WO1989006980A1

WO1989006980A1 - Radiopharmaceuticals

Info

Publication number: WO1989006980A1
Application number: PCT/AU1989/000025
Authority: WO
Inventors: Jeff J. Jacobs; Desmond J. Maddalena; J. Gerald Wilson
Original assignee: University Of Queensland; Australian Nuclear Science & Technology Organisati
Priority date: 1988-01-29
Filing date: 1989-01-27
Publication date: 1989-08-10

Abstract

Complexes between aminobenzyliminodiacetic acid derivatives and pharmaceutically acceptable radionuclides such as 99mTechnetium, 67Gallium and 111Indium are described. The complexes, when administered to a human or animal, concentrate in any tumours present enabling their size and location to be determined by scintigraphic imaging methods.

Description

TITLE: RADIOPHARMACEUTICALS

TECHNICAL FIELD

THIS INVENTION is related to radiopharmaceuticals. In particular, it is directed to the complexes between aminobenzyliminodiacetic acid derivatives and pharmaceutically acceptable radionuclides. These complexes are of use for the detection of tumours in animals and humans.

BACKGROUND ART

Although radionuclides such as ^99mVechnetium, Gallium and Indium are freely available, there are very few labelled radiopharmaceuticals currently available that will selectively seek a broad range of tumours. The only widely accepted clinically useful tumour diagnostic radiopharmaceutical is ⁶⁷Gallium citrate.

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide clinically useful radiopharmaceuticals which, when administered to a human or animal suspected of having one or more tumours within the body, will concentrate in the tumours enabling their size and location to be determined by scintigraphic imaging methods.

According to a first aspect of the present invention, there are provided complexes between:

(a) the compounds of the general formula (1):

wherein:

(a) R¹ and R² are radicals separately selected from the group consisting of hydrogen and alkyl;

R³, R⁴, R⁵, R⁶ and R⁷ are radicals separately selected from the group consisting of hydrogen, alkyl, alkoxy, halogen and the radical -NR⁸R⁹;

R⁸ and R⁹ are radicals separately selected from the group consisting of hydrogen, oxygen and alkyl;

with the proviso that at least one of R³ to R⁷ must represent the radical -NR⁸R⁹; and

(b) any pharmaceutically acceptable radionuclide.

The compounds of the general formula (1) are either (a) known and can be prepared, for example, by the procedure described by Awaluddin et al in Appl. Radiat: Isot., 38, 671-674 (1987); or (b) can be prepared from known compounds by standard reactions in the art.

Thus according to a second aspect of the present invention, there are provided compounds of the general formula (1) wherein R¹ to R⁹ are radicals as hereinbefore described.

Also, for novel compounds, as a third aspect of the present invention, there is provided a method for preparing compounds of the general formula (1), said method comprising the following steps: the reaction of compounds of general formula (2)

R¹O₂CH₂NHCH₂O₂R²

with a compound of general formula (3):

to produce a compound of general formula (1):

which if necessary, can be subjected to further selective reduction or hydrolysis processes; wherein X is a leaving group and the radicals R¹ to R⁹ are radicals as hereinbefore described.

Compounds of the general formulae (2) and (3) are either known or can be prepared from known compounds by standard reactions well known in the art.

Preferred compounds of general formula (1) are those in which R¹ and R² are radicals separately selected from the group consisting of hydrogen, methyl and ethyl; only one of R ³ to R⁷ represents -NR⁸R⁹, the remainder being radicals separately selected from the group consisting of hydrogen, methyl, ethyl, methoxy, chlorine , bromine and iodine; and R⁸ and R⁶ are either both hydrogen or both oxygen.

Of these preferred compounds, R¹ and R² are preferably either both hydrogen or both alkyl (most preferably, methyl) and the said remainder of R³ to R⁶ are either hydrogen or chlorine.

The radical X is any suitable leaving group known in the art such as tosylate or halogen. Preferably, the leaving group is halogen, more preferably, chloro.

The preferred radionuclides are ^99mTechnetium. Gallium and ¹¹¹Indium. The most preferred radionuclide is ^99mTechnetium.

According to a fourth aspect of the present invention there is provided a process for the preparation of a complex between a compound of general formula (1) as hereinbefore described and a pharmaceutically acceptable radionuclide comprising the reaction of a compound of general formula (1) and a salt of the radionuclide in the presence of a reducing agent.

The choice of reaction conditions for the process is dependent, in part, on the selection of the radionuclide. The most important aspects of the process are to change (if necessary) the valency state of the radionuclide so that it will complex with a compound of the general formula (1) and to control the pH at which this complexing occurs. This can be achieved with a variety of reducing agents, such as stannous, titanium and borohydride ions, following procedures well known in the art. The ions may not necessarily be in free solution but may be bound to solid substrates such as resins, plastics or glass. Consequently, the counter ion is relatively unimportant although it must be pharmaceutically acceptable.

Preferably, the radionuclide is ^99mTechnetium in the form of its sodium pertechnetate salt, and the preferred reducing agent is stannous chloride.

The complexes of the present invention, when administered to an animal (including humans) can be used to detect various types of tumours in the animal body.

Therefore, according to a fifth aspect of the present invention, there is provided a composition containing at least one complex as hereinbefore defined, in association with any pharmaceutical-type carrier or diluent which is suitable for its administration.

Also, according to a sixth aspect of the present invention, there is provided a method of detecting a tumour within an animal body, said method comprising:

1) administering to the animal suspected of having one or more tumours within its body a complex or a composition, both as hereinbefore defined; and 2) determining the location and size of any tumours by any suitable scintigraphic imaging method.

The present invention is particularly suited for the detection of EMT-6 sarcoma and the human melanoma strain MM96.

If looking for a particular type of tumour which may require a certain residence time for the radiopharmaceutical in the body, the rate and mode of excretion therefrom can be altered by controlling the lipophilicity of the complexes of the invention by the degree of substitution on the aromatic ring of the compounds of general formula (1). The presence of halogen radicals on this ring is particularly suited to this aspect of the invention.

DETAILED DESCRIPTION OF EMBODIMENTS

Specific details of the complexes of the present invention and their use in the detection of tumours are illustrated by the following examples. In these examples, technical terms have the usual meaning in the art. Crude reaction products can be purified by the means described herein, or by other means known in the art.

Example 1.

3-aminobenzyliminodiacetic acid dimethyl ester and 4-aminobenzyliminodiacetic acid dimethyl ester were prepared following the procedure described by Awaluddin et al (ibid).

Example 2.

The remaining compounds of formula (1) were prepared by the following general procedure.

Compounds of formula (2) were prepared from iminodiacetic acid hydrochloride following general esterification processes well known in the art. The thus esterified compound of formula (2) was then reacted with a compound of formula (3) by adapting a procedure described by Schwarzenbach et al in Helv. Chim. Acta, 35, 1794. For compounds of formula (1) wherein R¹ and/or R are required to be hydrogen, conventional ester hydrolysis techniques were used. Similarly, the radical -NR⁸R⁹ wherein R⁸ and R⁹ are each oxygen can be converted to the radical -NR⁸R⁹ wherein R⁸ and R⁹ are each hydrogen by conventional hydrogenation techniques, for example, PtO₂/H₂.

By using or adapting this general procedure, the following compounds were prepared:

4-aminobenzyliminodiacetic acid;

3-aminobenzyliminodiacetic acid; 2-amino-4-chlorobenzyliminodiacetic acid dimethyl ester;

4-(N,N'-dimethylamino)benzyliminodiacetic acid;

4-nitro-benzyliminodiacetic acid

4-chloro-2-nitro-benzyliminodiacetic acid 3-nitro-benzyliminodiacetic acid

3-nitro-4-chloroberϊzyliminodiacetic acid

4-nitro-benzyliminodiacetic acid dimethyl ester

4-chloro-2-nitro-benzyliminodiacetic acid dimethyl ester

3-nitro-benzyliminodiacetic acid dimethyl ester 3-nitro-4-chlorobenzyliminodiacetic acid dimethyl ester

Example 3.

To 70 ml Water-for-Injection. B.P. in a clean sterilised conical flask constantly purged with sterile oxygen-free nitrogen gas, is added 5ml IM NaOH and 100 mg of 4-aminobenzyliminodiacetic acid. The solution is mixed until the compound is dissolved and then 2.0 ml stannous chloride solution (20 mg SnCl₂.2H₂O in 10 ml 1M HCl) is added, the pH is adjusted to pH 7.5 with 1M HCl and the resultant solution diluted to 100 ml.

One millilitre aliquots are dispensed through a 0.22 micrometre membrane filter into 2ml sterile glass vials set in trays of dry ice, the water is removed by freeze drying, the vials sealed under vacuum then stored in a refrigerator at 4°C until use.

The complex is prepared by adding 1ml of sodium (^99mTC) pertechnetate to the vial.

Example 4.

A 330 mm length of 1.0 mm internal diameter polyvinyl chloride cannula tubing is coated internally with stannous chloride by passing a solution of 10 mg stannous chloride dihydrate in 5 ml 1M HCl through it three times followed by a single wash of nitrogen purged isotonic saline and a stream of oxygen-free nirogen gas to dry it. The tube is used within two hours of preparation.

To 3 ml 0.1M NaOH in a 10 ml glass vial is added 5 mg of 4-aminobenzyliminodiacetic acid dimethyl ester. The solution is stirred gently for 1 hour, the pH is then adjusted to pH 7.5 with 0.1M HCl solution, sodium (^99mTC) pertechnetate added, the solution diluted to 5 ml with water and then passed three times through a catheter tube coated internally with stannous ions as described above and finally passed through a 0.22 micrometre membrane filter into a clean sterile vial for use.

Example 5.

To 5mg of 3-aminobenzyliminodiacetic acid dissolved in 3 ml 0.1M NaOH is added sodium (^99mTC) pertechnetate solution in a volume less than 1 ml and the pH adjusted to between 6.5 and 7.5 with 1M HCl. Reduction of the pertechnetate is accomplished by the slow dropwise addition of 0.2 ml of 3.3M sodium borohydride in 0.1M sodium hydroxide solution over a period of 5 minutes with continual stirring. The reaction mixture is stirred for a further 30 minutes, diluted to 5 ml and passed through a 0.22 micrometre membrane filter into a sterile vial for use.

Example 6.

The remaining complexes of the present invention can be prepared by the following general procedure.

A compound of formula (1) (10mg) was dissolved in 0.1M NaOH (3 ml) and stannous chloride solution (0.3 ml of SnCl₂.2H₂O (20 mg) in 0.1M HCl) was added and the pH_. adjusted to between 7.0 and 7.5 by the dropwise addition of 0.1M HCl. An aqueous solution (1 ml) of the appropriate salt of the selected radionuclide was added and the mixture diluted to 5 ml. The final solution was passed through a 0.22 micrometre membrane filter for sterilisation and then purged with nitrogen gas prior to use.

Testing of the complexes of the present invention has established that they can detect EMT-6 sarcoma growing in specific pathogen free balb/c mice and the human melanoma strain MM96 growing in specific pathogen free nude mice.

Each biodistribution study was undertaken on groups of 3-5 mice. The animals were injected intravenously, via a tail vein, whilst under light diethylether anaesthesia, with 100μl ^99mTc-complex or sodium pertechnetate as a control, and sacrificed by overdose of diethylether at time periods between 30 minutes and24 hours post injection for tumour and tissue samples. The ^99mTc activity in the samples was counted in a Packard automatic gamma counter at a suitable window. Typical results are given in Tables 1 to 6.

TABLE 1 para - aminobenzyliminodiacetic acid

Means and standard devs . for 3 balb /c mice - EMT6 Tumora

TISSUE %INJ SD %/G SD T/M SD T/B SD %RE C

LIVER 7.45 1.72 4.905 1.120 62.3 11.4 7.41 1.57 10.34

SPLEEN 0.37 0.15 2.593 0.966 31.9 7.5 3.88 1.34 0.52

KIDNEY 1.73 0.36 3.760 1.025 47.5 8.8 5.64 1.15 2.37

MUSCLE 0.93 0.23 0.080 0.019 1.0 0.0 0.12 0.02 1.29

SKIN 7.50 4.17 1.361 0.625 17.2 6.7 2.06 0.93 10.59 BONE 1.25 0.29 0.390 0.086 4.9 0.1 0.58 0.09 1.74 LUNGS 0.41 0.53 1.097 0.862 12.9 7.9 1.61 1.16 0.56 HEART 0.05 0.01 0.352 0.073 4.4 0.2 0.53 0.07 0.07 BLOOD 1.18 0.11 0.661 0.050 8.5 1.6 1.00 0.00 1.63 UR+BLAD 34.80 11.20 2.647 0.520 35.5 15.6 4.06 1.08 46.89 TOT.GUT 13.26 2.27 13.261 2.265 174.9 65.0 20.15 4.00 17.99 STOMACH 1.98 1.45 1.977 1.452 23.6 13.2 2.91 1.94 2.69 GUT1 13.26 2.27 13.261 2.265 174.9 65.0 20.15 4.00 17.99 TUMOUR 2.43 3.13 6.601 3.518 79.0 27.2 9.75 4.56 3.28 PANCRES 0.03 0.01 0.261 0.086 3.2 0.5 0.39 0.11 0.04

TABLE 2 meta - aminobenzyliminodiacetic acid

Means and standard devs. for 3 nude mice - Melanoma mm96

TISSUE %INJ SD %/G SD T/M SD T/B SD %RE

LIVER 5.80 1.67 5.524 0.826 35.4 4.4 20.56 11.01 6.84 SPLEEN 0.43 0.05 3.213 1.983 21.5 13.4 11.72 10.69 0.46

KIDNEY 1.06 0.39 3.773 1.066 23.7 2.3 14.52 9.04 1.32

MUSCLE 1.19 0.21 0.158 0.034 1.0 0.0 0.61 0.35 1.35

SKIN 3.59 0.70 1.020 0.138 6.7 2.3 3.57 1.47 3.41

BONE 1.03 0.45 0.490 0.219 3.1 0.9 1.87 1.40 1.27

LUNGS 0.18 0.11 1.063 0.493 6.5 1.8 4.34 3.13 0.25

HEART 0.05 0.01 0.518 0.163 3.2 0.4 2.00 1.29 0.06

BLOOD 0.42 0.29 0.348 0.218 2.4 2.1 1.00 0.00 0.26

UR+BLAD 29470.89 50957.78 2.255 1.337 15.6 10.5 7.52 5.98 73.0

TOT.GUT 7.57 4.38 4.726 2.729 29-6 13.5 17.68 15.42 9.28

STOMACH 1.73 1.00 10.285 5.859 63.1 24.7 40.19 34.17 2.31

GUT1 7.57 4.38 4.726 2.729 29.6 13.5 17.68 15.42 9.28

TUMOUR 0.09 0.03 0.692 0.326 4.2 1.1 2.77 2.07 0.11

TUMOUR 2 0.02 0.01 1.772 0.901 10.8 3.3 7.17 5.51 0.01

Table 3

3-amino-4chloro-benzyliminodiacetic acid

Means and standard deviations for 3 balb/c mice -EMT6 TUMOR Four Hours TISSUE %INJ SD %/G SD T/M SD T/B SD %REC

LIVER 13.14 0.57 8.738 0.914 47.3 3.4 7.88 0.23 15.17

SPLEEN 0.37 0.04 2.998 0.731 16.0 2.5 2.67 0.41 0.43KIDNEY 5.50 0.75 9.593 1.343 52.7 12.6 8.75 1.80 6.25

MUSCLE 2.56 0.29 0.186 0.024 1.0 0.0 0.17 0.01 2.98

SKIN 6.34 0.68 0.991 0.124 5.3 0.0 0.89 0.04 7.36

BONE 2.49 0.34 0.649 0.096 3.5 0.2 0.58 0.03 2.89 LUNGS 0.23 0.01 1.109 0.081 6.0 0.4 1.00 0.03 0.27

HEART 0.07 0.01 0.411 0.042 2.2 0.1 0.37 0.01 0.08

BLOOD 2.36 0.19 1.110 0.115 6.0 0.3 1.00 0.00 2.73

UR+

BLAD 33.67 15.70 0.422 0.396 2.2 2.1 0.36 0.34 37.26

SKM. 0.48 0.17 1.631 0.628 8.9 3.5 1.47 0.52 0.54

GUT 18.15 2.88 7.902 1.592 42.3 3.6 7.07 0.76 21.07

TUMOR 0.05 0.02 1.07 0.314 5.7 1.0 0.95 0.21 0.05

FAECES 2.45 1.84 6.791 5.041 34.9 25.5 5.84 4.19 2.91

Table 4 2-amino-4 chloro-benzyliminodiacetic acid

Means and standard deviations for 3 balb/c mice -EMT6 TUMOR

Four Hours

TISSUE %INJ SD %/G SD T/M SD T/B SD %REC

LIVER 11.96 2.49 9.185 2.126 38.5 12.1 4.73 1.27 15.33 SPLEEN 0.19 0.03 1.459 0.494 6.2 2.7 0.76 0.30 0.24

KINEY 4.64 0.73 8.529 0.646 35.6 5.9 4.40 0.60 5.98

MUSCLE 3.34 0.22 0.242 0.024 1.0 0.0 0.12 0.01 4.40

SKIN 4.82 0.87 0.759 0.171 3.1 0.6 0.39 0.05 6.25

BONE 2.16 0.35 0.560 0.067 2.3 0.5 0.29 0.06 2.83LUNGS 0.24 0.01 1.408 0.232 5.8 0.4 0.72 0.09 0.32

HEART 0.10 0.02 0.652 0.075 2.7 0.1 0.33 0.01 0.14

BLOOD 4.09 0.31 1.956 0.221 8.1 0.5 1.00 0.00 5.35

UR +

BLAD 21.92 5.30 0.231 0.196 0.9 0.8 0.12 0.10 28.10

STOM. 0.57 0.15 2.407 0.549 10.2 3.3 1.26 0.39 0.75

GUT 19.96 5.40 8.668 2.440 36.4 13.2 4.47 1.41 25.44

TUMOR 0.27 0.16 1.097 0.111 4.5 0.3 0.56 0.01 0.36FAECES 3.26 2.89 7.791 7.429 30.3 28.3 3.78 3.42 4.50

Table 5

4-nitro-benzyliminodiacetic acid

Means and standard deviations for 3 balb/c mice - EMT6 tumor 2 Hours Post Injection

%INJ SD %/G SD T/M SD T/B SD

LIVER 23.38 4.53 18.30 4.92 28.3 7.6 6.9 1.9

SPLEEN 0.56 0.22 3.30 0.17 5.1 0.3 1.2 0.1

KIDNEY 4.56 0.95 12.86 1.70 19.9 2.6 4.9 0.6

MUSCLE 6.56 1.98 0.65 0.22 1.0 0.3 0.2 0.1

SKIN 7.38 1.88 1.57 0.47 2.4 0.7 0.6 0.2

BONE 3.16 0.59 1.11 0.13 1.7 0.2 0.4 0.0

LUNGS 0.38 0.15 2.01 0.46 3.1 0.7 0.8 0.2

HEART 0.12 0.03 0.91 0.28 1.4 0.4 0.3 0.1

BLOOD 4.38 1.46 2.65 0.71 4.1 1.1 1.0 0.3

U R +

BLAD 4.16 0.96 0.00 0.00 0.0 0.0 0.0 0.0

STOM. 6.81 1.38 35.79 8.53 55.3 13.2 13.5 3.2

GUT 13.62 2.36 8.50 1.93 13.1 3.0 3.2 0.7

TUMOR 5.92 3.39 14.57 2.64 22.5 4.1 5.5 1.0

Table 6

2-nitro-4-chloro-benzyliminodiacetic acid

%INJ SD %/G SD T/M SD T/B SD

LIVER 18.13 2.69 11.38 0.72 28.3 1.8 3.8 1.9

SPLEEN 0.48 0.12 3.13 0.74 7.8 1.8 1.0 0.1

KIDNEY 2.37 0.38 4.28 0.71 10.6 1.8 1.4 0.6

MUSCLE 5.21 1.47 0.40 0.11 1.0 0.3 0.1 0.1

SKIN 8.26 1.99 1.38 0.30 3.4 0.8 0.5 0.2

BONE 3.79 0.92 1.05 0.22 2.6 0.5 0.3 0.0

LUNGS 0.4 0.1 1.93 0.49 4.8 1.2 0.6 0.2

HEART 0.15 0.03 0.93 0.18 2.3 0.5 0.3 0 .1

BLOOD 5.81 1.17 3.01 0.59 7.5 1.5 1.0 0.3

U R +

BLAD 2.64 2.86 0.00 0.00 0.0 0.0 0.0 0 .0

STOM. 7.1 0.87 31.07 6.80 77.3 16.9 10.3 3 .2

GUT 15.62 3.53 7.68 2.01 19.1 5.0 2.6 0.7

TUMOR 3.91 2.05 9.49 2.08 23.6 5.2 3.2 1.0

The complexes of the present invention, when used in conjunction with conventional scintigraphic imaging apparatus, can detect a number of types of tumours and have a greater affinity for concentrating in tumours than other radiopharmaceuticals currently available, particularly those that contain the radionuclide Technetium.

Since ^99mTechnetium has very good physical properties for scintigraphic imaging, is widely available and relatively inexpensive, the complexes of the present invention which utilise that particular radionuclide should provide a comparatively inexpensive diagnostic tool for the early detection of tumours in animals, including humans.

It will be appreciated that the above is given by way of exemplification of the invention only and that changes may be made to the details set out therein without departing from the scope of the invention as defined in the following claims.

Claims

Complexes between compounds of the general formula

wherein :

R¹, R⁴, R⁵, R⁶ and R⁷ are radicals separately selected from the group consisting of hydrogen, alkyl, alkoxy, halogen and the radical - NR⁸R⁹;

(b) any pharmaceutically acceptable radionuclide.

Compounds of the general formula (1) as defined in Claim 1.

3. Compounds of the general formula (1) as defined in Claim 1 or claim 2 wherein R¹ and R² are radicals separately selected from the group consisting of hydrogen, methyl and ethyl; only one of R³ to R⁷ represents -NR⁸R⁹, the remainder being radicals separately selected from the group consisting of hydrogen, methyl, ethyl, methoxy, chlorine, bromine and iodine; and R⁸ and R⁹ are either both hydrogen or both oxygen.

4. Compounds as defined in Claim 3 wherein R¹ and R² are either both hydrogen or both alkyl, and the said remainder of R³ to R⁶ are either hydrogen or chlorine.

5. Compounds of the general formula (1) as defined in Claim 4 wherein R¹ and R² are both methyl.

6. Compounds of the general formula (1) as defined in Claim 4 wherein R¹ and R² are both hydrogen.

7. 2-Amino-4-chlorobenzyliminodiacetic acid dimethylester.

8. 4-(N,N'-dimethylamino)benzyliminodiacetic acid.

9. 4-Nitro-benzyliminodiacetic acid

10. 4-Chloro-2-nitro-benzyliminodiacetic acid

11. 3-Nitro-benzyliminodiacetic acid

12. 3-Nitro-4-chlorobenzyliminodiacetic acid

13. 4-nitro-benzyliminodiacetic acid dimethyl ester

14. 4-chloro-2-nitro-benzyliminodiacetic acid dimethyl ester

15. 3-nitro-benzyliminodiacetic acid dimethyl ester

16. 3-nitro-4-chlorobenzyliminodiacetic acid dimethyl ester

17. Complexes as defined in Claim 1 comprising compounds of the general formula (1) as defined in any one of Claims 1 to 16 wherein the radionuclide is selected from 99mTechnetium, ⁶⁷Gallium and 1¹¹Indium.

18. Complexes as defined in Claim 17 wherein the radionuclide is ^99mTechnetium in the form of its sodium pertechnetate salt

19. A process for the preparation of a complex between a compound of general formula (1) and a pharmaceutically acceptable radionuclide comprising the reaction of a compound of general formula (1) and a salt of the radionuclide in the presence of a reducing agent.

20. A process as defined in Claim 19 wherein the reducing agent is stannous chloride.

21. A composition comprising at least one complex as defined in any one of Claims 1, 17 or 18 in association with a pharmaceutical carrier or diluent.

22. A method of detecting a tumour within an animal body, said method comprising:

1) administering to the animal suspected of having one or more tumours within its body a complex or a composition, both as hereinbefore defined; and

2) determining the location and size of any tumours by any suitable scintigraphic imaging method.

23. Complexes as defined in Claim 1 substantially as hereindescribed with reference to the Examples.

24. Compounds as the general formula (1) substantially as hereindescribed with reference to the Examples.

25. A process for the preparation of a complex between a compound of general formula (1) and a pharmaceutically acceptable radionuclide substantially as hereindescribed with reference to the Examples.