SODIUM SACCHARIN HAVING CONJUGATED LIGAND, DERIVATIVES THEREOF AND PROCESS FOR THE PREPARATION THEREOF

Abstract

The present invention provides sodium saccharin bound with a ligand, derivatives thereof, and a process for preparation thereof. According to the present invention, there is provided a compound which is specifically bonded to hypoxic tumors. Specifically, the compound having sodium saccharin (SAC) bound with a ligand compound, or derivatives thereof can be applied to a contrast agent for cancer diagnosis.

Claims

1. A compound having sodium saccharin (SAC) bound with a ligand compound, or derivatives thereof.

2. The compound or derivatives thereof according to claim 1, wherein the ligand comprises 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

3. The compound or derivatives thereof according to claim 1, wherein the compound comprises the structure of the following formula 1: ##STR00007##

4. The compound or derivatives thereof according to claim 3, wherein the compound comprises the structure of the following formula 2: ##STR00008## in the formula 2, Sodium saccharin (SAC) is ionically bonded to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and M* is a radio isotope which is coordinated to the NOTA.

5. The compound or derivatives thereof according to claim 4, wherein the radio isotope is .sup.68Ga.

6. A method for producing a compound or derivatives thereof, the method comprising the steps of: ionically binding sodium saccharin (SAC) with a ligand compound to prepare a sodium saccharin-ligand compound, and reacting the sodium saccharin-ligand compound with a radio isotope.

7. The method for producing a compound or derivatives thereof according to claim 6, wherein the ligand compound comprises 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

8. The method for producing a compound or derivatives thereof according to claim 6, wherein the radio isotope is .sup.68Ga.

9. A contrast agent comprising the compound according to claim 1 or derivatives thereof.

10. The contrast agent according to claim 9, wherein the contrast agent is used in Positron Emission Tomography (PET) for cancer diagnosis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 is a graph showing the result of thin-layer chromatography (TLC) on a compound according to the present invention.

[0030] FIG. 2 is an image of positron emission computed tomography (PET/CT) using a compound according to the present invention.

[0031] FIG. 3 is a PET/CT image for identification of tumor selectivity.

[0032] FIG. 4 is a graph showing the distribution of a compound according to the present invention in tissues.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Since various modifications and variations can be made in the present invention, particular embodiments are illustrated in the drawings and will be described in detail in the detailed description. It should be understood, however, that the invention is not intended to be limited to the particular embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. In the following description of the present invention, detailed description of known functions will be omitted if it is determined that it may obscure the gist of the present invention.

[0034] Hereinafter, a compound or derivatives thereof according to an embodiment of the present invention will be described in more detail.

[0035] The present invention provides a compound having sodium saccharin (SAC) bound with a ligand compound, or derivatives thereof.

[0036] According to one embodiment, the ligand compound may comprise 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). Such a metal affinity ligand compound can prevent radio isotopes from liberating in the body, thereby releasing the radioactive substance out of the body. From this, it can serve as a protecting agent by reducing cytotoxicity (Marouan Rami et al., Carbonic anhydrase inhibitors: Gd(III) complexes of DOTA- and TETA-sulfonamide conjugates targeting the tumor associated carbonic anhydrase isozymes IX and XII , New J. Chem., 2010, 34, 2139-2144; Silvio Aime et al., NMR relaxometric studies of Gd(III) complexes with heptadentate macrocyclic ligands, Magnetic Resonance in Chemistry (1998) Volume: 36, Issue: 51, Pages: S200-S208).

[0037] Specifically, the compound according to the present invention may comprise the compound of the following formula 1.

##STR00003##

[0038] In the above formula 1, sodium of saccharin and a carboxyl group of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) are bonded to each other.

[0039] According to one embodiment, the compound according to the present invention may comprise the structure of the following formula 2.

##STR00004##

[0040] In the formula 2,

[0041] Sodium saccharin (SAC) is ionically bonded to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and

[0042] M* is a radio isotope which is coordinated to the NOTA.

[0043] According to one embodiment, the radio isotope may be .sup.68Ga.

[0044] The NOTA can coordinate with the radio isotope to easily form a complex.

[0045] That is, as described above, the present invention can provide a compound having conjugated ligand in order to bind a radio isotope to sodium saccharin (SAC) stably.

[0046] According to other embodiment of the present invention, there is provided a method for producing a compound and derivatives thereof, the method comprising the steps of:

[0047] ionically binding sodium saccharin (SAC) with a ligand compound to prepare a sodium saccharin-ligand compound, and

[0048] reacting the sodium saccharin-ligand compound with a radio isotope.

[0049] According to one embodiment, the method of the present invention may comprise a step of reacting a solution of .sup.68Ga with a buffer solution of pH 5 to 6 and a compound of formula 1, for example, at 50 to 100 C., for example, at 80 C. to form a compound of formula 2. The buffer solution may include, but is not limited to, for example, sodium acetate.

[0050] According to one embodiment, the ligand compound may comprise 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0051] Specifically, the production process according to one embodiment of the present invention is shown in Scheme 1.

##STR00005##

[0052] As shown in Scheme 1, in the present invention, a ligand was used to coordinate sodium saccharin with a metallic radio isotope. The ligand compound includes a compound in which sodium of saccharin are bonded to a carboxyl group of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0053] Sodium saccharin (SAC) has a property of specifically binding to carbonic anhydrase IX (CA IX), which is not expressed in normal cells but expressed only in hypoxic tumors. Sodium saccharin (SAC) is labeled with a radio isotope through the ligand compound. Due to such targeting to hypoxic tumors, the substance according to the present invention can be applied to a contrast agent.

[0054] Specifically, a contrast agent comprising a compound according to the present invention or derivatives thereof can be used as a contrast agent of Positron Emission Tomography (PET) for hypoxic tumors.

[0055] According to one embodiment, the PET contrast agent according to the present invention can be formulated to an injection or the like.

[0056] According to one embodiment, when the compound according to the present invention is used as a contrast agent, a non-toxic solution, which is an isotonic component with blood, may be contained as a diluent. The diluent may include, for example, sodium chloride solution, potassium chloride solution, sodium bicarbonate solution, Hartmann's solution, glucose solution, glucose physiological saline solution and the like. Specific examples of the diluent include phosphate buffer solution of pH 7.4 or the like.

[0057] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

EXAMPLE 1

Preparation of Compound of Formula 1 (SAC-NOTA)

[0058] To prepare a compound of formula 1 (SAC-NOTA), a K.sub.2CO.sub.3 solution and 1 mg of a ligand compound, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) were added to a reaction vessel and dissolved therein. In another vessel, 1.83 mg (5 eq.) of sodium saccharin (SAC) was dissolved in distilled water (D.W) and then added to the reaction vessel. After stirring at room temperature for 24 hours, separation and purification were carried out using a column (Pharmacia Fine Chemicals, Sephadex G-25).

EXAMPLE 2

Preparation of Compound of Formula 2 (SAC-NOTA-.SUP.68.Ga)

[0059] To prepare a compound of formula 2 (SAC-NOTA-.sup.68Ga), gallium was used as a radio isotope. .sup.68Ga which was extracted from a .sup.68Ga generator was obtained as a colorless transparent solution dissolved in a dilute hydrochloric acid solution. After taking it properly according to the amount of radioactivity required for the experiment (above 0.1 mCi), the .sup.68Ga solution was placed in a suitable glass vessel and dried at 100 C. while blowing nitrogen therein. The dried .sup.68Ga is applied to the glass vessel in the form of a transparent film. 0.5 mL of the compound of formula 1 (SAC-NOTA) which was isolated and extracted in Example 1 was added to the reaction vessel coated with .sup.68Ga, and then the labeling reaction was allowed to proceed for 10 minutes at pH 5 to 6 at 80 C. Reaction according to Example 2 is shown in Scheme 2.

##STR00006##

[0060] The final compound, the compound of formula 2 (SAC-NOTA-.sup.68Ga), was adsorbed on a thin layer chromatography (TLC) plate and sufficiently developed with 0.1 M citrate buffer solution. From this, radiochemical yield and purity was identified. The results are shown in FIG. 1. FIG. 1 is a graph showing Radio-TLC (AR-2000, Eckert & Ziegler) results of the compound of formula 2 (SAC-NOTA-.sup.68Ga).

[0061] As shown in FIG. 1, the radiochemical yield and purity from the TLC image are confirmed to be 98% or more on average.

EXPERIMENTAL EXAMPLE 1

Evaluation of Stability

[0062] In order to evaluate stability for the compound of formula 2 (SAC-NOTA-.sup.68Ga) prepared in Example 2, stability in human serum and mouse serum was measured. 100 L of 0.4 mCi of the compound of formula 2 (SAC-NOTA-.sup.68Ga) was mixed with 0.5 mL of each of human serum and mouse serum, and then cultured at 37 C. Thin layer chromatography (TLC) was performed using radio-thin film chromatography (AR-2000, Eckert & Ziegler) equipment at the time to be measured (1 hour, 3 hours, 6 hours). The measurement results of stability of the compound of formula 2 (SAC-NOTA-.sup.68Ga) in the serum over time are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Item 1 h 3 h 6 h Compound of Human serum 100% .sup.100% 98% formula 2 Mouse serum 100% 98.26% 98.50%

[0063] As shown in Table 1, since the form of the compound of formula 2 as a target compound is maintained, that is, the purity is maintained, it can be confirmed that the compound of formula 2 is stable for 6 hours or longer in human serum and mouse serum.

EXPERIMENTAL EXAMPLE 2

PET Image Acquisition of SAC-NOTA-.SUP.68.Ga

[0064] Positron emission tomography (PET) images were taken with PET/CT equipment for small animals (INVEON, Simens Medical Solutions). U87MG cells, a malignant glioma cell line, were implanted subcutaneously in the left shoulder of a nude mouse to form a tumor model. PET/CT images were obtained at 30 minutes, 60 minutes, and 90 minutes by injecting 0.23 mCi of the compound of formula 2 (SAC-NOTA-.sup.68Ga) prepared in Example 2 into the tail vein of the tumor-transplanted mouse (nu/nu nude, 20 g). The results were shown in FIG. 2. As shown in FIG. 2, it can be observed that the signal increases at the tumor site after administration of the compound of formula 2 (SAC-NOTA-.sup.68Ga).

EXPERIMENTAL EXAMPLE 3

Identification of Tumor Selectivity of SAC-NOTA-.SUP.68.Ga

[0065] Experiments were carried out to identify selective specificity for receptors expressed in tumor cells of SAC-NOTA-.sup.68Ga prepared in Example 2. U87MG cells, a malignant glioma cell line, were injected subcutaneously into the shoulder of a nude mouse to form a tumor model. First, sodium saccharin (10 mg/kg) was injected through the tail vein of the anesthetized mouse to block the receptor. After 30 minutes, 0.2 mCi of the SAC-NOTA-.sup.68Ga was injected to acquire PET/CT images in the same manner as in Experimental Example 2. The results were shown in FIG. 3.

[0066] In FIG. 3, a PET/CT image (center) is taken by injecting SAC-NOTA-.sup.68Ga without blocking the receptor present in the tumor and a PET/CT image (right) is taken by injecting SAC-NOTA-.sup.68Ga after blocking the receptor present in the tumor by injecting sodium saccharin.

[0067] According to FIG. 3, it can be seen that the signal of the tumor site where the receptor is blocked is lower than the signal of the tumor site where the receptor is not blocked. From these results, it can be seen that SAC-NOTA-.sup.68Ga has selective specificity for receptors expressed in the tumor cell.

EXPERIMENTAL EXAMPLE 4

Identification of Distribution of SAC-NOTA-.SUP.68.Ga in Tissues

[0068] The distribution in mouse tissues of SAC-NOTA-.sup.68Ga prepared in Example 2 was confirmed. U87MG cells, a malignant glioma cell line, were injected subcutaneously into the shoulder of a nude mouse (body weight 20 g) to form a tumor model. 0.01 mCi of SAC-NOTA-.sup.68Ga was injected through the tail vein of the tumor-bearing mouse (n=4). After 30 minutes and 60 minutes of injection, the organs (blood, muscle, heart, lung, liver, spleen, stomach, intestine, kidney, bone, tumor) were extracted, respectively. The radioactivity of the tissue was measured with a gamma counter. The results were shown in FIG. 4.

[0069] FIG. 4 is a graph showing a ratio of the tissue radioactivity to the amount of injection (% ID/g) measured in various organs after injection of SAC-NOTA-.sup.68Ga over time.

[0070] From FIG. 4, it can be seen that the radioactivity in the tumor is maintained at 1.52% after 30 minutes of injection and at 0.69% after 60 minutes of injection. In addition, it can be seen that the radioactivity value of the liver tissue was kept low at 0.99% after 30 minutes and at 0.63% after 60 minutes of injection.

[0071] The above description is only illustrative of the technical idea of the present invention, and various changes and modifications may be made by those skilled in the art without departing from the essential characteristics of the present invention. It should be noted that the embodiments disclosed in the present invention are not intended to illustrate rather than limit the scope of the present invention. The scope of the present invention is not limited by these embodiments. The scope of protection of the present invention should be construed according to the following claims, and all technical ideas within the scope of equivalents should be construed as falling within the scope of the present invention.