WO2018133862A1 - 细胞色素bc1复合物抑制剂在制备医药组合物中的用途 - Google Patents

细胞色素bc1复合物抑制剂在制备医药组合物中的用途 Download PDF

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WO2018133862A1
WO2018133862A1 PCT/CN2018/073609 CN2018073609W WO2018133862A1 WO 2018133862 A1 WO2018133862 A1 WO 2018133862A1 CN 2018073609 W CN2018073609 W CN 2018073609W WO 2018133862 A1 WO2018133862 A1 WO 2018133862A1
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elq
cytochrome bcl
bcl complex
complex inhibitor
cytochrome
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PCT/CN2018/073609
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English (en)
French (fr)
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从玉文
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北京蔚蓝之源医药科技有限公司
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Priority to AU2018209571A priority Critical patent/AU2018209571B2/en
Priority to CN201880007758.4A priority patent/CN110545815A/zh
Priority to KR1020197023819A priority patent/KR102466932B1/ko
Priority to US16/479,531 priority patent/US11058690B2/en
Priority to CA3050828A priority patent/CA3050828C/en
Priority to EP18741427.1A priority patent/EP3574903A4/en
Priority to JP2019560444A priority patent/JP7146809B2/ja
Publication of WO2018133862A1 publication Critical patent/WO2018133862A1/zh

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Definitions

  • the present invention relates to the field of biomedicine, and in particular to the medical use of a cytochrome bcl complex inhibitor.
  • the invention relates to the use of a cytochrome bcl complex inhibitor for the treatment of a smooth muscle spasm-related disease, for the treatment of inflammatory diseases and for the relief of pain.
  • the mitochondrial cytochrome bc1 complex also known as the cytochrome c reductase, also known as the mitochondrial respiratory chain complex III, is an important component of the mitochondria and most of the bacterial respiration electron transport chains, catalyzing the transfer of electrons from ubiquinone to cells.
  • the reaction of pigment c cytochrome c2 in bacteria.
  • the cytochrome bcl complex has two functional binding sites: one is the oxidation site Q O on the side of the membrane gap, close to the heme b L ; one reduction site Q i is on the side of the matrix, close to the heme b H .
  • Cytochrome bcl complex inhibitors can therefore be divided into two categories: one is an inhibitor that binds to the Q i site located in the inner wall of the mitochondrial inner membrane, and such inhibitors are called Q i site inhibitors, such as antimycin and Cyanosole; the other is an inhibitor that binds to the Q O site located in the outer lining of the mitochondria. These inhibitors are called Q O site inhibitors, such as methoxy acrylate fungicides, imidacloprid. Wait.
  • the present invention provides a method for treating a smooth muscle spasm-related disease, treating an inflammatory disease, and/or relieving pain using a cytochrome bcl complex inhibitor, which is prepared for treating a smooth muscle spasm-related disease, treating an inflammatory disease, and/or Or use in a pain-relieving drug, and a pharmaceutical composition comprising a cytochrome bcl complex inhibitor.
  • the cytochrome bcl complex inhibitor is a cytochrome bcl complex Qo site inhibitor.
  • the cytochrome bcl complex inhibitor is a cytochrome bcl complex, a Qi site inhibitor.
  • the cytochrome bcl complex inhibitor is a methoxyacrylate cytochrome bcl complex inhibitor.
  • the methoxy acrylate cytochrome bcl complex inhibitor is selected from the group consisting of: pyracrypyrim (FAPM), azoxystrobin, trifloxystrobin, ether bacteria Kresoxim-methyl, pyraclostrobin, Picoxystrobin, Dimoxystrobin, Fluoxastrobin, and prodrugs thereof or pharmaceutically acceptable thereof salt.
  • the cytochrome bcl complex inhibitor is a pyridone cytochrome bcl complex inhibitor.
  • the pyridone cytochrome bcl complex inhibitor is selected from the group consisting of clopidogol, GW844520, and GSK932121, and prodrugs thereof or pharmaceutically acceptable salts thereof.
  • the cytochrome bcl complex inhibitor is a hydroxynaphthoquinone cytochrome bcl complex inhibitor.
  • the hydroxynaphthoquinone cytochrome bcl complex inhibitor is selected from the group consisting of Atovaquone, Parvaquone, Buparvaquone, S-10576, NQ3. (2-OH-3-(2-methyl-trifluorooctyl)-8-methyl-naphthoquinone), and their prodrugs or their pharmaceutically acceptable salts.
  • the cytochrome bcl complex inhibitor is a quinolone cytochrome bcl complex inhibitor.
  • the quinolone cytochrome bcl complex inhibitor is selected from the group consisting of RCQ06, Endochin, and Endochin-like quinolone (ELQ), such as ELQ-118, ELQ-120, ELQ-121, ELQ-136, ELQ-233, ELQ-245, ELQ-260, ELQ-271, ELQ-274, ELQ-300, ELQ-314, ELQ-316, ELQ-317, ELQ-319, ELQ-337, ELQ-338, ELQ- 351, ELQ-370, ELQ-372, ELQ-380, ELQ-384, ELQ-385, ELQ-388, ELQ-390, ELQ-400, ELQ-404, ELQ-428, P4Q-95 and
  • ELQ Endochin
  • the cytochrome bcl complex inhibitor is an acridinedione cytochrome bcl complex inhibitor.
  • the acridinedione cytochrome bcl complex inhibitor is selected from the group consisting of Fluxacrine, WR249685, WR243246, and prodrugs thereof or pharmaceutically acceptable salts thereof.
  • the cytochrome bcl complex inhibitor is a statin lactone cytochrome bcl complex inhibitor.
  • the statin lactone cytochrome bcl complex inhibitor is selected from the group consisting of Simvastatin, Cerivastatin, Rosuvastatin, and Pravastatin. ), pitavastatin, mevastatin, lovastatin, fluvastatin, atorvastatin, and prodrugs thereof or pharmaceutically acceptable salts thereof .
  • the cytochrome bcl complex inhibitor is selected from the group consisting of antimycin A, Lansoprazole, Lansoprazole sulfide, omeprazole (Omeprazole), Pentamidine, and their prodrugs or their pharmaceutically acceptable salts.
  • the smooth muscle spasm-related disease is selected from the group consisting of dysmenorrhea, airway sputum related diseases such as bronchial asthma, asthmatic bronchitis, chronic obstructive pulmonary disease or airway hyperresponsiveness, vasospasm related diseases such as cerebral vasospasm , migraine or cluster headache, gastrointestinal tract related diseases such as acute gastritis, chronic gastritis, gastric ulcer, duodenal ulcer, acute enteritis, chronic enteritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis or Intestinal tuberculosis, hypertension, coronary artery spasm-related diseases such as variant angina, glaucoma, postoperative intestinal obstruction, urinary frequency, fibromyalgia, sexual pain, irritable bowel syndrome, neck muscles and eyelids, overactive bladder , postoperative eye inflammation and respiratory distress syndrome.
  • airway sputum related diseases such as bronchial asthma, asthmatic bronchitis, chronic
  • the inflammatory disease is selected from the group consisting of rheumatoid arthritis, osteoarthritis, osteolysis, tendinitis, synovitis, and inflammatory respiratory diseases such as chronic obstructive pulmonary disease, fibrosis, emphysema Acute respiratory distress syndrome.
  • the pain is inflammatory pain.
  • FIG. 1 shows the chemical structure of FAPM and its derivatives.
  • Figure 2 Shows the inhibitory effect of FAPM and its derivatives on PGF2 ⁇ -induced contraction of rat uterine smooth muscle.
  • Figure 3 Shows the inhibitory effect of cytochrome bcl complex Qo site inhibitor on PGF2 ⁇ -induced rat uterine smooth muscle contraction.
  • Figure 4 Shows the inhibitory effect of FAPM on rat uterine smooth muscle contraction induced by oxytocin in rats.
  • Figure 5 Shows the inhibitory effect of FAPM on acetylcholine-induced contraction of rat uterine smooth muscle.
  • Figure 6 Shows the inhibitory effect of FAPM on potassium chloride-induced contraction of rat uterine smooth muscle.
  • Figure 7 Shows the inhibition of atropine on PGF2 ⁇ -induced contraction of rat uterine smooth muscle.
  • IC50 8.44 ⁇ 0.7 ⁇ M.
  • Figure 8 Shows the inhibitory effect of FAPM on PGF2 ⁇ -induced phosphorylation of rat smooth muscle cells MLC20.
  • cytochrome bcl complex inhibitor can inhibit smooth muscle contraction in vivo and can be used for treating a smooth muscle spasm-related disease.
  • the present invention provides a method of treating a smooth muscle spasm-related disease in a subject comprising administering to the subject a therapeutically effective amount of a cytochrome bcl complex inhibitor.
  • cytochrome bcl complex inhibitors may inhibit smooth muscle contraction by preventing phosphorylation of MLC20.
  • Myosin light chain kinase MLCK
  • MLC20 Myosin Light Chain
  • MLC20 Myosin Light Chain
  • MLCK-mediated phosphorylation of MLC20 is required to initiate smooth muscle contraction.
  • the present inventors have surprisingly found that a cytochrome bcl complex inhibitor can prevent phosphorylation of MLC20, thereby treating a smooth muscle spasm-related disease by inhibiting smooth muscle contraction.
  • smooth muscle includes, but is not limited to, uterine smooth muscle, bladder smooth muscle, iris muscle, genital smooth muscle, fallopian tube smooth muscle, bronchial smooth muscle, vascular smooth muscle, and gastrointestinal smooth muscle.
  • smooth muscle spasm-related disease includes, but is not limited to, a disease selected from the following 1)-8):
  • Obstetrics and gynecology diseases (uterine smooth muscle): dysmenorrhea; premature delivery; premature rupture of fetal membranes.
  • urinary system diseases urethral smooth muscle, bladder smooth muscle: benign prostatic hyperplasia; urinary stones, such as kidney stones, ureteral stones, etc.; overactive bladder; frequent urination; erectile dysfunction.
  • Digestive diseases esophageal smooth muscle, sphincter, gastrointestinal smooth muscle: esophageal achalasia; irritable bowel syndrome; Oddi sphincter dysfunction; gastrointestinal spasm, such as gastritis, gastric ulcer, duodenum Ulcer, ulcerative colitis, Crohn's disease, acute and chronic appendicitis; cholelithiasis; acute and chronic cholecystitis; acute and chronic pancreatitis; acute and chronic peritonitis; intestinal obstruction.
  • Respiratory diseases trachea, bronchial smooth muscle: asthma, such as chronic bronchitis, allergic asthma, drug-induced asthma, senile asthma, cough variant asthma, chronic asthma, sports asthma, childhood asthma; trachea Inflammation; acute and chronic bronchitis; chronic obstructive pulmonary disease; respiratory distress syndrome.
  • cardiovascular disease vascular smooth muscle: hypertension; portal hypertension; atherosclerosis; angina pectoris, such as variant angina, unstable angina, stable angina, etc.; myocardial infarction; hypertrophic cardiomyopathy.
  • cerebral vasospasm related diseases ischemic cerebrovascular disease, such as cerebral infarction, transient ischemic attack, cerebral thrombosis, etc.; vascular dementia; cerebral arteriosclerosis; dizziness; headache; intractable hiccup.
  • peripheral vasospasm related diseases migraine; intermittent claudication; Raynaud's syndrome; glaucoma; sudden deafness; tinnitus; dizziness;
  • the "smooth muscle spasm-related disease” may be selected from the group consisting of dysmenorrhea, airway sputum related diseases such as bronchial asthma, asthmatic bronchitis, chronic obstructive pulmonary disease or airway hyperresponsiveness, vasospasm related diseases such as cerebral vasospasm, pulmonary artery High blood pressure, vascular deafness, migraine or cluster headache, gastrointestinal spasm related diseases such as cholecystitis, cholangitis, biliary calculi, acute gastritis, chronic gastritis, gastric ulcer, duodenal ulcer, acute enteritis, chronic enteritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis or intestinal tuberculosis, hypertension, coronary artery spasm-related diseases such as variant angina, cystitis, ureteral stones, renal colic and glaucoma.
  • airway sputum related diseases such as bronchial asthma, asthmatic
  • the "smooth muscle spasm-related disease” may also include, for example, postoperative intestinal obstruction, urinary frequency, fibromyalgia, dyspareunia, irritable bowel syndrome, neck muscles and eyelids, overactive bladder, Postoperative eye inflammation, respiratory distress syndrome, etc.
  • treatment with a cytochrome bcl complex inhibitor can result in at least partial smooth muscle cell relaxation, resulting in an increase in arterial diameter of the aorta (eg, aorta) and middle artery, lowering blood pressure, and increasing the middle artery (eg, coronary artery)
  • the diameter of the arteries reduces angina pectoris.
  • Relaxing pulmonary bronchial smooth muscle cells cause an increase in airway diameter and attenuate asthma symptoms.
  • the catheter system of the relaxed eye causes an increase in intraocular pressure such as an increase in the diameter of the lacrimal duct, thereby reducing glaucoma and vision.
  • the risk of loss, relaxation of the fallopian tubes and smooth muscle cells of the uterus leads to relaxation of the muscle tissue to improve fertility and / or reduce the symptoms of dysmenorrhea, smooth muscle cells of the bile duct, ureter and urethra increase the diameter of the catheter and reduce the gallstone or kidney stone
  • the risk of paralysis is caused by relaxing the smooth muscles of the gastrointestinal tract and reducing, for example, spastic colic.
  • the smooth muscle spasm associated disease is dysmenorrhea. In a further preferred embodiment, the smooth muscle spasm associated disease is primary dysmenorrhea.
  • cytochrome bcl complex inhibitors can significantly inhibit inflammatory symptoms in vivo.
  • Example 5 shows that a cytochrome bcl complex inhibitor is capable of inhibiting xylene-induced mouse auricle swelling and carrageenan-induced paw swelling in rats.
  • the present invention provides a method of treating an inflammatory disease in a subject comprising administering to the subject a therapeutically effective amount of a cytochrome bcl complex inhibitor.
  • cytochrome bcl complex inhibitors may inhibit inflammation by inhibiting the production of nitric oxide (NO).
  • NO is an important small-molecule substance that regulates life activities and is synthesized by L-arginine by nitric oxide synthase (NOS).
  • NOS nitric oxide synthase
  • the present inventors have surprisingly found that cytochrome bcl complex inhibitors can inhibit the production of nitric oxide in macrophages and thus can be used to treat inflammatory diseases.
  • Example 3 demonstrates that cytochrome bcl complex inhibitors are capable of inhibiting NO production in mouse monocyte macrophages.
  • inflammatory diseases which can be treated by the method of the invention include rheumatoid arthritis, osteoarthritis, osteolysis, tendonitis, synovitis, gout, Alzheimer's disease, glomerulonephritis, pyelonephritis, atherosclerosis Sclerosing diseases, vasculitis, and inflammatory respiratory diseases (such as chronic obstructive pulmonary disease, fibrosis, emphysema, acute respiratory distress syndrome, etc.).
  • the inflammatory disease is a non-infectious inflammatory disease.
  • cytochrome bcl complex inhibitors have an analgesic effect in vivo.
  • administration of a cytochrome bcl complex inhibitor can significantly inhibit the number of writhing in mice caused by acetic acid, diethylstilbestrol, and PGF2 ⁇ .
  • the invention provides a method of relieving pain in a subject, comprising administering to the subject a therapeutically effective amount of a cytochrome bcl complex inhibitor.
  • the pain is inflammatory pain.
  • the inflammatory mediator released by the inflammation site initiates a series of signal transduction pathways that activate or sensitize the pain receptors, causing pain.
  • the cytochrome bcl complex inhibitor is a cytochrome bcl complex Qo site inhibitor. In other embodiments of various aspects of the invention, the cytochrome bcl complex inhibitor is a cytochrome bcl complex, a Qi site inhibitor.
  • the cytochrome bcl complex inhibitor is a methoxyacrylate cytochrome bcl complex inhibitor.
  • methoxy acrylate-based cytochrome bcl complex inhibitors include, but are not limited to, Fluacrypyrim (FAPM), azoxystrobin, Trifloxystrobin, and Etherin (Kresoxim- Methyl), pyroclostrobin, Picoxystrobin, Dimoxystrobin, Fluoxastrobin, and prodrugs thereof or pharmaceutically acceptable salts thereof.
  • the cytochrome bcl complex inhibitor is a pyridone cytochrome bcl complex inhibitor.
  • pyridone cytochrome bcl complex inhibitors include, but are not limited to, Clopidol, GW844520, and GSK932121, and prodrugs thereof or pharmaceutically acceptable salts thereof.
  • the cytochrome bcl complex inhibitor is a hydroxynaphthoquinone cytochrome bcl complex inhibitor.
  • hydroxynaphthoquinone cytochrome bcl complex inhibitors include, but are not limited to, Atovaquone, Parvaquone, Buparvaquone, S-10576, NQ3, and their Prodrugs or their pharmaceutically acceptable salts.
  • the cytochrome bcl complex inhibitor is a quinolone cytochrome bcl complex inhibitor.
  • quinolone cytochrome bcl complex inhibitors include, but are not limited to, RCQ06, Endochin, and Endochin-like quinolone (ELQ), such as ELQ-118, ELQ-120, ELQ-121, ELQ-136, ELQ-233, ELQ-245, ELQ-260, ELQ-271, ELQ-274, ELQ-300, ELQ-314, ELQ-316, ELQ-317, ELQ-319, ELQ-337, ELQ-338, ELQ-351, ELQ- 370, ELQ-372, ELQ-380, ELQ-384, ELQ-385, ELQ-388, ELQ-390, ELQ-400, ELQ-404, ELQ-428, P4Q-95 and P4Q
  • the cytochrome bcl complex inhibitor is an acridinedione cytochrome bcl complex inhibitor.
  • acridinedione cytochrome bcl complex inhibitors include, but are not limited to, Floxacrine, WR249685, WR243246, and prodrugs thereof or pharmaceutically acceptable salts thereof.
  • the cytochrome bcl complex inhibitor is a statin lactone cytochrome bcl complex inhibitor.
  • statin-based cytochrome bcl complex inhibitors include, but are not limited to, Simvastatin, Cerivastatin, Rosuvastatin, Pravastatin, and Pinyrus Pitavastatin, mevastatin, lovastatin, fluvastatin, atorvastatin, and prodrugs thereof or pharmaceutically acceptable salts thereof.
  • the cytochrome bcl complex inhibitor may be selected from the group consisting of Antimycin A, Lansoprazole, Lansoprazole sulfide, Omera Omeprazole, Pentamidine, and prodrugs thereof or pharmaceutically acceptable salts thereof.
  • cytochrome bcl complex inhibitors as described herein and other cytochrome bcl complex inhibitors useful herein can be found, for example, in Proc Natl Acad Sci U S A. 2015 Jan 20, 112(3): 755-60; Antimicrob Agents Chemother.2016Jul 22,60(8):4972-82;Am J Trop Med Hyg.2015Jun,92(6):1195-201;PLoS One.2013Aug 12,8(8):e71726;Nat Commun.2015Jul 9, 6:7659; Cell Metabolism 22, 399-407, September 1, 2015; Nat Chem Biol. 2015 Nov, 11(11): 834-6; J. Med. Chem. 2015, 58, 937-9381; ACS Med.
  • the present invention also encompasses derivatives of the compounds described above having a cytochrome bcl complex inhibition function.
  • the term "subject” refers to a mammal, preferably a primate, more preferably a human.
  • the present invention also provides the use of a cytochrome bcl complex inhibitor for the preparation of a medicament for the treatment of a smooth tendon-related disease, for the treatment of an inflammatory disease and/or for the relief of pain.
  • the present invention provides a pharmaceutical composition for treating a smooth muscle spasm-related disease, treating an inflammatory disease, and/or relieving pain, comprising a cytochrome bc1 complex inhibitor as an active ingredient, and a pharmaceutically acceptable Accepted carrier.
  • compositions of the present invention comprise an effective amount of one or more cytochrome bcl complex inhibitors dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an undesirable, allergic or other untoward reaction when administered to an animal, such as a human, as desired.
  • the preparation of a pharmaceutical composition comprising at least one cytochrome bcl complex inhibitor is known to those skilled in the art in light of this disclosure and is exemplified in "Remington: The Science and Practice of Pharmacy," 21st Edition, 2005. , which is incorporated herein by reference.
  • human administration it should be understood that the preparation should also meet the criteria for sterility, pyrogenicity, overall safety, and purity required by the drug approval authority.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, antioxidants, salts, coatings, surfactants, preservatives (eg, methyl or propyl paraben, sorbic acid, antibacterial).
  • Agent antifungal agent
  • isotonic agent such as paraffin
  • adsorbent for example, kaolin, bentonite
  • drug stabilizer for example, sodium lauryl sulfate
  • gel adhesive
  • adhesive eg, syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, carboxymethylcellulose, alginate
  • excipients eg, lactose, polyethylene glycol
  • disintegrants eg Agar, starch, lactose, calcium phosphate, calcium carbonate, alginic acid, sorbitol, glycine
  • wetting agents eg, cetyl alcohol, glyceryl monostearate
  • lubricants eg, quarters) Ammonium salt
  • edible oil eg, almond oil, coconut oil, oily ester or propylene glycol
  • sweeteners eg, flavoring agents, coloring agents, fillers (eg, starch, lac
  • the composition can comprise a plurality of antioxidants to retard oxidation of one or more components.
  • antioxidants include ascorbic acid, cysteine hydrochloride, sodium sulfite, sodium hydrogensulfite, sodium metabisulfite, ascorbyl palmitate, butylated hydroxytoluene, butylated hydroxyanisole, lecithin, propyl gallate, and tocopherol.
  • microorganisms can be achieved by the use of preservatives such as various antibacterial and antifungal agents including, but not limited to, parabens (for example, methylparaben, p-hydroxyl Propyl benzoate), chlorobutanol, phenol, sorbic acid, thimerosal or a combination thereof.
  • preservatives such as various antibacterial and antifungal agents including, but not limited to, parabens (for example, methylparaben, p-hydroxyl Propyl benzoate), chlorobutanol, phenol, sorbic acid, thimerosal or a combination thereof.
  • Pharmaceutically acceptable salts include acid addition salts such as those formed with the free amino groups of the protein component or with mineral acids (for example, hydrochloric acid, hydrobromic acid or phosphoric acid) or organic acids (for example, acetic acid, oxalic acid, tartaric acid, benzene). a salt formed from formic acid, lactic acid, phosphonic acid, citric acid, maleic acid, fumaric acid, succinic acid, naphthalenesulfonic acid, clavulanic acid, stearic acid or mandelic acid.
  • mineral acids for example, hydrochloric acid, hydrobromic acid or phosphoric acid
  • organic acids for example, acetic acid, oxalic acid, tartaric acid, benzene.
  • the salt formed with the free carboxyl group may also be derived from an inorganic base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, magnesium hydroxide or iron hydroxide or an organic base (eg, isopropylamine, trimethylamine, Histidine or procaine).
  • an inorganic base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, magnesium hydroxide or iron hydroxide or an organic base (eg, isopropylamine, trimethylamine, Histidine or procaine).
  • the carrier can be a solvent or dispersion medium including, but not limited to, water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), liquid ( For example, triglycerides, vegetable oils, liposomes, and combinations thereof.
  • a coating such as lecithin
  • a surfactant For example, hydroxypropyl cellulose
  • compositions can be administered by any suitable method known to those of ordinary skill in the art (see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005).
  • Pharmaceutical compositions can be administered intravenously, intramuscularly, intraperitoneally Administration within the inner, cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes.
  • the cytochrome bcl complex inhibitor When administered orally, may take the form of a tablet, capsule, sachet, vial, powder, granule, lozenge, reconstitutable powder or liquid preparation.
  • a sterile injectable solution is prepared, if necessary, by incorporating the required amount of the active compound into a suitable solvent containing a plurality of the other ingredients described above, followed by filtration sterilization.
  • dispersions are prepared by incorporating a plurality of sterile active ingredients into a sterile vehicle containing a base dispersion medium and/or such other ingredients.
  • the preferred method of preparation is a vacuum drying or freeze drying technique which produces the active ingredient from a previously sterilized liquid medium Add any other powder of the desired ingredients. If necessary, the liquid medium should be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose prior to injection. It is also contemplated to prepare highly concentrated compositions for direct injection, where it is contemplated that the use of DMSO as a solvent results in extremely rapid penetration, delivering high concentrations of active agent to small areas.
  • terapéuticaally effective amount refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, block or partially arrest the symptoms of a disease or condition.
  • the actual dosage of a composition of the invention administered to a patient can be determined according to the following physical and physiological factors: body weight, sex, severity of symptoms, type of disease being treated, prior or current therapeutic intervention, unknown etiology of the patient, time of administration, The excretion rate of the specific compound and the route of administration. In any event, the concentration of the active ingredient in the composition and the appropriate dosage for the subject will be determined by the medical personnel responsible for administration.
  • the cytochrome bcl complex inhibitor is 1-150 mg/kg body weight, for example 1-100 mg/kg body weight, 1-50 mg/kg body weight, 1-30 mg/kg body weight, 1-20 mg/ A dose of kg body weight, 1-15 mg/kg body weight, 1-10 mg/kg body weight, 1-5 mg/kg body weight or 3-5 mg/kg body weight is administered.
  • the cytochrome bcl complex inhibitor or the pharmaceutical composition is administered once daily, twice daily, three times daily, or every two days, every three days, every four days, every five days. Apply once every day, every six days or every seven days.
  • prolonged absorption of the injectable compositions can be brought about by the use of agents that delay absorption (e.g., aluminum monostearate, gelatin, or a combination thereof) in the compositions.
  • agents that delay absorption e.g., aluminum monostearate, gelatin, or a combination thereof
  • Example 1 Inhibition of rat smooth muscle contraction by cytochrome bcl complex inhibitor
  • Wistar rats Female, weighing 230-270 g, were provided by the Experimental Animal Center of the Academy of Military Medical Sciences, MD1-3039.
  • Oxytocin injection (Oxytocin Nanjing Biochemical Pharmaceutical Factory, batch number 051069). Diethylstilbestrol (Diethylstibestrol Beijing Yimin Pharmaceutical Co., Ltd., batch number 0506150).
  • PGF2 ⁇ , kresoxim-methyl, acetylcholine, azoxystrobin, Fluoxastrobin, Stigmatellin, Myxothiazol, and Atovaquone were purchased from Sigma (St. Louis, MO).
  • Trifloxystrobin, Pyraclostrobin, Picoxystrobin, and Dimoxystrobin were purchased from Santa Cruz Biotechnology. Fluocrypyrim (FAPM) and derivatives are synthesized in the laboratory (Int J Cancer. 2010 Sep 1; 127(6): 1259-70)
  • Pclab biological signal acquisition and processing system purchased from Beijing WeChat Star Technology Development Co., Ltd.
  • CS502-3C digital super thermostat purchased from Chongqing Si Da Experimental Instrument Co., Ltd., temperature fluctuation ⁇ ⁇ 0.5 ° C
  • GTS biosignal Sensor purchased from Beijing WeChat Star Technology Development Co., Ltd.
  • JZ101 muscle tension transducer Garnier Xinhang Electromechanical Equipment Co., Ltd.
  • Wistar female healthy rats were used for the experiment.
  • the body weight was 230-270 g.
  • the diethylstilbestrol suspension was intraperitoneally injected with 0.1 mg/kg twice a day before the experiment, resulting in artificial estrus.
  • the rats were sacrificed by cervical dislocation and the uterus was removed by laparotomy and immediately placed in a glass dish containing Tyrode's solution. Carefully peel off the connective tissue and adipose tissue attached to the wall of the uterus, cut the uterus along the line of the mesentery, lay it flat on the glass plate, and then divide the uterus with a surgical blade to divide the uterus along the smooth muscle.
  • the left and right uterine muscle strips are ligated with two ends of the uterine muscle strips respectively, and transferred to a nutrient tube containing 5 ml of Tai's solution containing oxygen and pre-warmed to 37.2 ⁇ 0.5 ° C, and the lower end is fixed. The upper end is connected to the tension sensor. Rest for 20-30 minutes, change the solution 2-3 times, then give the uterus load 1g and keep it for 30-60 minutes. After the regular contraction wave of the uterus, give KCL solution 40mM for 10 minutes, evaluate the function of uterine muscle strip, then relax The muscle strips were washed with Tyrode's solution for 2 to 3 times and then allowed to stand for 30 minutes.
  • the uterine muscle strips were loaded with 1 g for 30-60 minutes, and the uterine muscle strips contracted smoothly (ie, the regular contraction waveform appeared in the microcomputer display).
  • the changes of the contraction tension, amplitude, and area under the contraction curve of the uterine smooth muscle strip were recorded, and the given drugs were evaluated.
  • the working temperature of Tyrode's solution was always maintained at 37.2 ⁇ 0.5°C, and oxygen was continuously supplied to the nutrition tube, 1-2 bubbles per second.
  • 5 ⁇ l per administration was sequentially added from a low concentration to a high concentration, and the final concentration of the drug refers to the concentration of the drug dissolved in 5 ml of Tyrode's solution in the tube.
  • Fluacrypyrim is the first methoxy acrylate acaricide developed by BASF and developed by Japan's Soda Corporation. It is a Qo site inhibitor of mitochondrial cytochrome bcl complex.
  • HTFAPM, FAPMA and IFAPM are three derivatives of FAPM synthesized by the inventors' laboratory.
  • Fig. 2A under normal circumstances, rat uterine smooth muscle strips spontaneously contracted spontaneously, capable of autonomous rhythmic contraction. When PGF2 ⁇ (450 nM) was added to the nutrient tube, the uterine smooth muscle strips were significantly enhanced. Shrinkage amplitude and frequency.
  • the IC50 values of FAPM and IFAPM were 1.84 ⁇ 0.08 ⁇ M and 18.5 ⁇ 3.0 ⁇ M, respectively.
  • Trifloxystrobin 1.77 ⁇ 0.05 Kresoxim-methyl 4.08 ⁇ 0.29 Pyraclostrobin 1.05 ⁇ 0.01 Picoxystrobin 1.20 ⁇ 0.02 Dimoxystrobin 1.65 ⁇ 0.12 Fluoxastrobin 2.63 ⁇ 0.30 Stigmatellin 89.9nM Myxothiazol 214.7nM
  • Oxytocin is a uterine smooth muscle stimulant that causes rhythmic contraction of uterine smooth muscle by activating oxytocin receptors.
  • Acetylcholine is a neurotransmitter that activates smooth muscle M-type choline receptors to cause rhythmic contraction of uterine smooth muscle.
  • Potassium chloride can depolarize smooth muscle cell membrane. High concentration of potassium chloride (40 mM) can cause uterine smooth muscle tonic contraction, and low concentration of potassium chloride (16 mM) can cause uterine smooth muscle rhythm contraction.
  • FAPM dose-dependently inhibits uterine smooth muscle contraction induced by oxytocin (1 mU/ml), acetylcholine (0.25 ⁇ M), and potassium chloride (16 mM) with IC50 values of 1.6, respectively. ⁇ 0.08 ⁇ M, 1.36 ⁇ 0.04 ⁇ M, and 1.21 ⁇ 0.07 ⁇ M.
  • Atovaquone is a hydroxy 1,4-naphthoquinoline, a homolog of coenzyme Q with broad-spectrum antiprotozoal activity.
  • FDA US Food and Drug Administration
  • PCP Pneumocystis carinii pneumonia
  • IDSA American Society for Infectious Diseases
  • Atovaquone binds directly to the protozoal cytochrome bc1 complex, and inhibition of cytochrome bc1 complex activity is a molecular pharmacological mechanism for its broad-spectrum antiprotozoal effect (Siregar JE, Kurisu G, Kobayashi T, et al. Direct evidence For the atovaquone action on the Plasmodium cytochrome bc1 complex. Parasitol Int.2015 Jun;64(3):295-300). Based on this, we observed the inhibitory effect of atovaquone on PGF2 ⁇ -induced contraction of rat uterine smooth muscle. It was found that atovaquone can inhibit PGF2 ⁇ -induced contraction of rat uterine smooth muscle in a dose-dependent manner with an IC50 value of 8.44 ⁇ 0.7. ⁇ M ( Figure 7).
  • Wistar rats Female, weighing 230-270 g, were provided by the Experimental Animal Center of the Academy of Military Medical Sciences, MD1-3039.
  • WB 2 ⁇ SDS loading buffer, 5 ⁇ Tris-glycine running buffer, electrotransfer buffer, and separation gel buffer are all self-dispensed (see solution preparation), among which reagents and X-ray photographic film are purchased from Beijing Chemical Reagent Factory; Monoclonal antibody against phospho-myosin light chain-2 (ser19), horseradish peroxidase-labeled secondary antibody, horseradish peroxidase chromogenic substrate Substrates were purchased from Cell Signaling Technology.
  • the DNM-9602G microplate reader was purchased from Beijing Pulang New Technology Co., Ltd.
  • the stabilized current source, protein electrophoresis apparatus, protein electrotransformer, and protein molecular weight Marker were purchased from New England Biotech.
  • Tissue block culture method Diethylstilbestrol-treated rats were dissected and sacrificed, and immersed in 75% alcohol for disinfection. Open the abdominal cavity under aseptic conditions, separate the uterus, transfer it into the plate, cut the uterus to remove the uterine adventitia, gently scrape off the endometrium with a blade, gently remove the endometrium with tweezers, and cut the intact smooth muscle into 1 ⁇ 1 ⁇ 1mm 3 or less, evenly attached to the wall of the culture bottle, add the culture solution to the bottom, 5% CO 2 , culture at 37 ° C for 3-4 hours, gently flip, immersed in RPMI-1640 medium (including 20 % calf serum) is cultured for about one week, and the cells can be ligated into pieces to form monolayer cultured cells.
  • RPMI-1640 medium including 20 % calf serum
  • the culture medium in the bottle can be poured, the tissue block in the bottle is removed twice by PBS, and an appropriate amount of 0.25% trypsin is added, and observed under a microscope.
  • the reaction is stopped by adding calf serum, and the liquid PBS is washed twice.
  • Add RPMI-1640 in an appropriate amount gently pipette the eluted cells with a pipette to prepare a cell suspension for use.
  • the cell concentration was adjusted to 10 5 /ml, and inoculated into a 6-well plate at 4 ⁇ 10 5 /well. After adhering overnight, the cells were cleared for 24 hours, and then pretreated with different concentrations of FAPM, 5 minutes after stimulation with PGF 2 ⁇ . The cells were collected, washed three times with ice-cold PBS, and lysed with 2 ⁇ SDS loading buffer. The collected cell lysate was boiled for 10 min, then centrifuged at 12000 r/min for 5 min, and the supernatant was taken and stored in - 80 ° C spare.
  • the cell lysate was dispensed, thawed at room temperature, and subjected to SDS-PAGE gel electrophoresis. Carefully add the sample to the comb hole with a micro-syringe, adjust the 100V to the bromophenol blue strip to remove the separation gel, and then adjust the voltage to 200V. After electrophoresis, the membrane was transferred under the condition of an ice bath at a constant flow of 160 mA for 1 h to transfer the protein onto the nitrocellulose membrane.
  • Example 3 Inhibition of cytochrome bcl complex inhibitor on nitric oxide production in macrophages
  • Mouse mononuclear macrophages RAW 264.7 were purchased from the Concord Cell Bank.
  • Lipopolysaccharide was purchased from Sigma, RPMI1640 medium, penicillin, streptomycin, fetal bovine serum were purchased from Invitrogen; nitric oxide detection kit was purchased from Biyuntian; NM-9602G microplate reader was purchased from Beijing Pu Long New Technology Co., Ltd.
  • Mouse mononuclear macrophages were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100 ⁇ g ⁇ mL-1 streptomycin, 100 units ⁇ mL-1 penicillin, and incubated in a constant temperature incubator at 37 ° C, 5% CO 2 . Growing, passed down every other day.
  • the content of NO in the sample was determined by the Griess method.
  • the mouse peritoneal macrophage cell line RAW264.7 is one of the commonly used cell models for studying inflammation. LPS was used to stimulate NO production in RAW264.7 cells, and the inhibitory effects of various cytochrome bc1 complex inhibitors on NO production by macrophages were observed. As shown in Table 2, cytochrome bc1 complex inhibitors azoxystrobin, trifloxystrobin, kresoxim-methyl, and FAPM significantly inhibited NO production with IC50 of 1.89 ⁇ 0.35 ⁇ M, 0.64 ⁇ 0.21 ⁇ M, 2.77 ⁇ 0.69 ⁇ M, and 3.80 ⁇ M, respectively. The inhibition rate is above 80%, and its effective concentration is equivalent to its inhibition of uterine smooth muscle contraction.
  • Stigmatellin and Myxothiazol inhibited the contractile activity of uterine smooth muscle, and the IC50 of NO production was the lowest, 6.24 ⁇ 1.15nM and 2.8nM, respectively.
  • the maximum inhibition rates were 88.3 ⁇ 3.9% and 95%, respectively.
  • Example 4 In vivo analgesic effect of cytochrome bcl complex inhibitor
  • Oxytocin injection (Oxytocin, Nanjing Biochemical Pharmaceutical Factory, batch number 051069). Diethylstilbestrol (Diethylstibestrol Beijing Yimin Pharmaceutical Co., Ltd., batch number 0506150). Indomethacin, PGF2 ⁇ , kresoxim-methyl, and azoxystrobin were purchased from Sigma (St. Louis, MO). Trifloxystrobin, Picoxystrobin and Dimoxystrobin were purchased from Santa Cruz Biotechnology. FAPM is synthesized in the laboratory of the inventors (Int J Cancer. 2010 Sep1; 127(6): 1259-70).
  • mice peritonitis pain model was replicated.
  • Adult healthy Kunming male mice were randomly divided into groups of 6-10 each.
  • Different types and doses of cytochrome bc1 complex inhibitor were administered intraperitoneally or intragastrically 1 hour before the experiment.
  • Indomethacin 50 mg/kg was intraperitoneally injected as a positive control, followed by intraperitoneal injection of 0.6% glacial acetic acid 0.1.
  • M1/10g the time to start writhing (reaction latency) and the number of writhing in 30 min were observed.
  • Pain is an unpleasant feeling and emotional experience caused by acute or potential tissue damage.
  • the pain model made by chemical stimulation such as glacial acetic acid is the most widely studied and widely used pain model. These inflammatory stimulants cause acute inflammatory pain through neutrophil chemotaxis, while mediating macrophage infiltration causing persistent pain.
  • the mouse acetic acid writhing test is suitable for screening non-narcotic analgesics, especially a sensitive and simple method for screening analgesic effects of steroidal anti-inflammatory drugs.
  • mice were intraperitoneally injected with glacial acetic acid (0.6%, 0.1m1/10g) within 30 minutes, the number of writhings was about 40 times, the incubation period was 2-3 minutes, and the cytochrome was injected intraperitoneally 1 hour before the experiment.
  • the writhing time was significantly prolonged in the FAPM group and the Trifloxystrobin group, and there was a dose effect, while the Azoxystrobin group did not significantly prolong the writhing time.
  • 100 mg/Kg of FAPM, Trifloxystrobin and Azoxystrobin are equivalent to 50 mg/Kg indomethacin.
  • mice At the dose of 200mg/Kg, the inhibitory effects of cytochrome bc1 complex inhibitors Dimoxystrobin, Kresoxim-methyl and Picoxystrobin on acetic acid-induced writhing in mice were compared. The number of mice writhing, but the time to start writhing was not extended.
  • Dysmenorrhea is one of the most common gynecological symptoms. It is divided into primary dysmenorrhea and secondary dysmenorrhea.
  • Primary dysmenorrhea refers to dysmenorrhea without organic disease in genital organs, accounting for more than 90% of dysmenorrhea; secondary dysmenorrhea refers to Dysmenorrhea caused by pelvic organic diseases.
  • the occurrence of primary dysmenorrhea is mainly related to the increase of endometrial prostaglandin content during menstruation. Elevated levels of PGF2 ⁇ are the main cause of dysmenorrhea.
  • reference literature was used to establish a mouse model of dysmenorrhea induced by two-step sensitization of diethylstilbestrol and PGF2 ⁇ .
  • Oxytocin is a uterine smooth muscle stimulant.
  • Indomethacin, dexamethasone, and carrageenan xylene were purchased from Sigma (St. Louis, MO). FAPM is synthesized in the laboratory of the inventors (Int J Cancer. 2010 Sep1; 127(6): 1259-70).
  • mice 50 healthy adult male mice were randomly divided into 5 groups.
  • the experimental mice were intraperitoneally injected with different doses of FAPM (50, 100 and 200 mg/kg) once, and the control group was injected with the same amount of solvent.
  • Indomethacin (50 mg/kg) was intraperitoneally injected as a positive control.
  • 1 hour after administration 20 ⁇ L of xylene was evenly applied to both sides of the right ear of the mouse, and the left ear was not coated with xylene self-control; after 1 hour, the ears were cut, and the 6 mm punch was used to remove the two parts of the same ear.
  • Ears placed on a precision balance, weighed, calculated swelling (right ear weight - left ear weight) and swelling inhibition rate [(right ear weight - left ear weight) / left ear weight ⁇ 100%) .
  • Inflammation is an important defense mechanism produced by the body for noxious stimuli.
  • the inflammatory response is the most basic anti-injury reaction of the body. Inflammation helps to promote wound healing, but it can also cause a lot of damage, such as causing arthritis, asthma and body disorders. Inflammation can be classified into infectious inflammation, aseptic inflammation (non-specific inflammation), and allergic inflammation depending on the cause of the disease. Xylene-induced mouse ear swelling test and carrageenan-induced paw swelling in rats is the most commonly used method for evaluating and screening anti-inflammatory drugs.

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Abstract

细胞色素bc1复合物抑制剂在制备治疗平滑肌痉挛相关疾病、治疗炎症疾病和缓解疼痛药物组合物中的用途。

Description

[根据细则37.2由ISA制定的发明名称]　细胞色素BC1复合物抑制剂在制备医药组合物中的用途 技术领域
本发明涉及生物医药领域，特别涉及细胞色素bc1复合物抑制剂的医药用途。具体而言，本发明涉及细胞色素bc1复合物抑制剂用于治疗平滑肌痉挛相关疾病、治疗炎症疾病和缓解疼痛的用途。
背景技术
线粒体细胞色素bc1复合物，也叫细胞色素c还原酶，同时称为线粒体呼吸链复合体III，是线粒体以及大多数细菌呼吸作用电子传递链中的重要组成部分，催化电子从泛醌传递到细胞色素c(细菌中是细胞色素c2)的反应。细胞色素bc1复合物有两个醌的功能结合位点：一个是氧化位点Q O位于膜间隙一侧，靠近血红素b L；一个还原位点Q i位于基质一侧，靠近血红素b H。细胞色素bc1复合物抑制剂因此可分为两类：一类是与位于线粒体内膜内壁Q i位点结合的抑制剂，这类抑制剂称为Q i位点抑制剂，如抗霉素和氰霜唑；另一类是与位于线粒体内膜外壁的Q O位点结合的抑制剂，这类抑制剂称为Q O位点抑制剂，如甲氧基丙烯酸酯类杀菌剂、咪唑菌酮等。
已有的关于细胞色素bc1复合物抑制剂的研究主要关注其杀菌和杀虫作用，很少关于这些抑制剂治疗人类内源性疾病的报道。
发明简述
本发明提供使用细胞色素bc1复合物抑制剂治疗平滑肌痉挛相关疾病、治疗炎症疾病和/或缓解疼痛的方法，细胞色素bc1复合物抑制剂在制备用于治疗平滑肌痉挛相关疾病、治疗炎症疾病和/或缓解疼痛的药物中的用途，以及包含细胞色素bc1复合物抑制剂的药物组合物。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是细胞色素bc1复合物Qo位点抑制剂。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是细胞色素bc1复合物Qi位点抑制剂。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是甲氧基丙烯酸酯类细胞色素bc1复合物抑制剂。在一些具体实施方案中，所述甲氧基丙烯酸酯类细胞色素bc1复合物抑制剂选自嘧螨酯(Fluacrypyrim，FAPM)、腈嘧菌酯(azoxystrobin)、肟菌酯(Trifloxystrobin)、醚菌酯(kresoxim-methyl)、吡唑醚菌酯(Pyraclostrobin)、啶氧菌酯(Picoxystrobin)、醚菌胺(Dimoxystrobin)、氟嘧菌酯(Fluoxastrobin)，以及它们的前药或它们的可药用盐。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是吡啶酮类细胞色素bc1复合物抑制剂。在一些具体实施方案中，所述吡啶酮类细胞色素bc1复合物抑制剂选自氯羟吡啶(Clopidol)、GW844520和GSK932121，以及它们的前药或它们的可药用盐。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是羟萘醌类细胞色素bc1复合物抑制剂。在一些具体实施方案中，所述羟萘醌类细胞色素bc1复合物抑制剂选自阿托伐醌(Atovaquone)、帕伐醌(Parvaquone)、布帕伐醌(Buparvaquone)、S-10576、NQ3(2-OH-3-(2-methyl-trifluorooctyl)-8-methyl-naphthoquinone)，以及它们的前药或它们的可药用盐。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是喹诺酮类细胞色素bc1复合物抑制剂。在一些具体实施方案中，所述喹诺酮类细胞色素bc1复合物抑制剂选自RCQ06，Endochin，以及Endochin-样喹诺酮(ELQ)，例如ELQ-118、ELQ-120、ELQ-121、ELQ-136、ELQ-233、ELQ-245、ELQ-260、ELQ-271、ELQ-274、ELQ-300、ELQ-314、ELQ-316、ELQ-317、ELQ-319、ELQ-337、ELQ-338、ELQ-351、ELQ-370、ELQ-372、ELQ-380、ELQ-384、ELQ-385、ELQ-388、ELQ-390、ELQ-400、ELQ-404、ELQ-428、P4Q-95和P4Q-391等，以及它们的前药或它们的可药用盐。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是吖啶二酮类细胞色素bc1复合物抑制剂。在一些具体实施方案中，所述吖啶二酮类细胞色素bc1复合物抑制剂选自氟克吖啶(Floxacrine)、WR249685、WR243246，以及它们的前药或它们的可药用盐。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是他汀内酯类细胞色素bc1复合物抑制剂。在一些具体实施方案中，所述他汀内酯类细胞 色素bc1复合物抑制剂选自辛伐他汀(Simvastatin)、西立伐他汀(Cerivastatin)、瑞舒伐他汀(Rosuvastatin)、普伐他汀(Pravastatin)、匹伐他汀(Pitavastatin)、美伐他汀(Mevastatin)、洛伐他汀(Lovastatin)、氟伐他汀(Fluvastatin)、阿托伐他汀(Atorvastatin)，以及它们的前药或它们的可药用盐。
在一些具体实施方案中，所述细胞色素bc1复合物抑制剂选自抗霉素A(Antimycin A)、兰索拉唑(Lansoprazole)、兰索拉唑硫化物(Lansoprazole sulfide)、奥美拉唑(Omeprazole)、潘他米丁(Pentamidine)，以及它们的前药或它们的可药用盐。
在一些具体实施方案中，所述平滑肌痉挛相关疾病选自痛经，气道痉挛相关疾病如支气管哮喘、喘息性支气管炎、慢性阻塞性肺疾病或气道高反应，血管痉挛相关疾病如脑血管痉挛、偏头痛或丛集性头痛，胃肠道痉挛相关疾病如急性胃炎、慢性胃炎、胃溃疡、十二指肠溃疡、急性肠炎、慢性肠炎、克隆氏病、炎症性肠病、溃疡性结肠炎或肠道结核，高血压，冠状动脉痉挛相关疾病如变异型心绞痛，青光眼，术后肠梗阻，尿频，纤维肌痛，性交疼，肠易激综合征，颈部肌肉和眼睑痉挛，膀胱过度活动症，术后眼炎症和呼吸窘迫综合征。
在一些具体实施方案中，所述炎症疾病选自类风湿关节炎、骨关节炎、骨质溶解、肌腱炎、滑膜炎、和炎症性呼吸道疾病如慢性阻塞性肺病、纤维化、肺气肿、急性呼吸窘迫综合征。
在一些具体实施方案中，所述疼痛是炎症性疼痛。
附图简述
图1.示出FAPM及其衍生物的化学结构。
图2.示出FAPM及其衍生物对PGF2α引起的大鼠子宫平滑肌收缩的抑制作用。
图3.示出细胞色素bc1复合物Qo位点抑制剂对PGF2α引起的大鼠子宫平滑肌收缩的抑制作用。
图4.示出FAPM对大鼠缩宫素引起的大鼠子宫平滑肌收缩的抑制作用。
图5.示出FAPM对乙酰胆碱引起的大鼠子宫平滑肌收缩的抑制作用。
图6.示出FAPM对氯化钾引起的大鼠子宫平滑肌收缩的抑制作用。
图7.示出阿托他醌对PGF2α引起的大鼠子宫平滑肌收缩的抑制作用。IC50＝8.44±0.7μM。
图8.示出FAPM对PGF2α引起的大鼠平滑肌细胞MLC20磷酸化的抑制作用。
发明详述
已知在不同器官中发生的多种明显不同的疾病是由平滑肌细胞的生化机能障碍引起的。平滑肌细胞合适地发挥功能是在所有平滑肌细胞起重要功能的器官中避免健康问题的关键。
本发明人令人惊奇地发现，细胞色素bc1复合物抑制剂可以在体内抑制平滑肌收缩，从而可以用于治疗平滑肌痉挛相关疾病。
因此，在第一方面，本发明提供了一种治疗对象中平滑肌痉挛相关疾病的方法，包括给所述对象施用治疗有效量的细胞色素bc1复合物抑制剂。
不受任何理论束缚，细胞色素bc1复合物抑制剂可能通过阻止MLC20的磷酸化而抑制平滑肌收缩。肌球蛋白轻链激酶(myosin light chain kinase，MLCK)是钙离子和钙调蛋白依赖的专一磷酸化肌球蛋白Ⅱ的调节轻链(Myosin Light Chain，MLC20)的蛋白激酶，MLC20磷酸化有利于肌球蛋白粗丝和肌动蛋白细丝相互作用。MLCK介导的MLC20磷酸化是启动平滑肌收缩所必需。本发明人令人惊奇地发现，细胞色素bc1复合物抑制剂可以阻止MLC20的磷酸化，从而可以通过抑制平滑肌收缩而治疗平滑肌痉挛相关疾病。
如本文所用，“平滑肌”包括但不限于子宫平滑肌、膀胱平滑肌、虹膜肌、生殖道平滑肌、输卵管平滑肌、支气管平滑肌、血管平滑肌和胃肠道平滑肌等。
如本文所用，“平滑肌痉挛相关疾病”包括但不限于选自以下1)-8)的疾病：
1)妇产科疾病(子宫平滑肌)：痛经；早产；未足月胎膜早破等。
2)泌尿系统疾病(尿道平滑肌、膀胱平滑肌)：良性前列腺增生症；泌尿系结石，如肾结石，输尿管结石等；膀胱过度活动症；尿频；勃起障碍等。
3)消化系统疾病(食管平滑肌、括约肌、胃肠道平滑肌)：食管贲门失弛缓症；肠易激综合征；Oddi括约肌功能障碍；胃肠痉挛性腹痛，如胃炎，胃溃疡，十二指肠溃疡，溃疡性结肠炎，克隆氏病，急、慢性阑尾炎等；胆石症；急、慢性胆囊炎；急、慢性胰腺炎；急、慢性腹膜炎；肠梗阻。
4)呼吸系统疾病(气管、支气管平滑肌)：哮喘病，如慢性支气管炎哮喘、过敏性哮喘、药物性哮喘、老年性哮喘、咳嗽变异性哮喘、慢性哮喘、运动性哮喘、儿童性哮喘；气管炎；急、慢性支气管炎；慢性阻塞性肺疾病；呼吸窘迫综合征。
5)心血管疾病(血管平滑肌)：高血压；门脉高压；动脉粥样硬化；心绞痛，如变异型心绞痛，不稳定型心绞痛，稳定型心绞痛等；心肌梗死；肥厚性心肌病等。
6)脑血管痉挛相关性疾病：缺血性脑血管病，如脑梗塞，短暂性脑缺血发作、脑血栓形成等；血管性痴呆；脑动脉硬化症；头晕；头痛；顽固性呃逆等。
7)外周血管痉挛相关疾病：偏头痛；间歇性跛行；雷诺综合征；青光眼；突发性耳聋；耳鸣；眩晕；晕动病。
8)其他：眼脸痉挛。
例如，所述“平滑肌痉挛相关疾病”可以选自痛经，气道痉挛相关疾病如支气管哮喘、喘息性支气管炎、慢性阻塞性肺疾病或气道高反应，血管痉挛相关疾病如脑血管痉挛、肺动脉高压、血管性耳聋、偏头痛或丛集性头痛，胃肠道痉挛相关疾病如胆囊炎、胆管炎、胆道结石、急性胃炎、慢性胃炎、胃溃疡、十二指肠溃疡、急性肠炎、慢性肠炎、克隆氏病、炎症性肠病、溃疡性结肠炎或肠道结核，高血压，冠状动脉痉挛相关疾病如变异型心绞痛，膀胱炎，输尿管结石，肾绞痛和青光眼等。在一些实施方式中，所述“平滑肌痉挛相关疾病”还可以包括例如术后肠梗阻、尿频、纤维肌痛、性交疼、肠易激综合征、颈部肌肉和眼睑痉挛、膀胱过度活动症、术后眼炎症、呼吸窘迫综合征等。
例如，用细胞色素bc1复合物抑制剂进行治疗可以导致至少部分平滑肌细胞明显松驰，导致大动脉(如，主动脉)和中动脉的动脉直径的增加而降低血压，增加中动脉(如，冠状动脉)的动脉直径而使心绞痛减少，松驰肺细支气管平滑肌细胞导致气道直径增加而使哮喘症状减弱，松驰眼的导管系 统导致如泪管直径的增加而减小眼压从而降低青光眼和视觉缺失的危险，松驰输卵管和子宫的平滑肌细胞导致肌组织松驰而改善生育力和/或减弱痛经症状，松驰胆管、输尿管和尿道的平滑肌细胞增加导管的直径而降低由胆结石或肾结石引起的痉挛危险，松弛胃肠道平滑肌而减轻例如痉挛性肠绞痛。
在一优选的实施方式中，所述平滑肌痉挛相关疾病是痛经。在一进一步优选的实施方式中，所述平滑肌痉挛相关疾病是原发性痛经。
此外，本发明人还令人惊奇地发现，细胞色素bc1复合物抑制剂可以在体内显著抑制炎症症状。例如，实施例5显示细胞色素bc1复合物抑制剂能够抑制二甲苯致小鼠耳廓肿胀和角叉菜胶致大鼠足跖肿胀。
因此，在第二方面，本发明提供了一种治疗对象中炎症疾病的方法，包括给所述对象施用治疗有效量的细胞色素bc1复合物抑制剂。
不受任何理论束缚，细胞色素bc1复合物抑制剂可能通过抑制一氧化氮(NO)的产生来抑制炎症。NO是一种重要的调节生命活动的小分子物质，由L-精氮酸通过一氧化氮合成酶(NOS)合成。NOS同功酶有三种亚型，即在正常状态下表达的神经元型一氧化氮合酶(nNOS)和内皮型一氧化氮合酶(eNOS)以及在损伤后诱导表达的诱导型一氧化氮合酶(iNOS)。巨噬细胞在宿主防御系统、免疫和炎症反应方面发挥了重大的作用；在病理情况下，巨噬细胞iNOS表达显著增加，合成的大量NO参与机体各种急、慢性炎症反应。本发明人令人惊奇地发现，细胞色素bc1复合物抑制剂可以抑制巨噬细胞的一氧化氮生成，从而可用于治疗炎症疾病。例如，实施例3证明了细胞色素bc1复合物抑制剂能够抑制小鼠单核巨噬细胞的NO生成。
可以由本发明方法治疗的炎症疾病的实例包括类风湿关节炎、骨关节炎、骨质溶解、肌腱炎、滑膜炎、痛风、阿尔兹海默病、肾小球肾炎、肾盂肾炎、动脉粥样硬化性疾病、脉管炎和炎症性呼吸道疾病(如慢性阻塞性肺病、纤维化、肺气肿、急性呼吸窘迫综合征等)。优选地，所述炎症疾病是非感染性炎症疾病。
本发明人还令人惊奇地发现，细胞色素bc1复合物抑制剂在体内具有镇痛作用。例如，在本申请实施例4，施用细胞色素bc1复合物抑制剂能够显著抑制醋酸、己烯雌酚和PGF2α引起的小鼠扭体次数。
因此，在第三方面，本发明提供了一种在对象中缓解疼痛的方法，包 括给所述对象施用治疗有效量的细胞色素bc1复合物抑制剂。在一些实施方式中，所述疼痛是炎症性疼痛。例如是当机体产生炎症后，由炎症部位所释放的炎症介导因子启动一系列信号转导通路，激活或增敏痛觉感受器，从而引起的疼痛
在本发明各个方面的一些实施方案中，所述细胞色素bc1复合物抑制剂是细胞色素bc1复合物Qo位点抑制剂。在本发明各个方面的另一些实施方案中，所述细胞色素bc1复合物抑制剂是细胞色素bc1复合物Qi位点抑制剂。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是甲氧基丙烯酸酯类细胞色素bc1复合物抑制剂。甲氧基丙烯酸酯类细胞色素bc1复合物抑制剂的实例包括但不限于，嘧螨酯(Fluacrypyrim，FAPM)、腈嘧菌酯(azoxystrobin)、肟菌酯(Trifloxystrobin)、醚菌酯(kresoxim-methyl)、吡唑醚菌酯(Pyraclostrobin)、啶氧菌酯(Picoxystrobin)、醚菌胺(Dimoxystrobin)、氟嘧菌酯(Fluoxastrobin)，以及它们的前药或它们的可药用盐。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是吡啶酮类细胞色素bc1复合物抑制剂。吡啶酮类细胞色素bc1复合物抑制剂的实例包括但不限于，氯羟吡啶(Clopidol)、GW844520和GSK932121，以及它们的前药或它们的可药用盐。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是羟萘醌类细胞色素bc1复合物抑制剂。羟萘醌类细胞色素bc1复合物抑制剂的实例包括但不限于，阿托伐醌(Atovaquone)、帕伐醌(Parvaquone)、布帕伐醌(Buparvaquone)、S-10576、NQ3，以及它们的前药或它们的可药用盐。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是喹诺酮类细胞色素bc1复合物抑制剂。喹诺酮类细胞色素bc1复合物抑制剂的实例包括但不限于，RCQ06，Endochin，以及Endochin-样喹诺酮(ELQ)，例如ELQ-118、ELQ-120、ELQ-121、ELQ-136、ELQ-233、ELQ-245、ELQ-260、ELQ-271、ELQ-274、ELQ-300、ELQ-314、ELQ-316、ELQ-317、ELQ-319、ELQ-337、ELQ-338、ELQ-351、ELQ-370、ELQ-372、ELQ-380、ELQ-384、ELQ-385、ELQ-388、ELQ-390、ELQ-400、ELQ-404、ELQ-428、P4Q-95和P4Q-391等，以及它们的前药或它们的可药用盐。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是吖啶二酮类细 胞色素bc1复合物抑制剂。吖啶二酮类细胞色素bc1复合物抑制剂实例包括但不限于，氟克吖啶(Floxacrine)、WR249685、WR243246，以及它们的前药或它们的可药用盐。
在一些实施方案中，所述细胞色素bc1复合物抑制剂是他汀内酯类细胞色素bc1复合物抑制剂。他汀内酯类细胞色素bc1复合物抑制剂的实例包括但不限于，辛伐他汀(Simvastatin)、西立伐他汀(Cerivastatin)、瑞舒伐他汀(Rosuvastatin)、普伐他汀(Pravastatin)、匹伐他汀(Pitavastatin)、美伐他汀(Mevastatin)、洛伐他汀(Lovastatin)、氟伐他汀(Fluvastatin)、阿托伐他汀(Atorvastatin)，以及它们的前药或它们的可药用盐。
在另外一些实施方案中，所述细胞色素bc1复合物抑制剂可以选自抗霉素A(Antimycin A)、兰索拉唑(Lansoprazole)、兰索拉唑硫化物(Lansoprazole sulfide)、奥美拉唑(Omeprazole)、潘他米丁(Pentamidine)，以及它们的前药或它们的可药用盐。
本文上述的具体细胞色素bc1复合物抑制剂和可用的本发明的其他细胞色素bc1复合物抑制剂可见于，例如，Proc Natl Acad Sci U S A.2015Jan 20,112(3):755-60；Antimicrob Agents Chemother.2016Jul 22,60(8):4972-82；Am J Trop Med Hyg.2015Jun,92(6):1195-201；PLoS One.2013Aug 12,8(8):e71726；Nat Commun.2015Jul 9,6:7659；Cell Metabolism 22,399-407,September 1,2015；Nat Chem Biol.2015 Nov,11(11):834-6；J.Med.Chem.2015,58,9371-9381；ACS Med.Chem.Lett.,2012,3(12),pp 951-951；J.Agric.Food Chem.2015,63,3377-3386；J.Phys.Chem.B 2016,120,2701-2708；Mol Biochem Parasitol.2011 May,177(1):12-9；US2015/0203445 A1；和WO2012070015 A1。
本发明还涵盖上文所述化合物的具有细胞色素bc1复合物抑制功能的衍生物。
如本文所用，术语“对象”是指哺乳动物，优选灵长类动物，更优选人。
在第四方面，本发明还提供了细胞色素bc1复合物抑制剂在制备用于治疗平滑肌痉挛相关疾病、治疗炎症疾病和/或缓解疼痛的药物中的用途。
在第五方面，本发明还提供了一种用于治疗平滑肌痉挛相关疾病、治疗炎症疾病和/或缓解疼痛的药物组合物，其包含作为活性成分的细胞色素 bc1复合物抑制剂，以及药学可接受的载体。
本发明的药物组合物包含溶于或分散于药学可接受的载体中的有效量的一种或多种细胞色素bc1复合物抑制剂。短语“药学可接受的”是指当按照需要施用于动物(例如人)时不会产生不良的、变应性的或其它不利的反应的分子实体和组合物。对包含至少一种细胞色素bc1复合物抑制剂的药物组合物的制备是本领域技术人员根据本公开内容已知的，并示例于“Remington：The Science and Practice of Pharmacy，”第21版，2005，其通过参考并入本文中。另外，对于人类施用来说，应当理解，制备还应满足药物审批机构所要求的对无菌性、热原性、整体安全性以及纯度的标准。
本文使用的“药学可接受的载体”包括任何和所有的溶剂、分散介质、抗氧化剂、盐、包衣、表面活性剂、防腐剂(例如对羟基苯甲酸甲酯或丙酯、山梨酸、抗菌剂、抗真菌剂)、等渗剂、溶液阻滞剂(例如石蜡)、吸附剂(例如，高岭土、膨润土)、药物稳定剂(例如，十二烷基硫酸钠)、凝胶、粘合剂(例如，糖浆、阿拉伯胶、明胶、山梨醇、黄芪胶、聚乙烯吡咯烷酮、羧甲基纤维素、藻酸盐)、赋形剂(例如，乳糖、聚乙二醇)、崩解剂(例如琼脂、淀粉、乳糖、磷酸钙、碳酸钙、海藻酸、山梨醇、甘氨酸)、润湿剂(例如，十六烷醇、单硬脂酸甘油酯)、润滑剂、吸收促进剂(例如，季铵盐)、可食用油(例如，杏仁油、椰油、油性酯或丙二醇)、甜味剂、调味剂、着色剂、填充剂(例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇、硅酸)、压片润滑剂(例如，硬脂酸镁、淀粉、葡萄糖、乳糖、白垩)、吸入用载体(例如，烃抛射剂)、缓冲剂或诸如此类的物质及其组合，如本领域普通技术人员所了解地(参见例如，“Remington：The Science and Practice of Pharmacy，”第21版，2005)。将任何除了与所述活性成分不相容的常规载体以外的常规载体用于所述治疗性或药物组合物也在考虑范围内。
在任何情形中，所述组合物可包含多种抗氧化剂以阻滞一种或多种组分的氧化。抗氧化剂的实例包括抗坏血酸、盐酸半胱氨酸、亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、抗坏血酸棕榈酸酯、丁羟甲苯、丁羟茴醚、卵磷脂、没食子酸丙酯和生育酚。另外，防止微生物作用可通过使用防腐剂来实现，所述防腐剂例如多种抗菌剂和抗真菌剂，其包括但不限于对羟基苯甲酸酯类(例如，对羟基苯甲酸甲酯、对羟基苯甲酸丙酯)、氯丁醇、苯酚、山梨酸、硫柳汞或其组合。
可药用的盐包括酸加成盐，例如与蛋白质组分的游离氨基形成的盐或者与无机酸(例如，盐酸、氢溴酸或磷酸)或有机酸(例如，乙酸、草酸、酒石酸、苯甲酸、乳酸、膦酸、柠檬酸、马来酸、富马酸、琥珀酸、萘磺酸、克拉维酸、硬脂酸或杏仁酸)形成的盐。与游离羧基形成的盐还可衍生自无机碱(例如氢氧化钠、氢氧化钾、氢氧化铵、氢氧化钙、氢氧化镁或氢氧化铁)或者有机碱(例如，异丙胺、三甲胺、组氨酸或普鲁卡因)。
在所述组合物为液体形式的一些实施方案中，载体可以是溶剂或分散介质，其包括但不限于水、乙醇、多元醇(例如，甘油、丙二醇、液体聚乙二醇等)、液体(例如，甘油三酯、植物油、脂质体)及其组合。可维持适当的流动性，例如通过使用包衣(如卵磷脂)来实现；通过分散于载体(例如，液体多元醇或脂类)中而维持所需粒径来实现；通过使用表面活性剂(例如，羟丙基纤维素)来实现；或者这些方法的组合。在许多情形中，优选包含等渗剂(例如，糖、氯化钠或其组合)。
本发明可通过本领域普通技术人员已知的任何适当方法进行施用(参见例如，Remington：The Science and Practice of Pharmacy，”第21版，2005)。药物组合物可通过静脉内、肌肉内、腹腔内、脑脊髓内、皮下、关节内、滑膜腔内、鞘内、口服、局部或吸入途径施用。
当口服施用时，所述细胞色素bc1复合物抑制剂可采用以下形式：片剂、胶囊、小袋(sachet)、小瓶、粉剂、颗粒剂、锭剂、可重构的粉剂或液体制备物。根据需要，通过将所需量的所述活性化合物掺入含有多种上述其它成分的合适溶剂中来制备无菌注射用溶液，然后进行过滤除菌。通常，通过将多种无菌活性成分掺入含有基本分散介质和/或所述其它成分的无菌载体中来制备分散体系。在用于制备无菌注射用溶液、混悬液或乳剂的无菌粉剂的情形中，优选的制备方法为真空干燥或冷冻干燥技术，所述技术从先前过滤除菌的液体介质中产生活性成分加任何其它所需成分的粉末。必要时，所述液体介质应进行适当缓冲，并且在注射前用足够的盐水或葡萄糖首先使液体稀释剂等渗。制备用于直接注射的高度浓缩组合物也在考虑范围内，其中可以想到使用DMSO作为溶剂会导致极其快速的渗透、将高浓度的活性药剂递送至小的区域。
如本文所用，“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此， 其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。
对患者施用本发明组合物的实际剂量可根据以下身体和生理因素来确定：体重、性别、症状严重程度、所治疗疾病的类型、先前或当前的治疗干预、患者的未知病因疾病、施用时间、具体化合物的排泄率以及施用途径。在任何情况下，将由负责施用的医务人员确定组合物中活性成分的浓度以及用于个体对象的合适剂量。在一些具体实施方案中，所述细胞色素bc1复合物抑制剂以1-150mg/kg体重，例如1-100mg/kg体重、1-50mg/kg体重、1-30mg/kg体重、1-20mg/kg体重、1-15mg/kg体重、1-10mg/kg体重、1-5mg/kg体重或3-5mg/kg体重的剂量施用。在一些具体实施方案中，所述细胞色素bc1复合物抑制剂或所述药物组合物每天施用一次、每天施用两次、每天施用三次，或每两天、每三天、每四天、每五天、每六天或每七天施用一次。
在一些具体的实施方案中，可通过在所述组合物中使用延迟吸收的药剂(例如，单硬脂酸铝、明胶或其组合)来实现注射用组合物的延长吸收。
实施例。
通过参考在此给出的一些具体实施例可获得对本发明的进一步的理解，这些实施例仅用于说明本发明，其无意于对本发明的范围做出任何限制。显然，可以对本发明作出多种改动和变化而不脱离本发明的实质，因此，这些改动和变化同样在本申请要求保护的范围内。
实施例1、细胞色素bc1复合物抑制剂对大鼠平滑肌收缩的抑制作用
1.实验材料与方法
1.1实验动物：
Wistar大鼠，雌性，体重230-270g，由军事医学科学院实验动物中心提供，医动字D01-3039。
1.2实验试剂：
缩宫素注射液(Oxytocin南京生物化学制药厂，批号051069)。己烯雌酚(Diethylstibestrol北京益民药业有限公司，批号0506150)。PGF2α、醚菌酯(kresoxim-methyl)、乙酰胆碱(acetylcholine)、腈嘧菌酯(azoxystrobin)、氟嘧菌酯(Fluoxastrobin)、Stigmatellin、粘噻唑(Myxothiazol)和阿托伐醌 (Atovaquone)均购自Sigma(St.Louis,MO)。肟菌酯(Trifloxystrobin)、吡唑醚菌酯(Pyraclostrobin)、啶氧菌酯(Picoxystrobin)和醚菌胺(Dimoxystrobin)均购自Santa Cruz Biotechnology。嘧螨酯(Fluacrypyrim，FAPM)及衍生物为实验室内合成(Int J Cancer.2010Sep 1；127(6):1259-70)
1.3实验仪器：
Pclab生物信号采集处理系统(购自北京微信斯达科技发展有限责任公司)；CS502-3C数字超级恒温器(购自重庆四达实验仪器有限公司，温度波动度≤±0.5℃)；GTS生物信号传感器(购自北京微信斯达科技发展有限责任公司)；JZ101型肌肉张力换能器(高碑店市新航机电设备有限公司)。
1.4实验方法
实验选用Wistar雌性健康大白鼠，体重230-270g，在实验前2天给予己烯雌酚悬浊液腹腔内注射0.1mg/kg一次，造成人工动情期。用颈椎脱臼法处死大鼠，剖腹取出子宫，立即置于盛有台氏液的玻璃平皿中。小心剥离附于子宫壁上的结缔组织和脂肪组织，沿系膜线下将子宫剖开，平铺于玻璃平皿低，而后用手术刀片将子宫沿平滑肌走行的子宫收缩带将一侧子宫分为左右两子宫肌条，将一侧子宫肌条的两端分别用线结扎，转移通入有氧气的盛有5ml台氏液的并已被预温到37.2±0.5℃的营养管中，下端固定，上端与张力传感器相连。静息20-30分钟，换液2-3次，然后给予子宫负荷1g并保持30-60分钟，至子宫出现规律的收缩波后，给予KCL溶液40mM 10分钟，评价子宫肌条功能，然后放松肌条并用台氏液冲洗2～3次后静置30分钟，静置后给予子宫肌条负荷1g并保持30-60分钟，待子宫肌条收缩平稳(即微机显示屏中出现规律的收缩波形)后开始给药并观察，记录子宫平滑肌条收缩张力、幅度、收缩曲线下面积等相关数据变化，对所给药物进行评价。在实验过程中，台氏液的工作温度始终保持在37.2±0.5℃，营养管中持续通入氧气，每秒1-2个气泡。采用累积加药法，每次给药5μl，依次由低浓度加到高浓度，药物终浓度指药物溶解于麦氏管中5ml台氏液的浓度。
1.5统计学处理
所有实验数据以“x±s”表示，采用SAS软件做单因素多水平T检验进行统计学分析，作图采用originpro7.5软件。图片处理采用Photoshop CS2软件。
2.实验结果
2.1FAPM及其衍生物对PGF2α引起的大鼠平滑肌收缩的抑制作用
嘧螨酯(fluacrypyrim，FAPM)是由巴斯夫公司研制、日本曹达公司开发的第一个甲氧基丙烯酸酯类杀螨剂，是线粒体细胞色素bc1复合物Qo位点抑制剂。如图1所示，HTFAPM、FAPMA和IFAPM是本发明人实验室合成的FAPM三个衍生物。如图2A所示，正常情况下，大鼠离体子宫平滑肌肌条偶发自律性收缩，能够自主的产生节律性收缩，当在营养管中加入PGF2α(450nM)后能明显增强子宫平滑肌肌条的收缩幅度和频率。实验发现FAPM可剂量依赖性地抑制PGF2α引起的大鼠子宫平滑肌收缩的幅度和频率，在相同剂量下，IFAPM部分抑制PGF2α引起的大鼠子宫平滑肌收缩，而HTFAPM和FAPMA没有抑制作用。分别计算药物干预前及不同剂量药物干预后10分钟平滑肌收缩曲线下面积，以PGF2α引起子宫平滑肌收缩曲线下面积为100％，作药物剂量-效应曲线，推算药物抑制子宫平滑肌收缩的半数有效剂量(IC50值)，如图2B所示，FAPM和IFAPM的IC50值分别为1.84±0.08μM和18.5±3.0μM。构效关系分析揭示FAPM的O14的甲氧基丙烯酸酯是其抑制子宫平滑肌收缩的关键效应基团。
2.2甲氧基丙烯酸酯类杀菌剂对PGF2α引起的大鼠平滑肌收缩的抑制作用
本文在PGF2α引起的大鼠子宫平滑肌收缩实验模型上，系统比较了多种甲氧基丙烯酸酯类杀菌剂如Azoxystrobin、Trifloxystrobin、kresoxim-methyl、Fluoxastrobin、Pyraclostrobin、Picoxystrobin和Dimoxystrobin对子宫平滑肌收缩的抑制作用。如图3和表1所示，这些化合物均能剂量依赖地抑制PGF2α引起的大鼠子宫平滑肌收缩，其IC50值位于1.05和4.08μM之间，与FAPM效果相当。Stigmatellin和Myxothiazol是报道最强的细胞色素bc1复合物呼吸抑制剂，是研究细胞色素bc1复合物最常用的小分子化合物探针，发现二者抑制PGF2α引起的大鼠子宫平滑肌收缩作用也最强，IC50值在纳摩尔水平。
表1.细胞色素bc1复合物抑制剂对PGF2α引起的大鼠子宫平滑肌收缩的抑制作用
抑制剂 IC 50(μM)
FAPM 1.69±0.16
azoxystrobin 1.70±0.12
trifloxystrobin 1.77±0.05
kresoxim-methyl 4.08±0.29
Pyraclostrobin 1.05±0.01
Picoxystrobin 1.20±0.02
Dimoxystrobin 1.65±0.12
Fluoxastrobin 2.63±0.30
Stigmatellin 89.9nM
Myxothiazol 214.7nM
2.3FAPM对缩宫素、乙酰胆碱和氯化钾引起的大鼠平滑肌收缩的抑制作用
缩宫素是子宫平滑肌兴奋药，通过激活缩宫素受体引起子宫平滑肌节律性收缩。乙酰胆碱是一种神经递质，可激活平滑肌M型胆碱受体引起子宫平滑肌节律性收缩。氯化钾可使平滑肌细胞膜去极化，高浓度氯化钾(40mM)可引起子宫平滑肌强直收缩，低浓度氯化钾(16mM)可引起子宫平滑肌节律收缩。如图4-6所示，FAPM可剂量依赖性地抑制缩宫素(1mU/ml)、乙酰胆碱(0.25μM)和氯化钾(16mM)引起的大鼠子宫平滑肌收缩，其IC50值分别为1.6±0.08μM、1.36±0.04μM和1.21±0.07μM。
2.4阿托伐醌(Atovaquone)对PGF2α引起的大鼠平滑肌收缩的抑制作用
阿托伐醌(atovaquone)为羟基1,4-萘喹啉，是辅酶Q的同系物，具有广谱抗原虫的活性。美国食品监督管理局(FDA)2011年批准用于治疗疟原虫属、卡氏肺孢子菌肺炎(PCP)和弓形虫等感染；美国感染性疾病协会(IDSA)指南2006年推荐阿托伐醌用于阿贝虫病治疗。阿托伐醌可直接结合于原虫细胞色素bc1复合物，抑制细胞色素bc1复合物活性是其发挥广谱抗原虫疗效的分子药理学机制(Siregar JE,Kurisu G,Kobayashi T,et al.Direct evidence for the atovaquone action on the Plasmodium cytochrome bc1 complex.Parasitol Int.2015 Jun；64(3):295-300)。基此，本文观察了阿托伐醌对PGF2α引起的大鼠子宫平滑肌收缩的抑制作用，发现阿托伐醌可剂量依赖性地抑制PGF2α引起的大鼠子宫平滑肌收缩，其IC50值为8.44±0.7μM(图7)。
实施例2、细胞色素bc1复合物抑制剂FAPM对大鼠平滑肌细胞MLC20磷酸化的抑制作用
1.实验材料与方法
1.1实验动物
Wistar大鼠，雌性，体重230-270g，由军事医学科学院实验动物中心提供，医动字D01-3039。
1.2实验试剂：
WB 2×SDS上样缓冲液、5×Tris-甘氨酸电泳缓冲液、电转移缓冲液、分离胶缓冲液均自配(见溶液配制)其中试剂以及X光感光胶片均购自北京化工试剂厂；抗phospho-myosin light chain-2(ser19)单克隆抗体，辣根过氧化物酶标记的二抗、辣根过氧化物酶显色底物
Figure PCTCN2018073609-appb-000001
substrate均购自Cell Signaling technology公司。
1.3实验仪器：
DNM-9602G型酶标仪购自北京普朗新技术有限公司。稳压稳流电源、蛋白电泳仪，蛋白电转装置、蛋白分子量Marker购自New England Biotech公司。
1.4大鼠离体子宫平滑肌细胞培养：
采用组织块培养法：将己烯雌酚处理的大鼠脱臼处死后，浸泡于75％酒精中消毒。无菌条件下打开腹腔，分离子宫，移入平皿中，剪开子宫去除子宫外膜，用刀片轻轻刮取除去子宫内膜，用镊子轻轻撕去子宫外膜，将完整的平滑肌剪成1×1×1mm 3以下的小块，均匀贴于培养瓶壁，底部向上加入培养液，5％CO 2、37℃培养3～4小时后轻轻翻转，浸于RPMI-1640培养液(含20％小牛血清)培养约一周左右，细胞可连成片，形成单层培养细胞。此时可倾倒瓶中培养液，PBS冲洗两次除去瓶中组织块，加入适量0.25％胰蛋白酶，显微镜下观察，当多数细胞收缩变圆时加小牛血清终止反应，倾倒液体PBS洗两次，加RPMI-1640适量，用吸管轻轻吹打洗脱细胞，制备细胞悬浮液待用。
1.5全细胞蛋白的提取：
将细胞浓度调整至10 5/ml，以4×10 5/孔接种于6孔板中，贴壁过夜后，缺血清24小时，而后再用不同浓度FAPM预处理，PGF 2α刺激后5分钟收集细胞，用冰冷的PBS洗细胞三次，用2×SDS上样缓冲液裂解细胞，收集的细胞裂解液，煮沸10min，随后12000r/min，离心5min，取上清液，分装后贮存于-80℃备用。
1.6免疫印迹检测(Western Blotting杂交)
取分装细胞裂解液，室温解冻后进行SDS-PAGE凝胶电泳。用微量注射器小心将样品加入梳孔中，稳压100V至溴酚兰条带泳出分离胶，随后将电压调至200V。电泳后转膜，条件为冰浴稳流160mA 1h，将蛋白转印到硝酸纤维素膜上。将膜转入平皿中，5％的脱脂牛奶室温封闭1h，TBS/T洗3次×5min，适当比例的一抗稀释液孵育过夜，次日用TBS/T洗4次×5min，相应的适当比例二抗稀释液再孵育1h，TBS/T洗4次×5min，
Figure PCTCN2018073609-appb-000002
底物避光孵育1min，在暗室中X光片感光显影。如有必要用其它抗体重新标记膜，可将膜用清除缓冲液(见溶液配制)于65℃孵育30min，清除后用TBS/T洗膜，封闭后再标记抗体。
1.7统计学处理
所有实验数据以“x±s”表示，采用SAS软件做单因素多水平T检验进行统计学分析，作图采用originpro7.5软件。图片处理采用Photoshop CS2软件。
2.结果
如图8所示，PGF2α刺激可显著增强大鼠子宫平滑肌细胞MLC20磷酸化，提前给予不同浓度FAPM预处理后，MLC20磷酸化程度有明显降低。通过灰度分析，发现5μM和10μM的FAPM可显著抑制PGF2α引起的大鼠子宫平滑肌细胞MLC20磷酸化。这一结果表明FAPM等细胞色素bc1复合物抑制剂可能通过阻止MLC20磷酸化而抑制平滑肌收缩。
实施例3、细胞色素bc1复合物抑制剂对巨噬细胞一氧化氮生成的抑制作用
1.材料与方法
1.1细胞株
小鼠单核巨噬细胞RAW 264.7购自协和细胞库。
1.2试剂及仪器
脂多糖(LPS)购自Sigma公司,RPMI1640培养基、青霉素、链霉素、胎牛血清购自Invitrogen公司；一氧化氮检测试剂盒购自碧云天；NM-9602G型酶标仪购自北京普朗新技术有限公司。
1.3细胞培养
小鼠单核巨噬细胞培养于含10％胎牛血清、100μg·mL-1链霉素、100 单位·mL-1青霉素的RPMI1640培养基,于37℃、5％CO2的恒温培养箱中孵育生长，隔天传代。
1.4 NO释放量的测定
采用Griess法测定样品中NO含量。将RAW 264.7细胞制成含5×10 5个/mL的单细胞悬液，接种于96孔细胞培养板(200μL/孔)。在37℃、5％CO2的培养箱里孵育12h后，每孔加入不同浓度的测试样品，孵育30分钟加入LPS(终浓度1μg·mL-1)。同时设LPS组和空白对照组,每个样品3个重复孔。在培养箱里孵育24h后,吸取培养液上清100μL至酶标板中,加入等体积的Griess试剂，室温反应10min后测定540nm的吸光值。抑制率计算公式为:抑制率＝[OD] LPS-[OD] LPS+样品/[OD] LPS-[OD] 空白×100％。
2.结果
小鼠腹腔巨噬细胞细胞系RAW264.7是常用的研究炎症的细胞模型之一。应用LPS刺激RAW264.7细胞生成NO，观察多种细胞色素bc1复合物抑制剂对巨噬细胞生成NO的抑制作用。如表2所示，细胞色素bc1复合物抑制剂azoxystrobin、trifloxystrobin、kresoxim-methyl和FAPM均显著抑制NO生产，IC50分别为1.89±0.35μM、0.64±0.21μM、2.77±0.69μM和3.80μM，最大抑制率均在80％以上，其有效浓度与其抑制子宫平滑肌收缩相当。Stigmatellin和Myxothiazol抑制子宫平滑肌收缩活性最强，其抑制NO生产的IC50也最低，分别为6.24±1.15nM和2.8nM，最大抑制率分别为88.3±3.9％和95％。
表2、细胞色素bc1复合物抑制剂对巨噬细胞一氧化氮生成的抑制作用
Figure PCTCN2018073609-appb-000003
实施例4、细胞色素bc1复合物抑制剂的体内镇痛作用研究
1.材料与方法
1.1实验动物
清洁级昆明种雌性和雄性小鼠，体重22-28g，由军事医学科学院实验 动物中心提供，医动字D01-3039。
1.2实验药品及试剂
缩宫素注射液(Oxytocin，南京生物化学制药厂，批号051069)。己烯雌酚(Diethylstibestrol北京益民药业有限公司，批号0506150)。消炎痛(indomethacin)、PGF2α、kresoxim-methyl和azoxystrobin均购自Sigma(St.Louis,MO)。Trifloxystrobin、Picoxystrobin和Dimoxystrobin购自Santa Cruz Biotechnology。FAPM为本发明人实验室内合成(Int J Cancer.2010 Sep1；127(6):1259-70)。
1.3小鼠醋酸扭体实验
参照徐淑云《药理实验方法学》小鼠醋酸扭体法，复制小鼠腹膜炎疼痛模型。成年健康昆明雄性小鼠，随机分组，每组6-10只。小鼠实验前1小时腹腔或灌胃给予不同种类、不同剂量的细胞色素bc1复合物抑制剂，腹腔注射吲哚美辛(50mg/kg)为阳性对照，随后小鼠腹腔注射0.6％冰醋酸0.1m1/10g，观察记录小鼠开始扭体时间(反应潜伏期)及30min内扭体次数。
1.4小鼠痛经模型建立
参考文献资料建立小鼠痛经模型(Yang L.,Cao Z.,Yu B.& Chai C.An in vivo mouse model of primary dysmenorrhea.Exp Anim.2015,Aug 64,295-303)。成年健康昆明雌性小鼠，随机分组，每组10只。实验小鼠每日灌胃己烯雌酚0.2mg，连续12d。于末次给药30min后，腹腔或灌胃给予不同种类、不同剂量的细胞色素bc1复合物抑制剂，腹腔注射吲哚美辛(50mg/kg)为阳性对照，1h后分别腹腔注射PGF2α(1.3mg/kg)或缩宫素(20U/kg)，观察记录小鼠开始扭体时间(反应潜伏期)及30min内扭体次数。
1.5统计学处理
所有实验数据以“x±s”表示，采用SAS软件做单因素多水平T检验进行统计学分析。
2.结果
2.1细胞色素bc1复合物抑制剂对醋酸致小鼠扭体的抑制作用
疼痛是一种由急性或潜在组织损伤引起的不愉快的感觉及情感体验。化学刺激如冰醋酸等制造的疼痛模型是目前研究最多、应用最广泛的一类疼痛模型。这些炎性刺激药物通过中性粒细胞趋化作用而引起急性炎性疼痛，同时介导巨噬细胞浸润引起持续性疼痛。小鼠醋酸扭体实验适于筛选 非麻醉性镇痛药，尤其是筛选甾体抗炎药镇痛作用的一种敏感而简便的一种方法。如表3至6所示，小鼠腹腔注射冰醋酸(0.6％，0.1m1/10g)后30分钟内，扭体次数达40次左右，潜伏期2-3分钟，实验前1小时腹腔注射细胞色素bc1复合物抑制剂FAPM(50、100和200mg/Kg)和Trifloxystrobin(50、100和200mg/Kg)或灌胃给予Azoxystrobin(50、100、200和400mg/Kg)均能剂量依赖地抑制小鼠扭体次数，同对照组相比，FAPM组和Trifloxystrobin组开始扭体时间(反应潜伏期)显著延长，并存在剂量效应，而Azoxystrobin组开始扭体时间没有明显延长。100mg/Kg的FAPM、Trifloxystrobin和Azoxystrobin与50mg/Kg吲哚美辛疗效相当。在200mg/Kg的给药剂量下，比较了细胞色素bc1复合物抑制剂Dimoxystrobin、Kresoxim-methyl和Picoxystrobin灌胃给药对醋酸致小鼠扭体的抑制作用，发现三种抑制剂均能明显抑制小鼠扭体次数，但开始扭体时间没有延长。
表3、FAPM腹腔给药对醋酸致小鼠扭体的抑制作用
Figure PCTCN2018073609-appb-000004
与移植对照组比较 **P<0.01。
表4、Trifloxystrobin腹腔给药对醋酸致小鼠扭体的抑制作用
Figure PCTCN2018073609-appb-000005
与移植对照组比较 *P<0.05； **P<0.01。
表5、Azoxystrobin灌胃给药对醋酸致小鼠扭体的抑制作用
Figure PCTCN2018073609-appb-000006
与移植对照组比较 **P<0.01。
表6、Dimoxystrobin、Kresoxim-methyl和Picoxystrobin灌胃给药对醋酸致小鼠扭体的抑制作用
给药剂量 动物数 动物扭体次数 开始扭体时间
溶剂对照组 0.2ml 10 41.3±3.8 3.1±0.7
Dimoxystrobin 200mg/Kg 10 31.5±4.7 ** 3.3±0.5
Kresoxim-methyl 200mg/Kg 10 33.0±2.9 ** 3.4±1.0
Picoxystrobin 200mg/Kg 10 29.3±4.7 ** 3.3±1.0
与移植对照组比较 **P<0.01。
2.2细胞色素bc1复合物抑制剂FAPM和Azoxystrobin对痛经模型小鼠的镇痛作用
痛经为最常见的妇科症状之一，分为原发性痛经和继发性两类，原发性痛经指生殖器官无器质性病变的痛经，占痛经90％以上；继发性痛经指由盆腔器质性疾病引起的痛经。原发性痛经的发生主要与月经时子宫内膜前列腺素含量增高有关。PGF2α含量升高是造成痛经的主要原因。基于痛经的病理生理机制，参考文献资料，建立了已烯雌酚致敏和PGF2α致痛两步法诱导的小鼠痛经模型。
如表7和表8所示，已烯雌酚致敏后，腹腔注射PGF2α(1.3mg/kg)后30分钟内，小鼠扭体次数达20次左右，潜伏期1-2分钟，实验前1小时腹腔注射细胞色素bc1复合物抑制剂FAPM(50、100和200mg/Kg)或灌胃给予Azoxystrobin(50、100和200mg/Kg)均能剂量依赖地抑制小鼠扭体次数，各剂量组与同对照组相比均有统计学差异。除200mg/Kg FAPM组开始扭体时间有明显延长外，其他各组开始扭体时间与对照组没有明显差异。50mg/Kg的FAPM和Azoxystrobin与50mg/Kg吲哚美辛的疗效相当。
缩宫素是子宫平滑肌兴奋药，如图9所示，已烯雌酚致敏和缩宫素致痛两步法诱导的小鼠痛经模型，小鼠扭体次数近40次；灌胃给予Azoxystrobin(50、100和200mg/Kg)能剂量依赖地抑制小鼠扭体次数，各剂量组与对照组相比均有统计学差异。在该模型上，50mg/Kg的Azoxystrobin与50mg/Kg吲哚美辛的疗效相当。
表7、FAPM腹腔给药对PGF2a致小鼠痛经的镇痛作用
Figure PCTCN2018073609-appb-000007
与移植对照组比较 **P<0.01。
表8、Azoxystrobin灌胃给药对PGF2a致小鼠痛经的镇痛作用
Figure PCTCN2018073609-appb-000008
与移植对照组比较 **P<0.01。
表9、Azoxystrobin灌胃给药对缩宫素致小鼠痛经的镇痛作用
Figure PCTCN2018073609-appb-000009
与移植对照组比较 **P<0.01。
实施例5、细胞色素bc1复合物抑制剂FAPM的体内抗炎作用研究
1.材料与方法
1.1实验动物
清洁级昆明种雌性小鼠，体重22-28g，SD大鼠，体重120-140g，由军事医学科学院实验动物中心提供，医动字D01-3039。
1.2实验药品及试剂
消炎痛(indomethacin)、地塞米松和角叉菜胶二甲苯均购自Sigma(St.Louis,MO)。FAPM为本发明人实验室内合成(Int J Cancer.2010 Sep1；127(6):1259-70)。
1.3二甲苯致小鼠耳廓肿胀实验
健康成年雄性小鼠50只，随机分为5组。实验小鼠分别腹腔注射不同剂量FAPM(50,100 and 200mg/kg)一次，对照组注射等量的溶剂，腹腔注射吲哚美辛(50mg/kg)作为阳性对照。给药后1小时，用二甲苯20μL均匀涂于小鼠右耳两侧,左耳不涂抹二甲苯自身对照；1小时后剪下两耳,用6mm打孔器于两耳相同部位取下两耳片,置于精密天平上称重,计算肿胀度(右耳片重-左耳片重)和肿胀抑制率[(右耳片重-左耳片重)/左耳片重×100％)。
1.4角叉菜胶致大鼠足跖肿胀实验
健康成年雄性小鼠18只，随机分为3组。FAPM(100mg/kg)腹腔给药，每天一次，连续3天，对照组注射等量的溶剂，阳性药地塞米松(4mg/kg)灌胃给药，每天一次，连续3天。于末次给药后30min，在大鼠右后足跖皮下注射1％角叉菜胶每只0.1mL，并于注射前及注射后1、2和3h测量足跖厚度。3小时后脱臼处死大鼠，从膝关节下剪下双足，置于精密天平上称重，计算足跖肿胀度(右足重-左足重)。
1.5统计学处理
所有实验数据以“x±s”表示，采用SAS软件做单因素多水平T检验进行统计学分析。
2.结果
炎症是机体对有害刺激所产生的一种重要防御机制，炎症反应是机体最基本的抗损伤反应。炎症有助于促进伤口的愈合，但也能带来很多伤害,如引起关节炎、哮喘以及机体失调等。炎症根据发病的原因可分为感染性炎症、无菌性炎症(非特异性炎症)及变态反应性炎症。二甲苯致小鼠耳廓肿胀实验和角叉菜胶致大鼠足跖肿胀实验是评价和筛选抗炎药物最为常用的方法。如表10至表12所示，100mg/kg FAPM腹腔给药可显著抑制二甲苯致小鼠耳廓肿胀和角叉菜胶致大鼠足跖肿胀，同对照组相比均有统计学差异。阳性药吲哚美辛(10mg/Kg)组耳廓肿胀程度虽较对照组轻，但无统计学差异。阳性药地塞米松(4mg/kg)可显著抑制角叉菜胶致大鼠足跖肿胀，其疗效略强于FAPM。
表10、FAPM腹腔给药对二甲苯致小鼠耳肿胀的抑制作用
给药剂量 动物数 耳肿胀(mg)
溶剂对照组 0.2ml 10 14.18±1.53
吲哚美辛 10mg/Kg 10 12±3.74
FAPM 100mg/Kg 10 7.83±3.85 **
与对照组比较 **P<0.01。
表11、FAPM腹腔给药对角叉菜胶致大鼠足跖肿胀厚度的影响
给药剂量 动物数 1h(mm) 2h(mm) 3h(mm)
溶剂对照组 0.2ml 6 6.09±0.09 6.87±0.4 7.16±0.67
地塞米松 4mg/Kg 6 5.52±0.39 5.65±0.39 ** 6.11±0.15
FAPM 100mg/Kg 6 5.72±0.29 5.92±0.19 ** 6.24±0.20
与对照组比较 **P<0.01。
表12、FAPM腹腔给药对角叉菜胶致大鼠足跖肿胀的抑制作用
给药剂量 动物数 足跖肿胀(g)
溶剂对照组 0.2ml 6 1.441±0.28
地塞米松 4mg/Kg 6 0.518±0.05 **
FAPM 100mg/Kg 6 0.834±0.32 **
与对照组比较 **P<0.01。

Claims (19)

  1. 细胞色素bc1复合物抑制剂在制备用于治疗平滑肌痉挛相关疾病、治疗炎症疾病和/或缓解疼痛的药物中的用途。
  2. 权利要求1的用途，其中所述细胞色素bc1复合物抑制剂是细胞色素bc1复合物Qo位点抑制剂。
  3. 权利要求1的用途，其中所述细胞色素bc1复合物抑制剂是细胞色素bc1复合物Qi位点抑制剂。
  4. 权利要求1的用途，其中所述细胞色素bc1复合物抑制剂是甲氧基丙烯酸酯类细胞色素bc1复合物抑制剂。
  5. 权利要求4的用途，其中所述甲氧基丙烯酸酯类细胞色素bc1复合物抑制剂选自嘧螨酯(Fluacrypyrim，FAPM)、腈嘧菌酯(azoxystrobin)、肟菌酯(Trifloxystrobin)、醚菌酯(kresoxim-methyl)、吡唑醚菌酯(Pyraclostrobin)、啶氧菌酯(Picoxystrobin)、醚菌胺(Dimoxystrobin)、氟嘧菌酯(Fluoxastrobin)，以及它们的前药或它们的可药用盐。
  6. 权利要求1的用途，其中所述细胞色素bc1复合物抑制剂是吡啶酮类细胞色素bc1复合物抑制剂。
  7. 权利要求6的用途，其中所述吡啶酮类细胞色素bc1复合物抑制剂选自氯羟吡啶(Clopidol)、GW844520和GSK932121，以及它们的前药或它们的可药用盐。
  8. 权利要求1的用途，其中所述细胞色素bc1复合物抑制剂是羟萘醌类细胞色素bc1复合物抑制剂。
  9. 权利要求8的用途，其中所述羟萘醌类细胞色素bc1复合物抑制剂选自阿托伐醌(Atovaquone)、帕伐醌(Parvaquone)、布帕伐醌(Buparvaquone)、S-10576、NQ3，以及它们的前药或它们的可药用盐。
  10. 权利要求1的用途，其中所述细胞色素bc1复合物抑制剂是喹诺酮类细胞色素bc1复合物抑制剂。
  11. 权利要求10的用途，其中所述喹诺酮类细胞色素bc1复合物抑制剂选自RCQ06，Endochin，以及Endochin-样喹诺酮(ELQ)，例如ELQ-118、ELQ-120、ELQ-121、ELQ-136、ELQ-233、ELQ-245、ELQ-260、ELQ-271、ELQ-274、ELQ-300、ELQ-314、ELQ-316、ELQ-317、ELQ-319、ELQ-337、 ELQ-338、ELQ-351、ELQ-370、ELQ-372、ELQ-380、ELQ-384、ELQ-385、ELQ-388、ELQ-390、ELQ-400、ELQ-404、ELQ-428、P4Q-95和P4Q-391等，以及它们的前药或它们的可药用盐。
  12. 权利要求1的用途，其中所述细胞色素bc1复合物抑制剂是吖啶二酮类细胞色素bc1复合物抑制剂。
  13. 权利要求12的用途，其中所述吖啶二酮类细胞色素bc1复合物抑制剂选自氟克吖啶(Floxacrine)、WR249685、WR243246，以及它们的前药或它们的可药用盐。
  14. 权利要求1的用途，其中所述细胞色素bc1复合物抑制剂是他汀内酯类细胞色素bc1复合物抑制剂。
  15. 权利要求14的用途，其中所述他汀内酯类细胞色素bc1复合物抑制剂选自辛伐他汀(Simvastatin)、西立伐他汀(Cerivastatin)、瑞舒伐他汀(Rosuvastatin)、普伐他汀(Pravastatin)、匹伐他汀(Pitavastatin)、美伐他汀(Mevastatin)、洛伐他汀(Lovastatin)、氟伐他汀(Fluvastatin)、阿托伐他汀(Atorvastatin)，以及它们的前药或它们的可药用盐。
  16. 权利要求1的用途，其中所述细胞色素bc1复合物抑制剂选自抗霉素A(Antimycin A)、兰索拉唑(Lansoprazole)、兰索拉唑硫化物(Lansoprazole sulfide)、奥美拉唑(Omeprazole)、潘他米丁(Pentamidine)，以及它们的前药或它们的可药用盐。
  17. 权利要求1-16中任一项的用途，其中所述平滑肌痉挛相关疾病选自痛经，气道痉挛相关疾病如支气管哮喘、喘息性支气管炎、慢性阻塞性肺疾病或气道高反应，血管痉挛相关疾病如脑血管痉挛、肺动脉高压、血管性耳聋、偏头痛或丛集性头痛，胃肠道痉挛相关疾病如胆囊炎、胆管炎、胆道结石、急性胃炎、慢性胃炎、胃溃疡、十二指肠溃疡、急性肠炎、慢性肠炎、克隆氏病、炎症性肠病、溃疡性结肠炎或肠道结核，高血压，冠状动脉痉挛相关疾病如变异型心绞痛，膀胱炎，输尿管结石，肾绞痛，青光眼，术后肠梗阻，尿频，纤维肌痛，性交疼，肠易激综合征，颈部肌肉和眼睑痉挛，膀胱过度活动症，术后眼炎症和呼吸窘迫综合征。
  18. 权利要求1-16中任一项的用途，其中所述炎症疾病选自类风湿关节炎、骨关节炎、骨质溶解、肌腱炎、滑膜炎、痛风、阿尔兹海默病、肾小球肾炎、肾盂肾炎、动脉粥样硬化性疾病、脉管炎和炎症性呼吸道疾病 如慢性阻塞性肺病、纤维化、肺气肿、急性呼吸窘迫综合征。
  19. 权利要求1-16中任一项的用途，其中所述疼痛是炎症性疼痛。
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