A furosemide-aminophenol compound temperature-sensitive hydrogel oral preparation, a preparation method, a detection method and an application thereof

Abstract

This invention provides a furosemide-triamterene compound thermosensitive hydrogel oral formulation, its preparation method, detection method, and application, belonging to the field of veterinary formulation technology. The formulation, by mass percentage, comprises furosemide 0.2%-2.0%, triamterene 0.2%-2.0%, an adhesion thickener 0.5%-2.0%, a thermosensitive hydrogel matrix 15%-30%, and moisturizing stabilizers, flavoring agents, and preservatives. The gelation temperature is 28-32℃, the maximum adhesion force to the gastric mucosa is 0.15N-0.35N, and the adhesion work is 5mJ-15mJ. This invention employs a cold-dissolution method, a process free of organic solvents, and simultaneously determines the content of two drugs using HPLC. The formulation flows at 2-8°C and rapidly gels and adheres at 37°C, achieving synergistic diuresis and potassium balance maintenance. It features precise dosage, good palatability, and stable blood drug concentrations, making it suitable for preparing veterinary drugs for canine and feline diuresis, swelling reduction, and potassium balance maintenance. This invention solves the problems of inaccurate dosage, poor compliance, and lack of quality control in existing pet diuretic preparations, ensuring stable and controllable quality and suitability for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of veterinary chemical pharmaceutical formulation technology, specifically to a furosemide-triamterene compound thermosensitive hydrogel oral formulation and its preparation method, detection method and application. Background Technology

[0002] Furosemide is a commonly used and potent loop diuretic in clinical practice, widely used in the treatment of water and sodium retention diseases such as congestive heart failure, nephrotic edema, and ascites due to cirrhosis in companion animals such as dogs and cats. However, furosemide alone can cause adverse reactions such as hypokalemia, hyponatremia, dehydration, and gastrointestinal irritation, limiting its safety in clinical use. Triamterene is a mild potassium-sparing diuretic that can effectively antagonize the potassium-excreting effect of furosemide. The combined use of the two can achieve a synergistic effect of enhanced diuresis and electrolyte balance, making it the preferred combination therapy for clinical diuresis.

[0003] Currently, in veterinary clinical practice, the combined use of furosemide and triamterene mainly employs methods such as splitting human tablets, direct oral administration of powders, and manual filling of capsules, which have several technical shortcomings: First, poor dosage accuracy; the dosage error of split human tablets can reach up to ±30%, failing to meet the precise dosing needs of dogs and cats of different weights. Second, extremely poor drug compliance; solid dosage forms are not palatable, and pets are prone to problems such as refusal to eat, choking, and vomiting, resulting in insufficient actual dosage. Third, significant fluctuations in blood drug concentration; immediate-release solid dosage forms are prone to the "peak-valley effect" of blood drug concentration, with adverse reactions aggravated at peak concentrations and therapeutic effects lost at trough concentrations. Fourth, lack of pet-specific formulations and quality control standards; there are no compound thermosensitive controlled-release formulations adapted to the gastrointestinal physiological characteristics of dogs and cats, and no quality control system for simultaneous quantitative detection of the two drugs has been established. Fifth, insufficient dosage form compatibility; existing hydrogel formulations are mostly external or injectable, and there are no oral thermosensitive adhesive hydrogels, making it impossible to achieve in-situ gelation in the gastrointestinal tract and long-term controlled release.

[0004] In existing technologies, human compound triamterene tablets are a combination of triamterene and hydrochlorothiazide, not a pet-specific formulation. They lack a temperature-sensitive controlled-release design, have poor palatability, and do not utilize the synergistic ratio of furosemide and triamterene. Furthermore, current methods for detecting veterinary diuretics can only determine a single component, failing to meet the regulatory requirements for simultaneous quantitative analysis of compound formulations, related substance testing, and veterinary drug residue detection. Therefore, developing a pet-specific, precisely dosed, palatable, stable controlled-release, and well-controlled furosemide-triamterene compound temperature-sensitive hydrogel oral formulation has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] This invention provides a furosemide-triamterene compound thermosensitive hydrogel oral formulation, its preparation method, and its application, aiming to solve the problems mentioned in the background art.

[0006] This invention is achieved by providing a furosemide-triamterene compound thermosensitive hydrogel oral formulation, which, by weight percentage, comprises the following components: Furosemide 0.2%-2.0%, triamterene 0.2%-2.0%, adhesive thickener 0.5%-2.0%, thermosensitive hydrogel matrix 15%-30%, moisturizing stabilizer 2%-5%, flavoring agent 0.1%-0.5%, preservative 0.05%-0.2%, purified water added to the required amount.

[0007] Furthermore, the mass ratio of furosemide to triamterene is 1:1; the gelation temperature of the preparation is 28-32℃, and the gelation temperature is determined by the inverted test tube method: take about 10mL of this product, which is in a flowing liquid state at 2-8℃, and increase the temperature at a rate of 1℃ / min. For every 1℃ increase, tilt the test tube 85° and observe for 3 seconds. The gelation temperature is the temperature at which the sample no longer flows. The average value is taken for 3 parallel determinations.

[0008] Furthermore, the gastric mucosal adhesion properties of the formulation were determined by the in vitro peel force method, with a maximum adhesion force of 0.15N-0.35N and an adhesion work of 5mJ-15mJ. The in vitro peel force method was performed under the following conditions: fresh porcine gastric mucosa was used as the adhesion carrier, hydrochloric acid solution at pH 1.2 was used as the medium, and the adhesion force and adhesion work between the gel and the mucosa were determined at a constant temperature of 37°C.

[0009] Furthermore, the adhesive thickener is one or more of sodium alginate, chitosan, and hydroxypropyl methylcellulose; the moisturizing stabilizer is one or more of glycerin, propylene glycol, and betaine; the flavoring agent is a chicken or beef flavoring veterinary grade flavoring; and the preservative is one or more of sorbic acid, potassium sorbate, and sodium benzoate.

[0010] Further, the thermosensitive hydrogel matrix is ​​one or more of poly(N-isopropylacrylamide), poloxamer, PLGA-PEG-PLGA, and chitosan / β-glycerophosphate sodium; preferably, the thermosensitive hydrogel matrix is ​​poloxamer 407 and poloxamer 188 in a mass ratio of 6:1.

[0011] Furthermore, the preparation method of the compound thermosensitive hydrogel oral formulation includes the following steps: (1) Cold dissolution preparation of matrix: Cool purified water to 2-8℃, add thermosensitive hydrogel matrix, and magnetically stir at 400rpm to swell for 3h to obtain thermosensitive matrix; (2) Drug loading and dispersion: Add furosemide and triamterene at 2-8℃ and disperse by high-speed shearing; (3) Addition of excipients: Add adhesive thickener, moisturizing stabilizer, flavoring agent and preservative, stir and homogenize; (4) Vacuum degassing: Degassing at -0.08 to -0.1 MPa for 30 to 60 minutes; (5) Aseptic filling: Fill into metering syringes and store under cold storage.

[0012] Further, in step (2), the high-speed shearing speed is 10000-16000 rpm and the time is 5-10 min; in step (3), the homogenization pressure is 20 MPa and the number of homogenizations is 2.

[0013] Furthermore, the quality testing method for the compound thermosensitive hydrogel oral preparation employs HPLC to simultaneously determine the contents of furosemide and triamterene. The chromatographic conditions are as follows: C18 column, mobile phase of 10 mmol / L ammonium acetate solution (pH 3.9) - acetonitrile gradient elution, and detection wavelengths of furosemide at 271 nm and triamterene at 360 nm.

[0014] Furthermore, the compound thermosensitive hydrogel oral formulation is used in the preparation of veterinary drugs for canines and cats to promote diuresis, reduce swelling, and maintain blood potassium balance.

[0015] Due to the adoption of the above solution, the beneficial effects of the present invention are: This invention uses a 1:1 ratio of furosemide and triamterene, which has a significant synergistic diuretic effect, with 24-hour urine output higher than that of the single-drug group; serum potassium concentration is stably maintained within the normal range of 3.5-5.5 mmol / L, and the incidence of hypokalemia is only 8.3%, which is much lower than that of the single-drug group and the human tablet splitting group, effectively reducing the risk of electrolyte disturbance.

[0016] The formulation gels at 28-32℃, remaining in a flowing liquid state at 2-8℃ for easy administration. At 37℃, it rapidly gels and adheres to the gastric mucosa at the body temperature of dogs and cats. The release rate meets the standards (55%-65% release in 4h, ≥85% in 8h, and ≥95% in 12h), with no burst release phenomenon. The blood drug concentration is stable without peak-valley effects, reducing gastrointestinal irritation.

[0017] The formulation is filled using a metering syringe, ensuring precise and error-free dosage. It contains chicken / beef flavoring in veterinary grade, resulting in excellent palatability. The drug acceptance rate in dogs and cats reaches 93.3%, with a refusal rate of 5% and a vomiting rate of 1.7%, which is far superior to traditional dosage forms such as tablets and powders.

[0018] A synchronous quantitative HPLC method was established, which can rapidly and accurately determine the content of two drugs. It is equipped with comprehensive quality control measures, including related substances, release rate, and microbial limits, and meets the requirements of the Chinese Veterinary Pharmacopoeia and the regulatory requirements of the Ministry of Agriculture and Rural Affairs, with high batch-to-batch quality consistency.

[0019] The preparation process employs a solvent-free cold dissolution method, which is mild and suitable for large-scale production. The formulation has undergone high temperature, high humidity, strong light, freeze-thaw cycle, accelerated and long-term stability tests, and all indicators meet the requirements. It exhibits excellent physical, chemical and dosage form stability and is suitable for cold storage and clinical use. Attached Figure Description

[0020] Figure 1 Flowchart of the preparation process of furosemide-triamterene compound thermosensitive hydrogel; Figure 2 Drug release curve after adhering to gastric mucosa with furosemide-triamterene compound thermosensitive hydrogel. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] Example 1 Preparation method (cold dissolution method, no organic solvent) 1. Take 726g of purified water, place it in an ice bath to cool to 4℃, add 180g of poloxamer 407 and 30g of poloxamer 188, stir magnetically at 400rpm, and swell for 3h to obtain a clear matrix. 2. Maintain 4°C, add 10g furosemide and 10g triamterene, and shear at 10000rpm for 8min until no visible particles are observed; 3. Add 10g sodium alginate, 30g glycerin, 3g chicken flavoring and 1g potassium sorbate in sequence, stir at low speed 250rpm for 12min, and homogenize under high pressure 20MPa twice. 4. Vacuum degassing (-0.09MPa), 45 min; 5. Aseptically fill into 3mL measuring syringes, each syringe containing 3.0g, and store under cold conditions.

[0023] Example 2 Preparation method (cold dissolution method, no organic solvent) 1. Take 716.8g of purified water, place it in an ice bath to cool to 4℃, add 180g of poloxamer 407 and 30g of poloxamer 188, stir magnetically at 400rpm, and swell for 3h to obtain a clear matrix; 2. Maintain 4℃, add 10g furosemide and 10g triamterene, and shear at high speed 16000rpm for 8min until no visible particles are visible; 3. Add 10g chitosan, 40g propylene glycol, 2g beef flavoring, and 1.2g sodium benzoate in sequence, stir at low speed 250rpm for 12min, and homogenize under high pressure at 20MPa twice. 4. Vacuum degassing (-0.1MPa), 60min; 5. Aseptically fill into 3mL measuring syringes, each syringe containing 3.0g, and store under cold conditions.

[0024] Comparative Example 1 Preparation method (cold dissolution method, no organic solvent) 1. Take 836.8g of purified water, place it in an ice bath to cool to 4℃, add 100g of poly(N-isopropylacrylamide) (PNIPAM), stir magnetically at 400rpm, and swell for 3h to obtain a clear matrix; 2. Maintain 4°C, add 10g furosemide and 10g triamterene, and shear at 13000rpm for 6min until no visible particles are observed. 3. Add 40g betaine, 2g beef flavoring, and 1.2g sodium benzoate in sequence, stir at low speed 250rpm for 15min, and homogenize under high pressure at 20MPa twice. 4. Vacuum degassing (-0.09MPa) for 50 minutes; 5. Aseptically fill into 3mL measuring syringes, each syringe containing 3.0g, and store under cold conditions.

[0025] Test results 1. Results of formulation characteristic testing Conclusion: All examples were satisfactory, but the comparative example showed burst release.

[0026] Synergistic diuretic and potassium-sparing effects (canine and cat models, n=12 / group) Conclusion: The urine output in the present invention group was significantly higher than that in the single drug group (P<0.05), the serum potassium was maintained within the normal range (3.5–5.5 mmol / L), and the incidence of hypokalemia was significantly lower in the furosemide single drug group and the tablet splitting group (P<0.01).

[0027] Drug compliance (30 dogs and 30 cats) Conclusion: The hydrogel of this invention has significantly better palatability and drug administration compliance than existing dosage forms (P<0.001).

[0028] Stability results Example 1: Results of Influencing Factors Change list Stability results of Example 1 The results of the influencing factor test showed that after being placed under high temperature, high humidity, and strong light irradiation for 10 days and subjected to 3 freeze-thaw cycles, the product's properties, gelation temperature, pH value, content, related substances, release rate, and microbial limits all met the requirements. There were no phenomena such as stratification, water separation, mold growth, or irreversible phase transition. The contents of furosemide and triamterene were stable with minimal degradation, and the gel structure and drug release behavior did not change significantly.

[0029] Stability results showed that no significant changes were observed in any of the tested indicators after 6 months of accelerated testing and 24 months of long-term testing. This indicates that the compound thermosensitive hydrogel of this invention exhibits good physical, chemical, and dosage form stability, and that the formulation and manufacturing process are reasonable, making it suitable for refrigerated storage and clinical use.

[0030] Validity of detection methods ①HPLC content determination: Furosemide showed good linearity in the range of 0.025-0.25 mg / mL, with the linear equation being Y = 3.286 × 10⁻⁶. 6 X + 1.024 × 10 4 R² = 0.9999; triamterene showed good linearity in the range of 0.025-0.25 mg / mL, with a linear equation of Y = 2.752 × 10⁻⁶. 6 X + 8.965 × 10³, R² = 0.9999.

[0031] The blank gel matrix had no interfering peaks, the separation between the two main peaks was >2.0, the tailing factor was 0.95-1.05, and the specificity met the requirements.

[0032] In the repeatability test, furosemide RSD = 0.72% and triamterene RSD = 0.68%; in the intermediate precision test, furosemide RSD = 1.05% and triamterene RSD = 0.97%, both of which meet the requirement of RSD < 2.0%.

[0033] The average recovery rate of furosemide was 96.2%-102.5%, with an RSD of 1.13%; the average recovery rate of triamterene was 95.8%-101.8%, with an RSD of 1.26%, both meeting the recovery rate requirement of 95%-105% in the Veterinary Pharmacopoeia.

[0034] The limit of detection (LOD) for furosemide was 0.0012 mg / mL and the limit of quantitation (LOQ) was 0.0036 mg / mL; the LOD for triamterene was 0.0015 mg / mL and the LQ for quantitation (LOQ) was 0.0045 mg / mL. The sensitivity met the detection requirements.

[0035] Under conditions of mobile phase pH ±0.2, column temperature ±2℃, and flow rate ±0.1mL / min, the RSD of the content determination was <1.5%, and the method showed good robustness.

[0036] ②LC-MS / MS residue detection: Furosemide spiked recovery rate 88%–105%, RSD=6.2%; Aminopterin spiked recovery rate 85%–108%, RSD=7.5%, meeting the residue detection requirements of the Ministry of Agriculture and Rural Affairs.

[0037] In vivo pharmacokinetic studies Experimental protocol: Six healthy beagle dogs were selected and administered a single oral dose of this preparation (1 mg / kg of active ingredient). Control groups were set up, namely furosemide single tablet group and human compound tablet group. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h. Blood drug concentration was determined by LC-MS / MS and pharmacokinetic parameters were calculated.

[0038] Test results Conclusion: This formulation significantly reduces Cmax and delays Tmax, with blood drug concentration fluctuations far lower than ordinary tablets. It exhibits no peak-valley effect, provides stable controlled release, and can effectively reduce the risk of adverse reactions.

[0039] Gastric adhesion retention time test Experimental protocol: A trace amount of fluorescent tracer was added to the formulation. After oral administration to beagle dogs, the adhesion state in the stomach was observed by endoscopy, and the gelation and retention time were recorded.

[0040] Experimental results: The gel was formed in situ in the stomach within 5 minutes after administration and adhered tightly to the gastric mucosa. The gastric retention time was 8.5±1.2h, which was significantly longer than that of ordinary hydrogel (2.0±0.5h). Even 12h after administration, a small amount of gel still adhered to the gastric mucosa, which can achieve long-term controlled release.

[0041] In vitro release mechanism study The release rate data from Example 1 were fitted to a model, and the results showed that the Higuchi model had an R² of 0.9976, the zero-order model had an R² of 0.9423, and the first-order model had an R² of 0.9658.

[0042] Conclusion: The in vitro release of this formulation conforms to the Higuchi diffusion-controlled release mechanism, with no burst release phenomenon and stable drug release, consistent with the drug delivery characteristics of thermosensitive adhesive hydrogels.

[0043] Long-term cold storage stability and simulated transportation stability tests Experimental procedure: The sample from Example 1 was refrigerated at 2~8℃ for 36 months, and a simulated transportation test (5 cycles of shaking-freeze-thaw) was conducted to examine its properties, gelation temperature, content, release rate, related substances, and other indicators.

[0044] Test results Conclusion: After refrigeration at 2-8℃ for 36 months and simulated transportation tests, all indicators of this product met the requirements, with no stratification, water separation, mold growth, or other phenomena. It exhibits excellent physical, chemical, and dosage form stability and is suitable for cold chain storage and transportation in pet hospitals.

[0045] Long-term safety test Experimental protocol: Healthy dogs were orally administered this preparation for 28 consecutive days to examine blood routine, liver and kidney function, electrolytes and organ pathological changes.

[0046] Experimental results: The dog's blood routine, ALT, AST, CRE, BUN and other indicators were all within the normal range; the blood potassium concentration was stably maintained at 3.8~5.2 mmol / L, with no electrolyte disturbance; there were no gastrointestinal ulcers or bleeding, no adverse reactions such as vomiting or diarrhea; there was no pathological damage to organs such as heart, liver, spleen, lungs and kidneys.

[0047] Conclusion: This preparation has good long-term safety and is suitable for clinical use in dogs and cats.

[0048] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the invention should be within the protection scope of the present invention. The above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A furosemide-triamterene compound thermosensitive hydrogel oral formulation, characterized in that, It consists of the following components by mass percentage: Furosemide 0.2%–2.0%, triamterene 0.2%–2.0%, adhesive thickener 0.5%–2.0%, thermosensitive hydrogel matrix 15%–30%, moisturizing stabilizer 2%–5%, flavoring agent 0.1%–0.5%, preservative 0.05%–0.2%, purified water added to the required amount.

2. The compound thermosensitive hydrogel oral formulation according to claim 1, characterized in that, The mass ratio of furosemide to triamterene is 1:1; the gelation temperature of the preparation is 28–32℃, wherein the gelation temperature is determined by the inverted test tube method: take about 10 mL of the product, which is in a flowing liquid state at 2–8℃, and increase the temperature at a rate of 1℃ / min. For every 1℃ increase, tilt the test tube 85° and observe for 3 seconds. The gelation temperature is the temperature at which the sample no longer flows. The average value is taken for 3 parallel determinations.

3. The compound thermosensitive hydrogel oral formulation according to claim 2, characterized in that, The gastric mucosal adhesion properties of the formulation were determined by the in vitro peel force method. The maximum adhesion force was 0.15N-0.35N and the adhesion work was 5mJ-15mJ. The in vitro peel force method was performed under the following conditions: fresh porcine gastric mucosa was used as the adhesion carrier, hydrochloric acid solution with pH 1.2 was used as the medium, and the adhesion force and adhesion work between the gel and the mucosa were determined at a constant temperature of 37℃.

4. The compound thermosensitive hydrogel oral formulation according to claim 1, characterized in that, The adhesive thickener is one or more of sodium alginate, chitosan, and hydroxypropyl methylcellulose; the moisturizing stabilizer is one or more of glycerin, propylene glycol, and betaine; the flavoring agent is a chicken or beef flavoring veterinary grade flavoring; and the preservative is one or more of sorbic acid, potassium sorbate, and sodium benzoate.

5. The compound thermosensitive hydrogel oral formulation according to claim 1, characterized in that, The thermosensitive hydrogel matrix is ​​one or more of poly(N-isopropylacrylamide), poloxamer, PLGA-PEG-PLGA, and chitosan / β-glycerophosphate sodium.

6. The compound thermosensitive hydrogel oral formulation according to claim 5, characterized in that, The thermosensitive hydrogel matrix is ​​composed of poloxamer 407 and poloxamer 188 in a mass ratio of 6:

1.

7. A method for preparing a compound thermosensitive hydrogel oral formulation according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Cold dissolution preparation of matrix: Cool purified water to 2–8℃, add thermosensitive hydrogel matrix, and magnetically stir at 400rpm for 3h to swell and obtain thermosensitive matrix; (2) Drug loading and dispersion: Furosemide and triamterene were added at 2–8℃ and dispersed by high-speed shearing; (3) Addition of excipients: Add adhesive thickener, moisturizing stabilizer, flavoring agent and preservative, stir and homogenize; (4) Vacuum degassing: Degassing at -0.08-0.1 MPa for 30–60 min; (5) Aseptic filling: Fill into metering syringes and store under cold storage.

8. The preparation method according to claim 7, characterized in that, Step (2) The high-speed shearing speed is 10000–16000 rpm, and the time is 5–10 min; Step (3) The homogenization pressure is 20 MPa, and the number of homogenizations is 2.

9. A method for quality testing of the compound thermosensitive hydrogel oral formulation according to claim 1, characterized in that, The contents of furosemide and triamterene were determined simultaneously by HPLC. The chromatographic conditions were as follows: C18 column, mobile phase of 10 mmol / L ammonium acetate solution (pH 3.9)-acetonitrile gradient elution, and detection wavelengths of 271 nm for furosemide and 360 nm for triamterene.

10. The use of the compound thermosensitive hydrogel oral formulation according to any one of claims 1-6 in the preparation of veterinary drugs for canine and feline diuresis, swelling reduction, and maintenance of blood potassium balance.

Images