PSMA BINDER AND USE THEREOF

Abstract

The present invention discloses a prostate specific membrane antigen (PSMA) binding compound, a radioactive isotope complex thereof, and the use thereof in nuclear medicine as a tracer and an imaging agent for different disease states of prostate cancer.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, prodrug or ester thereof, wherein the formula (I) is: ##STR00035## wherein: m is an integer from 0 to 5; n is an integer from 0 to 5; f and g each is 0 or 1; R and R′ each is independently selected from H, alkyl, halogen, —CN, —OH, —NH.sub.2, alkoxy or cycloalkyl; Q is —COOH, —SOOH, —SO.sub.3H, —SO.sub.4H, —POOH, —PO.sub.3H or —PO.sub.4H.sub.2; X is an optionally substituted aryl or an optionally substituted heteroaryl, which is substituted by at least one R group; Y is an optionally substituted aryl, an optionally substituted heterocyclic aryl, an optionally substituted cycloalkyl, or an optionally substituted heterocycloalkyl, which is substituted by at least one R group; AA.sub.1 is a natural or non-natural amino acid, or —CH.sub.2CH.sub.2—.

2. The compound of formula (I) or the pharmaceutically acceptable salt, prodrug or ester thereof according to claim 1, wherein the formula (I) is a compound represented by formula (I-1): ##STR00036##

3. The compound of formula (I) or the pharmaceutically acceptable salt, prodrug or ester thereof according to claim 1, wherein: the R and R′ each is independently selected from H and C.sub.1-C.sub.10 alkyl; the X is an optionally substituted phenyl, naphthyl, biphenyl, indolyl, benzothiazolyl or quinolinyl; the optionally substituted heterocycloalkyl is selected from N-piperidinyl or N-methylated piperidinium.

4. The compound of formula (I) or the pharmaceutically acceptable salt, prodrug or ester thereof according to claim 1, wherein: the AA.sub.1 is ##STR00037## wherein ##STR00038## and R′═H, COOH, CH.sub.2COOH, C.sub.2H.sub.4COOH, CH(COOH).sub.2, CH(CH.sub.2COOH).sub.2, CH(COOH)(CH.sub.2COOH), CH.sub.2CH(COOH).sub.2 or SO.sub.3H; i=1-3; and R═H or CH.sub.3.

5. The compound according to claim 1, having the following structures: ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046##

6. A .sup.99mTc complex of the compound of formula (I) according to claim 1, having a structure of formula (II): ##STR00047## wherein Q, R, X, f, Y, g, m, R′, AA.sub.1 and n are as defined in claim 1, and L is N-tris(hydroxymethyl)methylglycine, ethylenediamine-N,N′-diacetic acid, triphenylphosphine-3,3′,3″-trisulfonic acid trisodium, disodium 3,3′-(phenylphosphinediyl)bis(benzene-1-sulphonate), sodium diphenylphosphinobenzene-3-sulfonate, nicotinic acid, glucoheptonate, glucosamine, mannitol, or diphenylphosphinobenzoic acid.

7. The complex according to claim 6, wherein the complex is a compound represented by formula (II-1): ##STR00048##

8. The complex according to claim 6, wherein: the R and R′ each is independently selected from H and C.sub.1-C.sub.10 alkyl; the X is an optionally substituted phenyl, naphthyl, biphenyl, indolyl, benzothiazolyl or quinolinyl; the optionally substituted heterocycloalkyl is selected from N-piperidinyl or N-methylated piperidinium.

9. The complex according to claim 6, wherein: the AA.sub.1 is ##STR00049## wherein ##STR00050## and R′═H, COOH, CH.sub.2COOH, C.sub.2H.sub.4COOH, CH(COOH).sub.2, CH(CH.sub.2COOH).sub.2, CH(COOH)(CH.sub.2COOH), CH.sub.2CH(COOH).sub.2, or SO.sub.3H; i=1-3; and R═H or CH.sub.3.

10. The complex according to claim 6, wherein the complex is selected from the following compounds: ##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058##

11. A preparation method of the complex according to claim 6, comprising: formulating 0.5-2 ml of a mixture containing 1-100 μg of the compound according to claim 1, 0-500 μg of stannous chloride, 1-50 mg of a ligand L, 20-50 mg of disodium succinate, 5-30 mg of succinic acid, and 0-100 mg of mannitol in a 10-mL vial; adding 0.5-2 mL of Na.sup.99mTcO.sub.4 solution (10-100 mCi); heating the vial in a 100° C. water bath to carry out reaction for 10-20 minutes; and cooling at room temperature for 10 minutes after the reaction is completed; thus obtaining the complex according to claim 6; wherein the ligand L is selected from N-tris(hydroxymethyl)methylglycine, ethylenediaminediacetic acid, triphenylphosphine-3,3′,3″-trisulfonate, disodium 3,3′-(phenylphosphinediyl)bis(benzene-1-sulphonate), sodium diphenylphosphinobenzene-3-sulfonate, nicotinic acid, glucoheptonate, glucosamine, mannitol, or diphenylphosphinobenzoic acid.

12. A pharmaceutical composition comprising the compound according to claim 1, or the pharmaceutically acceptable salt, prodrug or ester thereof, and a pharmaceutically acceptable carrier, wherein the complex having a structure of formula (II): ##STR00059## wherein Q, R, X, f, Y, g, m, R′, AA.sub.1 and n are as defined in claim 1, and L is N-tris(hydroxymethyl)methylglycine, ethylenediamine-N,N′-diacetic acid, triphenylphosphine-3,3′,3″-trisulfonic acid trisodium, disodium 3,3′-(phenylphosphinediyl)bis(benzene-1-sulphonate), sodium diphenylphosphinobenzene-3-sulfonate, nicotinic acid, glucoheptonate, glucosamine, mannitol, or diphenylphosphinobenzoic acid.

13. Use of the compound according to claim 1 or a complex of the compound according to claim 1 or the pharmaceutically acceptable salt, prodrug or ester thereof in the preparation of a reagent for imaging in a patient, wherein the complex having a structure of formula (II): ##STR00060## wherein Q, R, X, f, Y, g, m, R′, AA.sub.1 and n are as defined in claim 1, and L is N-tris(hydroxymethyl)methylglycine, ethylenediamine-N,N′-diacetic acid, triphenylphosphine-3,3′,3″-trisulfonic acid trisodium, disodium 3,3′-(phenylphosphinediyl)bis(benzene-1-sulphonate), sodium diphenylphosphinobenzene sulfonate, nicotinic acid, glucoheptonate, glucosamine, mannitol, or diphenylphosphinobenzoic acid.

14. Use of the compound according to claim 1 or a complex of the compound according to claim 1 or the pharmaceutically acceptable salt, prodrug or ester thereof in the preparation of a reagent for diagnosing prostate cancer and/or its metastases, wherein the complex having a structure of formula (II): ##STR00061## wherein Q, R, X, f, Y, g, m, R′, AA.sub.1 and n are as defined in claim 1, and L is N-tris(hydroxymethyl)methylglycine, ethylenediamine-N,N′-diacetic acid, triphenylphosphine-3,3′,3″-trisulfonic acid trisodium, disodium 3,3′-(phenylphosphinediyl)bis(benzene-1-sulphonate), sodium diphenylphosphinobenzene-3-sulfonate, nicotinic acid, glucoheptonate, glucosamine, mannitol, or diphenylphosphinobenzoic acid.

15. Use of the compound according to claim 1 or a complex of the compound according to claim 1 or the pharmaceutically acceptable salt, prodrug or ester thereof in the preparation of a drug for treating prostate cancer and/or its metastases, wherein the complex having a structure of formula (II): ##STR00062## wherein Q, R, X, f, Y, g, m, R′, AA.sub.1 and n are as defined in claim 1, and L is N-tris(hydroxymethyl)methylglycine, ethylenediamine-N,N′-diacetic acid, triphenylphosphine-3,3′,3″-trisulfonic acid trisodium, disodium 3,3′-(phenylphosphinediyl)bis(benzene-1-sulphonate), sodium diphenylphosphinobenzene-3-sulfonate, nicotinic acid, glucoheptonate, glucosamine, mannitol, or diphenylphosphinobenzoic acid.

16. Use of the pharmaceutical composition according to claim 12 in the preparation of a reagent for imaging in a patient.

17. Use of the pharmaceutical composition according to claim 12 in the preparation of a reagent for diagnosing prostate cancer and/or its metastases.

18. Use of the pharmaceutical composition according to claim 12 in the preparation of a drug for treating prostate cancer and/or its metastases.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 shows an HPLC UV-vis spectrum of HYNIC-PSMA-XL-2.

[0034] FIG. 2 shows a mass spectrum of HYNIC-PSMA-XL-2.

[0035] FIG. 3 shows an HPLC UV spectrum of HYNIC-PSMA-XL-3.

[0036] FIG. 4 shows a mass spectrum of HYNIC-PSMA-XL-3.

[0037] FIG. 5 shows a Radio-HPLC spectrum of .sup.99mTc-HYNIC/EDDA-PSMA-XL-2.

[0038] FIG. 6 shows a Radio-HPLC spectrum of .sup.99mTc-HYNIC/EDDA-PSMA-XL-3.

[0039] FIG. 7 shows a Radio-HPLC spectrum of .sup.99mTc-HYNIC/TPPTS-PSMA-XL-2.

[0040] FIG. 8 shows a Radio-HPLC spectrum of .sup.99mTc-HYNIC/TPPTS-PSMA-XL-3.

[0041] FIG. 9 shows the stability of .sup.99mTc-HYNIC/EDDA-PSMA-XL-2 and .sup.99mTc-HYNIC/EDDA-PSMA-XL-3 in vitro.

[0042] FIG. 10 shows an SPECT/CT image of .sup.99mTc-HYNIC/EDDA-PSMA-XL-2 imaging under a LNCaP tumor model.

[0043] FIG. 11 shows an SPECT/CT image of .sup.99mTc-HYNIC/EDDA-PSMA-XL-3 imaging under the LNCaP tumor model.

[0044] FIG. 12 shows an SPECT/CT image of .sup.99mTc-HYNIC/EDDA-PSMA-XL-2 imaging under a PC-3 tumor model.

[0045] FIG. 13 shows an SPECT/CT image of .sup.99mTc-HYNIC/EDDA-PSMA-XL-3 imaging under the PC-3 tumor model.

[0046] FIG. 14 shows the in vivo distribution of .sup.99mTc-HYNIC/EDDA-PSMA-XL-3 under the LNCaP tumor model.

[0047] FIG. 15 shows a target-to-background ratio (TBR) image of different tissues undergoing SPECT/CT imaging after injection of .sup.99mTc-HYNIC-PSMA-XL-3 into a prostate cancer patient.

[0048] FIG. 16 shows typical images of SPECT/CT imaging after injection of .sup.99mTc-HYNIC-PSMA-XL-3 into a patient with primary prostate cancer.

[0049] FIG. 17 shows typical images of SPECT/CT imaging after injection of .sup.99mTc-HYNIC-PSMA-XL-3 into a prostate cancer patient with multiple systemic metastases.

[0050] FIG. 18 shows pictures of .sup.99mTc-HYNIC-PSMA-XL-3 SPECT/CT imaging tumors and corresponding immunohistochemical pictures of surgical specimens in a prostate cancer patient. The results show that the .sup.99mTc-HYNIC-PSMA-XL-3 SPECT/CT imaging tumors in a prostate cancer patient were highly consistent with the immunohistochemical PSMA expression of the corresponding surgical specimens.

[0051] FIG. 19 shows primary tumors of a prostate cancer patient. The commonly used PSMA molecular probes .sup.99mTc-HYNIC-ALUG and .sup.68Ga-PSMA1 1 in clinic both have high physiological distributions in the bladder (A/B in FIG. 19), which affects the distinction between prostate tumors and bladder physiological regions. However, the .sup.99mTc-HYNIC-PSMA-XL-3 of the present invention has a less distribution of radioactivity in the bladder (C in FIG. 19), therefore, distinguishing prostate cancer tumors from normal tissue structures clearly is achieved, and laying the foundation for further clinical puncture, surgery and other applications.

DETAILED DESCRIPTION

[0052] The following embodiments describe the present invention in detail, and these embodiments are provided for illustration only, they should not to be construed in any way as limitation of the present invention.

Embodiment 1

Synthesis of Glu-Urea-Lys-2Nal-AMB-Glu-Glu-HYNIC (HYNIC-PSMA-XL-2)

[0053] It was synthesized by solid-phase synthesis using 2 mmol of tert-butyl ester-protected glutamic acid immobilized on 2-CTC resin as starting material, wherein 2 mol of N,N′-carbonyldiimidazole was added and then reacted at room temperature overnight. The unreacted N,N′-carbonyldiimidazole was eluted with DMF, methyl trifluoromethanesulfonate and triethylamine were added to react for 1 h, and then Fmoc-Lys(OtBu)-NH2 was added to react for 2 h. Then 1.96 mmol of HOBt and 2 mmol of DIC were used as amidation catalysts in DMF, followed by adding 2 mmol of Fmoc-2Nal-OH, Fmoc-4-aminomethyl-benzoic acid, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH and Boc-HYNIC successively. Finally, the 2-CTC resin and the tert-butyl ester were removed with a mixture consisting of trifluoroacetic acid, triisopropylsilane and water (95/2.5/2.5) to obtain a crude product.

[0054] The obtained crude product was separated and purified by preparative RP-HPLC, and then the purified product was analyzed by analytical RP-HPLC and LC-MS. The HPLC spectrum was shown in FIG. 1, and the mass spectrum was shown in FIG. 2.

##STR00027## ##STR00028##

Embodiment 2

Synthesis of Glu-Urea-Lys-2Nal-AMB-Glu-HYNIC (HYNIC-PSMA-XL-3)

[0055] It was synthesized by solid-phase synthesis using 2 mmol of tert-butyl ester-protected glutamic acid immobilized on 2-CTC resin as starting material, wherein 2 mol of N,N′-carbonyldiimidazole was added and then reacted at room temperature overnight. The unreacted N,N′-carbonyldiimidazole was eluted with DMF, methyl trifluoromethanesulfonate and triethylamine were added to react for 1 h, and then Fmoc-Lys(OtBu)-NH2 was added to react for 2 h. After that, 1.96 mmol of HOBt and 2 mmol of DIC were used as amidation catalysts in DMF, followed by adding 2 mmol of Fmoc-2Nal-OH, Fmoc-4-aminomethyl-benzoic acid, Fmoc-Glu(OtBu)-OH and Boc-HYNIC successively. Finally, the 2-CTC resin and the tert-butyl ester were removed with a mixture consisting of trifluoroacetic acid, triisopropylsilane and water (95/2.5/2.5) to obtain a crude product.

[0056] The obtained crude product was separated and purified by preparative RP-HPLC, and then the purified product was analyzed by analytical RP-HPLC and LC-MS. The HPLC spectrum was shown in FIG. 3, and the mass spectrum was shown in FIG. 4.

##STR00029## ##STR00030##

Embodiment 3

HYNIC-PSMA-XL-2 and HYNIC-PSMA-XL-3 Affinity Assay

[0057] The binding capacity of HYNIC-PSMA-XL-2 and HYNIC-PSMA-XL-3 to PSMA protein was determined by Surface Plasmon Resonance (SPR). Since the dissociations of HYNIC-PSMA-XL-2 and HYNIC-PSMA-XL-3 were both very slow with a kd beyond the detection limit of the instrument (1E−6), the final calculated KD of the binding of HYNIC-PSMA-XL-2 and PSMA protein should be less than 6.43 pM, and the calculated KD of the binding of HYNIC-PSMA-XL-3 and PSMA protein should be less than 4.857 pM based on the ka results. In order to compare with the reported PSMA inhibitors, the binding capacity of PSMA11, .sup.19F-PSMA1007, 2-PMPA and HYNIC-ALUG compounds publicly reported by our research group were determined by the same method. The specific KD values are listed in Table 1.

TABLE-US-00001 TABLE 1 Affinity KD values of different PSMA inhibitors to PSMA KD value Compound (SPR method) HYNIC-PSMA-XL-2 <6.43 pM HYNIC-PSMA-XL-3 <4.897 pM 2-PMPA 9.868 nM PSMA11 1.255 nM .sup.19F-PSMA1007 64.92 nM HYNIC-ALUG 299.4 nM

Embodiment 4

Preparation of .SUP.99m.Tc-HYNIC/EDDA-PSMA-XL-2 Complex

[0058] 1 μg of HYNIC-PSMA-XL-2, 5 mg of ethylenediamine-N,N′-diacetic acid, 50 mg of disodium succinate, 30 mg of succinic acid, 20 mg of Tricine, 100 mg of mannitol and 30 μg of stannous chloride were dissolved in 1 ml of sterile water for injection, then 0.5 ml of Na.sup.99mTcO.sub.4 eluent (1100 MBq) was added, after that the mixture was reacted in a boiling water bath for 20 min to obtain the target compound .sup.99mTc-HYNIC/EDDA-PSMA-XL-2. The target compound has a radiochemical purity greater than 99% determined by Radio-HPLC. The relevant analytical spectrum was shown in FIG. 5.

##STR00031##

Embodiment 5

Preparation of .SUP.99m.Tc-HYNIC/EDDA-PSMA-XL-3 Complex

[0059] 10 μg of HYNIC-PSMA-XL-3, 50 mg of ethylenediamine-N,N′-diacetic acid, 20 mg of disodium succinate, 10 mg of succinic acid, 1 mg of Tricine, 40 mg of mannitol and 500 μg of stannous chloride were dissolved in 0.5 ml of sterile water for injection, then 2 ml of Na.sup.99mTcO.sub.4 eluent (1100 MBq) was added, after that the mixture was reacted in a boiling water bath for 20 min to obtain the target compound .sup.99mTc-HYNIC/EDDA-PSMA-XL-3. The target compound has a radiochemical purity greater than 99% determined by Radio-HPLC. The relevant analytical spectrum was shown in FIG. 6.

##STR00032##

Embodiment 6

Preparation of .SUP.99m.Tc-HYNIC/TPPTS-PSMA-XL-2 Complex

[0060] 50 μg of HYNIC-PSMA-XL-2, 1 mg of triphenylphosphine-3,3′,3″-trisulfonic acid trisodium, 35 mg of disodium succinate, 5 mg of succinic acid, 15 mg of Tricine and 10 μg of stannous chloride were dissolved in 0.8 ml of sterile water for injection, then 1 ml of Na.sup.99mTcO.sub.4 eluent (1100 MBq) was added, after that the mixture was reacted in a boiling water bath for 20 min to obtain the target compound .sup.99mTc-HYNIC/TPPMS-PSMA-XL-2. The target compound has a radiochemical purity greater than 99% determined by Radio-HPLC. The relevant analytical spectrum was shown in FIG. 7.

##STR00033##

Embodiment 7

Preparation of the .SUP.99m.Tc-HYNIC/TPPTS-PSMA-XL-3 Complex

[0061] 100 μg of HYNIC-PSMA-XL-3, 25 mg of Triphenylphosphine-3,3′,3″-trisulfonic acid trisodium, 45 mg of disodium succinate, 20 mg of succinic acid, 50 mg of Tricine and 60 mg of mannitol were dissolved in 2 ml of sterile water for injection, then 1 ml of Na.sup.99mTcO.sub.4 eluent (1100 MBq) was added, after that the mixture was reacted in a boiling water bath for 20 min to obtain the target compound .sup.99mTc-HYNIC/TPPMS-PSMA-XL-3. The target compound has a radiochemical purity greater than 99% determined by Radio-HPLC. The relevant analytical spectrum was shown in FIG. 8.

##STR00034##

Embodiment 8

In Vitro Stability Experiments of the Complexes

[0062] A certain amount of .sup.99mTc-HYNIC/EDDA-PSMA-XL-2 and .sup.99mTc-HYNIC/EDDA-PSMA-XL-3 were added to PBS and fresh mouse serum respectively, and the radiochemical purity was determined at different time points to detect the stability of the two complexes. The results were shown in FIG. 9, the two complexes both have radiochemical purity greater than 90% and maintain high stability after 6 h.

Embodiment 9

Cell Uptake Experiments

[0063] Human prostate cancer LNCaP cells (PSMA positive) were cultured in a RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody under 37° C. with 5% CO.sub.2 and saturated humidity in the incubator. When the cells reach log phase, they were digested with 0.25% trypsin, then the cells were collected and washed twice with PBS, after that a cell suspension was obtained for further culture. A fixed number of cell lines (1.0×10.sup.6 cells/1 ml) was placed in each well of a 24-well cell culture dish, and the experiment was performed after the logarithmic growth phase.

[0064] The cells were divided into an experimental group and a blocking group. 0.5 μCi of the complex .sup.99mTc-HYNIC/EDDA-PSMA-XL-2 or .sup.99mTc-HYNIC/EDDA-PSMA-XL-3 was added to each well, wherein an excess of PSMA inhibitor 2-PMPA (1000-fold molar equivalent) was added to the blocking group half an hour in advance. 6 groups of experiments were carried out in parallel. After 1 h, the culture medium was aspirated, placed in a γ-counting tube, and then washed three times with PBS. The washing solution and the culture medium were combined for storage. Then the cells were trypsinized, collected in another counting tube, and the percentage of cell uptake was calculated by a γ-counting tube. The results were shown in Table 2:

TABLE-US-00002 TABLE 2 Results of LNCaP cell uptake experiments Compound Experimental group Blocking group HYNIX-PSMA-XL-2 16.54 ± 1.33% 3.07 ± 0.80% HYNIX-PSMA-XL-3 14.01 ± 1.11% 2.66 ± 0.51%

[0065] The results show that both .sup.99mTc-HYNIX-PSMA-XL-2 and .sup.99mTc-HYNIX-PSMA-XL-3 could highly specifically bind to PSMA-positive LNCaP cells.

Embodiment 10

SPECT Imaging Studies

[0066] Male SCID mice weighing 18-20 g, were provided by SHANGHAI SLAC LABORATORY ANIMAL CO. LTD, and were raised in the SPF class animal laboratory of the Laboratory Animal Department of Fudan University. After two days of adaptive feeding in the animal room, LNCaP human prostate cancer cells were injected subcutaneously into the armpit of nude mice with an injection volume of 0.2 ml (1×10.sup.7 cells/ml dispersed in 50% Matrigel). The feeding was continued for 4-6 weeks after injection, and the mice were used for imaging experiments when their solid tumor mass grows to 500-600 mm.sup.3.

[0067] 1 mCi/0.2 ml of the .sup.99mTc-HYNIC/EDDA-PSMA-XL-2 or .sup.99mTc-HYNIC/EDDA-PSMA-XL-3 complex was injected into tail vein of the tumor-bearing mice. 2 hours after injection, the experimental animals were imaged with Small-Animal SPECT/CT. The images were shown in FIGS. 10 and 11.

[0068] Male Balb/c nude mice weighing 18-20 g, were provided by SHANGHAI SLAC LABORATORY ANIMAL CO. LTD, and were raised in the SPF class animal laboratory of the Laboratory Animal Department of Fudan University. After two days of adaptive feeding in the animal room, PC-3 human prostate cancer cells were injected subcutaneously into the armpit of nude mice with an injection volume of 0.2 ml. The feeding was continued for 4-6 weeks after injection, and the mice were used for imaging experiments when the solid tumor mass grows to 500-600 mm.sup.3.

[0069] 1 MCi/0.2 ml of the .sup.99mTc-HYNIC/EDDA-PSMA-XL-2 or .sup.99mTc-HYNIC/EDDA-PSMA-XL-3 complex was injected into tail vein of the tumor-bearing mice. 2 hours after injection, the experimental animals were imaged with Small-Animal SPECT/CT. The images were shown in FIGS. 12 and 13.

Embodiment 11

In Vivo Distribution Study in Mice Under the Prostate Tumor Model

[0070] Male SCID mice weighing 18-20 g, were provided by SHANGHAI SLAC LABORATORY ANIMAL CO. LTD, and were raised in the SPF class animal laboratory of the Laboratory Animal Department of Fudan University. After two days of adaptive feeding in the animal room, LNCaP human prostate cancer cells were injected subcutaneously into the armpit of nude mice with an injection volume of 0.2 ml (1×10.sup.7 cells/ml dispersed in a 50% Matrigel). The feeding was continued for 4-6 weeks after injection, and the mice were used for in vivo distribution experiments when the solid tumor mass grows to 500-600 mm.sup.3.

[0071] 20 μCi/0.2 ml of the .sup.99mTc-HYNIC/EDDA-PSMA-XL-3 complex was injected into tail vein of tumor-bearing mice. 0.5, 1 and 2 hours after injection, the mice were sacrificed under anesthesia and dissected. Each organ tissue was weighed, and the radioactivity was measured to calculate the drug uptake of each tissue. The results were shown in FIG. 14.

Embodiment 12

Preparation of .SUP.99m.Tc-HYNIC-PSMA-XL-3 Lyophilized Kit

[0072] 1 mg of HYNIX-PSMA-XL-3, 1 g of disodium succinate, 0.3 g of succinic acid, 1 g of Tricine, 0.5 g of EDDA and 10 mg of SnCl.sub.2 were dissolve in sterile water for injection, and metered to 100 ml. The mixture solution was sterilized by a sterile Millipore filter for subsequent packaging.

[0073] 1 ml of the above solution was taken and placed in a 10 ml of sterile vial for split charging into 100 vials, then lyophilized in nitrogen atmosphere, and finally sealed for preservation.

[0074] Lyophilized kits were randomly selected and tested the sterility and bacterial endotoxin.

[0075] Embodiment 13

.SUP.99.mTc-HYNIC-PSMA-XL-3 for Human Use

[0076] 30-100 mCi of Na.sup.99mTcO.sub.4 solution was added to a vial of .sup.99mTc-HYNIC-PSMA-XL-3 lyophilized kit, and reacted at 100° C. for 10 min. The radiochemical purity of .sup.99mTc-HYNIX-PSMA-XL-3 was determined by radio-TLC. Radionuclide purity was controlled by half-life measurements as well as gamma spectroscopy. The pH, clarity, radioactive concentration, as well as sterility and bacterial endotoxin of product solution were tested.

Embodiment 14

Application of .SUP.99m.Tc-HYNIX-PSMA-XL-3 in Patients With Prostate Cancer

[0077] SPECT/CT imaging was performed on 10 patients with prostate cancer, including 5 patients with primary prostate cancer, 3 patients with biochemical recurrence, and 2 patients with hormone-resistant prostate cancer. The specific clinical information was shown in Table 3. The patients were injected with about 740MBq of .sup.99mTc-HYNIC-PSMA-XL-3, and 2 hours later, they were examined by a Discovery 670 (GE, USA) scanner. Taking the right obturator internus muscle as the background, the target-to-background ratio (TBR) was calculated, and the results were shown in FIG. 15. It can be seen that the distribution of radioactivity in the bladder is low, which is significantly lower than the tumor uptake, indicating excellent diagnostic performance for the primary tumors. A typical example was shown in FIG. 16. In addition, the tumor targeting is high and has a superior detection value for metastases such as lymph nodes/bone. A typical example was shown in FIG. 17.

TABLE-US-00003 TABLE 3 Clinical characteristics of 10 patients with prostate cancer PSA value Pathology before of primary Gleason imaging Tumor No. Age tumors Score State (ng/ml) location  1 77 acinar 5 + 4 before 9.9 prostate adenocarcinoma treatment (puncture)  4 56 acinar 4 + 3 before 15.23 prostate adenocarcinoma treatment (puncture)  5 71 ductal 4 + 3 before 27.82 prostate + adenocarcinoma treatment pelvic (puncture) lymph nodes  6 68 acinar 4 + 4 before 18.22 prostate + adenocarcinoma treatment pelvic (puncture) lymph nodes  2 62 acinar 3 + 4 before 34.8  prostate + adenocarcinoma treatment multiple (puncture) lymph nodes + bone  3 80 acinar 5 + 4 biochemical  0.78 T12 + L5 adenocarcinoma recurrence vertebral (radical surgery) body  7 72 acinar 4 + 5 biochemical  1.22 prostatic adenocarcinoma recurrence fossa (radical surgery)  8 69 acinar 4 + 4 biochemical  7.78 right iliac adenocarcinoma recurrence blood (radical surgery) vessels  9 59 acinar 4 + 5 hormone 34.91 multiple adenocarcinoma resistance bones (radical surgery) period 10 70 ductal 5 + 5 hormone 12.56 lymph adenocarcinoma resistance node + (puncture) period bone

[0078] Four of the five patients with primary prostate cancer underwent radical prostatectomy, and one of the three patients with biochemical recurrence prostate cancer underwent salvage lymph node dissection. Without knowing the results of .sup.99mTc-HYNIC-PSMA-XL-3 SPECT/CT, pathology specialists performed pathological analysis of samples. Representative sections were stained by immunohistochemistry method, deparaffinized in xylene and rehydrated in graded ethanol series. Antigen retrieval was performed with the aid of autoclave and retrieval buffer (Target Retrieval Solution, Dako). Mouse monoclonal antibody against PSMA (clone 3E6, Dako) was diluted at a 1:100 dilution and incubated overnight at 4° C., followed by immunodetection using the Histostain-Plus Detection Kit (Invitrogen). Stained sections were scanned using Nanozoomer 2.0-HT Scansystem (Hamamatsu Photonics) to generate digital overall images. Pathological PSMA expression was consistent with high uptake tumors in SPECT/CT, and a typical case was shown in FIG. 18.

Embodiment 15

[0079] Comparative Study of .sup.99mTc-HYNIC-PSMA-XL-3 and Existing PSMA Imaging Agents that Commonly used in Clinic

[0080] 15 patients with prostate cancer were randomly divided into 3 groups. One group underwent .sup.99mTc-HYNIC-ALUG SPECT/CT, one group underwent .sup.68Ga-PSMA11 PET/CT, and one group underwent .sup.99mTc-HYNIC-PSMA-XL3 SPECT/CT. The results showed that the bladders imaged by .sup.99mTc-HYNIC-ALUG SPECT/CT and .sup.68Ga-PSMA11 PET/CT both exhibited a higher radiophysiological distribution (as shown in A and B of FIG. 19), which affected the distinction between prostate tumors and bladder physiological regions. In contrast, the bladders imaged by .sup.99mTc-HYNIC-PSMA-XL3 SPECT/CT exhibited a very low distribution of radioactivity (as shown in C of FIG. 19), which can clearly distinguish prostate tumors from normal tissue structures, laying the foundation for further clinical puncture, surgery and other applications.

Embodiment 16

Comparison of .SUP.99m.Tc-HYNIC-PSMA-XL-3 SPECT/CT and .SUP.68.Ga-PSMA1 1 PET/CT

[0081] 10 patients with biochemical recurrent prostate cancer were randomly divided into two groups. The basic clinical information of the two groups was similar: they both have previously received radical prostatectomy, and the current PSA of them were 1-3 ng/ml, and the GS score of them were 8-9. One group underwent .sup.68Ga-PSMA11 PET/CT, and the other group underwent .sup.99mTc-PSMA-XL SPECT/CT.

TABLE-US-00004 TABLE 4 Clinical characteristics and examination methods of 10 patients with prostate cancer With or without Examination Number local No. PSA GS method of tumors recurrence  1 1.2 8 .sup.68Ga-PSMA11 2 no  2 2.2 8 .sup.68Ga-PSMA11 1 no  3 2.5 9 .sup.68Ga-PSMA11 4 yes  4 0.8 8 .sup.68Ga-PSMA11 0 no  5 1.7 9 .sup.68Ga-PSMA11 1 no  6 2.4 9 .sup.99mTc-PSMA-XL 3 yes  7 0.6 8 .sup.99mTc-PSMA-XL 1 no  8 2.1 8 .sup.99mTc-PSMA-XL 2 no  9 1.6 8 .sup.99mTc-PSMA-XL 0 no 10 0.7 9 .sup.99mTc-PSMA-XL 4 no

[0082] After the preliminary statistics of the 10 patients, the positive rates of .sup.68Ga-PSMA11 and .sup.99mTc-PSMA-XL imaging were both 80%, and at the same time, 1 patient in both .sup.68Ga-PSMA11 and .sup.99mTc-PSMA-XL imaging were found to have local recurrence in the prostatic fossa. .sup.99mTc-PSMA-XL imaging is not inferior to .sup.68Ga-PSMA11 imaging, which will be supported by designating prospective randomized controlled clinical trials and expanding the sample size in the future.

[0083] The meanings of the English abbreviations herein were shown in Table 5:

TABLE-US-00005 English Chinese abbreviations full names English full names Glu Glutamic acid Lys Lysine 2Nal 2  2-amino-3-(naphthalen-2- yl)propanoic acid AMB 4  4-aminobenzoic acid HYNIC 6- 6-HYDRAZINONICOTINIC ACID PSMA Prostate Specific Membrane Antigen EDDA  -N,N′- Ethylenediamine-N,N′-diacetic acid TPPTS Triphenylphosphine-3,3′,3′′- trisulfonic acid trisodium 2-PM PA 2-(Phosphonomethyl)pentanedioic acid PSMA11 They are all .sup.19F-PSMA1007 PSMA inhibitors HYNIC-ALUG synthesized based on Glu-Urea-Lys structure

[0084] Embodiments of the present invention are described herein, comprising the preferred mode of the present invention known to the inventors. After reading the foregoing description, variations of those embodiments may become apparent to those skilled in the art. The inventors expect that those skilled in the art can suitably utilize such variations, and the present invention can be implemented differently from that described herein. Thus, as permitted by law, the present invention includes all variations and equivalents of the subject matter described in the claims. Moreover, unless otherwise indicated herein or otherwise obviously contradictory to the context, the elements described above in any combination of all possible modifications are included in the present invention.