Edaravone and novel injection for synthesizing (+)-2-borneol and preparation method thereof

Abstract

The invention discloses a novel injection containing edaravone and high-purity synthesized (+)-2-borneol, because the (+)-2-borneol is prepared by a chemical synthesis process, the curative effect of the (+)-2-borneol is better than that of natural borneol and (+)-2-borneol extracted from the natural borneol under the same dosage, the toxic and side effects can be reduced, and the product quality is more stable; and the established GC-MS analysis method can more accurately control the content of each coexisting substance and better guarantee the shelf life quality. The injection is more suitable for improving the treatment effect on central nervous system diseases, oxidative stress injuries such as diabetes, retinopathy, diabetic peripheral neuropathy and neurodegenerative diseases and thrombus-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a novel injection composition of edaravone and high-purity synthetic (+)-2-borneol and a preparation method thereof. Background Art

[0002] Acute atherosclerotic cerebral infarction (ACI) is a common ischemic stroke in clinical practice. Current treatments mainly include ultra-early thrombolytic therapy and secondary prevention of antiplatelet aggregation. [2] Edaravone is a neuroprotective agent recommended for the treatment of acute ischemic stroke [3] Edaravone, also known as 3-methyl-1-phenyl-2-pyrazol-5-one, is a brain protective agent with a chemical formula of C 10 H 10 N2O is mainly used as a free radical scavenger. It has the advantages of wide distribution, short half-life, safety, and low toxicity. It is an effective first-line treatment for clinical ischemic stroke. It also has the effect of treating oxidative stress damage and thrombosis-related diseases such as diabetic retinopathy, diabetic peripheral neuropathy, and neurodegenerative diseases. [2] .

[0003] Natural borneol is a commonly used Chinese medicine. It is made from fresh branches and leaves of the Lauraceae plant Cinnamomum camphora, which is included in the Chinese Pharmacopoeia. It contains dextrorotatory borneol (C 10 H 18 O) shall not be less than 96.0% [7] .

[0004] Research shows [6] Natural borneol has a strong anti-inflammatory effect, which may be through inhibiting the activation of transcription factor NF-κB, inhibiting the expression of inflammatory proteins (iNOS and COX-2) and the release of inflammatory cytokines (TNF-α, IL-1β, etc.), thus protecting cells. One of the main components of natural borneol is (+)-2-borneol. [7] .

[0005] CN101848711B discloses a pharmaceutical composition of edaravone and borneol, indicating that the two active ingredients have a synergistic effect and good efficacy in treating cerebrovascular diseases. [4] , which was approved for marketing by the National Medical Products Administration (NMPA) in 2020 for the treatment of acute ischemic stroke. It contains two ingredients, edaravone and dexbrominol, mixed in a ratio of 4:1, and has antioxidant and anti-inflammatory effects [5] The dextroborneol in it is extracted from natural borneol. Phase III clinical trials have confirmed the efficacy of edaravone and dextroborneol in the treatment of acute ischemic stroke. [1] There are studies[3] Previous studies have shown that inflammatory response and oxidative stress are involved in the pathogenesis of ACI and affect the prognosis. [6] It has been shown that edaravone and dextroborneol can exert multi-target neuroprotective effects by reducing the expression of reactive oxygen free radicals (ROS), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), etc. [8-9] The results showed that human chitinase protein 40 (YKL-40) and human soluble CD40 (sCD40) played an important role in the oxidative stress and inflammatory response of ACI.

[0006] As an oxygen free radical scavenger, edaravone can reduce the damage of neurons and endothelial cells in ACI by inhibiting neurotoxicity and chronic inflammation, regulating the expression of endothelial and neuronal proteins, and reducing brain edema, thereby inhibiting delayed neuronal death and improving the prognosis of ischemic stroke. Dextroborneol has a neuroprotective effect in ischemia-reperfusion injury, such as reducing the generation of reactive oxygen species, inhibiting oxidative stress and reducing cell apoptosis. In addition, its high permeability can help other drugs easily penetrate the blood-brain barrier, thereby exerting their effects more effectively. [1，10] There are studies [1] Studies have shown that the treatment of ACI with edaravone and dextroborneol can reduce inflammatory response, inhibit oxidative stress, reduce serum free radical levels, and improve the degree of neurological deficit in ACI patients. Therefore, edaravone and dextroborneol, as a new type of neuroprotective agent, are believed to protect ischemic brain injury by reducing toxicity, oxidative stress, inflammatory response, apoptotic damage and other cell protection pathways [1]. The chemical structures of (+)-2-borneol and edaravone are as follows:

[0007]

[0008] However, adverse reactions such as abnormal liver function, jaundice, acute renal failure, thrombocytopenia, rash, and flushing have occurred in the clinical use of edaravone and dexamethasone injection. [1] ; Quality instability often occurs, such as the solution color tends to turn yellow, coexistence III and IV will be produced, and impurities grow rapidly during the stability period, and the solution color becomes darker. Therefore, there is an urgency in this field to further improve the quality of this product and ensure the safety of clinical medication. Summary of the invention

[0009] The invention discloses a novel injection of edaravone and high-purity synthetic (+)-2-borneol, wherein the synthetic (+)-2-borneol is obtained by chemical synthesis. The inventor unexpectedly found in the study that the injection prepared by using the fully chemically synthesized (+)-2-borneol and edaravone can significantly improve the symptoms of brain water content caused by ischemia-reperfusion, and has advantages over the combined use of edaravone and (+)-2-borneol extracted from natural borneol; the toxicity to the kidneys of rabbits: "edaravone / (+)-2-borneol prepared by extracting natural borneol 4:1" is greater than "edaravone / chemically synthesized (+)-2-borneol 4:1"; not only that, during the stability investigation of the injection, the coexistent III and IV also increase more slowly than the injection prepared by (+)-2-borneol extracted from natural borneol and edaravone, and the phenomenon of the solution color becoming darker is also well controlled. According to the present invention, it is expected that under the same dosage conditions, the use of chemically synthesized (+)-2-borneol will achieve better therapeutic effects than (+)-2-borneol extracted from natural borneol, and will have better synergistic effects when used in combination with edaravone, thereby improving the effects of treating oxidative stress damage and thrombosis-related diseases such as cerebrovascular diseases, diabetes, retinopathy, diabetic peripheral neuropathy, and neurodegenerative diseases.

[0010] The chemical structural formulas of coexistents I to VI are:

[0011]

[0012] In some embodiments, the present invention provides a novel injection solution of edaravone and high-purity synthetic (+)-2-borneol:

[0013] (1) The active ingredients of the injection are edaravone and synthetic (+)-2-borneol, and further contain one or more of coexistent substances I, II, III, and IV;

[0014] wherein the content of edaravone is 0.2-3 mg / ml (W / V), the content of synthetic (+)-2-borneol is 0.05-2 mg / ml (W / V); the content of coexistent I does not exceed 0.10% of the labeled amount of synthetic (+)-2-borneol, the content of coexistent II does not exceed 0.75% of the labeled amount of synthetic (+)-2-borneol, the content of coexistent III does not exceed 0.90% of the labeled amount of edaravone; and the content of coexistent IV does not exceed 0.25% of the labeled amount of edaravone;

[0015] (2) The inactive ingredients of the injection solution further include a cosolvent, an antioxidant and water for injection, wherein the amount of the cosolvent is 0.01 ml / ml to 0.2 ml / ml, and the amount of the antioxidant is 0.1 mg / ml to 1.5 mg / ml;

[0016] (3) The pH value of the injection solution is between 3.0 and 5.0;

[0017] (4) Sterilization process for injection: When the sterilization temperature is 115±3℃, the sterilization time is 30 to 48 minutes; or when the sterilization temperature is 121±3℃, the sterilization time is 8 to 16 minutes.

[0018] In some embodiments, the purity of the synthetic (+)-2-borneol is above 98%, the residual solvent n-heptane content in the synthetic (+)-2-borneol is ≤0.1%, the content of coexistent I is ≤0.15%, the content of coexistent II is ≤0.5%, the content of coexistent V is ≤0.1%, and the content of coexistent VI is ≤0.15%. The starting material of the synthetic route of the synthetic (+)-2-borneol can be α-pinene or D-camphor.

[0019] In some embodiments, the synthesis route of the (+)-2-borneol can be as shown in Formula 1:

[0020]

[0021] The synthesis steps are as follows: α-pinene is used as a starting material, oxalic acid is used to prepare an intermediate S1, and then the ester group is removed to obtain S2, S2 is subjected to derivatization and purification to obtain S3, and the ester group of S3 is removed to obtain (+)-2-borneol;

[0022] In formula S3, R is

[0023] It should be noted that the source of the chemically synthesized (+)-2-borneol of the present invention is not limited to the above-mentioned synthetic route, and is only illustrated here based on the actual research of the inventors to ensure that the present invention has better practicality. In fact, the (+)-2-borneol obtained by the semi-synthetic process route of several steps of chemical reactions using D-camphor as the starting material can also obtain the same effect under the premise of meeting the relevant quality requirements of the present invention.

[0024] In some embodiments, in the injection, the cosolvent is one or a combination of ethanol, propylene glycol, isopropanol, or polyethylene glycol, and the amount of the cosolvent is 0.01 ml / ml to 0.2 ml / ml, among which ethanol and propylene glycol are preferred, and the amount is preferably 0.01 ml / ml to 0.1 ml / ml.

[0025] In some embodiments, in the injection, the antioxidant is one or a combination of sodium sulfite, sodium metabisulfite, sodium thiosulfite, and vitamin C, among which sodium sulfite, sodium metabisulfite, sodium thiosulfite, and sodium thiosulfate can all achieve good antioxidant effects. The amount of the antioxidant is 0.1 mg / ml to 1.5 mg / ml, preferably 0.1 mg / ml to 1.2 mg / ml.

[0026] In some embodiments, the injection solution further comprises an isotonicity regulator, which comprises one or a combination of sodium chloride, potassium chloride, and glucose, preferably sodium chloride and glucose.

[0027] In some embodiments, in the injection, the preparation method comprises the following steps:

[0028] (1) Open the valves of the liquid distribution pipeline and the filling dispenser, introduce nitrogen (pressure control 0.02MPa ~ 0.10MPa), and continuously fill all liquid distribution pipelines, filters, and dispensers with nitrogen for 10 minutes to deoxygenate. After the nitrogen filling is completed, close the valves of all delivery pipelines and filters starting from the dispenser end, and maintain the nitrogen pressure for standby use;

[0029] (2) adding a cosolvent to the liquid preparation system, and after nitrogen filling below the liquid surface is used to control the dissolved oxygen to meet the requirement, adding edaravone, and after the dissolution is completed, closing the nitrogen filling below the liquid surface, opening the nitrogen filling above the liquid surface, and adding synthetic (+)-2-borneol to dissolve;

[0030] (3) After the dissolution is completed, add part of the water for injection, add the prescribed amount of antioxidant into the tank, continue to fill with nitrogen for protection, and after the dissolution, test the pH value. If necessary, add a pH adjuster to adjust the pH of the solution, add an isotonic adjuster to adjust the osmotic pressure of the solution, and then make up the volume;

[0031] (4) filtering, nitrogen filling, and terminal sterilization to obtain the injection solution.

[0032] In some embodiments, in the injection solution, the terminal sterilization adopts hot pressure sterilization, when the sterilization temperature is 115±3°C, the sterilization time is 30 to 48 minutes; or when the sterilization temperature is 121±3°C, the sterilization time is 8 to 16 minutes.

[0033] In some embodiments, in the injection solution, the pipeline is filled with nitrogen before preparation, the dissolved oxygen is controlled at 4.0 mg / L during liquid preparation, and the residual oxygen in the headspace is controlled to be below 3.0% during filling.

[0034] In some embodiments, the present invention provides, in addition to providing 5 ml small injections of edaravone and high-purity synthetic (+)-2-borneol, a class of intravenous drip solutions (also known as large infusions or large-volume injections) of edaravone and high-purity synthetic (+)-2-borneol. In addition to the aforementioned advantages of these small injections, this class of large infusions is more secure than marketed products: marketed products are concentrated solutions for injection, and in clinical use, three vials are required each time, cracked open, drawn, transferred to 100 ml of normal saline for dilution, and then intravenously dripped, and the dripping is completed within 30 minutes, twice a day, for 14 consecutive days of treatment.

[11] It can be seen that the dilution process is complicated and prone to microbial contamination, and a large amount of glass fragments will be generated during the opening of the glass ampoule, which will cause great safety risks after entering the vein. The large infusion product of the present invention does not need to be diluted and can be used directly, effectively avoiding the risks of microbial contamination and glass fragments.

[0035] The present invention also studies and establishes a more sensitive and exclusive detection method for the coexisting substances I, II, III, and IV that may exist in the product, so as to ensure that the (+)-2-borneol prepared by the chemical synthesis method has higher quality characteristics and is more in line with the requirements of the injection of the present invention, and to ensure the release quality and shelf life of the novel injection of edaravone and synthetic (+)-2-borneol of the present invention: the detection method for the content of coexisting substances I and II is the GC-MS method, the impurity reference substance external standard method is used for quantitative determination, and the programmed temperature is used for detection. The detector is a mass spectrometer, the injection port temperature is 250-300°C, the carrier gas is helium, the ion source is an EI source, the ion source temperature is 230°C, the selected ion detection (SIM), the quantitative ion is 95, the electron energy is 70eV, and the flow rate is 2-2.5ml per minute; the content detection method of coexistent III and coexistent IV adopts high performance liquid chromatography, self-control method or main component external standard method for quantitative, octadecylsilane bonded silica gel is used as a filler, gradient elution mode is adopted, and the detection wavelength is 248nm. The accuracy of the detection of each coexistent is guaranteed, the quality of (+)-2-borneol prepared by chemical synthesis and the quality of edaravone and synthetic (+)-2-borneol novel injection of the present invention are more accurately controlled, and the creativity of the present invention is further guaranteed.

[0036] The camphor mentioned herein refers to L-camphor, D-camphor and a mixture thereof, and the labeled amount refers to the content of the main drug specified in the preparation of the dosage form per unit dose. DETAILED DESCRIPTION

[0037] The present invention is further described below with reference to examples, but these examples are not limiting. Unless otherwise specified, the experimental solvents or reagents used in the following examples were purchased from the commercial market.

[0038] Example 1

[0039] Synthesis of (+)-2-borneol:

[0040]

[0041] Step 1: Preparation of S1

[0042] Weigh α-pinene (136.2 g, 1.0 mol), add 1000 ml of dry tetrahydrofuran, slowly add anhydrous oxalic acid (13.5 g, 0.15 mol), anhydrous boric anhydride (104.7 g, 1.5 mol) and acetic anhydride (5.2 g, 0.05 mol) under ice bath, heat to 60°C for reaction, add anhydrous oxalic acid (13.5 g, 0.15 mol) every 6 hours, for a total of three times, after the reaction is completed, slowly add 60°C hot water, continue stirring for 30 minutes, stand for 30 minutes and then separate the liquids, discard the aqueous layer, concentrate the organic layer at 40°C for 1 hour, and then concentrate at 55°C for 1 hour, and the residue is directly used in the next step.

[0043] Step 2: Preparation of S2

[0044] The above residue was added to 500 ml of sodium hydroxide (0.02 M) solution, and 500 ml of ethanol was added, and then heated to 50° C. for reaction for 10 hours. After the reaction was complete, distillation was performed, and the fractions were collected and filtered to obtain 26.2 g of crude product. The two-step yield was 17.1%.

[0045] Step 3:

[0046] Take 25.0 g of the above crude product and add it to 50 mL of n-hexane, heat to reflux until the product is completely dissolved, add 20 mL of water and continue stirring for 30 minutes, cool to room temperature, separate the aqueous layer, place the n-hexane solution in an ice bath for crystallization for 7 days, filter the resulting solid and add it again to 25 mL of n-hexane, heat to reflux until the product is completely dissolved, add 10 mL of water and continue stirring for 30 minutes, cool to room temperature, separate the aqueous layer, place the n-hexane solution in an ice bath for crystallization for 7 days, filter, and the resulting solid is placed in the shade at room temperature to obtain 3.4 g of (+)-2-borneol, with a yield of 13.6%.

[0047] Step 4: Preparation of S3

[0048] Take 25.0g of the above crude product and add it to 125mL of xylene, then add 24g of phthalic anhydride and 19.8g of N,N-dimethylaminopyridine, stir at room temperature for 24h, add 125mL of 5% sodium hydroxide aqueous solution, stir and separate, extract the water layer with 125mL of xylene, adjust the pH of the water layer to 4-5 with 1M hydrochloric acid, precipitate solid, filter, and dry to obtain 22g of white solid. Dissolve the above white solid in 220mL of chloroform, add 11.8g of quinine, reflux for 2h, concentrate to remove chloroform, add 220mL of ethyl acetate for recrystallization, and obtain 8g of white solid. Use it directly in the next step.

[0049] Step 5: Preparation of (+)-2-borneol

[0050] The white solid was dissolved in dichloromethane, washed three times with 1M hydrochloric acid, the aqueous phase was discarded, the dichloromethane phase was spin-dried and 100 mL of ethanol was added to dissolve it, and then 100 mL of sodium hydroxide aqueous solution (10%) was added, and the reaction was refluxed until completion, and the ethanol was concentrated to remove the ethanol, and the solid was filtered. The obtained solid was placed at room temperature and dried in the shade to obtain 3.8 g of (+)-2-borneol, with a two-step yield of 15.2%.

[0051] The (+)-2-borneol in the prepared synthetic (+)-2-borneol has a purity of more than 98%, a content of coexisting substance I of ≤0.15%, a content of coexisting substance II of ≤0.5%, a content of coexisting substance V of ≤0.1%, a content of coexisting substance VI of ≤0.15%, and a content of n-heptane of ≤0.1%.

[0052] Example 2

[0053] Weigh 2.5 g of synthetic (+)-2-borneol, add 400 ml of propylene glycol and an appropriate amount of 5 g of sodium bisulfite, stir to completely dissolve, slowly add water for injection to dissolve and add to 5000 ml to obtain the product.

[0054] Example 3

[0055] Weigh 10 g of edaravone, add 400 ml of propylene glycol and an appropriate amount of 5 g of sodium bisulfite, stir to completely dissolve, slowly add water for injection to dissolve and add to 5000 ml to obtain the product.

[0056] Example 4

[0057] Weigh 10 g of edaravone and 2.5 g of (+)-2-borneol prepared by extracting natural borneol, add 400 ml of propylene glycol and 5 g of appropriate amount of sodium bisulfite, stir to completely dissolve, slowly add water for injection to dissolve and add to 5000 ml to obtain.

[0058] Example 5

[0059] Weigh 10 g of edaravone and 1 g of synthetic (+)-2-borneol, add 400 ml of propylene glycol and an appropriate amount of 5 g of sodium bisulfite, stir to completely dissolve, slowly add water for injection to dissolve and add to 5000 ml to obtain the product.

[0060] Example 6

[0061] Weigh 10 g of edaravone and 2.5 g of synthetic (+)-2-borneol, add 400 ml of propylene glycol and 5 g of appropriate amount of sodium bisulfite, stir to completely dissolve, slowly add water for injection to dissolve and add to 5000 ml to obtain the product.

[0062] Example 7

[0063] Weigh 1g of edaravone and 10g of synthetic (+)-2-borneol, add 400ml of propylene glycol and 5g of appropriate amount of sodium bisulfite, stir to completely dissolve, slowly add water for injection to dissolve and add to 5000ml to obtain the product.

[0064] Example 8

[0065] Weigh 1.5 g of edaravone, 0.375 g of synthetic (+)-2-borneol, add 60 ml of propylene glycol, 0.75 g of appropriate amount of sodium metabisulfite, and 45 g of sodium chloride, stir to completely dissolve, slowly add water for injection to dissolve and add to 5750 ml to obtain.

[0066] Example 9

[0067] Weigh 1.5 g of edaravone and 0.375 g of synthetic (+)-2-borneol, add 400 ml of propylene glycol, 5 g of appropriate amount of sodium metabisulfite, and 45 g of sodium chloride, stir to completely dissolve, slowly add water for injection to dissolve and add to 5000 ml to obtain the product.

[0068] The preparation process of the above-mentioned embodiments 2 to 9 also includes the steps of liquid filtration, nitrogen filling, filling, sterilization, etc. The specific steps are as follows:

[0069] (1) Open the valves of the liquid distribution pipeline and the filling dispenser, and introduce nitrogen (pressure control 0.02MPa ~ 0.10MPa). Fill all liquid distribution pipelines, filters, and dispensers with nitrogen for 10 minutes to deoxygenate. After nitrogen filling, close the valves of all delivery pipelines and filters starting from the dispenser end, and keep nitrogen pressure for standby use.

[0070] (2) adding a cosolvent to the liquid preparation system, and filling nitrogen below the liquid surface to control the dissolved oxygen to the required level (4 mg / L), then adding edaravone, and after the dissolution is completed, closing the nitrogen filling below the liquid surface, opening the nitrogen filling above the liquid surface, and adding synthetic (+)-2-borneol to dissolve;

[0071] (3) After the dissolution is completed, add part of the water for injection, add the prescribed amount of antioxidant into the tank, continue to fill with nitrogen for protection, and after the dissolution, test the pH value. If necessary, add a pH adjuster to adjust the pH of the solution, add an isotonic adjuster to adjust the osmotic pressure of the solution, and then make up the volume;

[0072] (4) Filtration, nitrogen filling (residual oxygen is controlled to be below 3.0%), and terminal sterilization (121° C. for 12 min) to obtain the finished product.

[0073] Embodiment 10:

[0074] The inventors conducted a systematic investigation of the impurity stability, clarity and color of the preparation samples in the above examples.

[0075] Test process: The finished products of Example 4 and Example 6 were tested, and the finished products were placed under high temperature (60° C.), and samples were taken at 10 days to check the changes in the contents of coexisting substances I, II, III and IV. The test results are shown in Table 1.

[0076] Table 1 Experimental results of influencing factors (%)

[0077]

[0078] The above-mentioned coexistence I and II content detection methods are GC-MS method, impurity reference substance external standard method quantitative, program temperature rise mode, detector is mass spectrometer detector, injection port temperature is 250-300°C, carrier gas is helium, ion source is EI source, ion source temperature is 230°C, selected ion detection (SIM), quantitative ion is 95, electron energy is 70eV, flow rate is 2-2.5ml per minute; coexistence III and coexistence IV content detection methods adopt high performance liquid chromatography, self-reference method or main component external standard method quantitative, octadecylsilane bonded silica gel is used as filler, gradient elution mode is adopted, and detection wavelength is 248nm. The accuracy of each coexistence detection is guaranteed, and the quality of (+)-2-borneol prepared by chemical synthesis and the quality of edaravone and synthetic (+)-2-borneol novel injection of the present invention are more accurately controlled.

[0079] The above test results show that the injection solution prepared by (+)-2-borneol prepared by chemical synthesis process (Example 6) has lower content of coexistent substances I, II, III and IV than the injection solution prepared by (+)-2-borneol extracted from natural borneol (Example 4) under the same conditions, is more stable under high temperature conditions, grows more slowly, and the phenomenon of darkening of solution color is also well controlled, and the product quality is more stable.

[0080] Embodiment 11:

[0081] 1 Materials and methods

[0082] 1.1 Animals Healthy male Wistar rats, weighing 280-320 g.

[0083] 1.2 Drugs and Reagents The synthetic (+)-2-borneol injection (0.5 mg / ml) of Example 2, the edaravone injection (2 mg / ml) of Example 3, the combined administration group (Example 4, 2 mg / ml edaravone + 0.5 mg / ml (+)-2-borneol extracted from natural borneol), and the combined administration group (Example 6, 2 mg / ml edaravone + 0.5 mg / ml synthetic (+)-2-borneol).

[0084] 1.3 Methods

[0085] 1.3.1 Preparation of focal cerebral ischemia-reperfusion model. Main steps: 10% chloral hydrate (350 mg / kg) was intraperitoneally injected to anesthetize rats, and the right external carotid artery was separated, ligated and cut off. The nylon suture with a diameter of about 18 mm was slowly inserted from the stump of the external carotid artery along the common carotid and internal carotid arteries to block the entrance of the middle cerebral artery and cause ischemia. After 2 hours of ischemia, the suture was removed and reperfusion was performed for 24 hours; in the sham operation group, the suture only separated the blood vessels, and the rest of the steps were the same as those in the model group.

[0086] Model success criteria: Rats with scores of 2 to 3 points were included in the group according to the Longa method within 30 to 80 minutes after surgery, and animals with scores of 4 or less than 1 were excluded.

[0087] 1.3.2 Grouping and administration: Animal grouping and administration Experimental animals were randomly divided into 6 groups, namely sham operation group, model group and drug group. The experimental animals were intraperitoneally injected with the test drug twice, 30 minutes before ischemia and 12 hours after reperfusion. The sham operation group and model group were injected with equal volumes of normal saline.

[0088] 1.3.3 Determination of neurological deficit score, cerebral infarction volume and brain tissue water content

[0089] Neurological deficits were scored according to the Longa 5-point scoring system and behavioral evaluation was performed 24 hours after reperfusion.

[0090] For the determination of cerebral infarction volume, the animals were decapitated and the brains were removed after the last neurological deficit score. The olfactory brain, lower brain stem and cerebellum were removed, and the remaining parts were immediately weighed in wet weight. The brain was cut into 5 slices of basically equal thickness along the coronal plane on ice, each slice was 3 mm thick, and stained with 2% TTC. After staining, the infarction area will appear white, and the non-infarction area will appear red. The brain slices were then fixed in 10% formaldehyde, and the white tissue was carefully dug out and weighed. The percentage of the infarction tissue weight to the total brain weight was used as the infarction volume determination index.

[0091] Determination of brain water content: The brain water content was determined by the dry-wet method. After the animal was killed, the whole brain was taken out, the olfactory brain, lower brain and cerebellum were removed, and the wet weight of the brain was immediately weighed. The brain was baked in an oven at 120°C for about 18 hours and then the dry weight was weighed. The brain water content was calculated according to the following formula: brain water content (%) = (brain wet weight - brain dry weight) / brain wet weight × 100.

[0092] 2 Results

[0093] 2.1 Effects on cerebral infarct volume and neurological deficit scores after ischemia-reperfusion

[0094] Compared with the model group, the drug groups could significantly reduce the infarct volume of rats with cerebral ischemia-reperfusion (P<0.01); the effect on the neurological deficit symptoms of rats, the combination of edaravone and synthetic (+)-2-borneol can significantly improve the neurological deficit symptoms compared with the synthetic (+)-2-borneol group and the edaravone group, and has a slight advantage over the combination of edaravone and (+)-2-borneol extracted from natural borneol, indicating that the two drugs have synergistic or synergistic effects. The results are shown in Table 2.

[0095] Table 2 Effects on cerebral infarct volume and neurological deficit score after ischemia-reperfusion (mean ± SD)

[0096]

[0097] *P<0.05, **P<0.01, compared with the model group.

[0098] 2.2 Effects on brain water content in rats with cerebral ischemia-reperfusion

[0099] Compared with the model group, the drug groups can significantly reduce the brain water content caused by ischemia-reperfusion; the edaravone and synthetic (+)-2-borneol combined group can significantly improve the symptoms of brain water content caused by ischemia-reperfusion compared with the synthetic (+)-2-borneol group and the edaravone group, and has advantages over the combined use of edaravone and (+)-2-borneol extracted from natural borneol, indicating that the two drugs have synergistic or synergistic effects. The results are shown in Table 3.

[0100] Table 3 Effects of cerebral water content after ischemia-reperfusion

[0101]

[0102] *P<0.05, **P<0.01, compared with the model group.

[0103] Example 12: Comparative study on the toxicity of edaravone / (+)-2-borneol 4:1 and edaravone / (+)-2-borneol extracted from natural borneol 4:1

[0104] 1. Materials and methods

[0105] 1.1 Experimental animals

[0106] New Zealand rabbit, ordinary grade, weight 2.0~3.0kg, 36 pieces, ♀ 18 each.

[0107] 1.2 Experimental Grouping

[0108] Solvent group 0.9% sodium chloride injection 10ml / kg

[0109] Vehicle group 40% propylene glycol solution 10ml / kg

[0110] Edaravone / synthetic (+)-2-borneol 4:1

[0111] Edaravone / (+)-2-borneol extracted from natural borneol 4:1

[0112] The daily dosage of each rabbit in the above dosage groups was calculated according to the actual body weight of each rabbit, and the same volume of the test substance was given according to body weight.

[0113] 1.3 Experimental methods

[0114] Take 24 rabbits that meet the test requirements and number them, with males as odd numbers and females as even numbers. Randomly divide them into 4 test groups according to body weight, namely "Edaravone / synthetic (+)-2-borneol 4:1", "Edaravone / (+)-2-borneol prepared by natural borneol extraction 4:1" two dosage groups and a solvent group and a solvent group, with 6 rabbits in each group, 3 males and 3 females. Before the test, they were domesticated, observed and adapted for 3 days. During the test, the drug was administered once at the same time in the morning every day, 7 days a week, and continuously administered for 30 days (actually continuously administered for 3-20 days). Weigh once a week, and adjust the dosage as the weight increases or decreases. In the future, observe the time according to the inspection items and indicators. When it is impossible to administer the drug, the animal is killed, and the kidney and injection site are examined for gross and pathological histology. The toxicity of edaravone / synthetic (+)-2-borneol 4:1 and edaravone / (+)-2-borneol extracted from natural borneol 4:1 to rabbits was compared based on animal reactions, irritation at the injection site, and pathological histological examination results at the kidney and injection site.

[0115] 2. Experimental results

[0116] 2.1 General reaction

[0117] Continuous intravenous administration of "Edaravone / synthetic (+)-2-borneol 4:1" and "Edaravone / (+)-2-borneol prepared by natural borneol extraction 4:1" and 40% propylene glycol solution 10ml / kg were performed on the marginal ear vein of rabbits for 3-20 days. There was a significant stimulating effect on the marginal ear vein of rabbits, which was mainly manifested as congestion, embolism, edema and other phenomena at the injection site.

[0118] 2.2 Gross appearance and histopathological examination

[0119] (1) Gross examination: No obvious changes in the appearance of the kidneys were observed in the two combined dosage groups and the 40% propylene glycol solution 10 ml / kg dosage group.

[0120] (2) Pathological histological examination: The doses of "edaravone / (+)-2-borneol prepared by extracting natural borneol 4:1" and "edaravone / synthetic (+)-2-borneol 4:1" caused irritation changes such as vascular embolism to different degrees at the injection site of rabbits; there was also a certain degree of damage to the rabbit kidneys, mainly manifested as progressive renal lesions, such as tubular damage, interstitial inflammatory infiltration and glomerular lesions. The degree of pathological histological damage in the animals of the "edaravone / synthetic (+)-2-borneol 4:1" group was lighter than that of "edaravone / (+)-2-borneol prepared by extracting natural borneol 4:1". The dose of 10 ml / kg of 40% propylene glycol solution also caused irritation changes such as vascular embolism at the injection site of rabbits, and only mild tubular turbidity and vacuolar degeneration were manifested in the rabbit kidneys.

[0121] 2.3 Conclusion

[0122] Based on the test results, under the test dosage conditions:

[0123] (1) Irritation effect on the injection site of rabbits: The two compositions were basically similar.

[0124] (2) Toxicity to rabbit kidney: "Edaravone / (+)-2-borneol prepared by natural borneol extraction 4:1" is greater than "Edaravone / synthetic (+)-2-borneol 4:1".

[0125] Embodiment 13:

[0126] The test report of the synthetic (+)-2-borneol raw material drug used in the above-mentioned embodiment of the present invention is as follows:

[0127]

[0128] The detection method for coexistent substances I, V, and VI in the above-mentioned test report is as follows: determination by gas chromatography (General Rules 0521 of Part IV of the Chinese Pharmacopoeia 2020 Edition), quantitative calculation by peak area normalization method, and a capillary with polyethylene glycol as the stationary liquid as the chromatographic column. The starting temperature is 50°C, maintained for 1 minute, and heated to 240°C at a rate of 10°C per minute, maintained for 15 minutes. The injection port temperature is 250°C, the detector is a hydrogen flame ionization detector (FID), the detector temperature is 250°C, the carrier gas is nitrogen, and the column flow rate is 1.0 ml per minute.

[0129] The detection method for coexistence II in related substances is as follows: determination by gas chromatography (General Rules 0521 of Part IV of the Chinese Pharmacopoeia 2020 Edition), quantitative calculation by impurity peak area normalization method, a capillary column with β-cyclodextrin as the stationary liquid as the chromatographic column, the starting temperature is 40°C, maintained for 5 minutes, and heated to 180°C at a rate of 3.5°C per minute; the injection port temperature is 250°C; the detector is a hydrogen flame ionization detector (FID); the detector temperature is 300°C; the carrier gas is nitrogen; and the column head pressure is 15psi.

[0130] The detection method for the residual solvent n-heptane is as follows: determine according to the residual solvent determination method (Method 3 of Part IV of the Chinese Pharmacopoeia 2020 Edition, General Rules 0861), calculate by peak area according to the external standard method, and use a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary liquid as the chromatographic column. The starting temperature is 38°C, maintained for 6 minutes, and heated to 120°C at a rate of 20°C per minute, maintained for 3 minutes, and then heated to 240°C at a rate of 50°C per minute, and maintained for 8 minutes; the detector is a hydrogen flame ionization detector (FID), and the detector temperature is 250°C; the inlet temperature is 220°C, the carrier gas is nitrogen, and the column flow rate is 1.0 ml per minute.

[0131] The content detection method is: determined according to gas chromatography (General Rules 0521 of Part IV of the Chinese Pharmacopoeia 2020 Edition), calculated by peak area according to the internal standard method, using a capillary column with β-cyclodextrin as the stationary liquid as the chromatographic column, and the starting temperature is 120°C, heated to 140°C at a rate of 4°C per minute, maintained for 10 minutes, and then run at 220°C for 15 minutes; the inlet temperature is 250°C, the detector is a hydrogen flame ionization detector (FID), the detector temperature is 250°C, the carrier gas is nitrogen, and the column flow rate is 1.0 ml per minute.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0133] References:

[0134] 1.XU J, WANGA, MENG

[0135] 2. Chinese Society of Neurology, Chinese Society of Neurology Cerebrovascular Disease Group. Chinese Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke 2018[J]. Chinese Journal of Neurology, 2018, 51(9): 666-682.

[0136] 3. CHAMORROá, DIRNAGL U, URRAX, et al. Neuroprotection in acute stroke: targeting excitotoxicity, oxidative and nitrosative stress, and inflammation [J]. Lancet Neurol, 2016, 15(8): 869-881.

[0137] 4. Zhang Yingnan, Jiang Yang, Ren Li, et al. Effect of intravenous infusion of edaravone and dextroborneol on the improvement of brain damage in patients with acute anterior circulation cerebral infarction who underwent endovascular treatment[J]. Shandong Medicine, 2021, 61(18): 76-79.

[0138] 5.ZHANG XL, LIUY, CHENG

[0139] 6. LI L, HE G, SHI M, et al. Edaravone dexborneol ameliorates cognitive impairment by regulating the NF-κB pathway through AHR and promoting microglial polarization towards the M2 phenotype in mice with bilateral carotid artery stenosis (BCAS) [J]. Eur J Pharmacol, 2023, 957: 176036.

[0140] 7. Chinese Pharmacopoeia, 2020 edition, Volume 1, page 61.

[0141] 8.WANG Y, LI B, JIANG Y, et al. YKL-40 is associated with ultrasound-determined carotid atherosclerotic plaque instability[J]. Front Neurol, 2021, 12: 622869.

[0142] 9. SHAMIA, EDSFELDTA, BENGTSSON E, et al. Soluble CD40 levels in plasma are associated with cardiovascular disease and in carotid plaques with avulnerable phenotype [J]. J Stroke, 2021, 23(3): 367-376.

[0143] 10.HU R, LIANG J, DING L, et al. Edaravone dexborneol provides neuroprotective benefits by suppressing NLRP3 inflammasome-induced microglialpyroptosis in experimental ischemic stroke[J]. Int Immunopharmacol, 2022, 113(PtA): 109315.

[0144] 11. Instructions for use of Idarubicin and Dextromethorphan Concentrated Solution for Injection, [Usage and Dosage] section.

Claims

1. A novel injection containing edaravone and high-purity chemically synthesized (+)-2-borneol, characterized in that: (1) The active ingredients of the injection are edaravone and (+)-2-borneol derived from chemical synthesis, and the injection is characterized in that it also contains one or more of coexistent substances I, coexistent substances II, coexistent substances III, and coexistent substances IV; wherein the content of edaravone is 0.2-3 mg / ml (W / V), the content of synthetic (+)-2-borneol is 0.05-2 mg / ml (W / V); the content of coexistent I is not more than 0.10% of the labeled amount of synthetic (+)-2-borneol, the content of coexistent II is not more than 0.75% of the labeled amount of synthetic (+)-2-borneol, the content of coexistent III is not more than 0.90% of the labeled amount of edaravone; the content of coexistent IV is not more than 0.25% of the labeled amount of edaravone; (2) The inactive ingredients of the injection solution further include a cosolvent, an antioxidant and water for injection, wherein the amount of the cosolvent is 0.01 ml / ml to 0.2 ml / ml, and the amount of the antioxidant is 0.1 mg / ml to 1.5 mg / ml; (3) The pH value of the injection solution is between 2.5 and 7.0, preferably between 3.0 and 5.0; (4) The injection solution can withstand terminal sterilization process.

2. The injection according to claim 1, characterized in that: The (+)-2-borneol raw material drug derived from chemical synthesis is characterized by: purity of more than 98%, preferably more than 99.0%, content of coexistent I ≤0.15%, content of coexistent II ≤0.5%, content of coexistent V ≤0.1%, content of coexistent VI ≤0.15%, and content of n-heptane ≤0.1%; the starting material of the chemical synthesis route can be a-pinene or D-camphor.

3. The injection according to claim 2, characterized in that: The synthesis route of the chemically synthesized (+)-2-borneol API is shown in Formula 1: The synthesis steps are: using α-pinene as a starting material, first preparing an intermediate S1 with oxalic acid, then removing the ester group to obtain S2, S2 is derivatized and purified to obtain S3, and S3 is removed from the ester group to obtain the synthetic (+)-2-borneol; Among them, in S3, R is 4. The injection according to claim 1 or 2, characterized in that: The content detection method of the coexistent substances I and II is a gas chromatography-mass spectrometry detection method, with a capillary tube with β-cyclodextrin as the stationary liquid as the chromatographic column, a programmed temperature rising method, an EI source as the ion source of the mass spectrometer detector, selected ion monitoring (SIM), a quantitative ion of 95, an electron energy of 70 eV, and helium as the carrier gas.

5. The injection according to claim 1, characterized in that: The method for detecting the content of coexistent substance III and coexistent substance IV adopts high performance liquid chromatography, ultraviolet detector, main component self-control method or main component external standard method for quantification, uses octadecylsilane bonded silica gel as filler, triethylamine-phosphoric acid solution as mobile phase A, methanol as mobile phase B, and adopts linear gradient elution.

6. The injection according to claims 1 to 5, characterized in that: The chemical structural formulas of coexistents I to VI are:

7. The injection according to any one of claims 1 to 2, characterized in that: The cosolvent is one or more of ethanol, propylene glycol, isopropanol or polyethylene glycol.

8. The injection according to any one of claims 1 to 2, characterized in that: The antioxidant is one or more of sodium sulfite, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate and vitamin C.

9. The injection according to any one of claims 1 to 2, characterized in that: The injection solution also includes an isotonicity regulator, which includes one or more of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium lactate, glucose, sorbitol, xylitol, and dextran.

10. The injection according to any one of claims 1 to 2, characterized in that: The injection also includes a pH adjuster, which is one or more of sulfuric acid, hydrochloric acid, sodium hydroxide, disodium hydrogen phosphate or sodium bicarbonate.

11. A method for preparing the injection according to any one of claims 1 to 10, characterized in that: The following steps are involved: (1) Open the valves of the liquid distribution pipeline and the filling dispenser, introduce nitrogen gas to control the pressure at 0.02MPa to 0.10MPa, and continuously fill all liquid distribution pipelines, filters, and dispensers with nitrogen for 10 minutes to perform deoxygenation operations. After the nitrogen filling is completed, close the valves of all delivery pipelines and filters starting from the dispenser end, and maintain the nitrogen pressure for standby use; (2) adding a cosolvent to the liquid preparation system, and after nitrogen filling below the liquid surface is used to control the dissolved oxygen to meet the requirement, adding edaravone, and after the dissolution is completed, closing the nitrogen filling below the liquid surface, opening the nitrogen filling above the liquid surface, and adding synthetic (+)-2-borneol to dissolve; (3) After the dissolution is completed, add part of the water for injection, add the prescribed amount of antioxidant into the tank, continue to fill with nitrogen for protection, and after the dissolution, test the pH value. If necessary, add a pH adjuster to adjust the pH of the solution, add an isotonic adjuster to adjust the osmotic pressure of the solution, and then make up the volume; (4) filtering, nitrogen filling, and terminal sterilization to obtain the injection solution.

12. The preparation method according to claim 11, characterized in that: The terminal sterilization adopts hot pressure sterilization. When the sterilization temperature is 115±3°C, the sterilization time is 30 to 48 minutes; or when the sterilization temperature is 121±3°C, the sterilization time is 8 to 16 minutes.

13. The preparation method according to claim 11, characterized in that: Before preparation, the pipeline is filled with nitrogen, the dissolved oxygen is controlled at 4.0 mg / L during liquid preparation, and the residual oxygen in the headspace is controlled below 3.0% during filling.

14. Use of the injection according to any one of claims 1 to 10 or the injection prepared by the preparation method according to any one of claims 11 to 13 in the preparation of drugs for treating oxidative stress damage and thrombosis-related diseases such as cardiovascular and cerebrovascular diseases, diabetic retinopathy, diabetic peripheral neuropathy, and neurodegenerative diseases.

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