TW201340980A - Metal-polysaccharide conjugates: composition, synthesis and methods for cancer - Google Patents

Abstract

The current disclosure, in one embodiment, includes a polysaccharide conjugate. This conjugate has a polysaccharide and at least one liner covalently bound to the polysaccharide. The conjugate also has at least on 5 e metal conjugated by said linker. According to another embodiment, the disclosure provides a method of synthesizing a polysaccharide conjugate by covalently bonding a linker to a polysaccharide to obtain an intermediate and by conjugating said intermediate to a metal to form a polysaccharide conjugate. This conjugate has a higher relaxivity, so it is suitable to be used as a contrast medium for hybrid camera.

Description

Metal polysaccharide conjugate: a composition containing the same, a method for producing the same, and Its method of treating cancer

The present invention is primarily directed to a conjugate of a metal to a polysaccharide, and more particularly to a polysaccharide conjugate that induces cancer cell death, utilizes cancer therapy, and utilizes MRI to assess tumor angiogenesis.

Angiogenesis is a process involving increased vascular density and permeability within a tumor. With the understanding of this phenomenon and the understanding of cell cycle regulation and cell signaling, a variety of cancer treatments have opened a new page. Despite the many breakthroughs in angiogenesis, cancer, cancer, or other medical treatments involving angiogenesis still have many unmet problems. The use of cytotoxic drugs in vivo rather than the use of cytostatic drugs is one of the challenges that has not been overcome in the current field.

The efficacy of platinum conjugates in inhibiting tumor activity has been demonstrated. For example, cisplatin, a widely used anticancer drug, has been treated with breast cancer and ovarian cancer alone or in combination with other agents. Cisplatin, also known as cis-diamminedichloro-platinum (II) (CDDP), is a simple molecule that conjugates platinum to NH ₃ molecules. Cisplatin causes the cells to stop at the S phase and thereby arrest the mitosis of the proliferating cells. Cisplatin also reduces the expression of vascular endothelial growth factor (VEGF) and inhibits angiogenesis during chemotherapy. Cisplatin has an effect on the treatment of most solid tumors. However, cisplatin is clinically used such as significant nephrotoxicity, myelosuppression, drug resistance, gastrointestinal toxicity, neurotoxicity and other side effects (eg, vomiting, granulocytopenia, and The limitation of weight loss. In addition, the cisplatin dosage form requires the use of a large carrier with poor water solubility, thus reducing its efficacy. A variety of platinum conjugates have been chemically modified to increase their hydrophilicity, reduce their side effects, and increase their efficacy; however, these conjugates still have a number of serious drawbacks.

Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Positron emission tomography (PET), and Single photon emission computed tomography (SPECT) are near An important tool for diagnosing disease progression for several years. For example, cell photography of PET and SPECT provides valuable information for monitoring and guiding the clinical treatment of malignant tumors or other diseases. PET and SPECT agents can produce highly specific active reactions by generating images by nuclear transformation and using radioisotopes without carriers. On the other hand, computed tomography, nuclear magnetic resonance, and ultrasound scanning are prognostic tools because they do not provide information on the target cells; therefore, they are not ideal for assessing cancer treatment.

Recently, hybrid CT or MRI hybridization methods with PET or SPECT have been developed to provide better sensitivity and to dynamically quantify drug properties in real time. CT or MRI developers use blood-labeled molecules; therefore, there is a need in the art for a novel developer that can have a longer penetration/diffusion effect, or be the target cell.

In view of the disadvantages of the prior art, it is an object of the present invention to provide a developer which has a high retardivity and can be used for MRI/PET, MRI/SPECT, CT/PET, or CT/SPECT hybrids. Development and treatment.

In order to achieve the above object, the present invention provides a polysaccharide conjugate comprising: a polysaccharide; at least one linking group covalently bonded to the polysaccharide; and at least one metal which is linked by the foregoing The group is conjugated.

Preferably, the aforementioned polysaccharide has a molecular weight of 3,000 to 30,000 ampoules.

Preferably, the aforementioned polysaccharide system is selected from the group consisting of collagen, chondroitin, hyaluronic acid, chitosan (chitosan), and chitin. More preferably, the aforementioned polysaccharide body comprises chitosan.

Preferably, the polysaccharide has an amine group, and the aforementioned linking group is covalently bonded to the aforementioned amine group of the polysaccharide.

Preferably, the linking group has a carboxyl group or a monothiol group, and the metal is conjugated to the carboxyl group or the aforementioned thiol group of the linking group.

Preferably, the aforementioned linking group is a monomeric amino acid, a chelating agent or a modifier.

Preferably, the aforementioned chelating agent is ethylene diamine tetraacetic acid.

Preferably, the aforementioned linking group is an open-ring form of the modifier. More preferably, the aforementioned modifier is an imine sulfonate Butane (iminothiolane).

Preferably, the foregoing metal is selected from the group consisting of Tc-99m, Cu-60, Cu-61, Cu-67, In-111, Tl-201, Ga-67, Ga-68, As-72, Re-188, Ho -166, Y-90, Lu-177, Sm-153, Sr-89, Gd-157, Gd-158, Bi-212, Bi-213, Fe, Au, Ag, and Au/Ag composites Group.

Preferably, the aforementioned polysaccharide conjugate comprises 50 to 80% by weight of the aforementioned polysaccharide.

Preferably, the aforementioned polysaccharide conjugate comprises 10 to 40% by weight of the aforementioned linking group.

Preferably, the aforementioned polysaccharide conjugate comprises 10 to 30 wt% of the foregoing metal.

The present invention further provides a method for synthesizing a polysaccharide conjugate, comprising the steps of: covalently bonding a linking group to a polysaccharide to obtain an intermediate product; and conjugateing the intermediate product to a The metal forms a polysaccharide conjugate.

Preferably, the aforementioned method further comprises drying the aforementioned polysaccharide conjugate to form a powder.

The polysaccharide conjugate of the present invention not only has a prolonged time in the blood circulatory system, but is more likely to accumulate at the tumor site. Further, the polysaccharide conjugate of the present invention can be used as a developer required for MRI/PET, MRI/SPECT, CT/PET, or CT/SPECT hybrid development.

The present invention provides a polysaccharide conjugate which can be used as a hybrid Development, such as nuclear scanning or MRI, the desired developer (or macrocontrast medium). Further, when the polysaccharide conjugate is used as a developer, it has an extended period of time remaining in the circulatory system, so that minute lesions and hemodynamic changes can be detected.

The polysaccharide conjugate of the present invention comprises: a polysaccharide; at least one linking group covalently bonded to the polysaccharide; and at least one metal conjugated via the aforementioned linking group.

Some embodiments of the invention include metal polysaccharide conjugates, methods for their preparation, and uses thereof. Such applications include inducing cancer cell death, treating cancer, and diagnosing the course of the disease. Specifically, the polysaccharide conjugate comprises a polysaccharide having at least one linking group. The linking group can further conjugate the metal. In a preferred embodiment, the linking group is a monomolecular amino acid, a chelating agent, or a modifying agent. In a possible embodiment, the linking group is a monomolecular amino acid, and the linking group can be conjugated to the metal via an oxygen group (O-group) instead of via a nitrogen group (N- Group). In certain embodiments, the metal can be a transition metal. In many embodiments, the polysaccharide conjugate has a plurality of monomolecular amino acids in its structure, and thus can conjugate a plurality of metal groups. The conjugate can be of any size, but in certain embodiments, the conjugate is designed to have at least 10,000 otounds per molecule, for example, 10,000 amps to 50,000 amps, The conjugate is restricted from being excreted from the kidneys. In a particular embodiment, the polysaccharide conjugate has a molecular weight of about 20,000 ear swallows to 50,000 ear augmentation; more specifically, it has a molecular weight of about 26,000 amal to 30,000 auricular.

The polysaccharide used may be any kind of polysaccharide, but it is advantageous to use a polysaccharide system which is easy to vascular uptake. Specific Adhesive molecules such as proprotein, chondroitin, hyaluronic acid, chitosan, and chitin are particularly suitable as the aforementioned polysaccharide. In a specific embodiment, the aforementioned polysaccharide is chondroitin A. Although the invention is not limited to a particular in vivo mechanism, such polysaccharides may aid in absorption and delivery to cancer cells via blood vessels. This situation is particularly evident in the angiogenesis of most tumors. The molecular weight of the final product of the invention is 20,000 amps to 50,000 amps, which will aid in the progression of hematology therapy.

The aforementioned amino acid may be attached to the aforementioned polysaccharide in any stable manner, but in many embodiments of the invention, it is covalently bonded to the aforementioned polysaccharide. The aforementioned amino acid may be in a single molecular form, and each molecule is bonded to the aforementioned polysaccharide, respectively. The aforementioned amino acid may have an oxygen group which may be conjugated to the aforementioned metal; in particular, it may have two conjugated oxygen groups. The aforementioned metal may be conjugated to a single monomolecular amino acid or conjugated to two or more monomolecular amino acids. Examples of the amino acid monomer which may be used singly or in combination include glutamic acid, aspartic acid, a combination of glutamic acid and alanine, a combination of glutamic acid and aspartic acid, glutamic acid and A combination of branamide, a combination of glutamic acid and glycine, and a combination of aspartic acid and glycine. Aspartic acid is a preferred choice in view of the bond distance between the two carboxylic acid groups and better tumor cell uptake specificity. The aforementioned amino acid may be left-handed or right-handed, or a racemic mixture containing left-handed and right-handed. L-amino acid is advantageous because of its superior tumor uptake. The reason why aspartic acid can be selected is that a single aspartic acid molecule is sufficient to be conjugated to a metal. In addition, mammalian cells do not synthesize aspartic acid by themselves, so aspartic acid is an essential nutrient, making it more easily ingested by highly proliferating tumor cells.

The aforementioned amino acid may comprise from about 10 to about 50 of the aforementioned polysaccharide conjugate Weight percentage.

On the aforementioned polysaccharide molecule, the saturation of the position at which the aforementioned amino acid is linked may vary as the case may be. For example, as shown in Figure A, only one amino acid can be linked. Very low saturation, such as 5% or less, 10% or less, 20% or less, can be achieved. The first B diagram illustrates a conjugate having moderate saturation, for example, about 30%, about 40%, about 50%, about 70% saturation. The first C diagram illustrates a conjugate having a high degree of saturation, for example, about 80%, about 90%, about 95%, or higher, or substantially fully saturated. Although each of the amino acids in the first figure is conjugated with a metal, in many practical examples, there are a plurality of metal-conjugated amino acid saturations, for example, less than 5%, 10%. 20%, about 30%, about 40%, about 50%, about 70%, higher than 80%, 90%, 95%, or higher, or substantially fully saturated.

The aforementioned metal may be any metal atom, or an ion, or a compound containing a metal, and may be conjugated to the oxygen group of the aforementioned amino acid monomer. In a particular embodiment, the aforementioned metal can be a transition metal such as platinum, iron, ruthenium, osmium, manganese, cobalt, indium, gallium, or hafnium. The aforementioned metal may be a therapeutic metal. The aforementioned metal may be part of a large molecule, for example, a drug. The aforementioned metal may be conjugated to the aforementioned polysaccharide-amino acid skeleton by the oxygen group of the aforementioned amino acid monomer.

In one embodiment of the invention, the aforementioned metal comprises from 15 to 30% by weight based on the total weight of the polysaccharide conjugate.

In one embodiment of the invention, the aforementioned conjugate comprises chondroitin A, which is covalently bonded to an aspartic acid monomer. The aforementioned aspartic acid monomer is conjugated to platinum of a platinum-containing compound. A variation in the present invention In the above, the platinum may be divalent platinum; and in another variation of the invention, the platinum may be tetravalent platinum.

The aforementioned conjugate may be water soluble. For example, the aforementioned conjugates can have a solubility in water of at least about 20 mg (calculated as metal equivalents) per mL. The aforementioned conjugates can be provided in a number of different forms, for example, aqueous solutions or powders. The aforementioned conjugates and their formulations can be sterilized. For example, the aforementioned conjugate can be provided in the form of a sterilized powder.

The foregoing conjugates can be prepared in accordance with embodiments of the present teachings to covalently bond one or more amino acid monomers to a polysaccharide molecule, respectively. Next, a metal can be provided and conjugated to the aforementioned amino acid monomer. According to another embodiment of the present invention, the aforementioned metal may be first conjugated to the aforementioned amino acid monomer, and then one or more of the aforementioned amino acid monomers may be covalently bonded to the aforementioned polysaccharide.

The aforementioned conjugates of the invention can be used to kill cancer cells or tumor cells and thus treat cancer or tumors. The aforementioned conjugates can be labeled to tumors, especially solid tumors. This property can be verified, for example, via the aforementioned compound labeled with a radioisotope, such as a polysaccharide-amino acid backbone linked to ^99m Tc, which can be developed by an additive ray. A biotoxic agent containing a metal group can reduce its biological toxicity by being conjugated to the aforementioned polysaccharide-amino acid skeleton. For example, the biotoxic agent can be slowly released from the aforementioned polysaccharide to reduce fatal systemic toxicity. In addition, a drug having poor water solubility or poor target tumor ability can enhance its therapeutic index by conjugating the drug to the aforementioned polysaccharide-polymer backbone.

In certain embodiments, the platinum-containing conjugate inhibits cancer cell growth at a dose lower than that required for cisplatin. Further, a platinum-containing conjugate pair Cancer cells that are resistant to cisplatin also have an inhibitory effect, especially ovarian cancer cells.

The conjugate selected for use in the present invention kills or inhibits the growth of any type of cancer cell or tumor cell. However, these conjugates respond better to solid tumors. Examples of cancers that may be affected by the specific conjugate of the present invention are: ovarian cancer, cisplatin-resistant ovarian cancer, pancreatic cancer, breast cancer, connective tissue cell tumor, uterine cancer, and lymphoma.

In addition to cancer, certain conjugates of the invention may have targets and inhibit cells involved in the development and progression of diseases such as HIV, autoimmune diseases (eg, encephalomyelitis, leukoplakia, scleroderma, thyroiditis). , perforated collagen disease), genetic diseases (eg, xeroderma pigmentosum and glucose-6-phosphate dehydrogenase deficiency), metabolic diseases (eg diabetes), cardiovascular diseases, neuropsychiatric diseases (neuro/psychiatric diseases) ) and other disease conditions (eg hypoglycemia and cirrhosis).

As described above, the polysaccharide may be selected from any kind of polysaccharide. However, it is advantageous to select a polysaccharide having an amine group in consideration of easy adhesion to a linking group. More specifically, the aforementioned linking group is covalently bonded to the aforementioned amine group of the aforementioned polysaccharide. Further, the aforementioned polysaccharide preferably has a molecular weight of 10,000 to 30,000 aurants. More specifically, the aforementioned polysaccharide has a molecular weight of 10,000 to 15,000 auricular. Further, the aforementioned polysaccharide accounts for 50 to 80% of the total weight of the aforementioned polysaccharide conjugate.

In a selected embodiment of the invention, the aforementioned linking group is a chelating agent or a modifying agent, and the aforementioned polysaccharide conjugate may have a molecular weight of 10,000 to 40,000 amps.

In a preferred embodiment, the aforementioned linking group is via a carboxyl group or The thiol group is conjugated to the aforementioned metal.

In many embodiments, the aforementioned linking group can be a chelating agent or a modifying agent. The selection of the aforementioned chelating agent is not particularly limited as long as it is a chelating agent capable of sequestering a metal such as a trivalent to pentavalent metal. In the present invention, the aforementioned chelating agent may be chelated with the aforementioned metal via a nitrogen-, sulfur-, oxygen-, phosphorus-group or a combination thereof. In principle, the aforementioned chelating agent used in the present invention is ethylenediaminetetraacetic acid (EDTA).

The above "modifier" means a compound which modifies a functional group of the aforementioned polysaccharide. The selection of the above-mentioned modifier is not particularly limited as long as it can be made to have a thiol group after the reaction with the above polysaccharide. Such modifiers include, but are not limited to, imine cyclothiobutane (e.g., 2-imine cyclothiobutane). In a particular embodiment, the aforementioned polysaccharide (eg, chitosan) is modified with 2-imine cyclothiobutane to obtain a conjugate of a polysaccharide and a ring-opened 2-imine cyclothiobutane. (ie, a polysaccharide-thiol compound). The reaction scheme is as follows. The aforementioned metal is successively conjugated to the thiol group of the aforementioned polysaccharide-thiol compound to form a polysaccharide conjugate.

The aforementioned linking group may be 10 to 40% by weight based on the weight of the aforementioned polysaccharide conjugate.

In many embodiments of the present invention, the foregoing metal is selected from the group consisting of Tc-99m, Cu-60, Cu-61, Cu-67, In-111, Tl-20, Ga-67, Ga-68, As-72, Re-188, Ho-166, Y-90, Lu-177, Sm-153, Sr-89, Gd-157, Gd-158, Bi-212, Bi-213, Fe, Au, Ag, And a group consisting of Au/Ag complexes. Among the listed metals, Tc-99m, In-111, Tl-201, Ga-67, Ga-68, Re-188, Y-90, Lu-177, Gd-157, or Gd-158 are preferred. select. Further, the Au/Ag composite used in the present invention is preferably disclosed in the Patent Application No. 100111493 of the Republic of China, which is incorporated herein by reference.

The aforementioned metal may be 10 to 30% by weight based on the weight of the aforementioned polysaccharide conjugate. In particular, the aforementioned metal may comprise from 12 to 26% by weight based on the weight of the aforementioned polysaccharide conjugate.

The present invention also provides a method of synthesizing a polysaccharide conjugate comprising covalently bonding a linking group to a polysaccharide to obtain an intermediate product; and conjugateing the intermediate product to a metal to form A polysaccharide conjugate.

In a preferred embodiment, the method further comprises drying the aforementioned polysaccharide conjugate to form a powder.

Example

The following examples are presented to illustrate specific embodiments of the invention. It will be apparent to those skilled in the art that the technical aspects of the embodiments of the invention disclosed by the inventor of the present invention can be properly understood. However, based on the disclosure of the present invention, it is obvious to those skilled in the art that the present invention can be varied, and the same or similar results can be obtained without departing from the spirit and scope of the present invention.

Example 1: Synthesis of Platinum Derivatives (II) and (IV)-Polymer Conjugates Method A

Cis-1,2-Diaminocyclohexane sulfatoplatinum (II) (cis-1,2-DACH-Pt.SO ₄ ) was synthesized via a two-stage process. The cis-1,2-DACH-PtI ₂ complex was synthesized in the first stage. The filtered through a 120 ml of the K ₂ PtCl ₄ in deionized water (5.00g, 12 mmol) and a solution of KI (20.00g, in 12 ml of water, 120 mmol) were mixed and stirred for 5 minutes continuously. A further equivalent of cis-1,2-DACH (1.37 g, 1.487 ml, 12 mmol) was added to the solution. The reaction mixture was stirred at room temperature for 30 minutes. The resulting yellow solid product was isolated by filtration and washed with a small portion of DI water. The final product was dried in vacuo, it has obtained the yellow cis-1,2-DACH-Pt ( II) SO 4 (4.83g, 99%). Elemental analysis showed Pt: 44.6% (w/w).

Add N-hydroxythiosuccinimide (sulfo-NHS) (241.6 mg, 1.12 mmol) and 3-B in a stirred solution of chondroitin (1 g, MW. 30,000-35,000) and water (5 ml). 3-ethylcarbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl, EDC (218.8 mg, 1.15 mmol) (Pierce Chemical Company, Rockford, IL). Then L-aspartate sodium salt (356.8 mg, 1.65 mmol) was added. The mixture was stirred at room temperature for 24 hours. The mixture was then dialyzed for 48 hours using a Spectra/POR molecular porous membrane (Spectrum Medical Industries Inc., Huston, TX) having a cut-off of 10,000. After dialysis, the product was filtered and lyophilized using a lyophilizer (Labconco, Kansas City, MO). The product aspartate-chondroitin (polysaccharide) is a salt Form, the weight is 1.29g. Similar techniques can be used to prepare glutamic acid with alanine, glutamic acid and aspartic acid, glutamic acid and glutamine, glutamic acid and glycine, and glutamic acid and alanine. Chondroitin of aspartic acid conjugated to aspartic acid, branamide, or glycine.

cis-1,2-DACH-Pt(II)SO ₄ (500 mg, 1.18 mmol) was dissolved in 10 ml of deionized water, followed by a solution of aspartate-chondroitin (1.00 g in 15 ml of deionized water). The solution was stirred at room temperature for 24 hours. After dialysis (MW: 10,000) and lyophilization, the obtained cis-1,2-DACH-Pt(II)-polysaccharide was 1.1462 g.

The platinum-polysaccharide conjugate, cis-1,2-DACH-dichloro-Pt(IV)-aspartate-chondroitin (PC) was synthesized by the following procedure: the above-mentioned cis-1 was stirred for 24 hours. A solution of 2-DACH-Pt(II)SO ₄ and aspartic acid-chondroitin was added dropwise to 2.5 ml of a 30% aqueous hydrogen peroxide solution. After 24 hours, HCl (0.02 N, 75 mL) was added and stirred at room temperature for 24 hrs, and then dialyzed with deionized water (MW: 10,000) overnight and lyophilized under vacuum. The final product obtained was 1.15 g. Elemental analysis showed Pt: 21.87% (w/w). The synthetic flow chart is shown in the second figure.

Method B

Dissolve cis-1,2-DACH-Pt(II)SO ₄ or cis 1,2-DACH-dichloro-Pt(IV) (500mg, 1.18mmol) in 10ml of deionized water, then add one-day aspartate (67 mg, 0.5 mmol) was dissolved in 2 ml of deionized water. The solution was stirred at room temperature for 24 hours. After dialysis and freeze-drying, cis-1,2-DACH-Pt-aspartate and chondroitin (1 g, MW. 30,000-35,000) and sulfo-NHS (241.6 mg, 1.12 mmol) in water (5 ml) And EDC (218.8 mg, 1.15 mmol) (Pierce Chemical Company, Rockford, IL). The synthetic flow chart is shown in the third figure.

Example 2: In vitro cell culture assay

To evaluate the cytotoxicity of cisplatin with platinum(II)-polysaccharide conjugate (PC) prepared by Method A as described above for breast tumor cells, two human tumor cell lines were selected: 2008 cell line and its anti-platinum Cell sub-strain 2008-c13 (a cancer cell line resistant to platinum). All cells were cultured in RPMI 1640 medium (10% fetal bovine serum and 2 mM branamine) at 37 ° C in a humid atmosphere containing 5% CO ₂ . The 2008 cells or 2008.C13 cells were seeded in a 96-well culture dish (4,000 cells/well) and cultured in RPMI 1640 medium for 24 hours. Next, cells were treated with PC or CDDP at concentrations of 2.5, 5, 10, 20, 25 and 50 μg/mL for 48 and 72 hours. The control group was treated with DMSO or PBS. After the cells were treated, the cells were co-cultured with 20 μL of tetrazolium matrix at 37 ° C for 1 to 2 hours to measure cell growth and viability. The absorbance at 450 nm was measured using a 96-well Synergy HT-microplate reader (miro) (Biotek, Winooski, Vermont). The cell growth inhibition rate can be expressed as a percentage value as follows: % = 100 - (OD _{control group -} OD _{experimental group} ) / OD _{control group} . The experiment was repeated three times. The number of viable cells was measured using a methylene tetrazolium (MTT) staining assay. Cellular protein content was measured using the Lowry assay. A drug concentration that inhibits 50% of cell growth (IC-50) is then determined. Experimental data were presented as a percentage difference from the control group (OD of treated cells/OD of untreated cells). The fourth and fifth graphs show the resulting cell growth inhibition curves.

The results show that in anti-platinum ovarian cancer cell lines, cells exposed to IC-50 under platinum-polysaccharide conjugate (PC) are more sensitive than cells exposed to cisplatin (CDDP) at IC-50. The sensitivity is 5.7 times (fourth panel), and this condition does not appear in platinum-sensitive ovarian cell lines (2008) (fifth panel). In particular, in a 48-hour experiment, platinum and polysaccharide conjugates (PC) at concentrations of 2.5 and 5 μg/mL were increased by 5.9 and 4.6 times, respectively, compared to cisplatin (CDDP) (Fig. 4A). Cell death; in a 72-hour experiment, it was increased by 9.3 and 1.5 fold, respectively, compared to cisplatin (CDDP) (Fig. B). This data shows that low doses of platinum-polysaccharide conjugate (PC) significantly inhibited cell growth of anti-platinum ovarian cancer cells.

To measure the effect of platinum-polysaccharide conjugate (PC) against anti-platinum ovarian cancer cells, 2008-c13 cells (0.5 x 10 ^-6 ) were treated with platinum-polysaccharide conjugate (PC). The cells were trypsinized and then centrifuged at 2500 rpm for 5 minutes. After washing twice with 1× PBS, the cells were fixed overnight with 70% ethanol, washed twice with 1× PBS, and then resuspended in 1 mL of propidium iodide (PI) solution (1×10 ⁶ cells/ In mL). Then, after adding RNase solution (20 μg/mL), 1 mL of propidium iodide solution (PI, 50 μg/mL in PBS) was further added. The samples were incubated at 37 ° C for 15 minutes, followed by analysis of PI fluorescence using an EPIS XL analyzer. Compared to cisplatin (CDDP), platinum (IV)-polysaccharide conjugates significantly promoted apoptosis in anti-platinum ovarian cancer cells at low concentrations (2.5 and 5 μg/mL) (fifth and seventh) Figure).

These results were confirmed by the TUNEL assay, in which after 48 hours of treatment, it showed that the mortality of cells exposed to both drugs increased with increasing drug volume. However, in each dose compared, the experimental group treated with platinum-polysaccharide conjugate (PC) had more Dead cells (P < 0.05) (Fig. 8).

Example 3: Evaluation of anticancer effects using a rat model with breast tumors

The breast cancer cells (13762NF, 10 ⁶ cells / rats, after subcutaneous injection in the thigh) were seeded in female Fischer 344 rats (125-175g). After 15-20 days, when the tumor volume is 1 cm, the platinum-chondroitin (platinum-polysaccharide) conjugate (PC) or chondroitin is 10 mg Pt/kg (platinum (IV)-polysaccharide) or A dose of 45 mg/kg (chondroitin) was administered to a rat with a breast tumor. Tumor volume and body weight were recorded daily for 60 days. The volume of the tumor was measured in the manner of [length (l) x width (w) x thickness (h)]/2. A 15% weight loss is seen as a result of toxicity caused by chemicals. The data show that the platinum-polysaccharide conjugate (PC) can effectively combat the growth of breast tumors in vivo (Fig. 9). After treatment with the platinum-polysaccharide conjugate, the tumor tissue was dissected and soaked in formalin. The tumor tissue was fixed in paraffin and stained with tissue staining with hematoxylin and eosin. Large-scale necrosis was observed 94 hours after administration of the platinum-polysaccharide conjugate, but this phenomenon did not occur in the group administered with the polysaccharide alone (Fig. 10).

Example 4: Method for tumor cell death or inhibition

The 2008-c13 breast tumor cells were treated with a platinum-polysaccharide conjugate to analyze the effect of the platinum-polysaccharide conjugate of Example 1 on tumor cells, and then further using Western blot (Eleventh Figure) ) Analysis of its effect on intracellular proteins. Compared to cisplatin (CDDP), cells treated with platinum-polysaccharide conjugate (PC), lysed PARP (Poly ADP-ribose polymerase, adenosine diphosphate ribose polymerase) was significantly increased. It is apparent that the platinum-polysaccharide conjugate inhibits the growth of 2008-c13 cells by potentiating the apoptosis-dependent action of Caspase-3 protease.

The effect of this effect in the 2008-c13 cell line was tested by flow cytometry after 48 hours and 72 hours of administration. Flow cytometric analysis showed that the number of cells in the sub-G1 phase increased with the increase in drug volume after treatment with PC and CDDP, which represents the accumulation of hypodiploid cells and showed induction of apoptosis. However, at the same dose, the use of PC resulted in a more pronounced increase in cells in the sub-G1 phase compared to CDDP (Fig. 14).

The typical DNA cleavage phenomenon in apoptosis was further measured in three independent experiments using the TUNEL assay. After administration of the above two drugs, the number of apoptosis was observed to have a significant dose-dependent tendency. However, when each dose was compared, PC-treated cells had a higher degree of apoptosis (P < 0.05) (fifteenth panel).

To test whether apoptosis is induced via a Caspase-3 protease-dependent pathway associated with PARP cleavage, Western blotting was used to determine the amount of Caspase-3 protease and PARP after cleavage (which is activated by Caspase-3). Then formed). PARP is a 113-kDa nuclear protein that is known to be specifically cleaved into an 85-kDa fragment in apoptotic action dependent on Caspase-3 protease. After 48 hours of exposure of the cells to CDDP or PC, the cleavage of PARP occurred at each dose. In the CDDP-treated cells, the amount of cleavage of PARP increased with the dose of 2.5 μg/mL to 20 μg/mL, and the amount of cleaved Caspase-3 protease also had a similar trend. PC-treated cells were observed to be lower except in the group using 5 μg/mL PC. In addition to the performance, the amount of cleaved Caspase-3 protease corresponds to the amount of cleaved Caspase-3 protease in CDDP-treated cells. Although the amount of cleavage of PARP induced by high dose (20 μg/mL) PC was lower than that induced by low dose PC, the amount of cleaved Caspase-3 protease upstream of it did not show this difference. (Figure 16).

In a further test of in vitro 2008-c13 breast cancer cells, flow cytometric analysis showed that the cells apparently stayed in the S phase 48 hours after exposure to the platinum-polysaccharide conjugate (PC) (Fig. 12). The highest degree of S phase blockade occurred in the low dose (90.3% vs. 90.1%) experimental groups at 2.5 and 5 μg/mL. Compared to cisplatin (CDDP), the effect of platinum-polysaccharide conjugate (PC) on cell arrest in S phase is significantly different.

Specifically, after treating the 2008-c13 cells with PC or CDDP for 48 hours, the DNA content was analyzed by flow cytometry. Exposure to CDDP at 5 μg/mL dose induced cell arrest in S phase and increased the proportion of sub-G1 phase, but this did not occur in the lowest dose (2.5 μg/mL). The ratio of the number of cells resting in the S phase to the sub-G1 phase gradually increased with the increase of the CDDP dose, and the highest S phase (84.8%) occurred at a dose of 20 μg/mL. After 48 hours of exposure of the cells to PC, the highest degree of S-stage rest occurred at lower doses (2.5 μg/mL [90.3%] and 5 μg/mL [90.1%]). At higher doses (10 and 20 μg/mL), the degree of S-stage arrest was steadily decreasing with increasing proportion of sub-G1 phase. This can be explained by the fact that at high pressures of high doses of PC, the cells undergo rapid and direct apoptosis before they stop at S phase (Fig. 17).

To elucidate the mechanism of S-phase arrest caused by CDDP and PC in 2008-c13 cells, 48 hours after administration, at 2008-c13 The amount of p21 and cyclin A that play an important role in the cell cycle regulation of S phase in the cell. The amount of expression of p21 or cyclin A was not associated with the degree of S-stage arrest after CDDP treatment. However, the performance of p21 or cyclin A was directly related to the extent of S-stage arrest after PC treatment: after treatment with a low dose of PC, p21 was up-regulated to reach the highest degree of S-stage rest, but at a high This phenomenon does not occur after dose treatment; cyclin A is up-regulated after high-dose PC treatment and maintained at a low level after treatment with low-dose PC (Figure 17 B) .

When compared to cells treated with cisplatin (CDDP), the p21 expression in the 2008-c13 breast cancer cells treated with platinum-polysaccharide conjugate (PC) was either transcription (graph 13 A) or The extent of protein expression (Figure 13 B) has increased.

Example 5: Synthesis of Gd-EDTA-chitosan (i.e., polysaccharide conjugate of the present invention) material

The desired chitosan (85% deacetamidine and an average molecular weight of 3500 Da) was obtained from Dalwoo-ChitoSan (Korea) (http://members.tripod.com/~Dalwoo/index.htm). Ethylenediaminetetraacetic acid (EDTA) and dialysis membrane (MWcut off 3,500) were purchased from Fisher Scientific Company (Houston, TX). N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide.HCL) (EDC), Gd-DTPA and GdCl ₃ .6H ₂ O was purchased from Aldrich Chemical company (Milwaukee, WI).

The synthesis of Gd-EDTA-chitosan is carried out via the two-stage procedure described below.

The first stage: the synthesis of EDTA-chitosan

2.59 gm of chitosan was dissolved in 259 mL of water. Then, 8.1 gm (27.7 mmol) of ethylenediaminetetraacetic acid was added. The pH of the solution was adjusted to 6 with 5 M NaOH. After adjusting the pH, add 5.31 gm of EDC and adjust the pH to 6 again. The reaction mixture was stirred at room temperature overnight. The volume of the reaction mixture was reduced in a freeze-dried manner, and then the solution was dialyzed against water for 36 hours using a dialysis membrane (MWCO 3500). The dialyzed solution was freeze dried to obtain about 8.5 gm of EDTA-chitosan. The crude product, which was not further purified, was subjected to a guanidine chelate reaction (Gadolinium Chelation).

The second stage: chelation of EDTA-chitosan with gadolinium (Gd)

5.4 gm of EDTA-chitosan was dissolved in 45 mL of water. A cerium chloride solution having a concentration of 100 mg/mL was prepared and added dropwise to the EDTA-chitosan solution while maintaining the pH at 6 to 6.5 with 0.5 N NaOH. GdCl _{3 was} continuously added until the EDTA-chitosan solution turned cloudy. The reaction mixture was stirred at room temperature for about 4 hours and then centrifuged to separate the insoluble solids from water. The pH of the clear Gd-EDTA-chitosan solution was adjusted to 8, and then subjected to water dialysis for 36 hours with a dialysis membrane (MWCO 3500). Thereafter, it was freeze-dried to obtain 2.7 gm of Gd-EDTA-chitosan. The presence of free hydrazine was determined by using 4-(2-pyridylazo-resorcinol) as an indicator. According to the elemental analysis, the cerium content was 20.6% (w/w). The structure of Gd-EDTA-chitosan is shown in Fig. 18.

Example 6: In vitro experiment to determine the relaxation rate (Relaxivity)

Gd-EDTA-chitosan and Gd-DTPA aqueous solutions were prepared at concentrations of 0.01 mM, 0.02 mM, 0.04 mM, and 0.08 mM, respectively. Spin lattice (T1) relaxivity is measured at 7.0T. The R ₁ value at mM ^-1 s ^-1 was determined from the linear least-squares determination from the slope in the 1/T ₁ vs[Gd] plot. The R ₁ values of Gd-DTPA and Gd-EDTA-chitosan were measured at 3.61 and 9.59 mM ^-1 s ^{-1 ,} respectively.

Example 7: Living animal studies by nuclear magnetic resonance (MRI)

The MRI study was conducted in two models. In a rat model with tumor, 13762 breast cancer cells (sc, 10,000 cells/rat) were implanted from the hind thigh of the rat. MRI photography was performed when the tumor reached a size of 1.5 to 2 cm. Rats with breast cancer were anesthetized by adding 2 to 4% and 1 to 2% isoflurane to oxygen, respectively. The animal was placed on the developing table with the head facing downward, and the physiological state of the animal was monitored by introducing an anesthetic gas through a nose cone and wrapping the breathing bellows around the abdomen. A 7.0 T/30 cm MR scanner with a 400 mT/m, 12-cm inner diameter self-shielded gradient coil system (2,760 T/m/s slew rate) and a 72-cm inner diameter volumetric RF coil ( Bruker Biospin Corp., Billerica, MA) obtained a two-dimensional axis image. Tumor location was visualized via radial T2-weighted RARE scans (TE/TR 60 ms/3000 ms, field of view: 8 cm x 6 cm, matrix size: 256 x 128, 2 mm slices, 2 averages). Obtained on the matching face Tumor axial T1-weighted spin echo scan (TE/TR 9ms/1000ms, FOV 6 cm x 6 cm, 256 x 256 matrix, 2 mm slices) and T2-weighted RARE scan (TE/TR 60 ms/3000 ms, FOV 6 cm x 6 cm, 256 x 256 matrix, 2 mm slices, 4 averages). A fast spoiled gradient echo (fast spoiled gradient echo) was used from the first minute of injection of Gd-DTPA and Gd-EDTA-chitosan (0.14 mmol/kg) to the 6.5 minute period after injection. FSPGR) sequence) (TE/TR 1.7 ms/60 ms, FOV 6 cm x 6 cm, 128 x 96 matrix, 2 mm slices, 45 degree excitation angle, 80 repetitions) to obtain T1-weighted images continuously every 5.8 seconds. After the dynamic FSPGR image is completely acquired, the axial T1 weighted spin echo scan is repeated for one hour. Use the T1 weighted image and the T2 weighted image before the comparison to define the regions of interest (ROIs), and then calculate the point-to-point signal intensity values of the tumor and normal muscle tissue (the opposite leg) (pixel-by-pixel) Signal intensity; SI). Three consecutive images of the largest tumor tomographic section of each animal were analyzed. The tumor area of each section is estimated from all pixels and pixel sizes contained in each ROI. Moreover, in all aspects, the degree of signal intensity enhancement in the ROI is presented as the ratio of the SI values of the tumor to normal muscle tissue.

Figure 19 shows a live animal study by nuclear magnetic resonance (MRI) after injection of Gd-EDTA-chitosan. Figure 20 shows a live animal study by nuclear magnetic resonance (MRI) after injection of Gd-DTPA. Figures 19 and 20 show that the signal intensity of Gd-DTPA and Gd-EDTA-chitosan is comparable in tumor tissues, but Gd-EDTA-chitosan is higher in the relaxation rate in vitro. Gd-DTPA is high.

For a normal pig (20 kg), images were taken 15 minutes after administration of Gd-EDTA-chitosan (0.13 mmol/kg, iv). Bring Melanoma pigs (23 kg) are supine. MRI scans were performed from the baseline and repeated at 5, 30 and 60 minutes after administration of Gd-EDTA-chitosan (0.13 mmol/kg, iv). All functional MRI data were obtained by GE 1.5-T EchoSpeed scanner (256 x 128 matrix; ten 5-mm slices acquired every 11.04 seconds for 5.53 minutes; 22-to 36-cm axial field of view). The resulting images were sent to a central imaging analysis provider (VirtualScopics, LLC, Rochester, NY) for image quality assessment and final analysis. The voxels of the region of interest at each time point are averaged to produce a time curve of signal strength. The signal strength of each region of interest before the comparison is subtracted to normalize the curve. MRI images of normal pigs 15 minutes after administration of Gd-EDTA-chitosan showed vascular targeting as shown in Figures 21 to 23. MRI images of pigs with melanoma at 5, 30, and 60 minutes after administration of Gd-EDTA-chitosan showed that the tumor site was indeed visualized. MRI images of melanoma-bearing pigs at 5, 30, and 60 minutes after administration of Gd-EDTA-chitosan were presented in Figures 24, 25, and 26. The time intensity curve (signal intensity vs. time) of Gd-EDTA-chitosan absorbed by tumor cells of melanoma pigs is shown in Figure 27. According to the twenty-seventh figure, the strongest signal intensity was measured 30 minutes after the injection of Gd-EDTA-chitosan. The signal intensity shown in Fig. 17 is further converted into a percentage of enhancement of tumor tissue, and the converted result is as shown in Fig. 28. Figure 28 shows that the percentage of tumor enhancement falls within the range of 16.3 to 30.5% compared to the signal intensity before the comparison. The best boost signal was measured 30 minutes after the injection of Gd-EDTA-chitosan.

Example 8: Photographic Research

To confirm that Gd-EDTA-chitosan can be used in hybrid photography techniques such as nuclear photography or MRI, EDTA-chitosan is calibrated at ^{99 m} Tc in the presence of tin(II) chloride. Subcutaneous injections were made from the hind legs of Fischer 344 rats to implant breast cancer cells (10 ⁵ cells/rat). On the 14th day after injection, ^99m Tc-EDTA-chitosan (1 mg/rat) and ^99m Tc-EDTA (3 mg/rat) (300 μCi/rat, iv) were used as the developer at 0.5 and At 500 hours, M-camera (Siemens) was used to obtain 500,000 data. The region of tumor damage and the region of interest (ROI) relative to the normal normal muscle site were calculated by computer and used to determine the tumor-to-background count density ratios. . The results of planar scintigraphy scanning of rats with breast cancer at 0.5 or 1 hour after administration of ^99m Tc-EDTA or ^99m Tc-EDTA-chitosan are shown in Figure 29. Planar scintigraphic imaging result of having breast cancer in rats 0.5 hours showed that tumor / muscle ratio in absorption of ^99m Tc-EDTA- chitosan greater than ^{99m Tc-EDTA (3.85 vs 2.74} ).

Example 9: Application of Gd-EDTA-chitosan to computed tomography (CT)

In order to confirm that Gd-EDTA-chitosan can be applied to CT due to the characteristics of metal groups (such as Gd) contained therein, two concentrations (100 and 200 mM) of Gd-EDTA-chitosan were prepared. The spatial resolution test of these concentrations was performed by CT. The experimental results are shown in the thirty-th figure. The experimental results show that a high concentration of Gd-EDTA-chitosan (i.e., No. 3) exhibits a significant strength compared to the result before the comparison (i.e., No. 1). This one The results also confirmed that Gd-EDTA-chitosan can be applied to CT.

Example 10: Application of Gd-EDTA-chitosan in heat therapy and neutron capture therapy

Elemental analysis of Gd-EDTA-chitosan showed a sputum with 20.6% w/w. Comparison of different concentrations of Gd-EDTA-chitosan (125-250 mg/mL, Gd-eq 25-50 mg/mL) and Gd-DTPA (174 mg/mL, Gd-eq 50 mg/mL), Gd- The temperature change of EDTA-chitosan can be achieved by a 40 KHz ultrasonic bath (31st). By MRI, this technology platform quantifies the tumor's absorption of Gd-EDTA-chitosan, which contributes to the Gd-157(n,gamma)/Gd-158 response of neutron capture therapy.

Example 11: Synthesis of Au/Ag composite-thiol compound-chitosan conjugate

Synthesis of Au/Ag composite-thiol compound-chitosan conjugate by reacting a thiol compound-chitosan conjugate with an Au/Ag composite; wherein the thiol compound The chitosan conjugate is obtained by reacting chitosan with 2-imine cyclothiobutane, and the synthesis of the Au/Ag composite is as disclosed in the Patent Application No. 100111493 of the Republic of China. . The details of the synthesis of the Au/Ag composite-thiol compound-chitosan conjugate are as follows.

Stir well to dissolve 10 mg of chitosan in 1 mL of deionized water until the chitosan was completely dissolved to obtain an aqueous solution of chitosan. Next, 2 mg of 2-imine cyclothiobutane was added to the aqueous solution of chitosan and allowed to react at room temperature for 3 hours to obtain sulfur. An alcohol-modified chitosan aqueous solution (i.e., a thiol compound-chitosan conjugate) can be used directly in the subsequent step without further purification.

Because of the good bonding ability between the thiol group and gold (Au), the Au/Ag composite-thiol compound-chitosan conjugate can be easily passed via the thiol compound-chitosan The conjugate is prepared by mixing with an Au/Ag composite. 4 μL of 0.1 M ascorbic acid (reducing agent) was added per 10 mL of 10 mM HAuCl ₄ and 10 mM AgNO ₃ mixture. After allowing to react for 1 minute, 1 mL of the thiol compound-chitosan conjugate as described above was added to obtain a growth solution. Next, the growth solution was stably left for 1 minute to obtain an Au/Ag composite-thiol compound-chitosan conjugate.

Figure 32 shows the Fourier transform infrared spectroscopy of chitosan and thiol compound-chitosan conjugates. The results show that the FTIR images of chitosan and thiol compound-chitosan conjugates are very similar in transmittance, both of which have 3425 cm ^-1 (NH) and 1630 cm ^-1 ( (N) C=O), 2920 cm ^-1 (CH), and a peak of 1098 cm ^-1 (CO). Further, the thiol compound-chitosan conjugate has a peak of Fourier transform infrared spectroscopy having a peak of 3119 cm ^-1 (SH). The peak of 3119 cm ^-1 (SH) is derived from the reaction of a 2-imine cyclothiobutane with a functional group (amine group) at the end of chitosan, which means a thiol compound-chitosan The conjugate has been successfully synthesized.

In summary, the polysaccharide conjugate of the present invention has a property of prolonging the time in the blood circulation, and tends to accumulate in the tumor tissue because of the difference in vascular permeability between the normal tissue and the solid tumor tissue. In addition, the polysaccharide conjugate of the present invention can be utilized for detecting tumor blood vessels by MRI New phenomenon.

While the present invention has been described with respect to the exemplary embodiments of the present invention, it will be understood that those of ordinary skill in the art are able to make modifications and changes to the embodiments without departing from the spirit and scope of the invention. .

The following drawings are part of the specification and are included to provide further disclosure of the particular aspects of the invention. The invention may be more clearly understood by reference to one or more of the drawings and the description of the embodiments of the invention.

The first figure shows three metal-polysaccharide conjugates in accordance with an embodiment of the present invention. "AA" means an amino acid. "M" means a metal. In the first panel A, only one or a few amino acid groups are present to chelate with the metal. In the first panel B, there is a moderate amount of amino acid groups chelated with the metal. In the first panel B, there is a maximum or approximate maximum possible amount of amino acid groups chelated with the metal.

The second figure shows a method for synthesizing a platinum analog (II) and (IV)-polysaccharide conjugate according to an embodiment of the present invention (Method A).

The third panel shows another method of synthesizing platinum analog (II) and (IV)-polysaccharide conjugates according to an embodiment of the present invention (Method B).

The fourth panel shows the efficacy of a platinum-polysaccharide conjugate according to an embodiment of the present invention in inhibiting anti-platinum ovarian cancer cells (2008 c13) at 48 hours (A) and 72 hours (B). .

The fifth panel shows the efficacy of the platinum-polysaccharide conjugate according to an embodiment of the present invention in inhibiting platinum-sensitive ovarian cancer cells (2008 c13) at 48 hours (A) and 72 hours (B). .

Figure 6 shows the results of a flow cytometry experiment showing the cisplatin (CDDP) (A) and the platinum-polysaccharide conjugate (PC) (B) according to the invention in anti-platinum at a time point of 48 hours. The effect of apoptosis on ovarian cancer cell lines (2008-c13).

Figure 7 shows the results of flow cytometry experiments in which anti-platinum ovarian cancer cell lines (2008-c13) were administered 48 hours after administration of different concentrations of platinum-polysaccharide conjugate (PC) or cisplatin (CDDP). A) Percentage of apoptosis with 72 hours (B).

Figure 8 shows the experimental results of the TUNEL assay, which showed that anti-platinum ovarian cancer cell lines (2008-c13) were administered 48 hours after administration of different concentrations of platinum-polysaccharide conjugate (PC) or cisplatin (CDDP). , the percentage of apoptosis.

The ninth panel shows that the platinum-polysaccharide conjugate according to the embodiment of the present invention is administered in an in vivo experiment for 24 hours (A) and 94 hours (B) (single dose, Pt 10 mg/kg) to inhibit breast tumors. The effect of growth. The tumor line labeled DY was taken from an animal to which only chondroitin was administered. The DP-AP is indicated as being derived from an animal to which a platinum-polysaccharide conjugate is administered. In the ninth panel A, the volume of the tumor on the left side was 5.2591 cm ³ [(2.08 cm × 2.58 cm × 1.96 cm) / 2]; the volume of the tumor on the right side was 5.2661 cm ³ [(2.20 cm × 2.37 cm × 2.02cm)/2]. In the ninth panel B, the volume of the tumor on the left side was 14.3622 cm ³ [(2.99 cm × 3.29 cm × 2.92 cm) / 2]; the volume of the tumor on the right side was 0.8782 cm ³ [(1.11 cm × 1.84 cm × 0.86cm)/2].

The tenth graph is the result of H&E staining of the tumor, which shows the administration of the platinum-polysaccharide conjugate or chondroitin of the present invention 24 and 94 hours later. Cell necrosis. Figure 11A shows breast tumors (13762) 24 hours after administration of chondroitin. Figure 11B shows breast tumors (13762) 24 hours after administration of the platinum-polysaccharide conjugate. Figure 10C shows breast tumors (13762) 94 hours after administration of chondroitin. Figure 10D shows breast tumors (13762) 94 hours after administration of the platinum-polysaccharide conjugate.

The eleventh panel is a Western blot experiment showing the PARP protein of 2008-c13 cells treated with platinum-polysaccharide conjugate (PC) or cisplatin (CDDP).

Figure 12 is a flow cytometry experiment showing the cell cycle of 2008-c13 cells 48 hours after administration of polysaccharide conjugate (PC) or cisplatin (CDDP).

The thirteenth image is the experimental results of the northern ink dot and the western ink dot, which respectively show the p21 transcription amount of the 2008-c13 cells administered with a low dose of polysaccharide conjugate (PC) or cisplatin (CDDP) (tenth Figure 3) and performance (Figure 13 B).

Figure 14 is the flow cytometry experiment results. Figure 14A shows a dose-dependent increase in cells at sub-G1 48 hours after administration of the conjugate (PC or DDAP) or cisplatin (CDDP). At the same dose, PC substantially induced more anti-platinum 2008-c13 cells with a sub-G1 cycle compared to CDDP. Figure 14B shows the percentage of 2008-c13 cells in sub-G1 after 48 hours of administration of CDDP or PC (DDAP).

The fifteenth panel is the experimental result of the TUNEL assay, which shows apoptosis induced by exposure to cisplatin (CDDP) and platinum diamine dicarboxylate (PC or DDAP) for 48 hours. In Figure 15A, the pattern of apoptosis is indicated by brown particles. In Fifteenth Panel B, the percentage of cells having an apoptotic pattern is shown, wherein the strips are three independent sets of experimental results; and the bars on the strips are standard errors.

Figure 16 is a Western blot experiment showing the specific cleavage of cleaved caspase-3 protein and PARP in 2008-c13 cells administered with cisplatin (CDDP) and platinum diamine dicarboxylate (PC or DDAP). Type. GAPDH (glyceraldehydes-3-phosphate dehydrogenase).

Figure 17A shows the cell cycle distribution of the 2008-c13 cell line after 48 hours of treatment with cisplatin (CDDP) or platinum diamine dicarboxylate (PC or DDAP). G1, G2, M, and S represent different periods in the cell cycle, respectively. Figure 17B is a Western blot experiment showing the amount of p21 and cyclin A present in 2008-c13 cells after 48 hours of treatment with cisplatin (CDDP) or platinum diamine dicarboxylate (PC or DDAP). GAPDH (glyceraldehydes-3-phosphate dehydrogenase).

Fig. 18 is a view showing the structure of a polysaccharide conjugate obtained according to Example 5 of the present invention.

Fig. 19A shows a two-dimensional axial dynamic image which was obtained by an MR scanner to detect a breast cancer-bearing rat injected with Gd-EDTA-chitosan. Figure 19B shows a plot of time versus signal intensity for tumors and normal muscles, which is obtained by measuring regions of interest (ROIs) defined by T1 weighted data. Fig. 19C shows a two-dimensional axial static image which was obtained by an MR scanner to detect a rat with nasopharyngeal carcinoma after one hour of injection of Gd-EDTA-chitosan. Figure 19D shows a plot of time versus signal intensity for tumors and normal muscles, where the signal intensity is obtained by measuring the region of interest; this region of interest is derived from the use of Gd-EDTA-chitosan as a post- T1 weighted image of the MSME (multi-slice multi echo) sequence of the developer's MRI scanner.

Figure 20A shows a two-dimensional axial motion image with an MR scan The results of the test were carried out on rats with breast cancer injected with Gd-DTPA-chitosan. Fig. 20B shows a plot of time versus signal intensity for tumors and normal muscles, wherein the signal intensity is obtained by measuring regions of interest (ROIs) defined by T1 weighted data. Fig. 20C shows a two-dimensional axial static image which was obtained by an MR scanner to detect a rat with nasopharyngeal carcinoma after one hour of injection of Gd-DTPA-chitosan. Figure 20D shows a plot of time versus signal intensity for tumors and normal muscles, where the signal intensity is obtained by measuring the region of interest; this region of interest is derived from the use of Gd-DTPA-chitosan as a post- T1 weighted image of the MSME (multi-slice multi echo) sequence of the developer's MRI scanner.

Figure 21 shows MRI (1.5T) images of normal pigs (20 kg) after 15 minutes of Gd-EDTA-chitosan (0.13 mmol/kg, iv) injection.

Figure 22 shows MRI (1.5T) (angiographic) images of normal pigs (20 kg) after 15 minutes of Gd-EDTA-chitosan (0.13 mmol/kg, iv) injection.

Figure 23 shows MRI (1.5T) images of normal pigs (20 kg) after 15 minutes of Gd-EDTA-chitosan (0.13 mmol/kg, iv) injection.

Figure 24 shows MRI (1.5T) images of pigs with melanoma (23 kg) injected with Gd-EDTA-chitosan (0.13 mmol/kg, iv) for 5 minutes.

Figure 25 shows an MRI (1.5T) image of a pig with melanoma (23 kg) administered Gd-EDTA-chitosan (0.13 mmol/kg, iv) for 30 minutes.

Figure 26 shows that pigs with melanoma (23 kg) were injected with Gd-EDTA-chitosan (0.13 mmol/kg, iv) for 60 minutes. MRI (1.5T) image.

Figure 27 shows a time-intensity curve of tumors absorbing Gd-EDTA-chitosan (signal intensity) versus time in a pig with melanoma.

The twenty-eighth panel shows the percentage of tumor enhancement.

Figure 29A shows the results of planar scintigraphy scanning, which was observed for rats with breast cancer after administration of ^99m Tc-EDTA (3 mg, 300 μCi) for 0.5 hours. Figure 29B shows the results of planar scintigraphy scanning, which was observed for rats with breast cancer after administration of ^99m Tc-EDTA-chitosan (3 mg, 300 μCi) for 0.5 hours. Figure 29B shows the results of planar scintigraphy scanning, which was observed for rats with breast cancer after administration of ^99m Tc-EDTA-chitosan (3 mg, 300 μCi) for 1 hour. In addition, T/M represents the tumor to muscle intensity ratio (cpm/pixel).

Figure 30 shows the results of spatial resolution tests for different concentrations of Gd-EDTA-chitosan.

The thirty-first graph shows the time versus temperature curve obtained by experimenting with a 40 KHz ultrasonic bath.

Figure 32 shows the Fourier transform infrared spectrum of chitosan and a thiol compound-chitosan conjugate (chitosan-SH).

Claims (16)

A polysaccharide conjugate comprising: a polysaccharide; at least one linking group covalently bonded to the polysaccharide; and at least one metal conjugated via the linking group. The polysaccharide conjugate of claim 1, wherein the polysaccharide has a molecular weight of 3,000 to 30,000 ampoules. The polysaccharide conjugate according to claim 1, wherein the polysaccharide system is selected from the group consisting of collagen, chondroitin, hyaluronic acid, chitosan, and chitin. The polysaccharide conjugate of claim 1, wherein the polysaccharide comprises chitosan. The polysaccharide conjugate according to claim 1, wherein the polysaccharide has an amine group, and the linking group is covalently bonded to the aforementioned amine group of the polysaccharide. The polysaccharide conjugate according to claim 1, wherein the linking group has a carboxyl group or a thiol group, and the metal is conjugated via the carboxyl group or the thiol group of the linking group. The polysaccharide conjugate of claim 1, wherein the linking group is a monomeric amino acid, a chelating agent or a modifier. The polysaccharide conjugate of claim 7, wherein the chelating agent is ethylene diamine tetraacetic acid. The polysaccharide conjugate of claim 7, wherein the aforementioned The linking group is an open-ring form of the modifier. The polysaccharide conjugate of claim 9, wherein the modifier is iminothiolane. The polysaccharide conjugate according to claim 1, wherein the metal is selected from the group consisting of Tc-99m, Cu-60, Cu-61, Cu-67, In-111, Tl-201, Ga-67, Ga-68, As-72, Re-188, Ho-166, Y-90, Lu-177, Sm-153, Sr-89, Gd-157, Gd-158, Bi-212, Bi-213, Fe, A group of Au, Ag, and Au/Ag composites. The polysaccharide conjugate of claim 1, which comprises 50 to 80% by weight of the aforementioned polysaccharide. The polysaccharide conjugate of claim 1, which comprises 10 to 40% by weight of the aforementioned linking group. The polysaccharide conjugate of claim 1, which comprises 10 to 30% by weight of the aforementioned metal. A method for synthesizing a polysaccharide conjugate, comprising the steps of: covalently bonding a linking group to a polysaccharide to obtain an intermediate product; and conjugateing the intermediate product to a metal to form a Polysaccharide conjugate. The method of claim 15, further comprising drying the polysaccharide conjugate to form a powder.