WO2020045364A1 - Procédé et système d'analyse d'échantillon de peau pour déterminer le psoriasis - Google Patents

Procédé et système d'analyse d'échantillon de peau pour déterminer le psoriasis Download PDF

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WO2020045364A1
WO2020045364A1 PCT/JP2019/033364 JP2019033364W WO2020045364A1 WO 2020045364 A1 WO2020045364 A1 WO 2020045364A1 JP 2019033364 W JP2019033364 W JP 2019033364W WO 2020045364 A1 WO2020045364 A1 WO 2020045364A1
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amount
serine
psoriasis
amino acid
unit
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PCT/JP2019/033364
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Japanese (ja)
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洋介 東條
智恵子 水本
秀　道広
崇暢 菅
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株式会社 資生堂
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

Definitions

  • the present invention relates to a skin sample analysis method for determining psoriasis from a skin sample using the amount of D-serine as an index, a psoriasis test method, and a sample analysis system that outputs pathological information about psoriasis.
  • the determination of psoriasis can be made using not only the amount of D-serine but also parameters calculated from the amount of D-serine.
  • Psoriasis is an inflammatory skin disease with cutaneous keratinization associated with keratinocyte overgrowth, characterized primarily by erythema covered by scales. Although the cause of psoriasis has not yet been identified, genetic factors and multiple factors may contribute. For the treatment of psoriasis, topical therapy, phototherapy, internal therapy, surgery and the like are used. In particular, although immunosuppressants such as cyclosporine and infliximab and adalimumab, which are TNF- ⁇ antibodies, have high therapeutic effects, high drug prices place a heavy burden on patients, and apply these treatment methods after accurately diagnosing psoriasis. Is needed.
  • Diagnosis of psoriasis is made based on the appearance and distribution of lesions.Diseases characterized by scale-covered erythema include psoriasis, rose pityriasis, and pirates Different skin disorders are also known, such as rash, lichen pityriasis, lichen planus, and lichen sclerosus.
  • Diagnosis of psoriasis includes atopic dermatitis, seborrheic dermatitis, dermatophytosis, cutaneous lupus erythematosus, eczema, lichen planus, rose pityriasis, squamous cell carcinoma in the epidermis, chronic lichen simplest
  • Differential diagnosis from diseases such as second-stage syphilis is required, and the judgment of an experienced physician is required.
  • inflammatory markers such as TNF- ⁇ , IFN- ⁇ , IL-6, IL-8, IL-10 and IL-12 have been used as psoriasis markers (Patent Document 1).
  • inflammatory markers such as TNF- ⁇ , IFN- ⁇ , IL-6, IL-8, IL-10 and IL-12 have been used as psoriasis markers (Patent Document 1).
  • D-amino acids which were thought to be absent in mammalian organisms, exist in various tissues along with the development of detection technology (Patent Document 2). It was expected to carry. D-amino acids are broadly classified into bound forms, which are present as amino acid residues in proteins, and free forms, which dissolve in tissue fluids and cells. Some free D-amino acids in body fluids are independent of L-amino acids. Thus, it was found that the content fluctuated, and that the content fluctuated depending on the type of disease (Patent Document 3).
  • Non-Patent Document 1 Biotechnology (2014), Vol. 92, No. 12, pp. 653-656; Non-Patent Document 2: Fragrance ⁇ Journal ⁇ (2016), vol. 4, pages 14-18).
  • the present inventors have analyzed chiral amino acids in skin samples of patients with various skin diseases, and found that the amount of D-serine is reduced specifically for psoriasis, leading to the present invention.
  • the present invention relates to a method for analyzing a skin sample for determining psoriasis using the amount of D-serine as an index.
  • a decrease in the amount of D-serine can be associated with psoriasis.
  • the determination of psoriasis can be made using not only the amount of D-serine but also parameters calculated from the amount of D-serine.
  • Still another embodiment relates to a sample analysis system capable of performing the analysis method of the present invention.
  • a sample analysis system includes a storage unit, an input unit, a data processing unit, and an output unit.
  • the analysis result of the skin sample is input from the input unit, and the pathological information of psoriasis is output to the output unit. can do.
  • the present invention relates to a program that can be installed in the sample analysis system of the present invention and a storage medium that stores the program. Yet another aspect relates to a method of operating the sample analysis system of the present invention.
  • the present invention also relates to a kit for determining psoriasis, comprising an anti-D-serine antibody.
  • ⁇ Psoriasis markers different from conventionally known inflammatory markers can be provided.
  • FIG. 1 is a graph showing the ratio of D-serine in a skin sample obtained from a rash and a rash in psoriatic patients, atopic patients, and xeroderma patients.
  • FIG. 2 is an example of a configuration diagram of the analysis system of the present invention.
  • FIG. 3 is a flowchart illustrating an example of an operation for determining psoriasis.
  • the present invention relates to a method for analyzing a skin sample for determining psoriasis using the amount of D-serine in the skin sample as an index.
  • “Using the amount of D-serine as an index” refers to any embodiment in which the amount of D-serine is used as an index for determination. Therefore, it is not intended to use only the amount of D-serine, but includes using a parameter calculated from the amount of D-serine.
  • the analysis method of the present invention relates to an analysis method including a step of measuring the amount of D-serine in a skin sample and a step of relating a decrease in the amount of D-serine to psoriasis. Since psoriasis can be determined by the analysis method of the present invention, in another aspect, the present invention can also be referred to as a diagnostic method.
  • the psoriasis that can be determined in the present invention can be any disease classified into psoriasis such as psoriasis vulgaris, psoriatic arthritis, pustular psoriasis, and guttate psoriasis.
  • psoriasis vulgaris psoriasis vulgaris
  • psoriatic arthritis psoriatic arthritis
  • pustular psoriasis psoriasis
  • guttate psoriasis guttate psoriasis.
  • the analysis method of the present invention makes it possible to distinguish psoriasis from atopic dermatitis, which is an inflammatory skin disease, and xeroderma.
  • Psoriasis can also be specifically distinguished and determined from diseases.
  • the subject may be any subject, for example, a patient suffering from a skin disease of unknown etiology, the analysis method of the present invention may be performed, or a patient diagnosed clinically with psoriasis may be confirmed.
  • the analysis method of the present invention may be performed for diagnosis.
  • the target chiral amino acid may be measured alone, or may be measured together with another chiral amino acid.
  • chiral amino acids can be diagnostic markers for other diseases. Therefore, from the viewpoint of analyzing a plurality of diseases at once, it is preferable to collectively measure D-form and L-form for amino acids serving as diagnostic markers for other diseases, for example, 20 kinds of protein constituent amino acids.
  • D-serine, D-alanine, D-glutamic acid, and D-aspartic acid are present as D-amino acids in skin, and these D-amino acids and corresponding L-amino acids Is more preferably measured collectively.
  • a step of obtaining a skin sample and a step of processing the obtained skin sample may be performed.
  • the skin sample include a skin section and a stratum corneum sample obtained by a tape strip.
  • the skin sample may be obtained from the rash or the rash, but is preferably obtained from the rash to increase the accuracy of the determination of psoriasis. Since the rash of psoriasis is covered with scales, a horny layer sample may be obtained after performing a tape strip several times for the purpose of removing scales.
  • the stratum corneum sample obtained by the tape strip can be treated with a solvent such as methanol to extract amino acids contained in the sample.
  • the amount of each chiral amino acid can be measured by subjecting the extracted chiral amino acid to HPLC using a chiral column or immunological technique using an antibody capable of detecting the chiral amino acid.
  • HPLC is used in measuring the amount of D-serine
  • multi-step HPLC can be performed using another column before the chiral column.
  • the multi-step HPLC is performed by, after separation on a column, holding the separated fractions in a multi-loop unit and sequentially introducing the fractions into the next column.
  • the amount of D-serine in a sample may be measured using any method known to those skilled in the art.
  • D- and L-amino acids are derivatized stereoisomerically in advance with o-phthalaldehyde (OPA), N-tert-butyloxycarbonyl-L-cysteine (Boc-L-Cys) or another modifying reagent, and then A method of separating a mixture of 100 mM acetate buffer (pH 6.0) and acetonitrile by gradient elution using an analytical column such as ODS-80TsQA for simultaneous measurement of D-form and L-form of amino acids. Can be used.
  • OPA o-phthalaldehyde
  • Boc-L-Cys N-tert-butyloxycarbonyl-L-cysteine
  • D- and L-amino acids are previously derivatized with a fluorescent reagent such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and then ODS-80TsQA, Mightysil @ RP
  • a fluorescent reagent such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)
  • NBD-F 4-fluoro-7-nitro-2,1,3-benzoxadiazole
  • ODS-80TsQA ODS-80TsQA
  • Mightysil @ RP A method of separating each amino acid non-stereospecifically using an analytical column such as -18GP and the like, followed by optical resolution using a Pirkle type chiral stationary phase column (for example, Sumichiral @ OA-2500S or R), It can be used for the trace measurement of constituent amino acids (Kenji Hamase and Kiyoshi Zaitsu, Analytical Chemistry, 53, 677-690 (2004)).
  • the optical resolution column system in this specification refers to a separation analysis system using at least an optical resolution column, and may include a separation analysis using an analysis column other than the optical resolution column. More specifically, passing a sample containing a component having an optical isomer, together with a first liquid as a mobile phase, through a first column packing material as a stationary phase, and separating the components of the sample, Holding each of the components of the sample individually in a multi-loop unit, each of the components of the sample individually held in the multi-loop unit, together with a second liquid as a mobile phase, as a stationary phase Supplying the second column packing material having an optically active center through a channel to split the optical isomer contained in each of the components of the sample, and the optical isomer contained in each of the components of the sample.
  • a method for analyzing an optical isomer which comprises a step of detecting an isomer, the D- / L-amino acid concentration in a sample can be measured ( Patent No. 4,291,628).
  • D-amino acid can be alternatively quantified by an immunological technique using a monoclonal antibody that discriminates optical isomers of D-serine (JP-A-2009-184981).
  • detection can be performed by an ELISA method using a monoclonal antibody that binds to D-serine and a secondary antibody that binds to the antibody and has a label.
  • the step of associating with psoriasis may be performed using a parameter calculated from the amount of D-serine.
  • a parameter calculated from the amount of D-serine for example, a parameter obtained by normalizing the amount of D-serine using the amount of D-serine and an internal standard contained in a skin sample can be used.
  • the ratio or ratio between D-serine and the internal standard may be calculated, or an arbitrary constant may be added, subtracted, multiplied, or divided.
  • the amount of a skin sample, particularly a stratum corneum sample obtained by a tape strip, is not constant depending on the condition of the skin.
  • a stratum corneum sample obtained by a tape strip is not constant depending on the condition of the skin.
  • since the rash is covered with scale it is difficult to obtain a certain amount by using any technique. Therefore, by normalizing using the internal standard in the skin sample, a stable result can be obtained regardless of the technique of obtaining the skin sample.
  • Examples of the internal standard that can be used include a protein amount, a gene amount, a total amino acid amount, a total D-amino acid amount, a total L-amino acid amount, an arbitrary L-amino acid amount, a stratum corneum weight, and the like. Any substance can be used as long as the substance is contained at a certain ratio. In the present invention, it is particularly preferable to use the total amount of L-amino acids or the amount of individual L-amino acids as an internal standard. More preferably, L-serine can be used as an internal standard.
  • the presence or absence of psoriasis can be determined by comparing with a cutoff value of a parameter calculated from the amount of D-serine derived as described above. . Therefore, the judgment may be made by a medical assistant or the like who is not a doctor, or may be made by an analysis institution or the like. Therefore, the analysis method of the present invention can be said to be a preliminary method or an auxiliary method of diagnosis.
  • the analysis method of the present invention may include a step of calculating as a parameter for determining psoriasis instead of the step of associating a decrease in the amount of D-serine with psoriasis. By comparing a parameter calculated by this analysis method with a predetermined cutoff value, psoriasis can be determined.
  • the cutoff value for the determination of psoriasis can be arbitrarily determined by analyzing the cohort and performing statistical processing.
  • a method of the statistical processing a method well-known to those skilled in the art may be used.
  • ROC analysis, t-test, or the like may be used, and the average value, median value, X percentile value of a healthy group or a psoriasis patient group may be used.
  • any numerical value can be selected for X, and 3, 5, 10, 15, 20, 30, 40, 60, 70, 80, 85, 90, 95, and 97 can be appropriately used.
  • the cutoff value may be one, or the disease state may be classified according to the severity of the disease. Further, the cutoff value can be determined by comparison with a patient group having a skin disease other than psoriasis.
  • a treatment suitable for psoriasis is selected and performed.
  • At least one psoriasis treatment step selected from the group consisting of, but not limited to, topical therapy, phototherapy, oral therapy, injection therapy, surgery, and lifestyle improvement is further performed.
  • topical therapy include steroidal external preparations, immunosuppressant external preparations, and vitamin D3 and its derivatives.
  • Phototherapy includes ultraviolet irradiation, for example, irradiation with medium wavelength ultraviolet (UVB) and long wavelength ultraviolet (UVA).
  • UVB medium wavelength ultraviolet
  • UVA long wavelength ultraviolet
  • vitamin A derivatives such as retinoids, steroids, immunosuppressants such as cyclosporine, and PDE4 inhibitors are administered.
  • an antibody drug such as an antibody against an inflammatory cytokine is administered.
  • the treatment steps for psoriasis are not intended to be limited to those described above, and new therapies and agents may be used in accordance with medical advances.
  • anti-TNF- ⁇ monoclonal antibodies adalimumab and infliximab, anti-human IL-12 / 23p40 monoclonal antibody ustekinumab, anti-IL-23p19 monoclonal antibody guselkumab, anti-IL-17A monoclonal antibodies secukinumab and ixekizumab, anti-IL Brodalumab, a -17A receptor A monoclonal antibody, is effective.
  • the method of the present invention may include a therapeutic step including administering these antibody drugs after the determination or diagnosis.
  • FIG. 2 is a configuration diagram of the sample analysis system of the present invention.
  • a sample analysis system 10 includes a storage unit 11, an input unit 12, a data processing unit 14, and an output unit 15, and can analyze a target skin sample and output disease state information. More specifically, in the sample analysis system 10 of the present invention, The storage unit 11 stores a cutoff value of the amount of D-serine and pathological information of psoriasis for determining psoriasis input from the input unit 12, A measurement value of the amount of D-serine in the target skin sample is input from the input unit 12 and stored in the storage unit.
  • the data processing unit 14 determines the disease state information of the target psoriasis by comparing the measured value of the amount of D-serine with the cutoff value stored in the storage unit 11,
  • the output unit 15 can output the disease state information about the target psoriasis.
  • a further aspect of the present invention may relate to a program for operating such a sample analysis system, and also relates to a medium recording such a program.
  • FIG. 3 is a flowchart showing an example of an operation for determining psoriasis according to the program of the present invention.
  • Such programs include: An instruction to store the association between the cutoff value of the amount of D-serine and the pathological information of psoriasis in the storage unit 11; A command to read a measured value of the amount of D-serine in the target skin sample from the input unit 12 and store the measured value in the storage unit 11; A command to activate the data processing unit 14 to compare the measured value of the amount of D-serine stored in the storage unit with the cut-off value to determine pathological information of control psoriasis and store it in the storage unit 11; And a command to operate the output unit 15 to output the stored pathological information about psoriasis.
  • Such a program may be stored in a storage medium, or may be provided via an electric communication line such as the Internet or a LAN.
  • the storage unit 11 includes a memory device such as a RAM, a ROM, and a flash memory, a fixed disk device such as a hard disk drive, or a portable storage device such as a flexible disk and an optical disk.
  • the storage unit 11 includes data measured by the analysis and measurement unit, data and instructions input from the input unit 12, calculation results performed by the data processing unit 14, and other computer programs used for various processes of the information processing apparatus. Stores a database and the like.
  • the computer program may be installed via a computer-readable recording medium such as a CD-ROM, a DVD-ROM, or the Internet. The computer program is installed in the storage unit using a known setup program or the like.
  • the input unit 12 is an interface or the like, and includes an operation unit such as a keyboard and a mouse.
  • an operation unit such as a keyboard and a mouse.
  • the analysis / measurement unit 13 When the analysis / measurement unit 13 is provided outside, the measured data or the like can be input directly or via a network or a storage medium via an interface of the input unit.
  • the input unit can input data measured by the analysis and measurement unit 13, instructions for calculation processing performed by the data processing unit 14, and the like.
  • the analysis system of the present invention may include the analysis and measurement unit 13 via the input unit, or may include the analysis and measurement unit 13 without the input unit.
  • the analysis system of the present invention includes the analysis / measurement unit 13
  • a measurement value of the amount of D-serine in the target skin sample is input from the input unit 12 and stored in the storage unit.
  • 13 includes a step of measuring the amount of the chiral amino acid in the skin sample and storing it in the storage unit.
  • the program includes a command to operate the analysis and measurement unit 13 to determine the measured value of the amount of D-serine and store the measured value in the storage unit.
  • the analysis and measurement unit 13 has a configuration that enables separation and measurement of chiral amino acids.
  • Amino acids may be analyzed one by one, but some or all types of amino acids can be analyzed together.
  • the analysis and measurement unit 13 is not intended to be limited to the following, but may be, for example, a high-performance liquid chromatography system (HPLC) including a sample introduction unit, an optical separation column, and a detection unit.
  • HPLC is preferably a two-dimensional HPLC including a column for separating amino acids separately from the optical resolution column.
  • the analysis and measurement unit 13 may be included in the sample analysis system 10. Further, it may be configured separately from the sample analysis system 10, and the measured data or the like may be input via the input unit 12 using a network or a storage medium.
  • the data processing unit 14 is configured to determine psoriasis by comparing the measured amount of the chiral amino acid with the cutoff value stored in the storage unit 11.
  • the data processing unit 14 performs various arithmetic processes on the data measured by the analysis and measurement unit 13 and stored in the storage unit 11 according to the program stored in the storage unit 11.
  • the arithmetic processing is performed by a CPU included in the data processing unit 14.
  • the CPU includes a functional module that controls the analysis and measurement unit 13, the input unit 12, the storage unit 11, and the output unit 15, and can perform various controls. Each of these units may be configured by an independent integrated circuit, microprocessor, firmware, or the like.
  • the output unit 15 is configured to output a disease state index value and / or disease state information that is a result of performing the arithmetic processing in the data processing unit 14.
  • the calculation processing result in the data processing unit 14 may be directly output to the output unit 15 or may be stored in the storage unit 11 once and then output to the output unit 15 as needed.
  • the output unit 15 may be a display device such as a liquid crystal display that directly displays the result of the arithmetic processing, an output unit such as a printer, or an interface unit for outputting to an external storage device or outputting via a network. There may be.
  • a further aspect of the invention may relate to a kit for determining psoriasis.
  • a kit for determining psoriasis contains an antibody for detecting D-serine, and can detect D-serine in a sample.
  • Antibodies for detecting D-serine include anti-D-serine antibodies.
  • the antibody described in JP-A-2009-184981 can be used.
  • the composition further comprises a control antibody that allows detection of a substance present in the skin sample in an amount. Thereby, even if D-serine has not been detected, it can be determined whether or not the sample preparation has failed.
  • the kit for determining psoriasis may further include, in addition to the antibody, a tape for a tape strip and a solvent for amino acid extraction.
  • a kit may further include a substrate further provided with a detection portion, wherein the detection portion has immobilized thereon an antibody that captures D-serine.
  • the stratum corneum sample obtained by the tape strip is treated with a solvent for amino acid extraction, the solution is developed on a base material, and D-serine is supplemented to the antibody in the detection section. Thereafter, by developing a label capable of detecting the captured D-serine, the captured D-serine can be detected.
  • An example of such a label is an antibody labeled with colloidal gold or the like.
  • a horny layer sample was obtained by a tape strip method from the eruption part and the eruption part of 21 patients with psoriasis vulgaris, 31 patients with atopic dermatitis, and 8 patients with xeroderma. .
  • a horny layer sample was obtained from the skin of 42 healthy subjects as a control.
  • the severity of psoriasis patients was 8 in mild, 7 in moderate, 3 in severe, and 3 in unknown.
  • the horny layer sample was a corneal tape having a sampling area of 5.6 cm 2, and the tape was stripped three times at the same location, and the third sample was used.
  • the stratum corneum sample attached to the stratum corneum tape was treated with 1500 ⁇ l of 95% methanol, subjected to ultrasonic treatment, and centrifuged at 15000 rpm for 10 minutes. 250 ⁇ l of the supernatant was taken and dried under reduced pressure. A 200 mM aqueous sodium borate solution (pH 8.0: 30 ⁇ l) was added to the dried product to dissolve it. To the obtained solution, a 40 mM NBD-F acetonitrile solution (5 ⁇ l) was added, reacted at 60 ° C. for 2 minutes, and 2% (v / v) aqueous trifluoroacetic acid solution (90 ⁇ l) was added thereto to obtain a two-dimensional solution. It was subjected to HPLC analysis.
  • ML-750 Gradient elution using an aqueous mobile phase containing
  • the fraction of the target amino acid is automatically collected by using a multi-loop valve, and the optical resolution column (OA2500S-15250, 1.5 mmi.e. ⁇ 250 mm) (Distributor: Daicel Industries).
  • the mobile phase was a mixed solution of MeOH-MeCN containing citric acid or formic acid, and the fluorescence of NBD-amino acids was excited at 470 nm and detected at 530 nm. All quantitative data was obtained by fluorescence detection. HPLC-MS / MS was used to confirm the presence of D-amino acids in the actual biological matrix.
  • the concentration of D-serine and L-serine in the sample was measured, and the following formula: , The content of D-serine was measured.
  • the measurement results for each disease are shown in FIG.
  • the discrimination between a healthy subject and psoriasis can also be performed using another index including the amount of D-Ser in the calculation formula.
  • a parameter obtained by multiplying the amount of D-Ser by another amount of amino acid here, for example, D-Ser / D-Ala
  • a parameter (D-Ser / cm 2 ) obtained by normalizing the amount of D-Ser in terms of the amount per horny layer area is also useful for diagnosis and discrimination since a significant difference was observed between a healthy subject and a psoriatic eruption area. You can see that there is.

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Abstract

La présente invention concerne un procédé d'analyse d'un échantillon de peau pour déterminer le psoriasis chez un sujet, la quantité de D-sérine étant utilisée comme indice. La présente invention concerne également un système d'analyse d'un échantillon de peau pour déterminer le psoriasis chez un sujet, le système comprenant une unité de stockage, une unité d'entrée, une unité de traitement de données et une unité de sortie. En outre, la présente invention concerne une trousse pour déterminer le psoriasis, la trousse contenant un anticorps anti-D-sérine.
PCT/JP2019/033364 2018-08-27 2019-08-26 Procédé et système d'analyse d'échantillon de peau pour déterminer le psoriasis WO2020045364A1 (fr)

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Cited By (2)

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JP2023064024A (ja) * 2021-10-25 2023-05-10 良丹 孫 乾癬を評価するための評価システム及びその使用
CN117607334A (zh) * 2023-11-27 2024-02-27 南方医科大学南方医院 糖尿病皮肤病变风险筛查试剂盒及筛查方法

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