TWI852815B - Multivalent glyco-complex, imaging agent and uses thereof - Google Patents

Abstract

Disclosed herein relates to a multivalent glycol-complex, an imaging agent and uses thereof. The multivalent glycol-complex includes a plurality of glucose molecules, each of which connects to a central nitrogen atom through a linker, and a chelator group. The multivalent glycol-complex can be used to diagnose cancers and to evaluate the therapeutic effect of cancers.

Description

Polysaccharide complex, radioactive polysaccharide complex contrast agent and use thereof

The present disclosure relates to the field of medical imaging, and more particularly to a technique for performing imaging using a polysaccharide complex as represented by formula (10). Formula (10)

Malignant tumors are a major public health problem worldwide and one of the leading causes of death in the United States. The American Cancer Society reported on January 12, 2023 that, despite a steady decline in cancer mortality in the United States over the past 30 years, new cases of breast cancer, uterine cancer, and prostate cancer are increasing. The lifetime probability of a male in the United States being diagnosed with any invasive cancer is approximately 40.9%, and that of a female is approximately 39.1%. The report also estimates that in 2023, there may be nearly 2 million new cancer cases in the United States (equivalent to approximately 5,000 cases per day) and more than 600,000 deaths from cancer.

The latest (2020) cancer registration report released by the National Health Administration of the Ministry of Health and Welfare of Taiwan in 2023 pointed out that the number of new cancer cases was 121,979, an increase of 725 from 2019. The number of cancer deaths in 2020 was 50,161, accounting for 29.0% of the total number of deaths, with a mortality rate of 212.7 per 100,000 population; the standardized mortality rate was 117.3 per 100,000 population. Malignant tumors have been the leading cause of death among Taiwanese for the 41st consecutive year, and the "cancer clock" continues to speed up, 3 seconds faster than in 2010, with an average of one person suffering from cancer every 4 minutes and 19 seconds. The above data all show that the number of cancer patients and the scale of the related medical market are huge, and even the number of people suffering from cancer has a continuous upward trend.

The key to the treatment of malignant tumors lies in early diagnosis and early treatment. If the cancer can be discovered early and the patient is given appropriate treatment early, many cancer patients have the opportunity to significantly improve their survival rate. For example, if patients with colorectal cancer, breast cancer, etc. can receive appropriate treatment in the early stages of cancer, the patient usually recovers better.

In view of the high glucose utilization rate of most malignant tumors, glucose analogs such as ¹⁸ F-FDG (2-Deoxy-2-fluoro-D-glucose) are often used in clinical practice for cancer diagnosis. However, ¹⁸ F-FDG often encounters many limitations in its use. For example, ¹⁸ F-FDG is quite cumbersome to prepare. The production process requires the use of a cyclotron to produce F-18, and this equipment costs a lot and is not a basic equipment usually equipped in medical hospitals. In addition, the preparation process of ¹⁸ F-FDG requires the use of a synthesis box, and it is necessary to go through steps such as dehydration, fluorination, and deprotection to obtain ¹⁸ F-FDG, which takes a long time to synthesize.

Furthermore, since the tissues or organs that metabolize glucose in the body will absorb ¹⁸ F-FDG, when using this type of contrast agent for diagnosis, extremely high background values will be observed in the patient's brain and heart, making it difficult to use the contrast results of ¹⁸ F-FDG in these organs and their surrounding areas to distinguish normal and tumor tissues, which has its limitations in detection. What's worse, there will be a relatively high ¹⁸ F-FDG uptake in inflamed tissues, making it difficult to distinguish between tumors or inflamed tissues. It can be seen that the preparation of ¹⁸ F-FDG is complicated and time-consuming and has low specificity. In view of this, there is an urgent need in the art for a polysaccharide complex with better imaging identification rate to improve the defects of the prior art.

In order to let readers understand the basic meaning of the disclosure, the invention content provides a brief description of the disclosure. The invention content is not a complete description of the disclosure, and it is not intended to define the technical features or scope of rights of the invention.

In order to propose a more discriminative cancer diagnostic drug, in particular, a contrast agent for use in cancer diagnosis, the present disclosure proposes a polysaccharide complex in a new research result, which has a structure as shown in formula (10), which includes three glucose molecules, each of which is connected to a central nitrogen atom by a linker, and a chelator G (chelator G), wherein the linker includes at least a polyethylene glycol molecule (PEG), and the chelator G is connected to one of the linkers near the central nitrogen atom in the form of p-NCS-benzyl-chelator G. The chelating group G is 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (DTPA), acid, TETA) or 1,4,7-triazacyclononane phosphinic acid (TRAP). Formula (10)

In some embodiments, the chelating group G is 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), and the polysaccharide complex has a structure as shown in formula (3). Formula (3)

In any of the above embodiments, the polysaccharide complex further comprises a radioactive nuclide bound to the chelating group G to radiolabel the polysaccharide complex. In an optional embodiment, the radioactive nuclide can be niobium-188, technetium-99, indium-111, niobium-177, gallium-68, yttrium-90, fluorine-18 or copper-64.

In one embodiment, the chelating group G of the polysaccharide complex is 1,4,7-triazacyclononane-N, N', N''-triacetic acid (NOTA), and the radionuclide is gallium-68. The radiolabeled polysaccharide complex has a structure as shown in formula (4). Formula (4)

The present disclosure also relates to a contrast agent, which comprises the polysaccharide complex described in any of the above embodiments and a contrast agent.

The present disclosure also relates to a use of the polysaccharide complex described in any of the above embodiments in the preparation of a pharmaceutical product for diagnosing cancer.

In any of the above embodiments, the cancer is selected from the group consisting of lymphoma, multiple myeloma, testicular cancer, thyroid cancer, prostate cancer, pharyngeal cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, stomach cancer, head and neck cancer, esophageal cancer, rectal cancer, bladder cancer, kidney cancer, lung cancer, liver cancer, brain cancer, melanoma and skin cancer.

A person having ordinary knowledge in the technical field to which the present invention belongs can fully understand the central concept, the technical means adopted and various implementation modes of the present invention after referring to the following implementation modes.

In order to make the description of the disclosed content more detailed and complete, the following textual description is provided for the implementation aspects and specific embodiments of the present invention; however, the implementation aspects and specific embodiments of the present invention are not limited thereto.

Unless otherwise specified, the meanings of scientific and technical terms used in this specification are the same as those understood and used by those with ordinary knowledge in the technical field. Furthermore, the terms used in this specification include both the singular and plural forms of the terms, unless otherwise specified.

The term "individual" or "patient" in this specification refers to an animal that can receive the polysaccharide complex disclosed herein. In a preferred embodiment, the animal is a mammal, especially a human.

The "cancer" described in this specification may be a non-solid tumor or a solid tumor. For example, the cancer includes, but is not limited to, lymphoma, multiple myeloma, testicular cancer, thyroid cancer, prostate cancer, pharyngeal cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, stomach cancer, head and neck cancer, esophageal cancer, rectal cancer, bladder cancer, kidney cancer, lung cancer, liver cancer, brain cancer, melanoma and skin cancer.

As used in this specification, the word "about" generally refers to an actual value within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range. The word "about" herein means that the actual value falls within an acceptable standard error of the mean value, as determined by a person of ordinary skill in the art to which the invention belongs. Except for experimental exceptions, or unless otherwise expressly stated, it is understood that the ranges, quantities, values and percentages used herein are modified by "about". Therefore, unless otherwise stated, the values or parameters disclosed in this specification and the accompanying patent application are approximate values and may be changed as needed.

To solve the problems existing in the prior art, the present disclosure provides a more discriminative cancer diagnostic drug, particularly a contrast agent used in cancer diagnosis.

The following will exemplarily disclose multiple embodiments to illustrate various different implementations of the present disclosure, so that those with ordinary knowledge in the technical field to which the present disclosure belongs can implement the technical content disclosed in the present invention according to the disclosure of this specification. Therefore, the embodiments disclosed below cannot be used to limit the scope of rights of the present disclosure. Furthermore, all documents cited in this specification are deemed to be fully cited as part of this specification.

The present disclosure provides a polysaccharide complex having a structure as shown in formula (10). Formula (10)

The polysaccharide complex of formula (10) proposed in the present disclosure comprises three glucose molecules, each of which is connected to a central nitrogen atom by a linker, and a chelator G, wherein the linker comprises at least a polyethylene glycol molecule (PEG), and the chelator G is connected to one of the linkers near the central nitrogen atom in the form of p-NCS-benzyl-chelator G. The synthesis method of the polysaccharide complex of formula (10) proposed in the present disclosure will be described in detail in the following examples.

Example 1 Synthesis of the polysaccharide complex shown in formula (3) proposed in this disclosure

1.1 Preparation of the compound of formula (1) (NH ₂ -PEG-GLN)

Please refer to Figure 2, which is a synthetic flow chart of the compound of formula (1) ( _NH2 -PEG-GLN). The compound of formula (1) is represented as _NH2 -PEG-GLN, which is a precursor for synthesizing the polysaccharide complex disclosed herein. PEG is represented as a linker comprising polyethylene glycol (PEG), and the polyethylene glycol comprises at least 4 ethylene glycol monomer molecules. In addition, GLN is represented as a glucose molecule.

First, as shown in Figure 2, Amino-PEG-acid (650 mg, Sigma-Aldrich), Ethyl trifluoroacetate (Ethyl trifluoroacetate, Sigma-Aldrich, 2 ml) and Triethylamine (Triethylamine, Sigma-Aldrich, 2 ml) were dissolved in 20 ml of methanol (Sigma-Aldrich) and stirred at room temperature for 24 hours. Then, the solvent was removed by concentration under reduced pressure, and then separated by reverse-phase HPLC column, with an elution gradient of 10%-100% methanol, and the desired product was eluted at about 40% methanol in the gradient. After the product was collected, the solvent was removed by concentration under reduced pressure to obtain the intermediate product Tfa-PEG-COOH, which was about 210 mg of a light yellow oil with a yield of about 50%.

Next, Tfa-PEG-COOH (300 mg, 0.83 mmol) and N-hydroxysuccinimide (NHS, 110 mg, 0.9 mmol, Sigma-Aldrich) were dissolved in 10 ml of ethyl acetate, and N,N'-dicyclohexylcarbodiimide (DCC, 200 mg, 0.9 mmol, Sigma-Aldrich) was added. After stirring at room temperature for 24 hours, the insoluble matter was removed by filtering with filter paper, and the solvent was removed by concentrating under reduced pressure until the total volume was less than about 0.5 ml. The intermediate product Tfa-PEG-Osu was obtained after drying under vacuum. The molecular formula of the intermediate product is shown in Figure 2.

Next, Tfa-PEG-OSu (0.83 mmol), glucosamine (180 mg, 0.83 mmol, Sigma-Aldrich) and N,N-diisopropylethylamine (DIPEA, 1 ml, Sigma-Aldrich) were dissolved in dimethylformamide (DMF, 5 ml, Sigma-Aldrich), stirred at room temperature for 24 hours, and the solvent was removed by concentration under reduced pressure. The mixture was then separated by a reverse phase chromatography column with an elution gradient of 10%-100% methanol. The intermediate product Tfa-PEG-GLN was obtained by collecting the product. The reaction was then stirred in aqueous ammonia (pH = 11.3) at room temperature for 48 hours for hydrolysis to remove the Tfa protective group. The solvent was then removed by concentration under reduced pressure and then separated by a reverse phase chromatography column with an elution gradient of 10%-100% methanol. The collected product was confirmed to be the compound of formula (1), NH ₂ -PEG-GLN, about 218 mg, after mass spectrometry analysis (see Figure 3). Formula (1)

1.2 Preparation of the compound of formula (2) (NH ₂ -NTA-(PEG-GLN) ₃ )

Please refer to Figure 4, which is a synthetic flow chart of the compound of formula (2) (NH ₂ -NTA-(PEG-GLN) ₃ ). The compound of formula (2) is represented by NH ₂ -NTA-(PEG-GLN) ₃ , and its specific molecular structure is shown in Figure 4 . It is another precursor for synthesizing the polysaccharide complex disclosed herein.

The synthesis method is shown in Figure 4. First, N, N'-diisopropylcarbodiimide (DIC, Sigma-Aldrich), oxyma (Sigma-Aldrich) and NTA-Cbz are added to the compound of formula (1) ( _NH2 -PEG-GLN), and an amide bonding reaction is carried out at room temperature for 16 hours to obtain the compound of formula (2) ( _NH2 -NTA-(PEG-GLN) ₃ ).

1.3 Preparation of the polysaccharide complex (NOTA-(PEG-GLN) ₃ ) of formula (3) proposed in the present disclosure

Please refer to Figure 5, which is a synthetic flow chart of the polysaccharide complex (NOTA-(PEG-GLN) ₃ ) of formula (3) proposed in one embodiment of the present disclosure. As shown in the figure, triethylamine (Sigma-Aldrich), dimethylformamide (DMF, Sigma-Aldrich), and p-NCS-benzyl-NODA-GA (chematech) are added to the compound of formula (2) (NH ₂ -NTA-(PEG-GLN) ₃ ), and the amide bonding reaction is carried out at room temperature for 12 hours to obtain the polysaccharide complex (NOTA-(PEG-GLN) ₃ ) of formula (3), which is confirmed by mass spectrometry analysis (see Figure 6).

Example 2: Using lung cancer animal model to evaluate the efficacy of the polysaccharide complex disclosed herein after radionuclide labeling

2.1 Preparation of polysaccharide complex contrast agent ⁶⁸ Ga-NOTA-(PEG-GLN) ₃ (compound of formula (4))

Please refer to Figure 7, which is a flow chart of radiolabeling of the polysaccharide complex (NOTA-(PEG-GLN) ₃ ) of formula (3) to form the polysaccharide complex ( ⁶⁸ Ga-NOTA-(PEG-GLN) ₃ ) of formula (4) according to one embodiment of the present disclosure. First, 0.1N HCl is used to wash the ⁶⁸ Ge/ ⁶⁸ Ga generator to obtain a ⁶⁸ GaCl ₃ solution. Next, 0.5 mL of ⁶⁸ Ga (~185 MBq) and 0.15 mL of 1M HEPES buffer were added to 1 μg of the polysaccharide complex NOTA-(PEG-GLN) ₃ of formula (3), and the mixture was reacted at room temperature for 15 minutes to obtain the polysaccharide complex ⁶⁸ Ga-NOTA-(PEG-GLN) ₃ of formula (4) radiolabeled with radioactive nuclide gallium-68. The radiochemical purity (RCP) was determined by radio-thin layer chromatography (radio-TLC), and the results showed that the radiochemical purity (RCP) of ⁶⁸ Ga-NOTA-(PEG-GLN) ₃ was greater than 95%. Please refer to Figure 8 for the result of radio-thin layer chromatography.

2.2 Polysaccharide complex (Formula (4) ⁶⁸ Ga-NOTA-(PEG-GLN) ₃ ) contrast agent in positron emission tomography of lung cancer animals

The polysaccharide complex ⁶⁸ Ga-NOTA-(PEG-GLN) ₃ of formula (4) was dissolved in 0.1 mL of physiological saline at 11.1 MBq and injected into nude mice with NCI-H292 human lung cancer cells via the tail vein. Then, nanoPET/CT anesthesia was performed with 1.5% isoflurane. After 2 hours of dynamic angiography, the results are shown in Figure 9. As shown in the figure, the polysaccharide complex ⁶⁸ Ga-NOTA-(PEG-GLN) ₃ of formula (4) has obvious accumulation at the tumor site, and the tumor/muscle (radioactivity ratio) (tumor/muscle) ratio is 27, indicating that lung cancer cells can effectively take up the polysaccharide complex ⁶⁸ Ga-NOTA-(PEG-GLN) ₃ of formula (4), while on the contrary, the drug uptake in the brain is very low (see the image of Figure 9). It can be seen that the polysaccharide complex proposed in the present disclosure can specifically accumulate at the tumor site, and can effectively increase the tumor's uptake of this polysaccharide complex, and significantly reduce the radiation absorption dose in the brain.

The above examples clearly show that the polysaccharide complex shown in formula (10) proposed in the present disclosure has the following advantages:

1. The polysaccharide complex disclosed herein is used as a contrast agent because its molecular structure contains a chelator G, a linker, and multiple sugar molecules. It utilizes the high glucose utilization rate of most malignant tumors to enable the polysaccharide complex contrast agent to quickly enter tumor cells, enhance the signal contrast between the tumor and surrounding normal tissues, and increase detection efficiency. In addition to being used for the detection of malignant tumors, the polysaccharide complex contrast agent can also be used for cancer treatment efficacy evaluation, and the appropriateness of the treatment method can be evaluated in a non-invasive manner. If the treatment effect is found to be poor, the treatment method or drug can be changed immediately to avoid delaying the treatment schedule.

2. From the results of the above embodiments, the contrast agent of the polysaccharide complex disclosed in the present invention is different from ^{the 18} F-FDG which is most commonly used in clinical practice. Its molecule is significantly larger than ¹⁸ F-FDG, and its uptake in the normal brain and heart is significantly reduced. Therefore, there will be a lower background value in the brain and lungs, which can enhance the signal contrast between tumors and surrounding normal tissues and increase detection efficiency.

3. The polysaccharide complex contrast agent disclosed herein is used in positron emission tomography (PET) for imaging, and has the characteristics of non-invasiveness, high sensitivity, and high image resolution.

4. When the polysaccharide complex contrast agent disclosed herein is applied to the positron radioisotope gallium-68 (Ga-68), the radiation source can be obtained by a generator, so it is very convenient in clinical use and drug preparation without the need for a cyclotron.

5. The polysaccharide complex contrast agent disclosed herein can be prepared in a cryo-crystal kit, and after washing with a gallium-68 generator, gallium-68 is directly injected to complete rapid labeling at room temperature. No drug purification is required, and it is very convenient to use clinically, and can reduce drug costs and reduce radiation doses for operators.

6. According to the results of the above embodiments, it can be clearly seen that the contrast agent of the polysaccharide complex disclosed in the present invention (Formula (4) ⁶⁸ Ga-NOTA-(PEG-GLN) ₃ ) has obvious accumulation at the tumor site of the experimental animals tested, with a tumor/muscle ratio of 27, while the drug uptake at the brain site is very low, indicating that the polysaccharide complex proposed in the present invention can specifically accumulate at the tumor site, and can effectively increase the tumor's uptake of the polysaccharide complex, and significantly reduce the radiation absorption of the brain.

Although the present disclosure has been disclosed as above by way of embodiments, it is not intended to limit the present invention. A person having ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present invention shall be subject to the scope defined by the attached patent application.

without

In order to make the above and other purposes, features, advantages and embodiments of the present invention more clearly understandable, the drawings are described as follows: Figure 1 is a schematic diagram of the structure of a polysaccharide complex disclosed in the present invention. The polysaccharide complex has a structure as shown in formula (10). Figure 2 is a synthetic flow chart of the compound of formula (1). The compound of formula (1) is a precursor for synthesizing the polysaccharide complex disclosed in the present invention. Figure 3 is a mass spectrum analysis diagram of the compound of formula (1). Figure 4 is a synthetic flow chart of the compound of formula (2). The compound of formula (2) is another precursor for synthesizing the polysaccharide complex disclosed in the present invention. Figure 5 is a synthetic flow chart of a polysaccharide complex of an embodiment of the present invention, and the polysaccharide complex has a structure as shown in formula (3). FIG. 6 is a mass spectrum analysis diagram of a polysaccharide complex of an embodiment of the present disclosure, wherein the polysaccharide complex has a structure as shown in formula (3). FIG. 7 is a flow chart of radiolabeling of the polysaccharide complex of formula (3) of an embodiment of the present disclosure with radioactive nuclide gallium-68 to form a polysaccharide complex of formula (4). FIG. 8 is the result of radiochemical purity (RCP) determination of the compound of formula (4) by radio-thin layer chromatography (radio-TLC). FIG. 9 is the contrast imaging result of a polysaccharide complex of an embodiment of the present disclosure, which has a structure as shown in formula (4), in a lung cancer animal model by NanoPET/CT.

Claims (8)

A polysaccharide complex having a structure as shown in formula (10): Formula (10), wherein G is selected from 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (DTPA), The chelating group of 1,4,7-triazacyclononane phosphinic acid (TETA) or 1,4,7-triazacyclononane phosphinic acid (TRAP) is shown in FIG. The polysaccharide complex as claimed in claim 1, wherein the chelating group G is 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), and the polysaccharide complex has a structure as shown in formula (3): Formula (3). The polysaccharide complex as described in claim 1 further comprises a radionuclide bound to the chelating group G to radiolabel the polysaccharide complex, wherein the radionuclide is rhodium-188, technetium-99, indium-111, nickel-177, gallium-68, yttrium-90, fluorine-18 or copper-64. The polysaccharide complex as described in claim 3, wherein the radionuclide is gallium-68. The polysaccharide complex as described in claim 3, wherein the chelating group G is 1,4,7-triazacyclononane-N,N’,N’’-triacetic acid (NOTA), and the radionuclide is gallium-68. A contrast agent comprising: a polysaccharide complex as shown in any one of claims 1 to 5; and a contrast agent. A use of a polysaccharide complex in preparing a pharmaceutical for diagnosing cancer, wherein the polysaccharide complex is as shown in any one of claims 1-5. The use as described in claim 7, wherein the cancer is selected from the group consisting of: lymphoma, multiple myeloma, testicular cancer, thyroid cancer, prostate cancer, pharyngeal cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, head and neck cancer, esophageal cancer, rectal cancer, bladder cancer, kidney cancer, lung cancer, liver cancer, brain cancer, melanoma and skin cancer.