WO2017133523A1 - 吡咯喹啉醌、其衍生物和/或盐在干燥综合征中的用途以及药用组合物 - Google Patents

吡咯喹啉醌、其衍生物和/或盐在干燥综合征中的用途以及药用组合物 Download PDF

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WO2017133523A1
WO2017133523A1 PCT/CN2017/072130 CN2017072130W WO2017133523A1 WO 2017133523 A1 WO2017133523 A1 WO 2017133523A1 CN 2017072130 W CN2017072130 W CN 2017072130W WO 2017133523 A1 WO2017133523 A1 WO 2017133523A1
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syndrome
sjogren
vitamin
pqq
group
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French (fr)
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温传俊
孙奋勇
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南京舒鹏生物科技有限公司
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Priority to US16/074,449 priority patent/US10702519B2/en
Priority to EP17746843.6A priority patent/EP3412292B8/en
Publication of WO2017133523A1 publication Critical patent/WO2017133523A1/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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to the use of pyrroloquinoline quinone, its derivatives and/or salts in Sjogren's syndrome and pharmaceutical compositions, in particular to pyrroloquinoline quinone, its derivatives and/or salts in the preparation for treatment and/or The use of a drug that prevents Sjogren's syndrome, as well as dry mouth, dry eyes, and other exocrine glands and other organs in the gland other than Sjogren's syndrome.
  • Sjogren's syndrome is a chronic inflammatory autoimmune disease that primarily affects exocrine glands. The inflammatory response is mainly manifested in the epithelial cells of the exocrine glands. Clinically, in addition to the loss of salivary gland and lacrimal gland damage, dry mouth and dry eyes, there are other symptoms of multiple systemic damage caused by the involvement of other exocrine glands and other organs in the gland. There are a variety of autoantibodies and hyperimmune globulinemia in the patient's serum. The disease is divided into two categories: primary and secondary. Primary Sjogren's syndrome is a global disease with a prevalence of 0.3% to 0.7% in the Chinese population.
  • More than 90% of the disease is female, the ratio of male to female is 1:9 to 1:20, and the age of onset is 40-50 years old. Since the earliest description of Sjogren's syndrome at the end of the 19th century, its research history has been more than a hundred years. Although its pathogenesis has not yet been fully elucidated, after the 1990s, with the development of immunology and molecular biology techniques, the pathogenesis of SS has been studied. Some progress has been made, and its etiology may be related to factors such as genetics, immunity, endocrine and viral infections. At present, a consensus has been reached. Most researchers believe that SS has genetic susceptibility.
  • Non-steroidal anti-inflammatory drugs are mainly used to treat SS muscles, joint pain, mild serositis, and fever. They generally work faster and are effective within a few days after administration. But only symptomatic treatment can be done. Side effects include digestive tract reactions (even bleeding), kidney damage, and myelosuppression. They can also cause liver damage, occasionally rash, cytopenia, or pancreatitis.
  • glucocorticoids When patients with Sjogren's syndrome have visceral multiple system damage, such as nervous system, blood system, severe interstitial lung disease, vasculitis, liver damage, myositis, etc., generally need to be treated with glucocorticoids, critically ill, available hormone shock treatment. It should be noted that the use of long-term glucocorticoids may occur as follows Roles, such as: iatrogenic adrenal hyperfunction, induce and aggravate infection, induce and aggravate peptic ulcer, osteoporosis, aseptic osteonecrosis. For patients with rapid progression, immunosuppressive agents such as cyclophosphamide and azathioprine can be used.
  • Tumor necrosis factor (TNF- ⁇ ) promotes lysis of gland cells and alters the adhesion of endothelial cells (Adv Exp Med Biol, 1998, 438: 909-915).
  • Interleukin regulates cell growth and differentiation in SS exocrine glandular lesions, affects the behavior and characteristics of various cells, participates in inflammatory responses, and regulates immune responses (Arthritis Rheum, 1997, 40: 987-990).
  • Fox et al (Cur Opi Rheum, 2000, 12: 391-398) found that SGEC produced by SS patients produced 40-fold more IL-1 ⁇ , 6 and TNF- ⁇ mRNA levels than normal SGEC.
  • IL-1 ⁇ , IL-6, IL-10, TNF- ⁇ and IFN- ⁇ were improved by ELISA (Lab Invest. 1999.12: 1719-1726). Therefore, cytokine-mediated inflammatory responses are important pathogenesis of SS.
  • Nuclear factor kappa B is a DNA-binding protein that regulates gene expression. It regulates the expression of many important cytokines, adhesion molecules, and chemokine genes. Physiological and pathological processes, the most important of which are immune and inflammatory responses. NF- ⁇ B has been shown to be a very important class of transcription factors that are ubiquitous in inflammation and immune response. NF- ⁇ B is a fast-reacting transcription factor that acts by expressing inflammatory mediators (IL-1 ⁇ , IL-6, IL-10, TNF- ⁇ , etc.), adhesion molecules, and enzymes in the inflammatory response.
  • NF- ⁇ B is involved in the activation of macrophages and leukocytes, and controls the expression of many cytokines, inflammatory proteins, and pro-inflammatory factors. Loss of control in this regulatory phase will lead to amplification of the inflammatory response and even tissue damage. Studies have shown (Arthritis Res Ther. 2012 Mar 14; 14(2): R64) that activation of TOLL-like receptor 2 (TLR 2) induces IL-23/IL-17 expression via NF- ⁇ B This process is closely related to the formation of SS.
  • TLR 2 TOLL-like receptor 2
  • LisiSi et al also found that decreased expression of nuclear factor-kappa B inhibitory factor alpha (I ⁇ B ⁇ ) up-regulates the NF- ⁇ B pathway, increases SS-related cytokine production and inflammatory responses, leading to SS (Lisi et al., Pathology. 2012 Oct; 44(6): 557-61).
  • I ⁇ B ⁇ nuclear factor-kappa B inhibitory factor alpha
  • Oxidative Stress refers to the imbalance between oxidation and anti-oxidation in the body.
  • High-activity molecules such as reactive oxygen species (ROS) and reactive nitrogen radicals (RNS) are excessively produced, and the degree of oxidation exceeds the removal of oxides. , resulting in inflammatory infiltration of neutrophils and tissue damage.
  • ROS include superoxide anion, hydroxyl radical and hydrogen peroxide;
  • RNS includes nitric oxide, nitrogen dioxide and peroxynitrite Wait.
  • Representative biomarkers of oxidative stress are 8-hydroxydeoxyguanosine (8-OHdG) and thioredoxin (TRX).
  • 8-OHdG is a sensitive marker of oxidative stress DNA damage. Studies have reported that 8-OHdG is found to increase in the saliva of SS patients, but not in other salivary gland dysfunction patients and normal individuals (Ryo et al, Pathobiology. 2006; 73 (5): 252-60); SS patients Two protein oxidation markers PC (protein carbonyl) and APOO (higher oxidized protein product) were increased (Free Radic Res.
  • TRX thioredoxin
  • Antioxidants inhibit the inflammatory response of inflammatory factors and apoptosis, thereby protecting the salivary glands of SS patients from tissue damage caused by inflammatory reactions. This also indicates that oxidative stress can participate in the pathogenesis of SS by inducing inflammatory reactions in the gland, causing tissue damage.
  • antioxidants can eliminate oxidative stress and inhibit inflammatory damage in the pathological process of SS caused by oxidative stress, thereby preventing the pathological process of SS.
  • the inflammatory injury process may be carried out by activating the NF- ⁇ B pathway. Therefore, anti-oxidation may become a new treatment route for SS. Therefore, finding suitable antioxidants may be an important direction for future research and development.
  • antioxidants are superoxide dismutase, catalase, thioredoxin, N-acetylcysteine, ergothione, vitamin C, vitamin D, vitamin E, glutathione, melatonin.
  • Vitamin D has a wide range of effects including inhibition of Th17 cell-mediated autoimmunity (Mol. Cell. Biol. 2011, 31(17): 3653).
  • Vitamin D deficiency (VitD) is common in SS patients, especially female SS patients who are at high risk of VitD deficiency (Erten et al, Int J Rheum Dis. 2015 Jan; 18(1): 70-5).
  • Low vitamin D levels in patients with SS may be associated with serious complications such as lymphoma and peripheral neuropathy.
  • Vitamin D supplementation may be an additional tool for SS optimal treatment. Vitamin D may play a role in neuropathic pathology and disease expression in SS patients and may be used to monitor and treat such complications.
  • vitamin D deficiency and severe SS complications. It is recommended that vitamin D supplements should be given to each SS patient (BMC Med. 2013 Apr 4; 11: 93).
  • vitamin D itself is not biologically active and needs to be further metabolized in the body to hydroxylate to active 1,25-dihydroxyvitamin D3 (1,25-Dihydroxyvitamin D3), which has a large clinical application dose, and sometimes needs to be injected to ensure efficacy. Dosing.
  • N-acetylcysteine is a precursor for the synthesis of glutathione in the body and is an important antioxidant.
  • a randomized double-blind trial was performed in 26 patients with primary or secondary Sjogren's syndrome. The experimental group and the control group were treated with N-acetylcysteine (NAC) and placebo for 4 days. It was found that after treatment with NAC, the pain, sensitivity, and bad breath of the eyes were improved during the day. This suggests that NAC has a real therapeutic effect on the eye symptoms of SS patients and deserves further study (Walters et al, Scand J Rheumatol Suppl. 1986; 61: 253-8).
  • NAC is not stable in vivo and requires a large dose to ensure efficacy; at the same time, NAC is an acidic substance that is dosed up to 1.2 grams per day in human trials (Altern Med) Rev.2000Oct;5(5):467-71), this dose may cause gastrointestinal symptoms in some patients; and, according to the 2010 edition of the Pharmacopoeia of the People's Republic of China, NAC is still known as a known phlegm drug. A variety of other adverse reactions, including serious poisoning.
  • the technical problem to be solved by the present invention is that the above-mentioned existing drugs have disadvantages in the method for treating SS, and the stability of the known drugs for treating SS, VitD and NAC, is not high, and the pharmaceutically active activity is single, and a larger dose is required to ensure Efficacy and various adverse reactions caused by it.
  • R 1 , R 2 and R 3 are the same or different and each represents a lower alkyl group, a lower alkenyl group, a lower alkynyl group or an aralkyl group, respectively.
  • the purified tricarboxylic acid form of PQQ is brick red with a molecular weight of 330.21. Since the PQQ structure was confirmed in 1979, extensive research has been carried out on its natural distribution, biosynthesis, and biological functions.
  • PQQ is produced by certain Gram-negative bacteria, glutamic acid and tyrosine are precursors for their biosynthesis, and the genes required for synthesis have also been cloned. PQQ is widely present in a variety of microorganisms, plants and animals at trace levels. PQQ is a water-soluble anionic complex that acts as an electron acceptor or donor involved in the enzymatic reaction of oxidoreductases. PQQ exists in the early stages of biogenesis and evolution. Many microorganisms can synthesize PQQ and play an important role in their own growth.
  • PQQ is a novel vitamin
  • PQQ has a unique o-benzoquinone structure, it has physiological properties not found in other coenzymes, and is relatively stable in chemical properties. This property allows it to participate in 20,000 redox cycles (Altern Med Rev. 2009Sep; 14(3) ): 268-77).
  • PQQ PQQ
  • the main understandings include: (1) stimulating the growth of microorganisms, plants, animals and human cells; (2) trophic factors necessary for animal growth, development and reproduction; (3) removal Excessive free radicals protect the body from oxidative damage; (4) Provide neurotrophic and protective effects.
  • Human experiments have shown that PQQ can improve sleep and cognition and reduce the expression of inflammatory factors IL-6 and C-reactive protein (CRP) in plasma.
  • CRP C-reactive protein
  • PQQ can significantly improve the salivary secretion function of NOD mice in the dryness model. This effect is more pronounced when PQQ is combined with active vitamin D, and the same dose of active vitamin is used. d has no effect.
  • PQQ and PQQ combined with active vitamin D can significantly inhibit the oxidative stress level and lymphocyte infiltration in the submandibular gland of NOD mice.
  • the activity of NF-kappaB signaling pathway was significantly decreased in important inflammatory signaling pathways, and the expression of cytokines such as Il-1, Il-6, and gamma interferon was significantly reduced.
  • antioxidants mentioned above include N-acetyl-L-cysteine, resveratrol, epigallocatechin gallate, curcumin, anthocyanins, tea polyphenols, vitamin B12, vitamin E, vitamins. C or vitamin D.
  • the present invention provides a novel medicinal use for pyrroloquinoline quinone, its derivatives and/or salts, and provides a pharmaceutical composition containing pyrroloquinoline quinone, its derivatives and/or salts as active ingredients.
  • a drug using pyrroloquinoline quinone, a derivative thereof and/or a salt as an active ingredient, and a combined active VitD or NAC have the following advantages and advances as compared with a drug having an active VitD or NAC as an active ingredient alone:
  • PQQ has a unique o-benzoquinone structure and is relatively stable in chemical properties. This property allows it to participate in 20,000 redox cycles, and the free radical scavenging activity is significantly higher than NAC or vitamin D;
  • the dosage is small.
  • the dose of PQQ used by the rats in this drug regimen is 1 mg per kilogram of body weight per day, which is far less than the amount of NAC in animal experiments.
  • the daily volume per kilogram of body weight is 44-1300 mg (The American Journal of Pathology, Vol. 175) , No. 1, July 2009. Fertil Steril_2010; 94: 2905–8.);
  • PQQ has a significant synergistic effect with active vitamin D, which greatly reduces the amount of active vitamin D used and reduces the side effects of active vitamin D.
  • the combination of the drug has a better prospect for the treatment of Sjogren's syndrome.
  • Pyrroloquinoline quinone its derivatives and/or salts are used for the preparation of the treatment and/or prevention of Sjogren's syndrome, as well as dry mouth, dry eyes and other exocrine glands and other organs in the gland caused by Sjogren's syndrome. Drugs with multiple systemic damage have not been reported. Moreover, its therapeutic effect on exocrine gland symptoms such as dry eye and dry mouth of Sjogren's syndrome is better than VitD and NAC.
  • a first aspect of the invention relates to pyrroloquinoline quinone and derivatives and/or salts thereof for the preparation of a dry mouth, dry eye and other exocrine glands and glands for the treatment and/or prevention of Sjogren's syndrome and caused by Sjogren's syndrome Use of drugs that cause multiple systemic damage in the presence of other organs in vitro;
  • a second aspect of the invention relates to the use of the first aspect, characterized in that the Sjogren's syndrome comprises primary Sjogren's syndrome, secondary Sjogren's syndrome.
  • a third aspect of the invention relates to the use of the first aspect, characterized in that the multi-system damage caused by the involvement of other organs in the gland includes the chronic fatigue caused by the dry syndrome.
  • a fourth aspect of the invention relates to the use of the first aspect, characterized in that the pyrroloquinoline quinone, a derivative or a salt thereof is represented by the following structural formula (I):
  • R 1 , R 2 and R 3 are the same or different and each independently represent a lower alkyl group, a lower alkenyl group, a lower alkynyl group, an aralkyl group, an alkylaryl group, a phenyl group, a hydrogen atom, a sodium atom or Potassium atom.
  • a fifth aspect of the invention relates to a pharmaceutical composition characterized by using a therapeutically and/or prophylactically effective amount of pyrroloquinoline quinone, a derivative thereof and/or a salt thereof, in any one of the first to fourth aspects As an active ingredient.
  • a sixth aspect of the invention relates to the pharmaceutical composition of the fifth aspect, characterized by further comprising active vitamin D (1,25-Dihydroxyvitamin D3).
  • a seventh aspect of the invention relates to the pharmaceutical composition of the fifth aspect, characterized in that the pyrroloquinoline quinone is in the form of a tricarboxylic acid.
  • An eighth aspect of the invention relates to the pharmaceutical composition of the fifth aspect, characterized in that the pyrroloquinoline quinone is in the form of a disodium salt.
  • a ninth aspect of the invention relates to the pharmaceutical composition of the fifth aspect, characterized by further comprising any one or a mixture of one or more of the following: N-acetyl-L-cysteine, white Resveratrol, epigallocatechin gallate, curcumin, anthocyanin, tea polyphenols, vitamin B12, vitamin E, vitamin C or vitamin D.
  • Figure 1 is a graph showing the expression of NF- ⁇ B-related proteins in different PQQ and active vitamin D treatment groups.
  • Figure 2 is a graph showing the synergistic inhibition of inflammatory factors Il-1, Il-6, and gamma interferon by PQQ and active vitamin D.
  • Figure 3 is a graph showing the effect of PQQ and active vitamin D treatment on infiltration of submandibular gland lymphocytes in mice.
  • FIG. 4 shows the effect of PQQ and active vitamin D treatment on salivary flow (SFR) in mice.
  • Figure 5 shows the amount of water consumed during the first 8 weeks of PQQ and active vitamin D treatment in mice.
  • PQQ pure tricarboxylic acid form pure product
  • active vitamin d (1,25-Dihydroxyvitamin D3)
  • active vitamin D mother liquor concentration was 1mg/ml
  • the active vitamin D mother liquor concentration was 1 ⁇ g/ml.
  • PQQ disodium salt form (PQQ-Na 2 , donated by Mitsubishi Gas Chemical MGC, Japan), see Example 6, dissolved in water, the mother liquor concentration was 1 mg/ml.
  • Example 1 PQQ prevention and treatment of dry syndrome in experimental animals
  • NOD mice are a genetically deficient model of spontaneous diabetes. This female mouse develops diabetes from 14 weeks, and the incidence can be as high as 80% by 30 weeks. NOD mice are also an animal model of Sjogren's syndrome. The mice develop inflammatory cell infiltration from the submandibular gland at 12 weeks of age, accompanied by increased expression of various inflammatory factors, and decreased salivary secretion and increased salivary salt concentration at 16-20 weeks of age.
  • mice 8-week-old female NOD mice were purchased from Shanghai Slack Laboratory Animal Company and divided into control group, active vitamin d group, PQQ group and PQQ combined active vitamin D. There were four different treatment groups, 10 in each group. Saliva flow rates were measured at 12, 24, and 36 weeks of age. The amount of PQQ and active vitamin D was 1 mg per kg body weight per day and 0.1 microgram per kg body weight per day.
  • avertin Choinese name: tribromoethanol
  • injection dose 0.2ml/10g body weight
  • formula 10.4g avertin dissolved in 0.25ml tert-amyl alcohol, shake for 12 hours, completely dissolved; 2 added 19.75 ml of 0.9% NaCl solution gave 1.2% avertin. Mildly anesthetize mice.
  • 4X SDS loading buffer according to the amount of protein sample: 4X SDS is 3:1, mix well, boil at 95 °C boiling water for 5 min; prepare 10% separation gel and 5% concentrated gel Perform SDS-PAGE electrophoresis, transfer the membrane, and block the PVDF membrane with 5% skim milk in TBST for 1 h or 4 °C overnight; dilute the antibody with blocking solution, incubate at room temperature for 2 h or 4 ° C overnight; wash the membrane, incubate with secondary antibody, and then After washing the membrane, the A and B liquids in the luminescent kit were mixed in a medium volume of the container, and the mixed solution was evenly dropped on the PVDF membrane, and the reaction was 3-5 min in the dark.
  • the submandibular gland tissue was taken and Western Blot was used to detect the activation of NF- ⁇ B in the tissue, indicating that there was no significant change in the Vit D group compared with the control group, but the PQQ group and the PQQ+ active vitamin d group.
  • the phosphorylation level of P65 is significantly weakened, and P65 is the protein subunit of NF- ⁇ B, and its phosphorylation level directly reflects the activity of NF- ⁇ B.
  • I ⁇ B prevents the latter from entering the nucleus by binding to NF- ⁇ B, and is degraded once I ⁇ B is phosphorylated, releasing NF- ⁇ B.
  • the phosphorylation level of I ⁇ B in the pure PQQ group and the PQQ+ active vitamin d group was significantly attenuated, indicating that the activation level of NF- ⁇ B was significantly inhibited. see picture 1.
  • Example 3 PQQ synergizes with active vitamin D to inhibit the expression level of inflammatory factors in tissues
  • the reverse transcription primers were Oligo dT and Random Hexamer.
  • Realtime quantitative PCR reaction system 2 ⁇ PCR premix 10 ⁇ l, Primers 0.8 ⁇ l, cDNA 1 ⁇ l, H 2 0 fill volume 20 ⁇ l; reaction conditions: 95 ° C for 10 min, 95 ° C for 15 s, 60 ° C for 60 s, plate reading, a total of 50 cycles; Melting curve analysis: temperature 55 ° C -95 ° C, read once per minute. Set 3 duplicate holes for each sample.
  • Quantitative PCR was used to analyze the expression of inflammatory cytokines in submandibular gland of mice. It was found that PQQ treatment significantly inhibited the expression levels of inflammatory cytokines IL-1 ⁇ , IL-6, IL-10 and TNF- ⁇ at 36 weeks (p ⁇ 0.05). However, the treatment with active vitamin D alone did not detect the effect, and the effect of PQQ combined with active vitamin D was more obvious, and there was a significant difference (P ⁇ 0.05) with the use of PQQ alone, see Figure 2.
  • Detection of MDA content The submandibular submandibular gland tissue of the experimental mice was taken, and the tissue homogenate was prepared with ice physiological saline, and the protein content was quantified by BCA method. 1 ml of the homogenate was taken, and 1 ml of 30% trichloroacetic acid and 1 ml of 0.67% TBA were sequentially added thereto, and the mixture was cooled to room temperature in a boiling water bath at 30 rnin. Centrifuge at 3000g for 10min, measure the absorbance (A) of the supernatant at 535nm, calculate the MDA content according to the standard curve, and the result is expressed in nmol/mg organization.
  • SOD content detection The submandibular gland tissue of the experimental mice was taken, and the tissue homogenate was prepared with ice physiological saline, and the protein content was quantified by BCA method. Refer to the kit operating instructions. Take 0.2ml sample and mix with 1.3ml test solution, react in water bath at 37 °C for 40min, add 2ml color developer, leave it at room temperature for 10min, measure OD value at 550m. The results are expressed by U/mg protein, which stipulates that the amount of SOD corresponding to 50% of SOD inhibition per mg of protein in a reaction solution is one SOD activity unit.
  • MDA malondialdehyde
  • Submandibular gland tissues of 36-week-old mice were isolated, embedded in paraffin, stained with hematoxylin-eosin staining, and photographed by inverted microscope.
  • Lymphocyte infiltration in the gland showed a mass. As shown in Figure 3, normal C57 mice had no symptoms of xerostomia, no lymphocyte mass infiltration, and NOD mice had obvious lymphocytic infiltration in the submandibular gland, PQQ treatment group. The area of lymphocyte infiltration was significantly reduced. PQQ combined with active vitamin D group, the area of lymphocyte infiltration was further reduced compared with PQQ group.
  • the experiment was carried out in the form of PQQ disodium salt (PQQ-Na 2 ), and the other methods were the same as in Example 1, and the administration time was 24 weeks.
  • mice were tested weekly for drinking water.
  • the amount of water per week was measured in cages and converted into milliliters per gram of body weight per day.
  • the results showed that the drinking water volume of the control mice increased significantly compared with the drug-treated group at the 8th week.
  • the onset of onset of symptoms began to be normal, while the medication group was normal. See Figure 4 and Figure 5.
  • the animal experiment PQQ is 1 mg/kg/day. This dose is the usual dose in animal experiments, corresponding to about 60 mg per day for adults. If the difference in metabolic rate between human and mouse is taken into account, it is about 10 mg per day for adults (60 kg/person for adults, the same below).
  • Active vitamin D dose 0.1 ⁇ g / kg / day, corresponding to an adult of about 0.6 micrograms per day, It is a safe dose that the body can tolerate.
  • the above drugs are all taken in an oral dosage form, that is, formulated into an aqueous solution, mixed with drinking water, and finely adjusted according to the daily drinking water of the rats.
  • Those skilled in the art based on the disclosure of the present application, can determine a suitable dosage range and a suitable oral dosage form by any limited experiment without any inventive effort, and thus any dosage range suitable for the purpose of the present invention and a suitable oral dosage form also fall within the scope of the present application. Within the scope of the invention.

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Abstract

本发明涉及式(I)吡咯喹啉醌、其衍生物和/或盐(PQQ)在制备用于治疗和/或预防原发性干燥综合征、继发性干燥综合征以及由干燥综合征引起的口干、眼干以及其他外分泌腺及腺体外其他器官的受累而出现多系统损害的药物中的应用，其中，式(I)中，R1、R2和R3相同或不同，分别单独表示低级烷基、低级烯基、低级炔基、芳烷基、烷芳基、苯基、氢原子、钠原子或钾原子；以及PQQ或PQQ联合活性维生素d、NAC、白藜芦醇、表没食子儿茶素没食子酸酯、姜黄素、花青素、维生素E、维生素C或维生素D等的药物组合，该药物及药物组合可以治疗干燥综合征以及与干燥综合征相关的适应症。本发明能够清除体内多余的氧自由基，抑制氧化应激导致的NF-kB激活，抑制自身免疫，从而减少干燥综合征的炎症反应。

Description

吡咯喹啉醌、其衍生物和/或盐在干燥综合征中的用途以及药用组合物 技术领域
本发明涉及吡咯喹啉醌、其衍生物和/或盐在干燥综合征中的用途以及药用组合物，具体涉及吡咯喹啉醌、其衍生物和/或盐在制备用于治疗和/或预防干燥综合征、以及由干燥综合征引起的口干、眼干以及其他外分泌腺及腺体外其他器官的受累而出现多系统损害的药物中的应用。
背景技术
干燥综合征(Sjogren’s syndrome，SS)是一种主要累及外分泌腺体的慢性炎症性自身免疫性疾病。其炎症反应主要表现在外分泌腺体的上皮细胞。临床上除有唾液腺和泪腺受损功能下降而出现口干、眼干外，尚有其他外分泌腺及腺体外其他器官的受累而出现多系统损害的症状。患者血清中有多种自身抗体和高免疫球蛋白血症。本病分为原发性和继发性两类。原发性干燥综合征属全球性疾病，在中国人群的患病率为0.3％～0.7％。本病90％以上为女性，男女之比为1:9～1:20，发病年龄多在40-50岁。从19世纪末干燥综合征最早被描述以来，其研究历史己超过百年，虽然其发病机制至今尚未完全阐明，但进入90年代后，随着免疫学、分子生物学技术的发展，SS发病机制的研究取得了一定的进展，其病因可能与遗传、免疫、内分泌和病毒感染等因素有关。目前已基本达成共识，多数学者认为SS存在遗传易感性，在此基础上，局部非特异性炎症，会诱发细胞因子IFN-γ、TNF-a的表达，引发一系列免疫反应(Ann Rheum Dis,2003,62(4):359-62.Clin Rev Allergy Immunol,2007,32(3):252–64)。由于患者免疫调节机制的紊乱，炎症迁延为慢性并损伤外分泌腺，因此其病理改变主要为淋巴细胞和浆细胞浸润腺体组织并引起进行性破坏，导致唾液和泪液分泌减少，出现口、眼干燥症状。
本病目前尚无安全有效的治疗方法。主要是采取措施改善症状，控制和延缓因免疫反应而引起的组织器官损害的进展以及继发性感染。非甾体抗炎药(NSAIDS)主要用于治疗SS肌肉、关节痛、轻度浆膜炎以及发热等症状，一般起效较快，用药后数天即有效。但只能进行对症治疗。副作用有消化道反应(甚至出血)、肾损伤及骨髓抑制等，亦可引起肝功能损害，偶尔出现皮疹、血细胞减少或胰腺炎。当干燥综合征患者出现内脏多系统损害，如神经系统、血液系统、严重的肺间质病变、血管炎、肝损、肌炎等，一般需糖皮质激素使用治疗，病情危重者，可用激素冲击治疗。需要注意的是，长期糖皮质激素使用可出现以下副 作用，如：医源性肾上腺皮质机能亢进症，诱发和加重感染，诱发和加重消化性溃疡，骨质疏松，无菌性骨坏死等。对于病情进展迅速者可合用免疫抑制剂，如环磷酰胺、硫唑嘌呤等。
对SS的研究历史虽已超过百年，但其病因及发病机制至今仍不明确。免疫、遗传、环境、感染、异常神经调节等因素可能与SS发病有关。研究表明(Journal of Autoimmunity,2001，17：141-153)，细胞因子与SS之间存在广泛而密切的联系，在SS的发病过程中起了重要作用。如干扰素既能抑制唾液腺上皮细胞(SGEC)的生长和分化，又可诱导SGEC分离和凋亡(J.Imrnunol，2000，164：1277-1285)。肿瘤坏死因子(TNF-α)能促进腺细胞裂解，改变内皮细胞的粘附性能(Adv Exp Med Biol，1998，438：909-915)。白介素在SS外分泌腺组织病变中调节细胞生长分化，影响多种细胞的行为和特征，参与炎症反应，调节免疫应答(Arthritis Rheum，1997，40：987-990)。Fox等(Cur Opi Rheum，2000，12：391-398)发现，SS病人的SGEC产生的IL-1α，6和TNF-αmRNA水平是正常SGEC的40多倍。用ELISA法检测发现，SS病人腮腺中的IL-1β、IL-6、IL-10、TNF-α和IFN-γ水平都有提高(Lab Invest.1999.12：1719-1726)。因此，细胞因子介导的炎症反应是SS重要的发病机制。
核因子-κB(nuclear factor kappa B，NF-κB)是一种调控基因表达的DNA结合蛋白，它通过调控许多重要的细胞因子、粘附分子和趋化因子基因的表达，参与机体的各种生理、病理过程，其中最重要的是免疫和炎症反应。已证实NF-κB是在炎症和免疫反应过程中普遍存在的一类很重要的转录因子。NF-κB是一个快速反应的转录因子，在炎症反应中通过表达炎症介质(IL-1β、IL-6、IL-10、TNF-α等)、粘附分子和酶等发挥作用。在炎症反应过程中，NF-κB参与巨噬细胞及白细胞的活化，并控制着许多细胞因子和炎症蛋白、促炎因子的基因表达。这一调节环节的失控将导致炎症反应的放大乃至组织损伤。研究表明(Arthritis Res Ther.2012Mar 14；14(2):R64)，TOLL样受体2(toll-like receptor 2，TLR 2)的激活可以通过NF-κB诱导IL-23/IL-17的表达，此过程与SS的形成密切相关。LisiSi等人也发现核因子κB抑制因子α(IκBα)的表达下降能够上调NF-κB通路，增加与SS有关的细胞因子产量和炎症反应，从而导致SS的发生(Lisi等人，Pathology.2012Oct；44(6):557-61)。
氧化应激(Oxidative Stress，OS)是指体内氧化与抗氧化作用失衡，高活性分子，如活性氧自由基(ROS)和活性氮自由基(RNS)产生过多，氧化程度超出氧化物的清除，从而导致中性粒细胞炎性浸润以及组织损伤。ROS包括超氧阴离子、羟自由基和过氧化氢等；RNS包括一氧化氮、二氧化氮和过氧化亚硝酸盐 等。氧化应激具有代表性的生物标志物有8-羟化脱氧鸟苷(8-OHdG)、硫氧还原蛋白(TRX)。
8-OHdG是敏感的氧化应激DNA损害标志物。有研究报道，8-OHdG被发现在SS患者唾液中增高，而在其他唾液腺功能紊乱患者和正常个体中不增高(Ryo等人，Pathobiology.2006；73(5):252-60)；SS患者两种蛋白氧化反应标志物PC(蛋白羰基)和APOO(高级氧化蛋白产物)均增高(Free Radic Res.2012Feb；46(2):141-6)；有研究者通过泪试验、荧光素清除试验、终止时间(BUT scores)和视角面疾病指数(OSDI)在干眼症动物模型和SS患者结膜上皮细胞中比较了有干眼症患者与无干眼症者的氧化应激水平(Graefes Arch Clin Exp Ophthalmol.2015Mar；253(3):425-30)，发现有干眼症的患者比无干眼症者氧化应激水平增高；在SS的NOD(NOD.B10.Sn-H2)小鼠模型、胰腺外分泌腺细胞以及人工培养的人唾液腺细胞中，抗氧化剂表没食子儿茶素通过促分裂原活化蛋白激酶信号途径增加了防御性的抗氧化能力(Autoimmunity.2014 May；47(3):177-84)。以上文献均表明氧化应激参与SS中唾液腺组织的破坏，参与SS的病理进程。
最近研究(Pagano等人，Free Radic Res.2013Feb；47(2):71-3)发现SS患者血浆中，蛋白氧化、髓过氧化物酶活性、TNF-α、硝基络氨酸，和谷胱甘肽水平均明显变化。SS患者细胞内线粒体发生改变，线粒体功能障碍的发生导致氧化应激相关的紊乱，参与氧化应激的形成。
此外有报道显示，硫氧还蛋白(TRX)在SS中发挥对抗唾液腺氧化应激组织损伤的保护作用(J Rheumatol.2007Oct；34(10):2035-43)，在SS患者的唾管细胞中产生大量的8-OHdG和TRX，且TRX与唾液流率明显负相关。此外，TRX作用于人唾液腺细胞后，显著抑制了IFN-γ诱导的IL-6表达和Fas调控的细胞凋亡。TRX为细胞内重要的抗氧化剂，即使TRX单独存在也可表现出对单态氧和羟自由基的消除作用。而IL-6表达和Fas调控的细胞凋亡是典型的炎症反应的病理过程，抗氧化剂抑制炎症因子和细胞凋亡的炎症反应，从而保护SS患者唾液腺免受炎症反应造成的组织损伤。这也说明氧化应激能够通过引发腺体的炎症反应，造成组织损伤，从而参与SS的发病机制。
综上所述，抗氧化剂可以消除氧化应激，抑制氧化应激导致的SS病理过程中的炎症损伤，从而阻止SS的病理进程。其中，炎症损伤过程可能是通过激活NF-κB通路而进行的。因此，抗氧化可能成为新的SS治疗途径，因此，寻找到合适的抗氧化剂可能是今后研发的重要方向。
常见的抗氧化剂有超氧化物歧化酶、过氧化氢酶、硫氧还蛋白、N-乙酰半胱氨酸、麦角硫因、维生素C、维生素D、维生素E、谷胱甘肽、褪黑素、α-硫辛 酸、类胡萝卜素、微量元素铜、锌、硒(Se)等。
维生素d具有广泛的作用，其中包括抑制Th17细胞介导的自身免疫(Mol.Cell.Biol.2011,31(17):3653)。维生素D(VitD)缺乏在SS患者中很常见，特别是女性SS患者有很高的VitD缺乏的风险(Erten等人，Int J Rheum Dis.2015Jan；18(1):70-5)。SS患者低维生素D水平可能与严重的并发症相关，如淋巴瘤和外周神经病。补充维生素D可能是SS最优化治疗的额外工具。维生素D可能在SS患者神经病病理进程和疾病表达中起作用，并且可能用于监测和治疗这种并发症。维生素D缺乏症和严重SS并发症的明显相关，推荐维生素D的补充应该给与每一位SS患者(BMC Med.2013Apr 4；11:93)。但是，维生素D本身无生物活性，需经体内进一步代谢才能羟化为活性的1,25-二羟维生素D3(1,25-Dihydroxyvitamin D3)，其临床应用剂量较大，为保证疗效有时需注射给药。
N-乙酰半胱氨酸(NAC)是体内合成谷胱甘肽的前体，是重要的抗氧化剂。有研究选取了26例原发或继发性干燥综合征患者进行随机双盲试验。实验组和对照组分别予以4天的N-乙酰半胱氨酸(NAC)和安慰剂治疗。结果发现NAC进行治疗后，眼睛的痛苦、敏感以及口臭白天口渴均有改善。这表明NAC对SS患者的眼睛症状有真实的治疗效果，值得进一步研究(Walters等人，Scand J Rheumatol Suppl.1986；61:253-8)。但是，NAC的临床应用有着明显的不足：NAC在体内稳定性不高，需要较大的剂量才能保证疗效；同时，NAC是一种酸性物质，在人体试验中每天服用剂量高达1.2克(Altern Med Rev.2000Oct；5(5):467-71)，这一剂量可能引起部分患者的消化道症状；而且，根据2010版中华人民共和国药典的记载，NAC作为一种公知的化痰药物，还存在多种其它不良反应，严重的包括中毒。
因此，有必要寻找更加稳定、安全，功能强大的药学活性物质，或者药物组合，用于治疗干燥综合症。
发明内容
本发明要解决的技术问题就是上述现有药物治疗SS的方法所存在的缺陷，以及已知的治疗SS的药物VitD、NAC所存的稳定性不高，药学活性单一，需较大的剂量才能保证疗效以及由此带来的多种不良反应等问题。
吡咯喹啉醌(Pyrroloquinoline quinone，简称PQQ)，见下式(I)：
Figure PCTCN2017072130-appb-000001
是一个含三个羧酸基团的邻位醌，结构式(I)中，R1、R2和R3相同或不同，分别单独表示低级烷基、低级烯基、低级炔基、芳烷基、烷芳基、苯基、氢原子、钠原子或钾原子。纯化的三羧酸形式的PQQ呈砖红色，分子量为330.21。自从1979年确认PQQ结构以来，对其自然分布、生物合成以及生物学功能进行了广泛的研究。PQQ由某些革兰氏阴性菌产生，谷氨酸和酪氨酸是其生物合成的前体，合成所需的基因也已得到克隆。PQQ以痕量水平广泛存在于各种微生物、植物和动物体内。PQQ是一种水溶性的阴离子复合物，可作为电子的受体或供体参与氧化还原酶的酶促反应。PQQ在早期生物发生和进化的过程中就存在，许多微生物可以合成PQQ，并对其自身的生长具有重要作用。尽管目前对于PQQ是否是一种新型维生素还存在争议(Bishop等人，Nutr Rev,1998,56(10):287-93)，但不可否认PQQ在人类和动物体内发挥了多种重要的生理功能，如营养作用，防止酒精性肝脏损伤，保护心脏免受缺血再灌注损伤，清除自由基，保护机体免受自由基损害，增加星形胶质细胞中神经生长因子水平等(Matsushita等人，Appl Microbiol Biotechnol,2002,58(1):13-22)。由于PQQ具有独特的邻苯醌结构，使其具有其它辅酶所没有的生理特性，在化学性质方面相对稳定，这种特性使其能参与20000次氧化还原循环(Altern Med Rev.2009Sep；14(3):268-77)。
目前，对于PQQ的生物学功能尚处于探索阶段，主要认识包括：(1)刺激微生物、植物、动物及人体细胞生长；(2)作为动物生长、发育和繁殖必需的营养因子；(3)清除过多的自由基，保护机体免受氧化损伤；(4)提供神经营养和保护作用。人体实验显示，PQQ能够改善睡眠和认知，降低血浆中炎症因子IL-6和C-反应蛋白(CRP)的表达。有学者提出将PQQ作为第14种维生素归入B族维生素行列，现已作为保健品在美国和日本上市。
申请人通过大量的研究和实验，惊奇的发现：PQQ能够显著改善干燥症模型NOD小鼠的唾液分泌功能，PQQ联合活性维生素D时，这一改善作用更加显著，而单纯采用相同剂量的活性维生素d则没有作用。
申请人通过进一步的研究和实验，还惊奇的发现：PQQ、PQQ联合活性维生素D能够显著抑制NOD小鼠颌下腺组织的氧化应激水平和淋巴细胞的浸润， 重要的炎症信号通路NF-kappaB信号通路活性明显降低，Il-1,Il-6,γ干扰素等细胞因子的表达明显减少。
前述其他抗氧化剂还包括N-乙酰基-L-半胱氨酸、白藜芦醇、表没食子儿茶素没食子酸酯、姜黄素、花青素、茶多酚、维生素B12、维生素E、维生素C或维生素D。
本发明为吡咯喹啉醌、其衍生物和/或盐提供了新的药用用途，并为人们提供了一种以吡咯喹啉醌、其衍生物和/或盐为有效成分的药物组合物。采用吡咯喹啉醌、其衍生物和/或盐为有效成分的药物以及联合活性VitD或NAC与单纯以活性VitD或NAC为有效成分的药物相比，具有如下优点和进步：
1)稳定，PQQ具有独特的邻苯醌结构，在化学性质方面相对稳定，这种特性使其能参与20000次氧化还原循环，自由基清除活性显著高于NAC或维生素D；
2)用量小，本药物方案中大鼠所用PQQ剂量为每天每公斤体重1毫克，远远小于NAC在动物实验中的用量每天每公斤体重44-1300毫克(The American Journal of Pathology,Vol.175,No.1,July 2009.Fertil Steril_2010；94:2905–8.)；
3)安全，PQQ的大鼠半数致死剂量为1000-2000毫克/公斤体重，每天每公斤体重100毫克连续14天，未发现任何不良反应(Regul Toxicol Pharmacol.2014Oct；70(1):107-21)；
4)PQQ与活性维生素d有明显协同作用，大大减低了活性维生素d的使用量，降低了活性维生素d的副作用，该药物组合治疗干燥综合征有更好的前景。
将吡咯喹啉醌、其衍生物和/或盐用于制备治疗和/或预防干燥综合征、以及由干燥综合征引起的口干、眼干以及其他外分泌腺及腺体外其他器官的受累而出现多系统损害的药物，目前未见报道。而且，其对干燥综合征眼干、口干等外分泌腺症状的治疗效果较VitD和NAC更好。
本发明的第一方面，涉及吡咯喹啉醌及其衍生物和/或盐在制备用于治疗和/或预防干燥综合征以及由干燥综合征引起的口干、眼干以及其他外分泌腺及腺体外其他器官的受累而出现多系统损害的药物中的应用；
本发明的第二方面，涉及第一方面的应用，其特征在于所述的干燥综合征包括原发性干燥综合征、继发性干燥综合征。
本发明的第三方面，涉及第一方面的应用，其特征在于所述的腺体外其他器官的受累而出现多系统损害包括干燥综合症引起的慢性疲劳。
本发明的第四方面，涉及第一方面的应用，其特征在于所述的吡咯喹啉醌、其衍生物或盐由以下结构式(I)表示：
Figure PCTCN2017072130-appb-000002
结构式(I)中，R1、R2和R3相同或不同，分别单独表示低级烷基、低级烯基、低级炔基、芳烷基、烷芳基、苯基、氢原子、钠原子或钾原子。
本发明的第五方面，涉及一种药物组合物，其特征在于采用治疗和/或预防有效量的第一至第四方面任一项应用中的吡咯喹啉醌、其衍生物和/或盐作为有效成分。
本发明的第六方面，涉及第五方面的药物组合物，其特征在于还包含活性维生素D(1,25-Dihydroxyvitamin D3)。
本发明的第七方面，涉及第五方面的药物组合物，其特征在于所述的吡咯喹啉醌为三羧酸形式。
本发明的第八方面，涉及第五方面的药物组合物，其特征在于所述的吡咯喹啉醌为二钠盐形式。
本发明的第九方面，涉及第五方面的药物组合物，其特征在于还包含以下物质中的任意一种或一种以上任意比例的混合物：N-乙酰基-L-半胱氨酸、白藜芦醇、表没食子儿茶素没食子酸酯、姜黄素、花青素、茶多酚、维生素B12、维生素E、维生素C或维生素D。
附图说明
图1为不同PQQ与活性维生素d处理组NF-κB相关蛋白表达图。
图2为PQQ与活性维生素d协同抑制炎症因子Il-1,Il-6,γ干扰素的表达图。
图3为PQQ和活性维生素d处理对小鼠颌下腺淋巴细胞浸润的影响图。
图4为PQQ与活性维生素d处理对小鼠唾液流量(SFR)的影响。
图5为PQQ与活性维生素d处理小鼠用药的前8周饮水量。
具体实施方式
实验化合物来源及处理
PQQ(三羧酸形式纯品)，见实施例一至五；活性维生素d(1,25-Dihydroxyvitamin D3)，均购自于sigma公司，分别溶解于双蒸水，PQQ母液浓度为1mg/ml，活性维生素d母液浓度为1μg/ml。PQQ二钠盐形式 (PQQ-Na2，由日本三菱瓦斯化学MGC赠送)，见实施例六，溶解于水，母液浓度为1mg/ml。
实施例一：PQQ预防与治疗实验动物的干燥综合征
方法：
模型：NOD小鼠模型。NOD(Non-obese Diabetes)小鼠是一种遗传缺陷的自发性糖尿病动物模型。该种雌性小鼠从14周开始发生糖尿病，至30周发病率可高达80％。NOD小鼠同时也是干燥综合征动物模型，小鼠从12周龄开始颌下腺出现炎症细胞浸润，伴有多种炎症因子的表达增加，16-20周龄开始出现唾液分泌减少和唾液盐浓度增加。
实验分组：8周龄雌性NOD小鼠购自上海斯莱克实验动物公司，分成对照组、活性维生素d组、PQQ组和PQQ联合活性维生素d，共四个不同处理组，每组10只，分别在12，24，36周龄检测唾液流速。PQQ和活性维生素d用量分别是1毫克每公斤体重每天和0.1微克每公斤体重每天。
唾液流量检测：
a.腹腔注射1.2％avertin(中文名：三溴乙醇)，注射剂量：0.2ml/10g体重，配方：①0.4g avertin溶于0.25ml叔戊醇，摇12小时，使其完全溶解；②加入19.75ml 0.9％的NaCl溶液，即得到1.2％avertin。温和麻醉小鼠。
b.颈部皮下注射毛果芸香碱，注射剂量：0.5mg/kg＝0.01mg/20g体重，母液浓度：0.5g/10ml生理盐水＝50mg/ml＝0.05×103mg/ml，使用液浓度：将母液稀释1000倍，即0.05mg/ml，刺激唾液分泌。
c.5min后开始收集唾液，即将无菌脱脂棉球制成干重约8mg左右的棉球，放入1.5mL EP管中称干重，测唾液分泌时将棉球放入小鼠后颊部，10min后取出棉球放入EP管中，电子天平上称湿重。唾液分泌量＝棉球湿重-棉球干重。
结果：
治疗至12周龄时，各实验组与对照组相比，唾液流量都没有明显差异；治疗至24周龄时，PQQ+活性维生素d组与对照组相比，唾液流量明显增加(P﹤0.05)，而单纯活性维生素d组和PQQ组与对照组相比较差异不明显；治疗至36周时，PQQ+活性维生素d组与对照组相比，以及单纯PQQ组与对照组相比，唾液流量都明显增加，其差异有显著统计学意义(P﹤0.05)，而且PQQ+活性维生素d组与单纯PQQ组相比，效果更加明显(P﹤0.05)。单纯活性维生素d组与对照组相比较，无统计学差异。上述结果充分说明，PQQ能有有效缓解干燥综合症的症状，但小剂量活性维生素d单独使用却没有治疗效果，二者的协同作用明显。
表1.PQQ与活性维生素d处理对小鼠唾液流量(SFR)的影响
Figure PCTCN2017072130-appb-000003
注：*表示各组与对照组进行比较，P﹤0.05；#表示PQQ+活性维生素d组与PQQ组进行比较，P<0.05
实施例二：PQQ抑制NF-κB的激活
方法：
Western Blot检测小鼠颌下腺颌下腺组织中NF-κB的激活
每个样品取相同总蛋白量，按照蛋白样品量：4X SDS为3：1的比例加入4X SDS上样缓冲液，混匀后，95℃沸水煮沸5min；配制10％分离胶和5％浓缩胶进行SDS-PAGE电泳，转膜，用含5％脱脂牛奶的TBST溶液室温封闭PVDF膜1h或4℃过夜；用封闭液稀释抗体，室温孵育2h或4℃过夜；洗膜，二抗孵育，再洗膜，将发光试剂盒中的A和B液在容器中等体积混合后，将混合液均匀滴加在PVDF膜上，避光反应3-5min。
结果：
治疗至36周时，取小鼠颌下腺组织，Western Blot检测组织中NF-κB的激活状态，表明单纯Vit D组与对照组相比没有明显的变化，而单纯PQQ组以及PQQ+活性维生素d组因中，P65的磷酸化水平明显减弱，P65是NF-κB的蛋白亚基，其磷酸化水平直接反映NF-κB的活性。此外，IκB通过与NF-κB结合而阻止后者入核发挥活性，一旦IκB被磷酸化后即被降解，释放出NF-κB。此处，单纯PQQ组以及PQQ+活性维生素d组中IκB的磷酸化水平明显减弱，表明NF-κB的激活水平收到明显抑制。见图1。
实施例三：PQQ与活性维生素d协同抑制组织中炎症因子的表达水平
方法：
取实验小鼠颌下腺组织，Trizol抽提RNA，Superscript Ⅲ进行逆转录，42℃反应30分钟，逆转录引物采用Oligo dT与Random Hexamer。Realtime定量PCR反应体系：2×PCR premix 10μl,Primers 0.8μl,cDNA 1μl，H20补平体积20μl；反应条件：95℃10min,95℃15s，60℃60s，读板，共50个循环；融解曲线分 析：温度55℃-95℃，每分钟读1次。每个样设置3个复孔。
结果：
采用定量PCR技术对小鼠颌下腺组织中的炎症因子表达水平进行分析，发现PQQ处理36周能够明显抑制炎症因子IL-1β,IL-6,IL-10,TNF-α的表达水平(p<0.05)，而单独使用活性维生素d处理没有检测到作用，而PQQ与活性维生素d联合使用的作用更加明显，与单纯使用PQQ处理又有显著差异(p<0.05)，见图2。
实施例四：氧自由基检测
方法：
MDA含量检测：取实验小鼠颌下腺颌下腺组织，用冰生理盐水制备组织匀浆，BCA法定量蛋白含量。取1ml匀浆，依次加入1ml 30％三氯乙酸和1ml 0.67％TBA，沸水浴30rnin后冷却至室温。3000g离心10min，在535nm处测上清液吸光度(A)值，按标准曲线计算MDA含量，结果以nmol/mg组织表示
SOD含量检测：取实验小鼠颌下腺组织，用冰生理盐水制备组织匀浆，BCA法定量蛋白含量。参照试剂盒操作说明取0.2ml样品与1.3ml检测液混合，37℃水浴反应40min，加入2ml显色剂，室温放置10min，550m测定OD值。结果用U/mg蛋白表示，规定：每毫克蛋白在l ml反应液中SOD抑制率达50％时所对应的SOD量为一个SOD活力单位。
结果：
氧自由基参与并通过脂质过氧化反应是导致炎症反应的重要因素，氧自由基参与脂质过氧化反应，并产生脂质过氧化物，如丙二醛(MDA)，脂质过氧化反应和脂质过氧化物的分解产物都能引起细胞膜结构受损、炎症介质大量释放。脂质过氧化物也参与某些炎症介质的产生过程，如通过激活环加氧酶，促进花生四烯酸向前列腺素的合成。通过检测MDA来反映脂质过氧化物程度，间接评价细胞组织受损程度。SOD能有效地清除氧自由基，从而抑制肠组织中的脂质过氧化反应，并能稳定细胞膜SOD活性，是反映细胞膜功能和机体抗炎症反应的主要指标。
结果表明，PQQ单独处理36周时，与对照组相比，MDA的含量明显降低(P<0.05)，SOD的含量则显著增高；Vit D单独处理时，却无明显差异；但二者联用时，与对照组相比，MDA与SOD的含量都有明显变化，而且呈现明显的协同作用(P<0.05)。充分说明，PQQ能够有效抑制氧自由基造成脂质过氧化反应，并与Vit D协同，充分保护细胞免除氧自由基的破坏。
表2.PQQ及活性维生素d处理对36周龄小鼠颌下腺组织中MDA、SOD的影响
Figure PCTCN2017072130-appb-000004
注：*表示各组与对照组进行比较，P﹤0.05；#表示PQQ+活性维生素d组与PQQ组进行比较，P<0.05
实施例五：小鼠颌下腺组织切片检测
方法：
分离36周龄小鼠颌下腺组织，石蜡包埋切片，苏木精-伊红染色法染色，倒置显微镜拍照。
结果：
腺体中淋巴细胞浸润呈现出团块状，如附图3所示，正常C57小鼠无干燥症症状，无淋巴细胞团块状浸润，NOD小鼠颌下腺有明显淋巴细胞浸润，PQQ治疗组，淋巴细胞浸润面积显著减少，PQQ联合活性维生素d组，相比较PQQ组，淋巴细胞浸润的面积进一步减少。
实施例六：PQQ预防与治疗实验动物的干燥综合征
材料与方法：
本实验采用PQQ二钠盐形式(PQQ-Na2)外，其他方法与实施例一相同，用药时间为24周。
用药后小鼠每周饮水检测，以笼为单位，检测每周饮水量，换算成毫升每天每克体重，结果提示第8周开始，对照小鼠饮水量与用药组相比较明显增加，提示对照组第8周干燥症开始发病，而用药组正常。见图4和图5。
剂量与剂型
PQQ剂量：
在本发明中，动物实验PQQ采用1毫克/公斤/天。该剂量是动物实验中常用剂量，对应成人约60毫克每天，如果考虑到人鼠代谢率的差异，约为成人每天10毫克(成人以60公斤/人计，下同)。
活性维生素D剂量：采用0.1微克/公斤/天，对应成人约为每天0.6微克， 是人体能够耐受的安全剂量。
上述药物均采用口服剂型，即配制成水溶液，掺入饮水，根据大鼠每天饮水量作微调。本领域技术人员根据本申请的公开内容，通过有限的实验，无需任何创造性劳动即可确定合适的剂量范围及合适的口服剂型，因此任何适合本发明目的的剂量范围及合适的口服剂型也落入本发明保护范围内。

Claims (9)

  1. 吡咯喹啉醌及其衍生物和/或盐在制备用于治疗和/或预防干燥综合征、以及由干燥综合征引起的口干、眼干以及其他外分泌腺及腺体外其他器官的受累而出现多系统损害的药物中的应用。
  2. 如权利要求1所述的应用，其特征在于所述的干燥综合征包括原发性干燥综合征、继发性干燥综合征。
  3. 如权利要求1所述的应用，其特征在于所述的腺体外其他器官的受累而出现多系统损害包括干燥综合症引起的慢性疲劳。
  4. 如权利要求1所述的应用，其特征在于所述的吡咯喹啉醌、其衍生物或盐由以下结构式(I)表示：
    Figure PCTCN2017072130-appb-100001
    结构式(I)中，R1、R2和R3相同或不同，分别单独表示低级烷基、低级烯基、低级炔基、芳烷基、烷芳基、苯基、氢原子、钠原子或钾原子。
  5. 一种药物组合物，其特征在于采用治疗和/或预防有效量的权利要求1-4任意一项所述的应用中的吡咯喹啉醌、其衍生物和/或盐作为有效成分。
  6. 如权利要求5所述的药物组合物，其特征在于还包含活性维生素D(1,25-Dihydroxyvitamin D3)。
  7. 如权利要求5所述的药物组合物，其特征在于所述的吡咯喹啉醌为三羧酸形式。
  8. 如权利要求5所述的药物组合物，其特征在于所述的吡咯喹啉醌为二钠盐形式。
  9. 如权利要求5所述的药物组合物，其特征在于还包含以下物质中的任意一种或一种以上任意比例的混合物：N-乙酰基-L-半胱氨酸、白藜芦醇、表没食子儿茶素没食子酸酯、姜黄素、花青素、茶多酚、维生素B12、维生素E、维生素C或维生素D。
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