A method for constructing an animal model of light anesthesia via sublingual tablet administration

Abstract

This invention discloses a method for constructing a light anesthesia lozenge administration animal model, belonging to the field of biotechnology. The method involves administering propofol to animals, and after the righting reflex disappears, administering a lozenge to obtain a light anesthesia lozenge administration animal model. This method has good reproducibility, the experimental animals exhibit good tolerance, and it accurately simulates the clinical lozenge administration process, solving the key problem of discrepancies between the gavage administration route and clinical practice in traditional lozenge animal experiments.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for constructing an animal model of light anesthesia via sublingual tablet administration. Background Technology

[0002] Establishing effective administration methods for lozenge formulations in animal experiments presents significant technical challenges in preclinical studies. Since experimental animals cannot autonomously administer lozenges, there are currently no specific methodologies for animal experiments targeting lozenge administration. Researchers typically use the alternative of dissolving the lozenges and administering them via gavage. However, this method differs fundamentally from human lozenge administration in terms of drug absorption kinetics. Drug absorption through the gastrointestinal tract involves the first-pass effect, degradation by digestive juices, and slow intestinal wall penetration, resulting in significant differences in onset time, bioavailability, and plasma concentration profiles compared to lozenge administration. Therefore, data obtained via gavage are insufficient to accurately predict the actual behavior of lozenges in humans.

[0003] Light anesthesia as an adjunct to drug administration is considered a potential solution. However, several key scientific issues remain to be addressed: anesthetic drugs may interfere with the animal's physiological state, thus affecting the assessment of drug absorption and metabolism; simultaneously, standardized protocols for controlling the depth of anesthesia and standardized drug administration procedures are currently lacking. Therefore, systematically studying suitable anesthetics for animal administration of sublingual tablets, and the safe and tolerable dosage range of anesthetics in rats, further validating the precision, accuracy, and reproducibility of this method, ensuring the reliability and reproducibility of experimental data, and establishing an animal drug administration method that conforms to the actual characteristics of sublingual tablet use, has significant scientific and practical value. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for constructing an animal model of light anesthesia lozenge administration. This method has good reproducibility, the animal model has good overall tolerance, and there are no adverse symptoms such as salivation or bleeding that affect the administration of the lozenge.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing a light anesthesia sublingual tablet administration animal model, comprising the following steps: administering propofol to the animal, and administering the sublingual tablet after the animal's righting reflex disappears, thereby obtaining a light anesthesia sublingual tablet administration animal model.

[0006] Preferably, the propofol is a propofol solution, and the dosage of the propofol solution is 0.02-0.15 mL / 100 g, applied once a day for 8-12 consecutive days.

[0007] Preferably, the preparation steps of the propofol solution include: mixing propofol, Tween-80, and sodium chloride solution evenly.

[0008] Preferably, the volume ratio of propofol, Tween-80, and sodium chloride solution is (0.2-0.8):(0.5-1.5):(2-5).

[0009] Preferably, the sodium chloride solution has a mass-volume percentage concentration of 0.7-1.1%.

[0010] Preferably, the animal includes a normal rat or a model rat.

[0011] Preferably, the model rat includes a pharyngitis model rat.

[0012] Preferably, the righting reflex disappears 2-10 minutes after the application of propofol.

[0013] This invention provides a method for administering a lozenge to animals under light anesthesia, comprising the following steps: administering propofol to the animal, and after the animal's righting reflex disappears, administering the lozenge orally.

[0014] Preferably, the propofol is a propofol solution, the dosage of which is 0.02-0.15 mL / 100 g, and the administration method is intraperitoneal injection. The propofol solution is administered once a day followed by oral lozenge administration for 8-12 consecutive days.

[0015] The beneficial effects of this invention are: This invention proposes for the first time a method for constructing an animal model of light anesthesia sublingual tablet administration, and innovatively develops a standardized rat sublingual tablet administration method based on controllable anesthesia. This invention precisely controls the depth of anesthesia, keeping the anesthesia time within 1 hour, and avoids adverse symptoms such as salivation and bleeding that could affect sublingual tablet administration. The method involves administering propofol solution to animals, and after the righting reflex disappears, administering the sublingual tablet to obtain an anesthetized sublingual tablet administration animal model. This method has good reproducibility, and the experimental animals exhibit good tolerance, achieving accurate simulation of the clinical sublingual tablet administration process and solving the key problem of discrepancies between the gavage administration route and clinical practice in traditional sublingual tablet animal experiments. The establishment of this method provides a scientifically reliable drug delivery technology for preclinical pharmacological studies of sublingual tablet formulations, and is of great significance for enhancing the translational value of new drug development. Attached Figure Description

[0016] Figure 1 This is the result of a rat test for tolerance to anesthetics. IL-6 is interleukin-6; TNF-α is tumor necrosis factor-α; and COX-2 is cyclooxygenase-2.

[0017] Figure 2 These are the results of drug intervention in rats with acute pharyngitis. Among them, IL-6 is interleukin-6; TNF-α is tumor necrosis factor-α; and COX-2 is cyclooxygenase-2. Detailed Implementation

[0018] This invention provides a method for constructing a light anesthesia sublingual tablet administration animal model, comprising the following steps: administering propofol to the animal, and after the animal's righting reflex disappears, administering the sublingual tablet to obtain a light anesthesia sublingual tablet administration animal model. This invention utilizes propofol to provide light anesthesia to animals, enabling effective sublingual tablet administration and solving the key problem of discrepancies between traditional sublingual tablet animal experiments and clinical practice.

[0019] In this invention, the propofol is a propofol solution, and the dosage of the propofol solution is 0.02-0.15 mL / 100g, administered once daily for 8-12 consecutive days; preferably, the dosage of the propofol solution is 0.04-0.1 mL / 100g, administered once daily for 9-11 consecutive days; more preferably, the dosage of the propofol solution is 0.06 mL / 100g, administered once daily for 10 consecutive days. The propofol solution of this invention is administered once daily followed by oral lozenges for 8-12 consecutive days. The anesthetic and anesthesia method used in this invention are well tolerated by animals and do not affect the physiological function of the oral mucosa. This invention establishes the optimal anesthetic dose range for maintaining the swallowing reflex while inhibiting active rejection through stepwise dose experiments, ensuring that animals are in a stable, lightly anesthetized state with minimal physiological disturbance.

[0020] In this invention, the preparation step of the propofol solution includes: mixing propofol, Tween-80, and sodium chloride solution evenly. The volume ratio of propofol, Tween-80, and sodium chloride solution in this invention is (0.2-0.8):(0.5-1.5):(2-5), preferably (0.4-0.6):(0.7-1.3):(3-4.5), and more preferably 0.5:1.0:3.5. This invention utilizes the above preparation method to induce propofol to form a stable micelle or microemulsion structure by leveraging the surface activity of Tween-80. This method not only solves the problem of uneven dispersion of lipid-soluble propofol in the aqueous phase, significantly improving the stability, homogeneity, and dosage accuracy of the solution; simultaneously, the microemulsified propofol can effectively reduce chemical irritation to the peritoneum, simplifying the intraperitoneal injection procedure, thereby improving the safety and convenience of clinical use. The sodium chloride solution of the present invention has a mass-volume percentage concentration of 0.7-1.1% (0.7-1.1g sodium chloride dissolved in 100ml water), preferably 0.8-1.0%, and more preferably 0.9%.

[0021] In this invention, the animals include normal rats or model rats. The model rats in this invention include rats with pharyngitis.

[0022] In this invention, the righting reflex disappears 2-10 minutes after the application of propofol, preferably 2-5 minutes or 5-10 minutes.

[0023] This invention provides a method for administering a lozenge to animals under light anesthesia, comprising the following steps: administering propofol to the animal, and after the animal's righting reflex disappears, administering a lozenge orally. The propofol used in this invention is a propofol solution, the dosage of which is 0.02-0.15 mL / 100g, administered via intraperitoneal injection. The propofol solution is administered once daily, followed by oral lozenge administration, for 8-12 consecutive days. This invention systematically establishes for the first time a quantifiable and reproducible method for adjunctive administration of light anesthesia. It overcomes existing technical bottlenecks through the following core technical means, providing a standardized execution scheme that can withstand scientific verification: obtaining an anesthetic agent with good animal tolerance and without affecting the physiological function of the oral mucosa through comparative experiments, solving the problem of uncontrolled anesthetic interference; and establishing the optimal anesthetic dose range for maintaining the swallowing reflex while inhibiting active rejection through stepwise dose experiments, ensuring the animal is in a stable state of light anesthesia with minimal physiological interference, and determining the safety window dose. Meanwhile, the present invention verified the good reproducibility of the method through repeatability experiments; at the same time, by comparing the key physiological parameters such as the level of inflammatory factors in the light anesthesia group and the normally fed animals, the results showed that the type and dosage of anesthetic used in the present invention had little interference with the overall physiological state of the animals and had good biocompatibility and experimental applicability.

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Unless otherwise specified, the following embodiments are all conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1 A method for constructing an animal model of light anesthesia via lozenge administration includes the following steps: 0.06 mL / 100 g of propofol solution is injected intraperitoneally into the animal daily. After the righting reflex disappears, a lozenge is administered orally for 10 consecutive days to obtain the animal model of light anesthesia via lozenge administration.

[0028] The propofol solution is prepared as follows: Take 0.8 mL of propofol and place it in a 15 mL centrifuge tube. Add 1.5 mL of Tween-80 and vortex for 2 min. Then add 0.9% (w / v) sodium chloride injection to 8 mL and vortex for 3 min to obtain a 10% propofol solution by volume. Wrap the solution in aluminum foil to protect it from light.

[0029] Example 2 A method for constructing an animal model of light anesthesia via lozenge administration includes the following steps: 0.02 mL / 100 g of propofol solution is injected intraperitoneally into the animal daily. After the righting reflex disappears, the lozenge is administered orally for 8 consecutive days to obtain the animal model of light anesthesia via lozenge administration.

[0030] The propofol solution is prepared as follows: Take 0.6 mL of propofol and place it in a 15 mL centrifuge tube, add 0.5 mL of Tween-80, vortex for 2 min, then add 0.9% (w / v) sodium chloride injection to 6 mL, vortex for 3 min, and obtain a 10% propofol solution by volume. Wrap the solution in aluminum foil to protect it from light.

[0031] Example 3 A method for constructing a light anesthesia animal model includes the following steps: injecting propofol solution into the peritoneum of animals at a rate of 0.1 mL / 100 g daily. After the righting reflex disappears, administer lozenges orally. Continue this injection for 12 consecutive days to obtain a light anesthesia lozenge-administered animal model.

[0032] The propofol solution is prepared as follows: Take 0.8 mL of propofol and place it in a 15 mL centrifuge tube. Add 1.5 mL of Tween-80 and vortex for 2 min. Then add 0.9% (w / v) sodium chloride injection to 8 mL and vortex for 3 min to obtain a 10% propofol solution by volume. Wrap the solution in aluminum foil to protect it from light.

[0033] Example 4 I. Materials and Reagents: Teletamine hydrochloride-zoprazepam hydrochloride for injection (batch number zoletil50), manufactured by VIRBAC (France); Xylazine hydrochloride injection (batch number 070031777), manufactured by Dunhua Shengda Animal Pharmaceutical Co., Ltd.; Propofol (batch number M10385), manufactured by Shanghai Mairui Chemical Reagent Co., Ltd.; Ethyl carbamate (batch number U108629), manufactured by Shanghai Aladdin Biochemical Technology Co., Ltd. Experimental Animals: SPF grade SD rats.

[0034] II. Detection Methods 1. Preliminary screening of anesthetics and determination of anesthetic dosage range Grouping: Twelve SD rats were housed at room temperature (22-24℃) and relative humidity (40-60%) with 12-hour / 12-hour day / night lighting. They were randomly divided into four groups of six rats each, labeled as follows: Telatamine Hydrochloride-Zolazepam Hydrochloride Injection Group, Xylazine Hydrochloride Injection Group, Propofol Group, and Urethane (Ethyl Carbamate) Group, for preliminary experiments. Based on references and dosages provided by reagent manufacturers, the dosage ranges for the four anesthetics were preliminarily determined.

[0035] Experimental methods: Before anesthesia, the body weight of each rat was weighed and the dosage of anesthetic was calculated (the first dose shown in Table 1) and administered via intraperitoneal injection. After the righting reflex of the anesthetized rats disappeared, the rats were placed on a warming pad, and the rats' condition, anesthesia induction time, maintenance time, and recovery time were observed and recorded.

[0036] After the rats recovered, they rested for 1 hour and were then injected intraperitoneally with the second dose shown in Table 1. The first dose procedure was repeated, and so on, until the highest anesthetic dose (anesthetic time 1-2 hours) and the lowest anesthetic dose (anesthetic time approximately 20 minutes) suitable for sublingual administration were preliminarily determined (based on the Chinese Pharmacopoeia 0921 disintegration time test method, which stipulates that the sublingual tablet must completely disintegrate or melt within 10 minutes to determine the anesthetic time). The results are shown in Table 1.

[0037] Observation indicators: Routine observation indicators refer to the changes in vital signs such as respiration, heartbeat and body temperature of rats during the entire anesthesia period, as well as behavioral changes such as limb movement, gait, whisker touch response, salivation, muscle relaxation and tremor, and recording the mortality rate of rats in each group.

[0038] Time-related anesthesia monitoring indicators: Anesthesia induction time refers to the time from the injection of anesthetic drugs into the rat to the rat collapsing and losing pain sensation; Anesthesia maintenance time refers to the time from the rat losing pain sensation to the rat regaining pain sensation; Anesthesia recovery time refers to the time from the rat regaining pain sensation to the rat turning over on its own; Total anesthesia time = Anesthesia induction time + Anesthesia maintenance time + Anesthesia recovery time.

[0039] Criteria for judging loss of pain sensation: The rat's whole body muscles are relaxed, breathing is smooth, and when the tip of the hind limb toe is pressed with forceps, the rat has no pain response.

[0040] Criteria for assessing the recovery of pain sensation: The rat's respiratory rate increases, and the rat shows a pain response when the tips of its hind toes are pressed with forceps.

[0041] Table 1. Effects of different anesthetics on the anesthesia time of rats.

[0042] In the dose screening experiment of anesthetics used in a sublingual administration model, Table 1 shows that the injection of teletamine hydrochloride-zoprazepam hydrochloride, xylazine hydrochloride injection, and ethyl carbamate all had a certain impact on sublingual administration in mice after anesthesia. At a dose of 0.01 mL / 100 g of propofol, the anesthesia time was suitable, the recovery was slightly longer, but sublingual administration could still be completed; at a dose of 0.005 mL / 100 g, the anesthesia time was suitable, the recovery was faster, and sublingual administration could be completed; at a dose of 0.004 mL / 100 g, the anesthesia time was shortest and the recovery was fastest, but the anesthesia state was unstable in some animals, and sublingual administration was still possible. All three doses met the requirements for sublingual administration. Therefore, further investigation was conducted on the tolerance of animals to a dose range of 0.004-0.01 mL / 100 g of propofol (the prepared propofol solution was 0.04-0.1 mL / 100 g).

[0043] 2. Animal tolerance to anesthetics Grouping: Twenty-four male SD rats, weighing 200-220g, were housed at room temperature (22-24℃) and relative humidity (40%-60%), with 12h / 12h day / night lighting. They were randomly divided into four groups of six rats each: a control group, a high-dose propofol group (0.1mL / 100g propofol solution injected into mice), a medium-dose propofol group (0.06mL / 100g propofol solution injected into mice), and a low-dose propofol group (0.04mL / 100g propofol solution injected into mice). The propofol solution used was prepared as described in Example 1.

[0044] Experimental Methods: The control group received an intraperitoneal injection of 0.2 mL of 0.9% sodium chloride solution daily. Other groups of animals were anesthetized once daily with the corresponding anesthetic and dosage for 10 consecutive days. After the rats recovered from anesthesia, they were kept in a food-free environment for 12 hours, and 2-3 mL of whole blood was collected. The blood was centrifuged at 3000 rpm for 10 minutes, and the serum was collected for later use. Evaluation Indicators: The levels of inflammatory factors IL-6, TNF-α, and COX-2 in the serum were detected using ELISA. Table 2 Results of rat tolerance to anesthetics ( - χ±s, n=6)

[0045] The results are as follows Figure 1 As shown in Table 2, the results indicate that during the 10-day observation period, compared with the saline control group, the serum levels of inflammatory factors IL-6, TNF-α, and COX-2 in rats anesthetized with the selected dose of propofol showed no significant changes, indicating that this anesthesia regimen had a mild effect on the systemic inflammatory response of the animals and was well tolerated overall. Therefore, this propofol anesthesia regimen can be used as a feasible adjunct in rat sublingual administration studies.

[0046] Example 5 I. Establishment of an acute pharyngitis model in rats Select male SD rats, weighing 200-220g. Experimental materials and reagents involved: 25% ammonia solution, purified water, throat applicator, ultrasonic cleaner, propofol. Preparation of 20% ammonia solution: Add 8mL of 25% ammonia solution to the throat applicator, add 2mL of purified water, mix thoroughly using an ultrasonic cleaner, and wrap with aluminum foil to protect from light before use.

[0047] Modeling process (3 consecutive days): ① Male SD rats were sprayed with 20% ammonia water into their throats using a throat applicator, 3 puffs per rat each time, once in the morning and once in the afternoon. ② From the second day onwards, the animals' behavior and mental state were observed, and changes in water intake and weight were recorded. The condition of the pharynx (mucosal color, shape, etc.) was observed to determine the success of the model. ③ After the modeling was completed, the water intake was set at 500 mL per group, and the food intake was 200 g per group.

[0048] Experimental results: The rats exhibited tangled fur, dullness, increased saliva secretion, dry cough, hoarseness, weight loss, increased water intake, and decreased food intake, indicating successful model establishment.

[0049] II. Drug intervention in rats with acute pharyngitis Experimental materials and reagents involved: 25% ammonia solution, purified water, throat applicator, ultrasonic cleaner, compound herbal lozenges, propofol. Preparation of 2.5% ammonia solution: Take 1 mL of 25% ammonia solution, add 9 mL of purified water to the throat applicator, mix well using an ultrasonic cleaner, and wrap with aluminum foil to protect from light before use.

[0050] Grouping: Control group: normal rats; Model group: rats with acute pharyngitis model constructed above; No anesthesia + lozenge group: rats with acute pharyngitis model + lozenge administration; Anesthesia + lozenge group: rats with acute pharyngitis model + propofol anesthesia + lozenge administration. Six rats per group.

[0051] Experimental Procedure (10 consecutive days): In the morning, 2.5% ammonia solution was applied to the throat of each group using sterile cotton swabs; in the afternoon, each group was administered Compound Herba Sarcandrae Lozenges at a dose of 0.55 g / kg. Administration Method: For anesthetized lozenge administration: Rats were intraperitoneally injected with propofol solution at 0.06 mL / 100 g daily. After 2-5 minutes, the rats' righting reflex disappeared, and Compound Herba Sarcandrae Lozenges (divided into appropriate doses using a cutter) were administered. Propofol solution and Compound Herba Sarcandrae Lozenges were administered continuously for 10 days. Anesthetized rats could suck on the lozenges normally without vomiting. For unanesthetized pharyngitis model rats, Compound Herba Sarcandrae Lozenges (divided into appropriate doses using a cutter) were administered directly for 10 consecutive days. During this period, because the model rats were conscious, they vomited the medication after administration. The propofol solution mentioned was prepared as described in Example 1.

[0052] Sampling and testing: After the last administration, the rats were fasted but allowed to drink water for 12 hours. 2-3 mL of whole blood was collected, centrifuged at 3000 r / min for 10 min, and the serum was collected for ELISA detection to detect the levels of inflammatory factors IL-6, TNF-α, and COX-2 in the serum.

[0053] Table 3. Results of drug intervention in rats with acute pharyngitis ( - χ±s, n=6)

[0054] Note: Compared with the control group, # P <0.05, ## P <0.01; compared with the model group, P <0.05, P <0.05.

[0055] Experimental results are as follows Figure 2 As shown in Table 3, the results indicate that the established propofol light anesthesia regimen ensures stable and successful administration of lozenges in animals. Simultaneously, experimental data show that the serum levels of inflammatory factors (IL-6, TNF-α, and COX-2) in the anesthesia + lozenge group were significantly lower than those in the model group, consistent with the expected efficacy trend. This indicates that the regimen did not interfere with the pharmacological activity of the lozenges, effectively relieving symptoms of acute pharyngitis and effectively solving the technical challenge of difficult lozenge administration. It can serve as a reliable, mild, and non-invasive anesthetic adjunctive method suitable for pharmacodynamic studies of animal lozenge formulations.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of light anesthesia via sublingual anesthesia, characterized in that, The procedure includes the following steps: administering propofol to the animal, and after the animal's righting reflex disappears, administering a lozenge to obtain a light anesthesia lozenge-administered animal model.

2. The construction method as described in claim 1, characterized in that, The propofol is a propofol solution, and the dosage of the propofol solution is 0.02-0.15 mL / 100 g, applied once a day for 8-12 consecutive days.

3. The construction method as described in claim 2, characterized in that, The preparation steps of the propofol solution include: mixing propofol, Tween-80, and sodium chloride solution evenly.

4. The construction method as described in claim 3, characterized in that, The volume ratio of propofol, Tween-80, and sodium chloride solution is (0.2-0.8):(0.5-1.5):(2-5).

5. The construction method as described in claim 3, characterized in that, The sodium chloride solution has a mass-volume percentage concentration of 0.7-1.1%.

6. The construction method as described in claim 1, characterized in that, The animals include normal rats or model rats.

7. The construction method as described in claim 6, characterized in that, The model rats include rats with pharyngitis.

8. The construction method as described in claim 1, characterized in that, The righting reflex disappeared 2-10 minutes after the application of propofol.

9. A method for administering a sublingual tablet to lightly anesthetized animals, characterized in that, The procedure includes the following steps: administer propofol to the animal, and after the animal's righting reflex disappears, administer a lozenge orally.

10. The method for administering a sublingual tablet to lightly anesthetized animals as described in claim 9, characterized in that, The propofol is a propofol solution, and the dosage of the propofol solution is 0.02-0.15 mL / 100 g. The administration method is intraperitoneal injection. The propofol solution is administered once a day followed by oral lozenge administration for 8-12 consecutive days.

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