WO2022105895A1 - 一种预防银屑病复发的内源性代谢物组合 - Google Patents

一种预防银屑病复发的内源性代谢物组合 Download PDF

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WO2022105895A1
WO2022105895A1 PCT/CN2021/131923 CN2021131923W WO2022105895A1 WO 2022105895 A1 WO2022105895 A1 WO 2022105895A1 CN 2021131923 W CN2021131923 W CN 2021131923W WO 2022105895 A1 WO2022105895 A1 WO 2022105895A1
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acid
long
ufa
sfa
unsaturated fatty
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PCT/CN2021/131923
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French (fr)
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WO2022105895A9 (zh
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乐云辰
郑捷
李霞
陈利红
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上海交通大学医学院附属瑞金医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids

Definitions

  • the invention relates to the technical field of medical biological detection, in particular to an endogenous metabolite combination for preventing the recurrence of psoriasis.
  • Psoriasis commonly known as psoriasis, is a chronic inflammatory skin disease with a long course of disease and a tendency to recur easily.
  • the disease mainly occurs in young adults and has a great impact on the physical health and mental status of patients.
  • the clinical manifestations are mainly erythema and scales, which can occur all over the body, and are more common on the scalp and extensor sides of the limbs, which are mostly aggravated in winter.
  • the purpose of the present invention is to provide a long-chain unsaturated fatty acid for inhibiting IL-17 and/or preventing and/or treating IL-17-related diseases.
  • Another object of the present invention is to provide the use of long-chain unsaturated fatty acids and long-chain saturated fatty acids or their detection reagents for prognosis of psoriasis patients.
  • the first aspect of the present invention provides the use of a long-chain unsaturated fatty acid for preparing a composition for (i) inhibiting IL-17; and/or (ii) preventing and/or treating and IL-17 related diseases;
  • the long-chain unsaturated fatty acid is selected from the group consisting of: linoleic acid (C18:2, Linoleic acid), oleic acid (C18:1, Oleic acid), palmitoleic acid (C16:1, Palmitoleic acid), or its combination.
  • the second aspect of the present invention provides the use of long-chain unsaturated fatty acids and long-chain saturated fatty acids or detection reagents thereof, for preparing detection reagents or detection kits, and the detection reagents or detection kits are used to detect silver flakes patient prognosis;
  • Described long chain unsaturated fatty acid is selected from following group: linoleic acid (C18:2, Linoleic acid), oleic acid (C18:1, Oleic acid), palmitoleic acid (C16:1, Palmitoleic acid) , or a combination thereof;
  • the long-chain saturated fatty acid is selected from the group consisting of palmitic acid (C16:0, Palmitic acid), stearic acid (C18:0, Stearic acid), or a combination thereof.
  • the IL-17-related disease includes psoriasis.
  • the psoriasis includes patients with plaque psoriasis.
  • the plaque psoriasis includes patients with moderate to severe plaque psoriasis.
  • the prevention includes preventing the recurrence of psoriasis.
  • the prognosis includes the prognosis after the drug for psoriasis treatment is stopped.
  • the prognosis includes the prognosis after the drug for psoriasis treatment is stopped, and the drug for psoriasis treatment has been treated for 6-8 weeks, preferably 8 weeks.
  • the psoriasis therapeutic drug includes IL-17 antibody.
  • the prognosis includes the prognosis after the IL-17 antibody is stopped.
  • the IL-17A antibody includes ixekizumab.
  • the IL-17 antibody includes a monoclonal antibody or a polyclonal antibody.
  • the composition is a pharmaceutical composition.
  • the composition further includes a pharmaceutically acceptable carrier.
  • the dosage form of the composition is a solid preparation, a semi-solid preparation or a liquid preparation.
  • the dosage form of the composition is an oral preparation, an injection preparation or an external preparation.
  • the dosage form of the composition is an external skin preparation or a subcutaneous injection preparation.
  • the prognosis includes predicting the recurrence rate after drug withdrawal.
  • the prognosis is in vitro prognosis.
  • the psoriasis patients for prognosis include patients after IL-17A antibody treatment is discontinued.
  • the sample for prognosis is a blood sample.
  • the sample for prognosis is a blood sample.
  • the blood sample includes serum, plasma or blood.
  • the prognosis is performed by detecting the ratio of UFA/SFA in the sample.
  • the sample includes serum, plasma or blood.
  • the sample is derived from a patient with psoriasis.
  • the detection kit also includes a label or an instruction manual, and the label or instruction manual records the following content:
  • each substitution value of linoleic acid, oleic acid, palmitoleic acid, palmitic acid and stearic acid is the serum concentration value.
  • each fatty acid substitution value is its peak area value in a gas chromatography-mass spectrometry (GC-MS) device.
  • GC-MS gas chromatography-mass spectrometry
  • the ratio of long-chain unsaturated fatty acid (UFA) to long-chain saturated fatty acid (SFA) is the ratio when psoriasis patients are treated with psoriasis drug for 6-8 weeks, preferably 8 weeks.
  • a detection kit in a third aspect of the present invention, includes:
  • Described long-chain unsaturated fatty acid is selected from following group: linoleic acid, oleic acid, palmitoleic acid, or its combination;
  • the long-chain saturated fatty acid is selected from the group consisting of palmitic acid, stearic acid, or a combination thereof.
  • a fourth aspect of the present invention provides a use of the detection kit according to the third aspect of the present invention, for preparing a diagnostic kit for prognosing psoriasis patients.
  • the diagnostic kit further includes a label or an instruction manual, and the label or instruction manual records the following content:
  • each substitution value of linoleic acid, oleic acid, palmitoleic acid, palmitic acid and stearic acid is the serum concentration value.
  • each fatty acid substitution value is its peak area value in a gas chromatography-mass spectrometry (GC-MS) device.
  • GC-MS gas chromatography-mass spectrometry
  • the ratio of long-chain unsaturated fatty acid (UFA) to long-chain saturated fatty acid (SFA) is the ratio when psoriasis patients are treated with psoriasis drug for 6-8 weeks, preferably 8 weeks.
  • the detection sample of the diagnostic kit is a blood sample.
  • the blood sample includes serum, plasma or blood.
  • a fifth aspect of the present invention provides a device or system, the device or system comprising:
  • detection module described detection module is used to measure the content or content ratio (UFA/SFA) of long-chain unsaturated fatty acid (UFA) and long-chain saturated fatty acid (SFA) in the sample;
  • the output module outputs the content or content ratio (UFA/SFA) of the long-chain unsaturated fatty acid (UFA) and long-chain saturated fatty acid (SFA) in the sample according to the detection module to silver Information on the prognosis of patients with psoriasis.
  • UFA/SFA content or content ratio
  • the information output by the output module includes:
  • each substitution value of linoleic acid, oleic acid, palmitoleic acid, palmitic acid and stearic acid is the serum concentration value.
  • each fatty acid substitution value is its peak area value in a gas chromatography-mass spectrometry (GC-MS) device.
  • GC-MS gas chromatography-mass spectrometry
  • the ratio of long-chain unsaturated fatty acid (UFA) to long-chain saturated fatty acid (SFA) is the ratio when psoriasis patients are treated with psoriasis drug for 6-8 weeks, preferably 8 weeks.
  • the sample includes serum, plasma or blood.
  • the sample is derived from a patient with psoriasis.
  • the device is a gas chromatography-mass spectrometer (GC-MS) or a high performance liquid chromatograph.
  • GC-MS gas chromatography-mass spectrometer
  • the device or system further includes a solvent delivery module, a sample injection module, a separation module, a detector, a data processing and recording module.
  • the solvent delivery module includes a liquid reservoir, a degasser, an infusion pump and/or an elution device.
  • the sample injection module includes a sample injector.
  • the separation module includes a chromatographic column.
  • the device or system is a high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the purpose of the present invention is to provide an understanding that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other, thereby forming a new the preferred or preferred technical solution. Due to space limitations, it is not repeated here.
  • FIG. 1 Partial representative flow cytometry plots showing that different fatty acids have different regulatory abilities on IL-17A-secreting T cells in the blood of patients with psoriasis.
  • Unstimulated means unstimulated T cells
  • Stimulated means T cells that add stimulating agents to enhance the ability of IL-17A secretion, and the proportion of cells secreting IL-17A is significantly increased (hereinafter referred to as Sti);
  • Sti+DMSO means that when cells are stimulated Dimethyl sulfoxide (DMSO) was added.
  • DMSO Dimethyl sulfoxide
  • Sti+Palmitoleic acid indicated that palm oil was added when stimulating T cells.
  • Acid C16:1, Palmitoleic acid
  • Sti+Azelaic acid means adding azelaic acid when stimulating T cells, at this time the proportion of T cells secreting IL-17A is slightly Decreased
  • Sti+Stearic acid means adding stearic acid (C18:0, Stearic acid) when stimulating T cells, and the proportion of T cells secreting IL-17A is basically unchanged.
  • the figure shows that the unsaturated fatty acids linoleic acid (C18:2, Linoleic acid), oleic acid (C18:1, Oleic acid) and palmitoleic acid (C16:1, Palmitoleic acid) have significant inhibitory effects on IL-17A production.
  • Figure 3 Standard curve for determination of serum major fatty acid levels in patients with psoriasis.
  • Figures 4A and 4B Viability of human peripheral blood mononuclear cells was not affected at a concentration of 12.5 ⁇ g/mL of long-chain fatty acids determined by in vitro assays. In vitro experiments showed that the viability of human peripheral blood mononuclear cells was reduced when the fatty acid concentration in the medium was 200 ⁇ g/mL, 50 ⁇ g/mL and 25 ⁇ g/mL. Cell viability was not affected when the fatty acid concentration was reduced to 12.5 ⁇ g/mL.
  • the regulation effect of fatty acid on IL-17A-producing cells can be avoided to avoid the decrease of IL-17A due to the decrease of cell viability, and at the same time, to ensure that the change of IL-17A secretion capacity is entirely due to the change of IL-17A secretion in a certain fatty acid.
  • the normal physiological response of cells to the environment can be avoided to avoid the decrease of IL-17A due to the decrease of cell viability, and at the same time, to ensure that the change of IL-17A secretion capacity is entirely due to the change of IL-17A secretion in a certain fatty acid.
  • Figures 5A and 5B Cytoflow analysis of changes in the ability of peripheral blood mononuclear cells (PBMCs) from 10 psoriasis patients to secrete IL-17A under different long-chain fatty acid environments.
  • the rows in the figure represent the analysis data for each patient, the columns represent the analysis data under different treatment conditions, and the numbers in each panel represent the proportion of IL-17A-secreting T cells in PBMCs.
  • Figure 6 Psoriasis patients with high UFA/SFA ratios relapse later after 8 weeks of ixekizumab treatment (W8), whereas patients with low ratios relapse earlier.
  • Figure 7 ROC analysis shows that the critical value of UFA/SFA ratio in serum of patients with early and late recurrence after treatment is 0.364, that is, the ratio of UFA/SFA below 0.364 is early recurrence, and higher than 0.364 is late recurrence.
  • Figure 8 The boxplot of the ratio of UFA/SFA after treatment obtained by ROC analysis, the red line is the best critical value of 0.364, the ratio of UFA/SFA below 0.364 is early recurrence, and higher than 0.364 is late recurrence.
  • Figure 9 UFA/SFA ratio in serum after 8 weeks (W8) of IL-17A mAb ixekizumab treatment.
  • the present inventors discovered for the first time that long-chain unsaturated fatty acids linoleic acid, oleic acid and/or palmitoleic acid can effectively inhibit IL-17 and prevent and/or treat IL-17-related diseases such as psoriasis.
  • the present invention also unexpectedly finds that the ratio of UFA/SFA can be used as a judgment index for the prognosis of psoriasis patients, so as to reduce the recurrence risk by formulating a targeted treatment plan for psoriasis patients with high recurrence risk after treatment. On this basis, the inventors have completed the present invention.
  • the terms “comprising,” “including,” and “containing” are used interchangeably to include not only closed definitions, but also semi-closed, and open definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
  • 3-HB is 3-hydroxybutyric acid
  • UFA refers to long chain unsaturated fatty acids
  • SFA refers to long chain saturated fatty acids
  • the term "less than or equal to” includes less than and/or equal to.
  • the term "greater than or equal to” includes greater than and/or equal to.
  • IL-17A refers to interleukin-17A.
  • long-chain unsaturated fatty acids and long-chain saturated fatty acids or their detection reagents are interchangeable with “long-chain unsaturated fatty acids or their detection reagents, and long-chain saturated fatty acids or their detection reagents" use.
  • the long chain unsaturated fatty acid is selected from the group consisting of linoleic acid, oleic acid, palmitoleic acid, or a combination thereof.
  • the detection reagent of long-chain unsaturated fatty acid is not particularly limited, as long as it satisfies accurate detection.
  • the long-chain unsaturated fatty acid detection reagent can be an antibody or a detection reagent suitable for methods such as high performance liquid phase.
  • the long chain saturated fatty acid is selected from the group consisting of palmitic acid, stearic acid, or a combination thereof.
  • the detection reagent for long-chain saturated fatty acid is not particularly limited, as long as it satisfies accurate detection.
  • the long-chain saturated fatty acid detection reagent can be an antibody or a detection reagent suitable for methods such as high performance liquid phase.
  • Psoriasis commonly known as psoriasis, is a chronic inflammatory skin disease with a long course of disease and a tendency to recur easily.
  • the clinical manifestations are mainly erythema and scales, which can occur all over the body, and are more common on the scalp and extensor sides of the limbs, which are mostly aggravated in winter.
  • the psoriasis is preferably a patient with plaque psoriasis.
  • the plaque psoriasis includes patients with moderate to severe plaque psoriasis.
  • the detection samples of long-chain unsaturated fatty acids and long-chain saturated fatty acids or their detection reagents, detection kits, prognosis judgments, diagnostic reagents, devices or systems of the present invention are not particularly limited, and are preferably blood samples.
  • the blood sample comprises serum, plasma or blood
  • the sample of the present invention is derived from the psoriasis patient.
  • the present invention provides the use of a long-chain unsaturated fatty acid for the preparation of a composition for (i) inhibiting IL-17; and/or (ii) preventing and/or treating IL-17 related diseases.
  • the long-chain unsaturated fatty acid is selected from the group consisting of linoleic acid, oleic acid, palmitoleic acid, or a combination thereof.
  • the IL-17-related disease includes psoriasis.
  • the prevention includes preventing the recurrence of psoriasis.
  • prevention refers to a method of preventing the onset, recurrence of a disease and/or its attendant symptoms, or protecting a subject from acquiring the disease. "Prevention” as used herein also includes delaying the onset of the disease and/or its attendant symptoms and reducing the risk of the disease in a subject.
  • Treatment includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal.
  • the long-chain unsaturated fatty acids of the present invention inhibit IL-17 and reverse psoriasis, eg, by at least about 10%, at least About 30%, at least about 50%, or at least about 80%.
  • the present invention also provides the use of long-chain unsaturated fatty acids (UFA) and fatty acids and long-chain saturated fatty acids (SFA) or their detection reagents, for preparing detection reagents or detection kits, and the detection reagents or detection kits are used in for the prognosis of patients with psoriasis.
  • UFA long-chain unsaturated fatty acids
  • SFA long-chain saturated fatty acids
  • the long-chain unsaturated fatty acid is selected from the group consisting of linoleic acid, oleic acid, palmitoleic acid, or a combination thereof.
  • the long-chain saturated fatty acid is selected from the group consisting of palmitic acid, stearic acid, or a combination thereof.
  • the prognosis includes the prognosis after the drug for psoriasis treatment is stopped.
  • the prognosis includes the prognosis after the drug for psoriasis treatment is stopped, and the drug for psoriasis treatment has been treated for 6-8 weeks, preferably 8 weeks.
  • the psoriasis therapeutic drug includes IL-17 antibody.
  • the IL-17A antibody comprises ixekizumab.
  • the prognosis includes the prognosis following discontinuation of the IL-17 antibody.
  • the sample for prognosis is a blood sample, such as serum, plasma or blood.
  • the prognosis is performed by detecting the ratio of UFA/SFA in the sample.
  • the detection kit further includes a label or an instruction manual, and the label or instruction manual records the following content:
  • each substitution value of linoleic acid, oleic acid, palmitoleic acid, palmitic acid and stearic acid is the serum concentration value.
  • each fatty acid substitution value is its peak area value in a gas chromatography-mass spectrometry (GC-MS) device.
  • GC-MS gas chromatography-mass spectrometry
  • the ratio of long-chain unsaturated fatty acid (UFA) to long-chain saturated fatty acid (SFA) is the ratio when psoriasis patients are treated with psoriasis drug for 6-8 weeks, preferably 8 weeks.
  • the present invention also provides a detection kit, the detection kit includes:
  • Described long-chain unsaturated fatty acid is selected from following group: linoleic acid, oleic acid, palmitoleic acid, or its combination;
  • the long-chain saturated fatty acid is selected from the group consisting of palmitic acid, stearic acid, or a combination thereof.
  • the present invention also provides the use of a detection kit for preparing a diagnostic kit, which is used for prognosis of psoriasis patients.
  • the diagnostic kit further includes a label or an instruction manual, and the label or instruction manual records the following content:
  • each substitution value of linoleic acid, oleic acid, palmitoleic acid, palmitic acid and stearic acid is the serum concentration value.
  • each fatty acid substitution value is its peak area value in a gas chromatography-mass spectrometry (GC-MS) device.
  • GC-MS gas chromatography-mass spectrometry
  • the ratio of long-chain unsaturated fatty acid (UFA) to long-chain saturated fatty acid (SFA) is the ratio when psoriasis patients are treated with psoriasis drug for 6-8 weeks, preferably 8 weeks.
  • the detection sample of the diagnostic kit is a blood sample.
  • the blood sample includes serum, plasma or blood.
  • the present invention also provides a device or system, the device or system comprising:
  • detection module described detection module is used to measure the content or content ratio (UFA/SFA) of long-chain unsaturated fatty acid (UFA) and long-chain saturated fatty acid (SFA) in the sample;
  • the output module outputs the content or content ratio (UFA/SFA) of long-chain unsaturated fatty acids (UFA) and long-chain saturated fatty acids (SFA) in the sample to silver according to the detection module Information on the prognosis of patients with psoriasis.
  • UFA/SFA content or content ratio
  • the information output by the output module includes:
  • each substitution value of linoleic acid, oleic acid, palmitoleic acid, palmitic acid and stearic acid is the serum concentration value.
  • each fatty acid substitution value is its peak area value in a gas chromatography-mass spectrometry (GC-MS) device.
  • GC-MS gas chromatography-mass spectrometry
  • the ratio of long-chain unsaturated fatty acid (UFA) to long-chain saturated fatty acid (SFA) is the ratio when psoriasis patients are treated with psoriasis drug for 6-8 weeks, preferably 8 weeks.
  • the sample includes serum, plasma or blood.
  • the sample is derived from a patient with psoriasis.
  • the device is a gas chromatography-mass spectrometer (GC-MS) or a high performance liquid chromatograph.
  • GC-MS gas chromatography-mass spectrometer
  • the device or system further comprises a solvent delivery module, a sample injection module, a separation module, a detector, a data processing and recording module.
  • the solvent delivery module includes a liquid reservoir, a degasser, an infusion pump and/or an elution device.
  • Reservoir used to store enough mobile phase to meet the requirements. Equipped with a solvent filter to prevent particles in the mobile phase from entering the pump.
  • Degasser The purpose of degassing is to prevent the release of air bubbles into the detector when the mobile phase flows out of the chromatographic column, which will cause noise and cannot be detected normally.
  • Infusion pump The mobile phase in the liquid reservoir continuously enters the liquid circuit system in the form of high pressure, so that the sample can complete the separation process in the chromatographic column.
  • Elution device It is a device that increases the elution capacity by gradually changing the composition of the mobile phase during the separation process.
  • the sample injection module includes a sample injector.
  • the injector is a device that feeds the sample into the chromatographic column.
  • injection methods There are two types of injection methods: valve injection or automatic injection. More commonly used is the use of automatic sampler loading.
  • the separation module includes a chromatographic column.
  • the chromatographic column separates the sample and is the heart of the entire chromatographic system. Its quality directly affects the separation effect.
  • the detector converts the sample components continuously flowing out of the chromatographic column into easily measurable electrical signals, which are received by the data system to obtain a chromatogram of sample separation.
  • the data processing and recording module processes the chromatographic data and participates in the automatic control of the HPLC instrument.
  • the device or system is a high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • composition of the present invention is preferably a pharmaceutical composition.
  • compositions of the present invention may also include a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid, semisolid, liquid or gel fillers which are suitable for human or animal use and which must be of sufficient purity and low enough toxicity. "Compatibility” refers to the components in the pharmaceutical composition and the active ingredients of the drug and their intermingling with each other without significantly reducing the efficacy of the drug.
  • the pharmaceutically acceptable carrier is not particularly limited, and materials commonly used in the art can be selected, or prepared by conventional methods, or purchased from the market.
  • pharmaceutically acceptable carrier moieties include cellulose and its derivatives (such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffers, chelating agents, thickeners, pH adjusters, skin penetration enhancers, colorants, flavors, stabilizers, antioxidants, preservatives ,
  • cellulose and its derivatives such as
  • the dosage form of the composition of the present invention is not particularly limited, and can be a solid preparation, a semi-solid preparation or a liquid preparation.
  • the dosage form of the composition of the present invention is an oral preparation, an injection preparation or an external preparation.
  • the dosage form of the composition of the present invention is an external skin preparation or a subcutaneous injection preparation.
  • the drug formulation should match the mode of administration.
  • the agents of the present invention may also be used (including before, during or after use) with other synergistic therapeutic agents.
  • a safe and effective amount of the drug is administered to the desired subject (eg, a human or non-human mammal), which is usually at least about 10 micrograms per kilogram of body weight, and in most cases Not to exceed about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the present invention unexpectedly finds for the first time that unsaturated long-chain fatty acids (such as linoleic acid, oleic acid, palmitoleic acid) can significantly inhibit the production of lymphocyte IL-17A, thereby preventing and/or treating silver associated with IL-17A. Psoriasis.
  • unsaturated long-chain fatty acids such as linoleic acid, oleic acid, palmitoleic acid
  • the present invention unexpectedly finds for the first time that the recurrence of psoriasis can be effectively predicted by detecting the ratio of long-chain unsaturated fatty acid/long-chain saturated fatty acid (UFA/SFA).
  • Serum sample processing 50 ⁇ L of plasma samples were frozen and thawed at room temperature, 200 ⁇ L of pre-cooled methanol was added, vortexed for 5 minutes, and after standing for 10 minutes, centrifuged at 12 000 rpm for 15 minutes at 4°C. Pipette an appropriate amount of supernatant for metabolomic analysis.
  • Serum gas chromatography-mass spectrometry (GC-MS) metabolomic analysis The sample-treated serum supernatant was completely dried with nitrogen at 40 °C, and then 50 ⁇ L of methoxyamine pyridine solution (15 mg/mL) was added and vortexed. Spin for 3 minutes, oximation at 40°C for 1 hour, then add 40 ⁇ L of N-methyl-N-(trimethylsilyl) trifluoroacetamide derivatization reagent, vortex for 3 minutes and derivatize at 40°C 1 hour, then centrifuged at 12 000 rpm for 10 minutes, and the supernatant was run in an injection vial for GC-MS analysis
  • the GC-MS analysis system consisted of a 7890B gas phase instrument and a 5977A single quadrupole mass spectrometer detector (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation adopts DB-5MS capillary column (30m ⁇ 250 ⁇ m ⁇ 0.25 ⁇ m). The injection volume was 1 ⁇ L, the split ratio was 20:1, and the helium flow rate was 1.2 mL/min. The temperatures of the injection port, mass spectrometer interface and mass spectrometer detector were set to 300°C, 280°C and 230°C, respectively. The initial temperature of the column oven was kept at 70 °C for 3 min, and the gradient was heated to 300 °C (5 °C/min) for 5 min. The gain factor was set to 2.0, and the ion source was electron impact (70 eV). The scanning range of mass spectrometry was 33-600 m/z, and the solvent delay time was set to 6 min.
  • Cell metabolomic analysis The cell culture medium was discarded, the cells were washed three times with phosphate buffered saline (PBS), and then the cells were quenched by adding liquid nitrogen into the centrifuge tube. After the liquid nitrogen was naturally volatilized, 500 ⁇ l of pre-cooled 20 ⁇ g/mL tridecanoic acid methanol solution was added, and the cells together with the methanol solution were subjected to ultrasonic extraction for 30 minutes, centrifuged at 12 000 rpm for 10 minutes, and the supernatant was taken. Derivatization after drying.
  • PBS phosphate buffered saline
  • peripheral blood mononuclear cells The peripheral blood mononuclear cells isolated from patients were seeded in 96-well plates, and the cell density was about 1*10 6 cell/ mL of 1640 medium containing 10% fetal bovine serum (FBS) in a volume of 200 ⁇ L/well, and cultured at 37° C. for 6-8 h.
  • FBS fetal bovine serum
  • cell stimulator Cell Stimulation Cocktail, Cat#423304, Biolegend, San Diego, CA
  • 1 ⁇ L of 12.5-200 mg/mL DMSO solutions of various fatty acids were added so that the final concentration of lactic acid in the cell culture medium was 100 ⁇ g/mL, and A positive control, a negative control and a blank control group were established and incubated at 37°C for 4 h.
  • Collect cells into flow tubes add 1 mL of PBS to each tube, wash cells at 1500 rpm for 5 min, and discard the supernatant; add human Fc block 1 ⁇ L/tube to block FcR on the cell surface, and incubate at 4°C for 20 min in the dark to avoid non-specific staining; cell surface marker staining (CD3-PE, CD8-PE-Cy7, viability-FITC), add labeled antibodies to each tube, and incubate at 4°C for 30 min in the dark; after washing the cells, add 400 ⁇ L of fixative to fix the cells at room temperature for 20 min; add 1 mL of permeabilization to each tube buffer, wash twice at 1500rpm for 5min, and discard the supernatant; for intracellular IL-17A staining, add the labeled antibody to each tube and incubate at 4°C for 1h in the dark; add 1mL of PBS to each tube, wash the cells at 1500rpm for 5min, discard the supernatant
  • the psoriasis patient is a moderate to severe plaque psoriasis patient.
  • Unsaturated fatty acids are referred to as UFA; saturated fatty acids are referred to as SFA;
  • the substitution value of each fatty acid in the formula is its peak area value (representing its serum concentration level) in a gas chromatography-mass spectrometry (GC-MS) device.
  • GC-MS gas chromatography-mass spectrometry
  • the ratio (UFA/SFA) of monounsaturated fatty acids (UFAs, which includes palmitoleic, oleic, and linoleic acids) to saturated fatty acids (SFAs, which includes palmitic and stearic acids), is significantly lower in the skin of psoriasis patients in the normal healthy control group.
  • IL-17A monoclonal antibody (Eli Lilly (China), Taltz (Tokyo) ), ixekizumab) treatment in serum further decreased this ratio ( Figure 9), indicating that mAb treatment did not improve the UFA deficiency in the skin.
  • This example is the prediction of the proportion of unsaturated fatty acids on the recurrence of psoriasis after treatment
  • unsaturated fatty acids are referred to as UFA; saturated fatty acids are referred to as SFA;
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  • GC-MS gas chromatography-mass spectrometry

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Abstract

一种预防银屑病复发的内源性代谢物组合。一种长链不饱和脂肪酸的用途，用于制备组合物，组合物用于(i)抑制IL-17；和/或(ii)预防和/或治疗与IL-17相关疾病；长链不饱和脂肪酸选自下组：亚油酸、油酸、棕榈油酸，或其组合。一种2种长链不饱和脂肪酸和长链饱和脂肪酸或其检测试剂的用途，用于制备检测试剂或检测试剂盒，检测试剂或检测试剂盒用于对银屑病患者进行预后；长链不饱和脂肪酸(UFA)选自下组：亚油酸(C18:2,Linoleic acid)、油酸(C18:1,Oleic acid)、棕榈油酸(C16:1,Palmitoleic acid)，或其组合；长链饱和脂肪酸(SFA)选自下组：棕榈酸(C16:0,Palmitic acid)、硬脂酸(C18:0,Stearic acid)，或其组合。

Description

一种预防银屑病复发的内源性代谢物组合 技术领域
本发明涉及医学生物检测技术领域，具体地一种预防银屑病复发的内源性代谢物组合。
背景技术
银屑病俗称牛皮癣，是一种慢性炎症性皮肤病，病程较长，有易复发倾向，有的病例几乎终生不愈。该病发病以青壮年为主，对患者的身体健康和精神状况影响较大。临床表现以红斑，鳞屑为主，全身均可发病，以头皮，四肢伸侧较为常见，多在冬季加重。
银屑病反复发作给病人带来很大的痛苦，因此，本领域需要开发一种能够有效预防银屑病复发的方法。对治疗后复发风险较高的银屑病患者制定针对性的治疗方案可以降低复发风险。然而，现有技术中，缺乏对银屑病复发机制的认识，从而不利于患者预防复发。
因此，本领域需要开发一种能够有效预防银屑病复发的方法。银屑病患者的系统性代谢可能对其致病性的IL-17A分泌产生重要影响。通过研究患者系统性代谢在复发过程中的变化，有望寻找到一种安全有效的预防银屑病复发的内源性代谢物组合。
发明内容
本发明的目的在于提供一种长链不饱和脂肪酸用于抑制IL-17和/预防和/或治疗与IL-17相关疾病。
本发明的另一目的在于提供一种长链不饱和脂肪酸和长链饱和脂肪酸或其检测试剂的用途用于对银屑病患者进行预后。
本发明第一方面，提供一种长链不饱和脂肪酸的用途，用于制备组合物，所述的组合物用于(i)抑制IL-17；和/或(ii)预防和/或治疗与IL-17相关疾病；
所述的长链不饱和脂肪酸选自下组：亚油酸(C18:2,Linoleic acid)、油酸(C18:1,Oleic acid)、棕榈油酸(C16:1,Palmitoleic acid)，或其组合。
本发明第二方面，提供一种长链不饱和脂肪酸和长链饱和脂肪酸或其检测试剂的用途，用于制备检测试剂或检测试剂盒，所述的检测试剂或检测试剂盒用于对银屑病患者进行预后；
所述的长链不饱和脂肪酸(UFA)选自下组：亚油酸(C18:2,Linoleic acid)、油酸(C18:1,Oleic acid)、棕榈油酸(C16:1,Palmitoleic acid)，或其组合；
所述的长链饱和脂肪酸(SFA)选自下组：棕榈酸(C16:0,Palmitic acid)、硬脂酸(C18:0,Stearic acid)，或其组合。
在另一优选例中，所述的与IL-17相关疾病包括银屑病。
在另一优选例中，所述的银屑病包括斑块型银屑病患者。
在另一优选例中，所述的斑块型银屑病包括中至重度斑块型银屑病患者。
在另一优选例中，所述的预防包括预防银屑病的复发。
在另一优选例中，所述的预后包括银屑病治疗药物停药后的预后。
在另一优选例中，所述的预后包括银屑病治疗药物停药后的预后，且银屑病治疗药物已经治疗6-8周，优选为8周。
在另一优选例中，所述的银屑病治疗药物包括IL-17抗体。
在另一优选例中，所述的预后包括IL-17抗体停药后的预后。
在另一优选例中，所述的IL-17A抗体包括依奇珠单抗。
在另一优选例中，所述的IL-17抗体包括单克隆抗体或多克隆抗体。
在另一优选例中，所述的组合物为药物组合物。
在另一优选例中，所述的组合物还包括药学上可接受的载体。
在另一优选例中，所述的组合物的剂型为固体制剂、半固体制剂或液体制剂。
在另一优选例中，所述的组合物的剂型为口服制剂、注射制剂或外用制剂。
在另一优选例中，所述的组合物的剂型为皮肤外用制剂或皮下注射制剂。
在另一优选例中，所述的预后包括预测停药后复发快慢。
在另一优选例中，所述的预后为体外预后。
在另一优选例中，所述进行预后的银屑病患者包括IL-17A抗体治疗停药后患者。
在另一优选例中，所述进行预后的样品为血样。
在另一优选例中，进行预后的样品为血样。
在另一优选例中，所述的血样包括血清、血浆或血液。
在另一优选例中，所述的预后是通过检测样品中UFA/SFA的比例进行预后在另一优选例中，所述的样品包括血清、血浆或血液。
在另一优选例中，所述的样品来源于银屑病患者。
在另一优选例中，所述的检测试剂盒还包标签或使用说明书，所述的标签或使用说明书记载以下内容：
当长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例(UFA/SFA)小于0.364为较早复发，大于等于0.364为较晚复发；
其中，UFA/SFA的计算公式如下：
Figure PCTCN2021131923-appb-000001
在另一优选例中，在UFA/SFA的计算公式中，亚油酸、油酸、棕榈油酸、棕榈酸和硬脂酸的各代入值为血清浓度值。
在另一优选例中，在UFA/SFA的计算公式中，各脂肪酸代入值为其在气相色谱-质谱联用仪(GC-MS)装置中的峰面积值。
在另一优选例中，长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例为银屑病患者经银屑病治疗药物治疗6-8周，优选为8周时的比例。
本发明第三方面，提供一种检测试剂盒，所述的检测试剂盒包括：
(a)长链不饱和脂肪酸和长链饱和脂肪酸或其检测试剂；
所述的长链不饱和脂肪酸(UFA)选自下组：亚油酸、油酸、棕榈油酸，或其组合；
所述的长链饱和脂肪酸(SFA)选自下组：棕榈酸、硬脂酸，或其组合。
本发明第四方面，提供一种如本发明第三方面所述的检测试剂盒的用途，用于制备一诊断试剂盒，所述诊断试剂盒用于对银屑病患者进行预后。
在另一优选例中，所述的诊断试剂盒还包标签或使用说明书，所述的标签或使用说明书记载以下内容：
当长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例(UFA/SFA)小于0.364为较早复发，大于等于0.364为较晚复发；
其中，UFA/SFA的计算公式如下：
Figure PCTCN2021131923-appb-000002
在另一优选例中，在UFA/SFA的计算公式中，亚油酸、油酸、棕榈油酸、棕榈酸和硬脂酸的各代入值为血清浓度值。
在另一优选例中，在UFA/SFA的计算公式中，各脂肪酸代入值为其在气相色谱-质谱联用仪(GC-MS)装置中的峰面积值。
在另一优选例中，长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例为银屑病患者经银屑病治疗药物治疗6-8周，优选为8周时的比例。
在另一优选例中，所述诊断试剂盒的检测样品为血样。
在另一优选例中，所述的血样包括血清、血浆或血液。
本发明第五方面，提供一种装置或系统，所述装置或系统包括：
(1)检测模块，所述的检测模块用于对样品中的长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的含量或含量比例(UFA/SFA)进行测定；
(2)输出模块，所述的输出模块输出根据所述的检测模块对样品中的长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的含量或含量比例(UFA/SFA)对银屑病患者进行预后的信息。
在另一优选例中，所述的输出模块输出的信息包括：
当长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例(UFA/SFA)小于0.364为较早复发，大于等于0.364为较晚复发；
其中，UFA/SFA的计算公式如下：
Figure PCTCN2021131923-appb-000003
在另一优选例中，在UFA/SFA的计算公式中，亚油酸、油酸、棕榈油酸、棕榈酸和硬脂酸的各代入值为血清浓度值。
在另一优选例中，在UFA/SFA的计算公式中，各脂肪酸代入值为其在气相色谱-质谱联用仪(GC-MS)装置中的峰面积值。
在另一优选例中，长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例为银屑病患者经银屑病治疗药物治疗6-8周，优选为8周时的比例。
在另一优选例中，所述的样品包括血清、血浆或血液。
在另一优选例中，所述的样品来源于银屑病患者。
在另一优选例中，所述的装置为气相色谱-质谱联用仪(GC-MS)或高效液相色谱仪。
在另一优选例中，所述的装置或系统还包括溶剂输送模块、进样模块、分离模块、检测器、数据处理和记录模块。
在另一优选例中，所述的溶剂输送模块包括储液器、脱气器、输液泵和/或洗脱装置。
在另一优选例中，所述的进样模块包括进样器。
在另一优选例中，所述的分离模块包括色谱柱。
在另一优选例中，所述的装置或系统为高效液相色谱仪(HPLC)。
本发明的目的在于提供一种应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1:部分代表性细胞流式分析图，该图表明不同脂肪酸对银屑病患者血中分泌IL-17A的T细胞具有不同调控能力。图中Unstimulated表示未经过刺激的T细胞；Stimulated表示加入刺激试剂增强IL-17A分泌能力的T细胞，其分泌IL-17A的细胞比例显著增加(以下简称Sti)；Sti+DMSO表示在刺激细 胞时加入二甲基亚砜(DMSO)，DMSO为加入其他脂肪酸共同刺激细胞时的空白溶剂，这时T细胞分泌IL-17A的能力不受影响；Sti+Palmitoleic acid表示在刺激T细胞时加入棕榈油酸(C16:1,Palmitoleic acid)，这时分泌IL-17A的T细胞比例大幅降低；Sti+Azelaic acid表示在刺激T细胞时加入壬二酸，这时分泌IL-17A的T细胞比例略有降低；Sti+Stearic acid表示在刺激T细胞时加入硬脂酸(C18:0,Stearic acid)，这时分泌IL-17A的T细胞比例基本无变化。
图2:不同脂肪酸对银屑病患者外周血单个核细胞分泌IL-17A能力的调控作用(n＝10)。该图表明不饱和脂肪酸亚油酸(C18:2,Linoleic acid)、油酸(C18:1,Oleic acid)和棕榈油酸(C16:1,Palmitoleic acid)对IL-17A产生具有显著抑制作用。
图3：银屑病患者血清主要脂肪酸含量测定的标准曲线。
[根据细则91更正 14.03.2022]　
图4A和4B：通过体外试验确定长链脂肪酸浓度为12.5μg/mL时，人外周血单个核细胞的活力不受影响。体外实验表明，在培养基中脂肪酸浓度为200μg/mL、50μg/mL和25μg/mL时，人外周血单个核细胞的活力会降低。当脂肪酸浓度降为12.5μg/mL时，细胞活力不受影响。在细胞活力不受影响的条件下，研究脂肪酸对IL-17A产生细胞的调控作用，才能避免由于细胞活力下降导致的IL-17A降低，同时确保IL-17A分泌能力的变化完全是由于在一定脂肪酸环境下细胞作出的正常生理反应。
[根据细则91更正 14.03.2022]　
图5A和5B：10名银屑病患者外周血单个核细胞(PBMCs)在不同长链脂肪酸环境下分泌IL-17A能力变化的细胞流式分析图。图中行表示每位患者的分析数据，列表示不同处理条件下的分析数据，每张小图中的数字表示PBMCs中分泌IL-17A的T细胞比例。
图6：依奇珠单抗治疗8周(W8)后UFA/SFA比例高的银屑病患者较晚复发，而该比例低的患者较早复发。
图7:ROC分析显示治疗后较早复发和较晚复发患者血清中UFA/SFA比例的临界值为0.364，即UFA/SFA比例低于0.364为较早复发，高于0.364为较晚复发。
图8:ROC分析得到的治疗后UFA/SFA比例箱型图，红线处为最佳临界值 0.364，UFA/SFA比例低于0.364为较早复发，高于0.364为较晚复发。
图9：IL-17A单抗依奇珠单抗治疗8周(W8)后的血清中UFA/SFA比例。
具体实施方式
本发明人经过广泛而又深入的研究，首次意外发现长链不饱和脂肪酸亚油酸、油酸和/或棕榈油酸能够有效抑制IL-17和预防和/或治疗与IL-17相关疾病如银屑病。此外，本发明还意外发现UFA/SFA比例能够作为对银屑病患者进行预后的判断指标，从能够对治疗后复发风险较高的银屑病患者制定针对性的治疗方案来降低复发风险。在此基础上，发明人完成了本发明。
术语
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。
如本文所用，术语“3-HB”为3-羟基丁酸。
如本文所用，术语“UFA”指的是长链不饱和脂肪酸。
如本文所用，术语“SFA”指的是长链饱和脂肪酸。
如本文所用，术语“小于等于”包括小于和/或等于。
如本文所用，术语“大于等于”包括大于和/或等于。
如本文所用，术语“IL-17A”指的白介素-17A。
在本发明中应当理解是是，“长链不饱和脂肪酸和长链饱和脂肪酸或其检测试剂”与“长链不饱和脂肪酸或其检测试剂，和长链饱和脂肪酸或其检测试剂”可互换使用。
长链不饱和脂肪酸或其检测试剂
在本发明中，长链不饱和脂肪酸选自下组：亚油酸、油酸、棕榈油酸，或其组合。
在本发明中，长链不饱和脂肪酸的检测试剂并没有特别的限定，只要满足准确检测即可。例如，长链不饱和脂肪酸检测试剂可以为抗体或适用高效液相 等方法的检测试剂。
长链饱和脂肪酸或其检测试剂
在本发明中，长链饱和脂肪酸(SFA)选自下组：棕榈酸、硬脂酸，或其组合。
在本发明中，长链饱和脂肪酸的检测试剂并没有特别的限定，只要满足准确检测即可。例如，长链饱和脂肪酸检测试剂可以为抗体或适用高效液相等方法的检测试剂。
银屑病
银屑病俗称牛皮癣，是一种慢性炎症性皮肤病，病程较长，有易复发倾向，有的病例几乎终生不愈。临床表现以红斑，鳞屑为主，全身均可发病，以头皮，四肢伸侧较为常见，多在冬季加重。
在本发明中，所述的银屑病优选为斑块型银屑病患者。
在另一优选例中，所述的斑块型银屑病包括中至重度斑块型银屑病患者。
样品
本发明所述的长链不饱和脂肪酸和长链饱和脂肪酸或其检测试剂、检测试剂盒、预后判断、诊断试剂、装置或系统的检测样品并没有特别的限定，优选为血样。
优选地，所述的血样包括血清、血浆或血液
优选地，本发明所述的样品来源所述的银屑病患者。
用途
本发明提供一种长链不饱和脂肪酸的用途，其用于制备组合物，所述的组合物用于(i)抑制IL-17；和/或(ii)预防和/或治疗与IL-17相关疾病。
优选地，所述的长链不饱和脂肪酸选自下组：亚油酸、油酸、棕榈油酸，或其组合。
在本发明的一个优选例中，所述的与IL-17相关疾病包括银屑病。
在另一优选例中，所述的预防包括预防银屑病的复发。
在本发明中，术语“预防”表示预防疾病和/或它的附随症状的发作、复发或者保护对象免于获得疾病的方法。本文中使用的"预防"还包括延迟疾病和/或它的附随症状的发作和降低对象的得病的风险。
本发明所述的“治疗”包括延缓和终止疾病的进展，或消除疾病，并不需要100％抑制、消灭和逆转。在一些实施方案中，与不存在本发明所述长链不饱和脂肪酸观察到的水平相比，本发明所述长链不饱和脂肪酸抑制IL-17和逆转银屑病例如至少约10％、至少约30％、至少约50％、或至少约80％。
本发明还提供一种长链不饱(UFA)和脂肪酸和长链饱和脂肪酸(SFA)或其检测试剂的用途，用于制备检测试剂或检测试剂盒，所述的检测试剂或检测试剂盒用于对银屑病患者进行预后。
优选地，所述的长链不饱和脂肪酸(UFA)选自下组：亚油酸、油酸、棕榈油酸，或其组合。
优选地，所述的长链饱和脂肪酸(SFA)选自下组：棕榈酸、硬脂酸，或其组合。
在另一优选例中，所述的预后包括银屑病治疗药物停药后的预后。
在另一优选例中，所述的预后包括银屑病治疗药物停药后的预后，且银屑病治疗药物已经治疗6-8周，优选为8周。
代表性地，所述的银屑病治疗药物包括IL-17抗体。优选地，所述的IL-17A抗体包括依奇珠单抗。
典型地，所述的预后包括IL-17抗体停药后的预后。
在本发明的另一优选例中，所述进行预后的样品为血样，例如血清、血浆或血液。
在本发明的另一优选例中，所述的预后是通过检测样品中UFA/SFA的比例进行预后。
在本发明的另一优选例中，所述的检测试剂盒还包标签或使用说明书，所述的标签或使用说明书记载以下内容：
当长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例(UFA/SFA)小于0.364为较早复发，大于等于0.364为较晚复发；
其中，UFA/SFA的计算公式如下：
Figure PCTCN2021131923-appb-000004
在另一优选例中，在UFA/SFA的计算公式中，亚油酸、油酸、棕榈油酸、棕榈酸和硬脂酸的各代入值为血清浓度值。
在另一优选例中，在UFA/SFA的计算公式中，各脂肪酸代入值为其在气相色谱-质谱联用仪(GC-MS)装置中的峰面积值。
在另一优选例中，长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例为银屑病患者经银屑病治疗药物治疗6-8周，优选为8周时的比例。
检测试剂盒
本发明还提供一种检测试剂盒，所述检测试剂盒包括：
(a)长链不饱和脂肪酸和长链饱和脂肪酸或其检测试剂；
所述的长链不饱和脂肪酸(UFA)选自下组：亚油酸、油酸、棕榈油酸，或其组合；
所述的长链饱和脂肪酸(SFA)选自下组：棕榈酸、硬脂酸，或其组合。
本发明还提供一种检测试剂盒的用途，用于制备一诊断试剂盒，所述诊断试剂盒用于对银屑病患者进行预后。
在另一优选例中，所述的诊断试剂盒还包标签或使用说明书，所述的标签或使用说明书记载以下内容：
当长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例(UFA/SFA)小于0.364为较早复发，大于等于0.364为较晚复发；
其中，UFA/SFA的计算公式如下：
Figure PCTCN2021131923-appb-000005
在另一优选例中，在UFA/SFA的计算公式中，亚油酸、油酸、棕榈油酸、棕榈酸和硬脂酸的各代入值为血清浓度值。
在另一优选例中，在UFA/SFA的计算公式中，各脂肪酸代入值为其在气相色谱-质谱联用仪(GC-MS)装置中的峰面积值。
在另一优选例中，长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例为银屑病患者经银屑病治疗药物治疗6-8周，优选为8周时的比例。
在另一优选例中，所述诊断试剂盒的检测样品为血样。
在另一优选例中，所述的血样包括血清、血浆或血液。
装置或系统
本发明还提供一种装置或系统，所述的装置或系统包括：
(1)检测模块，所述的检测模块用于对样品中的长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的含量或含量比例(UFA/SFA)进行测定；
(2)输出模块，所述的输出模块输出根据所述的检测模块对样品中的长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的含量或含量比例(UFA/SFA)对银屑病患者进行预后的信息。
在另一优选例中，所述的输出模块输出的信息包括：
当长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例(UFA/SFA)小于0.364为较早复发，大于等于0.364为较晚复发；
其中，UFA/SFA的计算公式如下：
Figure PCTCN2021131923-appb-000006
在另一优选例中，在UFA/SFA的计算公式中，亚油酸、油酸、棕榈油酸、棕榈酸和硬脂酸的各代入值为血清浓度值。
在另一优选例中，在UFA/SFA的计算公式中，各脂肪酸代入值为其在气相色谱-质谱联用仪(GC-MS)装置中的峰面积值。
在另一优选例中，长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例为银屑病患者经银屑病治疗药物治疗6-8周，优选为8周时的比例。
在另一优选例中，所述的样品包括血清、血浆或血液。
在另一优选例中，所述的样品来源于银屑病患者。
在另一优选例中，所述的装置为气相色谱-质谱联用仪(GC-MS)或高效液相色谱仪。
在另一优选例中，所述的装置或系统还包括溶剂输送模块、进样模块、分 离模块、检测器、数据处理和记录模块。
在另一优选例中，所述的溶剂输送模块包括储液器、脱气器、输液泵和/或洗脱装置。
储液器：用来贮存数量足够、符合要求的流动相。配有溶剂过滤器，以防止流动相中的颗粒进入泵内。
脱气器：脱气的目的是为了防止流动相从色谱柱内流出时释放出气泡进入检测器，从而引起噪声，不能正常检测。
输液泵：将储液器中的流动相连续不断地以高压形式进入液路系统，使样品在色谱柱中完成分离过程。
洗脱装置：是在分离过程中通过逐渐改变流动相的组成增加洗脱能力的一种装置。
在另一优选例中，所述的进样模块包括进样器。
进样器是将样品送入色谱柱的装置，进样方式可以分为两种：阀进样或自动进样。比较常用的是采用自动进样器装样。
在另一优选例中，所述的分离模块包括色谱柱。
色谱柱是对样品进行分离，是整个色谱系统的心脏，它的质量优劣直接影响到分离的效果。
检测器是将色谱柱连续流出的样品组分转变成易于测量的电信号，被数据系统接收，得到样品分离的色谱图。
数据处理和记录模块是对色谱数据进行处理，并参与HPLC仪器的自动控制。
在另一优选例中，所述的装置或系统为高效液相色谱仪(HPLC)。
组合物
本发明所述的组合物优选为药物组合物。本发明所述的组合物还可以包括药学上可接受的载体。
如本文所用“药学上可接受的载体”是指一种或多种相容性固体、半固体、液体或凝胶填料，它们适合于人体或动物使用，而且必须有足够的纯度和足够 低的毒性。“相容性”是指药物组合物中的各组分和药物的活性成分以及它们之间相互掺和，而不明显降低药效。
应理解，在本发明中，所述的药学上可接受的载体没有特别的限制，可选用本领域常用材料，或用常规方法制得，或从市场购买得到。药学可接受的载体部分例子有纤维素及其衍生物(如甲基纤维素、乙基纤维素、羟丙甲基纤维素、羧甲基纤维素钠等)、明胶、滑石粉、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油、等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温)、润湿剂(如十二烷基硫酸钠)、缓冲剂、螯合剂、增稠剂、pH调节剂、透皮促进剂、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、抑菌剂、无热原水等。
本发明所述的组合物的剂型并没有特别的限制，可以为为固体制剂、半固体制剂或液体制剂。
优选地，本发明所述的组合物的剂型为口服制剂、注射制剂或外用制剂。
代表性地，本发明所述的组合物的剂型为皮肤外用制剂或皮下注射制剂。
药物制剂应与给药方式相匹配。本发明药剂还可与其他协同治疗剂一起使用(包括之前、之中或之后使用)。使用药物组合物或制剂时，是将安全有效量的药物施用于所需对象(如人或非人哺乳动物)，所述安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点在于：
1.本发明首次意外发现不饱和长链脂肪酸(如亚油酸、油酸、棕榈油酸)能够显著抑制淋巴细胞IL-17A的产生，从而能够预防和/或治疗与IL-17A相关的银屑病。
2.本发明首次意外发现通过检测长链不饱和脂肪酸/长链饱和脂肪酸(UFA/SFA)的比例能够有效对银屑病的复发进行有效预测。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明 本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例
血清样本处理：50μL血浆样本室温下冻融，加入200μL预冷的甲醇，涡旋5分钟，静置10分钟后，4℃条件下12 000rpm速度离心15分钟。吸取适量上清液进行代谢组学分析。
血清气相色谱-质谱(GC-MS)代谢组学分析：经过样本处理的血清上清液在40℃条件下氮气将其完全吹干，然后加入甲氧胺吡啶溶液(15mg/mL)50μL后涡旋3分钟，在40℃条件下肟化1小时，再加入N-甲基-N-(三甲基硅烷基)三氟乙酰胺衍生化试剂40μL，涡旋3分钟后在40℃下衍生化1小时，然后在12 000rpm转速下离心10分钟，取上清液于进样小瓶中运行GC-MS分析
GC-MS分析系统由7890B气相仪和5977A单四级杆质谱检测器(Agilent Technologies,Santa Clara,CA,USA)组成。色谱分离采用DB-5MS毛细管色谱柱(30m×250μm×0.25μm)。进样量1μL，分流比20:1，氦气流速1.2mL/min。进样口、质谱接口和质谱检测器的温度分别设置为300℃、280℃和230℃。柱温箱起始温度70℃保持3min，梯度升温至300℃(5℃/min)保持5min.Gain factor设置为2.0，离子源为electron impact(70eV)。质谱扫描范围33-600m/z，溶剂延迟时间设为6min。
细胞代谢组学分析：细胞培养基弃去，磷酸缓冲盐溶液(PBS)冲洗细胞3次，随后将液氮加入离心管中淬灭细胞。待液氮自然挥发以后，加入预冷的20μg/mL十三酸(tridecanoic acid)甲醇溶液500微升，细胞连同甲醇溶液一起经过30分钟超声提取，在12 000rpm转速下离心10min，取上清液吹干后进行衍生化处理。加入甲氧胺吡啶溶液(15mg/mL)50μL后涡旋3分钟，在40℃条件下肟化1小时，再加入N-甲基-N-(三甲基硅烷基)三氟乙酰胺衍生化试剂40μL，涡旋3分钟后在40℃下衍生化1小时，然后在12 000rpm转速下离心10分钟，取上清液于进样小瓶中运行GC-MS分析。
脂肪酸对人外周血单个核细胞(PBMCs)分泌IL-17A能力的影响实验：将从病人分离得到的外周血单个核细胞(PBMCs)种于96孔板，细胞密度约为1*10 6cell/mL含有10％胎牛血清(FBS)的1640培养基，体积为200μL/孔，37℃培养6-8h。同时加入1μL细胞刺激剂(Cell Stimulation Cocktail,Cat#423304,Biolegend,San Diego,CA)和1μL 12.5-200mg/mL不同脂肪酸的DMSO溶液，使得细胞培养基中乳酸的终浓度为100μg/mL，并设立阳性对照、阴性对照和空白对照组，37℃培养4h。收集细胞至流式管，每管加入1mL PBS，1500rpm 5min洗涤细胞，弃上清；加入human Fc block 1μL/管封闭细胞表面FcR，4℃避光孵育20min，避免非特异性染色；细胞表面标记染色(CD3-PE，CD8-PE-Cy7,viability-FITC),分别将标记抗体加入各管，4℃避光孵育30min；洗涤细胞后加入固定剂400μL常温下固定细胞20min；每管加入1mL破膜buffer，1500rpm 5min洗涤2次，弃上清；胞内IL-17A染色,将标记抗体加入各管，4℃避光孵育1h；每管加入1mL PBS，1500rpm 5min洗涤细胞，弃上清，洗去未结合的多余抗体；加入200μL PBS上机检测。
实施例1
向银屑病患者皮肤中补充UFA至合适浓度可以治疗并预防银屑病的复发
在本实施例中，银屑病患者为中至重度斑块型银屑病患者。
不饱和脂肪酸简称UFA；饱和脂肪酸简称SFA；
UFA/SFA比例的计算方法为：
Figure PCTCN2021131923-appb-000007
公式各脂肪酸代入值为其在气相色谱-质谱联用仪(GC-MS)装置中的峰面积值(代表其血清浓度水平)。
银屑病患者皮肤中单不饱和脂肪酸(UFA，其包括棕榈油酸、油酸和亚油酸)与饱和脂肪酸(SFA，其包括棕榈酸和硬脂酸)的比例(UFA/SFA)显著低于正常健康对照组。IL-17A单抗(礼来制药(中国)，Taltz(拓咨
Figure PCTCN2021131923-appb-000008
)，依奇珠单抗)治疗后血清中该比例进一步下降(图9)，表明单抗治疗并没有改善皮肤中UFA缺乏的状况。且银屑病患者经过单抗治疗后，血清中UFA比例高的患者更不容易 复发(图6-图8)。银屑病人皮肤中的UFA缺乏，可能是UFA由于某种原因游离至血液和皮肤外导致的。由此造成皮肤中的IL-17A无法得到抑制，成为银屑病复发的关键因素，因此需要向皮肤中补充UFA治疗和预防银屑病复发。体外试验表明，在12μg/mL浓度下，UFA可以显著抑制淋巴细胞IL-17A的产生而不影响细胞的活性(图1-图5)。因此，通过皮肤外用UFA(包括棕榈油酸、油酸和亚油酸)的方法向银屑病患者皮肤中补充UFA至合适浓度，可以治疗并预防银屑病的复发。
实施例2
本实施例为不饱和脂肪酸比例对银屑病治疗后复发情况的预测
首先考察不饱和脂肪酸对IL-17A通路的作用，加入一定浓度脂肪酸以观察对IL-17产生细胞的作用。用Phorbol-12-myristate-13-acetate(PMA)和ionomycin激活银屑病患者PBMCs，使产生IL-17A的细胞数量扩增。同时加入不同脂肪酸进行共刺激，观察不同脂肪酸对IL-17A产生细胞数量的影响。共刺激时添加的脂肪酸在血清中的浓度均为12.5μg/mL，该浓度下细胞的活性不受影响(图4)。结果显示，不饱和长链脂肪酸(亚油酸、油酸、棕榈油酸)显著减少产生IL-17A细胞的数量，而饱和脂肪酸、壬二酸和3-HB(3-羟基丁酸)等脂肪酸以及溶剂DMSO(二甲基亚砜)都没有显著抑制IL-17A产生的作用(图1-图2)。
此外还发现，血清中的UFA/SFA在IL-17A单抗治疗时也是与停药(IL-17A单抗礼来制药(中国)，Taltz(拓咨
Figure PCTCN2021131923-appb-000009
)，依奇珠单抗)治疗)后复发相关的指标。即不饱和脂肪酸较高的患者复发时间较晚(图6-图8)，表明较高比例的不饱和脂肪酸水平可能有助于缓解IL-17A导致的银屑病复发。
其中，不饱和脂肪酸简称UFA；饱和脂肪酸简称SFA；
UFA/SFA比例的计算方法为：
Figure PCTCN2021131923-appb-000010
公式各脂肪酸代入值为其在气相色谱-质谱联用仪(GC-MS)装置中的峰面积值(代表其血清浓度水平)。
从图6-图8中得知，UFA/SFA的比例在治疗后第8周时，UFA/SFA比例低 于0.364为较早复发，高于0.364为较晚复发。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种长链不饱和脂肪酸的用途，其特征在于，用于制备组合物，所述的组合物用于(i)抑制IL-17；和/或(ii)预防和/或治疗与IL-17相关疾病；
    所述的长链不饱和脂肪酸选自下组：亚油酸(C18:2,Linoleic acid)、油酸(C18:1,Oleic acid)、棕榈油酸(C16:1,Palmitoleic acid)，或其组合。
  2. 一种长链不饱和脂肪酸和长链饱和脂肪酸或其检测试剂的用途，其特征在于，用于制备检测试剂或检测试剂盒，所述的检测试剂或检测试剂盒用于对银屑病患者进行预后；
    所述的长链不饱和脂肪酸(UFA)选自下组：亚油酸(C18:2,Linoleic acid)、油酸(C18:1,Oleic acid)、棕榈油酸(C16:1,Palmitoleic acid)，或其组合；
    所述的长链饱和脂肪酸(SFA)选自下组：棕榈酸(C16:0,Palmitic acid)、硬脂酸(C18:0,Stearic acid)，或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述的与IL-17相关疾病包括银屑病。
  4. 如权利要求2所述的用途，其特征在于，所述的预后包括IL-17抗体停药后的预后；
    优选地，所述的IL-17A抗体包括依奇珠单抗。
  5. 如权利要求1所述的用途，其特征在于，所述的组合物的剂型为皮肤外用制剂或皮下注射制剂。
  6. 如权利要求2所述的用途，其特征在于，所述进行预后的样品为血样。
  7. 如权利要求2所述的用途，其特征在于，所述的检测试剂盒还包标签或使用说明书，所述的标签或使用说明书记载以下内容：
    当长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的比例(UFA/SFA)小于0.364为较早复发，大于等于0.364为较晚复发；
    其中，UFA/SFA的计算公式如下：
    Figure PCTCN2021131923-appb-100001
  8. 一种检测试剂盒，其特征在于，所述的检测试剂盒包括：
    (a)长链不饱和脂肪酸和长链饱和脂肪酸或其检测试剂；
    所述的长链不饱和脂肪酸(UFA)选自下组：亚油酸、油酸、棕榈油酸，或其组合；
    所述的长链饱和脂肪酸(SFA)选自下组：棕榈酸、硬脂酸，或其组合。
  9. 一种如权利要求8所述的检测试剂盒的用途，其特征在于，用于制备一诊断试剂盒，所述诊断试剂盒用于对银屑病患者进行预后。
  10. 一种装置或系统，其特征在于，所述装置或系统包括：
    (1)检测模块，所述的检测模块用于对样品中的长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的含量或含量比例(UFA/SFA)进行测定；
    (2)输出模块，所述的输出模块输出根据所述的检测模块对样品中的长链不饱和脂肪酸(UFA)与长链饱和脂肪酸(SFA)的含量或含量比例(UFA/SFA)对银屑病患者进行预后的信息。
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