KR101480393B1 - gadolinium complexs for contrast agent and contrast agent for diagnosing hepatoma - Google Patents

Abstract

The present invention relates to a gadolinium complex for contrast agent for diagnostic of liver cancer, a method for preparing the same, and a diagnostic agent for liver cancer using the same. More particularly, the present invention relates to a method for producing liver gadolinium complex Gadolinium complex for contrast agent, a method for producing the same, and a diagnostic contrast agent for liver cancer using the same.
The gadolinium complex of the present invention is characterized in that gadolinium (Gd) is bonded to a DTPA-HA intermediate formed by reacting diethylene triamine pentaacetic acid (DTPA) with hyaluronic acid (HA) .

Description

Gadolinium Complex for Contrast Agent for Diagnosis of Liver Cancer and Gadolinium Complex for Contrast Agents for Diagnosing Hepatoma

The present invention relates to a gadolinium complex for contrast agent for diagnosis of liver cancer, and more particularly to a contrast agent for diagnostic liver cancer using hyaluronic acid, And a method for producing the same, and a contrast agent for diagnosing liver cancer using the same.

Contrast agents are used to obtain differentiated images between lesions and surrounding tissues such as tumors. The purpose of this study is to observe contrast, contrast, and location of lesions and surrounding tissues.

The nuclear magnetic resonance imaging technique (MRI) shows superiority and stability in distinguishing the lesion tissue from the surrounding tissue. Although there are many ways to observe the body inside and MRI technology is the most recently developed technology, the applicability and the usage of MRI are increasing rapidly, This is because it is safer.

Since methods such as X-ray, CT, and PET require the human body to inject radioactivity that can not be regarded as harmless to human body, it is not applicable to patients who are concerned about genetic mutation, especially cancer patients and pregnant women. However, MRI is an imaging technique that does not depend on shortcomings such as radioactivity and limitations of applications.

These MRI images can be shaped through the use of contrast agents. MRI contrast agents are preparations that increase the contrast of images by shortening the relaxation times of T1 and T2 of human tissues. The preparations using paramagnetic or superparamagnetic materials It is mainstream. By using contrast agents, it is possible to maximize the contrast of contrast by amplifying signals of whole or partial tissues of a desired organ, or weakening the signals of surrounding tissues. MRI contrast agents are largely gadolinium (Gd) preparations used as T1 contrast agents and iron oxide formulations used as T2 contrast agents.

Gadolinium, which is mainly used as T1 contrast agent, has a very small molecular weight and toxicity, and thus has many problems in using it as a contrast agent. This led to the development of DTPA-Gd complexes (Magnevist ^™ , Bayer) formed by coordination of gadolinium and diethylene triamine pentaacetic acid (DTPA).

However, the DTPA-Gd complex has an extremely short half-life of about 14 minutes and is rapidly released into the urine after administration, making it difficult to diagnose various parts of the body by a single injection. In addition, it is difficult to acquire images that are transmitted nonspecifically to normal tissues and lesion tissues and have apparent contrast.

And the promoist product of Gd-EOB-DTPA (Primovist ^TM , Schering AG, Germany) as an MRI liver specific contrast agent in which DTPA-Gd is combined with ethoxybenzyl is widely used. The PROMOBYST product provides a dynamic image such as another typical gadolinium-containing extracellular fluid contrast agent, and enhances a healthy liver parenchyma in the image 10 to 20 minutes after the injection, and thus plays a liver-specific contrast agent. Gd-EOB-DTPA has been supplemented with ethoxy benzyl (EOB) to enter hepatocytes via membrane-bound carriers (organic anion transport polypeptides). EOB, which is a lipophilic moiety, affects the pharmacokinetics of this gadolinium chelate. The promo-biost products are normal hepatocytes, except for the absence of hepatic cell function or mild lesions (cysts, metastasis, most hepatocellular carcinoma) through membrane-bound carriers belonging to the group of organic-anion-transporing polypeptide OATP1 (2-5) Lt; / RTI > However, Primobist products have limitations in diagnosing cancer in patients with liver cirrhosis. The liver of patients with liver cirrhosis does not have normal hepatic cell function, and Gd-EOB-DTPA can not be introduced efficiently, making it difficult to diagnose liver cancer.

Hepatocellular carcinoma, which is common in Asian countries including Korea, occurs in patients with liver cirrhosis. Patients with severe liver cirrhosis have poor liver function, so the product of Primobist is less absorbed into liver parenchyma. Therefore, it is difficult to diagnose hepatocellular carcinoma because it has a weak contrast with hepatocellular carcinoma.

Accordingly, the present inventors have completed the present invention by developing a contrast agent that can improve the limitations of the conventional MRI T1-specific contrast agent in cirrhosis patients.

The present invention relates to a gadolinium complex for contrast agent capable of enhancing the liver cancer-producing ability in liver cirrhosis patients by effectively transferring gadolinium having a T1-contrast effect to hepatocytes by using hyaluronic acid having liver specificity by various receptors, And to provide a diagnostic contrast agent for liver cancer using the same.

Yet another object of the present invention is to maximize liver absorption of the contrast agent to minimize the amount of the contrast agent, thereby providing an economic effect.

In order to accomplish the above object, the present invention provides a gadolinium complex for contrast agent, which comprises a DTPA-HA intermediate formed by reacting diethylene triamine pentaacetic acid (DTPA) with hyaluronic acid (HA) gadolinium, Gd) are combined.

The DTPA-HA intermediate material is characterized in that the carboxyl group of the diethylene triamine pentaacetic acid and the hydroxy group of the hyaluronic acid are formed by an ester bond.

The gadolinium complex is characterized in that the gadolinium is coordinately bound to diethylene triamine pentaacetic acid of the DTPA-HA intermediate.

In order to accomplish the above object, the present invention provides a method for preparing a gadolinium complex for contrast agent, comprising the steps of: a) reacting diethylene triamine pentaacetic acid (DTPA) with hyaluronic acid (HA) - producing an HA intermediate; b) binding gadolinium (Gd) to the DTPA-HA intermediate material.

The organic solvent in step a) is dimethylsulfoxide (DMSO).

The step b) comprises dissolving the DTPA-HA intermediate in water and then adding a gadolinium aqueous solution to coordinate the gadolinium on the diethylene triamine pentaacetic acid of the DTPA-HA intermediate.

In order to achieve the above object, the present invention provides a contrast agent for diagnosing liver cancer, which comprises the gadolinium complex.

In the present invention, hyaluronic acid is introduced into the DTPA-Gd complex structure to specifically transfer gadolinium to hepatocytes, thereby effectively enhancing the contrast of tumor tissue and normal hepatocytes, thereby providing more accurate image information.

In particular, the present invention can introduce gadolinium into the cirrhotic tissue by hyaluronic acid, so that a contrast image between the hepatocellular carcinoma and liver cirrhotic tissue in the liver cirrhosis patient can be obtained, thereby improving the liver cancer imaging ability.

Further, the present invention is administered through intravenous infusion, and the contrast agent which is not absorbed into the hepatocytes is rapidly released to the body after about 1 hour, and is safe for the human body.

1 is a graph showing electrical characteristics of the gadolinium complex of the present invention,
FIG. 2 is an FT-IR graph for confirming the formation of DTPA-HA intermediate material,
3 is a photograph showing the intensity of MRI signal for the gadolinium complex of the present invention,
4 to 6 are MRI photographs of liver, kidney, and bladder of the mouse according to time.

Hereinafter, a gadolinium complex for contrast agent for diagnosis of liver cancer according to a preferred embodiment of the present invention, a method for producing the complex, and a contrast agent for diagnosing liver cancer using the same will be described in detail.

The gadolinium complex for contrast agent for diagnosis of liver cancer according to an embodiment of the present invention is characterized in that gadolinium is bound to the DTPA-HA intermediate formed by reacting diethylenetriamine pentaacetic acid with hyaluronic acid. A complex is defined as a group of atoms consisting of several different atomic molecules or atomic groups centering on one or more atoms or ions and having a directional and cubic coordination.

In the present invention, diethylene triamine pentaacetic acid (DTPA) includes anhydrous DTPA (anhydrous DTPA). Since DTPA is a metal chelating agent, it has a strong binding property with metals, so it can form stable bonds with metals used in contrast agents, and can easily bind physiologically active substances.

DTPA can be represented by the following formula.

Since DTPA has a carboxyl group, it can easily form an ester bond with the hydroxy group of hyaluronic acid. DTPA also forms a complex with gadolinium by coordination bonds.

And DTPA may have a dianhydride form as represented by the following formula.

BACKGROUND ART Hyaluronic acid (HA) is a biocompatible hydrophilic polymer, and is composed of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine Lt; RTI ID = 0.0 > heteropolysaccharide. ≪ / RTI > HA is a linear polymer having a molecular weight in the range of about 3 to 13,000 kDa, depending on the raw material and the preparation method. HA is naturally present in pericellular gels, the underlying material of connective tissue in vertebrates, the lubrication of joints, the vitreous humor and the umbilical cord. HA plays an important role in biological organisms, such as physical support for cells in many tissues such as skin, tendons, muscles, and cartilage.

HA can be represented by the following formula.

Since HA has a hydroxy group, it forms an ester bond with the carboxyl group of DTPA. In addition, HA plays a role in introducing gadolinium into hepatocytes by HA receptor expressed in hepatocytes through blood flow.

HA is known to have hepatic specificity by a variety of receptors, which can induce intracellular infiltration similar to that of general liver in the liver cirrhosis model and cirrhosis by staying longer in the liver cirrhosis model (ACS NANO, 4 , 3005-3014, 2010).

Thus HA induces gadolinium in cirrhotic tissue by HA receptor, thereby enhancing contrast by increasing contrast of hepatocellular carcinoma and cirrhotic tissue in liver cirrhosis patients.

The suitable molecular weight of HA applied in the present invention is 3 to 150 kDa. Within the above range, HA of low molecular weight (about 50 kDa or less) is slightly weaker in binding ability to hepatocytes than HA of high molecular weight, but is small in size and can be easily released from the body. On the other hand, HA having a molecular weight of less than 3 kDa has a problem that binding ability with hepatocytes is remarkably lowered.

Within the above range, high molecular weight (about 50 kDa or more) of HA has strong binding ability with hepatocytes, so that a high quality image can be obtained. On the other hand, HA having a molecular weight of more than 150 kDa is large in size and therefore has a disadvantageous emission in the body.

DTPA-HA intermediates in which the carboxyl group of DTPA and the hydroxy group of HA are ester-bonded vary in the degree of substitution of DTPA. In the present invention, the DTPA-HA intermediate includes all substances produced depending on the degree of substitution of DTPA.

The DTPA-HA intermediate can be represented, for example, by the following formula: In the following formula, n may be an integer of 7 to 375.

The gadolinium complex (Gd-DTPA-HA) of the present invention having gadolinium bonded to the DTPA-HA intermediate may be represented by the following formula. In the above formula, n is an integer of 7 to 375.

The gadolinium complex of the present invention is formed by coordinating gadolinium on the DTPA of the DTPA-HA intermediate. In addition, as in the above formula, the gadolinium complex of the present invention may have a form in which sodium ion is combined with gadolinium. Bonding of sodium ions is to impart electrical stability of the final material. In other words, Gd-DTPA, which has a negative charge, is given a neutral electrical property to prevent side effects due to unnecessary binding with substances having a positive charge in the blood.

The gadolinium complex of the present invention can selectively transfer gadolinium to hepatocytes by introducing hyaluronic acid into the conventional DTPA-Gd complex structure, thereby effectively enhancing the contrast of tumor tissue and normal hepatocytes. The gadolinium complex of the present invention is primarily delivered to the liver, which binds to normal liver tissues but not to tumor tissues, so that an MRI image can be obtained that can distinguish liver lesion sites and normal tissue sites. In particular, the gadolinium complex of the present invention can provide accurate cancer diagnosis data by enhancing the contrast of cirrhosis tissue and cancer tissue. In addition, the gadolinium complex of the present invention is rapidly released into the body and is safe for the human body when about one hour has elapsed after the injection without being absorbed in the liver.

Hereinafter, a method for producing the gadolinium complex of the present invention will be described.

First, DTPA and HA are reacted in an organic solvent to form a DTPA-HA intermediate. DTPA and HA can be manufactured in various ratios, and the range can be DTPA: HA = 1 to 10: 1 in mole ratio. Further, DTPA: HA = 50 to 150: 1.

The organic solvent may be used without limitation as long as it can dissolve DTPA and HA. For example, dimethyl sulfoxide (DMSO), dicyclohexylcarbodiimide (DCC), dimethylaminopurine (DMAP), and sulfolane may be used. A preferred organic solvent is dimethylsulfoxide. Since dimethylsulfoxide is mixed well with water in an organic solvent, it is easy to remove unreacted materials after the reaction. It is also suitable for chemical resistance of dialysis membranes.

DTPA and HA are dissolved in an organic solvent and then stirred and reacted for 6 to 48 hours. The reaction generates DTPA-HA intermediates in which DTPA and HA are esterified. After the reaction, the organic solvent and the unreacted material are removed by distilled water dialysis to remove the generated intermediate material, and the moisture is removed by freeze drying.

Next, a gadolinium complex is prepared by bonding gadolinium to the DTPA-HA intermediate. To this end, the DTPA-HA intermediate is dissolved in distilled water and reacted by adding a gadolinium-containing solution. Here, gadolinium is possible in the form of various gadolinium compounds such as oxides and chlorides. For example, gadolinium oxide (Gd ₂ O ₃ ), gadolinium chloride (GdCl ₃ ), gadolinium fluoride (GdF ₃ ), gadolinium bromide (GdBr ₃ ) As an example, the gadolinium compound used in the present invention is gadolinium chloride. The mixing ratio of gadolinium chloride and DTPA-HA is determined by the ratio of the initial DTPA to the HA. DTPA-HA: GdCl ₃ = 1: 50: 1 when the molar ratio DTPA: HA = 1 to 10: 1 and DTPA-HA: GdCl ₃ = 1: 150.

The reaction is carried out for 6 to 48 hours while maintaining a pH of 7.0 using 1N sodium hydroxide solution. After the reaction, the unreacted material is removed through distilled water dialysis, and then the water is removed through freeze-drying to prepare a solid gadolinium complex.

Contrast agents containing the gadolinium complex of the present invention are used to obtain MRI images for the diagnosis of liver lesions, particularly liver cancer.

The contrast agents of the present invention may be formulated according to conventional formulation methods for preparing contrast agents known in the art. For example, it can be formulated in oral or parenteral dosage forms. Preferably as an intravenous injection. The contrast agent of the present invention can be used in the form of a suspension or solution in a solvent such as distilled water for injection, physiological saline, and Ringer's solution. If necessary, pharmacologically acceptable additives such as carriers, excipients and the like may be added. The additives vary depending on the mode of administration, route of administration, and the like. Specific examples of injection administration include buffers, antibacterial agents, stabilizers, solubilizers and excipients, which can be used alone or in combination.

The contrast agent of the present invention has a pH of 6.0 to 8.0, preferably 6.5 to 7.5. The contrast agent of the present invention is appropriately determined depending on the mode of administration, route of administration, and dosage depending on body weight, condition, and site. The specific dose at the time of intravenous administration varies depending on the age, physique, site to be contrasted, etc. of the patient to be administered, but the amount of the gadolinium complex contained is preferably 25 占 퐉 ol / kg to 100 占 퐉 ol / kg. The contrast agent of the present invention can be suitably used as a contrast agent for various animals other than humans.

Hereinafter, the present invention will be described by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

(Example)

37 mg of DTPA dianhydride (Sigma-Aldrich, USA) and 125 mg of HA (MW 6,800, Bio Land, KOREA) were dissolved in 200 ml of dimethylsulfoxide (Sigma-Aldrich, USA) and reacted for 36 hours. After freeze-drying (Freezone 2.5 plus, LABCONCO, USA, USA), the unreacted materials and solvent were removed by dialysis against distilled water for 3 days using a molecular weight cut-off membrane (Spectra / Por-MWCO 3,500, Spectrum Laboratories, <0.001 mbar, -80 <0> C) to give the DTPA-HA intermediate in solid form.

113 mg of the obtained DTPA-HA intermediate was dissolved in 100 ml of distilled water, and then 10 ml of a 5.3 mg / ml GdCl ₃ .6H ₂ O solution was added. The reaction was carried out for 24 hours while maintaining the pH at 7.0 using 1N sodium hydroxide solution. After the reaction, unreacted material was removed by dialysis against molecular weight blocking membrane (Spectra / Por-MWCO 3,500, Spectrum Laboratories, Inc., USA) for 00 days and freeze-dried (Freezone 2.5 plus, LABCONCO, (gd-DTPA-HA) was prepared via solid-liquid extraction at -80 < 0 &gt; C. The reaction between the DTPA-HA intermediate and gadolinium in the above example can be represented as follows.

Experimental Example 1: Measurement of zeta potential [

The electrical properties of the gadolinium complex prepared in the above examples were tested. For this purpose, samples were prepared with distilled water at a concentration of 0.05 mM [Gd]. The electrical properties of the gadolinium complexes prepared using Zetasizer Nano Z (Malvern Instruments, Malvern, UK) were measured and the results are shown in FIG.

Referring to FIG. 1, the electrical properties of the gadolinium complex exhibit a weak negative charge value, which allows the gadolinium complex to stably exist in the blood and prevent the entry into non-specific body regions.

<Experimental Example 2: FT-IR analysis>

Experiments were conducted to determine the presence or absence of DTPA-HA intermediate formed in the above examples. For this purpose, KBr and 3 mg of DTPA-HA solid samples were mixed and compressed to prepare pellets, and then they were placed in a FT-IR infrared spectrophotometer (Galaxy 7020A, Mattson Instruments Ins.) And measured. 2.

Referring to FIG. 2, in the intermediate DTPA-HA, it is absent in HA, and CH ₂ No peak was observed, indicating that the DTPA-HA intermediate was well formed.

&Lt; Experimental Example 3: Signal strength >

Experiments were performed to confirm the MRI signal intensity of the gadolinium complex prepared in the above examples. For this purpose, a gadolinium complex was dissolved in distilled water to prepare samples having gadolinium concentrations of 0.5, 0.05, 0.005 and 0.0005 mM.

The signal intensity was checked using a magnetic resonance imager (Siemens magneto tim trio 3.0T, wrist coil, Germany). Primovist ^TM (Schering AG, Germany), a commercially available T1 contrast medium, was used as a control. The experimental results are shown in Fig.

Referring to FIG. 3, it can be seen that the signal intensity of the sample including the gadolinium complex of the present example is higher than that of the conventional primevalast contrast agent.

<Experimental Example 4: MRI photographing experiment 1>

Samples containing the gadolinium complex of the above examples were prepared for in vivo experiments. The gadolinium complex prepared in the above example was dissolved in distilled water to prepare a sample having a gadolinium concentration of 6.25 mM. Samples were administered via tail vein in healthy mice at 0.05 mmol [Gd] / kg [mouse weight]. For example, when the mouse weight is 25 g, 0.2 ml of a sample of 62.5 mM [Gd] is injected.

Time-of-repetition / time of echo: 5.6 / 1.5 ms, NEX: 2, field of view: 130 mm * 160 mm, section thickness using a magnetic resonance imager (Siemens magneto tim trio 3.0T, wrist coil, Germany) 1.53 mm, matrix No.:166x256, and the results are shown in FIG.

Referring to FIG. 4, it can be seen that the sample begins to bind to the hepatocytes at 2 minutes after the injection, and the liver appears white. This means that dynamic contrast enhancement imaging is possible within 5 minutes after the injection of the sample and that the hepatocellular carcinoma can be diagnosed rapidly. At the same time as injection, almost maximal signal intensity was reached and visualization was possible within a short time, and apparent enhancement was observed even after 1 hour.

On the other hand, the kidneys of the mice were photographed using the same sample, and the results are shown in FIG. Referring to FIG. 5, it was confirmed that most of the samples not absorbed by the hepatocytes were discharged to the outside of the body in about 1 hour. Consistent with this, in the bladder photograph of FIG. 6, it can be seen that a large amount is discharged around 1 hour. Therefore, it is possible to prevent side effects caused by the contrast agent that may be present in the body.

<Experimental Example 5: MRI photographing experiment 2>

In order to examine the actual contrast effect on liver cancer, the time-dependent contrast results using the mouse liver metastasis cancer model are shown in FIGS. 7 and 8. FIG. The gadolinium complexes prepared in the above examples were administered to mice by the same method as in Experimental Example 4, and photographed.

Referring to FIGS. 7 and 8, all of the mice in which cancer had developed were all found to be cancer one hour after administration. In the figure, CNR is the contrast to noise ratio and is used as an index to evaluate the contrast agent performance. It shows the difference in contrast between the liver cancer site and the normal site. The CNR value was calculated by the following equation.

CNR = (SI _{Tumor -} SI _Normal ) / noise,

_(Tumor SI: signal intensity of tumor part, _Normal SI: signal intensity of normal liver part, noise: noise of machine)

From the above results, it can be seen that the present invention can improve the contrast enhancement ability of the hepatocarcinoma because the contrast of the hepatocarcinoma area and the normal area can be increased.

<Experimental Example 6: MRI photographing experiment 3>

The contrast effect of the gadolinium complex prepared in the above example and the product of Primovist ( ^TM ) (Schering AG, Germany), which is a T1 contrast agent, was compared using a healthy mouse. The mice were administered by the same method as Experimental Example 4 and photographed, and the results are shown in FIG.

Referring to FIG. 9, the conventional contrast medium, Primobist, was known to have an optimal diagnosis time point of 10 minutes, and the gadolinium complex contrast agent of the present invention showed the strongest signal intensity after 3 hours. And the signal intensity after 3 hours is about 1.4 times higher than the strongest signal strength of Primobist and similar signal intensity appears after 1 hour. Thus, it can be confirmed that the present invention can continuously monitor images even after 1 hour from administration. This means that MRI can be taken regardless of time because the diagnosis time is free.

<Experimental Example 7: MRI photographing experiment 4>

In order to compare the effect of the gadolinium complex of the examples with the effect of the primobist contrast agent, the time-dependent imaging results are shown in FIG. The mice were administered by the same method as Experimental Example 4 and photographed.

Referring to FIG. 10, after 10 minutes, both drugs were able to differentiate cancer, but it was difficult to clearly distinguish cancer sites of mice administered with primobist after 1 hour. On the other hand, in the mouse to which the gadolinium complex of the present invention was administered, the cancer site was more clearly distinguished. At 24 hours, it was confirmed that both drugs were not accumulated and released stably. From the above results, it can be seen that the present invention is more effective than the Primobist contrast agent.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Accordingly, the true scope of protection of the present invention should be determined only by the appended claims.

Claims (7)

A gadolinium complex formed by bonding gadolinium (Gd) to a DTPA-HA intermediate formed by reacting diethylenetriaminepentaacetic acid (DTPA) with hyaluronic acid (HA)
Wherein the DTPA-HA intermediate material is a material represented by the following formula (1) wherein the carboxyl group of the diethylene triamine pentaacetic acid and the hydroxy group of the hyaluronic acid are formed by an ester bond,
Wherein the gadolinium complex is formed by coordination bonding of gadolinium to diethylene triamine pentaacetic acid of the DTPA-HA intermediate and is a substance represented by the following formula (2).
(Formula 1)

In the above formula, n is an integer of 7 to 375.
(2)

In the above formula, n is an integer of 7 to 375. delete delete delete delete delete A contrast agent for diagnosis of liver cancer, which comprises the gadolinium complex according to claim 1.

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