CN114886897A

CN114886897A - Pharmaceutical composition containing theophylline acetaldehyde or hydrate thereof, and preparation method and application thereof

Info

Publication number: CN114886897A
Application number: CN202210522932.7A
Authority: CN
Inventors: 杨凌; 马红红; 李小东; 马红; 陈国良; 赵晓华; 石雅洁; 付晓玲
Original assignee: Wuxi Eryun Technology Co ltd
Current assignee: Wuxi Eryun Technology Co ltd
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2022-08-12

Abstract

The invention discloses a pharmaceutical composition containing theophylline acetaldehyde or hydrate thereof, and a preparation method and application thereof, wherein the pharmaceutical composition comprises the theophylline acetaldehyde or hydrate thereof and NAD, wherein the molar mass ratio of the theophylline acetaldehyde or hydrate thereof to the NAD is (1-2): (0.5-1): 2-5) ⁺ And ALDHs. (1) The pharmaceutical composition can be applied to the field of preparing medicaments for organ injury (including heart, liver, lung, kidney and other injuries), eye diseases, inflammation, asthma and the like caused by antioxidant stress by increasing the generation of intracellular antioxidant NADH (nicotinamide adenine dinucleotide), further inhibiting the generation and development of oxidative stress through multiple paths, enriches the types of relevant medicaments for preparing the antioxidant stress, the inflammation resistance and the like, and provides brand-new selection and strategies for preparing the medicaments for the oxidative stress organ injury and the inflammatory diseases; (2) the pharmaceutical composition has simple components, low toxicity and side effects.

Description

Pharmaceutical composition containing theophylline acetaldehyde or hydrate thereof, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and relates to a new application of a xanthine aldehyde compound, in particular to a pharmaceutical composition containing theophylline acetaldehyde or hydrate thereof, and a preparation method and an application thereof.

Background

Oxidative stress is thought to be caused by a lack of balance between pro-oxidants and antioxidants, a certain number of ROS are necessary for cell signaling and maintenance of redox homeostasis, but excessive ROS/RNS production can disrupt the organism's defense system, which is called oxidative stress. Sustained attack of endogenous and exogenous ROS results in altered conformation and oxidation state of key biomolecules, such as oxidative modifications of key biomolecules by oxidative stress: lipid peroxidation, protein carbonylation, carbonyl (aldehyde/ketone) adduct formation, nitration, sulfoxidation, DNA damage (such as strand breakage or nucleobase oxidation), and the like. An antioxidant is a compound that prevents or retards oxidation, and its mechanism of action can be divided into various types, such as by inhibiting the formation of free radical species to prevent the oxidation process. These antioxidants act very rapidly and can neutralize any free radicals which may lead to the generation of other free radicals or any chemical species which may be converted into free radicals, e.g. alsoThe prototype Nicotinamide Adenine Dinucleotide (NADH) has the reduction function of directly eliminating part of free radicals, and experiments also prove that the NADH can eliminate hydrogen peroxide in nerve cells and help prevent neurodegenerative diseases. Free radical scavenging antioxidants are also a mechanism of antioxidant stress in which the scavenging of ROS is exerted by electron transfer, typically hydrophilic antioxidants (ascorbic acid, glutathione, uric acid) and lipophilic antioxidants (alpha-tocopherol and panthenol). Repair following free radical damage is also an antioxidant mechanism, and such antioxidants are also known as "de novo" antioxidants. This group consists of enzymes that repair damaged DNA, lipids and proteins. NADH, as mentioned above, releases biohydrogen which is oxidized to Nicotinamide Adenine Dinucleotide (NAD) after supplying energy for ATP synthesis ⁺ ) Complement cellular NAD ⁺ A library. NAD (nicotinamide adenine dinucleotide) ⁺ Poly [ ADP-ribose as DNA repair enzyme]The only coenzyme for polymerase 1(PARP1) is an essential substance involved in DNA repair. The body also has adaptive mechanisms for free radicals, and in the body, the signals generated by free radicals induce sufficient antioxidant synthesis and transport to the appropriate sites.

Oxidative stress is also implicated in a number of diseases, such as the formation of ROS in the lung, which are an integral part of the molecular mechanism of asthma, and the oxidizing agents that mediate the inflammatory response activate pro-inflammatory cytokine and chemokine genes to promote the up-regulation of adhesion molecules and the release of pro-inflammatory mediators, which in turn are concomitantly produced by ROS. In addition, ROS can alter the morphological and functional properties of endothelial cells, such as increased permeability and expression of adhesion molecules, leading to adhesive interactions between inflammatory cells and endothelial cells, contributing more to the manifestation of inflammatory symptoms. Meanwhile, ROS can also regulate the expression of transcription factors, such as nuclear factor-k B (NF-k B) and activin-1 (AP-1) in epithelial cells, and the gene expression of a plurality of proinflammatory cytokines, enzymes and adhesion molecules can be caused by the activation of the transcription factors so as to induce inflammatory reaction, which indicates that a dynamic relation exists between the molecular mechanism of ROS and the expression of asthma symptoms. Oxidative stress also plays a great role in the pathogenesis of ocular diseases, such as age-related macular degeneration cataract, uveitis, premature retinopathy, keratitis, and ocular inflammation. Elevated levels of oxidized protein are associated with the high inflammatory activity associated with these diseases. Increased ROS generation can be induced on the ocular surface due to sustained exposure to atmospheric oxygen and inadequate support of antioxidants due to instability in the tear film. Oxidative stress can severely damage the heart, both during Ischemia Reperfusion (IR) in acute myocardial infarction and during chronic remodeling after myocardial infarction. Reactive Oxygen Species (ROS) produced within mitochondria are specific drivers of mechanisms leading to IR damage, including induction of mitochondrial permeability transition or oxidative damage to structures and molecules within mitochondria.

Methylxanthines are molecular compounds which have therapeutic effects on chronic obstructive pulmonary disease, emphysema and chronic bronchitis, and have the effects of contracting bronchi, promoting airflow, relieving inflammation and relaxing airways, and studies have shown that methylxanthines also have antioxidant stress effects, e.g. caffeine is an effective scavenger of hydroxyl radicals and alkoxy groups, and can prevent cell damage by reducing lipid peroxidation, and its protective effects on alzheimer's disease and lens from photochemical damage by ROS have been demonstrated, while TA, which has a similar parent nucleus structure, therefore, has equivalent antioxidant stress effects, and for aldehyde safety, generally only α - β unsaturated aldehydes can form an adduct with DNA to produce toxic effects, e.g. 4-hydroxynonenal (4-HNE), but TA does not have the structural characteristics of the above-mentioned highly reactive aldehydes, therefore, TA has no toxicity associated with highly reactive aldehydes. The in vitro and in vivo anti-inflammatory effects of doxofylline have also been demonstrated. However, the prior art does not find that TA and xanthine aldehyde compounds thereof can generate the effect of resisting oxidative stress through aldehyde disproportionation reaction, and inflammatory injury of ROS excitation mechanism is almost an important process of all oxidative stress injuries regardless of the causes. The principle of the medicinal composition for treating oxidative stress organ injury such as acute ischemic myocardial infarction, myocardial ischemia-reperfusion injury, inflammatory diseases, asthma and the like caused by ROS is as follows: TA with aldehyde oxidase (ALDHs), NAD ⁺ The basic reaction is that xanthine aldehyde compounds (such as theophylline acetaldehyde and TA) are mainly subjected to neutralizationALDHs (e.g. ALDH1A1 and ALDH2) with NAD ⁺ Oxidative metabolism takes place to give the corresponding oxidation product (TAA). In this case, the xanthine aldehyde compound TA primarily acts as a hydrogen donor to transfer hydrogen to NAD ⁺ The above steps generate NADH, and further play an antioxidation role, which shows that the combination is an antioxidant stress medicine combination with great development potential.

Disclosure of Invention

The technical problem to be solved is as follows: in order to overcome the defects of the prior art, the invention develops a brand-new pharmaceutical composition with anti-oxidative stress effect which is completely different from the action mechanism of the prior xanthine drugs, and the composition can directly produce the anti-oxidant reduced Nicotinamide Adenine Dinucleotide (NADH); the antioxidant has effects of resisting asthma, resisting oxidation and relieving stress in vivo and in vitro models, and can be used for treating various related diseases, such as endogenous and exogenous asthma, bronchial asthma, airway epithelial injury and inflammatory asthma subtype with increased inflammatory factor, or diseases with high oxidative stress state, such as acute ischemic myocardial infarction, myocardial ischemia reperfusion injury, etc. Meanwhile, the formula of the composition is simplified, the side effect and the toxicity of the composition are reduced, and the drug effect of the composition is improved; in view of this, the invention provides a pharmaceutical composition containing TA or hydrate thereof, and a preparation method and application thereof.

The technical scheme is as follows: a pharmaceutical composition containing Theophylline Acetaldehyde (TA) or hydrate thereof comprises TA or hydrate thereof and Nicotinamide Adenine Dinucleotide (NAD) in a molar mass ratio of (1-2) to (0.5-1) to (2-5) ⁺ ) And aldehyde dehydrogenases (ALDHs);

wherein, the structural formula of the theophylline acetaldehyde is as follows:

the structural formula of the theophylline acetaldehyde hydrate is as follows:

the ALDHs are ALDH1A1 or ALDH 2.

The preparation method of the pharmaceutical composition containing TA or hydrate thereof adopts bromo-acetaldehyde dimethyl acetal to attack imidazole ring of theophylline electrophilically, and then obtains TA and hydrate thereof after demethylation by potassium bisulfate, and comprises the following specific steps:

synthesis of S1 and 7- (2, 2-dimethoxyethyl) -1, 3-dimethyl-3, 7-dihydro-1H-purine-2, 6-dione

Theophylline (TP) is used as a reaction substrate, the molar ratio of TP to bromoacetaldehyde dimethyl acetal is 1: 1-5, and the preferable ratio is 1: 1.3-1.6, wherein the molar ratio of TP to potassium carbonate is 1: 1-10, and the ratio is preferably 1: 2-3, wherein the molar ratio of TP to potassium iodide is 1: 0.1-3, preferably 1: 0.5-1, wherein the molar ratio of TP to triethylamine is 1: 1-6, preferably 1: 2-2.5, wherein the volume ratio of the mass of theophylline to N, N-Dimethylformamide (DMF) is 1 g: 3-30 mL, preferably 1 g: (8-10) mL, performing oil bath reaction, performing rotary evaporation to remove DMF, adding water and Dichloromethane (DCM) for extraction, washing, drying overnight, performing suction filtration, evaporating to obtain a brown solid, dispersing in diethyl ether, pulping, and performing suction filtration to obtain an off-white solid;

synthesis of S2 and (2, 2-dihydroxyethyl) -1, 3-dimethyl-3, 7-dihydro-1H-purine-2, 6-dione

Adding potassium bisulfate, acetone and water into the S1 product. The molar ratio of the S1 product to potassium bisulfate is 1: 2-15, preferably 1: 7-10, wherein the volume ratio of the mass of the S1 product to acetone is 1 g: 8-35 mL, preferably 1 g: (15-20) mL, wherein the mass-to-water volume ratio of the S1 product is 1 g: 5-50 mL, preferably 1 g: (7.5-10) mL, performing oil bath reaction, cooling, performing suction filtration, evaporating filtrate, and recrystallizing water to obtain a white solid;

s3, mixing TA or hydrate thereof, NAD + and ALDHs according to the molar mass ratio of (1-2) to (0.5-1) to (2-5) to prepare the pharmaceutical composition containing TA or hydrate thereof.

The application of the medicine and the medicine combination in preparing the anti-oxidative stress medicine comprises TA or hydrate thereof in a molar mass ratio of (1-2) to (0.5-1) to (2-5): NAD +: the ALDH1A1 recombinant single enzyme is mainly used for the application of ROS-induced oxidative stress organ injury, inflammatory diseases and asthma medicaments.

Preferably, the medicament and the medicament combination are used for preparingThe application of the antioxidant stress medicament comprises TA or hydrate thereof in a molar mass ratio of 2:1: 5: NAD (nicotinamide adenine dinucleotide) ⁺ : the ALDH1A1 recombinant single enzyme is mainly used for diseases of high oxidative stress state caused by ROS, such as acute ischemic myocardial infarction and myocardial ischemia-reperfusion injury.

Preferably, the pharmaceutical composition comprises theophylline acetaldehyde, NAD, as topical drops for the treatment of dry eye due to oxidative stress against water deficit, cataract due to oxidative stress ⁺ The ALDHs are mainly used for treating eye diseases caused by ROS, such as xerophthalmia and cataract, and the molar ratio of the ALDHs is (1-2): (0.5-1): 2-5).

Preferably, the application of the medicine and the medicine combination in preparing the anti-inflammatory medicine comprises the following components in parts by mole mass ratio (1-2): (0.5-1): 2-5) of TA or hydrate thereof: NAD (nicotinamide adenine dinucleotide) ⁺ : the ALDH1A1 recombinant single enzyme is mainly used for inflammatory diseases with raised TNF-alpha.

Preferably, the application of the medicine, the medicine combination and the combined medicine in preparing the anti-asthma medicine comprises the following components in a molar mass ratio of TA or hydrate thereof of 2:1: 5: NAD (nicotinamide adenine dinucleotide) ⁺ : the ALDH1A1 recombinant single enzyme is mainly used for treating recurrent wheezing, dyspnea, chest distress or genetically caused asthma.

Preferably, the drug or the drug combination is used for preparing drugs for reducing airway hyperreactivity, drugs for reducing the inflammatory degree of lung tissues and drugs for inhibiting the activation of lung mast cells, and is characterized by comprising TA or hydrates thereof in a molar mass ratio of 2:1: 5: NAD (nicotinamide adenine dinucleotide) ⁺ : the ALDH1A1 recombinant single enzyme is mainly used for diseases which are in an overreaction state due to airway inflammation, show hypersensitive bronchial smooth muscle contraction reaction, cause airway stenosis and airway resistance increase, and further cause symptoms such as cough, chest distress, dyspnea and wheezing, and the like, such as bronchial asthma.

Preferably, the application of the medicine, the medicine combination and the combined medicine in preparing the medicine for relieving the exogenous and endogenous allergic asthma is characterized by comprising the following components in a molar mass ratio of (0.5-1) 5: NAD (nicotinamide adenine dinucleotide) ⁺ : the ALDH1A1 recombinant single enzyme is mainly used for inducing allergen such as mite and pollenThe acute asthma and fever, hypodynamia and the like caused by infection, the symptoms can cause abnormal respiratory regulation, and finally, neurogenic inflammation occurs, thereby causing the attack of asthma.

Preferably, the application of the medicine, the medicine combination and the combined medicine in the medicines for inhibiting the IgE level in serum and the increase of the levels of cytokines IL-4, IL-5, IL-13 and the like is characterized by comprising TA or hydrate thereof in a molar mass ratio of 2:0.5: 5: NAD (nicotinamide adenine dinucleotide) ⁺ : the ALDH1A1 recombinant single enzyme is mainly used for treating airway epithelial injury and asthma subtype characterized by the increase of IL-4, IL-5, IL-13 and IgE level caused by Th2 cell activation and mast cell activation.

Preferably, the drug or drug combination is TA, NAD ⁺ And ALDH1A1, and pharmaceutically acceptable adjuvants.

Preferably, the pharmaceutical preparation is drops, cream, gel, tablet, capsule, spray, oral liquid or injection.

Preferably, the preparation method of the injection comprises the following steps: precisely weighing 1-10g of each compound, placing the compound in a beaker, adding 50mL of 50% ethanol, adding 70g of hydroxypropyl beta cyclodextrin, fully stirring the mixture until the mixture is completely dissolved, stirring the mixture for 1h, concentrating the mixture on a rotary evaporator under reduced pressure until the mixture is dried, and sieving the dried mixture with a 60-mesh sieve to obtain the compound, wherein the compound in the formula is independently prepared into a preparation with equal molar concentration, and the dosage is adjusted according to the required proportion when the compound is used.

Preferably, the preparation method of the oral solid preparation comprises the following steps: precisely weighing 1-10g of each pharmaceutical composition, placing in a beaker, adding 50mL of 50% ethanol, adding 7 times of hydroxypropyl-beta-cyclodextrin, stirring fully until the mixture is completely dissolved, stirring for 1h, concentrating on a rotary evaporator under reduced pressure until the mixture is dried, and sieving with a 80-mesh sieve to obtain the pharmaceutical composition. The compounds in the formula are independently prepared into preparations with equimolar concentration, and the dosage is adjusted according to the required proportion when the preparation is used. Weighing 80g of dry, crushed and sieved hydroxypropyl-beta-cyclodextrin, adding 50g of starch, 20g of microcrystalline cellulose and 2.5g of magnesium stearate, mixing uniformly,

preferably, the pharmaceutical formulation is an eye drop for topical application, and the pharmaceutical composition comprises TA, NAD ⁺ Catalytic heavyThe single enzyme group ALDH1A1, the mol ratio is (1-2): (0.5-1): 2-5); the composition is placed in a viscous vehicle comprising a deionized water solution, 0.01% ethylenediaminetetraacetic acid, sodium chloride and sodium hydroxide to adjust the viscosity and pH of the eye drops to 7.4, 1% ascorbic acid, which acts as an antioxidant preservative for the mixture.

Has the advantages that: (1) the pharmaceutical composition increases the generation of intracellular antioxidant NADH through xanthine aldehyde metabolic reaction, further inhibits the generation and development of oxidative stress through multiple paths, can be applied to the field of preparing medicaments for treating high oxidative stress organ injury caused by ROS, such as myocardial ischemia reperfusion injury, eye diseases, inflammation, asthma and the like, enriches the types of preparing relevant medicaments for resisting oxidative stress, inflammation and the like, and provides brand new selection and strategy for preparing medicaments for treating oxidative stress organ injury and inflammatory diseases; (2) the pharmaceutical composition has simple components, low toxicity and side effects.

Drawings

FIG. 1 shows TA and its hydrates ¹ HNMR spectrogram

FIG. 2 is a toxicity evaluation of TA on HepG2 cells, and the results show that 5-60. mu.M of TA had no effect on the survival rate of HepG2 cells within 48 h.

FIG. 3 shows equimolar TA and NAD ⁺ Or NAD ⁺ The combination of ALDH1A1(1: 1:1) can obviously increase the content of NADH in cells.

FIG. 4 is an evaluation of the anti-inflammatory activity of TA, indicating that TNF- α was significantly elevated in the LPS group, indicating model success, while 10 μ M TA +20 μ M NAD ⁺ The TNF- α release was significantly reduced in the +20 μ M ALDH1A1 group, indicating TA, NAD ⁺ The combination of ALDH1A1(1:2:2) inhibited LPS-induced TNF- α release.

FIG. 5 is an evaluation of the antioxidant stress effect of TA in combination with NAD + or NAD +, ALDH1A1 for H inhibition ₂ O ₂ Induced intracellular ROS content.

FIG. 6 is the result of detecting IgE in serum of each administration group of asthmatic rats, which indicates that IgE in the model group is obviously increased and IgE in the administration group is reduced, indicating that TA and NAD ⁺ Or NAD ⁺ The combination of ALDH1A1 can be used for inhibiting asthmaIgE release in rat serum

FIG. 7 shows the results of detection of IL-5 in serum of each administration group of asthmatic rats, indicating TA and NAD ⁺ Or NAD ⁺ The combination of the ALDH1A1 can obviously inhibit the release of IL-5 in the serum of asthmatic rats.

FIG. 8 shows the results of detection of IL-5 in alveolar lavage fluid of each administration group of asthmatic rats, indicating TA and NAD ⁺ Or NAD ⁺ The combination of ALDH1A1 can obviously inhibit the release of IL-5 in BALF of asthmatic rats.

FIG. 9 shows the results of detection of corneal epithelial cell apoptosis in each administration group of xerophthalmia mice, which indicates TA and NAD ⁺ The combination of the alpha-glucosidase and ALDH1A1 recombinant single enzyme (1-2) (0.5-1) (2-5) can inhibit xerophthalmia of asthmatic mice caused by oxidative stress.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

The pharmaceutical composition comprises (1-2) by molar mass, (0.5-1) by molar mass, (2-5) by molar mass, theophylline acetaldehyde or hydrate thereof, and NAD ⁺ And ALDHs;

wherein, the structural formula of the theophylline acetaldehyde is as follows:

the structural formula of the theophylline acetaldehyde hydrate is as follows:

the ALDHs are ALDH1A1 or ALDH 2.

Adding potassium bisulfate, acetone and water into the S1 product. The molar ratio of the S1 product to potassium bisulfate is 1: 2-15, preferably 1: 7-10, wherein the volume ratio of the mass of the S1 product to acetone is 1 g: 8-35 mL, preferably 1 g: (15-20) mL, wherein the mass-to-water volume ratio of the S1 product is 1 g: 5-50 mL, preferably 1 g: (7.5-10) mL, performing oil bath reaction, cooling, performing suction filtration, evaporating filtrate, and recrystallizing water to obtain a white solid. The relevant information on the hydrogen spectrum is as follows: 1H-NMR (400MHz, CDCl3), δ:7.95(s,1H),6.17(s,2H),5.05(t, J ═ 4.8Hz,1H),4.16(d, J ═ 5.4Hz,2H),3.43(s,3H),3.23(s,3H). the spectrum is shown in fig. 1.

Example 2

The pharmaceutical composition of example 1 can significantly increase intracellular NADH content to achieve antioxidant stress or anti-inflammatory effects, with the following results:

(1) toxicity evaluation of TA (CCK8 experiment)

HepG2 cells were counted at 5X10 ⁴ Spreading in 96-well plate at a density of one/mL, culturing for 24 hr, washing with sterile PBS to remove the culture medium, and adding 5-60 μ M basic culture mediumTA solution, and after adding medicine for 48h, detecting the cell survival rate by using CCK8, and evaluating the toxicity of theophylline acetaldehyde.

(2) The combined use of TA, NAD + and ALDH1A1 recombinant single enzymes can increase the intracellular NADH content

HepG2 cells were counted at 5X10 ⁵ Plating the cells/mL in a 6-well plate, culturing for 24h, washing the complete medium with sterile PBS, adding 100. mu.M TA and 100. mu.M NAD in a basal medium ⁺ 100 mu M ALDH1A1 and the mixed drug solution of the three, and NAD is adopted after adding drugs for 48 hours ⁺ NADH in cells is extracted by an NADH detection kit (Biyunyan, S0175) and the content of the NADH is detected.

(3) Evaluation of anti-inflammatory Activity of TA drug combinations

RAW264.7 cells were counted as 1X10 ⁶ Spreading the culture medium in 6-well plate at a density of one/mL, culturing for 4 hr, replacing basic culture medium, culturing for 2 hr, adding 10 μ M TA and 20 μ M NAD ⁺ 20 μ M ALDH1A1 was cultured for 4 hours, after 4 hours, LPS (final concentration: 100ng/mL) was added to each fraction, cells were cultured for 4 hours, cell culture supernatant was collected, and TNF-. alpha.release level in the supernatant was measured using Elisa kit

(4) TA and NAD ⁺ Or NAD ⁺ The combination of ALDH1A1 can reduce the content of ROS in cells

HepG2 cells were counted at 5X10 ⁵ The cells were plated in 6-well plates at a density of one cell/mL, and after 24 hours of culture, the complete medium was washed with sterile PBS, and 200. mu.M TA and 200. mu.M NAD were added to the medium prepared in the basal medium ⁺ 600. mu.M H ₂ O ₂ 、200μM TA+600μM H ₂ O ₂ 、200μM NAD ⁺ +600μM H ₂ O ₂ 、200μM TA+200μM NAD ⁺ +600μM H ₂ O ₂ And 200. mu.M TA + 200. mu.M NAD ⁺ +200μM ALDH1A1+600μM H ₂ O ₂ And after adding the medicine for 48 hours, detecting the ROS content in the cells by adopting a fluorescent probe DCFH-DA.

Example 3 TA and NAD ⁺ Or NAD ⁺ ALDH1A1 combination for treatment of asthmatic rats

Adaptive feeding of ratsNourishing for 1 week, numbering from small to large according to body constitution, and completely randomly classifying into normal group, model group, TA group, and NAD ⁺ Group, model + TA aldehyde group, model + TA + NAD ⁺ Group, model + ALDH1A1, TA + NAD ⁺ Groups of 10 each.

Asthma models were prepared at 1d and 8d of the official start experiment by administering model group, TA group, NAD ⁺ Group, model + TA + NAD ⁺ Group rats were sensitized by intraperitoneal injection of 1mL of Ovalbumin (OVA) mixture (OVA 100mg, aluminum hydroxide 100mg plus physiological saline 1 mL) and 2 weeks later (day 15), the rats were placed in a transparent closed container and inhaled by 2% OVA nebulization for 15min to induce asthma attack, 1 time every other day to induce asthma for 2 weeks, 1mL of physiological saline was used for intraperitoneal injection in the normal group, 15min by physiological saline nebulization and inhalation in the day 15, 1 time every other day to induce asthma, and the same amount of physiological saline was intravenously injected every day, TA group and NAD group in the normal group and the model group, respectively ⁺ Group, model + TA + NAD ⁺ Group TA or TA and ALDH1A1, NAD were given 30min before each OVA challenge ⁺ The composition solution was injected into the tail vein (TA dose: 30mg/kg, NAD) ⁺ Dosage: 30mg/kg, ALDH1A1 dose: 60 mg/mL). Rats were sacrificed 24 hours after the last challenge to study TA and ALDH1A1, NAD ⁺ The composition has therapeutic effect on asthma rat.

After the last atomization excitation for 24h, 10% chloral hydrate (10mL/kg) is injected into the abdominal cavity to anaesthetize the rat, the orbital vein is taken out of blood, after standing, the blood is centrifuged at 4 ℃ (3000rpm/min) for 10min, the serum is separated, and the blood is placed at-80 ℃ for standby after being subpackaged. Laryngoscope and trachea cannula, fixing the laryngoscope and lung function instrument, determining lung function, the result is shown in table 1, the lung function result shows that the OVA model group rats I (airway hyperresponsiveness), Rn (central airway resistance) and G (tissue damping) are all increased compared with the Control group, and TA and NAD are given ⁺ Or with ALDH1A1, while Crs (dynamic compliance) did not change significantly between groups, indicating TA and NAD ⁺ Or ALDH1A1, NAD ⁺ The combination of (a) significantly improves airway hyperresponsiveness while reducing central airway resistance and tissue damping.

TABLE 1 pulmonary function test results in rats

Alveolar Lavage Fluid (BALF) was collected from each group of rats, and the content of cytokines in BALF was measured by ELISA. Rat orbital blood was collected and the content of cytokine in serum was measured by ELISA. The results of IgE detection in serum of each administration group are shown in FIG. 6, which shows that IgE in the model group is obviously increased, and IgE in the administration group is reduced, which shows that TA and NAD are increased ⁺ Or NAD ⁺ The combination of ALDH1A1 can inhibit the release of IgE in the serum of asthmatic rats. The results of IL-5 detection in serum and BALF of each administration group are shown in FIGS. 7 and 8, indicating TA and NAD ⁺ Or NAD ⁺ The combination of ALDH1A1 can obviously inhibit the release of IgE in the serum and BALF of asthmatic rats.

Example 4 treatment of dry eye mice with TA eyedrops

The mice are fed adaptively for 1 week, 60 mice are numbered according to the body mass from small to large, and are divided into a blank control group, a blank TA group, a blank NAD + group, a DED model + NAD + group, a DED model + TA group and a DED model + TA eye drop group by a completely random method, wherein each group comprises 3 mice. (wherein TA: 200. mu. M, NAD ⁺ 200 mu M, ALDH1A1 single enzyme 0.05mg/mL)

The DED mouse model was prepared at 1w and 6w of the official start experiment, and benzalkonium chloride solution with a concentration of 0.2% was added dropwise to both eyes (5 μ L per eye, 1 time per day, 6 weeks after continuous eye drop) in the DED model group, the DED model + TA group, the DED model + NAD + group, and the DED model + TA eye drop group, and the amount of tear secretion of the mouse was measured with tear detection filter paper to evaluate the success or failure of the dry eye model.

After the model is successful, blank matrix solution is given to the blank control group, and blank matrix solution + TA and blank NAD are given to the blank TA group ⁺ Blank matrix solution + NAD ⁺ DED model group administration of blank matrix solution, DED model + NAD ⁺ Group administration of blank matrix solution and NAD ⁺ The DED model + TA group was dosed with the blank matrix solution and TA, and the DED model + TA eye drop group was dosed with TA eye drops (all experiments, 5 μ L per eye, 3 times daily for 6 consecutive days).To avoid errors due to evaporation of the test reagents, all test reagents were applied directly to the corneal apex by pipette. After sacrifice, the cornea was dissected and tissue samples collected to assess histological changes to drug treatment. While dry eye disease caused by oxidative stress and inflammation leads to increased tear osmolality and then to apoptosis of corneal epithelial cells, we found experimentally that apoptosis of corneal epithelial cells was also significantly reduced after treatment with TA eye drops in the DED model compared to the blank group (the proportion of nuclear staining of TUNEL was reduced compared to the DED group). See figure 9 for details

Asterisks indicate statistically significant differences between the treatment groups and the DED group (. p <0.05,. p <0.005,. p < 0.001).

Claims

1. A pharmaceutical composition containing theophylline acetaldehyde or hydrate thereof is characterized by comprising TA or hydrate thereof and NAD in a molar mass ratio of (1-2) to (0.5-1) to (2-5) ⁺ And ALDHs;

wherein, the structural formula of TA is as follows:

the structural formula of the TA hydrate is as follows:

the ALDHs are ALDH1A1 or ALDH 2.

2. The method for preparing the pharmaceutical composition containing theophylline acetaldehyde or its hydrate according to claim 1, wherein bromo-acetaldehyde dimethyl acetal is used to attack electrophilic imidazole ring of theophylline, and then potassium bisulfate is used to remove methyl group to obtain TA and its hydrate, and the specific steps are as follows:

Taking TP as a reaction substrate, wherein the molar ratio of TP to bromoacetaldehyde dimethyl acetal is 1: 1-5, the molar ratio of TP to potassium carbonate is 1: 1-10, the molar ratio of TP to potassium iodide is 1: 0.1-3, the molar ratio of TP to triethylamine is 1: 1-6, the volume ratio of theophylline to DMF is 1 g: 3-30 mL, performing oil bath reaction, performing rotary evaporation to remove DMF, adding water and DCM for extraction, washing, drying overnight, performing suction filtration, evaporating to dryness to obtain a brown solid, dispersing in diethyl ether, pulping, and performing suction filtration to obtain an off-white solid;

Adding potassium bisulfate, acetone and water into the S1 product, wherein the molar ratio of the S1 product to the potassium bisulfate is 1: 2-15, wherein the volume ratio of the mass of the S1 product to the volume of acetone is 1 g: 8-35 mL, wherein the volume ratio of the mass of the S1 product to water is 1 g: 5-50 mL, performing oil bath reaction, cooling, performing suction filtration, evaporating filtrate, and recrystallizing water to obtain a white solid;

s3, mixing TA or its hydrate, NAD according to the molar mass ratio (1-2): (0.5-1): 2-5) ⁺ And ALDHs, and preparing the pharmaceutical composition containing TA or hydrate thereof.

3. The method for preparing a pharmaceutical composition comprising theanine acetaldehyde or its hydrate as claimed in claim 2, wherein the molar ratio of TP to bromoacetaldehyde dimethyl acetal in S1 is 1: 1.3-1.6, wherein the molar ratio of TP to potassium carbonate is 1: 2-3, wherein the molar ratio of TP to potassium iodide is 1: 0.5-1, wherein the molar ratio of TP to triethylamine is 1: 2-2.5, wherein the volume ratio of the mass of theophylline to DMF is 1 g: 8-10 mL.

4. The process for preparing a pharmaceutical composition comprising theophylline acetaldehyde or hydrate thereof as claimed in claim 2, wherein the molar ratio of the S1 product to potassium bisulfate is 1: 7-10, wherein the volume ratio of the mass of the S1 product to the volume of acetone is 1 g: 15-20 mL, wherein the volume ratio of the mass of the S1 product to water is 1 g: 7.5-10 mL.

5. Use of the pharmaceutical composition comprising theophylline acetaldehyde or a hydrate thereof according to claim 1 for the preparation of a medicament for the treatment of ROS-induced oxidative stress organ damage, inflammatory diseases and asthma.

6. The use according to claim 5, wherein the ROS-induced oxidative stress organ injury is myocardial ischemia reperfusion injury, or oxidative stress-induced water-deficient dry eye and cataract in which TA or its hydrate, NAD ⁺ And ALDHs in a molar mass ratio of 2:1: 5.

7. Use according to claim 5, wherein the inflammatory disease is an inflammatory disease with elevated TNF- α, when TA or hydrate thereof, NAD ⁺ And ALDHs in a molar mass ratio of 1-2: 0.5-1: 2-5.

8. Use according to claim 5, wherein the asthma is bronchial asthma or endogenous or exogenous asthma when TA or hydrate thereof, NAD ⁺ And ALDHs in a molar mass ratio of 1-2: 0.5-1: 5.

9. The use of claim 5, wherein the medicament is a pharmaceutical formulation prepared by mixing TA, NAD +, ALDHs, and pharmaceutically acceptable excipients.

10. The use according to claim 9, wherein the pharmaceutical formulation is drops, creams, gels, tablets, capsules, sprays, oral liquids and injections.