Antiviral-agent resistant virus detection system

Abstract

An oseltamivir analog and nanoparticles having the analog bound thereto, of the present invention, strongly bind to oseltamivir-resistant influenza virus, and thus, the use of the same can allow detection of oseltamivir-resistant influenza viruses quickly and conveniently with the naked eye. Therefore, the present invention can be favorably utilized in promptly establishing a therapeutic schedule for a patient infected with influenza viruses.

Claims

1. A compound of an oseltamivir hexylthiol represented by the following formula 2 or an oseltamivir hexylamine represented by the following formula 3: ##STR00009##

2. A nanoparticle for detecting an oseltamivir-resistant influenza virus comprising H275Y mutation, the nanoparticle having bound thereto a compound of an oseltamivir hexylthiol represented by the following formula 2 or an oseltamivir hexylamine represented by the following formula 3: ##STR00010##

3. The nanoparticle of claim 2, wherein the nanoparticle is a gold nanoparticle, a silver nanoparticle, or a fluorescent nanoparticle.

4. The nanoparticle of claim 2, wherein the nanoparticle has a higher binding affinity for the oseltamivir-resistant influenza virus comprising H275Y mutation than for an oseltamivir-sensitive influenza virus.

5. A kit for detecting an oseltamivir-resistant influenza virus comprising H275Y mutation, the kit comprising the compound of claim 1.

6. A method for treating influenza, comprising: treating a sample obtained from a subject with the nanoparticle of claim 2; confirming whether or not an oseltamivir-resistant influenza virus comprising H275Y mutation is present in the sample; and administering a therapeutically effective amount of oseltamivir phosphate to the subject when the oseltamivir-resistant influenza virus is not detected in the sample or when the sample contains a very small amount of the oseltamivir-resistant influenza virus.

7. The method of claim 6, wherein the confirming is performed using colorimetry.

8. The method of claim 6, wherein the confirming is performed visually.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the results of measuring the extent to which oseltamivir hexylamine and oseltamivir hexylthiol inhibited the neuraminidase activities of oseltamivir-sensitive virus and oseltamivir-resistant virus at the virus level.

(2) FIG. 2 is a graph showing the results obtained by treating each of oseltamivir-sensitive virus and oseltamivir-resistant virus with oseltamivir analogue-gold nanoparticles and measuring absorbance.

(3) FIG. 3 is a schematic view showing the patterns of binding of the oseltamivir hexylamine and oseltamivir hexylthiol of the present invention to oseltamivir-sensitive virus and oseltamivir-resistant virus. The oseltamivir hexylthiol whose formula is shown in the left lower portion of FIG. 3 showed about 250-fold higher G.sub.bind to oseltamivir-resistant virus.

(4) FIG. 4 shows absorbance graphs (left), visual observation of a color difference (right upper) and a graph showing the color difference (right bottom), which resulted when oseltamivir hexylthiol-bound gold nanoparticles were allowed to react with each of oseltamivir-sensitive virus and oseltamivir-resistant virus.

(5) FIG. 5 is a graph showing the results obtained by treating each of oseltamivir-sensitive virus and oseltamivir-resistant virus with oseltamivir hexylthiol-bound nanoparticles and measuring absorbance.

(6) FIG. 6 is a schematic view showing the configuration of a rapid kit for detection of antiviral agent-resistant virus, prepared using oseltamivir analogue-gold nanoparticles of the present invention.

(7) FIG. 7 shows the results of detecting antiviral agent-sensitive/resistant virus on a diagnostic rapid kit strip of the present invention.

MODE FOR INVENTION

(8) Hereinafter, preferred examples of the present invention will be described in order to facilitate understanding of the present invention. However, these examples are provided only for the purpose of facilitating understanding of the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Preparation of Nanoparticles

1-1: Synthesis of Oseltamivir Hexylthiol

(9) Oseltamivir hexylthiol was synthesized according to the following scheme. In the following description, the numbers in the parentheses after the compound names refer to the numbers shown at the bottom of the compound formulas in the following scheme.

(10) ##STR00005##

Synthesis of S-6-hydroxyhexyl Ethanethioate (2)

(11) At room temperature, potassium ethanethioate (6.31 g, 55.2 mmol) was added slowly dropwise to a solution of 1-bromohexanol (1) (5.00 g, 27.6 mmol) in DMF (50 mL) while the solution was stirred. The reaction mixture as stirred for 12 hours, and then diluted with distilled water (30 mL). The mixture was extracted with Et.sub.2O (330 mL), and the organic layers were combined, dried with anhydrous MgSO.sub.4, filtered, and then concentrated. The concentrate was separated by column chromatography (hexane:EtOAc=2:1-1:1) to obtain compound 2 (4.20 g, 86%) as a light yellow liquid.

Synthesis of (3R,5S)-ethyl-4-acetamido-5-(tert-butoxycarbonylamino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate (4)

(12) At room temperature, di-tert-butyl dicarbonate (7.84 mL, 34.1 mmol) and triethylamine (6.80 mL, 48.8 mmol) were added dropwise to a solution of oseltamivir phosphate salt (3) (10.0 g, 24.4 mmol) in MeOH (50 mL) while the solution was stirred. The reaction mixture was stirred at room temperature for 12 hours. Distilled water (100 mL) was added to the mixture which was then stirred for 1 hour. Next, the produced white solid was filtered and washed with distilled water. The filtrate was dried in a vacuum oven to obtain compound 4 (5.71 g, 56%) as a white solid.

Synthesis of (3R,5S)-4-acetamido-5-(tert-butoxycarbonylamino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylic Acid (5)

(13) At room temperature, NaOH (663 mg, 16.6 mmol) was added to a solution of compound 4 (5.70 g, 13.8 mmol) in THF/H.sub.2O (10:1, v/v, 30 mL) while the solution was stirred. The reaction mixture was stirred for 24 hours, and then concentrated to remove the reaction solvent. The concentrate was diluted with distilled water (20 mL), and the reactor was cooled to 0 C. The mixture was acidified to a pH 5 by addition of 1M HCl aqueous solution, and then stirred for 1 hour. The produced white solid was filtered and washed with distilled water. The filtrate was dried in a vacuum oven to obtain compound 5 (4.0 g, 75%) as a white solid.

Synthesis of (3R,5S)-6-(acetylthio)hexyl-4-acetamido-5-(tert-butoxycarbonylamino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate (6)

(14) At room temperature, compound 2 (2.20 g, 12.5 mmol), 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (2.79 g, 14.6 mmol), 4-(dimethylamino)pyridine (1.52 g, 12.5 mmol) and triethylamine (2.90 mL, 20.8 mmol) were sequentially added to a solution of compound 5 (4.00 g, 10.4 mmol) in CH.sub.2Cl.sub.2 (30 mL) while the solution was stirred. After stirring at room temperature for 24 hours, distilled water was added to the reaction mixture to stop the reaction. The mixture was extracted with CH.sub.2Cl.sub.2 (330 mL), and the organic layers were combined, dried with anhydrous Na.sub.2SO.sub.4, filtered, and then concentrated. The concentrate was separated by column chromatography (hexane:EtOAc=2:1-1:1) to obtain compound 6 (3.51 g, 62%) as a colorless liquid.

Synthesis of Oseltamivir Hexylthiol (7)

(15) At room temperature, concentrated hydrochloric acid (2.15 mL, 25.8 mmol) was added slowly dropwise to a solution of compound 6 (3.50 g, 6.45 mmol) in MeOH (30 mL) while the solution was stirred. The reactor was heated at 50 C. for 72 hours. The reaction mixture was cooled to room temperature, and then concentrated to remove the reaction solvent. The concentrate was diluted with MeOH (5 mL), and Et.sub.2O was added slowly dropwise thereto with stirring. The produced white solid was filtered and washed with Et.sub.2O. The filtrate was dried on a vacuum oven to obtain compound 7 (oseltamivir hexylthiol) (560 mg, 20%) as a white solid.

(16) The oseltamivir hexylthiol is represented by the following formula 2:

(17) ##STR00006##

1-2: Synthesis of Oseltamivir Hexylamine

(18) Oseltamivir hexylamine was synthesized according to the following scheme. In the following description, the numbers in the parentheses after the compound names refer to the numbers shown at the bottom of the compound formulas in the following scheme.

(19) ##STR00007##

Synthesis of tert-butyl 6-hydroxyhexylcarbamate (9)

(20) At room temperature, di-tert-butyl dicarbonate (6.47 mL, 28.2 mmol) was added dropwise to a solution of compound 8 (3.00 g, 25.6 mmol) in MeOH (30 mL) while the solution was stirred. The reaction mixture was stirred at room temperature for 6 hours, and then concentrated. The concentrate was separated by column chromatography (hexane:EtOAc=2:1-1:1) to obtain compound 9 (4.10 g, 74%) as a light yellow liquid.

Synthesis of (3R,5S)-6-(tert-butoxycarbonylamino)hexyl-4-acetamido-5-(tert-butoxycarbonylamino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate (10)

(21) At room temperature, compound 9 (2.18 g, 10.0 mmol), 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (2.44 g, 12.7 mmol), 4-(dimethylamino)pyridine (1.33 g, 10.9 mmol) and triethylamine (2.54 mL, 18.2 mmol) were sequentially added to a solution of compound 5 (3.50 g, 9.10 mmol) in DMF (20 mL). After stirring at room temperature for 24 hours, distilled water was added to the reaction mixture to stop the reaction. The mixture was extracted with EtOAc (320 mL), and the organic layers were combined, dried with anhydrous Na.sub.2SO.sub.4, filtered, and then concentrated. The concentrate was separated by column chromatography (hexane:EtOAc=2:1-1:1) to obtain compound 10 (2.41 g, 45%) as a yellow liquid.

Synthesis of Oseltamivir Hexylamine (11)

(22) At room temperature, concentrated hydrochloric acid (1.75 mL, 21.0 mmol) was added slowly dropwise to a solution of compound 10 (2.45 g, 4.20 mmol) in MeOH (20 mL) while the solution was stirred. After stirring at room temperature for 24 hours, the reaction mixture was concentrated to remove the reaction solvent. The concentrate was diluted with MeOH (5 mL), and Et.sub.2O was added slowly dropwise thereto with stirring. The produced white solid was filtered and washed with Et.sub.2O. The filtrate was dried in a vacuum oven to obtain compound 11 (oseltamivir hexylamine) (1.15 g, 65%) as a white solid.

(23) The oseltamivir hexylamine is represented by the following formula 3:

(24) ##STR00008##

1-3: Synthesis of Gold Nanoparticles

(25) 1 wt % HAuCl.sub.4 solution (1 mL) was added to 100 mL of distilled water, and the solution was vigorously stirred at 95 C. In this state, 1 wt % of sodium citrate (5 mL) was immediately added slowly to the solution, and was reacted under the same conditions for 30 minutes.

1-4: Nanoparticle-Oseltamivir Analogue Binding

(26) 5 mL of the gold nanoparticle solution synthesized in 1-3 above was centrifuged at 15000 rpm for 10 minutes to remove an excess of sodium citrate. The supernatant was discarded, and the residue was resuspended in 1 mL of distilled water, and then centrifuged at 15000 rpm for 10 minutes. This process was repeated twice more. In the last step, the supernatant was discarded, and 1 mL of the oseltamivir hexylamine or oseltamivir hexylthiol (6 mg/mL) suspended in distilled water was added to the residue. Next, the solution was vortexed for more than 12 hours so that the oseltamivir hexylamine or oseltamivir hexylthiol was bound to the gold nanoparticles. Then, the solution was centrifuged at 15000 rpm for 20 minutes, and the supernatant was removed and the residue was resuspended in 100 L of distilled water.

Example 2: Comparison of Neuraminidase Activities of Viruses

2-1: Comparison of the Effects of Oseltamivir Hexylthiol and Oseltamivir Hexylamine on Neuraminidase Activities of Viruses

(27) Using NA-Fluor Influenza Neurmaminidase Assay Kit (AB Applied biosystem, Prod No. 4457091), NA enzyme activities were measured.

(28) Specifically, solution A was prepared by dissolving NA-Fluor (480 L) in a working solution (5.52 mL) in the kit. A virus solution was prepared such that 100 or 1000 viruses would be contained in each well. As a wild type (antiviral agent-sensitive virus), pandemic H1N1 virus (A/04/2009/California) (pandemic H1N1) was used, and as a mutant type (antiviral agent-resistant virus), Influenza A/Korea/2785/2009 (H275Y mutation) was used. 19.3 mg of oseltamivir hexylamine or oseltamivir hexylthiol was prepared in 2 mL of distilled water (solution C). 50 L of solution A was added to each well of a 96-well plate, and each virus sample solution was added to each well. 50 L of solution C was added to each well at varying concentrations (only distilled water was added in a control group). Namely, solution A+each virus solution (wild type or mutant type)+solution C was added to each well. After 1 hour of incubation at 37 C., 50 L of NA-Fluor stop buffer solution was added to each well. NA activity at the protein level was measured by measuring fluorescence at an excitation wavelength (EX) of 360 nm and an emission wavelength (EM) of 450 nm. Higher NA activity indicates higher fluorescence intensity.

(29) The results of the measurement are shown in FIG. 1. From the left side (oseltamivir hexylamine) and right side (oseltamivir hexylthiol) of FIG. 1, it can be seen that high concentrations of oseltamivir hexylamine and oseltamivir hexylthiol inhibited the NA activity of the mutant-type virus (H275Y mutation) more significantly than the NA activity of the wild-type virus.

2-2: Measurement of Change in Absorbance after Treatment of Viral Neuraminidase Protein with Oseltamivir Analogue-Gold Nanoparticles

(30) Neuraminidase protein isolated from the wild-type or mutant-type virus was added to each well of a 96-well plate in an amount of 0.1 mg (100 g)/well, and the oseltamivir hexylthiol-bound gold nanoparticles synthesized in Example 1-4 above were added to each well in an amount of 100 L (1.22 mg in terms of gold ion concentration), after which the absorbance of each well was measured at a wavelength of 400 to 750 nm.

(31) The results of the measurement are shown in FIG. 2. As can be seen in FIG. 2, the absorbance wavelength for the mutant type (dotted line) was longer than the absorbance wavelength for the wild type (full line), indicating that the oseltamivir hexylthiol-bound gold nanoparticles show different absorbance wavelengths for the neuraminidase proteins of the wild type and the mutant type.

Example 3: Analysis of the Binding Affinity of Oseltamivir Analogue for Oseltamivir-Resistant Virus

(32) The binding energy (G.sub.bind) of the oseltamivir hexylthiol of the present invention for the neuraminidase site of oseltamivir-sensitive virus (wild type) and oseltamivir-resistant virus (mutant type) was calculated.

(33) As a result, it was shown that the binding energy of the oseltamivir hexylthiol for the wild type was 24.33 kcal/mol, but the binding energy of the oseltamivir hexylthiol for the mutant type was 27.62 kcal/mol (see FIG. 3). This suggests that the oseltamivir hexylthiol binds to the mutant type with about 250-fold higher affinity than to the wild type.

Example 4: Development of Antiviral Agent-Resistant Virus Detection System Using Oseltamivir Analogue-Gold Nanoparticles and Evaluation of Performance Thereof

(34) The oseltamivir analogue-gold nanoparticles of the present invention bind to oseltamivir-resistant virus with high affinity. Thus, when the nanoparticles are added to oseltamivir-resistant virus, a color change occurs so that the virus can be visually detected.

(35) The virus was prepared to contain 0, 10, 100 or 1000 viruses per each well of a 96-well plate. As a wild type (antiviral agent-sensitive virus), pandemic H1N1 virus (A/04/2009/California) (pandemic H1N1) was used, and as a mutant type (antiviral agent-resistant virus), Influenza A/Korea/2785/2009 (H275Y mutation) was used. 100 L (1.22 mg in terms of gold ion concentration) of the oseltamivir hexylthiol-gold nanoparticles synthesized in Example 1 were added to each well, after which the absorbance of each well was measured at a wavelength of 400 to 750 nm to detect antiviral agent-sensitive/resistant virus by a color change.

(36) The results of the measurement are shown in FIG. 4. As can be seen in the left side of FIG. 4, even when the number of the oseltamivir-sensitive viruses (wild type) increased, there was little or no change in the absorbance wavelength of the oseltamivir hexylthiol-gold nanoparticles, but as the number of the oseltamivir-resistant viruses (mutant type) increased, the absorbance wavelength of the oseltamivir hexylthiol-gold nanoparticles was shifted. This is a phenomenon caused by a color change from the original color due to an increase in the number of oseltamivir hexylthiol-gold nanoparticles bound to the oseltamivir-resistant viruses, and can also be visually detected (the right upper portion of FIG. 4). This phenomenon is graphically shown in the right bottom side of FIG. 4. From the graph, it can be clearly seen that the absorbance wavelength was shifted.

(37) In addition, FIG. 5 shows the results of measuring absorbance in the case in which 1000 wild type viruses and mutant type viruses were added to each well in the above experiment. From the left bottom of FIG. 5, it can be visually seen that a color change appeared (the color in FIG. 5 was different from that in FIG. 4 because the background light for the plate was brighter)

Example 5: Development of Rapid Diagnostic Kit for Antiviral Agent-Resistant Virus Detection Based on Oseltamivir Analogue-Gold Nanoparticles

(38) Using the oseltamivir analogue-gold nanoparticles prepared in Example 1, a rapid kit for oseltamivir-resistant influenza virus detection was prepared (FIG. 6). Specifically, oseltamivir-bound bovine serum albumin (BSA) (test line) and 3.3 mg/mL of the neuraminidase protein of an oseltamivir-resistant influenza virus comprising H275Y mutation (control line) were dropped onto a nitrocellulose membrane, thereby preparing a strip having a size of 4 mm50 mm. Next, oseltamivir hexylthiol-gold nanoparticles were dispensed onto the conjugate pad of the strip, and then dried. The rapid kit of the present invention is configured such that when a sample is moved through a sample pad, the oseltamivir hexylthiol-gold nanoparticles on the conjugate pad are moved together with the sample and adsorbed onto an adsorption pad through the nitrocellulose membrane.

Example 6: Detection of Oseltamivir-Resistant Influenza Virus by Use of Rapid Diagnostic Kit Based on Oseltamivir Analogue-Gold Nanoparticles

(39) The ability of the rapid kit (prepared in Example 5) to detect oseltamivir-sensitive/resistant influenza virus was evaluated. When a buffer sample containing no influenza virus was added to the rapid kit, a line appeared only in the control line, and did not appear in the test line. Furthermore, when a buffer containing oseltamivir-sensitive virus (wild type) was added to the rapid kit, a line also appeared only in the control line. However, when a buffer containing oseltamivir-resistant virus (mutant type) was added to the rapid kit, a line appeared in both the control line and the test line (FIG. 7). Such results suggest that oseltamivir-resistant influenza virus can be efficiently detected using a rapid kit comprising the oseltamivir analogue-gold nanoparticles of the present invention.