KR20010053908A - A liver cancer-treating composition comprising diaminedithiol-radiorhenium complex and lipiodol, a process for its preparation and a kit for its preparation - Google Patents

Abstract

PURPOSE: A composition for the treatment of liver cancer containing diaminedithiol-radioactive and a kit for producing the same are provided. Therefore, the composition exhibits excellent effect on treating liver cancer without side effects when injected to liver cancer patients through the hepatic artery. CONSTITUTION: Diaminedithiol, a reducing agent and an auxiliary agent are reacted with radioactive rhenium to produce a radioactive rhenium complex, which is dissolved in lipiodol to produce the titled composition represented by formula 1, wherein the reducing agent and auxiliary agent are tin chloride and tartaric acid respectively. In formula, R1 to R12 are each independently H or C1-5 lower alkyl.

Description

A liver cancer-treating composition comprising diaminedithiol-radiorhenium complex and lipiodol, a process for its preparation and a kit for its preparation }

Radioactive rhenium is a radioisotope that is used medically for the treatment of various diseases. Recently, the development of generators is increasing. The half-life is relatively short (17 hours), resulting in a rapid therapeutic effect and fast dissipation in the body. In addition, 155 keV gamma rays are emitted at a rate of 15% to image the accumulation sites in the body, and because they emit 2.1 MeV beta rays, they are very effective in treatment. In particular, since it can be produced in a generator and can be obtained at a much lower price than other therapeutic isotopes, it will be widely used in the future.

Liver cancer is a serious problem, especially in Korea and in Asian countries. Solid carcinoma, which accounts for more than 90% of liver cancers, is difficult to treat and has a very poor prognosis. Most of the cases are due to the fact that there is no symptom at the beginning, and progressed beyond treatment at the time of discovery. This is because most people start with cirrhosis and die from complications. The most important method in the treatment of liver cancer is surgical removal. If surgery cannot remove the cancer, liver transplantation, hepatic artery embolization, percutaneous ethanol injection, and systemic chemotherapy are used. Of these, Lipiodol can be used for hepatic artery embolization for good effect.

Lipiodol is a fat-soluble contrast agent obtained by iodide and ethyl esterification of fatty acids of poppy plant seeds. Contains 38% by weight of iodine and high viscosity at room temperature can easily cause capillary embolism. Normal liver tissue is supplied through the portal vein more than 90% of the blood, liver cancer tissue is supplied through the hepatic artery more than 90% of blood. Therefore, when Lipiodol is injected into the hepatic artery on the side of liver cancer, most of it flows toward liver cancer, causing embolism in the capillaries of liver cancer and accumulating. The selective deposition of Lipiodol in liver cancer was discovered by Nakamura in 1983. Since then, Lipiodol has been used to diagnose liver cancer. Lipiodol alone was used to treat liver cancer, but in order to enhance the effect, it was used in the treatment of liver cancer in combination with anticancer drugs.

Lipiodol has been labeled with radioisotopes and administered to the hepatic artery to treat liver cancer. There have been reports of the accumulation of liver cancer in patients with I-131-labeled Lipiodol (M. Nakajo, H. Kobayashi, K. Shimabukuro, K. Shirono, H. Sakata, M. Taguchi, N). Uchiyama, T. Sonoda, and S. Shinohara, "Biodistribution and in vivo kinetics of iodine-131 lipiodol infused via the hepatic artery of patients with hepatic cancer." J. Nucl. Med., 29 : 1066-1077, 1988). In vivo Distribution of Lipiodol Labeled with Y-90 (S.-J. Wang, W.-Y. Lin, M.-N. Chen, L.-H. Shen, Z.-T. Tsai , G. Ting, "Preparation and biodistribution of yttrium-90 lipiodol in rats following hepatic arterial injection." Eur. J. Necl. Med. 22 : 233-236, 1995). However, I-131 is not a suitable isotope for treatment, and Y-90 is expensive and difficult to image, so its use is limited. To overcome this drawback, a paper labeled Re-188 was also published (S.-J. Wang, W.-Y. Lin, M.-N. Chen, B.-T. Hsieh, L.-H). Shen, Z.-T. Tsai, G. Ting, FF Knapp, Jr. "Radiolabelling of lipiodol with generator-produced ¹⁸⁸ Re for hepatic tumor therapy." Appl. Radiat. Isot. 47 : 267-271, 1996; S -J. Wang, W.-Y. Lin, M.-N. Chen, B.-T. Hsieh, L.-H. Shen, Z.-T. Tsai, G. Ting, FF Knapp, Jr. "Biodistribution of rhenium-188 lipiodol infused via the hepatic artery of rats with hepatic tumours." Eur. J. Nucl. Med. 23 : 13-17, 1996). However, the methods presented in these papers were in need of improvement due to complex labeling methods and low labeling efficiency and stability.

Korean Unexamined Patent Publication No. 96-33472 allows the preparation of various radioisotope complexes in chitosan rather than Lipiodol, which can be used to treat liver cancer.The principle of accumulation of radioisotope complexes of chitosan in liver cancer is also the same It is a phenomenon that causes embolism in capillaries of liver cancer. However, chitosan can not be traced by X-rays when administered to liver cancer, not labeled with rhenium, and has a side effect of suppressing bone marrow due to accumulation of holmium that falls off when labeled with holium in bone.

On the other hand, diaminedithiol is a substance having a structure represented by the following formula, and various substituted anions of Tc-99m as elongation imaging agent in EP 135,160, published by Davidson et al. On March 27, 1985. Sex diaminedithiol complexes have been described:

One

[Wherein, R ₁ to R ₁₂ are each independently hydrogen, alkyl having 1 to 3 carbon atoms or -COOR wherein R is -CH ₃ , -C ₂ H ₅ or -C ₃ H ₇ ; Provided that only R ₃ , R ₅ , and R ₇ to R _{10 in the} substituents R ₁ to R ₁₂ may be -COOR.]

Korean Patent Publication No. 92-2167 also reports on ester substituted diaminedithiol containing kits that are neutral fat soluble. This is described by Blondeau et al, Can. J. Chem. 45:49, 1967 to synthesize a ligand by the reductive dimerization of thiazolidine to form N, N'-1, 2-ethylenebis-L-cysteine derivatives and esters thereof, followed by Tc-99m. The label relates to creating a neutral fat-soluble complex and using it for imaging the blood flow in the brain.

It is an object of the present invention to provide a composition for treating liver cancer using a radioactive material which is easy to prepare and stable, and has a high accumulation rate in liver cancer tissues, thereby effectively obtaining liver cancer treatment effects without side effects.

1 shows the formation efficiency of the diaminedithiol-radioactive rhenium complex with heating time in the preparation of the diaminedithiol-radioactive rhenium complex,

Figure 2 shows the stability test results of the composition for treating liver cancer prepared in Example 4,

3 is an autoradiograph showing diaminedithiol-radioactive rhenium complexes accumulated in liver cancer tissues.

The present invention is a liver cancer treatment composition comprising a diaminedithiol-radioactive rhenium complex and Lipiodol; Its manufacturing method; And kits for producing the same.

Specifically, the present invention relates to a composition for treating liver cancer comprising a complex of diaminedithiol and radioactive rhenium represented by the following Chemical Formula 1, and Lipiodol:

One

Wherein R ₁ to R ₁₂ are each independently hydrogen or lower alkyl (1 to 5 carbon atoms).

Lipiodol, among the components, is a fat-soluble substance found to be selectively deposited on liver cancer tissue by Nakamura et al in 1983 as described in the art. Therefore, the composition of the present invention can accumulate the active substance diaminedithiol-radioactive rhenium complex in liver cancer tissue by including Lipiodol as a medium.

The amount of Lipiodol in the composition of the present invention may be any amount so long as it can dissolve the formed diaminedithiol-radioactive rhenium complex, and is preferably in the range of 0.5 ml to 50 ml per 1 mg of diaminedithiol.

The diaminedithiol-radioactive rhenium complex, which is the active substance in the composition of the present invention, is fat-soluble due to the properties of diaminedithiol, so that it is more soluble in Lipiodol, an oily X-ray contrast agent, than water. Therefore, when administered to the hepatic artery with Lipiodol as a medium, Lipiodol may accumulate together when embolism occurs due to embolism in capillaries of liver cancer tissues. Since diaminedithiol-radioactive rhenium complex is fat-soluble, it is continuously dissolved in Lipiodol and does not spread to surrounding tissues, so that Lipiodol stays together as long as it exists in cancer tissues and eventually irradiates liver cancer tissues selectively to remove only cancer cells. can do.

Radioactive rhenium constituting the diaminedithiol-radioactive rhenium complex means ¹⁸⁶ Re and ¹⁸⁸ Re.

Another aspect of the present invention provides a method for preparing a composition for treating liver cancer, the method comprising: reacting a diaminedithiol, a reducing agent and other auxiliaries with radioactive rhenium to form a radioactive rhenium complex; Dissolving it in Lipiodol.

The reducing agent here is for reducing radioactive rhenium to bind to diaminedithiol. Examples include tin chloride, vitamin C, iron chloride, dithionite, sodium sulfite, and the like, preferably tin chloride.

In addition, other auxiliaries are additives for improving quality such as chemical and physical stability, including additives for suppressing the production of by-products and increasing labeling efficiency in radioisotope labeling. For example, tartaric acid, gluconic acid, glucaric acid, glucoheptonic acid, lactic acid, MDP, lactose, EDTA, etc. are mentioned, Preferably it is tartaric acid.

In this process, conventional materials and methods that are not diaminedithiol (S.-J. Wang, W.-Y. Lin, M.-N. Chen, B.-T. Hsieh, L.-H. Shen, Z .-T. Tsai, G. Ting, FF Knapp, Jr. "Radiolabelling of lipiodol with generator-produced ¹⁸⁸ Re for hepatic tumor therapy." Appl. Radiat. Isot. 47 : 267-271, 1996). In case of labeling, the solution should be boiled with ammonium carbonate solution, add acetic acid, evaporate through nitrogen gas, and evaporate for more than 5 hours. However, labeling of radioactive rhenium for diaminedithiol used in the present invention, that is, diaminedithiol The preparation of radioactive rhenium complexes can be prepared within one hour by the production process according to the invention and the resulting complexes also exhibit good stability.

In a specific production method, first, diaminedithiol is reacted with radioactive rhenium in the presence of a reducing agent and an adjuvant to form a diaminedithiol-radioactive rhenium complex. Lipiodol is added to the aqueous solution of the formed complex and mixed well so that the fat-soluble complex is extracted from Lipiodol and centrifuged to form two layers. By extracting the Lipiodol layer forming the lower layer with a syringe can be obtained the composition for treating liver cancer of the present invention. Examples of more specific manufacturing methods are described in the Examples.

Another aspect of the present invention is a kit for preparing a composition for treating liver cancer, comprising: a first container containing lyophilized diaminedithiol, a reducing agent and an adjuvant; And a second vessel containing Lipiodol and capable of directly performing centrifugation operations. Reducing agents and auxiliaries herein are as defined above. Radioactive rhenium may also be supplied from the outside immediately before use.

In addition, since the lipidodol in the kit for preparing the composition for treating liver cancer can be supplied from the outside, the kit includes a first container containing lyophilized diaminedithiol, a reducing agent and an adjuvant, and the diaminedithi in the composition for treating liver cancer. It may also be used in the form of a kit for producing an all-radioactive rhenium complex.

The following examples and experimental examples specifically illustrate the present invention, but the scope of the present invention is not limited to these examples.

Example 1 Synthesis of Diaminedithiol Compound

29 g of 2,2'-dithio-bis (2-methylpropanal) was dissolved in 280 ml of benzene containing 50 mg of tosic acid, and 12.6 g of 2-methyl-1,2-diaminopropane was added dropwise while mixing well. . The reaction mixture was refluxed with water for 2 hours and the solvent was removed under reduced pressure. Low boiling petroleum ether was added to the residue to dissolve it, the activated carbon was treated, and the filtrate was concentrated until precipitation started. The crystals were collected by filtration and washed with cold petroleum ether to give light yellow crystals. The obtained crystals were dissolved in ethanol and added with an equal amount of sodium borohydride slowly stirring. After mixing for 2 hours at room temperature, acetone was added, stirred for 20 minutes, and the solvent was dried under a reduced pressure. Pentane was added to the residue to dissolve the residue, and the filtrate was dried and purified by column chromatography. This was dissolved in tetrahydrofuran and reduced with lithium aluminum hydride, followed by reaction of excess lithium aluminum hydride with ammonium chloride. The solution was completely dried and then dissolved in anhydrous ethanol, passed through hydrogen chloride, made into hydrochloride, and dried to form the final diaminedithiol. Yield 60% and confirmed the structure of formula 2 by NMR and GC / MS. ¹ H NMR (D ₂ O) 1.5 (s, 6 H, (CH ₃ ) ₂ ), 3.3 (s, 2 H, CH ₂ ), 3.6 (s, 2 H, CH ₂ ).

2

Example 2: Preparation of Liver Cancer Treatment Composition

All of the following procedures were carried out under the conditions of preparation of a pharmaceutical formulation that can be administered to a human body in which no sterile and pyrogenic substances were detected.

0.1 ml of diaminedithiol aqueous solution (10 mg / ml), 0.1 ml of tin chloride aqueous solution (100 mg / ml), and 1 ml of tartaric acid aqueous solution (200 mg / ml) were placed in a sterile glass container and lyophilized. A diaminedithiol-radioactive rhenium complex was prepared by adding 3 ml of a 10 mCi equivalent to a radioactive rhenium aqueous solution and warming it for 30 minutes in a 100 ° C water bath. 3 ml of Lipiodol was placed in a sterile glass vessel capable of centrifugation, and an aqueous solution containing the complex formed above was added and mixed well. Centrifugation at 3000 rpm for 10 minutes to separate the water layer and Lipiodol layer, and then using a long needle and syringe to take the Lipiodol layer in the lower layer to prepare a composition for treating liver cancer according to the present invention.

Example 3: Preparation of kit for preparing a composition for treating liver cancer

All of the following procedures were carried out under the conditions of preparation of a pharmaceutical formulation that can be administered to a human body in which no sterile and pyrogenic substances were detected.

0.1 ml of diaminedithiol solution (10 mg / ml), 0.1 ml of tin chloride solution (100 mg / ml) and 1 ml of tartaric acid solution (200 mg / ml) were placed in a sterile glass container (the first container) and lyophilized. After sealing with a rubber stopper.

Meanwhile, another sterile glass vessel (second vessel) capable of centrifugation was added 3 ml of Lipiodol and sealed with a rubber stopper.

The two containers were packaged together in a paper box to complete the kit.

When using the kit prepared in this way, the radioactive rhenium solution was added to the first vessel, depending on the purpose, 0.5 to 5000 mCi equivalent, and warmed for 30 minutes in a 100 ℃ water bath, then transferred to the second vessel and mixed well. The complex thus formed is carried over to the Lipiodol layer. Centrifugation takes only the Lipiodol layer, which is ready for administration to liver cancer patients. If necessary, it may be further diluted with Lipiodol.

Example 4: Preparation of a composition for treating liver cancer using a kit for preparing a composition for treating liver cancer

The radioactive rhenium aqueous solution contained in the third vessel of the diaminedithiol kit prepared in Example 3 was added to 3 ml of 10 mCi equivalent, and added to the first vessel and warmed for 30 minutes in a 100 DEG C water bath to form a complex. The reaction solution was transferred to the second container containing Lipiodol using a syringe, and the complex formed by shaking the container several times was extracted into the Lipiodol layer. After centrifugation at 3000 rpm for 10 minutes to separate the water layer and Lipiodol layer, using a long needle and a syringe to extract the layer of Lipiodol in the lower layer.

Experimental Example 1: Formation efficiency of diaminedithiol-radioactive rhenium complex

Using the diamine dithiol kit prepared in Example 3 to prepare a composition for treating liver cancer. The preparation method was the same as in Example 4, and the boiling time for forming the complex was prepared by 0, 5, 15, 30, 60, 90 and 120 minutes, respectively.

The formation efficiency of the diaminedithiol-radioactive rhenium complex was evaluated by labeling efficiency of the diaminedithiol with the radioactive rhenium. The labeling efficiency and purity were measured by chromatography using Gelman's ITLC-SG as the fixed phase and ethyl acetate as the mobile phase. The change in labeling efficiency with heating time is shown in Fig. 1. After 15 minutes of heating time, the labeling efficiency reached about 80%, indicating no significant change.

Experimental Example 2: Stability of the composition for treating liver cancer

The stability of the composition for treating liver cancer according to the present invention was evaluated.

Samples were taken every certain time while leaving the liver cancer treatment composition prepared in Example 4 at room temperature as it was, and the content of diaminedithiol-radioactive rhenium complex was measured by the chromatographic method as in Experimental Example 1.

In addition, in order to evaluate the stability after administration in vivo, 0.4 ml of the liver cancer treatment composition prepared in Example 4 was put in 2 ml of human serum and incubated for 48 hours in a 5% carbon dioxide gas incubator at 37 ° C. The content of the diaminedithiol-radioactive rhenium complex was measured by the chromatographic method as described above.

The results are shown in FIG. The composition for treating liver cancer of the present invention was very stable up to 48 hours upon administration to the body and remained stable for 24 hours even when left at room temperature. Since the composition of the present invention is a preferred radioisotope preparation, it is confirmed that the above-described stability is sufficient because it is used within 24 hours at room temperature after preparation.

Experimental Example 3: Confirmation of Liver Cancer Accumulation by Animal Experiment

The liver cancer-induced rat liver artery is administered directly to the liver cancer treatment composition according to the present invention, if accumulated selectively in liver cancer is direct evidence that it can be effectively used for the treatment of liver cancer. Therefore, the accumulation of liver cancer was confirmed as follows.

Liver cancer was induced by injection of 2 × 10 ⁶ N1-S1 liver cancer cells in the liver of Sprague-Dawley rats and growing for 10 days. 40 μl of a Lipiodol solution of diaminedithiol-radioactive rhenium prepared in Example 4 was injected into the hepatic artery and livers were removed after 5 minutes and 1 hour. The radioactivity of the liver cancer site and the normal liver site was measured with a gamma scintillation counter to determine the ratio. Liver collected at 1 hour and 24 hours after administration was frozen and then sliced and subjected to autoradiography.

The results are shown in Table 1 and FIG. It was confirmed that more radioactivity accumulates in the liver cancer region than in the normal region.

The rate of accumulation between liver cancer and normal in Lipiodol solution of diaminedithiol-radioactive rhenium. Elapsed time after dosing 5 minutes 1 hours Rate of radioactivity (liver cancer / normal to normal) 7.9 ± 4.9 7.3 ± 5.8

* Each value represents the mean ± standard deviation.

The active substance diaminedithiol-radioactive rhenium complex in the composition for treating liver cancer according to the present invention is easy to manufacture, stable in medium Lipiodol, and high liver cancer tissue when injected into liver cancer through the hepatic artery in the form of the composition of the present invention. Since the accumulation rate is shown, when the composition for treating liver cancer according to the present invention is applied to the treatment of liver cancer, an effective liver cancer treatment effect can be obtained without side effects.

Claims (6)

A composition for treating liver cancer comprising a complex of diaminedithiol and radioactive rhenium represented by Formula 1 and Lipiodol. One (Wherein, R ₁ to R ₁₂ are each independently hydrogen or a lower alkyl group having 1 to 5 carbon atoms.) Reacting diaminedithiol, a reducing agent and an adjuvant with the radioactive rhenium to form a radioactive rhenium complex; Method for preparing a composition for treating liver cancer according to claim 1, comprising dissolving it in Lipiodol. A first container containing lyophilized diaminedithiol, a reducing agent and an adjuvant; And a second container containing Lipiodol, the kit for preparing a composition for treating liver cancer according to claim 1. The kit for producing a composition for treating liver cancer according to claim 2 or 3, wherein the reducing agent and the adjuvant are tin chloride and tartaric acid, respectively. A kit for the preparation of a complex of diaminedithiol and radiorenium in a composition for treating liver cancer according to claim 1, comprising a first container containing lyophilized diaminedithiol, a reducing agent and an adjuvant. The kit for producing a complex of diaminedithiol and radiorenium according to claim 5, wherein the reducing agent and the auxiliary agent are tin chloride and tartaric acid, respectively.