WO2017007204A1 - Antiviral-agent-resistant virus detection system - Google Patents

Antiviral-agent-resistant virus detection system Download PDF

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WO2017007204A1
WO2017007204A1 PCT/KR2016/007191 KR2016007191W WO2017007204A1 WO 2017007204 A1 WO2017007204 A1 WO 2017007204A1 KR 2016007191 W KR2016007191 W KR 2016007191W WO 2017007204 A1 WO2017007204 A1 WO 2017007204A1
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oseltamivir
formula
influenza virus
nanoparticles
mutation
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PCT/KR2016/007191
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French (fr)
Korean (ko)
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정주연
임은경
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한국생명공학연구원
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Priority claimed from KR1020160048823A external-priority patent/KR101788454B1/en
Application filed by 한국생명공학연구원 filed Critical 한국생명공학연구원
Priority to US15/741,577 priority Critical patent/US10336693B2/en
Priority to CN201680044850.9A priority patent/CN107850588B/en
Publication of WO2017007204A1 publication Critical patent/WO2017007204A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/02—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
    • C07C233/04—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C233/07—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to an antiviral agent-resistant virus detection system.
  • Influenza (flu) is a respiratory disease transmitted by the influenza virus through the respiratory system of humans and animals (birds, pigs, dogs, horses, etc.). Human influenza occurs in 10-20% of the world's population each year, and is highly contagious and tends to spread on a global scale every year. Symptoms of influenza include respiratory diseases such as high fever, headache, muscle pain, sore throat, sore throat, and cough, and in severe cases, death of the elderly, chronic disease holders, and the like.
  • influenza infections are suspected, they should be treated promptly to prevent the development of dangerous situations and to prevent further transmission to others.
  • oseltamivir phosphate Tamiflu
  • Methods for distinguishing viruses have been reported in a number of documents, for example Marin MJ et al. Discloses a method for distinguishing between human influenza virus and avian influenza virus.
  • no effective method has been developed to determine whether a patient suspected of influenza infection has been infected with oseltamivir resistant virus.
  • Non-Patent Document 1 Marin MJ et al. (Glyconanoparticles for the plasmonic detection and discrimination between human and avian influenza, Org Biomol Chem. 2013 Nov 7; 11 (41): 7101-7)
  • the present invention aims to provide a oseltamivir derivative compound, preferably oseltamivir hexylthiol or oseltamivir hexylamine, which is a novel compound that can be used for the detection of oseltamivir resistant virus.
  • An object of the present invention is to provide a nanoparticle for detecting oseltamivir resistant virus in which the oseltamivir derivative compound, preferably oseltamivir hexylthiol or oseltamivir hexylamine is bound.
  • the oseltamivir derivative compound preferably oseltamivir hexylthiol or oseltamivir hexylamine is bound.
  • An object of the present invention is to provide a method for detecting a virus exhibiting resistance to oseltamivir using the nanoparticles.
  • An object of the present invention is to provide a kit for detecting oseltamivir resistant virus comprising the nanoparticles.
  • the present invention provides a method for treating influenza by detecting a virus that is resistant to oseltamivir and administering a therapeutically effective amount of oseltamivir phosphate in a subject that does not contain a virus that is resistant to oseltamivir using the nanoparticles. To provide.
  • the present invention provides an oseltamivir derivative compound represented by the following Chemical Formula 1.
  • R 1 is thiol ( ) Or amine ( )being.
  • the oseltamivir derivative compound is oseltamivir hexylthiol represented by Formula 2 or oseltamivir hexylamine represented by Formula 3 below.
  • the present invention provides a nanoparticle for detecting oseltamivir resistant influenza virus, to which an oseltamivir derivative compound represented by Chemical Formula 1 is bound.
  • the present invention provides nanoparticles for detecting oseltamivir resistant influenza virus, in which oseltamivir hexylthiol represented by Formula 2 or oseltamivir hexylamine represented by Formula 3 is combined.
  • Nanoparticles of the present invention may be any nanoparticles that can be identified by the naked eye or absorption / fluorescence equipment as particles having a diameter of 1 to 100 nanometers, for example, gold nanoparticles, silver nanoparticles, fluorescent nanoparticles, Fluorescent dyes;
  • the gold nanoparticles and silver nanoparticles refer to particles of various shapes (spherical, polygonal, etc.) having a diameter of 100 nanometers or less.
  • the fluorescent nanoparticles are nanoparticles of 100 nanometers or less, which exhibit fluorescence properties, and even if the same material is used, the fluorescence wavelength varies depending on the size of the particles, thereby obtaining fluorescence in various wavelength bands.
  • fluorescent nanoparticles include various nanoparticles containing fluorescent dyes and quantum dots (quantum dot particles), and those consisting of a core having a size of about 2 to 10 nm and a shell composed mainly of ZnS.
  • Group II-VI or III-V compounds forming the quantum dots are, for example, CdSe, CdSe / ZnS, CdTe / CdS, CdTe / CdTe, ZnSe / ZnS, ZnTe / ZnSe, PbSe, PbS InAs, InP, InGaP, InGaP / It may be selected from the group consisting of ZnS and HgTe (in the form of a single core or core / shell).
  • Fluorescent dyes are for example fluorescent organic molecules (e.g., pyrene or derivatives thereof), cyanine, Cy series, Alexa Fluor series, BODIPY series, DY series, Rhodamine or derivatives thereof, Fluorescein or derivatives thereof, coumarin or derivatives thereof, Acridine homodimer or derivatives thereof, Acridine Orange or thereof Derivatives, 7-aminoactinomycin D (7-AAD) or derivatives thereof, actinomycin D or derivatives thereof, ACMA, 9-amino-6-chloro-2 -methoxyacridine or derivatives thereof, DAPI or derivatives thereof, dihydroethidium or derivatives thereof, ethidium bromide or derivatives thereof, ethidium homodimer-1 or EthD-1 this Derivatives, ethidium homodimer-2 (EthD-2) or derivatives thereof, ethidium monoazide or derivatives thereof, hexidium iodide or derivatives thereof
  • the nanoparticles may be gold nanoparticles, but is not limited thereto.
  • Oseltamivir resistant influenza virus is commonly known to undergo mutation of H275Y. Meanwhile, in the art, H275Y may also be described as H274Y depending on the manner of expression, and mutations of H274Y and H275Y are treated as being substantially the same. Hereinafter, the H275Y mutation is used herein to have the same meaning as H274Y.
  • Oseltamivir binds to the influenza virus and kills the virus in a way that reduces neuraminidase (NA) activity. When the H275Y mutation occurs, oseltamivir is difficult to bind to the virus.
  • NA neuraminidase
  • the oseltamivir hexylamine or oseltamivir hexylthiol bound to the nanoparticles of the present invention binds with a binding force about 1000 times stronger to the virus showing resistance to oseltamivir due to H275Y mutation than the oseltamivir sensitive virus.
  • a detection method using a compound that binds to oseltamivir sensitive virus but not to oseltamivir resistant virus only reveal whether or not oseltamivir susceptible virus is present in the sample, such as the presence of other pathogens other than the influenza virus, the presence of other serotype or mutant strains of influenza, or the absence of the pathogen.
  • oseltamivir resistant virus was indistinguishable from the presence of oseltamivir resistant virus, and the disadvantage of having to further confirm the presence of oseltamivir resistant virus in a sample using other means such as specific antibodies and genetic analysis. there was.
  • the presence of oseltamivir susceptible virus can be immediately confirmed, which enables accurate and simple detection compared to the conventional methods.
  • the present invention provides a method for detecting oseltamivir resistant influenza virus using the nanoparticles.
  • nanoparticles of the present invention are bound to a virus can be confirmed by visually comparing the colors (colorimetric method) or by absorbance measurement. Depending on the type and concentration of the nanoparticles used, and the amount of virus contained in the sample may vary, a person skilled in the art can easily distinguish between the presence and absence of oseltamivir resistant influenza virus in the sample. For example, when the nanoparticles of the present invention are treated with oseltamivir resistant influenza virus, as shown in the upper right of FIG.
  • the concentration of the treated nanoparticles increases, the color becomes significantly darker (virus and
  • the nanoparticles of the present invention are treated with the oseltamivir susceptible influenza virus, it can be seen that light colors appear even when the concentration of the nanoparticles is increased.
  • the method of the present invention comprises the steps of: 1) contacting a sample isolated from a subject with oseltamivir hexylthiol represented by formula (2) or oseltamivir hexylamine represented by formula (3). step; 2) When the same color that appears when the nanoparticle is contacted with a sample containing oseltamivir resistant influenza virus appears in the sample of step 1), it is determined that oseltamivir resistant influenza virus is present in the sample. Steps.
  • “same color” means a color that can be regarded as substantially the same even if there are slight differences in chroma, brightness, and hue in reaction conditions, sample conditions, and the like. Means.
  • the method of the present invention 1) a sample isolated from the subject and oseltamivir hexylthiol represented by the formula (2) or oseltamivir hexylamine represented by the formula (3) and Contacting; 2) If a color different from the color that appears when the nanoparticle is contacted with a sample without oseltamivir resistant influenza virus appears in the sample of step 1), it is determined that oseltamivir resistant influenza virus is present in the sample. It includes a step.
  • the subject may be a human or other animal, such as a bird or a mammal.
  • the sample may be whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsy, lymph, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid (CSF), semen, saliva, mucus, sputum, sweat or urine.
  • CSF cerebrospinal fluid
  • the present invention provides a kit for detecting oseltamivir resistant influenza virus comprising the nanoparticles.
  • the kit of the present invention may further include other items necessary for detection, and may further include instructions for use.
  • the detection kit of the present invention can be implemented in various forms.
  • a detection solution kit a rapid kit (a small amount of sample dropped into the kit using a lab-on-a-chip), or the sample can be moved on the kit to quickly check the results.
  • a rapid kit a small amount of sample dropped into the kit using a lab-on-a-chip
  • the sample can be moved on the kit to quickly check the results.
  • the detection kit of the present invention may be a rapid diagnostic kit in the form of a diagnostic strip.
  • the detection kit of the present invention includes a sample pad, a conjugate pad, a nitrocelluose membrane, and an adsorption pad.
  • the conjugate pad is supported by oseltamivir hexylthiol-gold nanoparticles or oseltamivir hexylamine-gold nanoparticles,
  • the oseltamivir hexylthiol-gold nanoparticles or oseltamivir hexylamine-gold nanoparticles supported on the conjugate pad are developed together with the sample into the nitrocellulose membrane and the adsorption pad.
  • the nitrocellulose membrane is provided with a control line and a test ine separated from each other.
  • control line is fixed neuramiminids of oseltamivir resistant influenza virus comprising H275Y mutation or oseltamivir resistant influenza virus comprising H275Y mutation
  • test line is oseltamivir represented by Formula 2 Hexylthiol or oseltamivir hexylamine represented by Formula 3 may be fixed.
  • the present invention provides a method for detecting oseltamivir resistant influenza virus using the detection kit.
  • the method is
  • the color change appears only in the control line when the oseltamivir resistant influenza virus is not present in the sample, but the color change appears in both the control line and the test line when present.
  • the degree of color change of the test line is large, it can be determined that the amount of oseltamivir resistant influenza virus contained in the sample is large.
  • a method of treating influenza comprising administering a therapeutically effective amount of oseltamivir phosphate to a subject for which no oseltamivir resistant influenza virus is detected or containing a trace amount of oseltamivir resistant influenza virus.
  • sample without oseltamivir resistant influenza virus or containing a trace amount of oseltamivir resistant influenza virus is free of oseltamivir resistant influenza virus or the level at which oseltamivir phosphate may have a therapeutic effect in a subject.
  • therapeutically effective amount is a reasonable oseltamivir phosphate dosage applicable to any medical treatment, and refers to the amount of therapeutic agent that is statistically related to a particular therapeutic effect when administered to a population of subjects.
  • the detection kit of the present invention is not limited to the above.
  • the oseltamivir analogs of the present invention and the nanoparticles to which the analogs bind are strongly bound to the oseltamivir resistant influenza virus, so that the oseltamivir resistant influenza virus can be detected quickly and conveniently with the naked eye. Therefore, it can be useful to plan treatment of patients who are infected with influenza virus quickly.
  • Figure 1 confirms the degree of oseltamivir hexylamine and oseltamivir hexylthiol inhibit the neuraminidase enzyme activity of oseltamivir-sensitive and oseltamivir resistant virus at the virus level.
  • FIG. 2 is a graph showing the results obtained by treating oseltamivir analogues-gold nanoparticles with oseltamivir-sensitive virus and oseltamivir-resistant virus, respectively, and monitoring the absorbance.
  • Figure 3 is a schematic diagram showing the binding mode of oseltamivir hexylamine and oseltamivir hexylthiol and oseltamivir-sensitive virus and oseltamivir resistant virus of the present invention.
  • ⁇ G bind was about 250 times higher for oseltamivir resistant virus.
  • FIG. 5 is a graph showing the results obtained by treating oseltamivir hexylthiol-bound nanoparticles with oseltamivir-sensitive virus and oseltamivir-resistant virus, respectively, and monitoring the absorbance.
  • Figure 6 is a schematic diagram of the rapid kit and configuration for the detection of antiviral resistant virus prepared using the oseltamivir analog-gold nanoparticles of the present invention.
  • Oseltamivir hexylthiol was synthesized with the following scheme.
  • the numbers indicated in parentheses after the compound names refer to numbers indicated at the bottom of the compound in the following scheme.
  • the oseltamivir hexylthiol is represented by the following formula (2).
  • Oseltamivir hexylamine was synthesized with the following scheme.
  • the numbers indicated in parentheses after the compound names refer to numbers indicated at the bottom of the compound in the following scheme.
  • the oseltamivir hexylamine is represented by the following formula (3).
  • HAuCl 4 solution 1 wt% was added to 100 mL of distilled water and stirred vigorously at 95 ° C. In that state, 1 wt% of sodium citrate (5 mL) was immediately injected slowly and reacted under the same conditions for 30 minutes.
  • Excess sodium citrate was removed by centrifuging 5 mL of the gold nanoparticle solution synthesized in 1-3 at 15000 rpm for 10 minutes. The supernatant was discarded, redispersed in 1 mL of distilled water, and then centrifuged at 15000 rpm for 10 minutes. This process was repeated two more times. In the last step, the supernatant was discarded and 1 mL of oseltamivir hexylamine or oseltamivir hexylthiol (6 mg / mL) dispersed in distilled water was added. And, by voltexing for more than 12 hours, to be bonded to the gold nanoparticles. After centrifugation at 15000 rpm for 20 minutes, the supernatant was removed, and redispersed in 100 ⁇ L of distilled water.
  • NA enzyme activity was measured using NA-FluorTM Influenza Neurmaminidase Assay Kit (AB Applied biosystem, Prod No. 4457091).
  • Solution A was prepared by dissolving Na-fluor (480 ⁇ L) in a working solution (5.52 mL) in the kit.
  • Virus solutions were prepared to contain 100 or 1000 viruses per well, respectively.
  • the wild type (antiviral susceptible virus) used pandemic H1N1 virus (A / 04/2009 / California) (pandemic H1N1), and the mutant type (infectious virus resistant virus) used Influenza A / Korea / 2785/2009 (H275Y mutation).
  • Solution A 50 ⁇ L was added to each well of a 96 well plate, and a virus sample solution was added to each well.
  • Solution C was added to each well prepared at different concentrations by 50 ⁇ L (only distilled water was added in the comparison group). That is, solution A + each virus solution (wild type or mutant type) + solution C was added to each well. After incubation at 37 ° C. for 1 hour, 50 ⁇ L of Na-flour stop buffer solution was added to each well. Fluorescence was measured at Ex: 360 nm and Em: 450 nm to determine NA activity at the protein level. Higher NA enzyme activity means higher fluorescence intensity.
  • Each well of a 96 well plate was prepared to contain 0.1 mg (100 ⁇ g) / well of neuraminidase protein isolated from the wild type or the mutant type, and oseltamivir hexyl synthesized in 1-4 in each well. After adding 100 ⁇ L (1.22 mg of gold ion concentration) to the thiol-bound gold nanoparticles, the absorbance was measured in the 400-750 nm range.
  • the binding energy ( ⁇ G bind ) of the neuramiminidase site of oseltamivir-sensitive virus (wild type) and oseltamivir resistant virus (mutant type) and oseltamivir hexylthiol of the present invention was calculated.
  • the binding energy of oseltamivir hexylthiol to wild type was -24.33 kcal / mol, but the binding energy to mutant type was -27.62 kcal / mol (refer to the schematic diagram of FIG. 3).
  • Example 4 Development and efficacy evaluation of antiviral resistant virus detection system using oseltamivir analogues-gold nanoparticles
  • the oseltamivir analogue-nanoparticle of the present invention binds to the oseltamivir resistant virus with strong binding force, and thus the color change occurs when the nanoparticle is added to the oseltamivir resistant virus.
  • Each well of a 96 well plate was prepared with 0, 10, 100, or 1000 viruses.
  • the wild type (anti-viral susceptible virus) used pandemic H1N1 virus (A / 04/2009 / California) (pandemic H1N1), and the mutant type (anti-viral resistant virus) used Influenza A / Korea / 2785/2009 (H275Y mutation).
  • 100 ⁇ L of oseltamivir hexylthiol-gold nanoparticles prepared in Example 1 (1.22 mg based on gold ion concentration) was added, and then the absorbance was measured in the range of 400-750 nm to determine antiviral sensitivity / resistance. Viruses were detected through color change.
  • oseltamivir analogue-gold nanoparticles prepared in Example 1, a rapid kit for detecting oseltamivir resistant influenza virus was prepared (FIG. 6). Specifically, 3.3 mg / mL of the neuraminidase protein of oseltamivir resistant influenza virus containing a oseltamivir-bound BSA (test line) and an H275Y mutant was prepared using a nitrocellulose membrane. Membrane strips were prepared in 4 mm x 50 mm. Then, oseltamivir hexylthiol-gold nanoparticles were dispensed to the conjugate pad and dried to prepare.
  • the rapid kit of the present invention has a structure in which the oseltamivir hexylthiol-gold nanoparticles of the conjugation pad are developed together with the sample and absorbed by the adsorption pad through the nitrocellulose membrane when the sample is developed through the sample pad. have.
  • Example 5 Using the rapid kit prepared in Example 5, the detection ability of oseltamivir-sensitive / resistive influenza virus was confirmed.
  • a buffer containing no influenza virus was added to the Rapid Kit as a sample, a line appeared only in the control line and no line in the test line.
  • a buffer containing oseltamivir susceptible virus wild type
  • lines also appeared only in the control line.
  • a buffer containing oseltamivir resistant virus (mutant type) was added to the rapid kit, lines appeared in both the control line and the test line (FIG. 7). From the above results, it was verified that the oseltamivir resistant influenza virus can be efficiently detected using the rapid kit including the oseltamivir analog-gold nanoparticles of the present invention.

Abstract

An oseltamivir analog and nanoparticles having the analog bound thereto, of the present invention, strongly bind to oseltamivir-resistant influenza viruses, 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.

Description

항바이러스제-저항성 바이러스 검출 시스템Antiviral-resistant virus detection system
본 발명은 항바이러스제-저항성 바이러스 검출 시스템에 관한 것이다.The present invention relates to an antiviral agent-resistant virus detection system.
인플루엔자(influenza, 독감)는 인플루엔자 바이러스(Influenza virus)에 의해 사람 및 동물(조류, 돼지, 개, 말 등)의 호흡기를 통해 전파되는 호흡기성 질병이다. 사람 인플루엔자(Human influenza)의 경우 매년 전 세계 10-20% 인구에서 발생하며, 전염성이 높아 매년 세계적 규모로 유행하는 경향이 있다. 인플루엔자의 증상은 고열, 두통, 근육통, 인후의 염증, 통증, 기침 등의 호흡기질환을 수반하며 심한 경우 노약자, 만성질환보유자 등의 사망을 유발할 수 있다.Influenza (flu) is a respiratory disease transmitted by the influenza virus through the respiratory system of humans and animals (birds, pigs, dogs, horses, etc.). Human influenza occurs in 10-20% of the world's population each year, and is highly contagious and tends to spread on a global scale every year. Symptoms of influenza include respiratory diseases such as high fever, headache, muscle pain, sore throat, sore throat, and cough, and in severe cases, death of the elderly, chronic disease holders, and the like.
인플루엔자 감염이 의심되면 신속하게 치료하여 위험한 상황이 초래되는 것을 막고 다른 사람들에게 추가적으로 전파되는 것을 방지해야 한다. 현재 인플루엔자 감염의 치료에는 오셀타미비르 포스페이트(타미플루)가 주로 이용되고 있는데, 근래에 오셀타미비르에 저항성을 나타내는 바이러스 변이주 발생이 증가하고 있다. 바이러스를 구별할 수 있는 방법에 대해서는 다수의 문헌에서 보고된 바 있는데, 예를 들어 Marin MJ et al.은 인간 인플루엔자 바이러스와 조류 인플루엔자 바이러스를 구별하는 방법에 대해 개시하고 있다. 그러나 인플루엔자 감염이 의심되는 환자가 오셀타미비르 저항성 바이러스에 감염되었는지 여부를 확인할 수 있는 효과적인 방법은 현재까지 개발된 바 없다.If influenza infections are suspected, they should be treated promptly to prevent the development of dangerous situations and to prevent further transmission to others. Currently, oseltamivir phosphate (Tamiflu) is mainly used for the treatment of influenza infection, and recently, the occurrence of virus mutants that are resistant to oseltamivir is increasing. Methods for distinguishing viruses have been reported in a number of documents, for example Marin MJ et al. Discloses a method for distinguishing between human influenza virus and avian influenza virus. However, no effective method has been developed to determine whether a patient suspected of influenza infection has been infected with oseltamivir resistant virus.
[선행기술문헌][Preceding technical literature]
[비특허문헌][Non-Patent Documents]
(비특허문헌 1)Marin MJ et al. (Glyconanoparticles for the plasmonic detection and discrimination between human and avian influenza, Org Biomol Chem. 2013 Nov 7; 11(41):7101-7)(Non-Patent Document 1) Marin MJ et al. (Glyconanoparticles for the plasmonic detection and discrimination between human and avian influenza, Org Biomol Chem. 2013 Nov 7; 11 (41): 7101-7)
본 발명은 오셀타미비르 저항성 바이러스 검출을 위해 사용할 수 있는 신규한 화합물인 오셀타미비르 유도체 화합물, 바람직하게 오셀타미비르 헥실티올 또는 오셀타미비르 헥실아민을 제공하는 것을 목적으로 한다.The present invention aims to provide a oseltamivir derivative compound, preferably oseltamivir hexylthiol or oseltamivir hexylamine, which is a novel compound that can be used for the detection of oseltamivir resistant virus.
본 발명은 상기 오셀타미비르 유도체 화합물, 바람직하게 오셀타미비르 헥실티올 또는 오셀타미비르 헥실아민이 결합된 오셀타미비르 저항성 바이러스 검출용 나노입자를 제공하는 것을 목적으로 한다.An object of the present invention is to provide a nanoparticle for detecting oseltamivir resistant virus in which the oseltamivir derivative compound, preferably oseltamivir hexylthiol or oseltamivir hexylamine is bound.
본 발명은 상기 나노입자를 이용하여 오셀타미비르에 저항성을 나타내는 바이러스를 검출하는 방법을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a method for detecting a virus exhibiting resistance to oseltamivir using the nanoparticles.
본 발명은 상기 나노입자를 포함하는 오셀타미비르 저항성 바이러스 검출용 키트를 제공하는 것을 목적으로 한다. An object of the present invention is to provide a kit for detecting oseltamivir resistant virus comprising the nanoparticles.
본 발명은 상기 나노입자를 이용하여 오셀타미비르에 저항성을 나타내는 바이러스를 검출하고 오셀타미비르에 저항성을 나타내는 바이러스를 포함하지 않는 대상체에서 오셀타미비르 포스페이트의 치료학적 유효량을 투여하여 인플루엔자를 치료하는 방법을 제공한다.The present invention provides a method for treating influenza by detecting a virus that is resistant to oseltamivir and administering a therapeutically effective amount of oseltamivir phosphate in a subject that does not contain a virus that is resistant to oseltamivir using the nanoparticles. To provide.
본 발명은 하기 화학식 1로 기재되는 오셀타미비르 유도체 화합물을 제공한다. The present invention provides an oseltamivir derivative compound represented by the following Chemical Formula 1.
[화학식 1][Formula 1]
Figure PCTKR2016007191-appb-I000001
Figure PCTKR2016007191-appb-I000001
상기 화학식 1에서, In Chemical Formula 1,
R1은 티올(
Figure PCTKR2016007191-appb-I000002
) 또는 아민(
Figure PCTKR2016007191-appb-I000003
)임.R 1 is thiol (
Figure PCTKR2016007191-appb-I000002
) Or amine (
Figure PCTKR2016007191-appb-I000003
)being.
바람직하게 상기 오셀타미비르 유도체 화합물은 하기 화학식 2로 기재되는 오셀타미비르 헥실티올(oseltamivir hexylthiol) 또는 하기 화학식 3로 기재되는 오셀타미비르 헥실아민(oseltamivir hexylamine)이다.Preferably, the oseltamivir derivative compound is oseltamivir hexylthiol represented by Formula 2 or oseltamivir hexylamine represented by Formula 3 below.
[화학식 2][Formula 2]
Figure PCTKR2016007191-appb-I000004
Figure PCTKR2016007191-appb-I000004
[화학식 3][Formula 3]
Figure PCTKR2016007191-appb-I000005
Figure PCTKR2016007191-appb-I000005
본 발명은 화학식 1로 기재되는 오셀타미비르 유도체 화합물이 결합된, 오셀타미비르 저항성 인플루엔자 바이러스 검출용 나노입자를 제공한다. 바람직하게, 화학식 2로 기재되는 오셀타미비르 헥실티올(oseltamivir hexylthiol) 또는 화학식 3으로 기재되는 오셀타미비르 헥실아민(oseltamivir hexylamine)이 결합된, 오셀타미비르 저항성 인플루엔자 바이러스 검출용 나노입자를 제공한다.  The present invention provides a nanoparticle for detecting oseltamivir resistant influenza virus, to which an oseltamivir derivative compound represented by Chemical Formula 1 is bound. Preferably, the present invention provides nanoparticles for detecting oseltamivir resistant influenza virus, in which oseltamivir hexylthiol represented by Formula 2 or oseltamivir hexylamine represented by Formula 3 is combined.
본 발명의 나노입자는 직경이 1~100 나노미터 단위인 입자로서 육안 또는 흡/형광 장비로 확인이 가능한 임의의 나노입자가 될 수 있으며, 예를 들어 금나노입자, 은나노입자, 형광 나노입자, 형광 염색약 등이 될 수 있다. 상기 금나노입자 및 은나노입자는 100 나노미터 이하의 직경을 가진 다양한 형태(구형, 다각형 등)의 입자를 말한다. 상기 형광 나노입자는 형광 특성을 보이는 100 나노미터 이하의 나노 입자로, 동일한 물질이라 하더라도 입자의 크기에 따라 형광파장이 달라져 다양한 파장대의 형광을 얻을 수 있다. 형광 나노입자의 예는 형광 염색약이 포함된 다양한 나노 입자 및 Quantum dots (양자점 입자)로, 약 2～10nm 크기의 중심(core)과 주로 ZnS 등으로 이루어진 껍질(shell)로 구성된 것을 들 수 있다. 상기 양자점을 이루는 Ⅱ-Ⅵ 또는 Ⅲ-Ⅴ족 화합물은 예를 들어 CdSe, CdSe/ZnS, CdTe/CdS, CdTe/CdTe, ZnSe/ZnS, ZnTe/ZnSe, PbSe, PbS InAs, InP, InGaP, InGaP/ZnS 및 HgTe로 구성된 군에서 선택될 수 있다(단일 코어(core) 또는 코어(core)/쉘(shell) 형태). 형광 염색약은 예를 들어 형광성을 보이는 유기 분자 (예, 파이렌 (Pyrene) 또는 이의 유도체), 시아닌(Cyanine, Cy) 시리즈, 알렉사플루오르(Alexa Fluor) 시리즈, 보디피 (BODIPY) 시리즈, DY 시리즈, 로다민 (rhodamine) 또는 이의 유도체, 플루오레신(Fluorescein) 또는 이의 유도체, 쿠마린 (coumarin) 또는 이의 유도체, 아크리딘 호모다이머 (Acridine homodimer) 또는 이의 유도체, 아크리딘 오렌지(Acridine Orange) 또는 이의 유도체, 7-아미노액티노마이신 D(7-aminoactinomycin D, 7-AAD) 또는 이의 유도체, 액티노마이신 D(Actinomycin D) 또는 이의 유도체, 에이씨엠에이(ACMA, 9-amino-6-chloro-2-methoxyacridine) 또는 이의 유도체, 디에이피아이(DAPI) 또는 이의 유도체, 디하이드로에티듐(Dihydroethidium) 또는 이의 유도체, 에티듐 브로마이드(Ethidium bromide) 또는 이의 유도체, 에티듐 호모다이머-1(EthD-1) 또는 이의 유도체, 에티듐 호모다이머-2(EthD-2) 또는 이의 유도체, 에티듐 모노아자이드(Ethidium monoazide) 또는 이의 유도체, 헥시디움 아이오다이드(Hexidium iodide) 또는 이의 유도체, 비스벤지마이드(bisbenzimide, Hoechst 33258) 또는 이의 유도체, 호에크스트 33342(Hoechst 33342) 또는 이의 유도체, 호에크스트 34580(Hoechst 34580) 또는 이의 유도체, 하이드로옥시스티바미딘(hydroxystilbamidine) 또는 이의 유도체, 엘디에스 751(LDS 751) 또는 이의 유도체, 프로피디움 아이오다이드(Propidium Iodide, PI) 또는 이의 유도체, 칼세인(Calcein) 또는 이의 유도체, 오레건 그린(Oregon Green) 또는 이의 유도체, 마그네슘 그린(Magnesium Green) 또는 이의 유도체, 칼슘 그린(Calcium Green) 또는 이의 유도체, JOE 또는 이의 유도체, 테트라메틸로다민(Tetramethylrhodamine) 또는 이의 유도체, TRITC 또는 이의 유도체, TAMRA 또는 이의 유도체, 피로닌 Y(Pyronin Y) 또는 이의 유도체, 리싸민(Lissamine) 또는 이의 유도체, ROX 또는 이의 유도체, 칼슘크림선(Calcium Crimson) 또는 이의 유도체, 텍사스 레드(Texas Red) 또는 이의 유도체, 나일 레드(Nile Red) 또는 이의 유도체, 티아디카복시아닌(Thiadicarbocyanine) 또는 이의 유도체, 단실아마이드(dansylamide) 또는 이의 유도체, 캐스캐이드 블루 (cascade blue), DAPI(4',6-diamidino-2-phenylindole), FITC, Cy3, Cy5, Cy5.5, Cy7 등)이 있다. 그러나 나노입자의 종류는 이에 제한되지 않는다. 각 나노입자는 공지의 방법에 의해 합성될 수 있다.Nanoparticles of the present invention may be any nanoparticles that can be identified by the naked eye or absorption / fluorescence equipment as particles having a diameter of 1 to 100 nanometers, for example, gold nanoparticles, silver nanoparticles, fluorescent nanoparticles, Fluorescent dyes; The gold nanoparticles and silver nanoparticles refer to particles of various shapes (spherical, polygonal, etc.) having a diameter of 100 nanometers or less. The fluorescent nanoparticles are nanoparticles of 100 nanometers or less, which exhibit fluorescence properties, and even if the same material is used, the fluorescence wavelength varies depending on the size of the particles, thereby obtaining fluorescence in various wavelength bands. Examples of fluorescent nanoparticles include various nanoparticles containing fluorescent dyes and quantum dots (quantum dot particles), and those consisting of a core having a size of about 2 to 10 nm and a shell composed mainly of ZnS. Group II-VI or III-V compounds forming the quantum dots are, for example, CdSe, CdSe / ZnS, CdTe / CdS, CdTe / CdTe, ZnSe / ZnS, ZnTe / ZnSe, PbSe, PbS InAs, InP, InGaP, InGaP / It may be selected from the group consisting of ZnS and HgTe (in the form of a single core or core / shell). Fluorescent dyes are for example fluorescent organic molecules (e.g., pyrene or derivatives thereof), cyanine, Cy series, Alexa Fluor series, BODIPY series, DY series, Rhodamine or derivatives thereof, Fluorescein or derivatives thereof, coumarin or derivatives thereof, Acridine homodimer or derivatives thereof, Acridine Orange or thereof Derivatives, 7-aminoactinomycin D (7-AAD) or derivatives thereof, actinomycin D or derivatives thereof, ACMA, 9-amino-6-chloro-2 -methoxyacridine or derivatives thereof, DAPI or derivatives thereof, dihydroethidium or derivatives thereof, ethidium bromide or derivatives thereof, ethidium homodimer-1 or EthD-1 this Derivatives, ethidium homodimer-2 (EthD-2) or derivatives thereof, ethidium monoazide or derivatives thereof, hexidium iodide or derivatives thereof, bisbenzimide, Hoechst 33258) or derivatives thereof, Hoechst 33342 or derivatives thereof, Hoechst 34580 or derivatives thereof, hydroxystilbamidine or derivatives thereof, LDS 751 (LDS 751) or Derivatives thereof, propidium iodide (PI) or derivatives thereof, calcein (Calcein) or derivatives thereof, Oregon Green or derivatives thereof, magnesium green or derivatives thereof, calcium green ( Calcium Green) or derivatives thereof, JOE or derivatives thereof, tetramethylrhodamine or derivatives thereof, TRITC or derivatives thereof, TAMRA or its derivatives Derivatives, Pyronin Y or derivatives thereof, Lissamine or derivatives thereof, ROX or derivatives thereof, Calcium Crimson or derivatives thereof, Texas Red or derivatives thereof, nile red (Nile Red) or derivatives thereof, thiadicarbocyanine or derivatives thereof, dansylamide or derivatives thereof, cascade blue, DAPI (4 ', 6-diamidino-2-phenylindole) , FITC, Cy3, Cy5, Cy5.5, Cy7, etc.). However, the type of nanoparticles is not limited thereto. Each nanoparticle can be synthesized by a known method.
본 발명의 일 구현예로서, 상기 나노입자는 금나노입자가 될 수 있으나, 이에 제한되지 않는다. In one embodiment of the present invention, the nanoparticles may be gold nanoparticles, but is not limited thereto.
오셀타미비르 저항성 인플루엔자 바이러스는 통상적으로 H275Y의 돌연변이가 일어나는 것으로 알려져 있다. 한편 당업계에서 H275Y는 표현 방식에 따라 H274Y로도 기기재될 수 있으며, H274Y와 H275Y의 돌연변이는 사실상 동일한 것으로 취급된다. 이하 본 명세서에서 H275Y 돌연변이는 H274Y와 동일한 의미를 갖는 것으로 사용된다. 오셀타미비르는 인플루엔자 바이러스에 결합하여 뉴라미니데이즈(neuraminidase, NA) 활성을 감소시키는 방식으로 바이러스를 사멸시키는데, H275Y 돌연변이가 일어나면 오셀타미비르가 바이러스에 결합하기가 어렵다. Oseltamivir resistant influenza virus is commonly known to undergo mutation of H275Y. Meanwhile, in the art, H275Y may also be described as H274Y depending on the manner of expression, and mutations of H274Y and H275Y are treated as being substantially the same. Hereinafter, the H275Y mutation is used herein to have the same meaning as H274Y. Oseltamivir binds to the influenza virus and kills the virus in a way that reduces neuraminidase (NA) activity. When the H275Y mutation occurs, oseltamivir is difficult to bind to the virus.
본 발명의 나노입자에 결합된 오셀타미비르 헥실아민 또는 오셀타미비르 헥실티올은 오셀타미비르 감수성 바이러스보다 H275Y 돌연변이가 일어나 오셀타미비르에 저항성을 나타내는 바이러스에 대하여 약 1000 배 더 강한 결합력으로 결합한다. 오셀타미비르 감수성 바이러스에 결합하고 오셀타미비르 저항성 바이러스에는 결합하지 않는 화합물을 이용한 검출 방법에 대해 기존에 보고된 바 있다. 그러나 이러한 방법에 의하면 시료 내에 오셀타미비르 감수성 바이러스가 존재하는지 여부만을 알 수 있어, 시료 내에 인플루엔자 바이러스 외의 다른 병원체가 존재하거나 다른 혈청형 또는 변이주의 인플루엔자 바이러스가 존재하거나 또는 병원체가 존재하지 않는 경우와 오셀타미비르 저항성 바이러스가 존재하는 경우를 구별할 수 없는 단점이 있었고, 결국 특이적 항체, 유전자 분석 등 다른 수단을 이용하여 시료 내에 오셀타미비르 저항성 바이러스의 존재를 확진하는 단계를 더 거쳐야 하는 단점이 있었다. 그러나 본 발명을 이용하면 오셀타미비르 감수성 바이러스의 존재를 바로 확인할 수 있어 기존의 방법에 비해 정확하고 간편한 검출이 가능하다. The oseltamivir hexylamine or oseltamivir hexylthiol bound to the nanoparticles of the present invention binds with a binding force about 1000 times stronger to the virus showing resistance to oseltamivir due to H275Y mutation than the oseltamivir sensitive virus. There has been previously reported a detection method using a compound that binds to oseltamivir sensitive virus but not to oseltamivir resistant virus. However, these methods only reveal whether or not oseltamivir susceptible virus is present in the sample, such as the presence of other pathogens other than the influenza virus, the presence of other serotype or mutant strains of influenza, or the absence of the pathogen. The disadvantage of oseltamivir resistant virus was indistinguishable from the presence of oseltamivir resistant virus, and the disadvantage of having to further confirm the presence of oseltamivir resistant virus in a sample using other means such as specific antibodies and genetic analysis. there was. However, by using the present invention, the presence of oseltamivir susceptible virus can be immediately confirmed, which enables accurate and simple detection compared to the conventional methods.
본 발명은 상기 나노입자를 이용하여 오셀타미비르 저항성 인플루엔자 바이러스를 검출하는 방법을 제공한다. The present invention provides a method for detecting oseltamivir resistant influenza virus using the nanoparticles.
본 발명의 나노입자가 바이러스에 결합했는지 여부는 육안으로 색상을 비교함으로써(비색법) 또는 흡광도 측정에 의해 확인할 수 있다. 사용한 나노입자의 종류 및 농도, 시료 내 포함된 바이러스의 양에 따라 다르게 나타날 수 있으나, 통상의 기술자는 시료 내에 오셀타미비르 저항성 인플루엔자 바이러스가 존재하는 경우와 그러지 않은 경우를 용이하게 구별할 수 있다. 예를 들어 본원 명세서 도 5의 우측 상단 도에 나타난 바와 같이, 오셀타미비르 저항성 인플루엔자 바이러스에 본 발명의 나노입자를 처리한 경우에는 처리한 나노입자의 농도가 높아질수록 현저히 색상이 진해지지만(바이러스와 나노입자의 결합이 증가하여 나타나는 현상), 오셀타미비르 감수성 인플루엔자 바이러스에 본 발명의 나노입자를 처리한 경우에는 처리한 나노입자의 농도가 높아져도 연한 색상이 나타남을 알 수 있다.Whether the nanoparticles of the present invention are bound to a virus can be confirmed by visually comparing the colors (colorimetric method) or by absorbance measurement. Depending on the type and concentration of the nanoparticles used, and the amount of virus contained in the sample may vary, a person skilled in the art can easily distinguish between the presence and absence of oseltamivir resistant influenza virus in the sample. For example, when the nanoparticles of the present invention are treated with oseltamivir resistant influenza virus, as shown in the upper right of FIG. 5 of the present specification, as the concentration of the treated nanoparticles increases, the color becomes significantly darker (virus and When the nanoparticles of the present invention are treated with the oseltamivir susceptible influenza virus, it can be seen that light colors appear even when the concentration of the nanoparticles is increased.
본 발명의 일 구현예로서, 본 발명의 방법은 1) 대상체로부터 분리된 시료를 화학식 2로 기재되는 오셀타미비르 헥실티올 또는 화학식 3으로 기재되는 오셀타미비르 헥실아민이 결합된 나노입자와 접촉시키는 단계; 2) 오셀타미비르 저항성 인플루엔자 바이러스가 존재하는 시료에 상기 나노입자를 접촉시킨 경우에 나타나는 색상과 동일한 색상이 상기 1) 단계의 시료에서 나타나는 경우 상기 시료 내에 오셀타미비르 저항성 인플루엔자 바이러스가 존재한다고 판단하는 단계를 포함한다. In one embodiment of the invention, the method of the present invention comprises the steps of: 1) contacting a sample isolated from a subject with oseltamivir hexylthiol represented by formula (2) or oseltamivir hexylamine represented by formula (3). step; 2) When the same color that appears when the nanoparticle is contacted with a sample containing oseltamivir resistant influenza virus appears in the sample of step 1), it is determined that oseltamivir resistant influenza virus is present in the sample. Steps.
상기 방법에 있어서, “동일한 색상”이라 함은 반응 조건, 시료의 상태 등에 채도(chroma), 명도(brightness), 색상(hue)에 있어 약간의 차이가 있더라도 실질적으로 동일하다고 볼 수 있는 정도의 색상을 의미한다.In the above method, "same color" means a color that can be regarded as substantially the same even if there are slight differences in chroma, brightness, and hue in reaction conditions, sample conditions, and the like. Means.
또한 본 발명의 다른 구현예로서, 본 발명의 방법은, 1) 대상체로부터 분리된 시료를 화학식 2로 기재되는 오셀타미비르 헥실티올 또는 화학식 3으로 기재되는 오셀타미비르 헥실아민이 결합된 나노입자와 접촉시키는 단계; 2) 오셀타미비르 저항성 인플루엔자 바이러스가 존재하지 않는 시료에 상기 나노입자를 접촉시킨 경우에 나타나는 색상과 상이한 색상이 상기 1) 단계의 시료에서 나타나는 경우 상기 시료 내에 오셀타미비르 저항성 인플루엔자 바이러스가 존재한다고 판단하는 단계를 포함한다.In another embodiment of the present invention, the method of the present invention, 1) a sample isolated from the subject and oseltamivir hexylthiol represented by the formula (2) or oseltamivir hexylamine represented by the formula (3) and Contacting; 2) If a color different from the color that appears when the nanoparticle is contacted with a sample without oseltamivir resistant influenza virus appears in the sample of step 1), it is determined that oseltamivir resistant influenza virus is present in the sample. It includes a step.
상기 대상체는 인간 또는 그 밖의 동물, 예컨대 조류 또는 포유류가 될 수 있다.The subject may be a human or other animal, such as a bird or a mammal.
상기 시료는 전혈, 혈청, 혈장, 혈액 세포, 내피 세포, 조직 생검, 림프액, 복수액, 간질액, 골수, 뇌척수액 (CSF), 정액, 타액, 점액, 객담, 땀 또는 소변일 수 있다.The sample may be whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsy, lymph, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid (CSF), semen, saliva, mucus, sputum, sweat or urine.
본 발명은 상기 나노입자를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스 검출용 키트를 제공한다. 본 발명의 키트는 기타 검출에 필요한 물품들을 더 포함할 수 있으며, 사용 지침(instruction)을 더 포함할 수 있다. The present invention provides a kit for detecting oseltamivir resistant influenza virus comprising the nanoparticles. The kit of the present invention may further include other items necessary for detection, and may further include instructions for use.
본 발명의 검출용 키트는 다양한 형태로 구현 가능하다. 예를 들어 검출 용액 키트, 래피드 키트(rapid kit)(랩온어칩을 이용하여 키트에 소량의 시료를 떨어뜨리면 시료가 키트 상에서 이동하여 신속하게 결과를 확인할 수 있는 키트) 또는 종이 스틱의 형태로 구현할 수 있다. The detection kit of the present invention can be implemented in various forms. For example, a detection solution kit, a rapid kit (a small amount of sample dropped into the kit using a lab-on-a-chip), or the sample can be moved on the kit to quickly check the results. Can be.
본 발명의 일 구현예로서, 본 발명의 검출용 키트는 진단 스트립(strip) 형태의 래피드 진단 키트가 될 수 있다. In one embodiment of the present invention, the detection kit of the present invention may be a rapid diagnostic kit in the form of a diagnostic strip.
예를 들어, 본 발명의 검출용 키트는 시료패드(sample pad), 컨주게이트 패드(conjugate pad), 니트로셀룰로오스 막(nitrocelluose membrane) 및 흡착패드(adsorption pad)를 구비하고 있으며, For example, the detection kit of the present invention includes a sample pad, a conjugate pad, a nitrocelluose membrane, and an adsorption pad.
상기 컨주게이트 패드는 오셀타미비르 헥실티올-금나노입자 또는 오셀타미비르 헥실아민-금나노입자가 담지되고, The conjugate pad is supported by oseltamivir hexylthiol-gold nanoparticles or oseltamivir hexylamine-gold nanoparticles,
시료패드를 통해 시료 전개시 상기 컨주게이트 패드에 담지된 오셀타미비르 헥실티올-금나노입자 또는 오셀타미비르 헥실아민-금나노입자는 시료와 함께 니트로셀룰로오스 막 및 흡착패드로 전개되고, When the sample is developed through the sample pad, the oseltamivir hexylthiol-gold nanoparticles or oseltamivir hexylamine-gold nanoparticles supported on the conjugate pad are developed together with the sample into the nitrocellulose membrane and the adsorption pad.
상기 니트로셀룰로오스 막에는 컨트롤 라인(control line) 및 테스트 라인(test ine)이 격리되어 구비되어 있고, The nitrocellulose membrane is provided with a control line and a test ine separated from each other.
여기에서 상기 컨트롤 라인은 H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스 또는 H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스의 뉴라미니데이즈가 고정되어 있고, 상기 테스트 라인은 화학식 2로 기재되는 오셀타미비르 헥실티올 또는 화학식 3으로 기재되는 오셀타미비르 헥실아민이 고정되어 있는 것일 수 있다. Wherein the control line is fixed neuramiminids of oseltamivir resistant influenza virus comprising H275Y mutation or oseltamivir resistant influenza virus comprising H275Y mutation, and the test line is oseltamivir represented by Formula 2 Hexylthiol or oseltamivir hexylamine represented by Formula 3 may be fixed.
본 발명은 상기 검출용 키트를 이용하여 오셀타미비르 저항성 인플루엔자 바이러스를 검출하는 방법을 제공한다. The present invention provides a method for detecting oseltamivir resistant influenza virus using the detection kit.
본 발명의 일 구현예로서, 상기 방법은 In one embodiment of the invention, the method is
1) 상기 검출용 키트의 컨주게이션 패드에 시료를 투입하여 전개시키는 단계;및1) putting a sample into the conjugation pad of the detection kit and deploying it; and
2) 상기 검출용 키트의 니트로셀룰로오스 막의 컨트롤 라인 및 테스트 라인으로부터 색상 변화를 확인하는 단계를 포함할 수 있다. 2) checking the color change from the control line and the test line of the nitrocellulose membrane of the detection kit.
본 발명의 방법에 있어서, 시료 내에 오셀타미비르 저항성 인플루엔자 바이러스가 존재하지 않는 경우 컨트롤 라인에서만 색상 변화가 나타나나, 존재하는 경우 컨트롤 라인 및 테스트 라인에서 모두 색상 변화가 나타난다.In the method of the present invention, the color change appears only in the control line when the oseltamivir resistant influenza virus is not present in the sample, but the color change appears in both the control line and the test line when present.
본 발명의 방법에 있어서, 테스트 라인의 색상 변화의 정도가 크면 시료 내 포함된 오셀타미비르 저항성 인플루엔자 바이러스의 양이 많다고 판단할 수 있다.In the method of the present invention, if the degree of color change of the test line is large, it can be determined that the amount of oseltamivir resistant influenza virus contained in the sample is large.
대상체로부터 수득한 시료에 화학식 1로 기재되는 오셀타미비르 유도체 화합물이 결합된 오셀타미비르 저항성 인플루엔자 바이러스 검출용 나노입자를 처리하는 단계;Treating the oseltamivir resistant influenza virus detection nanoparticles to which the oseltamivir derivative compound of Formula 1 is bound to a sample obtained from the subject;
상기 시료로부터 H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스를 확인하는 단계; 및 Identifying an oseltamivir resistant influenza virus comprising a H275Y mutation from the sample; And
오셀타미비르 저항성 인플루엔자 바이러스가 검출되지 않거나 미량의 오셀타미비르 저항성 인플루엔자 바이러스를 포함하는 시료를 가진 대상체에 오셀타미비르 포스페이트의 치료학적 유효량을 투여하는 단계를 포함하는, 인플루엔자의 치료 방법을 제공한다. A method of treating influenza, comprising administering a therapeutically effective amount of oseltamivir phosphate to a subject for which no oseltamivir resistant influenza virus is detected or containing a trace amount of oseltamivir resistant influenza virus.
상기에서 “오셀타미비르 저항성 인플루엔자 바이러스가 검출되지 않거나 미량의 오셀타미비르 저항성 인플루엔자 바이러스를 포함하는 시료”는 오셀타미비르 저항성 인플루엔자 바이러스가 없거나 또는 오셀타미비르 포스페이트가 대상체에서 치료 효과를 나타낼 수 있는 수준의 미량의 오셀타미비르 저항성 인플루엔자 바이러스를 포함한 상태를 의미한다.The above "sample without oseltamivir resistant influenza virus or containing a trace amount of oseltamivir resistant influenza virus" is free of oseltamivir resistant influenza virus or the level at which oseltamivir phosphate may have a therapeutic effect in a subject. Refers to a condition containing a small amount of oseltamivir resistant influenza virus.
상기에서 "치료학적 유효량"은 임의의 의학적 치료에 적용가능한 합리적인 오셀타미비르 포스페이트 투여량로, 대상의 모집단에 투여될 때 특정한 치료학적 효과와 통계학적으로 관련 있는 치료제의 양을 의미한다.As used herein, "therapeutically effective amount" is a reasonable oseltamivir phosphate dosage applicable to any medical treatment, and refers to the amount of therapeutic agent that is statistically related to a particular therapeutic effect when administered to a population of subjects.
본 발명의 검출용 키트, 이를 이용한 검출 방법 및 치료 방법은 상술한 바에 제한되지 않는다.The detection kit of the present invention, the detection method and the treatment method using the same are not limited to the above.
기존에 개발되어 있던 방법의 경우 특이적 항체의 경우 열과 pH에 약하여 유통과 사용에 제한이 있고, 유전자 분석의 경우 검출 기기가 필요하나, 본 발명의 경우 화합물을 이용하기 때문에 온도가 높은 지역에서도 사용할 수 있고 장기간 보관이 가능하며 여러 형태로 제품화할 수 있다는 장점이 있다. In the case of the conventionally developed methods, specific antibodies are weak in heat and pH, which limits their distribution and use, and in the case of genetic analysis, a detection device is required. It can be stored for a long time and can be commercialized in various forms.
본 발명의 오셀타미비르 유사체 및 상기 유사체가 결합된 나노입자는 오셀타미비르 저항성 인플루엔자 바이러스에 강하게 결합하므로, 이를 오셀타미비르 저항성 인플루엔자 바이러스를 육안으로 빠르고 편리하게 검출할 수 있다. 따라서 신속하게 인플루엔자 바이러스에 감염된 환자의 치료 계획을 세우는데 유용하게 활용될 수 있다. The oseltamivir analogs of the present invention and the nanoparticles to which the analogs bind are strongly bound to the oseltamivir resistant influenza virus, so that the oseltamivir resistant influenza virus can be detected quickly and conveniently with the naked eye. Therefore, it can be useful to plan treatment of patients who are infected with influenza virus quickly.
도 1은 바이러스 수준에서 오셀타미비르 헥실아민 및 오셀타미비르 헥실티올이 오셀타미비르 감수성 바이러스와 오셀타미비르 저항성 바이러스의 뉴라미니데이즈 효소 활성을 억제하는 정도를 확인한 것이다. Figure 1 confirms the degree of oseltamivir hexylamine and oseltamivir hexylthiol inhibit the neuraminidase enzyme activity of oseltamivir-sensitive and oseltamivir resistant virus at the virus level.
도 2는 오셀타미비르 유사체-금나노입자를 오셀타미비르 감수성 바이러스와 오셀타미비르 저항성 바이러스에 각각 처리하고 흡광도를 관찰한 결과를 나타낸 그래프이다.FIG. 2 is a graph showing the results obtained by treating oseltamivir analogues-gold nanoparticles with oseltamivir-sensitive virus and oseltamivir-resistant virus, respectively, and monitoring the absorbance.
도 3은 본 발명의 오셀타미비르 헥실아민 및 오셀타미비르 헥실티올과 오셀타미비르 감수성 바이러스 및 오셀타미비르 저항성 바이러스의 결합 양상을 나타낸 모식도이다. 좌측 하단에 화학식을 나타낸 오셀타미비르 헥실티올의 경우, △Gbind가 오셀타미비르 저항성 바이러스에 약 250배 더 높게 나타났다. Figure 3 is a schematic diagram showing the binding mode of oseltamivir hexylamine and oseltamivir hexylthiol and oseltamivir-sensitive virus and oseltamivir resistant virus of the present invention. In the case of oseltamivir hexylthiol represented by the formula at the bottom left, ΔG bind was about 250 times higher for oseltamivir resistant virus.
도 4는 오셀타미비르 헥실티올이 결합된 금나노입자를 오셀타미비르 감수성 바이러스와 오셀타미비르 저항성 바이러스와 각각 반응시켰을 때 흡광도를 확인한 그래프(좌측) 및 색상 차이를 육안으로 확인한 도(우측 상단) 및 이를 그래프로 나타낸 것(우측 하단)이다. 4 is a graph (left) and visually confirmed the difference in color (left) and a visual confirmation of absorbance when reacting oseltamivir hexylthiol-coupled gold nanoparticles with oseltamivir-sensitive virus and oseltamivir-resistant virus, respectively (upper right) And it is represented graphically (bottom right).
도 5는 오셀타미비르 헥실티올이 결합된 나노입자를 오셀타미비르 감수성 바이러스와 오셀타미비르 저항성 바이러스에 각각 처리하고 흡광도를 관찰한 결과를 나타낸 그래프이다. FIG. 5 is a graph showing the results obtained by treating oseltamivir hexylthiol-bound nanoparticles with oseltamivir-sensitive virus and oseltamivir-resistant virus, respectively, and monitoring the absorbance.
도 6은 본 발명의 오셀타미비르 유사체-금나노 입자를 이용해 제조한 항바이러스제 저항성 바이러스 검출을 위한 래피드 키트 및 구성에 대한 모식도이다. Figure 6 is a schematic diagram of the rapid kit and configuration for the detection of antiviral resistant virus prepared using the oseltamivir analog-gold nanoparticles of the present invention.
도 7은 본 발명의 래피드 키트 진단 스트립 상에서의 항바이러스 감수성/저항성 바이러스 검출을 나타낸 결과이다. 7 shows the results of antiviral susceptible / resistive virus detection on the rapid kit diagnostic strip of the present invention.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.
실시예 1. 나노입자의 제조Example 1. Preparation of Nanoparticles
1-1. 오셀타미비르 헥실티올(Oseltamivir Hexylthiol) 합성 1-1. Oseltamivir Hexylthiol Synthesis
오셀타미비르 헥실티올을 하기 scheme으로 합성하였다. 이하에서 화합물명 다음에 괄호 내에 표시한 숫자는 하기 scheme에서 화합물 하단에 표시한 숫자를 의미한다. Oseltamivir hexylthiol was synthesized with the following scheme. Hereinafter, the numbers indicated in parentheses after the compound names refer to numbers indicated at the bottom of the compound in the following scheme.
Figure PCTKR2016007191-appb-I000006
Figure PCTKR2016007191-appb-I000006
S-6-Hydroxyhexyl ethanethioate (2) 합성S-6-Hydroxyhexyl ethanethioate (2) Synthesis
실온에서 1-bromohexanol (1) (5.00 g, 27.6 mmol)의 DMF (50 mL) 용액을 교반하면서 potassium ethanethioate (6.31 g, 55.2 mmol)을 천천히 적가했다. 반응 혼합물을 12시간 동안 교반한 후 증류수 (30 mL)로 희석했다. 혼합물을 Et2O(3 x 30mL)로 추출하고, 유기 층을 합쳐 무수 MgSO4로 건조, 여과한 후 농축했다. 농축액을 컬럼 크로마토그래피 (Hexanes:EtOAc = 2:1 - 1:1)를 이용하여 분리해서 연한 노란색 액체의 화합물 2 (4.20 g, 86%)를 얻었다. Potassium ethanethioate (6.31 g, 55.2 mmol) was slowly added dropwise while stirring a solution of DMF (50 mL) of 1-bromohexanol (1) (5.00 g, 27.6 mmol) at room temperature. The reaction mixture was stirred for 12 hours and then diluted with distilled water (30 mL). The mixture was extracted with Et 2 O (3 × 30 mL), the organic layers were combined, dried over anhydrous MgSO 4 , filtered and concentrated. The concentrate was separated using column chromatography (Hexanes: EtOAc = 2: 1-1: 1) to give compound 2 as a pale yellow liquid. (4.20 g, 86%) was obtained.
(3R,5S)-Ethyl4-acetamido-5-(tert-butoxycarbonylamino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate (4) 합성(3R, 5S) -Ethyl4-acetamido-5- (tert-butoxycarbonylamino) -3- (pentan-3-yloxy) cyclohex-1-enecarboxylate (4) Synthesis
실온에서 Oseltamivir phosphate salt (3) (10.0 g, 24.4 mmol)의 MeOH (50 mL) 용액을 교반하면서 di-tert-butyl dicarbonate (7.84 mL, 34.1 mmol)과 triethylamine (6.80 mL, 48.8 mmol)을 적가했다. 반응 혼합물을 실온에서 12시간 동안 교반했다. 혼합물에 증류수 (100 mL)를 가해서 1시간 동안 교반한 후, 생성된 흰색 고체를 여과하고 증류수로 세척했다. 여과물을 진공 오븐에서 건조하여 흰색 고체의 화합물 4 (5.71 g, 56%)를 얻었다.Di-tert-butyl dicarbonate (7.84 mL, 34.1 mmol) and triethylamine (6.80 mL, 48.8 mmol) were added dropwise while stirring a solution of MeOH (50 mL) of Oseltamivir phosphate salt (3) (10.0 g, 24.4 mmol) at room temperature. . The reaction mixture was stirred at rt for 12 h. Distilled water (100 mL) was added to the mixture, followed by stirring for 1 hour. The resulting white solid was filtered and washed with distilled water. The filtrate was dried in a vacuum oven to give compound 4 (5.71 g, 56%) as a white solid.
(3R,5S)-4-acetamido-5-(tert-butoxycarbonylamino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylic acid (5) 합성(3R, 5S) -4-acetamido-5- (tert-butoxycarbonylamino) -3- (pentan-3-yloxy) cyclohex-1-enecarboxylic acid (5) Synthesis
실온에서 화합물 4 (5.70 g, 13.8 mmol)의 THF/H2O(10:1, v/v, 30mL)용액을 교반하면서 NaOH (663 mg, 16.6 mmol)을 가했다. 반응 혼합물을 24시간 동안 교반한 후, 농축하여 반응 용매를 제거했다. 농축액을 증류수 (20 mL)로 희석하고, 반응 용기를 0 ℃로 냉각했다. 혼합물에 1 M HCl 수용액을 가하여 pH 5까지 산성화한 후, 1시간 동안 교반했다. 생성된 흰색 고체를 여과하고 증류수로 세척했다. 여과물을 진공 오븐에서 건조하여 흰색 고체의 화합물 5 (4.0 g, 75%)를 얻었다. NaOH (663 mg, 16.6 mmol) was added while stirring a solution of Compound 4 (5.70 g, 13.8 mmol) in THF / H 2 O (10: 1, v / v, 30 mL) at room temperature. 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 reaction vessel was cooled to 0 ° C. An aqueous 1 M HCl solution was added to the mixture, acidified to pH 5, and stirred for 1 hour. The resulting white solid was filtered and washed with distilled water. The filtrate was dried in a vacuum oven to give compound 5 (4.0 g, 75%) as a white solid.
(3R,5S)-6-(Acetylthio)hexyl-4-acetamido-5-(tert-butoxycarbonylamino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate (6) 합성(3R, 5S) -6- (Acetylthio) hexyl-4-acetamido-5- (tert-butoxycarbonylamino) -3- (pentan-3-yloxy) cyclohex-1-enecarboxylate (6) Synthesis
실온에서 화합물 5 (4.00 g, 10.4 mmol)의 CH2Cl2(30mL)용액을 교반하면서 화합물 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)과 triethylamine (2.90 mL, 20.8 mmol)을 순차적으로 가했다. 실온에서 24시간 동안 교반한 후, 반응혼합물에 증류수를 가해서 반응을 종결했다. 혼합물을 CH2Cl2(3x30mL)로 추출하고, 유기 층을 합쳐 무수 Na2SO4 로 건조, 여과한 후 농축했다. 농축액을 컬럼 크로마토그래피 (Hexanes:EtOAc = 2:1 - 1:1)를 이용하여 분리하여 무색 액체의 화합물 6 (3.51 g, 62%)을 얻었다.Compound 2 (2.20 g, 12.5 mmol), 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride (2.79) while stirring a solution of CH 2 Cl 2 (30 mL) in compound 5 (4.00 g, 10.4 mmol) at room temperature. g, 14.6 mmol), 4- (dimethylamino) pyridine (1.52 g, 12.5 mmol) and triethylamine (2.90 mL, 20.8 mmol) were added sequentially. After stirring for 24 hours at room temperature, distilled water was added to the reaction mixture to terminate the reaction. The mixture was extracted with CH 2 Cl 2 (3 × 30 mL), the organic layers were combined, dried over anhydrous Na 2 SO 4 , filtered and concentrated. The concentrate was separated using column chromatography (Hexanes: EtOAc = 2: 1-1: 1) to give compound 6 (3.51 g, 62%) as a colorless liquid.
Oseltamivir Hexylthiol (7) 합성Oseltamivir Hexylthiol (7) Synthesis
실온에서 화합물 6 (3.50 g, 6.45 mmol)의 MeOH (30 mL) 용액을 교반하면서 진한 염산 (2.15 mL, 25.8 mmol)을 천천히 적가했다. 반응 용기를 50 ℃에서 72시간 동안 가열했다. 반응혼합물을 실온으로 냉각한 후 농축하여 반응 용매를 제거했다. 농축액을 MeOH (5 mL)로 희석하고 교반하면서 Et2O를 천천히 가하였다. 생성된 흰색 고체를 여과하고 Et2O로 세척했다. 여과물을 진공 오븐에서 건조하여 흰색 고체의 화합물 7(오셀타미비르 헥실티올) (560 mg, 20%)을 얻었다.A concentrated hydrochloric acid (2.15 mL, 25.8 mmol) was slowly added dropwise while stirring a solution of MeOH (30 mL) of compound 6 (3.50 g, 6.45 mmol) at room temperature. The reaction vessel was heated at 50 ° C. for 72 hours. The reaction mixture was cooled to room temperature and concentrated to remove the reaction solvent. The concentrate was diluted with MeOH (5 mL) and slowly added Et 2 O with stirring. The resulting white solid was filtered and washed with Et 2 O. The filtrate was dried in a vacuum oven to give white solid compound 7 (oseltamivir hexylthiol) (560 mg, 20%).
상기 오셀타미비르 헥실티올은 하기 화학식 2로 표시된다. The oseltamivir hexylthiol is represented by the following formula (2).
[화학식 2][Formula 2]
Figure PCTKR2016007191-appb-I000007
Figure PCTKR2016007191-appb-I000007
1-2. 오셀타미비르 헥실아민 (Oseltamivir Hexylamine) 합성1-2. Oseltamivir Hexylamine Synthesis
오셀타미비르 헥실아민을 하기 scheme으로 합성하였다. 이하에서 화합물명 다음에 괄호 내에 표시한 숫자는 하기 scheme에서 화합물 하단에 표시한 숫자를 의미한다. Oseltamivir hexylamine was synthesized with the following scheme. Hereinafter, the numbers indicated in parentheses after the compound names refer to numbers indicated at the bottom of the compound in the following scheme.
Figure PCTKR2016007191-appb-I000008
Figure PCTKR2016007191-appb-I000008
tert-Butyl 6-hydroxyhexylcarbamate (9) 합성tert-Butyl 6-hydroxyhexylcarbamate (9) Synthesis
실온에서 화합물 8 (3.00 g, 25.6 mmol)의 MeOH (30 mL) 용액을 교반하면서 di-tert-butyl dicarbonate (6.47 mL, 28.2 mmol)를 적가했다. 반응혼합물을 실온에서 6시간 동안 교반한 후 농축했다. 농축액을 column chromatography (Hexanes:EtOAc = 2:1 - 1:1)를 이용하여 분리해서 연한 노란색 액체의 화합물 9 (4.10 g, 74%)를 얻었다.Di-tert-butyl dicarbonate (6.47 mL, 28.2 mmol) was added dropwise while stirring a solution of MeOH (30 mL) of compound 8 (3.00 g, 25.6 mmol) at room temperature. The reaction mixture was stirred at room temperature for 6 hours and then concentrated. The concentrate was separated using column chromatography (Hexanes: EtOAc = 2: 1-1: 1) to give compound 9 (4.10 g, 74%) as a pale yellow liquid.
(3R,5S)-6-(tert-Butoxycarbonylamino)hexyl-4-acetamido-5-(tert-butoxycarbonylamino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate (10) 합성(3R, 5S) -6- (tert-Butoxycarbonylamino) hexyl-4-acetamido-5- (tert-butoxycarbonylamino) -3- (pentan-3-yloxy) cyclohex-1-enecarboxylate (10) Synthesis
실온에서 화합물 5 (3.50 g, 9.10 mmol)의 DMF (20 mL) 용액을 교반하면서 화합물 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)과 triethylamine (2.54 mL, 18.2 mmol)을 순차적으로 가했다. 실온에서 24시간 동안 교반한 후, 반응혼합물에 증류수를 가해서 반응을 종결했다. 혼합물을 EtOAc (3 x 20 mL)로 추출하고, 유기 층을 합쳐 무수 Na2SO4로 건조, 여과한 후 농축했다. 농축액을 컬럼 크로마토그래피(Hexanes:EtOAc = 2:1 - 1:1)를 이용하여 분리하여 연한 노란색 액체의 화합물 10 (2.41 g, 45%)을 얻었다.Compound 9 (2.18 g, 10.0 mmol), 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride (2.44 g, while stirring a solution of DMF (20 mL) of compound 5 (3.50 g, 9.10 mmol) at room temperature 12.7 mmol), 4- (dimethylamino) pyridine (1.33 g, 10.9 mmol) and triethylamine (2.54 mL, 18.2 mmol) were added sequentially. After stirring for 24 hours at room temperature, distilled water was added to the reaction mixture to terminate the reaction. The mixture was extracted with EtOAc (3 × 20 mL), the combined organic layers were dried over anhydrous Na 2 SO 4 , filtered and concentrated. The concentrate was separated using column chromatography (Hexanes: EtOAc = 2: 1-1: 1) to give compound 10 (2.41 g, 45%) as a pale yellow liquid.
Oseltamivir Hexylamine (11) 합성Oseltamivir Hexylamine (11) Synthesis
실온에서 화합물 10 (2.45 g, 4.20 mmol)의 MeOH (20 mL) 용액을 교반하면서 진한 염산 (1.75 mL, 21.0 mmol)을 천천히 적가했다. 실온에서 24시간 동안 교반한 후, 반응혼합물을 농축하여 반응 용매를 제거했다. 농축액을 MeOH (5 mL)로 희석하고 교반하면서 Et2O를 천천히 가하였다. 생성된 흰색 고체를 여과하고 Et2O로 세척했다. 여과물을 진공 오븐에서 건조하여 흰색 고체의 화합물 11(오셀타미비르 헥실아민)(1.15 g, 65%)을 얻었다. Concentrated hydrochloric acid (1.75 mL, 21.0 mmol) was slowly added dropwise while stirring a solution of MeOH (20 mL) of compound 10 (2.45 g, 4.20 mmol) at room temperature. After stirring for 24 hours at room temperature, the reaction mixture was concentrated to remove the reaction solvent. The concentrate was diluted with MeOH (5 mL) and slowly added Et 2 O with stirring. The resulting white solid was filtered and washed with Et 2 O. The filtrate was dried in a vacuum oven to give a white solid Compound 11 (Oseltamivir hexylamine) (1.15 g, 65%).
상기 오셀타미비르 헥실아민은 하기 화학식 3으로 표시된다.The oseltamivir hexylamine is represented by the following formula (3).
[화학식 3] [Formula 3]
Figure PCTKR2016007191-appb-I000009
Figure PCTKR2016007191-appb-I000009
1-3. 금나노입자 합성 1-3. Gold Nanoparticle Synthesis
100 mL의 증류수에 1 wt%의 HAuCl4 용액 (1 mL)를 넣고 95 oC에서 강렬하게 교반시켰다. 그 상태에서, 즉시 1 wt%의 Sodium citrate (5 mL)를 천천히 주입하고, 30분간 같은 조건에서 반응시켰다. 1 wt% of HAuCl 4 solution (1 mL) was added to 100 mL of distilled water and stirred vigorously at 95 ° C. In that state, 1 wt% of sodium citrate (5 mL) was immediately injected slowly and reacted under the same conditions for 30 minutes.
1-4. 나노입자-오셀타미비르 유사체 결합 1-4. Nanoparticle-Oseltamivir analog binding
1-3에서 합성된 금나노입자 용액의 5 mL을 15000 rpm, 10분간 원심분리하여 과량의 sodium citrate를 제거하였다. 상층액은 버리고, 1 mL의 증류수로 재 분산한 뒤, 15000 rpm, 10분간 원심분리하였다. 해당 과정을 2회 더 반복하였다. 마지막 단계에서 상층액은 버리고, 증류수에 분산된 오셀타미비르 헥실아민 또는 오셀타미비르 헥실티올(6 mg/mL) 1 mL 를 첨가하였다. 그리고, 12시간 이상 voltexing 하여, 금나노입자에 결합되도록 하였다. 그 다음 15000 rpm, 20분간 원심분리하고 상층액을 제거한 후, 증류수 100 μL에 재분산 시켰다. Excess sodium citrate was removed by centrifuging 5 mL of the gold nanoparticle solution synthesized in 1-3 at 15000 rpm for 10 minutes. The supernatant was discarded, redispersed in 1 mL of distilled water, and then centrifuged at 15000 rpm for 10 minutes. This process was repeated two more times. In the last step, the supernatant was discarded and 1 mL of oseltamivir hexylamine or oseltamivir hexylthiol (6 mg / mL) dispersed in distilled water was added. And, by voltexing for more than 12 hours, to be bonded to the gold nanoparticles. After centrifugation at 15000 rpm for 20 minutes, the supernatant was removed, and redispersed in 100 μL of distilled water.
실시예 2. 바이러스에서의 뉴라미니데이즈 활성 비교Example 2. Comparison of Neuraminidase Activity in Virus
2-1. 오셀타미비르 오셀타미비르 헥실티올과 오셀타미비르 헥실아민이 바이러스에서 뉴라미니데이즈 효소 활성에 미치는 영향 비교2-1. Comparison of Effects of Oseltamivir Oseltamivir Hexylthiol and Oseltamivir Hexylamine on Neuraminidase Enzyme Activity in Virus
NA-FluorTM Influenza Neurmaminidase Assay Kit (AB Applied biosystem, Prod No. 4457091)를 이용하여 NA 효소 활성도를 측정하였다. NA enzyme activity was measured using NA-FluorTM Influenza Neurmaminidase Assay Kit (AB Applied biosystem, Prod No. 4457091).
구체적으로, Kit 내의 working solution (5.52 mL)에 Na-fluor (480 μL)를 녹여 용액 A를 준비하였다. 바이러스 용액은 각 well 당 각각 100개, 또는 1000개의 바이러스가 들어 있도록 준비하였다. wild type (항 바이러스제 감수성 바이러스)은 pandemic H1N1 virus (A/04/2009/California) (pandemic H1N1), mutant type (항 바이러스제 저항성 바이러스)은 Influenza A/Korea/2785/2009 (H275Y mutation)를 사용하였다. 2 mL의 증류수에 오셀타미비르 헥실아민 또는 오셀타미비르 헥실티올을 19.3 mg을 녹여 준비하였다(용액 C). 96 well plate의 각 well에 용액 A를 50 μL 넣고, 각각 바이러스 시료 용액을 넣었다. 용액 C를 농도를 달리하여 준비된 well 에 50 μL 씩 첨가하였다(비교군에서는 증류수만 첨가). 즉, 각 well 당 용액 A+각 바이러스 용액(wild type 또는 mutant type)+용액 C를 넣었다. 37℃에서 1시간 배양한 뒤, 각 well 에 Na-flour stop buffer 용액을 50μL 씩 넣었다. Ex: 360 nm, Em: 450 nm 으로 형광을 측정하여 단백질 수준에서의 NA 활성도를 측정하였다. NA 효소 활성도가 높을수록 형광 강도(intensity)가 높음을 의미한다.Specifically, Solution A was prepared by dissolving Na-fluor (480 μL) in a working solution (5.52 mL) in the kit. Virus solutions were prepared to contain 100 or 1000 viruses per well, respectively. The wild type (antiviral susceptible virus) used pandemic H1N1 virus (A / 04/2009 / California) (pandemic H1N1), and the mutant type (infectious virus resistant virus) used Influenza A / Korea / 2785/2009 (H275Y mutation). . Dissolve 19.3 mg of oseltamivir hexylamine or oseltamivir hexylthiol in 2 mL of distilled water (solution C). 50 μL of Solution A was added to each well of a 96 well plate, and a virus sample solution was added to each well. Solution C was added to each well prepared at different concentrations by 50 μL (only distilled water was added in the comparison group). That is, solution A + each virus solution (wild type or mutant type) + solution C was added to each well. After incubation at 37 ° C. for 1 hour, 50 μL of Na-flour stop buffer solution was added to each well. Fluorescence was measured at Ex: 360 nm and Em: 450 nm to determine NA activity at the protein level. Higher NA enzyme activity means higher fluorescence intensity.
결과는 도 1에 나타내었다. 도 1의 좌측 도(오셀타미비르 헥실아민) 및 우측 도(오셀타미비르 헥실티올)에서 고농도 조건에서 오셀타미비르 헥실아민 및 오셀타미비르 헥실티올이 Wild type에서 NA 효소의 활성보다 Mutant type (H275Y mutation) 에서 NA 효소 활성을 훨씬 더 큰 정도로 억제함을 알 수 있다. The results are shown in FIG. In the left (Oseltamivir hexylamine) and right (Oseltamivir hexylthiol) of Fig. 1, oseltamivir hexylamine and oseltamivir hexylthiol at high concentration conditions than the activity of NA enzyme in the wild type (H275Y mutations inhibit NA enzyme activity to a much greater extent.
2-2. 오셀타미비르 유사체-금나노입자를 바이러스의 뉴라미니데이즈 단백질에 처리한 뒤 흡광 변화 확인2-2. Treatment of oseltamivir analogues-gold nanoparticles with the neuraminidase protein of the virus followed by changes in absorbance
96 well plate의 각 well 당 상기 wild type 또는 상기 mutant type에서 분리한 뉴라미니데이즈 단백질이 0.1 mg (100 μg)/well 로 들어 있도록 준비하고, 각 well에 상기 1-4에서 합성한 오셀타미비르 헥실티올이 결합된 금나노입자를 100 μL (금 이온 농도 기준 1.22 mg) 을 첨가한 뒤, 흡광도를 400-750 nm 범위에서 측정하였다. Each well of a 96 well plate was prepared to contain 0.1 mg (100 μg) / well of neuraminidase protein isolated from the wild type or the mutant type, and oseltamivir hexyl synthesized in 1-4 in each well. After adding 100 μL (1.22 mg of gold ion concentration) to the thiol-bound gold nanoparticles, the absorbance was measured in the 400-750 nm range.
결과는 도 2에 나타내었다. 도 2에서 mutant type (점선)에 대한 흡광도 파장이 wild type (실선) 보다 더 길게 나타나, 오셀타미비르 헥실티올이 결합된 금나노입자가 wild type과 mutant type의 뉴라미니데이즈 단백질에 대해 서로 상이한 흡광도 파장을 보임을 알 수 있다. The results are shown in FIG. In FIG. 2, the absorbance wavelength of the mutant type (dotted line) is longer than that of the wild type (solid line). It can be seen that the wavelength.
실시예 3. 오셀타미비르 유사체와 오셀타미비르 저항성 바이러스의 결합력 확인Example 3 Confirmation of Binding Force between Oseltamivir Analogues and Oseltamivir Resistant Viruses
오셀타미비르 감수성 바이러스(wild type) 및 오셀타미비르 저항성 바이러스(mutant type)의 뉴라미니데이즈 부위와 본 발명의 오셀타미비르 헥실티올의 결합 에너지(binding energy, △Gbind)를 계산하였다.The binding energy (ΔG bind ) of the neuramiminidase site of oseltamivir-sensitive virus (wild type) and oseltamivir resistant virus (mutant type) and oseltamivir hexylthiol of the present invention was calculated.
그 결과, 오셀타미비르 헥실티올의 wild type에 대한 결합 에너지는 -24.33 kcal/mol로 나타났으나, mutant type에 대한 결합 에너지는 -27.62 kcal/mol로 나타났다(도 3의 모식도 참조). 이는 오셀타미비르 헥실티올가 wild type보다 mutant type에 약 250배 높은 결합력으로 결합함을 의미한다. As a result, the binding energy of oseltamivir hexylthiol to wild type was -24.33 kcal / mol, but the binding energy to mutant type was -27.62 kcal / mol (refer to the schematic diagram of FIG. 3). This means that oseltamivir hexylthiol binds to the mutant type about 250 times higher than the wild type.
실시예 4. 오셀타미비르 유사체-금나노입자를 이용한 항바이러스제 저항성 바이러스 검출 시스템 개발 및 효능 평가 Example 4. Development and efficacy evaluation of antiviral resistant virus detection system using oseltamivir analogues-gold nanoparticles
본 발명의 오셀타미비르 유사체-나노입자는 오셀타미비르 저항성 바이러스에 강한 결합력으로 결합하여 상기 나노입자를 오셀타미비르 저항성 바이러스에 가하면 색상 변화가 일어나므로 육안으로 확인할 수 있게 된다. The oseltamivir analogue-nanoparticle of the present invention binds to the oseltamivir resistant virus with strong binding force, and thus the color change occurs when the nanoparticle is added to the oseltamivir resistant virus.
96 well plate의 각 well 당 바이러스가 0개, 10개, 100개, 또는 1000개의 바이러스가 들어 있도록 준비하였다. wild type (항 바이러스제 감수성 바이러스)은 pandemic H1N1 virus (A/04/2009/California) (pandemic H1N1), mutant type (항 바이러스제 저항성 바이러스)는 Influenza A/Korea/2785/2009 (H275Y mutation)을 사용하였다. 준비된 각 well 당 실시예 1에서 제조한 오셀타미비르 헥실티올-금나노입자 100 μL (금 이온 농도 기준으로 1.22 mg)을 첨가한 뒤, 흡광도를 400-750 nm 범위에서 측정하여 항바이러스제 감수성/저항성 바이러스를 색변화를 통하여 검출 하였다. Each well of a 96 well plate was prepared with 0, 10, 100, or 1000 viruses. The wild type (anti-viral susceptible virus) used pandemic H1N1 virus (A / 04/2009 / California) (pandemic H1N1), and the mutant type (anti-viral resistant virus) used Influenza A / Korea / 2785/2009 (H275Y mutation). . For each well prepared, 100 μL of oseltamivir hexylthiol-gold nanoparticles prepared in Example 1 (1.22 mg based on gold ion concentration) was added, and then the absorbance was measured in the range of 400-750 nm to determine antiviral sensitivity / resistance. Viruses were detected through color change.
결과는 도 4에 나타내었다. 도 4의 좌측 도에서 오셀타미비르 감수성 바이러스 (wild type) 수가 증가하여도 오셀타미비르 헥실티올-금나노입자의 흡광 파장은 거의 변화가 없었으나, 오셀타미비르 저항성 바이러스 (mutant type)의 수가 증가할수록 오셀타미비르 헥실티올-금나노 입자의 흡광 파장도는 이동하는 것을 알 수 있다. 이는 오셀타미비르 저항성 바이러스에 결합한 오셀타미비르 유사체-금나노입자가 증가하여 본래(original) 색으로부터 색 변화가 발생하여 나타난 현상으로서, 육안으로도 관찰할 수 있다(도 4의 우측 상단 도). 이를 도 4의 우측 하단에 그래프로 나타내었으며, 상기 그래프에서 흡광도의 파장이 이동함을 명확히 확인할 수 있다. The results are shown in FIG. Although the absorption wavelength of oseltamivir hexylthiol-gold nanoparticles was almost unchanged even though the number of oseltamivir-sensitive viruses (wild type) increased in the left figure of FIG. 4, the number of oseltamivir resistant viruses (mutant type) was increased. It can be seen that the absorption wavelength diagram of the oseltamivir hexylthiol-gold nanoparticles shifts as time goes by. This is a phenomenon caused by the increase in oseltamivir analogue-gold nanoparticles bound to oseltamivir resistant virus, resulting in a color change from the original color, which can also be observed with the naked eye (Fig. 4, upper right). This is shown in a graph on the lower right of Figure 4, it can be clearly seen that the wavelength of the absorbance shifts in the graph.
또한 상기 실험에서 wild type과 mutant type의 바이러스가 각각 well 당 1000개 포함된 경우의 흡광도 측정 결과를 도 5에 나타내었다. 도 5의 그래프 좌측 하단에서 육안으로 색 변화가 나타남을 확인할 수 있다(도 4와 색상이 다르게 나타난 것은 플레이트에 비춘 background light를 더 밝게 하여 촬영하였기 때문이다). In addition, the absorbance measurement results when 1000 wild-type and mutant-type viruses were included in each well in the experiment are shown in FIG. 5. In the lower left of the graph of Figure 5 it can be seen that the color change appears with the naked eye (the color is different from that of Figure 4 because the background light reflected on the plate was taken to lighter).
실시예 5. 오셀타미비르 유사체-금나노입자 기반 항바이러스제 저항성 바이러스 검출용 래피드(Rapid) 진단 키트 개발Example 5 Development of Rapid Diagnostic Kit for Detecting Oseltamivir Analogue-Gold Nanoparticle-Based Antiviral Resistant Virus
실시예 1에서 제조한 오셀타미비르 유사체-금 나노 입자를 이용해 오셀타미비르 저항성 인플루엔자 바이러스 검출용 래피드 키트를 제조하였다(도 6). 구체적으로, 오셀타미비르가 결합된 BSA(Bovine serum albumin)(테스트 라인) 및 H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스의 뉴라미니데이즈 단백질 3.3 mg/mL(컨트롤 라인)을 니트로셀룰로오스 막(nitrocellulose membrane)에 점적한 스트립을 4 mm× 50 mm로 준비하였다. 그 다음, 오셀타미비르 헥실티올-금나노입자를 컨주게이트 패드에 분주한 후 건조하여 준비하였다. 본 발명의 래피드 키트는 시료 패드(sample pad)를 통해 시료가 전개될 때 컨주게이션 패드의 오셀타미비르 헥실티올-금나노입자가 시료와 함께 전개되어 니트로셀룰로오스 막을 거쳐 흡착 패드로 흡수되는 구조로 되어 있다. Using the oseltamivir analogue-gold nanoparticles prepared in Example 1, a rapid kit for detecting oseltamivir resistant influenza virus was prepared (FIG. 6). Specifically, 3.3 mg / mL of the neuraminidase protein of oseltamivir resistant influenza virus containing a oseltamivir-bound BSA (test line) and an H275Y mutant was prepared using a nitrocellulose membrane. Membrane strips were prepared in 4 mm x 50 mm. Then, oseltamivir hexylthiol-gold nanoparticles were dispensed to the conjugate pad and dried to prepare. The rapid kit of the present invention has a structure in which the oseltamivir hexylthiol-gold nanoparticles of the conjugation pad are developed together with the sample and absorbed by the adsorption pad through the nitrocellulose membrane when the sample is developed through the sample pad. have.
실시예 6. 신규 선형 업컨버전 나노입자 기반 래피드(Rapid) 진단 키트를 이용한 오셀타미비르 저항성 인플루엔자 바이러스 검출Example 6 Oseltamivir Resistant Influenza Virus Detection Using a Novel Linear Upconversion Nanoparticle-Based Rapid Diagnostic Kit
실시예 5에서 제조한 래피드 키트를 이용해 오셀타미비르 감수성/저항성 인플루엔자 바이러스의 검출능을 확인하였다. 인플루엔자 바이러스가 포함되어 있지 않은 완충액(buffer)을 상기 래피드 키트에 시료(sample)로서 가하였을 때에는 컨트롤 라인에서만 선이 나타나고 테스트 라인에서는 선이 보이지 않았다. 또한 오셀타미비르 감수성 바이러스(wild type)가 포함된 완충액을 상기 래피드 키트에 가하였을 때에도 역시 컨트롤 라인에서만 선이 나타났다. 반면 오셀타미비르 저항성 바이러스(mutant type)가 포함된 완충액을 상기 래피드 키트에 가하였을 때에는 컨트롤 라인 및 테스트 라인에서 모두 선이 나타났다(도 7). 상기 결과로부터 본 발명의 오셀타미비르 유사체-금 나노 입자가 포함된 래피드 키트를 이용하여 오셀타미비르 저항성 인플루엔자 바이러스를 효율적으로 검출할 수 있음을 검증하였다. Using the rapid kit prepared in Example 5, the detection ability of oseltamivir-sensitive / resistive influenza virus was confirmed. When a buffer containing no influenza virus was added to the Rapid Kit as a sample, a line appeared only in the control line and no line in the test line. Also, when a buffer containing oseltamivir susceptible virus (wild type) was added to the rapid kit, lines also appeared only in the control line. On the other hand, when a buffer containing oseltamivir resistant virus (mutant type) was added to the rapid kit, lines appeared in both the control line and the test line (FIG. 7). From the above results, it was verified that the oseltamivir resistant influenza virus can be efficiently detected using the rapid kit including the oseltamivir analog-gold nanoparticles of the present invention.

Claims (13)

  1. 하기 화학식 1로 기재되는 오셀타미비르 유도체 화합물:Oseltamivir derivative compounds represented by Formula 1 below:
    [화학식 1][Formula 1]
    Figure PCTKR2016007191-appb-I000010
    Figure PCTKR2016007191-appb-I000010
    상기 화학식 1에서, In Chemical Formula 1,
    R1은 티올 또는 아민임.R 1 is thiol or amine.
  2. 제1항에 있어서, 상기 오셀타미비르 유도체는 하기 화학식 2로 기재되는 오셀타미비르 헥실티올(oseltamivir hexylthiol) 또는 하기 화학식 3으로 기재되는 오셀타미비르 헥실아민(oseltamivir hexylamine)인 오셀타미비르 유도체 화합물.The oseltamivir derivative compound of claim 1, wherein the oseltamivir derivative is oseltamivir hexylthiol represented by Formula 2 or oseltamivir hexylamine represented by Formula 3 below.
    [화학식 2][Formula 2]
    Figure PCTKR2016007191-appb-I000011
    Figure PCTKR2016007191-appb-I000011
    [화학식 3][Formula 3]
    Figure PCTKR2016007191-appb-I000012
    Figure PCTKR2016007191-appb-I000012
  3. 하기 화학식 1로 기재되는 오셀타미비르 유도체 화합물이 결합된, H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스 검출용 나노입자 :Nanoparticles for detecting oseltamivir resistant influenza virus comprising the H275Y mutation, to which the oseltamivir derivative compound represented by Formula 1 is bound:
    [화학식 1][Formula 1]
    Figure PCTKR2016007191-appb-I000013
    Figure PCTKR2016007191-appb-I000013
    상기 화학식 1에서, In Chemical Formula 1,
    R1은 티올 또는 아민임. R 1 is thiol or amine.
  4. 제3항에 있어서, 상기 오셀타미비르 유도체 화합물은 화학식 2로 기재되는 오셀타미비르 헥실티올(oseltamivir hexylthiol) 또는 하기 화학식 3으로 기재되는 오셀타미비르 헥실아민(oseltamivir hexylamine)인, H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스 검출용 나노입자. The oseltamivir derivative compound according to claim 3, wherein the oseltamivir derivative compound comprises an H275Y mutation, which is oseltamivir hexylthiol represented by Formula 2 or oseltamivir hexylamine represented by Formula 3 below. Nanoparticles for detecting oseltamivir resistant influenza virus.
    [화학식 2][Formula 2]
    Figure PCTKR2016007191-appb-I000014
    Figure PCTKR2016007191-appb-I000014
    [화학식 3][Formula 3]
    Figure PCTKR2016007191-appb-I000015
    Figure PCTKR2016007191-appb-I000015
  5. 제3항에 있어서, 상기 나노입자는 금나노입자, 은나노입자, 또는 형광 나노입자인 나노입자.The nanoparticle of claim 3, wherein the nanoparticles are gold nanoparticles, silver nanoparticles, or fluorescent nanoparticles.
  6. 제3항에 있어서, 상기 나노입자는 오셀타미비르 감수성 인플루엔자 바이러스보다 H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스에 대한 결합력이 더 높은 나노입자. The nanoparticle of claim 3, wherein the nanoparticle has a higher binding capacity to oseltamivir resistant influenza virus comprising an H275Y mutation than the oseltamivir sensitive influenza virus.
  7. 대상체로부터 수득한 시료에 제3항의 나노입자를 처리하는 단계를 포함하는, 제3항의 나노입자를 이용하여 H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스를 검출하는 방법.A method for detecting oseltamivir resistant influenza virus comprising an H275Y mutation using the nanoparticle of claim 3, comprising treating the nanoparticle of claim 3 with a sample obtained from a subject.
  8. 제7항에 있어서, 상기 검출은 비색법을 이용하는 방법. 8. The method of claim 7, wherein said detection uses colorimetric methods.
  9. 제7항에 있어서, 상기 검출은 육안으로 수행되는 방법.8. The method of claim 7, wherein said detection is performed with the naked eye.
  10. 제1항의 오셀타미비르 유도체 화합물, 또는 제3항의 나노입자를 포함하는, H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스 검출용 키트.A kit for detecting oseltamivir resistant influenza virus comprising the H275Y mutation, comprising the oseltamivir derivative compound of claim 1 or the nanoparticles of claim 3.
  11. 시료패드(sample pad), 컨주게이트 패드(conjugate pad), 니트로셀룰로오스 막(nitrocelluose membrane) 및 흡착패드(adsorption pad)를 구비하고 있으며, A sample pad, a conjugate pad, a nitrocelluose membrane and an adsorption pad,
    상기 컨주게이트 패드는 오셀타미비르 헥실티올-금나노입자 또는 오셀타미비르 헥실아민-금나노입자가 담지되고, The conjugate pad is supported by oseltamivir hexylthiol-gold nanoparticles or oseltamivir hexylamine-gold nanoparticles,
    시료패드를 통해 시료 전개시 상기 컨주게이트 패드에 담지된 오셀타미비르 헥실티올-금나노입자 또는 오셀타미비르 헥실아민-금나노입자는 시료와 함께 니트로셀룰로오스 막 및 흡착패드로 전개되고, When the sample is developed through the sample pad, the oseltamivir hexylthiol-gold nanoparticles or oseltamivir hexylamine-gold nanoparticles supported on the conjugate pad are developed together with the sample into the nitrocellulose membrane and the adsorption pad.
    상기 니트로셀룰로오스 막에는 컨트롤 라인(control line) 및 테스트 라인(test ine)이 격리되어 구비되어 있고, The nitrocellulose membrane is provided with a control line and a test ine separated from each other.
    여기에서 상기 컨트롤 라인은 H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스 또는 H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스의 뉴라미니데이즈가 고정되어 있고, 상기 테스트 라인은 하기 화학식 1로 기재되는 오셀타미비르 유도체 화합물이 고정되어 있는, Wherein the control line is fixed neuramiminids of oseltamivir resistant influenza virus comprising H275Y mutation or oseltamivir resistant influenza virus comprising H275Y mutation, and the test line is oseltami The bir derivative compound is fixed,
    H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스 검출용 키트 :Kit for detecting oseltamivir resistant influenza virus containing H275Y mutation:
    [화학식 1][Formula 1]
    Figure PCTKR2016007191-appb-I000016
    Figure PCTKR2016007191-appb-I000016
    상기 화학식 1에서, In Chemical Formula 1,
    R1은 티올 또는 아민임. R 1 is thiol or amine.
  12. 1) 제11항의 H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스 검출용 키트의 시료 패드에 시료를 투입하여 전개시키는 단계;및1) deploying the sample to the sample pad of the oseltamivir resistant influenza virus detection kit containing the H275Y mutation of claim 11; and
    2) 상기 키트의 니트로셀룰로오스 막의 컨트롤 라인 및 테스트 라인으로부터 색상 변화를 확인하는 단계를 포함하는, H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스를 검출하는 방법.2) A method for detecting oseltamivir resistant influenza virus comprising the H275Y mutation, comprising the step of identifying color change from the control line and the test line of the nitrocellulose membrane of the kit.
  13. 대상체로부터 수득한 시료에 제3항의 나노입자를 처리하는 단계;Treating the sample obtained from the subject with the nanoparticles of claim 3;
    상기 시료로부터 H275Y 돌연변이를 포함하는 오셀타미비르 저항성 인플루엔자 바이러스를 확인하는 단계; 및 Identifying an oseltamivir resistant influenza virus comprising a H275Y mutation from the sample; And
    오셀타미비르 저항성 인플루엔자 바이러스가 검출되지 않거나 미량의 오셀타미비르 저항성 인플루엔자 바이러스를 포함하는 시료를 가진 대상체에 오셀타미비르 포스페이트의 치료학적 유효량을 투여하는 단계를 포함하는, 인플루엔자의 치료 방법.A method of treating influenza comprising administering a therapeutically effective amount of oseltamivir phosphate to a subject for which no oseltamivir resistant influenza virus is detected or containing a trace amount of oseltamivir resistant influenza virus.
PCT/KR2016/007191 2015-07-03 2016-07-04 Antiviral-agent-resistant virus detection system WO2017007204A1 (en)

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