A mussel mygol warm-sensitive in-situ gel nasal preparation and application thereof

Abstract

This invention belongs to the field of biomedical technology and provides a mussel adhesive protein thermosensitive in situ gel nasal preparation and its application. The mussel adhesive protein thermosensitive in situ gel nasal preparation comprises the following components by weight percentage: 0.1%~5.0% recombinant mussel adhesive protein, 15%~25% poloxamer 407, 1%~10% poloxamer 188, pharmaceutically acceptable excipients, and the balance being water. The preparation has a viscosity of less than 100 mPa·s at 25°C and can transform into a semi-solid gel within a temperature range of 28°C to 35°C. This invention solves the technical problems of short drug residence time, low bioavailability, and frequent administration in existing postoperative nasal medication formulations. It can significantly prolong the drug's action time on the nasal wound. Relying on the anti-inflammatory, antibacterial, and tissue regeneration-promoting effects of mussel adhesive protein, it effectively promotes postoperative nasal mucosal repair and reduces complications such as adhesion and bleeding, thus having significant clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a mussel adhesive protein thermosensitive in situ gel nasal preparation and its application. Background Technology

[0002] Nasal and sinus surgeries, such as septoplasty, polypectomy, and functional endoscopic sinus surgery (FESS), are common treatments in otolaryngology. These surgeries are crucial for improving nasal ventilation and treating chronic nasal diseases. However, surgery inevitably causes damage to the nasal mucosa, and the postoperative wound healing process is complex, often accompanied by a series of complications such as nasal adhesions, postoperative bleeding, and infection, which seriously affect the final surgical outcome and the patient's quality of life. Statistics show that the incidence of nasal adhesions can be as high as 6%-44%, and it is one of the main causes of surgical failure and symptom recurrence.

[0003] Currently, clinical treatments for postoperative management of the nasal cavity mainly include systemic or local administration of steroids and antibiotics, nasal irrigation, and the use of absorbable nasal packing. Among these, nasal sprays are widely used as a convenient method of local drug delivery. However, existing sprays have a core technical drawback: due to the continuous mucociliary clearance function of the cilia of the nasal mucosa epithelial cells, the effective residence time of the sprayed solution in the nasal cavity is usually only 5-15 minutes. This results in extremely low drug bioavailability (approximately 15-20%), requiring frequent administration (e.g., 4-6 times daily) to maintain an effective therapeutic concentration. This not only causes inconvenience to patients but also reduces treatment adherence.

[0004] Mussel adhesive protein (MAP) is a type of natural biological protein extracted from the byssal glands of mussels. Its molecular structure is rich in 3,4-dihydroxyphenylalanine (DOPA) residues, which endows it with excellent adhesion in moist environments. Recent studies have found that, in addition to its adhesive properties, mussel adhesive protein also possesses significant anti-inflammatory, antibacterial, and wound-healing-promoting pharmacological activities, demonstrating its great application potential in tissue engineering and wound repair.

[0005] Thermosensitive in-situ gels are an advanced drug delivery system. These materials exist as low-viscosity liquids at room temperature, facilitating drug delivery via spraying or other methods. Upon contact with body temperature, they undergo a rapid phase transition, forming a semi-solid gel. This "in-situ gelation" characteristic allows them to adhere to the drug delivery site for extended periods, significantly prolonging drug retention time and enabling slow drug release. Poloxamer is a commonly used thermosensitive gel material approved by the US FDA, with the combination of poloxamer 407 (P407) and poloxamer 188 (P188) being widely used in constructing thermosensitive gel systems. By adjusting the concentration and ratio of these two components, the phase transition temperature of the gel can be precisely controlled to suit different clinical applications.

[0006] A search revealed that international patent WO2017028777A1 discloses the application of mussel adhesive protein in inhibiting mucosal inflammation, with dosage forms including liquids and gels, and indications covering rhinitis, otitis media, etc. However, this patent primarily discloses ordinary sprays or gels and does not involve temperature-sensitive technology capable of forming a gel in situ within the nasal cavity. Therefore, its spray form still cannot overcome the inherent limitation of short drug residence time. Furthermore, this patent does not specifically disclose the application of this technology to the specific and more demanding indication of postoperative nasal wound repair.

[0007] Therefore, there is a need for a mussel adhesive protein thermosensitive in situ gel nasal preparation and its application to solve the above problems. Summary of the Invention

[0008] The purpose of this invention is to provide a mussel adhesive protein thermosensitive in situ gel nasal preparation and its application, so as to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a mussel adhesive protein thermosensitive in situ gel nasal preparation, comprising the following components by weight percentage: 0.1%~5.0% recombinant mussel adhesive protein, 15%~25% poloxamer 407, 1%~10% poloxamer 188, pharmaceutically acceptable excipients, and the balance being water; the preparation has a viscosity of less than 100 mPa·s at 25°C and can be converted into a semi-solid gel in a temperature range of 28°C to 35°C.

[0010] In a further technical solution, the weight percentage of the recombinant mussel adhesive protein in the formulation is 1.0% to 3.0%. This concentration range can balance the biological activity of the recombinant mussel adhesive protein with the stability of the formulation's temperature-sensitive phase change characteristics, and avoid the protein concentration being too high, which would affect the gelation effect of the gel matrix.

[0011] In a further technical solution, the weight percentage of poloxamer 407 in the formulation is 18%~22%. As the main gelling component of the thermosensitive gel, this concentration can form a good synergistic effect with poloxamer 188, and accurately control the gel phase transition temperature.

[0012] In a further technical solution, the weight ratio of poloxamer 407 to poloxamer 188 is 9:1 to 8:2. This ratio is key to achieving dual optimization of the formulation's phase change temperature and gel mechanical strength, enabling the formulation to rapidly gel and adhere stably at the physiological temperature of the nasal cavity.

[0013] A further technical solution is that the gel transition temperature of the formulation is 30°C to 33°C, which is highly matched with the physiological temperature of 32-34°C in the nasal cavity, ensuring that the formulation can quickly complete the liquid-gel phase transition after entering the nasal cavity.

[0014] A further technical solution is that the storage modulus of the formulation at 32°C is greater than 1000 Pa. The sufficient storage modulus enables the formed semi-solid gel to have good mechanical strength and effectively resist the clearing action of nasal cilia.

[0015] In a further technical solution, the pharmaceutically acceptable excipients include buffers, isotonic regulators, and preservatives. Each type of excipient performs its own function, respectively achieving the functions of pH adjustment, osmotic pressure balance, and improved storage stability of the formulation.

[0016] A further technical solution is that the pH value of the preparation is 5.5-7.0, which is compatible with the physiological environment of the nasal mucosa and can reduce the irritation of the preparation to the nasal mucosa.

[0017] In a further technical solution, the buffer is a phosphate buffer solution, the isotonic regulator is sodium chloride, and the preservative is potassium sorbate or benzalkonium chloride. The phosphate buffer solution can stably adjust the pH of the preparation to the physiologically suitable range, the sodium chloride ensures that the osmotic pressure of the preparation is consistent with that of the nasal cavity fluid, and the potassium sorbate or benzalkonium chloride can effectively inhibit the growth of microorganisms and improve the storage stability of the preparation.

[0018] The application of a mussel adhesive protein thermosensitive in situ gel nasal preparation, applied to any of the mussel adhesive protein thermosensitive in situ gel nasal preparations described above, includes using the preparation to prepare a drug for preventing or treating mucosal damage after nasal or sinus surgery, wherein the nasal or sinus surgery includes septoplasty, nasal polyp removal, functional endoscopic sinus surgery, or turbinate reconstruction, and the mucosal damage includes poor postoperative wound healing, mucosal edema, inflammatory reaction, or nasal adhesions.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] This invention features intelligent phase transition, significantly extending the retention time: This formulation utilizes thermosensitive properties to form a gel in situ within the nasal cavity, effectively resisting the clearance action of nasal cilia. This significantly extends the effective retention time of the drug in the nasal cavity from 5-15 minutes of traditional sprays to several hours, increasing the retention half-life by approximately 7.5 times.

[0021] This invention improves bioavailability and enhances efficacy: by prolonging the contact time between the drug and the wound, this formulation significantly improves the bioavailability of mussel adhesive protein in the nasal mucosa, thereby more fully exerting its anti-inflammatory, antibacterial, and tissue regeneration-promoting pharmacological effects.

[0022] This invention utilizes multiple pharmacological mechanisms to effectively promote mucosal repair: This formulation combines the biological functions of mussel adhesive protein with the physical barrier effect of gel, which can effectively inhibit postoperative inflammation, prevent infection, and guide the orderly growth of epithelial cells, thereby significantly reducing the incidence of nasal adhesions and accelerating wound healing and the recovery of ciliary function.

[0023] This invention is convenient to use and improves patient compliance: the administration method of this formulation is simple, and due to its long-acting sustained-release characteristics, it can significantly reduce the number of daily doses, greatly improving the convenience and compliance of patients.

[0024] To more clearly illustrate the structural features and effects of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the thermosensitive phase transition of the formulation of the present invention. The formulation is a low-viscosity liquid that can be sprayed at room temperature of 25°C. After entering the nasal cavity, it completes the phase transition at physiological temperature, forming a semi-solid gel that adheres to the wound.

[0026] Figure 2 This is a comparison of the in vitro release curves of the formulation of the present invention and ordinary mussel adhesive protein solution, clearly showing the difference between the long-acting sustained-release characteristics of the formulation of the present invention and the rapid release of ordinary solution.

[0027] Figure 3 The diagram shows the nasal endoscopy scoring results of each group in the animal experiment of this invention, visually presenting the differences in wound healing scores among the groups on postoperative days 7 and 14.

[0028] Figure 4 These are representative HE-stained images of the nasal mucosa tissue from each group in the animal experiments of this invention (day 14), showing the histological differences in the nasal mucosa epithelium, ciliary regeneration, and inflammatory cell infiltration among the groups. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments.

[0030] The following embodiments are used to illustrate the present invention, but should not be used to limit the scope of protection of the present invention. The conditions in the embodiments can be further adjusted according to specific conditions, and simple improvements to the method of the present invention under the premise of the concept of the present invention are all within the scope of protection claimed by the present invention.

[0031] Example 1

[0032] like Figure 1-4 As shown in the figure, this invention provides a method for preparing a mussel adhesive protein thermosensitive in situ gel nasal preparation. The preparation is made by cold compounding. The preparation includes recombinant mussel adhesive protein, poloxamer 407, poloxamer 188, pharmaceutically acceptable excipients, and the balance water for injection. The excipients are sodium chloride and potassium sorbate. The pH is also adjusted using phosphate buffer.

[0033] In this embodiment, the prescribed amounts of poloxamer 407 and poloxamer 188 were accurately weighed and slowly dispersed in a specified amount of water for injection placed in a 4°C cold water bath. The mixture was continuously stirred until completely dissolved, forming a clear gel matrix solution. Then, recombinant mussel adhesive protein, sodium chloride, and potassium sorbate were added to the above solution, and stirring was continued until completely dissolved. Finally, the volume was adjusted to 100 mL with water for injection, and the pH was adjusted to 6.5 with phosphate buffer to obtain the formulation. The composition of each formulation is shown in Table 1 below.

[0034]

[0035] Example 2

[0036] The difference between this embodiment and Example 1 is that the physicochemical properties of the four formulations A, B, C, and D prepared in Example 1 were characterized, and the detection indicators included gel transition temperature, rheological properties, and spray properties. There were no adjustments to the formulation components or preparation steps.

[0037] In this embodiment, a rotational rheometer is used to perform temperature scanning on each formulation, using the storage modulus (G) as the metric. ， ) and loss modulus (G ，， The temperature corresponding to the intersection of the two points is recorded as the gel transition temperature. The viscosity at 25℃ and the G at 34℃ (simulated nasal cavity temperature) are also recorded. ，The physicochemical properties of each formulation are shown in Table 2 below. The optimal formulation B was selected and loaded into a commercially available quantitative nasal spray pump. Spray characteristics were tested at room temperature (25℃), including the spray angle and median particle size. The results showed that all prepared gels possessed ideal temperature-sensitive properties, exhibiting low viscosity and good flowability at room temperature. The gel transition temperature was within the range of 30-34℃, which matched the physiological temperature of the nasal cavity well. Formulation B had the most ideal gel transition temperature (Tsol-gel) of 32.8℃. At 34℃, all formulations formed elastic gels with sufficient mechanical strength (G'>5000Pa), effectively resisting the clearance action of nasal cilia. Formulation B had a spray angle of 55.6±4.8° and a median particle size (D50) of 65.2±5.5μm. The droplet size distribution met the optimal range for nasal administration (30-100μm), ensuring effective drug deposition on the nasal mucosa.

[0038]

[0039] Example 3

[0040] The difference between this embodiment and Example 2 is that the in vitro release characteristics of the optimal formulation B determined in Example 2 were studied using the Franz diffusion cell method, with a common mussel adhesive protein solution of the same concentration as a control, and no formulation-related adjustments were made.

[0041] In this embodiment, formulation B and ordinary mussel adhesive protein solution were placed in the drug delivery chamber of a Franz diffusion cell, while the receiving chamber was filled with phosphate buffer (pH 6.5) containing 0.9% sodium chloride. The diffusion cell temperature was maintained at 34°C to simulate the physiological temperature of the nasal cavity. Samples were taken at 0.5, 1, 2, 4, 8, 12, and 24 hours after drug administration to calculate the cumulative release rate. The results are as follows: Figure 2 As shown, the gel formulation of this invention continuously releases mussel adhesive protein within 24 hours, exhibiting a clear sustained-release characteristic; while the ordinary solution releases more than 90% within 2 hours; the kinetic model fitting of the release data shows that the release process best conforms to the Higuchi model (R²=0.9962), indicating that the drug release is mainly controlled by the diffusion mechanism.

[0042] Example 4

[0043] The difference between this embodiment and Example 3 is that: the optimal formulation B from Example 2 was subjected to biocompatibility and safety studies, and the tests included in vitro cytotoxicity, nasal mucosal irritation and ciliary toxicity experiments, without any formulation-related adjustments.

[0044] In this embodiment, the MTT assay was used to evaluate the in vitro cytotoxicity of formulation B on human nasal mucosal epithelial cells. The results showed that the cell survival rate was greater than 90%, indicating that the formulation had no significant cytotoxicity. Nasal mucosal irritation experiments were conducted on New Zealand white rabbits. After 7 consecutive days of administration, there was no redness, swelling, congestion, or erosion of the nasal mucosa on the administration side, and histopathological examination showed no inflammatory cell infiltration or tissue damage. The ciliary toxicity experiment was conducted using the isolated toad maxillary ciliary movement method. The results showed that the formulation of the present invention had no adverse effect on the ciliary function of the nasal mucosa. The above results demonstrate that the formulation of the present invention has good biocompatibility and safety.

[0045] Example 5

[0046] The difference between this embodiment and Example 4 is that: pharmacodynamic studies were conducted on the optimal formulation B from Example 2, including in vivo residence time determination and animal experiments on nasal mucosal damage repair, without any formulation-related adjustments.

[0047] In this embodiment, in vivo fluorescence imaging was used to compare the nasal retention capacity of Formulation B with that of ordinary mussel adhesive protein solution, commercially available budesonide nasal spray, and commercially available mometasone furoate nasal spray in an SD rat model. All formulations were fluorescently labeled with FITC. The fluorescence signal intensity in the nasal cavity was detected at 0.5, 1, 2, 4, 6, and 8 hours after administration. The percentage of drug retention was calculated and pharmacokinetic parameters were analyzed. The results are shown in Table 3 below. At the same time, a nasal mucosal injury model was established in New Zealand white rabbits and they were randomly divided into a blank control group, a matrix control group, a positive control group, the present invention group, and a commercially available spray group. They were administered the drug continuously for 14 days. Nasal endoscopy scoring was performed on the 7th and 14th days after surgery. On the 14th day, the animals were sacrificed for histological HE staining, immunohistochemistry, and inflammatory factor detection. The results showed that the nasal retention half-life of the formulation of the present invention was 4.52±0.38h, which was about 7.5 times longer than that of ordinary solution and about 10.8-11.9 times longer than that of commercially available sprays. The nasal endoscopy score showed that the present invention group was significantly lower than that of the blank, matrix and commercially available spray groups (p<0.01), and there was no significant difference from the positive control group. In addition, the mucosal epithelial layer of the present invention group was intact, the ciliary regeneration was good, the level of inflammatory factors was significantly reduced, and the positive expression rate of cell proliferation and angiogenesis markers was significantly increased.

[0048]

[0049] Example 6

[0050] The difference between this embodiment and Example 5 is that a stability study was conducted on the optimal formulation B from Example 2, including accelerated stability and long-term stability, without any formulation-related adjustments.

[0051] In this embodiment, formulation B was subjected to accelerated stability testing. It was placed at 40°C and 75%RH for 6 months, and the appearance, pH value, gel transition temperature and mussel adhesive protein content of the formulation were tested periodically. The results showed that there were no significant changes in any of the indicators. At the same time, a long-term stability test was conducted. It was stored at 2~8°C for 12 months and all indicators were stable. It is estimated that its shelf life is at least 12 months. Further long-term stability can be verified by extending the observation period.

[0052] Working principle and usage process of this invention:

[0053] The process revolves around a complete flow of "spray administration - in-situ phase transition - long-acting retention - slow release - synergistic repair - gel metabolism," with each step closely linked to fully leverage the physical properties and biological activity of the formulation.

[0054] 1. Preparation and spraying of the drug: The formulation is a low-viscosity liquid with a viscosity of less than 100 mPa·s at room temperature of 25°C. It has good fluidity and can be directly loaded into a metered nasal spray pump for spraying. The droplet size after atomization meets the requirements for nasal administration and can be accurately deposited on the surface of the nasal mucosa without being inhaled into the lungs.

[0055] 2. In-situ phase transition in the nasal cavity: After the formulation spray enters the nasal cavity, it comes into contact with the physiological temperature of the nasal cavity, which is 32-34°C. Since the gel transition temperature of the formulation is 30°C to 33°C, which is highly matched with the temperature of the nasal cavity, poloxamer 407 and poloxamer 188 in the formulation rapidly undergo intermolecular association, transforming from a random coil structure to a three-dimensional network structure. The liquid-gel phase transition is completed in a very short time, forming a semi-solid gel.

[0056] 3. Long-lasting retention on the wound: The semi-solid gel formed has a storage modulus greater than 1000Pa at 32℃, possessing sufficient mechanical strength and adhesiveness, and can stably adhere to the nasal cavity wound, effectively resisting the continuous clearance action of nasal cilia, achieving long-lasting retention of the drug on the wound, and overcoming the drawback of traditional sprays being cleared in a short time.

[0057] 4. Slow drug release: Recombinant mussel adhesive protein, as the active ingredient, is uniformly dispersed in the three-dimensional network structure of the gel. It is released slowly into the wound tissue through the pores of the gel via diffusion as the main release mechanism. The release process conforms to the Higuchi model and can be continuously released within 24 hours, ensuring that the wound is always within the effective drug concentration range.

[0058] 5. Synergistic Physical and Biological Repair: The gel forms a dense physical barrier on the wound surface, isolating external microorganisms and preventing wound infection, while providing support for epithelial cell growth; the slowly released recombinant mussel adhesive protein exerts anti-inflammatory, antibacterial, and tissue regeneration-promoting effects, inhibits the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α, reduces mucosal edema and inflammatory response, promotes Ki-67 positive cell proliferation and CD31 positive angiogenesis, guides orderly epithelial cell growth, and accelerates wound healing and ciliary function recovery.

[0059] 6. Gel metabolism: As the wound gradually heals, the gel matrix, as a biocompatible pharmaceutical excipient, slowly swells and degrades under the action of nasal fluid, and is excreted from the body through normal physiological metabolism of the nasal cavity, leaving no residue, causing no irritation, and will not cause secondary damage to the nasal mucosa.

[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mussel adhesive protein thermosensitive in-situ gel nasal preparation, characterized in that, The formulation comprises, by weight percentage, the following components: 0.1%–5.0% recombinant mussel adhesive protein, 15%–25% poloxamer 407, 1%–10% poloxamer 188, pharmaceutically acceptable excipients, and the balance being water; the formulation has a viscosity of less than 100 mPa·s at 25°C and can be converted into a semi-solid gel in the temperature range of 28°C to 35°C.

2. The mussel adhesive protein thermosensitive in-situ gel nasal preparation according to claim 1, characterized in that, The recombinant mussel adhesive protein constitutes 1.0% to 3.0% of the weight in the formulation.

3. The mussel adhesive protein thermosensitive in-situ gel nasal preparation according to claim 1, characterized in that, The weight percentage of poloxamer 407 in the formulation is 18% to 22%.

4. The mussel adhesive protein thermosensitive in-situ gel nasal preparation according to claim 1, characterized in that, The weight ratio of poloxamer 407 to poloxamer 188 is 9:1 to 8:

2.

5. The mussel adhesive protein thermosensitive in-situ gel nasal preparation according to claim 1, characterized in that, The gel transition temperature of the formulation is 30°C to 33°C.

6. The mussel adhesive protein thermosensitive in-situ gel nasal preparation according to claim 1, characterized in that, The formulation has a storage modulus greater than 1000 Pa at 32°C.

7. The mussel adhesive protein thermosensitive in-situ gel nasal preparation according to claim 1, characterized in that, Pharmaceutically acceptable excipients include buffers, isotonic modifiers, and preservatives.

8. The mussel adhesive protein thermosensitive in-situ gel nasal preparation according to claim 1, characterized in that, The pH value of the preparation is 5.5-7.

0.

9. The mussel adhesive protein thermosensitive in-situ gel nasal preparation according to claim 7, characterized in that, The buffer is a phosphate buffer, the isotonic regulator is sodium chloride, and the preservative is potassium sorbate or benzalkonium chloride.

10. The application of a mussel adhesive protein thermosensitive in-situ gel nasal preparation, used in any one of the mussel adhesive protein thermosensitive in-situ gel nasal preparations according to claims 1-9, characterized in that, This includes using the formulation to prepare a drug for the prevention or treatment of mucosal damage following nasal or sinus surgery, wherein the nasal or sinus surgery includes septoplasty, polypectomy, functional endoscopic sinus surgery, or turbinate reconstruction, and the mucosal damage includes poor wound healing, mucosal edema, inflammatory response, or nasal adhesions.

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