Nasal in-situ gel for recombinant human interferon alpha2b

Abstract

The invention discloses a recombinant human interferon alpha2b nasal in-situ gel. The in-situ gel is prepared from the following raw materials: recombinant human interferon alpha2b freeze-dried powder, a temperature response type gel matrix, an ion response type gel matrix, a biological mucosa adhesive, a preservative and water. Aiming at the current situations that a recombinant human interferon alpha2b anti-respiratory virus preparation is lacked in the market at present and only a common nasal drop solution is used for researching nasal delivery of the recombinant human interferon alpha2b, the invention develops recombinant human interferon alpha2b double-response nasal in-situ gel for resisting respiratory viruses. The problems that a nasal drop solution is easy to lose, low in bioavailability, required to be used for multiple times and the like are solved. Compared with a solution and single-response in-situ gel, the in-situ gel preparation has the advantages that the residence time of the medicine in the nasal cavity can be obviously prolonged, an in-situ medicine storage is formed, the release of the medicine is more stable and lasting, the improvement of the medicine effect of the medicine is facilitated, and the preparation process of the in-situ gel preparation is simple and feasible.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a recombinant human interferon alpha 2b nasal in-situ gel. Background Art

[0002] Interferon (IFN) is a broad-spectrum antiviral drug and one of the most important cytokines currently used in clinical treatment. When host cells are infected and stimulated by pathogens, IFN is produced. It has multiple biological functions, including regulating the innate and acquired immune responses after infection. Among them, type I IFN binds to cell surface receptors IFNAR1 and IFNAR2, activates cell genes, expresses multiple antiviral proteins through a series of biochemical processes such as signal transduction, and achieves an inhibitory effect on the virus. The respiratory mucosa contains α-IFN receptors. Nasal delivery of α-IFN can cause the upper respiratory mucosal system to produce a large amount of antiviral proteins under the action of IFN, putting the body in an antiviral state, thereby effectively preventing viral replication, inhibiting viral infection and proliferation, and playing a role in emergency prevention and early treatment. People with low immunity find it difficult to produce sufficient endogenous α-IFN in time, and may be more susceptible to viruses and gradually tend to become more severe. Therefore, supplementing exogenous α-IFN in advance can quickly improve the body's immunity and achieve the purpose of preventing and fighting viruses.

[0003] There have been studies on the nasal delivery of α-IFN. Patent CN1539500A discloses a recombinant human interferon α2b nasal drop and its preparation method, which verifies that nasal delivery of interferon can enhance the immunity of animals. However, the nasal drops stay in the nasal cavity for a short time, and the action time of the drug on the nasal mucosa is also shortened, affecting the efficacy of the drug.

[0004] Nasal in situ gel (NSG) is a new nasal administration dosage form that has attracted great attention at home and abroad in recent years, and is also one of the hot spots in the current formulation research field. NSG exists in the form of a low-viscosity liquid before administration. After administration, under the influence of factors such as temperature, pH and ions in the nasal physiological environment, it can quickly undergo a phase transition to form a high-viscosity semi-solid state. NSG has the advantages of convenient administration, easy dosage control, long intranasal retention time, high bioavailability and long-term sustained release. However, NSG formed by a single response mode has different disadvantages. When the in situ gel only uses a thermosensitive material (such as poloxamer 407) as a gel material, it has the disadvantages of low phase transition temperature, gel hysteresis and fast gel dissolution; when only pH-sensitive materials (such as carbomer) are used as gel materials, a large dose is required, which easily leads to excessive acidity and irritation to the mucosa; when only ion-sensitive gel materials (such as gellan gum) are used, there are problems of short gel duration and easy drug leakage. Currently, there are basically no products on the market for nasal delivery of IFN to fight respiratory viruses. Summary of the invention

[0005] One of the purposes of the present invention is to provide a recombinant human interferon α2b nasal in situ gel, the raw materials include, by weight percentage: 0.7% to 2.8% of recombinant human interferon α2b lyophilized powder, 14% to 16% of temperature-responsive gel matrix, 0.1% to 0.3% of ion-responsive gel matrix, 0.1% to 0.3% of biological mucosal adhesive, 0.01% of preservative, and the balance is water, and the sum of the weight percentages of all the raw materials is 100%.

[0006] Furthermore, the raw materials include, by weight percentage: 0.7% to 2.8% recombinant human interferon α2b freeze-dried powder, 15% temperature-responsive gel matrix, 0.2% ion-responsive gel matrix, 0.2% biomucosa adhesive, 0.01% preservative, and the remainder is water, and the sum of the weight percentages of all raw materials is 100%.

[0007] Furthermore, the temperature-responsive gel matrix is ​​poloxamer 407, and the ion-responsive gel matrix is ​​low-acyl gellan gum.

[0008] Furthermore, the biomucosal adhesive is hypromellose.

[0009] Furthermore, the preservative is selected from chlorhexidine acetate, benzalkonium chloride, methylparaben, and propylparaben.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned recombinant human interferon α2b nasal in situ gel, which comprises adding an ion-responsive gel matrix to a portion of the prescribed amount of water, adding a biological mucosal adhesive after complete dissolution, adding recombinant human interferon α2b lyophilized powder and a preservative after stirring and mixing, adding a temperature-responsive gel matrix after stirring and mixing, and replenishing the prescribed amount of water to obtain the gel.

[0011] In view of the current situation that there is a lack of recombinant human interferon α2b anti-respiratory virus preparations on the market, and the research on nasal delivery of recombinant human interferon α2b only has ordinary nasal solution, the present invention has developed a recombinant human interferon α2b dual-response nasal in-situ gel for anti-respiratory virus, which solves the problems of easy loss, low bioavailability, and multiple medication of nasal solution. Compared with solution and single-response in-situ gel, the in-situ gel preparation can significantly prolong the nasal retention time of the drug, form an in-situ drug reservoir, make the release of the drug more stable and lasting, help improve the drug efficacy, and its preparation process is simple and feasible.

[0012] The nasal in-situ gel of the present invention is composed of an ion-responsive gel matrix and a temperature-responsive gel matrix, which synergistically exert gelation under the temperature and calcium ions of the physiological environment of the nasal cavity. At room temperature, the preparation is a transparent liquid. After being dripped or sprayed into the nasal cavity, poloxamer 407 gels after reaching its critical gelation temperature due to the intensified entanglement and accumulation between micelles, and low-acyl gellan gum reacts with calcium ions in the nasal cavity to gel, thereby adhering to the nasal cavity. By adjusting the proportion of each component, it can be gelled at a suitable temperature and ionic strength to form an in-situ drug reservoir, maintain good drug release characteristics, thereby achieving long-term stable release, and extending the duration of the nasal immune barrier while forming a physical protective barrier to block viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a photograph of the ungelled state of the recombinant human interferon α2b dual-responsive nasal in situ gel.

[0014] Figure 2 This is a photo of the recombinant human interferon α2b dual-responsive nasal in situ gel after gelation.

[0015] Figure 3 This is the SEM image of the recombinant human interferon α2b dual-responsive nasal in situ gel.

[0016] Figure 4 This is a curve of shear stress versus shear rate of recombinant human interferon α2b dual-responsive nasal in situ gel.

[0017] Figure 5 This is a curve showing the change in viscosity of recombinant human interferon α2b dual-responsive nasal in-situ gel as a function of shear rate.

[0018] Figure 6 This is the viscosity rheology temperature scanning result curve of recombinant human interferon α2b dual-responsive nasal in-situ gel.

[0019] Figure 7 The results of nasal retention time of different preparations in rats. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0023] The recombinant human interferon α2b lyophilized powder used in the following examples was purchased from Shanghai Huaxin Biotech Co., Ltd.

[0024] Example 1

[0025] The ratio of the two gel matrices that play a gelling role was screened, and the best prescription was screened by measuring the gelling time of different prescription gel groups. The specific experimental plan is as follows: add 1 mL of in-situ gel to a 5 mL transparent glass bottle, place the prepared artificial nasal solution in a 32°C water bath to maintain constant temperature, take 0.2 mL of 32°C artificial nasal solution and add it to 1 mL of in-situ gel, record the time when the gel does not flow after inversion, which is the gelling time, and measure the gelling time of three groups in parallel for each prescription and take the average value.

[0026] The specific formulation composition and gel time test results are as follows:

[0027]

[0028] As shown in the table above, during the prescription screening process, different amounts of gel matrix will affect the gelation time of the in situ gel in a simulated nasal physiological environment. Considering the cost and the degree of time reduction, 15% poloxamer 407 and 0.2% low acyl gellan gum are the best choices.

[0029] After the prescription screening, the optimal proportions of each component of the prescription are as follows:

[0030]

[0031]

[0032] Take 1 mL of the dual-responsive in-situ gel prepared by the optimal prescription, place it in a 5 mL transparent glass bottle, add 32°C artificial nasal fluid, and the state before gelation is as follows: Figure 1 As shown, the gelled state is Figure 2 shown.

[0033] After freeze-drying the dual-responsive in-situ gel prepared by the optimal recipe, a dense network of macromolecular structures was observed by scanning electron microscopy, such as Figure 3 shown.

[0034] The rheological study of the dual-responsive in-situ gel prepared by the optimal formulation is as follows:

[0035] 1. Take the sample prepared with the optimal recipe, select the rotation test mode in the rheometer, and set the shear rate to 0.1-100s -1 The strain amplitude was 0.1%; the relationship between shear rate, shear stress and viscosity was measured at 4, 25 and 35°C respectively.

[0036] like Figure 4 and Figure 5 As shown, at different temperatures, the shear stress gradually increases with the increase of shear rate, while the viscosity decreases with the increase of shear rate. The in situ gel behaves as a pseudoplastic fluid.

[0037] 2. Take the sample prepared by the optimal recipe, select the oscillation test mode in the rheometer, set the scanning angular frequency to 10 rad / s, the strain amplitude to 0.1%; the temperature range is 4-37°C, the heating rate is 2°C / min, and the elastic modulus (G′) and viscous modulus (G″) are measured ( Figure 6 ).

[0038] like Figure 6 As shown, at 26°C, G″=G', indicating that this temperature is the "gel temperature", that is, the sample begins to change from a liquid state to a semi-solid colloidal state; when it is less than 26°C, G″>G', the loss part of the sample's deformation ability is greater than the storage part, the viscosity is low, and it has fluid characteristics; when it is greater than 26°C, G″<G', indicating that the gel has exceeded the gel temperature, and the gel exhibits semi-solid properties with elastic characteristics as the main feature.

[0039] Example 2

[0040] 1. Preparation of 25g human interferon α2b dual-response nasal in-situ gel (preparation group)

[0041]

[0042] Preparation method: Add the prescribed amount of low-acyl gellan gum to 20g of deionized water, stir in a 90°C water bath until completely dissolved, then add the prescribed amount of hydroxypropyl methylcellulose and stir to make it evenly dispersed, remove the water bath, cool at room temperature and continue to stir until the solution is clear, then add the prescribed amount of human interferon α2b lyophilized powder and preservatives and stir at a low speed until they are completely dissolved, finally add the prescribed amount of poloxamer 407 and make up with deionized water to the final mass, refrigerate at 4°C to swell and make it completely dissolved to obtain the final preparation.

[0043] 2. Preparation of 25g human interferon α2b thermosensitive nasal in situ gel (P407 group)

[0044]

[0045] Preparation method: Add the prescribed amount of hydroxypropyl methylcellulose gum to 20 g of deionized water, stir in a 90°C water bath to evenly disperse it, remove the water bath, cool at room temperature and continue stirring until the solution is clear, then add the prescribed amount of human interferon α2b lyophilized powder and preservatives and stir at a low speed until they are completely dissolved, finally add the prescribed amount of poloxamer 407 and make up to the final mass with deionized water, refrigerate at 4°C to swell and completely dissolve it to obtain the final preparation.

[0046] 3. Preparation of 25 g human interferon α2b calcium ion sensitive nasal in situ gel (DGG group)

[0047]

[0048] Preparation method: Add the prescribed amount of low-acyl gellan gum to 20g of deionized water, stir in a 90°C water bath until completely dissolved, then add the prescribed amount of hydroxypropyl methylcellulose and stir to disperse it evenly, remove the water bath, cool at room temperature and continue stirring until the solution is clear, then add the prescribed amount of human interferon α2b lyophilized powder and preservatives and stir at a low speed until they are completely dissolved, add deionized water to the final mass, and obtain the final preparation.

[0049] 4. Preparation of 25 g human interferon α2b nasal solution (solution group)

[0050]

[0051] Preparation method: Add the prescribed amount of hydroxypropyl methylcellulose gum to 20 g of deionized water, stir in a 90°C water bath to make it evenly dispersed, remove the water bath, cool at room temperature and continue stirring until the solution is clear, then add the prescribed amount of human interferon α2b lyophilized powder and preservatives and stir at a low speed until they are completely dissolved, and add deionized water to the final mass to obtain the final preparation.

[0052] After the above preparations were successfully prepared, Cy7.5 fluorescent dye was dispersed into different preparations at a concentration of 10 μg / mL. The rats were divided into 4 groups. After anesthesia with 1% sodium pentobarbital injected intraperitoneally, 10 μL of the above different preparations were slowly dripped into one nostril of the rats through a micro-syringe connected to a capillary hose inserted into the rats' nostrils. The same dosed rat was taken at 0.5, 1, 2, 3, 4, 6, and 8 h after the end of the administration, and transferred to a small animal in vivo imager after anesthesia. The Cy7.5 channel was selected, and the imaging was exposed to collect the fluorescence signal.

[0053] Depend on Figure 7 It can be seen that the fluorescence diffusion range of the solution group is large, and the fluorescence intensity only lasts for three hours, indicating that the solution is easy to lose and has a short nasal retention time; while the P407 group, DGG group and preparation group all formed gels after administration, and the fluorescence intensity and nasal retention time of the preparation group were significantly higher than those of the single gel group, indicating that the combination of the two gel matrices significantly enhanced the nasal retention time of the drug, which is beneficial to the improvement and maintenance of the efficacy.

Claims

1. A recombinant human interferon α2b nasal in situ gel, characterized in that: The raw materials include, by weight percentage: 0.7% to 2.8% recombinant human interferon α2b lyophilized powder, 14% to 16% temperature-responsive gel matrix, 0.1% to 0.3% ion-responsive gel matrix, 0.1% to 0.3% biological mucosal adhesive, 0.01% preservative, and the balance is water. The sum of the weight percentages of all the raw materials is 100%.

2. The recombinant human interferon α2b nasal in situ gel according to claim 1, characterized in that: The raw materials include, by weight percentage: 0.7% to 2.8% recombinant human interferon α2b lyophilized powder, 15% temperature-responsive gel matrix, 0.2% ion-responsive gel matrix, 0.2% biomucosa adhesive, 0.01% preservative, and the balance is water. The sum of the weight percentages of all the raw materials is 100%.

3. The recombinant human interferon α2b nasal in situ gel according to claim 1 or 2, characterized in that: The temperature-responsive gel matrix is ​​poloxamer 407, and the ion-responsive gel matrix is ​​low-acyl gellan gum.

4. The recombinant human interferon α2b nasal in situ gel according to claim 1 or 2, characterized in that: The biomucoadhesive agent is hypromellose.

5. The recombinant human interferon α2b nasal in situ gel according to claim 1 or 2, characterized in that: The preservative is selected from chlorhexidine acetate, benzalkonium chloride, methylparaben and propylparaben.

6. The method for preparing the recombinant human interferon α2b nasal in situ gel according to claim 1, characterized in that: The ion-responsive gel matrix is ​​added to a part of the prescribed amount of water, and after it is completely dissolved, a bio-mucosal adhesive is added, and after stirring and mixing, recombinant human interferon α2b lyophilized powder and a preservative are added, and after stirring and mixing, a temperature-responsive gel matrix is ​​added and the prescribed amount of water is supplemented to obtain the gel.

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