Patent Application
20220118124
The present invention relates to a radioactive isotope-labeled photocrosslinkable hydrogel and a method of preparing the same.
Cancer is one of the deadliest diseases faced by humans to date and although many advances have been made in early diagnosis and treatment, cancer still accounts for 30% of the world's deaths. Chemotherapy, one of the methods of treating cancer, is popularly used, but it is difficult to control the concentration of the drug and has a limitation of low targeting ability to the tumor site. External radiotherapy is a relatively well-controlled and regulated treatment method that induces radiation in the body through a device to destroy tumor cells, however problems such as non-specific destruction of normal tissue adjacent to the tumor, defects in the path of the ion beam, and the need for a high radiation dose to the tissue to be infiltrated remain. In contrast, internal radiotherapy is simpler than surgical and external radiotherapy and minimizes patient's pain. The destruction of tumor cells has been induced by intravenous injection of solutions containing radioactive elements such as 90Y, 67Cu, 188Re, 177Lu, 131I and the like.
Iodine radioactive isotope is a therapeutic agent used in internal radiotherapy that accumulates in the thyroid tissue due to the radiation ionization effect and causes apoptosis and necrosis of cancer cells. However, there is a side effect of rapidly spreading throughout the body after injection into the body and damaging unwanted organs or normal tissues. To solve this problem, iodine was labeled to or supported on the polymer to increase the retention time or target the desired site. However, the use of a carrier with low biocompatibility or a long manufacturing time lowered the efficiency and the effective retention at the desired site. Nanoparticles labeled with 131I in poly L-lactic acid (PLLA), an amphiphilic polymer, migrated to the tumor tissue, but local delivery was difficult due to radiation detection in organs other than target organs. In addition, the 131I-labeled chitosan microhydrogel containing doxorubicin therein, an anticancer drug, is locally maintained at the injection site, but the process of manufacturing the microhydrogel is complicated, making it difficult to apply it to the clinic.
In order to solve the above problems, the present invention provides a photocrosslinkable compound or a pharmaceutically acceptable salt thereof.
The present invention provides a photocrosslinkable hydrogel for radiotherapy comprising the compound or a salt thereof, and a method of preparing the same.
In addition, the present invention provides a pharmaceutical composition for radiotherapy, radiodiagnostic imaging or anticancer treatment comprising the hydrogel.
A compound or a pharmaceutically acceptable salt thereof may be represented by the following Chemical Formula 1:
In Chemical Formula 1, X may be selected from the group consisting of photocrosslinkable acrylate, methacrylate, glycidyl methacrylate and vinyl ester, and Y may be OH or the following Chemical Formula 2:
In Chemical Formulae 1 and 2, R1 and R2 may each be the same or different and may be selected from the group consisting of hydrogen; and imidazole, pyrrole, furan, thiophene, indole and 3,4-dihydroxyphenyl which are capable of labeling one or more radioactive isotopes, m1 and m2 may be integers from 0 to 2, and n may be 20 to 4,000.
The compound or a pharmaceutically acceptable salt thereof according to the present invention may be labeled with a radioactive isotope.
A photocrosslinkable hydrogel for radiotherapy according to the present invention may comprise the compound or a pharmaceutically acceptable salt thereof as an active ingredient.
A pharmaceutical composition for radiotherapy according to the present invention may comprise the hydrogel as an active ingredient.
A composition for radiodiagnostic imaging according to the present invention may comprise the hydrogel as an active ingredient.
A pharmaceutical composition for anticancer treatment according to the present invention may comprise the hydrogel and anticancer agent.
A method of preparing photocrosslinkable microhydrogel for radiotherapy may comprise preparing a photocrosslinkable compound; dissolving the prepared photocrosslinkable compound in a photoinitiator solution and then injecting it into an inlet of a microfluidic device; injecting an oil containing a surfactant into an outlet of the microfluidic device; forming microdroplets by centrifugation; and extracting the microdroplets, photocrosslinking and washing to form microgels.
The hydrogel according to the present invention is a micro-sized hydrogel in which a photocrosslinking property is given to hyaluronic acid and radionuclides are labeled, and it is excellent in staying in a local area requiring radiation treatment, so that it can be treated while minimizing damage to surrounding tissues. Therefore, the hydrogel may be used as a pharmaceutical composition for radiotherapy or a composition for radiodiagnostic imaging. In addition, it can be used in combination with an anticancer agent and used as a pharmaceutical composition for anticancer treatment, so that cancer can be effectively treated.
The hydrogel according to the present invention uses hyaluronic acid which is excellent in biodegradability and biocompatibility, and has almost no immune rejection, so it can be used as a safe therapeutic composition.
The hydrogel according to the present invention can be manufactured on site immediately and conveniently using a portable microfluidic system, thereby maximizing the radiation treatment effect.
Hereinafter, the present invention will be described in detail.
The present inventors produced a micro-sized hydrogel using a portable microfluidic system on site by imparting photocrosslinking properties to hyaluronic acid which is excellent in biodegradability and biocompatibility, and labeling the radionuclide 131I and injected in vivo and confirmed that it is not spread to other tissues and is locally maintained at the injected site, thereby completing the present invention.
As used herein, “radiation” refers to a phenomenon in which energy propagates through space or a material that mediates propagation, and may be emitted by various radionuclides.
As used herein, “radiotherapy” refers to a treatment that kills cancer cells, etc. through the action of causing chemical denaturation of nucleic acids, cell membranes, etc. essential for proliferation and survival of cells using the high energy radiation.
As used herein, “radioactivity” means the intensity of the radiation.
As used herein, “microhydrogel” refers to crosslinked particles that swell without dissolving in water at a level of several to several hundred micrometers (μm) that can be injected using a syringe, and it generally means swellable spherical or plate-shaped gel particles and may be expressed in terms such as “microgel” or “microgel”.
As used herein, “HAMA” refers to hyaluronic acid to which a methacrylate group is bonded, and may be expressed in terms such as hyaluronate methacrylate, methacrylated hyaluronic acid, etc.
As used herein, “HAMA-A” means what various kinds of cyclic compounds A are conjugated to hyaluronic acid to which a methacrylate group is bonded (HAMA).
As used herein, “HAMA-A-B” means what a radioactive isotope (B) is labeled on the cyclic compound A conjugated to the HAMA.
The present invention provides a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.
The compound or a pharmaceutically acceptable salt thereof may be represented by the following Chemical Formula 1:
In Chemical Formula 1, X may be selected from the group consisting of photocrosslinkable acrylate, methacrylate, glycidyl methacrylate and vinyl ester, and
Y may be OH or the following Chemical Formula 2:
In Chemical Formulae 1 and 2, R1 and R2 may each be the same or different and may be selected from the group consisting of hydrogen; and imidazole, pyrrole, furan, thiophene, indole and 3,4-dihydroxyphenyl which are capable of labeling one or more radioactive isotopes, m1 and m2 may be integers from 0 to 2, and n may be 20 to 4,000 (number average molecular weight is 10,000 to 2,000,000), preferably 100 to 400 (number average molecular weight is 50,000 to 200,000).
More specifically, the Y may be combined with any one selected from the group consisting N-(3-aminopropyl)-imidazole (API), 3-(1H-pyrrol-1-yl)-1-propanamine, 1-(3-furyl)methanamine, thienylmethylamine, tryptamine, 3,4-dihydroxyphenylalanine (DOPA) and derivatives thereof.
Accordingly, the compound may include hyaluronate methacrylate (hereinafter, HAMA)-API, HAMA-DOPA, HAMA-pyrrol, HAMA-furan, HAMA-thiophene, HAMA-indole and derivatives thereof.
The pharmaceutically acceptable salt according to the present invention may include either a pharmaceutically acceptable basic salt or an acidic salt.
The basic salt can be used in the form of either an organic base salt or an inorganic base salt, and it may be selected from the group consisting of sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethylaminium salt and a pyridinium salt, but it is not limited thereto.
As acidic salts, acid addition salts formed by free acids are useful. Inorganic acids and organic acids can be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, double phosphoric acid, nitric acid, etc. can be used as inorganic acids, and citric acid, acetic acid, maleic acid, malic acid, fumaric acid, glucoic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, stearic acid, and the like can be used as organic acids. Preferably, hydrochloric acid may be used as the inorganic acid and methanesulfonic acid may be used as the organic acid.
In addition, the compound according to the present invention may include all salts, hydrates and solvates that can be prepared by conventional methods, as well as pharmaceutically acceptable salts. For example, it can be prepared by a conventional method and the addition salt can be prepared by dissolving the compound in a water-miscible organic solvent such as acetone, methanol, ethanol or acetonitrile and adding an excessive amount of organic base or an aqueous base solution of an inorganic base, followed by precipitation or crystallization. Alternatively, it may be prepared by evaporating a solvent or an excess base and drying to obtain an addition salt, or by suction filtration of the precipitated salt.
In the compound or salt according to the present invention, the radioactive isotope may include a halogenated radioactive isotope, and may be at least one selected from the group consisting of 131I, 125I, 124I, 123I, 18F, 19F, 177Lu and 211At, preferably, it may be 131I, but it is not limited thereto.
The present invention provides a compound or a pharmaceutically acceptable salt thereof, wherein a radioisotope is labeled on the compound represented by the Chemical Formula 1 or a pharmaceutically acceptable salt thereof.
The radioactive isotope may include a halogenated radioactive isotope, and may be at least one selected from the group consisting of 131I, 125I, 124I, 123I, 18F, 19F, 177Lu and 211At, and preferably 131I, but it is limited thereto.
The radioactive isotopes may be labeled on at least one cyclic compound selected from the group consisting of imidazole, pyrrole, furan, thiophene, indole and 3,4-dihydroxyphenyl, which contained in the compound, and more specifically, may be labeled on a cyclic compound selected from the group consisting of N-(3-aminopropyl)-imidazole (API), 3-(1H-pyrrol-1-yl)-1-propanamine, 1-(3-furyl)methanamine, thienylmethylamine, tryptamine, OPA (3,4-dihydroxyphenylalanine) and derivatives thereof, to form HAMA-API-I, HAMA-DOPA-I, HAMA-pyrrol-I, HAMA-furan-I, HAMA-thiophene-I, HAMA-N-iodoindole and derivatives thereof. More details will be described later according to the following preparation examples.
The present invention provides a photocrosslinkable hydrogel for radiotherapy.
The photocrosslinkable hydrogel for radiotherapy according to the present invention may comprise a compound or a pharmaceutically acceptable salt thereof, which labels with a radioactive isotope, specifically at least one selected from the group consisting of 131I, 125I, 124I, 123I, 18F, 19F, 177Lu and 211At on the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof, as an active ingredient.
The hydrogel is a highly hydrated material composed of a hydrophilic polymer that forms a three-dimensional network organized in the crosslinking process so as to form a soft and porous structure.
The hydrogel may have an average particle size of a micrometer (μm) unit, and specifically, may be a microgel having an average particle size of 10 to 200 μm, preferably 50 to 100 μm. According to an experimental example of the present invention, the microgel may be injected into a human body for radiation treatment.
The present invention provides a pharmaceutical composition for radiotherapy.
The pharmaceutical composition for radiotherapy according to the present invention may comprise a photocrosslinkable hydrogel as an active ingredient.
In the present invention, “treatment (or therapy)” may be included without limitation as long as it is any action that improves or benefits a disease or disease such as cancer.
In the pharmaceutical composition according to the present invention, the type of disease to be subjected to radiation treatment is not particularly limited, but, for example, may be one or more cancers selected from the group consisting of thyroid cancer, breast cancer, biliary tract cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, gastric cancer, brain cancer, rectal cancer, lung cancer, bladder cancer, kidney cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, blood cancer and liver cancer, and preferably breast cancer, uterine cancer, prostate cancer or skin cancer.
The pharmaceutical composition of the present invention can be administered by parenteral administration including subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques, and preferably, it can be administered as an injection formulation.
Pharmaceutical compositions for parenteral administration include sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders prepared immediately before use as sterile solutions or suspensions. Examples of suitable sterile aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, saline, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and mixtures thereof, vegetable oils (e.g., olive oil), injectable organic esters (e.g., ethyloleate). For example, in the case of a dispersant and a suspending agent, a coating material such as lecithin is used to maintain an appropriate specific size and a surfactant can be used to maintain an appropriate fluidity.
In addition, parenteral compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. The sterilization of the injectable formulation can be performed, for example, by filtering through a sterile filter, or by pre-sterilizing the components of the mixture before mixing, at the time of manufacture or just before administration (as in the case of a double container syringe package).
The pharmaceutical composition according to the present invention may be injected around solid cancer alone for the treatment of diseases, or may be used to treat residual or scattered cancer after surgery. In addition, it can be used in combination with hormone therapy, drug therapy and methods using biological response modifiers.
The present invention provides a composition for radiodiagnostic imaging.
The composition for radiodiagnostic imaging according to the present invention may comprise the photocrosslinkable hydrogel as an active ingredient. The radiodiagnostic image refers to radiographic image data generated by passing through or injecting radiation into a subject for diagnosis of diseases or disorders. Through the image data, the presence or absence of a disease, the size and the location of the cancer may be identified.
In addition, the present invention provides a pharmaceutical composition for anticancer treatment.
The pharmaceutical composition for anticancer treatment according to the present invention may include the photocrosslinkable hydrogel and an anticancer agent. The anticancer agent may be one or more selected from the group consisting of taxane or a derivative thereof such as docetaxel, cabazitaxel, paclitaxel, and in addition to vinblastine, vincristine, cisplatin, actinomycin-D, 5-fluouracil, cyclophosphamide, Procarbazine, Rituximab, Imatinib, Gefitinib, Erlotinib, pharmaceutically acceptable salts and hydrates thereof, but it is not limited thereto.
It is possible to treat more effectively by using the radioactive isotope of the hydrogel in combination with the anticancer agent for anticancer treatment
The present invention provides a method of preparing a photocrosslinkable microhydrogel for radiotherapy. The method of preparing photocrosslinkable microhydrogel for radiotherapy according to the present invention comprises preparing a photocrosslinkable compound such as the compound; dissolving the photocrosslinkable compound in a photoinitiator solution and then injecting it into an inlet of a microfluidic device; injecting an oil containing a surfactant into an outlet of the microfluidic device; forming microdroplets by centrifugation; and extracting the microdroplets, photocrosslinking and washing to form microgels.
In the preparation method according to the present invention, a step of preparing a photocrosslinkable compound comprises reacting a biodegradable polymer selected from the group consisting of hyaluronic acid, a salt thereof and a combination thereof with methacrylate; conjugating one or more compounds selected from the group consisting of imidazole, pyrrole, furan, thiophene, indole and 3,4-dihydroxyphenyl capable of labeling one or more radioactive isotopes in a reacted reactant; and labeling a radioactive isotope on the conjugated compound.
The hyaluronic acid (HA) is a linear polysaccharide found in the extracellular matrix of soft tissues, and is a biopolymer having excellent biocompatibility and biodegradability. The “biodegradability” refers to a property that can be degraded when exposed to a physiological solution having a pH of 6 to 8, and preferably, it refers to a property that can be degraded by body fluids or microorganisms in the living body of mammals including humans.
The salt of hyaluronic acid may include inorganic salts such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, and organic salts such as tetrabutylammonium hyaluronate, but it is not limited thereto. In the present invention, hyaluronic acid itself or a salt thereof may be used alone, or two or more types of hyaluronic acid and a salt thereof may be used in combination.
In addition to the hyaluronic acid, the biodegradable polymer may include synthetic polymers such as poly(ethylene glycol) and poly(vinyl alcohol); polysaccharides or carbohydrates such as heparan sulfate, chondroitin sulfate, chitosan and alginate; natural proteins such as gelatin, collagen, albumin, and the like as a constituent element.
The methacrylate is a photocrosslinking agent, and reacts with the hyaluronic acid to obtain a photocrosslinkable hydrogel. The “photocrosslinking agent” refers to a compound capable of inducing a radical reaction in a reaction system by forming a radical by light irradiation, and a compound that is typically used as a photocrosslinker or a photoinitiator may be used as a photocrosslinker of the present invention, but it is more preferable to use a photocrosslinking agent or a photoinitiator that is non-toxic or poorly toxic in vivo.
The photocrosslinking technique enables crosslinking on site of the precursor solution and provides better spatial and temporal control over the crosslinking density than chemical crosslinking techniques. The photocrosslinking method using ultraviolet or visible light can produce a hydrogel having a wide range of mechanical properties and degradability.
The step of reacting the polymer and methacrylate may be performed by adding dropwise the methacrylate to the biodegradable polymer solution cooled to 0 to 10° C., preferably 3 to 7° C., for 30 minutes to 2 hours, preferably for 1 hour to 1 hour and 30 minutes, and maintained for 20 to 30 hours in the range of pH 7 to 11, preferably pH 8 to 10, and thus methacrylated hyaluronic acid (hereinafter, HAMA) can be formed. HAMA formed according to the method of the above step may have a degree of methacrylation (DM) of 100% or more, that is, a high degree of substitution of methacrylate groups, and the higher the methacrylation degree, the better the mechanical properties and strength. The HAMA with improved mechanical properties can be biodegraded slowly in a living body, so that radiation treatment can be performed for an appropriate time.
Next, in the conjugation step, a compound selected from the group consisting of imidazole, pyrrole, furan, thiophene, indole and 3,4-dihydroxyphenyl, capable of labeling one or more radioactive isotopes on the formed HAMA can be conjugated. More specifically, a cyclic compound selected from the group consisting of N-(3-aminopropyl)-imidazole (API), 3-(1H-pyrrol-1-yl)-1-propanamine, 1-(3-furyl)methanamine, thienylmethylamine, tryptamine, DOPA (3,4-dihydroxyphenylalanine) and derivatives thereof may be conjugated.
In the radioisotope labeling step, the conjugated compound may be labeled with a radioisotope, for example, at least one selected from the group consisting of 131I, 125I, 124I, 123I, 18F, 19F, 177Lu and 211At to form photocrosslinkable compounds.
In the preparation method according to the present invention, the photocrosslinkable compound may be contained in 1 to 20% by weight of the photoinitiator solution, and the centrifugation may be performed at 1000 to 2000 RPM, preferably 1400 to 1600 RPM. Accordingly, an appropriate amount of microhydrogel for radiation treatment can be prepared.
In the preparation method according to the present invention, the microgel may be prepared as a gel-type injection formulation, and may be injected into the body to remain at the injection site for 1 to 3 weeks. In addition, it can be used immediately by producing the microgel within 10 to 60 minutes, preferably within 10 to 30 minutes on site according to the preparation method, and can be appropriately prepared and used when radiation treatment is required.
Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. The examples of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.
Methacrylic anhydride, sodium hydroxide, N-(3-aminopropyl)-imidazole (API), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), chloramine T, sodium metabisulfite, sodium iodide, dimethyl sulfoxide (DMSO), acetaldehyde, cyclohexyl isocyanide, fluorescein-amine) was purchased from Sigma-Aldrich (St. Louis Mo., USA). Polydimethylsiloxane (PDMS, Sylgard 184) was purchased from Dow Corning. Pico-Surf™ 2 wt % in Novec™ 7500 was purchased from Sphere Fluidics (Cambridge, UK). Novec 7500 oil was purchased from 3M (St Paul, Minn.). Hyaluronic acid (HA) having a molecular weight of 90 KDa was purchased from Bioland (SK, Korea).
Referring to
HAMA-API was prepared by conjugating an API to hyaluronic acid through the amidation reaction of the carboxy group of HAMA and the amine group of N-(3-aminopropyl)-imidazole (hereinafter API). HAMA of 240 mg was dissolved in 40 mL of PBS (phosphate buffered saline, pH 7.4) at 25° C. for 4 hours. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (hereinafter, EDC) of 230 mg, 1.2 mmol and N-hydroxysuccinimide (hereinafter, NHS) of 276 mg, 2.4 mmol was added and stirred for 30 minutes to activate the carboxyl group. API (150 mg, 1.2 mmol) dissolved in 10 mL of PBS was added dropwise to this compound, and the reaction was performed for 12 hours. The finished compound was dialyzed in PBS and lyophilized to obtain HAMA-API.
The chloramine T method was used to replace iodine in the imidazole ring of HAMA-API. HAMA-API of 24 mg was added to a 2 mL centrifugation tube, dissolved in 500 μl of PBS, and 100 μl of NaI (Na131I) was added and left for 10 minutes. 10 μl (5 mg/mL) of chloramine T solution dissolved in PBS was added and vortexed for 10 minutes. To terminate the reaction, 10 μl (10 mg/mL) of a sodium bisulfite solution dissolved in PBS was added. To remove the residue, 1 mL of ethanol was added to the reactant and centrifuged at 5000 RPM for 20 minutes. After removing the supernatant, iodine-labeled HAMA was obtained.
The chemical properties of the hyaluronic acid-based conjugate were analyzed through 1H-NMR (600 MHz Agilent NMR System, Palo Alto, USA).
In order to minimize the decrease in radioactivity of isotope-labeled polymers, microgels must be quickly prepared on site. To this end, a HAMA microgel was prepared as a water-in-oil (W/O) emulsion using 131I-labeled photocrosslinkable hyaluronic acid through a portable centrifugal microfluidic system.
Referring to
After extracting the microgel formed in the outlet with a pipette, it was transferred to a microtube, and the crosslinking of HAMA was completed to form a microdroplet through UV light (350-500 nm, OhmiCure S1500, Exclitas, USA). To remove the oil from the crosslinked microgel, it was introduced in a mini cell strainer (pore size: 40 μm, Bio-Rev, Japan), and centrifugation was repeated for 3 minutes at 1000 RPM with ethanol to wash off the oil and the photocrosslinker. Subsequently, the washed HAMA microgel was dispersed in PBS.
A cell strainer (pore size: 70 μl, SPL) was put in a 6 well at room temperature, and a 0.3 mCi 131I-HAMA microgel was cast on the cell strainer. PBS (pH 7.4) of 5 mL was filled with in the wells filled with 131I-HAMA microgels and the radiation intensity of the 131I-HAMA microgels remaining in the cell strainer over time was measured by a CRC015R dose calibrator (Capintec Inc., Ramsey, USA).
Referring to
That is, the prepared radioactive microgel having a size of about 100 μm can be easily injected into a desired site through a syringe, and quantitative radiotherapy can be achieved. In addition, since the rate of decomposition and dissolution of microgels can vary depending on the degree of crosslinking, radiation treatment can be performed fora desired period of time, thereby minimizing the dose and maximizing radiation treatment to the local area, which can be expected to minimize tissue damage.
Male rats (12-13 weeks old) were purchased from SamTacho (Osan, Republic of Korea). All rats were managed at the Animal Resources Center at Pusan National University (Busan, Korea).
In order to evaluate the stability of HAMA microgels in vivo, HAMA bound with a fluorescent substance was prepared. 200 mg of HAMA was dissolved in 160 mL of DI water and 70 mL of dimethyl sulfoxide (DMSO) at 25° C. 100 μl of fluorescein-amine, acetaldehyde and cyclohexyl isocyanide were added to the mixture, and the amidation reaction was performed for 24 hours.
Referring to
Referring to
Referring to
An experiment was performed to confirm the distribution of 131I and how long the 131I-HAMA microgel was maintained at the site injected into the body. After injecting 0.2 mL of a solution of Na131I and 131I-HAMA microgel (1 mCi/mL) dispersed in PBS into the femoral muscle of the rat, gamma images were obtained over time with the SPECT imaging system (Infinia Hawkeye 4, GE Healthcare, USA). Within 30 minutes immediately after injection, the radioactivity at the injection site was measured with a counter per second in a dynamic mode. After 30 minutes, an image of the whole rat injected with 131I over time was obtained in static mode, and gamma counters of major tissues such as the injection site, thyroid gland and stomach were measured.
Referring to
Referring to
About 1.0 g of hyaluronic acid (HA, Mw: 90 kDa; SNvia, Korea) was dissolved in 12 mL distilled water, and the pH was adjusted to 8 using 1N sodium hydroxide. After cooling the hyaluronic acid solution to 5° C., 1 or 4 times the equivalent of methacrylic anhydride (MA) was added dropwise over 1 hour to the disaccharide unit of hyaluronic acid. At the same time, 1N sodium hydroxide was added to keep the pH between 8.0 and 10.0. After maintaining the temperature and pH for an additional 23 hours, the macromer solution was dialyzed with distilled water for 3 days (Cellu Sep, nominal molecular weight cutoff 3500 Da), frozen at −55° C., freeze-dried, and stored at 20° C. and then used. 1H NMR spectrum was obtained using a Bruker 600-NMR spectrometer. This confirms the binding of the methacrylate group to hyaluronic acid which is used to calculate the degree of methacrylation (hereinafter, DM) (n=3).
The photocrosslinkable methacrylate groups are generally incorporated into the hyaluronic acid polymer backbone by reacting it with methacrylic anhydride under aqueous basic conditions. The primary hydroxyl group of hyaluronic acid is considered the most reactive site for transesterification. Hyaluronic acid has 4 hydroxyl groups per disaccharide unit, and all of the 4 hydroxyl groups could be incorporated with methacrylate groups.
HAMAs with different DMs can be synthesized by varying the molecular weight of hyaluronic acid, the molar ratio of methacrylic anhydride to hyaluronic acid and the reaction time. There are also other parameters that determine DM, such as pH and temperature of the reaction mixture. Methacrylic anhydride undergoes hydrolyzed in an aqueous medium, especially above pH 10.0, catalyzed by hydroxide ions to form methacrylic acid, which does not react with hyaluronic acid. Hydrolysis of methacrylic anhydride to methacrylic acid at low temperatures is considered slower. However, at this temperature, methacrylic anhydride exists in a separate phase.
Referring to
Referring to
The molecular weight of hyaluronic acid and its derivatives were estimated with a gel permeation chromatography system. They showed similar polymer molecular weight distribution, indicating no premature crosslinking or significant chain cleavage during the reaction. Above 100% methacrylation suggests that at least one hydroxyl group was substituted when 4-equivalents of methacrylic anhydride were used.
That is, excessive hydrolysis of methacrylic anhydride was minimized by the slow addition of methacrylic anhydride over a period of 1 hour with vigorous stirring and maintaining the pH between pH 8.0 and 10.0 at 5° C. for 24 hours according to the Example 4, resulting in a high DM (>100%) can be obtained with a 4-fold excess of methacrylic anhydride.
Briefly, polymer precursor solutions at different concentrations of 5% (w/v), 10% (w/v) and 20% (w/v) were photocrosslinked to prepare a tensile test structure of a width of 5 mm, a length of 20 mm and a thickness of 1.5 mm.
The hydrogels were directly analyzed on a mechanical tester (AND 210, Korea). The strain rate was set to 1 mm min−1 for tensile testing. The ultimate tensile strengths of the samples were determined at the point of failure (fracture under tensile) of the hydrogel. The tensile strength was determined at the maximum point of the stress in the stress-strain curve.
The Young's modulus (tensile modulus) was calculated by obtaining the initial 5% of the slope in strain-stress curve. The elasticity was determined at the maximum point of the strain in the stress-strain curve.
The stress-strain curve of (A) showed that the higher the concentration of the HAMA solution with high DM, the larger the slope of the curve.
As the concentration of the HAMA solution increases from 5% to 20%, the tensile strength of (B) constantly increases from 3.31±0.61 to 21.22±6.48 kPa for the low DM, and from 8.83±2.99 to 44.24±7.09 kPa for the high DM.
The Young's modulus of (C) was also found to increase with higher DM and concentration.
The elongation of (D) was varied in the range of 6 to 17% depending on DM and concentration.
Reaction Scheme 1 shows the preparation process of HAMA-DOPA-I. After preparing HAMA with improved methacrylate substitution according to the Example 4, 100 mg of HAMA was dissolved in 10 mL PBS (pH 7.4) solution. After adjusting the pH to 5.5 using 1N HCl, 85.5 mg of EDC and 102.7 mg of NHS were added to the solution in the same manner as in the method of preparing HAMA-API of the Example 1, followed by stirring for 30 minutes. Thereafter, instead of the API, 126.9 mg of dopamine hydrochloride was added according to Reaction Scheme 1, and the pH of the reaction mixture was adjusted to 7 using 1N NaOH, followed by stirring at 25° C. for 24 hours. The reaction mixture was dialyzed in a pH 5.0 solution for 2 days (Cellu Sep, nominal molecular weight cutoff 3500 Da) and then dialyzed with distilled water for 1 day. The resulting solution was freeze-dried to obtain a product, and the resulting HAMA-DOPA solution was prepared, followed by labeling 131I. Since more 131I substitutions could be made than the HAMA-API according to the Example 1, it was expected that the radiation treatment effect would also be higher.
Reaction Scheme 2 shows the preparation process of HAMA-pyrrol-I. After preparing HAMA with improved methacrylate substitution according to the Example 4, 100 mg of HAMA was dissolved in 10 mL PBS (pH 7.4) solution. After adjusting the pH to 5.5 using 1N HCl, 85.5 mg of EDC and 102.7 mg of NHS were added to the solution in the same manner as in the method of preparing HAMA-API of the Example 1, followed by stirring for 30 minutes. Then, instead of the API, 110.7 mg of 3-(1H-pyrrol-1-yl)-1-propanamine was added according to the Reaction Scheme 2 and stirred at 25° C. for 24 hours. The reaction mixture was dialyzed for 2 days in a pH 5.0 solution, and then dialyzed for 1 day with distilled water. The resulting solution was freeze-dried to obtain a product, and the resulting HAMA-pyrrol solution was prepared, followed by labeling 131I.
Reaction Scheme 3 shows the preparation process of HAMA-furan-I. After preparing HAMA with improved methacrylate substitution according to the Example 4, 100 mg of HAMA was dissolved in 10 mL PBS (pH 7.4) solution. After adjusting the pH to 5.5 using 1N HCl, 85.5 mg of EDC and 102.7 mg of NHS were added to the solution in the same manner as in the method of preparing HAMA-API of Example 1, followed by stirring for 30 minutes. Thereafter, instead of the API, 119.2 mg of 3-(aminomethyl)furan hydrochloride was added according to the Reaction Scheme 3, followed by stirring at 25° C. for 24 hours. The reaction mixture was dialyzed for 2 days in a pH 5.0 solution, and then dialyzed for 1 day with distilled water. The resulting solution was freeze-dried to obtain a product, and the resulting HAMA-furan solution was prepared, followed by labeling 131I.
Reaction Scheme 4 shows the preparation process of HAMA-thiophene-I. After preparing HAMA with improved methacrylate substitution according to the Example 4, 100 mg of HAMA was dissolved in 10 mL PBS (pH 7.4) solution. After adjusting the pH to 5.5 using 1N HCl, 85.5 mg of EDC and 102.7 mg of NHS were added to the solution in the same manner as in the method of preparing HAMA-API of the Example 1, followed by stirring for 30 minutes. Thereafter, instead of the API, according to the Reaction Scheme 4, 100.9 mg of 2-thiophene methylamine was added, followed by stirring at 25° C. for 24 hours. The reaction mixture was dialyzed for 2 days in a pH 5.0 solution, and then dialyzed for 1 day with distilled water. The resulting solution was freeze-dried to obtain a product, and the resulting HAMA-thiophene solution was prepared, followed by labeling 131I.
Reaction Scheme 5 shows the preparation process of HAMA-N-iodoindole. After preparing HAMA with improved methacrylate substitution according to the Example 4, 100 mg of HAMA was dissolved in a 20 mL dimethyl sulfoxide (DMSO) solution. Then, 115.1 mg of EDC and 138.1 mg of NHS were added to the solution in the same manner as in the method of preparing HAMA-API of the Example 1, followed by stirring for 2 hours. Thereafter, instead of the API, 77.5 mg of N-ethyldiisopropylamine was added to the mixture according to the Reaction Scheme 5, and then 96.1 mg of tryptamine was added. The reaction was stirred under nitrogen for 36 hours. The reaction mixture was dialyzed in DMSO solution for 12 hours and then dialyzed with distilled water for 3 days. The resulting solution was freeze-dried to obtain a product, and the resulting HAMA-indole solution was prepared, and then 131I was labeled to prepare HAMA-N-iodoindole.
While the present invention has been particularly described with reference to specific examples thereof, it is apparent that this specific description is only a preferred example and that the scope of the present invention is not limited thereby to those skilled in the art. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0012906 | Jan 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/018094 | 12/19/2019 | WO | 00 |
