WO2020135872A1 - 免疫抑制药物组合物及其应用 - Google Patents

免疫抑制药物组合物及其应用 Download PDF

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WO2020135872A1
WO2020135872A1 PCT/CN2019/129945 CN2019129945W WO2020135872A1 WO 2020135872 A1 WO2020135872 A1 WO 2020135872A1 CN 2019129945 W CN2019129945 W CN 2019129945W WO 2020135872 A1 WO2020135872 A1 WO 2020135872A1
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forskolin
coch
compound
pharmaceutical composition
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PCT/CN2019/129945
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French (fr)
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巩晓明
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上海明隽生物技术有限公司
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Priority to US17/419,554 priority Critical patent/US20220079915A1/en
Priority to EP19901505.8A priority patent/EP3903778A4/en
Priority to AU2019414693A priority patent/AU2019414693A1/en
Priority to CA3125226A priority patent/CA3125226C/en
Priority to CN201980087113.0A priority patent/CN113226304B/zh
Publication of WO2020135872A1 publication Critical patent/WO2020135872A1/zh

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • 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
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/92—Naphthopyrans; Hydrogenated naphthopyrans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels

Definitions

  • the present invention relates to drugs and pharmaceutical compositions for immunosuppression and their applications, in particular to drugs, pharmaceutical compositions and methods for treating psoriasis using forskolin derivatives.
  • Psoriasis is a chronic skin inflammation that seriously affects the quality of human life and is an autoimmune disease. Common symptoms are red, scaly, patchy dermatoses distributed on the scalp, elbows, knees, etc., also known as psoriasis. The histological features of the lesion skin are marked thickening of the epidermis, invasion of immune cells in the epidermis, and an increase in the number of dilated blood vessels in the epithelium.
  • tumor necrosis factor ⁇ tumor necrosis factor ⁇
  • Interleukin 17A interleukin 17A
  • Tumor necrosis factor alpha alone or in combination with other pro-inflammatory factors such as interleukin 23 (Interleukin 23), interleukin 17 (Interleukin 17), etc., starts the downstream immune response. Therefore, tumor necrosis factor alpha, interleukin 17A, interleukin 23, etc. are all psoriasis clinical pathological indicators and high-value therapeutic targets.
  • TNF- ⁇ inhibitors are the first class of biological drugs used to treat psoriasis.
  • TNF- ⁇ receptor Fc fusion protein Since Etanercept was approved for marketing in 1998, it has become the standard reference for the development of psoriasis drugs; TNF- ⁇ monoclonal antibody adamu has also been upgraded from the second-line drug use in the clinical system to the first-line drug for moderate to severe chronic plaque psoriasis.
  • Interleukin 17A is a new target of immunomodulation that has been gaining attention since 2000. This is related to the discovery of T helper cell 17 (Th17) in 2005, a milestone in immunology. With the in-depth research and development of the pharmaceutical industry, from 2015 to 2016, Novartis' Secukinumab and Eli Lilly's Ixekizumab were the first to be listed as IL-17A monoclonal antibody drugs, and showed superior clinical efficacy and safety in the treatment of psoriasis.
  • the IL-17A antibody Compared with other target drugs, the IL-17A antibody showed a stronger clinical response in reaching the psoriasis area and inflammation index (Psoriasis area and severity index, PASI) PASI90 and PASI100 moderate to severe psoriasis responses, and The efficacy persists in both short-term and long-term treatment. According to the above performance, in 2015, "Nature" magazine evaluated "anti-interleukin 17 drugs will become the standard treatment for psoriasis.” Compared with other targets, IL-17A is currently recognized as a psoriasis treatment target with the highest pathological relevance, the most significant clinical response, superior efficacy and high safety.
  • macromolecular drugs such as antibodies
  • on the principle mechanism macromolecular drugs are easy to produce immunogenicity and are difficult to target intracellular targets
  • in production and application the preparation process of macromolecular drugs is complicated, the quality standards are difficult to unify, and production
  • the cost of transportation is high; most importantly, in terms of price and market, macromolecular drugs are expensive, especially not suitable for countries and regions with large populations and heavy medical security burdens, such as China. People who can tolerate injections.
  • small-molecule chemical drugs Although the potency of small-molecule chemical drugs is usually slightly lower than that of antibody drugs, it still has many advantages, such as usually oral administration, simple preparation process, low production cost, no immunogenicity, and can target intracellular targets, etc. Therefore, small molecule drugs that can effectively inhibit TNF- ⁇ or/and IL-17A are expected to become strong competitors or even market terminators of similar antibody drugs, and this field is due to many links in flux screening, medicinal chemistry, translational medicine, etc. There are extremely high professional barriers and practical challenges. At present, the market is basically blank.
  • immunosuppressive therapy with the basic characteristics and methods of reducing/suppressing pro-inflammatory factors is also widely applicable to many other diseases and symptoms, such as inflammation caused by infection or surgical trauma, including rheumatoid arthritis,
  • diseases and symptoms such as inflammation caused by infection or surgical trauma, including rheumatoid arthritis
  • autoimmune diseases including psoriatic arthritis
  • the market value of various autoimmune diseases based on immunosuppressive therapy totals more than US$60 billion.
  • foreign pathogen stimulation or pathological autoimmune response enhancement lymphatic T cells, macrophages, etc.
  • pro-inflammatory factors as key signaling molecules to initiate the body's inflammatory response
  • representative factors such as TNF- ⁇ , IL-17, Interleukin 6 (Interleukin 6), interferon ⁇ (Interferon- ⁇ ), etc. Therefore, the increased expression of pro-inflammatory factors is the main pathological diagnosis and cause of the inflammatory response and immune hyperstimulation process. It is the mainstream strategy of pharmaceutical research and development and clinical practice to achieve immunosuppression through the combination of antibody and drug inhibition of pro-inflammatory factors or their receptors.
  • immunosuppressor small molecule immunosuppressors used to eliminate inflammation (Anti-inflammation) and immune function suppression (Immunosuppression).
  • Clinically commonly used fast-acting immunosuppressants such as steroids
  • Dexamethasone Hydrocortisone
  • methylprednisolone Methylprednisolone
  • COX-2 cyclooxygenase 2
  • neutrophils and macrophages reduce the pro-inflammatory cytokine TNF- ⁇ , IL-1 ⁇ , etc.
  • have clinical practice and efficacy in the treatment of autoimmune diseases such as rheumatoid arthritis, asthma, Crohn's disease; in addition, methotrexate (Methotrexate), hydroxychloroquine (Hydroxychloroquine), etc.
  • Slower-acting immunosuppressive agents can inhibit the activity of immune cells by interfering with the synthesis of genetic material of immune cells and excessive cell proliferation, and can also reduce the level of proinflammatory factors such as IL-6. It is widely used in rheumatoid arthritis and systemic lupus erythematosus (Systemic Lupuserythematosus (SLE) and other autoimmune diseases are treated, but the above small molecule immunosuppressants all have several shortcomings, such as insufficient efficacy, easy to produce drug resistance, and long-term side effects.
  • the newly developed small-molecule drugs for immunosuppression also include JAK (Janus kinase) inhibitors, but they also have the defects of insufficient specificity against different subtypes of JAK and extensive side effects on immunomodulation.
  • JAK Japanese kinase
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof (also referred to herein as a forskolin derivative):
  • R 1 and R 2 is -COCH 2 CH 3 , -CO 2 CH 2 CH 3 , -COCH 2 OCHO or group
  • each R 4 and R 5 is independently hydrogen or lower alkyl, or R 4 and R 5 combine to form a lower alkylene chain, the lower alkylene chain contains or does not contain an oxygen atom or a nitrogen atom, m is an integer from 1 to 5;
  • the other of R 1 and R 2 is hydrogen or the group CO(CH 2 ) n X, where X is hydrogen or a group
  • R 6 and R 7 are independently hydrogen or lower alkyl, or R 6 and R 7 are combined to form a lower alkylene chain, the lower alkylene chain may or may not contain an oxygen atom or a nitrogen atom, n An integer from 1 to 5; or
  • R 1 is hydrogen or -COCH 2 CH 2 CO 2 H
  • R 2 is hydrogen, -COCH 3 , -COCH 2 CH 2 CH 2 CO 2 H or -COCH(OH)CH 2 OH, provided that R 1 is hydrogen
  • R 2 is -COCH 2 CH 2 CH 2 CO 2 H or -COCH(OH)CH 2 OH.
  • R 1 in formula I is hydrogen or a group Where m, R 4 and R 5 are as defined above.
  • R 1 in Formula I is -COCH 2 N(CH 3 ) 2 , -CO(CH 2 ) 2 N(CH 3 ) 2 , -CO(CH 2 ) 3 N(CH 3 ) 2 , Or -CO(CH 2 ) 3 NH 2 , and R 2 is -COCH 3 .
  • R 1 in formula I is hydrogen
  • R 2 is -COCH 2 CH 3 , -CO 2 CH 2 CH 3 or -COCH 2 OCHO
  • the pharmaceutical composition is administered in combination with a prostaglandin compound.
  • the pharmaceutical composition further includes prostaglandins.
  • the pharmaceutical composition is administered in combination with a glucocorticoid compound.
  • the pharmaceutical composition further includes a glucocorticoid compound.
  • the pharmaceutical composition is used for immunosuppression in a subject.
  • the pharmaceutical composition is used to treat autoimmune diseases in a subject.
  • the pharmaceutical composition is used as an anti-inflammatory drug.
  • the pharmaceutical composition is used to treat psoriasis in a subject.
  • the pharmaceutical composition is used to treat psoriatic arthritis (Psoriatic arthritis, PsA) in a subject.
  • Psoriatic arthritis PsA
  • the pharmaceutical composition is used to inhibit tumor necrosis factor alpha (TNF- ⁇ ) levels in a subject.
  • TNF- ⁇ tumor necrosis factor alpha
  • the pharmaceutical composition is used to inhibit interleukin 17A (IL-17A) levels in a subject.
  • IL-17A interleukin 17A
  • the present disclosure provides a method of immunosuppression in a subject, comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 and R 3 As defined above.
  • the method includes administering the prostaglandin compound to the subject.
  • the method includes administering to the subject in combination with a glucocorticoid compound.
  • the present disclosure provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for immunosuppression, wherein R 1 , R 2 and R 3 are as defined above.
  • the medicament is for co-administration with glucocorticoid compounds.
  • the medicament is for co-administration with prostaglandins.
  • the drug is an autoimmune disease drug.
  • the drug is a psoriasis drug.
  • the drug is a psoriatic arthritis drug.
  • the drug is an anti-inflammatory drug.
  • the compound of formula I is selected from 6-(4-aminobutyryl)forskolin, 6-[4-(dimethylamino)butyryl]forskolin, 6-[3 -Aminopropionyl]forskolin, 6-[3-(methylamino)propionyl]forskolin, 6-[3-(dimethylamino)propionyl]forskolin, and 6-[(piperidine) Acetyl]-7-deacetylated forskolin.
  • the pharmaceutically acceptable salt is the hydrochloride salt.
  • the pharmaceutically acceptable salt of the compound of Formula I is 6-[3-(dimethylamino)propionyl]forskolin hydrochloride.
  • the pharmaceutically acceptable salt of the compound of Formula I is 6-[3-(methylamino)propionyl]forskolin hydrochloride.
  • 6-[3-(dimethylamino)propionyl]forskolin hydrochloride is administered in combination with prostaglandin E2.
  • 6-[3-(dimethylamino)propionyl]forskolin hydrochloride is administered in combination with dexamethasone.
  • the compound of formula I or a pharmaceutically acceptable salt thereof works by reducing the expression of TNF- ⁇ of immune cells in the subject.
  • the compound of Formula I or a pharmaceutically acceptable salt thereof works by reducing IL-17A expression of immune cells in the subject.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising 1) forskolin; and 2) a prostaglandin compound or a glucocorticoid compound.
  • the pharmaceutical composition is used to treat psoriasis or psoriatic arthritis in a subject.
  • the present disclosure provides a method of immunosuppression in a subject, comprising administering 1) forskolin and 2) a prostaglandin or glucocorticoid compound to the subject.
  • the subject is a patient with psoriasis or a patient with psoriatic arthritis.
  • the present disclosure provides the use of forskolin in the preparation of a medicament administered in combination with prostaglandins or glucocorticoids.
  • the drug is a psoriasis drug or a psoriatic arthritis drug.
  • the prostaglandin compound is selected from the group consisting of prostaglandin E2, tromethamine dinoprost, carprost, tromethamine carprost, prostaglandin E1, bemeprost , Iloprost, limaprost, limaprost alpha cyclodextrin, misoprost, gemeprost, latanoprost, thioprostone, ornoprost and their pharmacologically acceptable Salt.
  • the glucocorticoid compound is selected from the group consisting of dexamethasone, hydrocortisone, prednisone, prednisolone, p-flumethasone, cortisone, betamethasone, and Prednisone, fludrocortisone, triamcinolone acetonide and their pharmaceutically acceptable salts.
  • Forskolin derivatives when used alone or in combination, are expected to become a new class of small-molecule drugs for the treatment of psoriasis and immunosuppression. Compared with first-line antibody drugs, they have significant process and price advantages, and are clinically first-line drug cards. Positrin betamethasone ointment has comparable efficacy and fewer side effects.
  • Figure 1 shows the inhibitory effect of different concentrations of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride on human mononuclear macrophage THP-1 secreted TNF- ⁇ induced by lipopolysaccharide (LPS) .
  • the error bar is STDEV, *: P ⁇ 0.05, T-test, compared with the group induced by LPS without 6-[3-(dimethylamino)propionyl]forskolin hydrochloride.
  • Figure 2 shows the inhibitory effect of forskolin on the secretion of TNF- ⁇ by TPS-1 cells induced by LPS.
  • the error bar is STDEV, *: P ⁇ 0.05, T-test, compared with the LPS-induced group without forskolin treatment.
  • the interval number on the abscissa indicates that the 80nM group was removed from the result due to an extra LPS error.
  • Figure 3 shows the viability level of THP-1 cells treated with 6-[3-(dimethylamino)propionyl]forskolin hydrochloride.
  • the error bar is STDEV.
  • Figure 4 shows the inhibitory effect of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and prostaglandin E2 alone and in combination on the secretion of TNF- ⁇ by THP-1 cells induced by lipopolysaccharide.
  • Cpd represents the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride, the error bar is STDEV, *: P ⁇ 0.05, T-test.
  • Figure 5 shows the viability level of THP-1 cells after 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and prostaglandin E2 treatment alone and in combination.
  • the error bar is STDEV
  • Cpd represents the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride.
  • Figure 6 shows the inhibitory effect of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and dexamethasone alone and in combination on the secretion of TNF- ⁇ by THP-1 cells induced by lipopolysaccharide.
  • Cpd represents the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride
  • HC represents dexamethasone
  • the error bar is STDEV
  • Figure 7 shows the viability of THP-1 cells after 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and dexamethasone alone and in combination.
  • the error bars are STDEV
  • Cpd represents the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride
  • HC represents dexamethasone.
  • Figure 8 shows different concentrations of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride on mouse spleen Inhibition of CD17+ T cells by IL-17A secretion by Th17 cells induced by cytokines. Error bars are SEM, *: P ⁇ 0.05, T-test, compared with the 6-[3-(dimethylamino)propionyl]forskolin hydrochloride treatment group induced by cytokines and the like.
  • Figure 9 shows 6-[3-(dimethylamino)propionyl]forskolin hydrochloride on mouse spleen CD4+ T cells have no significant toxic effects, and the error bar is SEM.
  • Figure 10 shows 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and prostaglandin E2 alone and in combination on the spleen of mice Inhibition of CD17+ T cells by IL-17A secretion by Th17 cells induced by cytokines.
  • Cpd represents the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride, the error bar is SEM, *: P ⁇ 0.05, T-test.
  • Figure 11 shows 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and prostaglandin E2 alone and in combination on the spleen of mice CD4+ T cells have no significant toxicity.
  • the error bars are SEM, and Cpd represents the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride.
  • Figure 12 shows 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and dexamethasone alone and in combination on the spleen of mice Inhibition of CD4+T cells to induce Th17 cells to differentiate and secrete IL-17A through cytokines.
  • Cpd represents the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride
  • HC represents dexamethasone
  • the error bar is SEM
  • Figure 13 shows 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and dexamethasone alone and in combination on the spleen of mice CD4+ T cells have no significant toxicity.
  • Error bars are SEM
  • Cpd represents the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride
  • HC represents dexamethasone.
  • Figure 14 shows the curve of the psoriasis-like index score on the back of mice over time.
  • the error bars are SEM, and the statistical significance analysis is shown in Table 10.
  • Fig. 15 shows a comparative photograph of the skin on the back of mice in each group at the 7th day of the end point of administration.
  • Fig. 16 shows a comparison photograph of the high-dose group and the model at 3 days of administration.
  • the right ear of the mouse is the site of modeling with imiquimod ointment, and the left ear is the untreated control.
  • Fig. 17 shows a graph of the results of skin inflammatory thickening of the right ear modeled in mice. The value is the thickness of the right ear minus the thickness of the left ear, the error line is SEM, the statistical significance analysis is shown in Table 11.
  • Fig. 18 shows a comparison photograph of the right ear of mice in each group at the 7th day of the end point of administration.
  • Figure 19 shows the body weight curve of mice over the course of administration.
  • Figure 20 shows the results of H&E staining analysis of skin histology in the model area of the back of the mouse (x4x objective lens, shown as a partial view, cut to the same grid size).
  • Forskolin is a compound extracted from the roots of Coleus forskohlii in the 1970s in the Indian Labiatae Coleus, which acts as an adenylate cyclase activator, It has the functions of strengthening heart and lowering blood pressure. Its structural formula is shown as formula II:
  • the present invention selects the structure-optimized adenylate cyclase agonist forskolin structural derivative, and demonstrates for the first time that the forskolin derivative can specifically reduce the pro-inflammatory factor TNF- in human mononuclear macrophage THP-1 in vitro alpha production, and in the mouse spleen CD4+ T cells induced differentiation in Th17, which can specifically reduce the production of pro-inflammatory factor IL-17A, and the above two inhibitory effects were synergistically enhanced when combined with prostaglandin E2 or dexamethasone.
  • the present invention also proved for the first time that the forskolin derivative has a therapeutic effect comparable to the clinical first-line drug calcipotriol betamethasone in the imiquimod mouse psoriasis model and has fewer side effects.
  • This disclosure is based on a series of compound in vitro pharmacodynamic evaluation studies, which for the first time revealed that the small molecule compound forskolin structure derivative can effectively reduce the level of tumor necrosis factor alpha secretion in the THP-1 human macrophage in vitro pharmacodynamic experiment.
  • Forskolin structural derivatives alone can reduce the background level of TNF- ⁇ secretion by up to 75%, and have a dose-response relationship (Concentration response).
  • it When combined with prostaglandin E2 or dexamethasone, it can be further Decreased TNF- ⁇ secretion by 90% of the background level, showing stronger pharmacodynamic activity.
  • the small molecule compound forskolin structural derivative CD4+ T cells can induce Th17 differentiation.
  • the forskolin derivative provided by the present disclosure can be represented by the following formula I:
  • R 1 and R 2 is -COCH 2 CH 3 , -CO 2 CH 2 CH 3 , -COCH 2 OCHO or group
  • each R 4 and R 5 is independently hydrogen or lower alkyl, or R 4 and R 5 combine to form a lower alkylene chain, the lower alkylene chain contains or does not contain an oxygen atom or a nitrogen atom, m is an integer from 1 to 5;
  • the other of R 1 and R 2 is hydrogen or the group CO(CH 2 ) n X, where X is hydrogen or a group
  • R 6 and R 7 are independently hydrogen or lower alkyl, or R 6 and R 7 are combined to form a lower alkylene chain, the lower alkylene chain may or may not contain an oxygen atom or a nitrogen atom, n An integer from 1 to 5; or
  • R 1 is hydrogen or -COCH 2 CH 2 CO 2 H
  • R 2 is hydrogen, -COCH 3 , -COCH 2 CH 2 CH 2 CO 2 H or -COCH(OH)CH 2 OH, provided that R 1 is hydrogen
  • R 2 is -COCH 2 CH 2 CH 2 CO 2 H or -COCH(OH)CH 2 OH.
  • R 1 in formula I is hydrogen or a group Where m, R 4 and R 5 are as defined above.
  • R 1 in Formula I is -COCH 2 N(CH 3 ) 2 , -CO(CH 2 ) 2 N(CH 3 ) 2 , -CO(CH 2 ) 3 N(CH 3 ) 2 , Or -CO(CH 2 ) 3 NH 2 , and R 2 is -COCH 3 .
  • R 1 in formula I is hydrogen
  • R 2 is -COCH 2 CH 3 , -CO 2 CH 2 CH 3 or -COCH 2 OCHO
  • lower alkyl refers to a linear or branched alkyl group containing 1 to 5 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, n-pentyl Base etc.
  • R 4 and R 5 combine to form a lower alkylene chain, it means that R 4 and R 5 combine with the nitrogen atom to which they are attached to form a five-, six-, or seven-membered ring, and the five-membered ring , Six-membered, or seven-membered ring may additionally or not contain oxygen atom or nitrogen atom.
  • R 6 and R 7 combine to form a lower alkylene chain, it means that R 6 and R 7 combine with the nitrogen atom to which they are attached to form a five-membered, six-membered, or seven-membered ring, and the five-membered , Six-membered, or seven-membered ring may additionally or not contain oxygen atom or nitrogen atom.
  • the forskolin derivative is 6-(4-aminobutyryl)forskolin (6-(4-aminobutyryl)forskolin), 6-[4-(dimethylamino)butyryl ]Forskolin (6-[4-(dimethylamino)butyryl]forskolin), 6-[3-(dimethylamino)propionyl]forskolin (6-[3-(dimethylamino)propiony]forskolin), 6- [3-(methylamino)butyryl]forskolin (6-[3-(methylamino)butyryl]forskolin), 6-[3-aminobutyryl]forskolin (6-[3-(amino)butyryl] forskolin), or 6-[(piperidine)acetyl]-7-deacetylated forskolin (6-[(piperidino)acetyl]-7-deacetylforskolin), etc.
  • HIL568 is also a forskolin derivative later developed by Hoechest Corporation for the treatment of glaucoma. It can be speculated that it also has the effect of inhibiting TNF- ⁇ and IL-17A as an activator of adenylate cyclase, so it is The therapeutic field still has some value, but because there is no follow-up development report, it is speculated that the compound has defects such as druggability.
  • 6-[3-(dimethylamino)propionyl]forskolin hydrochloride (CAS number: 138605-00-2) of the following formula III is used:
  • the present disclosure provides a medicament or pharmaceutical composition comprising a forskolin derivative or a pharmaceutically acceptable salt thereof.
  • the present disclosure provides a pharmaceutical composition including a forskolin derivative or a pharmaceutically acceptable salt thereof and a prostaglandin compound or glucocorticoid compound.
  • the present disclosure also contemplates a drug or pharmaceutical composition that includes a prodrug of a forskolin derivative and optionally a prostaglandin or glucocorticoid compound.
  • These drugs or pharmaceutical compositions can be used for immunosuppression in subjects in need.
  • “Pharmaceutically acceptable salt” here means an inorganic or organic acid addition salt that is substantially harmless to animals or humans, such as hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, Formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, enanthate, fumarate, glutamate, hydroxyethyl Salt, lactate, maleate, malonate, mandelate, mesylate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, Stearate, succinate, tartrate, p-toluenesulfonate, etc.
  • the salt of the forskolin derivative is its hydrochloride salt.
  • Such salts can be formed by methods well known to those skilled in the art.
  • prodrug of forskolin derivative includes compounds formed by modification on one or more reactive or derivatizable groups of forskolin derivative. Of particular interest are compounds modified on the carboxyl, hydroxyl, or amino groups. Examples of particularly suitable prodrugs are esters or amides of forskolin derivatives. These prodrugs are converted into forskolin derivatives or their salts in animals or humans, for example, under the action of enzymes.
  • the present disclosure provides a method of immunosuppression in a subject, which comprises administering to the subject a therapeutically effective amount of a forskolin derivative or a pharmaceutically acceptable salt thereof.
  • the method includes co-administering a forskolin derivative or a pharmaceutically acceptable salt thereof with a prostaglandin compound or glucocorticoid compound.
  • combined administration for example, for the pharmaceutical combination of forskolin derivative and prostaglandin compound, which includes the sequential administration of forskolin derivative and prostaglandin compound separately, for example, prostaglandin compound in forskine Administration before or after administration of corin derivatives also includes forskolin derivatives and prostaglandin compounds in the same pharmaceutical preparation or in separate pharmaceutical preparations.
  • the forskolin derivative and the prostaglandin compound usually coexist in the subject at least part of the time.
  • the forskolin derivative and the prostaglandin compound may have a synergistic effect, for example, the amount of one of the compounds is lower than the therapeutically effective amount when it is used alone, or preferably, both The dosage of the compound is lower than the therapeutically effective amount of the compound when used alone.
  • the present disclosure provides a pharmaceutical kit including a forskolin derivative or a pharmaceutically acceptable salt thereof, such as a hydrochloride salt, and a prostaglandin compound or glucocorticoid compound.
  • a pharmaceutical kit including a forskolin derivative or a pharmaceutically acceptable salt thereof, such as a hydrochloride salt, and a prostaglandin compound or glucocorticoid compound.
  • the pharmaceutical kit can be used for immunosuppression in a subject.
  • the forskolin derivative or a pharmaceutically acceptable salt thereof and the prostaglandin compound or glucocorticoid compound can be formulated in a pharmaceutical composition, for example, they are mixed and coexist in the same In dosage form or unit dosage form.
  • the forskolin derivative or a pharmaceutically acceptable salt thereof and the prostaglandin compound or glucocorticoid compound can be separately formulated and stored.
  • forskolin derivatives or their pharmaceutically acceptable salts and prostaglandins are in solution in different containers, or forskolin derivatives or their pharmaceutically acceptable salts are formulated as injections, and Prostaglandins are formulated as ointments.
  • the pharmaceutical kit provided by the present disclosure may include at least two separate kits, one of which includes a forskolin derivative or a pharmaceutically acceptable salt thereof, and the other includes a prostaglandin Compounds or glucocorticoids.
  • the pharmaceutical kit may also include instructions for using the two kits for administration to the recipient simultaneously or sequentially.
  • the forskolin derivative or a pharmaceutically acceptable salt thereof, as well as prostaglandin compounds and glucocorticoid compounds may be combined with a pharmaceutically acceptable carrier Preparation.
  • “Pharmaceutically acceptable carrier” here means solid or liquid diluents, fillers, antioxidants, stabilizers and the like that can be safely administered to animals or humans, and these substances are suitable for humans and/or The animal is administered without excessive adverse side effects, and is suitable for maintaining the vitality of the drug or active agent located therein.
  • Suitable administration routes include, for example, oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or inhalation, transdermal and other routes.
  • forskolin derivatives or pharmaceutically acceptable salts thereof, as well as prostaglandin compounds and glucocorticoid compounds, can be formulated with these pharmaceutically acceptable carriers into any clinically acceptable dosage forms, such as tablets Agents, granules, powders, capsules, injection preparations, suppositories, drops, externally applied ointments, ointments, medicated oils, or sprays, etc.
  • the present disclosure provides the use of forskolin derivatives or pharmaceutically acceptable salts thereof in the preparation of a medicament for immunosuppression.
  • the drug is administered in combination with a prostaglandin compound or glucocorticoid compound.
  • Subject refers to an individual (preferably a human) with or suspected of having a certain disease (eg, psoriasis), or, for example, when predicting the risk of a disease, "subject” may also include healthy individuals .
  • the term is often used interchangeably with “patient”, “test subject”, “treatment subject”, and the like.
  • a “therapeutically effective amount” refers to an amount sufficient to elicit a biological or medical response desired by a clinician in a subject, and can be generally determined by those skilled in the art according to the route of administration, the subject's weight, age, and condition And other factors. For example, a typical daily dosage may range from 0.01 mg to 100 mg of active ingredient per kg of body weight. This disclosure also considers other amounts.
  • the present disclosure provides a drug comprising 6-[3-(dimethylamino)propionyl]forskolin hydrochloride itself or a pharmaceutical composition thereof and prostaglandin E2 or dexamethasone, And their method or use for immunosuppression in a subject.
  • the present disclosure provides immunosuppressants including forskolin derivatives or pharmaceutically acceptable salts thereof, and optionally prostaglandin-like compounds or glucocorticoid-like compounds.
  • the immunosuppressive agent is a TNF- ⁇ inhibitor.
  • the immunosuppressive agent is an IL-17A inhibitor.
  • the immunosuppressive agent simultaneously inhibits the expression or secretion of TNF- ⁇ and IL-17A.
  • the prostaglandin compound is selected from prostaglandin E2 (Prostaglandin E2, PGE2), tromethamine Dinoprost (Droprost Tromethamine), carboprost (Carboprost), tromethamine carprost (Carboprost Tromethamine), prostaglandin E1 (Alprostadil), bematoprost (Bimatoprost), iloprost (Iloprost), Limaprost (Limaprost), Limaprost alpha cyclodextrin (Limaprostalfadex), miso Misoprostol, Gemeprost, Latanoprost, Sulprostone, Ornoprostil and their pharmaceutically acceptable salts.
  • prostaglandin E2 Prostaglandin E2, PGE2
  • tromethamine Dinoprost Droprost Tromethamine
  • carboprost Carboprost
  • the glucocorticoid compound is selected from dexamethasone, hydrocortisone (Hydrocortisone), prednisone (Prednisone), prednisolone (Prednisolone), parafluasone (Paramethasone), cortisone Cortisone, Betamethasone, Meprednisone, Fludrocortisone, Triamcinolone acetonide and their pharmaceutically acceptable salts.
  • the forskolin derivative or a pharmaceutically acceptable salt thereof functions by inhibiting the expression or secretion of TNF- ⁇ by immune cells, especially mononuclear macrophages. In some embodiments of the methods or uses of the present disclosure, the forskolin derivative or a pharmaceutically acceptable salt thereof works by inhibiting the secretion of IL-17A by immune cells, especially T lymphocytes. In some embodiments of the methods or uses of the present disclosure, the forskolin derivative or a pharmaceutically acceptable salt thereof works by inhibiting the expression or secretion of TNF- ⁇ and IL-17A of immune cells.
  • immunosuppression refers to the reduction of an undesirable immune response in a subject, including the production of certain cytokines such as TNF- ⁇ or IL-17A.
  • the subject in need of immunosuppression is a patient with autoimmune disease.
  • the subject in need of immunosuppression is a psoriasis patient.
  • the subject in need of immunosuppression is an inflammatory patient.
  • the drugs, or pharmaceutical compositions provided by the present disclosure are used as anti-inflammatory drugs.
  • Anti-inflammatory drugs are a class of drugs commonly used in medical practice to slow or eliminate acute and chronic inflammation, such as the glucocorticoid dexamethasone. It should be understood that all drugs that have an anti-inflammatory effect by reducing the body's own immune response rather than inhibiting the action of foreign pathogens (such as antibiotics) are essentially immunosuppressive agents. In this case, the concept of anti-inflammatory drugs is equivalent to Immunosuppressive agents are therefore within the scope of this disclosure.
  • the forskolin derivatives involved in this disclosure may have immunosuppressive function by inhibiting the production of pro-inflammatory cytokines TNF- ⁇ and/or IL-17A, and have potential value in treating or alleviating inflammation and various autoimmune diseases .
  • the forskolin derivative is expected to become a new type of immunosuppressant to make up for the shortage of existing drugs.
  • the present invention reveals for the first time that the small molecule compound Forskolin (Forskolin) structure derivative has a dual action mechanism of simultaneously reducing interleukin 17A and tumor necrosis factor alpha, and has multiple immunosuppressive effects compared to the existing single action mechanism drugs.
  • the compound itself has been fully optimized in structure and modified in nature, and has considerable drug-forming properties. It is also a rare small-molecule chemical drug in the same mechanism of action and related disease drug markets.
  • forskolin structural derivatives have great potential to effectively fill the existing market gaps in the field of psoriasis and immunosuppressive therapy.
  • THP-1 cells Resuscitate and expand THP-1 cells in a T75 culture flask.
  • the growth medium is 10 ml of 10% fetal bovine serum/RPMI1640 (containing 2mM glutamine)/1% double antibody per flask. Observe under an inverted microscope. Cells are in the logarithmic growth phase to start the following drug treatment experiments;
  • LPS lipopolysaccharide
  • Antibody Cocktail for enzyme-linked immunoassay.
  • Antibody dilution provided by Ab221825:
  • Antibody 2 18:1:1 volume ratio prepared for use, in which Antibody 1 is TNF- ⁇ capture antibody, Antibody 2 is TNF- ⁇ detection antibody, all Provided for kit Ab221825;
  • a series of gradient concentration samples of TNF- ⁇ standard products are configured with ultrapure water, which is used to prepare a standard curve of the reaction and determine the linear range of the detection signal;
  • reaction stop solution (provided by Ab221825), place on a horizontal shaker and mix at 400 rpm for 1 minute;
  • the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride can reduce the concentration of human mononuclear macrophages THP-1 in vitro after stimulation by lipopolysaccharide (LPS) TNF- ⁇ expression level, and the decrease in TNF- ⁇ is not due to the compound affecting THP-1 cell viability.
  • LPS lipopolysaccharide
  • THP-1 cells cultured in vitro showed a significantly increased level of TNF- ⁇ in the culture supernatant after 4 hours of stimulation with 100 ng/ml LPS, while the compound 6-[3-(dimethylamino)propane Acyl]forskolin hydrochloride can concentration-dependently reduce LPS-induced TNF- ⁇ secretion in the concentration range of 10 ⁇ M to 3 nM, at the highest concentration of 10 ⁇ M can inhibit about 75% of the positive control TNF- ⁇ level (induced by LPS TNF- ⁇ levels without drug treatment).
  • the compound forskolin of the same structure series which similarly showed the effect of reducing TNF- ⁇ (Figure 2), but the drug activity was slightly lower than the previous compounds.
  • TNF- ⁇ reducing effect is due to the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride dose-dependently attenuates THP-1 cell viability, in LPS and Representative compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride stimulated the end point of the experiment for 4 hours.
  • Prostaglandin E2 PGE2
  • 6-[3-(dimethylamino) propionyl] forskolin hydrochloride can strengthen 6-[3-(dimethylamino) propionyl] forskolin hydrochloride
  • the role of salt in reducing TNF- ⁇ levels PGE2 and 6-[3-(dimethylamino) propionyl] forskolin hydrochloride
  • a and B combined treatment expected value (A single treatment value / control value) * (B single treatment value / control value) * control value, A and B combined treatment expected value / actual A and B combination
  • the ratio of the treatment value ratio is the Synergy Index (Combination Index). If the Synergy Index is greater than 1, there is a synergistic effect between the A and B compounds, otherwise it is a simple additive effect.
  • the combined effect of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and PGE2 is a synergistic effect.
  • dexamethasone When dexamethasone is used in combination with 6-[3-(dimethylamino)propionyl]forskolin hydrochloride, it can strengthen the compound's effect of reducing TNF- ⁇ .
  • Dexamethasone is a glucocorticoid drug commonly used in clinical treatment of psoriasis and immunosuppression. We also investigated whether it has a synergistic effect on forskolin (or its derivatives). As shown in Figure 6, in the culture of THP-1 cells in vitro, we applied 5 ⁇ M and 10 ⁇ M of the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride to inhibit the increase of 100ng/ml LPS-induced TNF- ⁇ to 58% and 44% of the positive control level. To investigate whether dexamethasone has the effect of enhancing the effect of the compound, we used 30 ⁇ M dexamethasone in combination with 5 ⁇ M of the compound.
  • dexamethasone is an anti-inflammatory glucocorticoid drug commonly used in clinical practice, but in this experimental system, on the contrary, it has the effect of further stimulating the increase of inflammatory factor TNF- ⁇ . It has been previously reported that dexamethasone may have a biphasic effect (biphasic effect) when the time of administration and the time of inflammation are relatively different.
  • Our experimental system for inducing TNF- ⁇ factor expression in vitro uses dexamethasone or a test compound to pre-incubate the cells for 2 hours before the inflammatory stimulus lipopolysaccharide.
  • dexamethasone After the application of the inflammatory stimulant lipopolysaccharide, there may be dexamethasone at this time point to promote higher levels of TNF- ⁇ factor expression, to achieve the effect of enhancing the inflammatory response to completely resist the invasion of the inflammatory stimulus LPS, which just suggests that dexamethasone is used to treat chronic inflammation such as psoriasis There may be a mechanism defect in the long-term repeated medication.
  • Example 2 In vitro cytology experiment: the modulation effect of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride on IL-17A
  • Animal strain Adult C57BL/6 female mice, 8-12 weeks old.
  • splenocytes wash once with DPBS, resuspended in RoboSep TM buffer at a density of 1x10 8 / mL;
  • the primary CD4+ T cells are separated by the mouse primary CD4+ T cell separation kit
  • the isolated CD4+ T cells are resuspended in complete medium (RPMI 1640 medium + 10% inactivated FBS + 1% double antibody) with a density of 1x10 6 /mL;
  • the cells were cultured for 7 days under the above conditions, the culture conditions were 37 degrees Celsius, and 5% CO 2 concentration;
  • 1 stimulating factor complex (eBioscience, 500 ⁇ ) was added to the medium for 4 hours, and then the cell culture supernatant was taken for IL-17A ELISA kit detection;
  • the microplate reader reads the absorbance at OD450nm, and the standard curve is generated by 4-parameter logic fitting (4-PL) method.
  • reaction substrate solution (provided by the kit) to each well, incubate at room temperature for 20 minutes, protected from light;
  • the 450nm optical density value of each hole can be detected, and the corrected value can be deducted by the value of 540nm or 570nm.
  • LDH Lactate Dehydrogenase
  • the compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride can concentration-dependently reduce mouse primary CD4+ T cells in the cytokines TGF- ⁇ , IL-6, IL-23A induces the secretion and expression of IL-17A during the differentiation into Th17 cells under the combined action of IL-23, and the decrease of IL-17A is not due to the toxicity of the compound to Th17 cells.
  • the primary CD4+ T cells of mice showed a significantly increased level of IL-17A in the culture supernatant after 7 days of cytokine combination induction, while the compound 6-[3-(dimethylamino)propane Acyl]forskolin hydrochloride concentration-dependently reduces background-induced IL-17A secretion in the concentration range of 10 ⁇ M to 10 nM. At the highest concentration of 10 ⁇ M, it can inhibit about 97% of the positive control IL-17A level (via cytokines Combination induced IL-17A levels without drug treatment).
  • Prostaglandin E2 PGE2
  • 6-[3-(dimethylamino) propionyl] forskolin hydrochloride can strengthen 6-[3-(dimethylamino) propionyl] forskolin hydrochloride
  • the role of salt in reducing IL-17A levels PGE2 and 6-[3-(dimethylamino) propionyl] forskolin hydrochloride
  • Dexamethasone combined with 6-[3-(dimethylamino)propionyl]forskolin hydrochloride can strengthen 6-[3-(dimethylamino)propionyl]forskolin hydrochloride to reduce IL- The effect of 17A.
  • Dexamethasone is a glucocorticoid drug commonly used in clinical treatment of psoriasis and immunosuppression.
  • the factor combination induced increased IL-17A to 88% and 19% of the level of the positive control (no compound-treated group).
  • Animal strain 28 male BALB/c mice, 6-8 weeks old, SPF environment, 12-hour light-dark cycle, 24-26 degrees Celsius.
  • mice are anesthetized and shaved on the back with an area of about 2*3 cm;
  • Day1-7 Measure the body weight, left and right ear thickness, skin observation score of the mice every other day;
  • mice are anesthetized and shaved on the back with an area of about 2*3 cm;
  • Day1-7 Measure the body weight, left and right ear thickness, skin observation score of the mice every other day;
  • Day1-7 At a fixed time every day, apply 100 ⁇ l and 10 ⁇ l of 75% ethanol on the back and right ear modeling site, and massage briefly until the liquid evaporates;
  • Day1-7 Then apply 62.5mg imiquimod ointment to the shaved area on the back of each mouse, and apply 250 ⁇ g imiquimod ointment to the right ear on the inside and outside of the ear, and massage it against the direction of the hair to help absorption;
  • mice are anesthetized and shaved on the back with an area of about 2*3 cm;
  • Day1-7 Measure the body weight, left and right ear thickness, skin observation score of the mice every other day;
  • Day1-7 At a fixed time every day, apply 18 mg of calcipotriol betamethasone ointment to the shaved area on the back of each mouse, apply 1.8 mg of calcipotriol betamethasone ointment to the inside and outside of the right ear, and massage against the direction of the hair to help absorb;
  • mice are anesthetized and shaved on the back with an area of about 2*3 cm;
  • Day1-7 Measure the body weight, left and right ear thickness, skin observation score of the mice every other day;
  • Day1-7 At a fixed time every day, apply 100 ⁇ l and 10 ⁇ l of 75% ethanol on the back and ear modeling site, respectively, and massage briefly until the liquid evaporates;
  • Day1-7 Subsequent intraperitoneal injection of 3.5mg/kg in 200 ⁇ l sterile DPBS;
  • Day1-7 Then apply 62.5mg imiquimod ointment to the shaved area on the back of each mouse, apply 250 ⁇ g imiquimod ointment on the right ear to the inside and outside, and massage against the direction of the hair to help absorption;
  • mice are anesthetized and shaved on the back with an area of about 2*3 cm;
  • Day1-7 Measure the body weight, left and right ear thickness, skin observation score of the mice every other day;
  • Day1-7 At a fixed time every day, apply 100 ⁇ l and 10 ⁇ l of 75% ethanol on the back and ear modeling site, respectively, and massage briefly until the liquid evaporates;
  • Day1-7 Subsequent intraperitoneal injection of 0.8mg/kg in 200 ⁇ l sterile DPBS;
  • Day1-7 Then apply 62.5mg imiquimod ointment to the shaved area on the back of each mouse, apply 250 ⁇ g imiquimod ointment on the right ear to the inside and outside, and massage against the direction of the hair to help absorption;
  • mice are anesthetized and shaved on the back with an area of about 2*3 cm;
  • Day1-7 Measure the body weight, left and right ear thickness, skin observation score of the mice every other day;
  • Day1-7 At a fixed time every day, apply 100 ⁇ l and 10 ⁇ l of 0.0025% PGE2 (2.5 ⁇ g in the back of each mouse in 75% ethanol) to the back and ear modeling sites, and massage briefly until the liquid evaporates;
  • Day1-7 Subsequent intraperitoneal injection of 0.8mg/kg in 200 ⁇ l sterile DPBS;
  • Day1-7 1 hour after the administration of PGE2, apply 62.5 mg of imiquimod ointment to the shaved area on the back of each mouse, and apply 250 ⁇ g of imiquimod ointment to the inside and outside of the right ear, and massage it against the direction of the hair to help absorption;
  • the experimental index used is the psoriasis-like index score:
  • the area of the diseased skin is plaque, redness, and rash.
  • the psoriasis-like index of the skin in the modeling area on the back of the mouse gradually increases with the increase of the administration time, and the statistical significance analysis is shown in Table 10.
  • the building block is characterized by severe skin dandruff, redness, and rash (Figure 15).
  • the 6-[3-(dimethylamino)propionyl]forskolin hydrochloride high-dose group had a faster onset than the low-dose group, and showed a disease-reducing effect on the third day of administration, while the low-dose group started from the 5th The effect began to show on the day, but the difference in the efficacy of the high-dose group and the low-dose group had a tendency to narrow at the end of the 7th day, suggesting that although the high-dose group had a quick effect, the high-low-dose effect may converge under the long-term administration.
  • the effect of the combination medication group is superior to that of the high-dose group, which is consistent with the results of the in vitro experiments of the examples, and its improved efficacy is comparable to that of the positive medication calcipotriol betamethasone ointment.
  • the mice in the combined medication group had smooth skin on the back and no redness, and the skin redness was also lighter than that in the positive control group.
  • Table 10 is the statistical difference between the back psoriasis-like scores.
  • Figure 18 shows the degree of skin inflammation and redness in the right ear of the model in each treatment group at 7 days after the end of the administration.
  • Fig. 16 shows the comparison of redness of the left and right ears of the same mouse on the third day of the experiment, suggesting that the high-dose group may have a slowing effect early in the 3rd day of modeling.

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Abstract

提供了用于进行免疫抑制如治疗银屑病等的包括福司可林衍生物或其盐以及任选地***素类化合物或糖皮质激素类化合物的药物、药物组合物、或药物试剂盒。还提供了进行免疫抑制的方法，其包括以福司可林衍生物或其盐以及任选地***素类化合物或糖皮质激素类化合物给药。福司可林衍生物在单独或联合用药时有望成为用于免疫抑制如治疗银屑病的一类新机制小分子药物，相较一线抗体用药有显著工艺和价格优势，与临床一线化药卡泊三醇倍他米松软膏相比疗效相当、副作用更少。

Description

免疫抑制药物组合物及其应用 技术领域
本发明涉及进行免疫抑制的药物、药物组合物及其应用，尤其涉及利用了福司可林衍生物的药物、药物组合物以及治疗银屑病等的方法。
背景技术
银屑病(Psoriasis)是一种严重影响人类生活质量的慢性皮肤炎症，属自体免疫类疾病。常见的症状是分布在头皮、肘部、膝盖等处的发红、有鳞片的斑块状皮癣，亦称牛皮癣。病灶皮肤组织学特征为表皮显著增厚、免疫细胞在表皮入侵、上皮扩张血管数目增多等。
银屑病成年人发病率高达2～4％，全球有多达1.25亿患者，其中欧美地区发病率居前列。该病全球市场价值2016年达113亿美元，由于银屑病在全球有巨大未满足的医疗需求，其发病机理是近30年医学界的研究热点。诸多研究表明，先天免疫***和获得性免疫***的功能缺失，导致树突细胞激活、免疫T细胞失调、促炎症因子(Proinflammatory cytokine)分泌、角质形成细胞过度增殖和终末分化改变等，最终形成银屑病的皮下炎症和特征性斑块。其中，免疫T细胞等在病人血液和皮肤病灶存在高度富集，并进一步分泌高水平肿瘤坏死因子α(TNF-α)、白介素17A(Interleukin17A)等。肿瘤坏死因子α单独或与其他促炎症因子如白介素23(Interleukin 23)、白介素17(Interleukin 17)等联合发挥作用，启动下游免疫反应。因此，肿瘤坏死因子α、白介素17A、白介素23等都是银屑病的临床病理指征和高价值治疗靶点。
针对靶点TNF-α，全球市场已有以大分子药为主的成熟产品，例如以艾伯维的Adalimumab(阿达木单抗，修美乐)和安进的Etanercept(依那西普，恩利)为代表。小分子TNF-α抑制剂的开发存在药物化学上的挑战，目前仅有磷酸二酯酶抑制剂己酮可可碱(Pentoxifylline)上市，另外二次开发药物沙利度胺(Thalidomide)的抗炎疗效与降低TNF-α有关。TNF-α抑制剂是用于治疗银屑病的第一类生物药，其通过抑制TNF-α起效而在临床实践治疗银屑病中取得良好疗效，如：TNF-α受体Fc融合蛋白Etanercept自1998年批准上市至今，已成为治疗银屑病药物开发的标准参照；TNF-α单抗阿达木也从临床***二线用药批准升级成为中重度慢性斑块状银屑病一线用药。
白介素17A是自2000年起逐步受到关注的免疫调节新靶点，这与2005年发现T辅助细胞17(Th17 cell)这一免疫学里程碑事件有关。随着制药界研发深入，2015年至2016年，诺华的Secukinumab和礼来的Ixekizumab作为IL-17A单克隆抗体药率先上市，并在治疗银屑病上表现出优越的临床疗效和安全。相较于其他靶点药物，IL-17A抗体在达到银屑病面积和炎症指数(Psoriasis area and severity index，PASI)PASI90和PASI100的中重度银屑病响应上表现出更强的临床应答，且疗效在短期和长期治疗中都持续存在。根据上述表现，2015年《自然》杂志评价“抗白介素17药物将成为银屑病的标准治疗方案”。相较于其他靶点，白介素17A目前被公认为具备最高病理相关性、最显著临床应答、优越疗效和高安全性的银屑病治疗靶点。
尽管如此，不管是白介素17A抑制剂还是肿瘤坏死因子α抑制剂，单一因子的抑制均不能实现在100％的银屑病人中有满意应答,这提示该病涉及多重作用机理，存在其他可替代的炎症信号通路。因此，新近策略包含发展双特异抗体(Bispecific antibody)等其他药物形式实现同时抑制两个促炎症因子。同时靶向这两大促炎症因子被认为是可能实现更好疗效的策略，该策略目前尚无上市产品，是值得关注的高价值市场空白领域，仅有杨森在研COVA322为双特异抗体靶向IL-17A和TNF-α，目前在银屑病临床试验二期，值得期待。
值得强调的是，在上述领域全球上市药物中，大分子药占绝大多数。究其原因，促炎症因子相关信号通路错综复杂，制药界通常选择明确可行的抗体药开发策略，如通过抗体大分子直接结合促炎症因子，阻断其结合受体进而抑制下游功能等。但是，抗体等大分子药存在诸多缺陷：在原理机制上，大分子药易产生免疫原性、难以针对细胞内靶点；在生产应用上，大分子药制备工艺复杂，质量标准难统一、生产运输成本高；最重要的，在价格和市场上，大分子药价格昂贵，尤其不适合中国等人口众多、医疗保障负担沉重的国家和地区，另因无法口服给药，也不适合相当数量不耐受注射给药的人群。
小分子化学药虽效力(Potency)通常比抗体药略低，但仍具诸多优势，如通常可口服给药、制备工艺简单、生产成本低、无免疫原性、可针对细胞内靶点等，因此，能高效抑制TNF-α或/和IL-17A的小分子药物有望成为同类抗体药物的强有力竞争者甚至市场终结者，而该领域由于在通量筛选、药物化学、转化医学等诸多环节存在极高专业壁垒和现实挑战，目前基本为市场空白。
除针对银屑病治疗外，以降低/抑制促炎症因子为基本特征和方法的免疫抑制治疗也广泛适用其他多种疾病和症状，如传染或手术外伤等引发的炎症、包括类风湿关节炎、银屑病关节炎在内的其他自体免疫类疾病等，其中，基于免疫抑制治疗的多种自体免疫类疾病市场价值总和超过600亿美元。在有外来病原体刺激或病理性自体免疫反应增强的情况下，淋巴T细胞、巨嗜细胞等活化并分泌促炎症因子作为关键信号分子启动身体炎症反应，代表因子如TNF-α，IL-17，白介素6(Interleukin 6)，干扰素γ(Interferon-γ)等，因此促炎症因子的表达升高是炎症反应和免疫过激过程的主要病理指诊和原因。通过抗体药物结合抑制促炎症因子或其受体进而实现免疫抑制是制药研发和临床实践的主流策略，除前面提及的艾伯维(Abbvie)公司开发的靶向抑制TNF-α的Adalimumab和诺华(Norvatis)公司开发的靶向IL-17A的Secukinuma用于治疗类风湿性关节炎、银屑病等多种自体免疫疾病外，还有梯瓦(Teva)开发的靶向抑制IL-5的人源化抗体Reslizumab用于哮喘(Asthma)等，罗氏(Roche)和基因泰克(Genentech)公司开发的靶向抑制IL-6 Receptor抗体药Tocilizumab用于治疗类风湿性关节炎(Rheumatoid Arthritis)等，强生(J&J)开发的靶向抑制IL-12和IL-23的单克隆抗体药物Ustekinumab治疗克罗恩病(Crohn's disease)、银屑病等。
除上述抗体类免疫抑制药物外，还有诸多小分子免疫抑制剂(Immunosuppressor)用于消除炎症(Anti-inflammation)和免疫功能抑制(Immunosuppression)，临床常用快速起效的免疫抑制剂如甾体类消炎药***(Hydrocortisone)、甲泼尼龙(Methylprednisolone)等，可通过抑制环氧合酶2(COX-2)、抑制中性粒细胞和巨噬细胞活性，降低促炎症细胞因子TNF-α,IL-1β等，在治疗类风湿性关节炎、哮喘、克罗恩病等自体免疫类疾病中都有临床实践和疗效；另外，甲氨蝶呤(Methotrexate)、羟氯喹(Hydroxychloroquine)等起效较慢的免疫抑制剂，通过干扰免疫细胞遗传物质合成和细胞过度增殖抑制免疫细胞活性，亦可降低IL-6等促炎症因子水平，广泛用于类风湿关节炎、***性红斑狼疮(Systemic lupus erythematosus，SLE)等自 体免疫疾病治疗中，但上述小分子免疫抑制剂均存在若干缺点，例如疗效不足、易产生耐药性，长期使用副作用大等。新近开发用于免疫抑制的小分子药物还包括JAK(Janus激酶)抑制剂，但也存在针对JAK不同亚型作用特异性不够、对免疫调节副作用广泛的缺陷。
综上，基于当前全球银屑病药物和免疫抑制治疗的现有市场特征和未满足需求，能有效抑制IL-17A或/和TNF-α的小分子化学药将会成为市场同类疾病和相同机理药物中的稀有品种，其中，尤其是能同时高效抑制IL-17A和TNF-α、具备双重机制(Dual mechanisms)的小分子化合物将拥有极高的竞争优势和后续竞争壁垒，这将是银屑病和免疫抑制治疗领域极富前景的研发方向之一。
发明内容
在一方面，本公开提供了药物组合物，其包括式I的化合物或其药物上可接受的盐(在本文中也称为福司可林衍生物)：
Figure PCTCN2019129945-appb-000001
其中：
R 3为-CH＝CH 2、-CH 2CH 3、或环丙基；
R 1和R 2中的一个为-COCH 2CH 3、-CO 2CH 2CH 3、-COCH 2OCHO或基团
Figure PCTCN2019129945-appb-000002
其中每个R 4和R 5独立地为氢或低级烷基，或者R 4与R 5相结合而形成低级亚烷基链，所述低级亚烷基链含有或不含氧原子或氮原子，m为1至5的整数；R 1和R 2中的另一个为氢或基团CO(CH 2) nX，其中X为氢或基团
Figure PCTCN2019129945-appb-000003
其中R 6和R 7独立地为氢或低级烷基，或者R 6和R 7相结合而形成低级亚烷基链，所述低级亚烷基链中含有或不含有氧原子或氮原子，n为1至5中整数；或者
R 1为氢或-COCH 2CH 2CO 2H，R 2为氢、-COCH 3、-COCH 2CH 2CH 2CO 2H或-COCH(OH)CH 2OH，条件是当R 1为氢时，R 2是-COCH 2CH 2CH 2CO 2H或-COCH(OH)CH 2OH。
在一些实施方案中，式I中R 1为氢或基团
Figure PCTCN2019129945-appb-000004
其中m、R 4和R 5定义同上。
在一些实施方案中，式I中R 1为
Figure PCTCN2019129945-appb-000005
R 2为-CO(CH 2) nX，且R 3为-CH＝CH 2或-CH 2CH 3，其中R 4、R 5、m、n和X定义同上；或者R 1为氢或-COCH 2CH 2CO 2H，R 2为- COCH(OH)CH 2(OH)，且R 3为-CH＝CH 2。
在一些实施方案中，式I中R 1为-COCH 2N(CH 3) 2、-CO(CH 2) 2N(CH 3) 2、-CO(CH 2) 3N(CH 3) 2、或者-CO(CH 2) 3NH 2，且R 2为-COCH 3。
在一些实施方案中，式I中R 1为氢，R 2为-COCH 2CH 3、-CO 2CH 2CH 3或者-COCH 2OCHO，且R 3为-CH＝CH 2。
在一些实施方案中，所述药物组合物与***素类化合物联合给药。
在一些实施方案中，所述药物组合物还包括***素类化合物。
在一些实施方案中，所述药物组合物与糖皮质激素类化合物联合给药。
在一些实施方案中，所述药物组合物还包括糖皮质激素类化合物。
在一些实施方案中，所述药物组合物用于在受试者中进行免疫抑制。
在一些实施方案中，所述药物组合物用于在受试者中治疗自身免疫性疾病。
在一些实施方案中，所述药物组合物用作消炎药。
在一些实施方案中，所述药物组合物用于在受试者中治疗银屑病。
在一些实施方案中，所述药物组合物用于在受试者中治疗银屑病性关节炎(Psoriatic arthritis，PsA)。
在一些实施方案中，所述药物组合物用于在受试者中抑制肿瘤坏死因子α(TNF-α)水平。
在一些实施方案中，所述药物组合物用于在受试者中抑制白介素17A(IL-17A)水平。
另一方面，本公开提供了在受试者中进行免疫抑制的方法，包括以式I的化合物或其药物上可接受的盐向所述受试者给药，其中R 1、R 2和R 3如上文所定义。
在一些实施方案中，所述方法包括联合***素类化合物向所述受试者给药。
在一些实施方案中，所述方法包括联合糖皮质激素类化合物向所述受试者给药。
另一方面，本公开提供了式I的化合物或其药物上可接受的盐在制备用于进行免疫抑制的药物中的用途，其中R 1、R 2和R 3如上文所定义。
在一些实施方案中，所述药物用于与糖皮质激素类化合物联合给药。
在一些实施方案中，所述药物用于与***素类化合物联合给药。
在一些实施方案中，所述药物为自身免疫性疾病药物。
在一些实施方案中，所述药物为银屑病药物。
在一些实施方案中，所述药物为银屑病性关节炎药物。
在一些实施方案中，所述药物为消炎药。
在上述各个方面的一些实施方案中，式I的化合物选自6-(4-氨基丁酰)福司可林、6-[4-(二甲氨基)丁酰]福司可林、6-[3-氨基丙酰]福司可林、6-[3-(甲氨基)丙酰]福司可林、6-[3-(二甲氨基)丙酰]福司可林、和6-[(哌啶)乙酰基]-7-脱乙酰化福司可林。
在上述各个方面的一些实施方案中，所述药物上可接受的盐为盐酸盐。
在上述各个方面的一些实施方案中，式I的化合物的药物上可接受的盐为6-[3-(二甲氨基)丙酰]福司可林盐酸盐。
在上述各个方面的一些实施方案中，式I的化合物的药物上可接受的盐为6-[3-(甲氨基)丙酰]福司可林盐酸盐。
在上述各个方面的一些实施方案中，包括6-[3-(二甲氨基)丙酰]福司可林盐酸盐与***素E2联合给药。
在上述各个方面的一些实施方案中，包括6-[3-(二甲氨基)丙酰]福司可林盐酸盐与地塞 米松联合给药。
在上述各个方面的一些实施方案中，式I的化合物或其药物上可接受的盐通过在所述受试者中降低免疫细胞的TNF-α表达而起作用。
在上述各个方面的一些实施方案中，式I的化合物或其药物上可接受的盐通过在所述受试者中降低免疫细胞的IL-17A表达而起作用。
在另一方面，本公开提供了药物组合物，其包括1)福司可林；以及2)***素类化合物或糖皮质激素类化合物。
在一些实施方案中，所述药物组合物用于在受试者中治疗银屑病或银屑病性关节炎。
另一方面，本公开提供了在受试者中进行免疫抑制的方法，包括以1)福司可林和2)***素类化合物或糖皮质激素类化合物向所述受试者给药。
在一些实施方案中，所述受试者为银屑病患者或银屑病性关节炎患者。
另一方面，本公开提供了福司可林在制备与***素类化合物或糖皮质激素类化合物联合给药的药物中的用途。
在一些实施方案中，所述药物为银屑病药物或银屑病性关节炎药物。
在上述各个方面的一些实施方案中，所述***素类化合物选自***素E2、氨丁三醇地诺前列素、卡前列素、氨丁三醇卡前列素、***素E1、贝美前列素、伊洛前列素、利马前列素、利马前列素α环糊精、米索前列素、吉美前列素、拉坦前列素、硫前列酮、奥诺前列素以及它们的药物上可接受的盐。
在上述各个方面的一些实施方案中，所述糖皮质激素类化合物选自***、氢化可的松、***、***龙、对氟米松、可的松、倍他米松、甲基***、氟氢可的松、曲安奈德以及它们的药物上可接受的盐。
福司可林衍生物在单独或联合用药时有望成为治疗银屑病和用于免疫抑制等的一类新机制小分子药物，相较一线抗体用药有显著工艺和价格优势，与临床一线化药卡泊三醇倍他米松软膏相比疗效相当、副作用更少。
附图说明
图1显示了不同浓度6-[3-(二甲氨基)丙酰]福司可林盐酸盐对人单核巨噬细胞THP-1经脂多糖(LPS)诱导而分泌TNF-α的抑制作用。误差线为STDEV，*：P<0.05，T-test，与经LPS诱导且无6-[3-(二甲氨基)丙酰]福司可林盐酸盐处理组相比。
图2显示了不同浓度福司可林对THP-1细胞经LPS诱导而分泌TNF-α的抑制作用。误差线为STDEV，*：P<0.05，T-test，与经LPS诱导且无福司可林处理组相比。横坐标间隔号表示80nM组因误多加1倍LPS而从结果中去掉。
图3显示了经6-[3-(二甲氨基)丙酰]福司可林盐酸盐处理后的THP-1细胞的活力水平。误差线为STDEV。
图4显示了6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***素E2单独以及联合用药对THP-1细胞经脂多糖诱导而分泌TNF-α的抑制作用。Cpd代表化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐，误差线为STDEV，*：P<0.05，T-test。
图5显示了经6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***素E2单独以及联合处理后的THP-1细胞的活力水平。误差线为STDEV，Cpd代表化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐。
图6显示了6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***单独以及联合用药对THP-1细胞经脂多糖诱导而分泌TNF-α的抑制作用。Cpd代表化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐，HC代表***，误差线为STDEV，*：P<0.05，T-test。
图7显示了经6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***单独以及联合用药后的THP-1细胞的活力水平。误差线为STDEV，Cpd代表化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐，HC代表***。
图8显示了不同浓度6-[3-(二甲氨基)丙酰]福司可林盐酸盐对小鼠脾脏
Figure PCTCN2019129945-appb-000006
CD4+T细胞经细胞因子等诱导分化Th17细胞分泌IL-17A的抑制作用。误差线为SEM，*：P<0.05，T-test，与经细胞因子等诱导且无6-[3-(二甲氨基)丙酰]福司可林盐酸盐处理组相比。
图9显示了6-[3-(二甲氨基)丙酰]福司可林盐酸盐对小鼠脾脏
Figure PCTCN2019129945-appb-000007
CD4+T细胞无显著毒性作用，误差线为SEM。
图10显示了6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***素E2单独以及联合用药对小鼠脾脏
Figure PCTCN2019129945-appb-000008
CD4+T细胞经细胞因子等诱导分化Th17细胞分泌IL-17A的抑制作用。Cpd代表化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐，误差线为SEM，*：P<0.05，T-test。
图11显示了6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***素E2单独以及联合处理对小鼠脾脏
Figure PCTCN2019129945-appb-000009
CD4+T细胞无显著毒性。误差线为SEM，Cpd代表化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐。
图12显示了6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***单独以及联合用药对小鼠脾脏
Figure PCTCN2019129945-appb-000010
CD4+T细胞经细胞因子等诱导Th17细胞分化分泌IL-17A的抑制作用。Cpd代表化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐，HC代表***，误差线为SEM，*：P<0.05，T-test。
图13显示了6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***单独以及联合用药对小鼠脾脏
Figure PCTCN2019129945-appb-000011
CD4+T细胞无显著毒性。误差线为SEM，Cpd代表化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐，HC代表***。
图14显示了小鼠背部皮肤银屑病样指数评分随时间变化的曲线。误差线为SEM，统计显著性分析见表10。
图15显示了给药终点7天时的各个组的小鼠背部皮肤的对比照片。
图16显示了给药3天时高剂量组与造模组的对比照片。图中小鼠右耳为涂抹咪喹莫特软膏造模部位，左耳为未处理对照。
图17显示了小鼠造模右耳的皮肤炎症性增厚结果的曲线。数值为右耳厚度减左耳厚度，误差线为SEM，统计显著性分析见表11。
图18显示了给药终点7天时各个组的小鼠右耳对比照片。
图19显示了小鼠在给药时程中的体重变化曲线。
图20显示了小鼠背部造模区皮肤组织学H&E染色分析结果(x4倍物镜，所显示的为局部图，按相同网格尺寸裁剪)。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
福司可林(Forskolin)是20世纪70年代从印度唇形科锦紫苏属植物毛喉鞘蕊花(coleus forskohlii)根部提取的化合物，其作为腺甘酸环化酶激活剂(Adenylate cyclase activator)，具强心和降血压等作用，其结构式如式II所示：
Figure PCTCN2019129945-appb-000012
申请号为PCT/US84/00291的PCT专利申请报告在1985年观察到福司可林缓解了4名银屑病病人的病理症状这一现象，同时期也有较少研究表明福司可林是通过激活cAMP影响表皮细胞内cAMP与cGMP比例进而抑制了银屑病等表皮增生症状中的表皮细胞有丝***。需要指出，银屑病作为自体免疫类疾病，除表皮细胞增生外，病灶部位的免疫细胞入侵和反复持续炎症是最主要病理特征，但上述观察或研究始终缺乏对福司可林在银屑病病理性炎症的调节机制(Mechanism of Action)的阐述与发现，又因4例临床病人数目极为有限，缺乏显而易见的逻辑联系和充分的数据支持，正因如此，以福司可林为代表的腺苷酸环化酶激活剂在上述疾病的药物开发应用上在此后的30多年间始终处于停滞。福司可林化合物本身也有较多药效学研究，但其水溶性差限制了更一步发展成药。本发明选用结构优化的腺苷酸环化酶激动剂福司可林结构衍生物，并首次证明了福司可林衍生物在体外人单核巨噬细胞THP-1中能特异降低促炎症因子TNF-α的生成，且在小鼠脾脏
Figure PCTCN2019129945-appb-000013
CD4+T细胞诱导分化Th17中能特异降低促炎症因子IL-17A的生成，并且上述两种抑制作用都在联合使用***素E2或***时得到了协同增强。同时，本发明还首次证明了该福司可林衍生物在咪喹莫特小鼠银屑病模型中有与临床一线用药卡泊三醇倍他米松相当的疗效，且副作用更少。
本公开基于化合物的系列体外体内药效学评价研究，首次揭示了小分子化合物福司可林结构衍生物在THP-1人巨噬细胞体外药效实验中能高效降低肿瘤坏死因子α分泌水平，当福司可林结构衍生物单独用药时，最高可降低75％本底水平的TNF-α分泌，且具有量效依赖关系(Concentration response)，当与***素E2或***联合用药时，可进一步降低90％本底水平的TNF-α分泌，表现出更强的药效活性。同时，小分子化合物福司可林结构衍生物在小鼠脾脏
Figure PCTCN2019129945-appb-000014
CD4+T细胞诱导Th17分化体外药效实验中能有效降低白介素17A分泌水平，当福司可林结构衍生物单独用药时，最高可降低97％本底水平的IL-17A分泌，且具有量效依赖关系(Concentration response)，当与***素E2或***联合用药时，可进一步降低本底水平的IL-17A分泌，表现出更强的药效活性。进一步地，在咪喹莫特(Imiquimod)诱导小鼠银屑病模型中，福司可林结构衍生物表现出减缓多项疾病严重指标的效应，如银屑病样皮肤指诊评分、造模耳朵炎症性增厚、以及造模发炎皮肤组织学变化在高低剂量给药组和联合给药组均有相应改变。其中，与PGE2联合用药组的药效与目前临床外用一线用药卡泊三醇倍他米松相当，皮肤炎症性发红更弱，且没有卡泊三醇倍他米松会减低小 鼠体重的副作用等。上述发现提示福司可林结构衍生物在单独或联合用药时有望成为治疗银屑病和用于免疫抑制的一类新机制药物，且与当前临床一线用药相比疗效相当、副作用更少。
本公开提供的福司可林衍生物可由下式I表示：
Figure PCTCN2019129945-appb-000015
其中：
R 3为-CH＝CH 2、-CH 2CH 3、或环丙基；
R 1和R 2中的一个为-COCH 2CH 3、-CO 2CH 2CH 3、-COCH 2OCHO或基团
Figure PCTCN2019129945-appb-000016
其中每个R 4和R 5独立地为氢或低级烷基，或者R 4与R 5相结合而形成低级亚烷基链，所述低级亚烷基链含有或不含氧原子或氮原子，m为1至5的整数；R 1和R 2中的另一个为氢或基团CO(CH 2) nX，其中X为氢或基团
Figure PCTCN2019129945-appb-000017
其中R 6和R 7独立地为氢或低级烷基，或者R 6和R 7相结合而形成低级亚烷基链，所述低级亚烷基链中含有或不含有氧原子或氮原子，n为1至5中整数；或者
R 1为氢或-COCH 2CH 2CO 2H，R 2为氢、-COCH 3、-COCH 2CH 2CH 2CO 2H或-COCH(OH)CH 2OH，条件是当R 1为氢时，R 2是-COCH 2CH 2CH 2CO 2H或-COCH(OH)CH 2OH。
在一些实施方案中，式I中R 1为氢或基团
Figure PCTCN2019129945-appb-000018
其中m、R 4和R 5定义同上。
在一些实施方案中，式I中R 1为
Figure PCTCN2019129945-appb-000019
R 2为-CO(CH 2) nX，且R 3为-CH＝CH 2或-CH 2CH 3，其中R 4、R 5、m、n和X定义同上；或者R 1为氢或-COCH 2CH 2CO 2H，R 2为-COCH(OH)CH 2(OH)，且R 3为-CH＝CH 2。
在一些实施方案中，式I中R 1为-COCH 2N(CH 3) 2、-CO(CH 2) 2N(CH 3) 2、-CO(CH 2) 3N(CH 3) 2、或者-CO(CH 2) 3NH 2，且R 2为-COCH 3。
在一些实施方案中，式I中R 1为氢，R 2为-COCH 2CH 3、-CO 2CH 2CH 3或者-COCH 2OCHO，且R 3为-CH＝CH 2。
如本文所用，“低级烷基”指包含1至5个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、正丁基、叔丁基、正戊基等。
Figure PCTCN2019129945-appb-000020
中，当R 4与R 5相结合而形成低级亚烷基链时，指R 4和R 5与它们所连接的氮原子合起来形成五元、六元、或七元环，并且该五元、六元、或七元环可以另外含有或不 含有氧原子或氮原子。
Figure PCTCN2019129945-appb-000021
中，当R 6与R 7相结合而形成低级亚烷基链时，指R 6和R 7与它们所连接的氮原子合起来形成五元、六元、或七元环，并且该五元、六元、或七元环可以另外含有或不含有氧原子或氮原子。
在一些优选的实施方案中，该福司可林衍生物为6-(4-氨基丁酰)福司可林(6-(4-aminobutyryl)forskolin)、6-[4-(二甲氨基)丁酰]福司可林(6-[4-(dimethylamino)butyryl]forskolin)、6-[3-(二甲氨基)丙酰]福司可林(6-[3-(dimethylamino)propiony]forskolin)、6-[3-(甲氨基)丁酰]福司可林(6-[3-(methylamino)butyryl]forskolin)、6-[3-氨基丁酰]福司可林(6-[3-(amino)butyryl]forskolin)、或6-[(哌啶)乙酰基]-7-脱乙酰化福司可林(6-[(piperidino)acetyl]-7-deacetylforskolin)等，它们的结构式如表1中所示。另需要指出，HIL568也是Hoechest公司后来开发的福司克林衍生物用于青光眼治疗，可推测其作为腺甘酸环化酶激活剂同样有抑制TNF-α和IL-17A的效应，故在本发明所述治疗领域仍有一定价值，但因其并无后续发展报道，推测该化合物具有成药性等缺陷。
表1.优选的福司可林衍生物结构
Figure PCTCN2019129945-appb-000022
Figure PCTCN2019129945-appb-000023
Figure PCTCN2019129945-appb-000024
在特别优选的实施方案中，采用以下式III的6-[3-(二甲氨基)丙酰]福司可林盐酸盐(CAS号：138605-00-2)：
Figure PCTCN2019129945-appb-000025
在一些实施方案中，本公开提供了包括福司可林衍生物或其药物上可接受的盐的药物或药物组合物。
在一些实施方案中，本公开提供了包括福司可林衍生物或其药物上可接受的盐以及***素类化合物或糖皮质激素类化合物的药物组合物。
在一些实施方案中，本公开还考虑包括福司可林衍生物的前药(Prodrug)以及任选地***素类化合物或糖皮质激素类化合物的药物或药物组合物。
这些药物或药物组合物可用于在有需要的受试者中进行免疫抑制。
这里“药物上可接受的盐”指对于动物或人体基本无害的无机或有机酸加成盐，例如盐酸盐、氢溴酸盐、硝酸盐、高氯酸盐、磷酸盐、硫酸盐、甲酸盐、乙酸盐、阿康酸盐、抗坏血酸盐、苯磺酸盐、苯甲酸盐、肉桂酸盐、柠檬酸盐、庚酸盐、富马酸盐、谷氨酸盐、羟乙酸盐、乳酸盐、马来酸盐、丙二酸盐、扁桃酸盐、甲磺酸盐、萘-2-磺酸盐、邻苯二甲酸盐、水杨酸盐、山梨酸盐、硬脂酸盐、琥珀酸盐、酒石酸盐、对甲苯磺酸盐等。在一些实施方案中，尤其有利福司可林衍生物的盐为其盐酸盐。这类盐可以通过本领域技术人员所熟知的方法形成。
这里“福司可林衍生物的前药”包括在福司可林衍生物的一个或多个反应性或可衍生基团上进行修饰而形成的化合物。特别关注的是在羧基、羟基或氨基上进行修饰的化合物。特别 适合的前药实例是福司可林衍生物的酯或酰胺。这些前药在动物或人体内例如在酶的作用下会转化为福司可林衍生物或其盐。
在一些实施方案中，本公开提供了在受试者中进行免疫抑制的方法，其包括以治疗有效量的福司可林衍生物或其药物上可接受的盐向所述受试者给药。
在一些实施方案中，所述方法包括将福司可林衍生物或其药物上可接受的盐与***素类化合物或糖皮质激素类化合物联合给药。
术语“联合给药”，例如对于福司可林衍生物和***素类化合物的药物组合而言，其包括福司可林衍生物与***素类化合物顺序地分别给药，例如***素类化合物在福司可林衍生物给药之前或之后给药，还包括福司可林衍生物与***素类化合物在同一药物制剂中或以分开的药物制剂形式同时给药。在顺序给药的实施方案中，通常福司可林衍生物与***素类化合物至少在部分时间上共存于受试者体内。在联合给药的一些实施方案中，福司可林衍生物与***素类化合物可以具有协同效应，例如其中的一种化合物的用量低于其单独用药时的治疗有效量，或者优选地，两种化合物的用量均低于其单独用药时的治疗有效量。
在一些实施方案中，本公开提供了包括福司可林衍生物或其药物上可接受的盐，例如盐酸盐，以及***素类化合物或糖皮质激素类化合物的药物试剂盒。该药物试剂盒可用于在受试者中进行免疫抑制。
在该药物试剂盒的一些实施方案中，福司可林衍生物或其药物上可接受的盐与***素类化合物或糖皮质激素类化合物可以配制在药物组合物中，例如它们混合并共存于同一剂型或单位剂型中。在该药物试剂盒的另一些实施方案中，福司可林衍生物或其药物上可接受的盐与***素类化合物或糖皮质激素类化合物可以单独配制并保存。例如，福司可林衍生物或其药物上可接受的盐和***素类化合物均为溶液形式存在于不同的容器中，或者福司可林衍生物或其药物上可接受的盐配制为注射剂，而***素类化合物配制为软膏。
在一些实施方案中，本公开提供的药物试剂盒可以包括至少两个单独的试剂盒，其中一个试剂盒包括福司可林衍生物或其药物上可接受的盐，另一试剂盒包括***素类化合物或糖皮质激素类化合物。该药物试剂盒还可以包括用于将这两个试剂盒用于向受使者同时或顺序地给药的说明书。
在本公开提供的药物、药物组合物或药物试剂盒中，福司可林衍生物或其药物上可接受的盐、以及***素类化合物和糖皮质激素类化合物可以与药物上可接受的载体一起配制。这里“药物上可接受的载体”指对于动物体或人体而言，可以安全地给药的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于对人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。依照给药途径的不同，可以采用本领域众所周知的各种不同的载体，包括，但不限于糖类、淀粉、纤维素及其衍生物、麦芽糖、明胶、滑石、硫酸钙、植物油(例如蓖麻油)、合成油、多元醇、藻酸、磷酸缓冲液、乳化剂、等渗盐水、和/或无热原水等。适合使用的给药途径例如包括口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下，或经吸入、透皮等途径。相应地，福司可林衍生物或其药物上可接受的盐、以及***素类化合物和糖皮质激素类化合物可以与这些药物上可接受的载体一起配制为任何临床上可接受的剂型，例如片剂、颗粒、粉末、胶囊、注射制剂、栓剂、滴剂、外贴药膏、软膏、药油、或喷雾剂，等等。
在一些实施方案中，本公开提供了福司可林衍生物或其药物上可接受的盐在制备用于进行免疫抑制的药物中的用途。
在该用途的一些实施方案中，所述药物与***素类化合物或糖皮质激素类化合物联合给药。
这里所用的“受试者”指患有或者怀疑患有某种疾病(例如银屑病)的个体(优选人)，或者，例如在预测患病风险时“受试者”也可能包括健康个体。该术语通常可以和与“患者”、“检测对象”、“治疗对象”等互换使用。这里所用的“治疗有效量”指足以在受试者体内引起临床医师所期望的生物学或医学反应的的量，可通常由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg活性成分。本公开也考虑其他的用量。
在一些具体的实施方案中，本公开提供了包括6-[3-(二甲氨基)丙酰]福司可林盐酸盐自身的药物或其和***素E2或***的药物组合物，以及它们用于在受试者中进行免疫抑制的方法或用途。
在一些实施方案中，本公开提供了包括福司可林衍生物或其药物上可接受的盐，以及任选地***素类化合物或糖皮质激素类化合物的免疫抑制剂。在一些实施方案中所述免疫抑制剂为TNF-α抑制剂。在一些实施方案中所述免疫抑制剂为IL-17A抑制剂。在一些实施方案中，所述免疫抑制剂同时抑制TNF-α和IL-17A的表达或分泌。
在一些实施方案中，所述***素类化合物选自***素E2(Prostaglandin E2，PGE2)、氨丁三醇地诺前列素(Dinoprost Tromethamine)、卡前列素(Carboprost)、氨丁三醇卡前列素(CarboprostTromethamine)、***素E1(Alprostadil)、贝美前列素(Bimatoprost)、伊洛前列素(Iloprost)、利马前列素(Limaprost)、利马前列素α环糊精(Limaprostalfadex)、米索前列素(Misoprostol)、吉美前列素(Gemeprost)、拉坦前列素(Latanoprost)、硫前列酮(Sulprostone)、奥诺前列素(Ornoprostil)以及它们的药物上可接受的盐。
在一些实施方案中，所述糖皮质激素类化合物选自***、氢化可的松(Hydrocortisone)、***(Prednisone)、***龙(Prednisolone)、对氟米松(Paramethasone)、可的松(Cortisone)、倍他米松(Betamethasone)、甲基***(Meprednisone)、氟氢可的松(Fludrocortisone)、曲安奈德(Triamcinolone acetonide)以及它们的药物上可接受的盐。
在本公开的方法或用途的一些实施方案中，福司可林衍生物或其药物上可接受的盐通过抑制免疫细胞、尤其是单核巨噬细胞的TNF-α表达或分泌而起作用。在本公开的方法或用途的一些实施方案中，福司可林衍生物或其药物上可接受的盐通过抑制免疫细胞、尤其是T淋巴细胞的IL-17A分泌而起作用。在本公开的方法或用途的一些实施方案中，福司可林衍生物或其药物上可接受的盐通过抑制免疫细胞的TNF-α和IL-17A表达或分泌而起作用。
这里所用的“免疫抑制”指降低受试者体内不希望的免疫应答，包括一些细胞因子如TNF-α或IL-17A的生成。在一些实施方案中，需要进行免疫抑制的受试者为自身免疫性疾病患者。在一些实施方案中，需要进行免疫抑制的受试者为银屑病患者。在另一些实施方案中，需要进行免疫抑制的受试者为炎症患者。
在一些实施方案中，本公开提供的药物、或药物组合物作为消炎药使用。消炎药是医疗实践中常见的一类用于减缓或消除急慢性炎症的药品，如糖皮质激素***等。应当理解，凡是通过降低机体自身免疫应答而非抑制外来病原体活性(如抗生素)的作用机制而起到消炎作用的药物，本质上也属于免疫抑制剂，在这种情况下消炎药的概念等同于免疫抑制剂，因此也在本公开的范围内。
总之，本公开涉及的福司可林衍生物可通过抑制促炎症细胞因子TNF-α和/或IL-17A的生成而具有免疫抑制功能，有治疗或缓解炎症和多种自体免疫类疾病的潜在价值。所述福司可林衍生物基于新的分子靶点和机理，有望成为新型免疫抑制剂，以弥补现有药物不足。
本发明首次揭示了小分子化合物福司可林(Forskolin)结构衍生物具有同时降低白介素17A和肿瘤坏死因子α的双作用机制，比现有市场单一作用机制药物具备多重免疫抑制效应。同时，化合物本身已经充分结构优化和性质改造，具备相当的成药性，也是同类作用机制和相关疾病药物市场中稀有的小分子化学药品种。综上，福司可林结构衍生物具备有效填补银屑病和免疫抑制治疗领域现有市场空白的巨大潜力。
以下通过实施例来进一步说明本发明。
实施例
实施例1体外细胞学实验：6-[3-(二甲氨基)丙酰]福司可林盐酸盐对TNF-α的调节作用
1.1 实验材料与主要设备见下表2。
表2.体外细胞学实验所用材料和设备
材料 品牌 货号
RPMI1640培养基 Thermo 11835030
胎牛血清 Thermo 10099133
磷酸缓冲液(DPBS) Thermo 14190144
双抗 Thermo 15070063
人单核巨噬细胞(THP-1) BeNa Culture BNCC337680
脂多糖(LPS) Sigma L6529-1MG
佛波酯(PMA) Sigma P8139-1MG
人TNF-α免疫酶连试剂盒 Abcam ab181421
细胞活力检测试剂盒(MTT) 碧云天 C0009
***素E2(PGE2) MCE HY-101952
福司可林(Forskolin) Sigma F6886
NKH 477 Sigma N3290
***(Hydrocortisone) MCE HY-N0583
酶标仪 TECAN infinite M1000
细胞培养箱 Thermo Forma 371
超净工作台 BIOBASE BBS-V800
倒置显微镜 永新光学 NIB-100
离心机 Sigma 2-5
96孔培养板 Costar T3603
T75培养瓶 Corning 430641
1.2 实验步骤
1.2.1 细胞培养、药物处理及细胞活力检测：
1)复苏扩增THP-1细胞于T75培养瓶中，生长培养基为每瓶10毫升10％胎牛血清/RPMI1640(已含2mM谷氨酰胺)/1％双抗，倒置显微镜下观察，待细胞处于对数生长期开始以下药物处理实验；
2)将2*10e5/毫升的THP-1细胞培养于96孔培养板中，每孔100微升生长培养基(10％胎牛血清/RPMI1640/1％双抗)，添加100ng/ml PMA培育24小时；
3)用300微升新鲜10％胎牛血清/RPMI1640/1％双抗生长培养基洗脱未贴壁细胞1次；
4)加入200微升含有/不含试验化合物的10％胎牛血清/RPMI1640/1％双抗生长培养基于37度预孵育细胞2小时；
5)随后加入100ng/ml脂多糖(LPS)于37度孵育4小时；
6)收集50微升细胞培养基上清用于酶联免疫法检测相关细胞因子水平，或存于负80度冰箱待测；
7)每孔细胞补加10微升5mg/ml的MTT溶液，于细胞培养箱中继续孵育4小时；
8)每孔细胞加入100微升Formazan溶解液，于细胞培养箱中继续孵育4小时左右，待普通光学显微镜下观察Formazan晶体全部溶解；
9)以酶标仪检测570nm吸光度。
1.2.2、酶联免疫法检测细胞因子表达水平：
1)配制酶联免疫检测的1X洗脱缓冲液。以超纯水：10X洗脱缓冲母液＝9:1的体积比例配制待用；
2)配制酶联免疫检测的抗体混合液(Antibody Cocktail)。以抗体稀释液(Antibody Diluent，Ab221825提供):抗体1:抗体2＝18:1:1的体积比例配制待用，其中抗体1是TNF-α捕获抗体，抗体2是TNF-α检测抗体，均为试剂盒Ab221825提供；
3)用超纯水配置TNF-α标准品的系列梯度浓度样品，用于制作反应标准曲线，确定检测信号线性范围；
4)将50微升细胞培养液基上清/标准品和50微升上述抗体混合液加入酶学反应96孔板，薄膜密封后置于水平摇床，以400rpm速率室温孵育1小时，使抗体-抗原复合物在孔板底部充分结合偶联；
5)弃上清，用350微升1X洗脱缓冲液/孔洗脱3次，最后1次洗脱时将孔板倒扣于吸水纸充分吸洗干净；
6)每孔加入100微升TMB底物溶液(Ab221825提供)，置于水平摇床以400rpm速率于室温暗处孵育5分钟；
7)加入100微升反应中止液(Ab221825提供)，置于水平摇床以400rpm速率混匀1分钟；
8)以酶标仪检测450nm波长的吸光值。
1.3 实验结果
1.3.1 6-[3-(二甲氨基)丙酰]福司可林盐酸盐降低单核巨噬细胞的TNF-α表达
化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐(Colforsin daropate hydrochdeloride)可浓度依赖式地降低体外培养人单核巨嗜细胞THP-1经脂多糖(LPS)刺激后的TNF-α表达水平，并且TNF-α减少并非因该化合物影响THP-1的细胞活力所致。
如图1所示，体外培养THP-1细胞在100ng/ml的LPS刺激4小时后表现出显著升高的培养液上清TNF-α水平，而化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐在10μM到3nM的浓度范围内可浓度依赖式地降低LPS诱导的TNF-α分泌，在最高浓度10μM可抑制约75％的阳性对照TNF-α水平(经LPS诱导而无药物处理的TNF-α水平)。我们同时考察了同一结构系列的化合物福司可林，其类似表现为降低TNF-α的效应(图2)，但药物活性比前述化合物略低。为进一步考察这种TNF-α减少效应是否是因为化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐剂量依赖式地减弱了THP-1细胞活力造成的，在LPS和代表化合物6-[3-(二甲氨基) 丙酰]福司可林盐酸盐刺激4小时的实验终点，我们在培养细胞上清中加入10微升5mg/mL的MTT溶液检测细胞活力水平，最终确定了在有无化合物组之间细胞活力并无显著差异(图3)，从而证实6-[3-(二甲氨基)丙酰]福司可林盐酸盐降低TNF-α分泌的效应是经由特定免疫调控通路发生而非细胞活力受损间接导致。
1.3.2 6-[3-(二甲氨基)丙酰]福司可林盐酸盐与***素E2(PGE2)联合降低单核巨噬细胞的TNF-α表达
***素E2(PGE2)与6-[3-(二甲氨基)丙酰]福司可林盐酸盐联合使用时，可强化6-[3-(二甲氨基)丙酰]福司可林盐酸盐的降低TNF-α水平的作用。
如图4所示，在体外培养THP-1细胞中，我们施加5μM和10μM的化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐，分别使100ng/ml脂多糖诱导升高的TNF-α水平降至阳性对照的33％和27％。为考察该化合物与***素E2联合使用是否能够增强该化合物的上述效应，我们将10μM PGE2和5μM 6-[3-(二甲氨基)丙酰]福司可林盐酸盐联合使用，结果显示10μM PGE2的添加使得6-[3-(二甲氨基)丙酰]福司可林盐酸盐抑制TNF-α水平从单独使用时降低67％增强到可降低90％的TNF-α水平，并且该联合用药效果比单独使用更高浓度的10μM 6-[3-(二甲氨基)丙酰]福司可林盐酸盐的抑制效应(73％)效果更优，体现出联合使用相对于仅增加剂量的优势。同时，我们也检测了单独添加10μM的PGE2的效应，发现PGE2也有一定程度抑制TNF-α分泌表达的作用，我们推测这与其有类似提升细胞内cAMP水平有关。然而，与相同浓度的6-[3-(二甲氨基)丙酰]福司可林盐酸盐相比，单独的PGE2没有表现为更强的抑制效应，这提示PGE2单独用药缺乏优势，也进一步排除了PGE2联合用药增强6-[3-(二甲氨基)丙酰]福司可林盐酸盐抑制TNF-α的效应是由PGE2本身单方面主导。
为进一步明确上述6-[3-(二甲氨基)丙酰]福司可林盐酸盐与PGE2的联合作用是协同效应而非单纯叠加效应，根据参考文献【9-11】对化合物协同效应的判断原则：A和B联合处理预期值＝(A单独处理值/对照组值)*(B单独处理值/对照组值)*对照组值，A与B联合处理预期值/实际A和B联合处理值比值的比值为协同指数(Combination Index)，若协同指数>1,则A和B化合物存在协同作用，反之则为单纯叠加效应。根据该原则和下表3数据计算得到，6-[3-(二甲氨基)丙酰]福司可林盐酸盐与PGE2的联合作用是协同效应。
表3. 6-[3-(二甲氨基)丙酰]福司可林盐酸盐与PGE2的联合作用结果
Figure PCTCN2019129945-appb-000026
类似地，我们在上述实验药物刺激终点加入前述等量MTT溶液，数据显示细胞活力未受药物处理影响，不存在组间显著性差异(图5)，从而确认TNF-α表达调节是上述药物经由免疫调控相关信号通路调节的结果，而非细胞活力受损间接所致。
1.3.3 6-[3-(二甲氨基)丙酰]福司可林盐酸盐与***(Hydrocortisone，HC)联合降低单核巨噬细胞的TNF-α表达
***与6-[3-(二甲氨基)丙酰]福司可林盐酸盐联用时，可强化该化合物减少TNF-α的效应。
***是临床上常用的治疗银屑病和用于免疫抑制的糖皮质激素类药物，我们也考察了它是否与福司可林(或其衍生物)存在药效上的协同作用。如图6所示，在体外培养THP-1细胞中，我们分别施加5μM和10μM的化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐，抑制100ng/mlLPS诱导升高的TNF-α至阳性对照水平的58％和44％。为考察***是否有增强该化合物的效应的作用，我们将30μM***和5μM的该化合物联合使用，结果显示30μM***的添加使得6-[3-(二甲氨基)丙酰]福司可林盐酸盐抑制TNF-α的效果从单独5μM时降低42％进一步增强到联合用药降低88％的TNF-α的水平。该联合用药的效应比单独使用更高浓度的10μM 6-[3-(二甲氨基)丙酰]福司可林盐酸盐的抑制效应(56％)更优，体现出二者联合使用优于单一化合物增加剂量的效果。同时，我们也检测了单独30μM***本身的效应。有趣的是，***是临床实践常用的消炎类糖皮质激素药物，在本实验体系中却恰好相反体现出其有进一步刺激炎症因子TNF-α升高的效应。以前有报道，***在给药时间和炎症发生时间相对先后顺序不同时可能存在双向效应(Biphasic effect)。我们的体外诱导TNF-α因子表达实验体系采用的是***或测试化合物先于炎症刺激物脂多糖2小时预孵育细胞的“预防式”药物干扰，在之后施加炎症刺激物脂多糖后，可能存在***在该时间点促进更高水平TNF-α因子表达，实现增强炎症反应发生以彻底对抗炎症刺激物LPS侵入的作用，而这恰好提示***在治疗银屑病等慢性炎症的长期反复用药中可能存在机制上的缺陷。尽管如此，根据上述实验数据我们可以得出结论，***联合6-[3-(二甲氨基)丙酰]福司可林盐酸盐可增强6-[3-(二甲氨基)丙酰]福司可林盐酸盐降低TNF-α水平的效果，并且这一增强效应并不是由***本身主导的。类似地，我们通过下表4计算分析明确了6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***的联合作用为协同效应。
表4. 6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***联合作用结果
Figure PCTCN2019129945-appb-000027
我们同样在上述实验药物刺激终点加入前述等量MTT溶液，数据结果排除了细胞活力受药物处理影响的可能(图7)，肯定了TNF-α表达变化是上述药物经由免疫调控相关信号通路调节的结果。
实施例2体外细胞学实验：6-[3-(二甲氨基)丙酰]福司可林盐酸盐对IL-17A的调节作用
2.1 实验材料与主要设备见下表5。
表5.体外细胞学实验所用材料和设备
Figure PCTCN2019129945-appb-000028
Figure PCTCN2019129945-appb-000029
动物品系：成年C57BL/6雌性小鼠，8～12周龄。
2.2 实验步骤
2.2.1 小鼠原代T细胞分离培养、Th17细胞诱导分化和化合物处理
1)96孔板预包被2μg/mL anti-mouse CD3e孵育4摄氏度过夜；
2)从成年C57BL/6雌性小鼠(8～12周龄)分离新鲜脾脏，经70μm尼龙细胞滤网研磨过滤，得到单细胞悬液于预冷DPBS；
3)脾细胞用DPBS再洗一次，重悬于RoboSep TM缓冲液中，密度为1x10 8/mL；
4)原代CD4+T细胞用小鼠原代CD4+T细胞分离试剂盒要求操作分离；
5)分离的CD4+T细胞重悬于完全培养基(RPMI 1640培养基+10％灭活FBS+1％双抗)，密度为1x10 6/mL；
6)预包被anti-mouse CD3e的96孔板用DPBS洗两次，加入50μL细胞因子混合液(cytokine cocktail)，成分和终浓度如下表6所示，再加入50μL化合物溶液(DMSO终浓度为0.1％)每孔加入100μL细胞悬液，每孔最终细胞数为1Х10 5/100μL；
表6细胞因子混合液成分
Figure PCTCN2019129945-appb-000030
7)细胞在上述条件下培养7天，培养条件为37摄氏度，5％CO 2浓度；
8)7天后，1Х刺激因子复合物(stimulation cocktail，eBioscience 500Х)加入培养基中4小时，然后取细胞培养上清用于IL-17A酶联免疫试剂盒检测；
9)上清IL-17A浓度用小鼠IL-17DuoSet ELISA试剂盒完成；
10)酶标仪读取OD450nm吸光值，标准曲线用4参数逻辑拟合(4-PL)法生成。
2.2.2、酶联免疫法检测IL-17A细胞因子表达水平
1)用不含载体蛋白的PBS稀释捕获抗体(试剂盒提供)至工作浓度，即可以每孔100微升包被96孔板，封闭孔板室温孵育过夜；
2)吸取孔板的包被液体，400微升洗脱液(试剂盒提供)三次洗脱，每次尽可能吸尽液体；
3)每孔加入300微升稀释液(试剂盒提供)，室温平衡1小时；
4)如上述2)步骤洗脱孔板待样品加入使用；
5)每孔加入100微升样品或标准品于已加入的稀释液中，封闭孔板室温孵育2小时；
6)如上述2)步骤洗脱孔板；
7)每孔加入100微升检测抗体(试剂盒提供)，封闭孔板室温孵育2小时；
8)如上述2)步骤洗脱孔板；
9)每孔加入100微升Streptavidin-HRP工作液(试剂盒提供)，封闭孔板避光室温孵育20分钟；
10)如上述2)步骤洗脱孔板；
11)每孔加入100微升反应底物溶液(试剂盒提供)，室温孵育20分钟，避光；
12)每孔加入50微升终止液(试剂盒提供)，轻叩孔板确保液体混匀；
13)即可检测各孔450nm光密度值，可用540nm或570nm值扣减矫正数值。
2.2.3 化合物细胞毒性检测
1)从成年C57BL/6雌性小鼠(8～12周龄)分离新鲜脾脏，经70μm尼龙细胞滤网研磨过滤，得到单细胞悬液于预冷PBS；
2)原代CD4+T细胞接种于96孔板，密度为1Х10 5/90μL，培养基为1640培养基(无血清)
3)系列梯度稀释的化合物配于10μL体积加入到指定孔中；
4)细胞在37摄氏度5％CO 2培养条件下孵育4小时；
5)乳酸脱氢酶(Lactate Dehydrogenase，LDH)释放用CytoTox-one Homogeneous Membrane Integrity Assay Kit说明书检测。
2.3 实验结果
2.3.1 6-[3-(二甲氨基)丙酰]福司可林盐酸盐降低小鼠Th17细胞诱导分化的IL-17A表达
化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐(Colforsin daropate hydrochdeloride)可浓度依赖式地降低小鼠原代CD4+T细胞在细胞因子TGF-β、IL-6、IL-23等组合作用下向Th17细胞诱导分化中IL-17A的分泌表达，并且IL-17A减少并非因该化合物对Th17细胞存在毒性所致。
如图8所示，小鼠原代CD4+T细胞在细胞因子组合7天诱导下表现出显著升高的培养液上清IL-17A水平，而化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐在10μM到10nM的浓度范围内浓度依赖式地降本底诱导的IL-17A分泌，在最高浓度10μM可抑制约97％的阳性对照IL-17A水平(经细胞因子组合诱导而无药物处理的IL-17A水平)。为进一步考察这种IL-17A减少效应是否是因为化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐存在毒性减弱了CD4+T细胞活力造成的，我们单独测试了化合物对CD4+T细胞的毒性，将化合物稀释于培养基中与CD4+T细胞孵育4小时后检测释放到培养基的乳酸脱氢酶浓度，结果表明化合物对细胞不存在显著毒性(图9)，从而证实6-[3-(二甲氨基)丙酰]福司可林盐酸盐降低IL-17A分泌的效应是经由特定免疫调控通路发生而非细胞活力受损间接导致。
2.3.2 6-[3-(二甲氨基)丙酰]福司可林盐酸盐与***素E2(PGE2)联合降低小鼠Th17细胞诱导分化的IL-17A表达
***素E2(PGE2)与6-[3-(二甲氨基)丙酰]福司可林盐酸盐联合使用时，可强化6-[3-(二甲氨基)丙酰]福司可林盐酸盐的降低IL-17A水平的作用。
如图10所示，在小鼠原代CD4+T细胞诱导分化Th17中，我们施加0.5μM和1μM的化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐，分别使细胞因子组合诱导升高的IL-17A水平降至阳性对照(无化合物处理)的88％和19％。为考察该化合物与***素E2联合使用是否能够增强该化合物的上述效应，我们将3μM PGE2和0.5μM 6-[3-(二甲氨基)丙酰]福司可林盐酸盐联合使用，结果显示3μM PGE2的添加使得0.5μM 6-[3-(二甲氨基)丙酰]福司可林盐酸盐抑制IL-17A水平从单独使用时降低12％增强到可降低97％的IL-17A水平，并且该联合用药效果比单独使用更高浓度的1μM 6-[3-(二甲氨基)丙酰]福司可林盐酸盐的抑制效应(81％)效果更优，体现出联合使用相对于仅增加剂量的优势。同时，我们也检测了单独添加3μMPGE2的效应，发现PGE2也有一定程度抑制IL-17A分泌表达的作用，我们推测这与其有类似提升细胞内cAMP水平有关。然而，3μMPGE2的抑制效果与更低浓度的1μM的6-[3-(二甲氨基)丙酰]福司可林盐酸盐是相当的(分别抑制到本底水平的17％和18％)，表明单独PGE2作用没有更强的抑制效应，提示PGE2单独用药缺乏优势，也进一步排除了PGE2联合用药增强6-[3-(二甲氨基)丙酰]福司可林盐酸盐抑制IL-17A的效应是由PGE2本身单方面主导。
同样的，为进一步明确上述6-[3-(二甲氨基)丙酰]福司可林盐酸盐与PGE2在抑制IL-17A分泌上的联合作用是协同效应而非单纯叠加效应，与上文类似地，我们计算了相关协同指数，如下表所示。根据前述原则和下表7数据计算得到，6-[3-(二甲氨基)丙酰]福司可林盐酸盐与PGE2在降低IL-17A的联合作用是协同效应。
表7. 6-[3-(二甲氨基)丙酰]福司可林盐酸盐与PGE2联合作用结果
Figure PCTCN2019129945-appb-000031
类似地，我们在前述实验加药条件测试了化合物对小鼠CD4+T细胞的毒性影响，化合物与细胞孵育4小时检测上清中释放乳酸脱氢酶浓度，数据显示细胞活力未受药物处理影响，不存在组间显著性差异(图11)，从而确认IL-17A表达调节是上述药物经由免疫调控相关信号通路调节的结果，而非细胞活力受损间接所致。
2.3.3 6-[3-(二甲氨基)丙酰]福司可林盐酸盐与***(Hydrocortisone，HC)联合降低小鼠Th17细胞诱导分化的IL-17A表达
***与6-[3-(二甲氨基)丙酰]福司可林盐酸盐联用时，可强化6-[3-(二甲氨基)丙酰]福司可林盐酸盐减少IL-17A的效应。
***是临床上常用的治疗银屑病和用于免疫抑制的糖皮质激素类药物，我们也考察了它是否与福司可林衍生物是否存在药效上的协同作用。如图12所示，在小鼠CD4+T细胞诱导分化Th17过程中，我们分别施加0.5μM和1μM的化合物6-[3-(二甲氨基)丙酰]福司可林盐酸盐，抑制细胞因子联合诱导升高的IL-17A至阳性对照(无化合物处理组)水平的88％和19％。为考察***是否有增强该化合物的效应的作用，我们将3μM***和0.5 μM的该化合物联合使用，结果显示3μM***的添加使得6-[3-(二甲氨基)丙酰]福司可林盐酸盐抑制IL-17A的效果从单独0.5μM时降低12％进一步增强到联合用药降低61％的IL-17A的水平。同时，我们也检测了单独3μM***本身的效应为降低了本底水平IL-17A水平的38％。类似地，我们通过下表8计算分析明确了6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***在降低IL-17A分泌的联合作用为协同效应。
表8. 6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***联合作用结果
Figure PCTCN2019129945-appb-000032
我们同样在上述实验加药条件测试了化合物对小鼠CD4+T细胞的毒性影响，化合物与细胞孵育4小时检测上清中释放乳酸脱氢酶浓度，数据显示细胞活力未受药物处理影响，不存在组间显著性差异(图13)，从而确认IL-17A表达调节是上述药物经由免疫调控相关信号通路调节的结果，而非细胞活力受损间接所致。
实施例3体内疾病模型实验
3.1 实验材料与主要设备见表9。
表9.体内疾病模型实验所用的材料和设备。
材料 品牌 货号(或药品注册号)
5％咪喹莫特乳膏 Aldara H20160079
卡泊三醇倍他米松软膏 LEO H20160204
磷酸缓冲液(DPBS) Thermo 14190144
***素E2(PGE2) MCE HY-101952
75％乙醇 Greagent G73537N
4％多聚甲醛固定液 碧云天 P0099-100ML
数显千分测厚规 SYNTEK CLXL005
电动小鼠刮毛器 中科生命 3303
动物品系：28支雄性BALB/c小鼠，6-8周龄，SPF环境，12小时明暗周期，24-26摄氏度。
3.2 实验分组：
空白对照组：无处理，n＝3；
阳性药物组：卡泊三醇倍他米松软膏，n＝5；
造模组：5％咪喹莫特软膏，n＝5；
测试化合物高剂量组：3.5mg/kg药物腹腔注射+5％咪喹莫特软膏，n＝5；
测试化合物低剂量组：0.8mg/kg药物腹腔注射+5％咪喹莫特软膏，n＝5；
测试组合药物组：0.8mg/kg药物腹腔注射+0.0025％PGE2(于75％乙醇中表皮给药)+5％咪喹莫特软膏，n＝5；
3.3 实验步骤:
空白对照组：
Day0：小鼠麻醉，背部剃毛，面积约2*3厘米；
Day1-7：隔天测量小鼠体重、左右耳厚度、皮肤观察评分；
Day7：处死，背部皮肤取样固定于4％PFA溶液中，用于H&E染色；
咪喹莫特造模组：
Day0：小鼠麻醉，背部剃毛，面积约2*3厘米；
Day1-7：隔天测量小鼠体重、左右耳厚度、皮肤观察评分；
Day1-7：在每天固定时间，背部和右耳造模部位分别涂抹100μl和10μl的75％乙醇，短暂按摩至液体挥发；
Day1-7：随后腹腔注射200μl无菌DPBS做药物溶剂对照；
Day1-7：随后在每支小鼠背部剃毛区涂抹62.5mg咪喹莫特药膏，右耳涂抹250μg咪喹莫特药膏于耳朵内外面，适当逆毛发方向按摩帮助吸收；
Day7：处死，背部皮肤取样固定于4％PFA溶液中，用于H&E染色；
阳性药物组：
Day0：小鼠麻醉，背部剃毛，面积约2*3厘米；
Day1-7：隔天测量小鼠体重、左右耳厚度、皮肤观察评分；
Day1-7：在每天固定时间，每支小鼠背部剃毛区涂抹18mg卡泊三醇倍他米松软膏，右耳内外面涂抹1.8mg卡泊三醇倍他米松软膏，适当逆毛发方向按摩帮助吸收；
Day1-7：在卡泊三醇倍他米松软膏给药1小时肉眼观察完全吸收后，每支小鼠背部剃毛区涂抹62.5mg咪喹莫特药膏，右耳涂抹250μg咪喹莫特于内外面，适当逆毛发方向按摩帮助吸收；
Day7：处死，背部皮肤取样固定于4％PFA溶液中，用于H&E染色；
化合物高剂量组：
Day0：小鼠麻醉，背部剃毛，面积约2*3厘米；
Day1-7：隔天测量小鼠体重、左右耳厚度、皮肤观察评分；
Day1-7：在每天固定时间，背部和耳朵造模部位分别涂抹100μl和10μl的75％乙醇，短暂按摩至液体挥发；
Day1-7：随后腹腔注射给药3.5mg/kg于200μl无菌DPBS中；
Day1-7：随后每支小鼠背部剃毛区涂抹62.5mg咪喹莫特药膏，右耳涂抹250μg咪喹莫特药膏于内外面，适当逆毛发方向按摩帮助吸收；
Day7：处死，背部皮肤取样固定于4％PFA溶液中用于H&E染色；
化合物低剂量组：
Day0：小鼠麻醉，背部剃毛，面积约2*3厘米；
Day1-7：隔天测量小鼠体重、左右耳厚度、皮肤观察评分；
Day1-7：在每天固定时间，背部和耳朵造模部位分别涂抹100μl和10μl的75％乙醇，短暂按摩至液体挥发；
Day1-7：随后腹腔注射给药0.8mg/kg于200μl无菌DPBS中；
Day1-7：随后每支小鼠背部剃毛区涂抹62.5mg咪喹莫特药膏，右耳涂抹250μg咪喹莫特药膏于内外面，适当逆毛发方向按摩帮助吸收；
Day7：处死，背部皮肤取样固定于4％PFA溶液中用于H&E染色；
化合物和PGE2联合用药组：
Day0：小鼠麻醉，背部剃毛，面积约2*3厘米；
Day1-7：隔天测量小鼠体重、左右耳厚度、皮肤观察评分；
Day1-7：在每天固定时间，背部和耳朵造模部位分别涂抹100μl和10μl的0.0025％的PGE2(每只小鼠背部2.5μg，于75％乙醇中)，短暂按摩至液体挥发；
Day1-7：随后腹腔注射给药0.8mg/kg于200μl无菌DPBS中；
Day1-7：在PGE2给药1小时后，每支小鼠背部剃毛区涂抹62.5mg咪喹莫特药膏，右耳涂抹250μg咪喹莫特药膏于内外面，适当逆毛发方向按摩帮助吸收；
Day7：处死，背部皮肤取样固定于4％PFA溶液中，用于H&E染色；
3.4 实验结果
所采用的实验指标为银屑病样指数评分：
病变皮肤面积，斑块，发红，皮疹四个方面。面积在疾病模型中固定不计，其他三方面定量为：0＝无症状，1＝轻微，2＝中度，3＝显著，4＝非常显著；
耳朵皮肤增厚变化；
体重；
皮肤组织学变化(H&E染色)。
在咪喹莫特(Imiquimod)诱导小鼠银屑病模型实验中，我们采用6-[3-(二甲氨基)丙酰]福司可林盐酸盐的高剂量3.5mg/kg和低剂量0.8mg/kg组，同时设计了0.8mg/kg 6-[3-(二甲氨基)丙酰]福司可林盐酸盐与0.0025％***素E2(PGE2)的联合给药组。阳性处理组为目前临床一线外用组合药卡泊三醇倍他米松软膏。在连续7天给药中，我们观察到6-[3-(二甲氨基)丙酰]福司可林盐酸盐高低剂量组以及与PGE2联合用药组均能在一定时间段表现出减缓各项银屑病样指标的效果，且联合用药组与阳性对照一线用药相比在避免减轻小鼠体重减轻、皮肤发炎发红等指标上效果更优。
如图14所示，小鼠背部造模区皮肤的银屑病样指数随给药时间增加逐渐增大，统计显著性分析见表10。
表10小鼠背部皮肤银屑病样指数评分统计显著性分析，T-test，灰框为P<0.05。
Figure PCTCN2019129945-appb-000033
造模组表现为皮肤皮屑、发红和皮疹的严重化(图15)。6-[3-(二甲氨基)丙酰]福司可林盐酸盐高剂量组起效比低剂量组快，在给药第3天就表现出病情减缓效果，而低剂量组从第5天开始体现疗效，但在终点7天时高剂量组和低剂量组的药效差异有缩小的趋势，提示高剂量组虽起效快，但在长期给药情况下高低剂量的疗效可能趋同。另外，联合用药组效果优于高剂量组，这与实施例的体外实验结果一致，并且其改善疗效与阳性用药卡泊三醇倍他米松软膏相当。从图15可看出，相比起咪喹莫特造模组和单独的高、低剂量组，联合用药组小鼠背部皮肤光滑无斑块，皮肤发红情况也比阳性对照组更轻。表10为背部银屑病样评分的组间统计学差异，结合图14，我们可以得出结论，6-[3-(二甲氨基)丙酰]福司可林盐酸盐单独的高、低剂量组和联合用药组均能显著减缓小鼠背部造模皮肤银屑病样病理特征的发展，且联合用药组与当前一线用药卡泊三醇倍他米松疗效相当。
另外，如图16和图17所示，我们平行测量了小鼠的左右耳厚度，结果显示咪喹莫特造模右耳相较于未处理左耳会出现发炎性增厚和血管扩张皮肤发红的特征，该特征在咪喹莫特7天连续给药时间里是逐渐增强的。药物高、低剂量组和联合给药组都能在一定时间段内表现出减缓咪喹莫特诱导右耳发炎增厚和发红的效应，在给药终点7天时，组合用药效果最强(减缓52％)，与阳性用药(减缓53％)类似，高剂量组(减缓39％)与低剂量组(减缓30％)略弱，但高、低剂量组之间无显著差异，提示持续给药后期高、低剂量效果差异可能缩小，与背部评分实验结果类似。表11列出图17组间显著性差异分析数值。
表11小鼠耳部银屑病样指数评分统计分析显著性数据，灰框代表P<0.05。
Figure PCTCN2019129945-appb-000034
图18显示给药终点7天时各处理组的造模右耳不同程度的皮肤发炎发红情况。图16显示实验第3天时同一只小鼠的左右耳发红的比较，提示高剂量组可在造模3天时较早起到减缓疗效。
我们还分析了小鼠在给药期间的体重变化(图19)，除阳性对照组卡泊三醇倍他米松处理导致小鼠体重减轻，其他组小鼠均未出现体重明显变化。这与既有研究报道和本研究体系相关预实验结果一致，阳性组合药物中的激素类成分会减轻小鼠体重，且与其使用剂量正相关。这提示当前一线用药卡泊三醇倍他米松仍有其临床长期使用的副作用，而本发明的药物成分和组合未见影响体重，有望克服已有一线用药的副作用，成为更优用药选择。
背部造模区皮肤的组织学分析(图20)表明，造模组相较于空白组，小鼠背部皮肤的表皮和角质层均有增厚，而单独高低剂量、联合给药组和阳性对照组均有不同程度的厚度减轻。
以上实施例仅用以说明本发明的技术方案，而非对其限制；本领域的普通技术人员应当理解：可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围，其均应涵盖在本发明说明书的范围中。
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Claims (82)

  1. 药物组合物，其包括式I的化合物或其药物上可接受的盐：
    Figure PCTCN2019129945-appb-100001
    其中：
    R 3为-CH＝CH 2、-CH 2CH 3、或环丙基；
    R 1和R 2中的一个为-COCH 2CH 3、-CO 2CH 2CH 3、-COCH 2OCHO或基团
    Figure PCTCN2019129945-appb-100002
    其中每个R 4和R 5独立地为氢或低级烷基，或者R 4与R 5相结合而形成低级亚烷基链，所述低级亚烷基链含有或不含氧原子或氮原子，m为1至5的整数；R 1和R 2中的另一个为氢或基团CO(CH 2) nX，其中X为氢或基团
    Figure PCTCN2019129945-appb-100003
    其中R 6和R 7独立地为氢或低级烷基，或者R 6和R 7相结合而形成低级亚烷基链，所述低级亚烷基链中含有或不含有氧原子或氮原子，n为1至5中整数；或者
    R 1为氢或-COCH 2CH 2CO 2H，R 2为氢、-COCH 3、-COCH 2CH 2CH 2CO 2H或-COCH(OH)CH 2OH，条件是当R 1为氢时，R 2是-COCH 2CH 2CH 2CO 2H或-COCH(OH)CH 2OH。
  2. 如权利要求1所述的药物组合物，其中式I中R 1为氢或基团
    Figure PCTCN2019129945-appb-100004
    其中m、R 4和R 5定义同权利要求1。
  3. 如权利要求1或2所述的药物组合物，其中式I中R 1为
    Figure PCTCN2019129945-appb-100005
    R 2为-CO(CH 2) nX，且R 3为-CH＝CH 2或-CH 2CH 3，其中R 4、R 5、m、n和X定义同权利要求1；或者R 1为氢或-COCH 2CH 2CO 2H，R 2为-COCH(OH)CH 2(OH)，且R 3为-CH＝CH 2。
  4. 如权利要求1-3任一项所述的药物组合物，其中式I中R 1为-COCH 2N(CH 3) 2、-CO(CH 2) 2N(CH 3) 2、-CO(CH 2) 3N(CH 3) 2、或者-CO(CH 2) 3NH 2，且R 2为-COCH 3。
  5. 如权利要求1-4任一项所述的药物组合物，其中式I中R 1为氢，R 2为-COCH 2CH 3、-CO 2CH 2CH 3或者-COCH 2OCHO，且R 3为-CH＝CH 2。
  6. 如权利要求1-5任一项所述的药物组合物，其中式I的化合物选自6-(4-氨基丁酰)福司可林、6-[4-(二甲氨基)丁酰]福司可林、6-[3-氨基丙酰]福司可林、6-[3-(甲氨基)丙酰]福司可林、6-[3-(二甲氨基)丙酰]福司可林、和6-[(哌啶)乙酰基]-7-脱乙酰化福司可林。
  7. 如权利要求1-6任一项所述的药物组合物，其中所述药物上可接受的盐为盐酸盐。
  8. 如权利要求1-7任一项所述的药物组合物，其包括6-[3-(二甲氨基)丙酰]福司可林盐酸盐。
  9. 如权利要求1-8任一项所述的药物组合物，其包括6-[3-(甲氨基)丙酰]福司可林盐酸盐。
  10. 如权利要求1-9任一项所述的药物组合物，其与***素类化合物联合给药。
  11. 如权利要求1-10任一项所述的药物组合物，其还包括***素类化合物。
  12. 如权利要求1-11任一项所述的药物组合物，其中所述***素类化合物选自***素E2、氨丁三醇地诺前列素、卡前列素、氨丁三醇卡前列素、***素E1、贝美前列素、伊洛前列素、利马前列素、利马前列素α环糊精、米索前列素、吉美前列素、拉坦前列素、硫前列酮、奥诺前列素以及它们的药物上可接受的盐。
  13. 如权利要求1-12任一项所述的药物组合物，其与糖皮质激素类化合物联合给药。
  14. 如权利要求1-13任一项所述的药物组合物，其还包括糖皮质激素类化合物。
  15. 如权利要求1-14任一项所述的药物组合物，其中所述糖皮质激素类化合物选自***、氢化可的松、***、***龙、对氟米松、可的松、倍他米松、甲基***、氟氢可的松、曲安奈德以及它们的药物上可接受的盐。
  16. 如权利要求1-15任一项所述的药物组合物，其包括6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***素E2。
  17. 如权利要求1-16任一项所述的药物组合物，其包括6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***。
  18. 如权利要求1-17任一项所述的药物组合物，其用于在受试者中进行免疫抑制。
  19. 如权利要求1-18任一项所述的药物组合物，其用于在受试者中治疗自身免疫性疾病。
  20. 如权利要求1-19任一项所述的药物组合物，其用作消炎药或免疫抑制剂。
  21. 如权利要求1-20任一项所述的药物组合物，其用于在受试者中治疗银屑病。
  22. 如权利要求1-21任一项所述的药物组合物，其用于在受试者中治疗银屑病性关节炎。
  23. 如权利要求1-22任一项所述的药物组合物，其用于在受试者中抑制肿瘤坏死因子α(TNF-α)水平。
  24. 如权利要求1-23任一项所述的药物组合物，其用于在受试者中抑制白介素17A(IL-17A)水平。
  25. 如权利要求1-24任一项所述的药物组合物，其以片剂、颗粒、粉末、胶囊、注射制剂、栓剂、滴剂、外贴药膏、软膏、药油、或喷雾剂的形式存在。
  26. 在受试者中进行免疫抑制的方法，包括以式I的化合物或其药物上可接受的盐向所述受试者给药：
    Figure PCTCN2019129945-appb-100006
    其中：
    R 3为-CH＝CH 2、-CH 2CH 3、或环丙基；
    R 1和R 2中的一个为-COCH 2CH 3、-CO 2CH 2CH 3、-COCH 2OCHO或基团
    Figure PCTCN2019129945-appb-100007
    其中每个R 4和R 5独立地为氢或低级烷基，或者R 4与R 5相结合而形成低级亚烷基链，所述低级亚烷基链含有或不含氧原子或氮原子，m为1至5的整数；R 1和R 2中的另一个为氢或基团CO(CH 2) nX，其中X为氢或基团
    Figure PCTCN2019129945-appb-100008
    其中R 6和R 7独立地为氢或低级烷基，或者R 6和R 7相结合而形成低级亚烷基链，所述低级亚烷基链中含有或不含有氧原子或氮原子，n为1至5中整数；或者
    R 1为氢或-COCH 2CH 2CO 2H，R 2为氢、-COCH 3、-COCH 2CH 2CH 2CO 2H或-COCH(OH)CH 2OH，条件是当R 1为氢时，R 2是-COCH 2CH 2CH 2CO 2H或-COCH(OH)CH 2OH。
  27. 如权利要求26所述的方法，其中式I中R 1为氢或基团
    Figure PCTCN2019129945-appb-100009
    其中m、R 4和R 5定义同权利要求26。
  28. 如权利要求26或27所述的方法，其中式I中R 1为
    Figure PCTCN2019129945-appb-100010
    R 2为-CO(CH 2) nX，且R 3为-CH＝CH 2或-CH 2CH 3，其中R 4、R 5、m、n和X定义同权利要求26；或者R 1为氢或-COCH 2CH 2CO 2H，R 2为-COCH(OH)CH 2(OH)，且R 3为-CH＝CH 2。
  29. 如权利要求26-28任一项所述的方法，其中式I中R 1为-COCH 2N(CH 3) 2、-CO(CH 2) 2N(CH 3) 2、-CO(CH 2) 3N(CH 3) 2、或者-CO(CH 2) 3NH 2，且R 2为-COCH 3。
  30. 如权利要求26-29任一项所述的方法，其中式I中R 1为氢，R 2为-COCH 2CH 3、-CO 2CH 2CH 3或者-COCH 2OCHO，且R 3为-CH＝CH 2。
  31. 如权利要求26-30任一项所述的方法，其中式I的化合物选自6-(4-氨基丁酰)福司可林、6-[4-(二甲氨基)丁酰]福司可林、6-[3-氨基丙酰]福司可林、6-[3-(甲氨基)丙酰]福司可林、6-[3-(二甲氨基)丙酰]福司可林、和6-[(哌啶)乙酰基]-7-脱乙酰化福司可林。
  32. 如权利要求26-31任一项所述的方法，其中所述药物上可接受的盐为盐酸盐。
  33. 如权利要求26-32任一项所述的方法，其包括以6-[3-(二甲氨基)丙酰]福司可林盐酸盐向所述受试者给药。
  34. 如权利要求26-33任一项所述的方法，其包括以6-[3-(甲氨基)丙酰]福司可林盐酸盐向所述受试者给药。
  35. 如权利要求26-34任一项所述的方法，其包括联合***素类化合物向所述受试者给药。
  36. 如权利要求26-35任一项所述的方法，其中所述***素类化合物选自***素E2、氨丁三醇地诺前列素、卡前列素、氨丁三醇卡前列素、***素E1、贝美前列素、伊洛前列素、利马前列素、利马前列素α环糊精、米索前列素、吉美前列素、拉坦前列素、硫前列酮、奥诺前列素以及它们的药物上可接受的盐。
  37. 如权利要求26-36任一项所述的方法，其包括联合糖皮质激素类化合物向所述受试者给药。
  38. 如权利要求26-37任一项所述的方法，其中所述糖皮质激素类化合物选自***、氢化可的松、***、***龙、对氟米松、可的松、倍他米松、甲基***、氟氢可的松、曲安奈德以及它们的药物上可接受的盐。
  39. 如权利要求26-38任一项所述的方法，其包括以6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***素E2向所述受试者给药。
  40. 如权利要求26-39任一项所述的方法，其包括以6-[3-(二甲氨基)丙酰]福司可林盐酸盐和***向所述受试者给药。
  41. 如权利要求26-40任一项所述的方法，其中式I的化合物或其药物上可接受的盐与所述***素类化合物同时或顺序地以片剂、颗粒、粉末、胶囊、注射制剂、栓剂、滴剂、外贴药膏、软膏、药油、或喷雾剂形式向所述受试者给药。
  42. 如权利要求26-41任一项所述的方法，其中式I的化合物或其药物上可接受的盐与所述糖皮质激素类化合物同时或顺序地以片剂、颗粒、粉末、胶囊、注射制剂、栓剂、滴剂、外贴药膏、软膏、药油、或喷雾剂形式向所述受试者给药。
  43. 如权利要求26-42任一项所述的方法，其中所述受试者为自身免疫性疾病患者。
  44. 如权利要求26-43任一项所述的方法，其中所述受试者为银屑病患者。
  45. 如权利要求26-44任一项所述的方法，其中所述受试者为银屑病性关节炎患者。
  46. 如权利要求26-45任一项所述的方法，其中所述受试者为炎症患者。
  47. 如权利要求26-46任一项所述的方法，其中式I的化合物或其药物上可接受的盐通过在所述受试者中降低免疫细胞的TNF-α表达而起作用。
  48. 如权利要求26-47任一项所述的方法，其中式I的化合物或其药物上可接受的盐通过在所述受试者中降低免疫细胞的IL-17A表达而起作用。
  49. 式I的化合物或其药物上可接受的盐在制备用于进行免疫抑制的药物中的用途。
    Figure PCTCN2019129945-appb-100011
    其中：
    R 3为-CH＝CH 2、-CH 2CH 3、或环丙基；
    R 1和R 2中的一个为-COCH 2CH 3、-CO 2CH 2CH 3、-COCH 2OCHO或基团
    Figure PCTCN2019129945-appb-100012
    其中每个R 4和R 5独立地为氢或低级烷基，或者R 4与R 5相结合而形成低级亚烷基链，所述低级亚烷基链含有或不含氧原子或氮原子，m为1至5的整数；R 1和R 2中的另一个为氢或基团CO(CH 2) nX，其中X为氢或基团
    Figure PCTCN2019129945-appb-100013
    其中R 6和R 7独立地为氢或低级烷基，或者R 6和R 7相结合而形成低级亚烷基链，所述低级亚烷基链中含有或不含有氧原子或氮原子，n为1至5中整数；或者
    R 1为氢或-COCH 2CH 2CO 2H，R 2为氢、-COCH 3、-COCH 2CH 2CH 2CO 2H或-COCH(OH)CH 2OH，条件是当R 1为氢时，R 2是-COCH 2CH 2CH 2CO 2H或-COCH(OH)CH 2OH。
  50. 如权利要求49所述的药物组合物，其中式I中R 1为氢或基团
    Figure PCTCN2019129945-appb-100014
    其中m、R 4和R 5定义同权利要求49。
  51. 如权利要求49或50所述的用途，其中式I中R 1为
    Figure PCTCN2019129945-appb-100015
    R 2为-CO(CH 2) nX，且R 3为-CH＝CH 2或-CH 2CH 3，其中R 4、R 5、m、n和X定义同权利要求49；或者R 1为氢或-COCH 2CH 2CO 2H，R 2为-COCH(OH)CH 2(OH)，且R 3为-CH＝CH 2。
  52. 如权利要求49-51任一项所述的用途，其中式I中R 1为-COCH 2N(CH 3) 2、-CO(CH 2) 2N(CH 3) 2、-CO(CH 2) 3N(CH 3) 2、或者-CO(CH 2) 3NH 2，且R 2为-COCH 3。
  53. 如权利要求49-52任一项所述的用途，其中式I中R 1为氢，R 2为-COCH 2CH 3、-CO 2CH 2CH 3或者-COCH 2OCHO，且R 3为-CH＝CH 2。
  54. 如权利要求49-53任一项所述的用途，其中式I的化合物选自6-(4-氨基丁酰)福司可林、6-[4-(二甲氨基)丁酰]福司可林、6-[3-氨基丙酰]福司可林、6-[3-(甲氨基)丙酰]福司可林、6-[3-(二甲氨基)丙酰]福司可林、和6-[(哌啶)乙酰基]-7-脱乙酰化福司可林。
  55. 如权利要求49-54任一项所述的用途，其中所述药物上可接受的盐为盐酸盐。
  56. 如权利要求49-55任一项所述的用途，其中所述药物包括6-[3-(二甲氨基)丙酰]福司可林盐酸盐。
  57. 如权利要求49-56任一项所述的用途，其中所述药物包括6-[3-(甲氨基)丙酰]福司可林盐酸盐
  58. 如权利要求49-57任一项所述的用途，其中所述药物用于与***素类化合物联合给药。
  59. 如权利要求49-58任一项所述的用途，其中所述***素类化合物选自***素E2、氨丁三醇地诺前列素、卡前列素、氨丁三醇卡前列素、***素E1、贝美前列素、伊洛前列素、利马前列素、利马前列素α环糊精、米索前列素、吉美前列素、拉坦前列素、硫前列酮、奥诺前列素以及它们的药物上可接受的盐。
  60. 如权利要求49-59任一项所述的用途，其中所述药物用于与糖皮质激素类化合物联合给药。
  61. 如权利要求49-60任一项所述的用途，其中所述糖皮质激素类化合物选自***、氢化可的松、***、***龙、对氟米松、可的松、倍他米松、甲基***、氟氢可的松、曲安奈德以及它们的药物上可接受的盐。
  62. 如权利要求49-61任一项所述的用途，其中所述药物包括6-[3-(二甲氨基)丙酰]福司可林盐酸盐，并且用于与***素E2联合给药。
  63. 如权利要求49-62任一项所述的用途，其中所述药物包括6-[3-(二甲氨基)丙酰]福司可林盐酸盐，并且用于与***联合给药。
  64. 如权利要求49-63任一项所述的用途，其中所述药物为自身免疫性疾病药物。
  65. 如权利要求49-64任一项所述的用途，其中所述药物为银屑病药物。
  66. 如权利要求49-65任一项所述的用途，其中所述药物为银屑病性关节炎药物。
  67. 如权利要求49-66任一项所述的用途，其中所述药物为消炎药。
  68. 如权利要求49-67任一项所述的用途，其中所述药物以片剂、颗粒、粉末、胶囊、注射制剂、栓剂、滴剂、外贴药膏、软膏、药油、或喷雾剂的形式存在。
  69. 如权利要求49-68任一项所述的用途，其中式I的化合物或其药物上可接受的盐通过在所述受试者中降低免疫细胞的TNF-α表达而起作用。
  70. 如权利要求49-69任一项所述的用途，其中式I的化合物或其药物上可接受的盐通过在所述受试者中降低免疫细胞的IL-17A表达而起作用。
  71. 药物组合物，包括
    1)福司可林；以及
    2)***素类化合物或糖皮质激素类化合物。
  72. 如权利要求71所述的药物组合物，其中所述***素类化合物选自***素E2、氨丁三醇地诺前列素、卡前列素、氨丁三醇卡前列素、***素E1、贝美前列素、伊洛前列素、利马前列素、利马前列素α环糊精、米索前列素、吉美前列素、拉坦前列素、硫前列酮、奥诺前列素以及它们的药物上可接受的盐。
  73. 如权利要求71或72所述的药物组合物，其中所述糖皮质激素类化合物选自***、氢化可的松、***、***龙、对氟米松、可的松、倍他米松、甲基***、氟氢可的松、曲安奈德以及它们的药物上可接受的盐。
  74. 如权利要求71-73任一项所述的药物组合物，其用于在受试者中治疗银屑病或银屑病性关节炎。
  75. 在受试者中进行免疫抑制的方法，包括以1)福司可林和2)***素类化合物或糖皮质激素类化合物向所述受试者给药。
  76. 如权利要求75所述的方法，其中所述***素类化合物选自***素E2、氨丁三醇地诺前列素、卡前列素、氨丁三醇卡前列素、***素E1、贝美前列素、伊洛前列素、利马前列素、利马前列素α环糊精、米索前列素、吉美前列素、拉坦前列素、硫前列酮、奥诺前列素以及它们的药物上可接受的盐。
  77. 如权利要求75或76所述的方法，其中所述糖皮质激素类化合物选自***、氢化可的松、***、***龙、对氟米松、可的松、倍他米松、甲基***、氟氢可的松、曲安奈德以及它们的药物上可接受的盐。
  78. 如权利要求75-77任一项所述的方法，其中所述受试者为银屑病患者或银屑病性关节炎患者。
  79. 福司可林在制备与***素类化合物或糖皮质激素类化合物联合给药的药物中的用途。
  80. 如权利要求79所述的用途，其中所述***素类化合物选自***素E2、氨丁三醇地诺前列素、卡前列素、氨丁三醇卡前列素、***素E1、贝美前列素、伊洛前列素、利马前列素、利马前列素α环糊精、米索前列素、吉美前列素、拉坦前列素、硫前列酮、奥诺前列素以及它们的药物上可接受的盐。
  81. 如权利要求79或80所述的用途，其中所述糖皮质激素类化合物选自***、氢化可的松、***、***龙、对氟米松、可的松、倍他米松、甲基***、氟氢可的松、曲安奈德以及它们的药物上可接受的盐。
  82. 如权利要求79-81所述的用途，其中所述药物为银屑病药物或银屑病性关节炎药物。
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