0.52 0.19 13.77

(0.50) (1.66) (0.51) (0.06) (0.02) Liver 6.45 5.57 4.75 2.67 0.94 13.28

(1.74) (1.34) (1.41) (0.20) (0.05) Spleen 11.06 13.47 5.83 2.1.3 0.47 8.66

(1.87) (8.10) (1.18) (0.47) (0.46) Kidney- 9.93 9.56 5.17 2.28 0.60 10.38

(1.69) (4.10) (1.25) (0.19) (0.12) Intestine 7.72 12.56 13.42 11.42 1.75

(1.18) (4.69) (4.51) (1.40) (0.58) Blood 0.91 1.06 0.67 0.39 0.09 9.45

(0.18) (0.50) (0.14) (0.02) (0.01) Brain 8.31 6.50 3.11 0.99 0.14 7.09

(1.81) (2.32) (0.68) (0.14) (0.04) Pancreas 28.28 37.65 38.83 34.81 20.44 30.36

(4.78) (14.84) (7.77) (6.23) (7.55) Tumour 6.90 7.84 6.07 7.51 4.58 36.49

(3.09) (1.90) (1.26) (2.00) (2.26)

Tumour/ 7.6 7.4 9.1 19.3 50.9 Blood #

Long lived component

# Mean percent injected dose gram tumour / Mean percent injected dose per gram blood 21

Table 2

Biodistribution of 1-125 ERC 46 in C57BL/6CrlBR Black Mice Bearing B16F10 Melanoma Tumours

Mean Percent of Injected Dose Per Gram (Standard Deviation) , n=5

Time After

Iniection 2 Hours 24 Hours

Lungs 17.66 (1. .40) 0.70 (0.21)

Heart 1.83 (0. .32) 0.23 (0.07)

Liver 2.03 (0, .14) 0.41 (0.08)

Spleen 14.49 (2. .57) 0.83 (0.42)

Kidney- 10.97 (1. .42) 0.69 (0.18)

Intestine 9.63 (0. .69) 1.97 (0.44)

Blood 0.48 (0. , 07) 0.04 (0.01)

Brain 6.08 (0. .34) 0.31 (0.12)

Pancreas 28.37 (1. .20) 34.96 (7.70)

Tumour 3.46 (2. .08) 4.09 (0.73)

Tumour/Blood* 7.2 102.3

* Mean percent injected dose per gram tumour / Mean percent injected dose per gram blood

- 22 -

Table 3

Biodistribution of 1-125 ERC 90 in C57BL/6CrlBR Black Mice Bearing B16F10 Melanoma Tumours

Mean Percent of Injected Dose Per Gram (Standard Deviation) , n=5

Time After

Iniection 2 Hours 24 Hours

Lungs 17.53 (3, .62) 0.78 (0.13)

Heart 2.00 (0 .30) 0.19 (0.04)

Liver 2.82 (0. .41) 0.46 (0.11)

Spleen 13.62 (3. .35) 1.02 (0.35)

Kidney- 11.53 (1. .67) 0.68 (0.16)

Intestine 9.91 (0. .92) 1.93 (0.22)

Blood 0.60 (0. .04) 0.05 (0.02)

Brain 5.23 (0. .54) 0.25 (0.04)

Pancreas 25.08 (3, .61) 30.35 (6.-⁷4)

Tumour 4.15 (1, .64) 3.36 (0. 7)

Tumour/Blood* 6.9 67.2

- 23 -

Table 4

Biodistribution of 1-125 ERC 94 in C57BL/6CrlBR Black Mice Bearing B16F10 Melanoma Tumours

Mean Percent of Injected Dose Per Gram (Standard Deviation) , n=5

Time After

Iniection 2 Hours 24 Hours

Lungs 11.41 (2. .90) 0.72 (0. .11)

Heart 1.06 (0. .28) 0.10 (0, .01)

Liver 2.00 (0. .30) 0.43 (0. .04)

Spleen 9.66 (2. .63) 0.91 (1, .24)

Kidney 6.20 (1. .83) 0.50 (0. .05)

Intestine 8.33 (1. .03) 0.81 (0. .23)

Blood 0.47 (0. .07) 0.03 (0. 0)

Brain 3.64 (0. ,69) 0.10 (0. .05)

Pancreas 24.67 (5. ,11) 29.32 (5. .23)

Tumour 3.41 (1. .34) 3.10 (1. .18)

Tumour/Blood* 7.3 103.3

Claims

- 24 -We claim :

1. A method for detecting a tumour in a subject, the method comprising

(a) administering to the subject a compound of the formula:

wherein n is 1 to 10; X is a radioactive halogen atom; R_x and R₂ are the same or different and are hydrogen, hydroxyl or lower alkyl; R₃ is lower alkyl; and N' is a nitrogen atom having two lower alkyl substituents or a nitrogen atom forming part of a 4- to 8- embered heterocyclic ring containing one or two hetero atoms, one of which is the nitrogen, the heterocyclic ring being unsubstituted or substituted with one or more lower alkyl groups , or a pharmaceutically acceptable acid addition salt thereof, in an amount effective for diagnostic imaging; and

(b) scanning the subject with a scintigraphic imaging device to detect uptake of the compound by the tumour.

2. The method of claim 1 wherein the administered compound is a compound of the formula:

^R1 ^ ^R 2

N-(CH₂)_n- R,

wherein n is 1 to 10; X is a radioactive halogen atom; R]_ and R are the same or different and are hydrogen, hydroxyl or lower alkyl; R₃ is lower alkyl; and N' is a nitrogen atom having two lower alkyl substituents or a nitrogen atom forming part of a morpholino, piperidino or pyrrolidino ring which is unsubstituted or substituted with one or more lower alkyl - 25 -

groups or a pharmaceutically acceptable acid addition salt thereof .

3. The method of claim 1 or 2 wherein X is F, Br or I.

4. The method of claim 1 or 2 wherein X is selected from the group consisting of 1-121, 1-123, 1-124, 1-125, 1-131, F-18, Br-76 and Br-77.

5. The method of claim 1 wherein the compound is selected from the group consisting of:

(a) N[3 (4-morpholino) propyl] -N-methyl-2 -hydroxy-3-methyl-5- halobenzylamine ;

(b) N[3 (1-pyrrolidino) propyl] -N-methyl -2 -hydroxy- 3 -methyl -5- halobenzylamine;

(c) N[3 (1-piperidino) propyl] -N-methyl-2-hydroxy-3-methyl-5- halobenzylamine; and

(d) N[3 (diethylamino) propyl] -N-methyl-2-hydroxy-3-methyl-5- halobenzylamine ; and the halogen atom is selected from the group consisting of 1-121, 1-123, 1-124, 1-125, 1-131, F-18, Br-76 and Br-77.

6. The method of claim 1 wherein the compound is 1-123 or I- 131 N[3 (4-morpholino) propyl] -N-methyl -2 -hydroxy- 3 -methyl -5- iodobenzylamine.

7. The method of claim 1 wherein the compound is 1-123 or I- 131 N[3 (1-pyrrolidino) propyl] -N-methyl -2 -hydroxy-3 -methyl-5- iodobenzylamine .

8. The method of claim 1 wherein the compound is 1-123 or I- 131 N[3 (1-piperidino) propyl] -N-methyl-2-hydroxy-3 -methyl-5- iodobenzylamine . - 26 -

9. The method of claim 1 wherein the compound is 1-123 or I- 131 N[3 (diethylamino) propyl] -N-methyl-2-hydroxy-3 -methyl-5- iodobenzylamine .

10. The method of any of the preceding claims wherein the compound is administered by intravenous .injection.

11. The method of any of the preceding claims wherein step (b) employs SPECT imaging.

12. The method of any of the preceding claims wherein step (b) employs planar imaging.

13. The method of any of the preceding claims wherein the tumour is a melanoma.

14. The method of any of the preceding claims wherein the tumour is a multiple myeloma.

15. The method of any of the preceding claims wherein the subject is a human.

16. A composition for detecting a' tumour in a subject comprising a compound of the formula:

wherein n is 1 to 10; X is a radioactive halogen atom; R_╬╗ and R₂ are the same or different and are hydrogen, hydroxyl or lower alkyl; R₃ is lower alkyl; and N' is a nitrogen atom having two lower alkyl substituents or a nitrogen atom forming part of a 4- to 8-membered heterocyclic ring containing one or two hetero - 27 -

atoms, one of which is the nitrogen, the heterocyclic ring being unsubstituted or substituted with one or more lower alkyl groups , or a pharmaceutically acceptable acid addition salt thereof, in an amount and of a specific activity effective for diagnostic imaging and a pharmaceutically acceptable vehicle.

17. The composition of claim 16 comprising a compound of the formula :

wherein n is 1 to 10; X is a radioactive halogen atom; R_x and

R₂ are the same or different and are hydrogen, hydroxyl or lower alkyl; R₃ is lower alkyl; and N¹ is a nitrogen atom having two lower alkyl substituents or a nitrogen atom forming part of a morpholino, piperidino or pyrrolidino ring which is unsubstituted or substituted with one or more lower alkyl groups or a pharmaceutically acceptable acid addition salt thereof .

18. The composition of claim 16 or 17 wherein X is F, Br or I.

19. The composition of claim 16 or 17 wherein X is selected from the group consisting of 1-121, 1-123, 1-124, 1-125, I- 131, F-18, Br-76 and Br-77.

20. The composition of claim 16 wherein the compound is selected from the group consisting of :

(a) N[3 (4-morpholino) propyl] -N-methyl-2 -hydroxy-3 -methyl-5- halobenzylamine ; (b) N [3 (1-pyrrolidino) propyl] -N-methyl-2-hydroxy-3-methyl-5- - 28 -

halobenzylamine ;

(c) N[3 (1-piperidino) propyl] -N-methyl -2 -hydroxy-3 -methyl- 5- halobenzylamine; and

21. The composition of claim 16 wherein the compound is 1-123 or 1-131 [3 (4 -morpholino) propyl] -N-methyl -2 -hydroxy-3 -methyl -

5 - iodobenzylamine .

22. The composition of claim 16 wherein the compound is 1-123 or 1-131 N [3 (1-pyrrolidino) propyl] -N-methyl-2 -hydroxy- 3 - methyl -5- iodobenzylamine .

23. The composition of claim 16 wherein the compound is 1-123 or 1-131 N [3 (1-piperidino) propyl] -N-methyl -2 -hydroxy- 3 -methyl - 5 - iodobenzylamine .

24. The composition of claim 16 wherein the compound is 1-123 or 1-131 N [3 (diethylamino) propyl] -N-methyl -2 -hydroxy- 3 -methyl - 5 - iodobenzylamine .

25. A method for treating a tumour in a subject, the method comprising administering to the subject a compound of the formula:

^N

X wherein n is 1 to 10; X is a radioactive halogen atom; R_x and R₂ are the same or different and are hydrogen, hydroxyl or lower - 29 -

alkyl; R₃ is lower alkyl; and N' is a nitrogen atom having two lower alkyl substituents or a nitrogen atom forming part of a 4- to 8-membered heterocyclic ring containing one or two hetero atoms, one of which is the nitrogen, the heterocyclic ring being unsubstituted or substituted with one or more lower alkyl groups , or a pharmaceutically acceptable acid addition salt thereof, in an amount and of a specific activity effective to inhibit the tumour.

26. The method of claim 25 wherein the administered compound is a compound of the formula:

N-(CH₂)

wherein n is 1 to 10; X is a radioactive halogen atom; R_x and R₂ are the same or different and are hydrogen, hydroxyl or lower alkyl; R₃ is lower alkyl; and N' is a nitrogen atom having two lower alkyl substituents or a nitrogen atom forming part of a morpholino, piperidino or pyrrolidino ring which is unsubstituted or substituted with one or more lower alkyl groups or a pharmaceutically acceptable acid addition salt thereof .

27. The method of claim 25 or 26 wherein X is I, At or Br.

28. The method of claim 25 or 26 wherein X is selected from the group consisting of 1-131, At-211, Br-76 and Br-77.

29. The method of claim 25 wherein the compound is selected from the group consisting of: (a) [3 (4-morpholino) propyl] -N-methyl-2-hydroxy-3-methyl-5- - 30 -

halobenzylamine ;

(b) N[3 (1-pyrrolidino) propyl] -N-methyl -2 -hydroxy-3 -methyl -5- halobenzylamine ;

(c) N[3 (1-piperidino) propyl] -N-methyl-2 -hydroxy-3 -methyl-5- halobenzylamine; and

(d) N[3 (diethylamino) propyl] -N-methyl-2 -hydroxy-3 -methyl-5- halobenzylamine ; and the halogen atom is selected from the group consisting of 1-131, At-211, Br-76 and Br-77.

30. The method of claim 25 wherein the compound is 1-131 N[3(4- morpholino) propyl] -N-methyl-2-hydroxy-3 -methyl-5- iodobenzylamine .

31. The method of claim 25 wherein the compound is 1-131 N[3(l- pyrrolidino) propyl] -N-methyl-2-hydroxy-3 -methyl-5- iodobenzylamine .

32. The method of claim 25 wherein the compound is 1-131 N[3(l- piperidino) propyl] -N-methyl -2 -hydroxy-3 -methyl-5- iodobenzylamine .

33. The method of claim 25 wherein the compound is 1-131 N[3 (diethylamino) propyl] -N-methyl -2 -hydroxy-3 -methyl-5- iodobenzylamine.

34. The method of any of claims 25 to 33 wherein the compound is administered by intravenous injection.

35. The method of any of claims 25 to 33 wherein the tumour is a melanoma.

36. The method of any of claims 25 to 33 wherein the tumour is a multiple myeloma. 31

37. The method of any of claims 25 to 33 wherein the subject is a huma .

38. A pharmaceutical composition for treating a tumour in a subject comprising a compound of the for╬▒ula:

wherein n is 1 to 10; X is a radioactive halogen atom; R_x and R₂ are the same or different and are hydrogen, hydroxyl or lower alkyl; R₃ is lower alkyl; and N' is a nitrogen atom having two lower alkyl substituents or a nitrogen atom forming part of a 4- to 8-membered heterocyclic ring containing one or two hetero atoms, one of which is the nitrogen, the heterocyclic ring being unsubstituted or substituted with one or more lower alkyl groups, or a pharmaceutically acceptable acid addition salt thereof, in an amount effective to inhibit the tumour and a pharmaceutically acceptable vehicle.

39. The pharmaceutical composition of claim 38 comprising a compound of the formula:

wherein n is 1 to 10; X is a radioactive halogen atom; R and R₂ are the same or different and are hydrogen, hydroxyl or lower alkyl; R₃ is lower alkyl; and N' is a nitrogen atom having two lower alkyl substituents or a nitrogen atom forming part of a morpholino, piperidino or pyrrolidino ring which is - 32 -

unsubstituted or substituted with one or more lower alkyl groups or a pharmaceutically acceptable acid addition salt thereof .

40. The pharmaceutical composition of claim 38 or 39 wherein X is I, At or Br .

41. The pharmaceutical composition of claim 38 or 39 wherein X is selected from the group consisting of 1-131, At-211, Br-76 and Br-77.

42. The pharmaceutical composition of claim 38 wherein the compound is selected from the group consisting of :

(a) N[3 (4-morpholino) propyl] -N-methyl-2-hydroxy-3 -methyl-5- halobenzylamine;

(c) N[3 (1-piperidino) propyl] -N-methyl-2-hydroxy-3-methyl-5- halobenzylamine; and (d) N [3 (diethylamino) propyl] -N-methyl-2-hydroxy-3-methyl-5- halobenzylamine ; and the halogen atom is selected from the group consisting of 1-131, At-211, Br-76 and Br-77.

43. The pharmaceutical composition of claim 38 wherein the compound is 1-131 N [3 (4-morpholino) propyl] -N-methyl -2 -hydroxy- 3 -methyl - 5 - iodobenzylamine .

44. The pharmaceutical composition of claim 38 wherein the compound is 1-131 N [3 (1-pyrrolidino) propyl] -N-methyl-2- hydroxy-3 -methyl-5- iodobenzylamine .

45. The pharmaceutical composition of claim 38 wherein the compound is 1-131 N [3 (1-piperidino) propyl] -N-methyl -2 -hydroxy- - 33 -

3 -methyl-5 - iodobenzylamine .

46. The pharmaceutical composition of claim 38 wherein the compound is 1-131 N [3 (diethylamino) propyl] -N-methyl -2 -hydroxy- 3 -methyl-5-iodobenzylamine .