Compound oral film agent for protecting nerve cells

Abstract

The invention discloses a compound oral film preparation for nerve cell protection. The compound oral film preparation comprises a nerve protection type natural product, a compound lipid compound containing phosphoric acid, vitamin or a vitamin-like compound, a nano lipid carrier, a film forming material and pharmaceutically acceptable auxiliary materials. The preparation solves the problems of low absorption and metabolism efficiency and insufficient synergistic effect caused by physicochemical property difference among multiple components, and has the characteristics of convenience in taking and high safety. In-vitro cell experiments show that the film agent can significantly promote the growth of nerve cells, has a significant nerve protection effect, is helpful for treating neurodegenerative diseases and promoting brain health, and has a great application prospect in the aspects of improving memory and attention, and preventing memory degeneration or neural tube malformation.

Description

Technical Field

[0001] The present application relates to the technical field of biopharmaceutical preparations, and in particular to a compound oral thin film for protecting nerve cells. Background Art

[0002] As the aging population intensifies, the incidence of neurodegenerative diseases such as Alzheimer's and Parkinson's has increased significantly. Mitochondria, the core of cellular energy metabolism, have a significant impact on neurodegenerative diseases due to their dysfunction. Existing medications, such as compound levodopa preparations, dopamine receptor agonists, anticholinergics, and amantadine, primarily improve symptoms but do not prevent disease progression. Furthermore, the long-term dependence on medications for chronic diseases can worsen the condition over time, leading to deterioration or even failure of physiological functions.

[0003] Therefore, developing a multi-component synergistic drug preparation for neuronal cell protection, intervening in nerve damage through multiple mechanisms, activating mitochondrial function, improving nerve cell metabolism and repair and regeneration, effectively preventing nerve cell degeneration or neural tube defects, reducing neuroinflammation, and promoting brain health has broad market and clinical application prospects. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a compound oral film for protecting nerve cells, which overcomes the problems of low absorption and metabolic efficiency and lack of synergistic enhancement caused by differences in the physical and chemical properties of multiple components (such as solubility and stability). It is easy to take and has few toxic and side effects.

[0005] The present invention provides a compound oral thin film for protecting nerve cells, characterized in that it comprises the following components: a neuroprotective natural product, a complex lipid compound containing phosphoric acid, vitamins or vitamin-like compounds, a nano-lipid carrier, a film-forming material and pharmaceutically acceptable excipients, wherein the nano-lipid carrier comprises phospholipids and cholesterol, and the neuroprotective natural product, vitamins or vitamin-like compounds are encapsulated in the nano-lipid carrier and exist in the form of lipid nanoparticles.

[0006] In one embodiment, the weight ratio of phospholipid to cholesterol in the nanolipid carrier is 1-6:1.

[0007] In one embodiment, the compound oral thin film consists of a double-layer structure of a basement membrane and a covering membrane, wherein the basement membrane comprises neuroprotective natural product lipid nanoparticles, vitamin or vitamin-like compound lipid nanoparticles, a film-forming material and a pharmaceutically acceptable excipient, and the covering membrane comprises a phosphoric acid-containing complex lipid compound, a film-forming material and a pharmaceutically acceptable excipient.

[0008] In one embodiment, the weight ratio of the neuroprotective natural product to the nano lipid carrier in the neuroprotective natural product lipid nanoparticles is 1:1-20; the weight ratio of the vitamin or vitamin-like compound to the nano lipid carrier in the vitamin or vitamin-like compound lipid nanoparticles is 1:1-20.

[0009] In one embodiment, the weight ratio of the neuroprotective natural product to the nano lipid carrier in the lipid nanoparticles is 1:1-10; the weight ratio of the vitamin or vitamin-like compound to the nano lipid carrier is 1:1-10.

[0010] In one embodiment, the content of the neuroprotective natural product lipid nanoparticles is 1-15 weight %, the content of the phosphoric acid-containing complex lipid compound is 1-20 weight %, the content of the vitamin or vitamin-like compound lipid nanoparticles is 1-15 weight %, the content of the film-forming material is 20-70 weight %, the content of the excipient is 1-30 weight %, and the total content of each component is 100 weight %.

[0011] In one embodiment, the content of the neuroprotective natural product lipid nanoparticles is 5-15 weight%, the content of the phosphoric acid-containing complex lipid compound is 10-20 weight%, the content of the vitamin or vitamin-like compound lipid nanoparticles is 5-15 weight%, the content of the film-forming material is 30-70 weight%, the content of the excipient is 1-30 weight%, and the total content of each component is 100 weight%.

[0012] In one embodiment, the neuroprotective natural product is selected from one or more of emodin, chrysophanol, tanshinone, shikonin, ginkgolide, ginsenosides, astragalus polysaccharide, huperzine A, ferulic acid, urolithin, gastrodin, gastrodilutein, eugenol, kaempferol, resveratrol, and curcumin. Neuroprotective natural products exert anti-inflammatory, antioxidant, and anti-apoptotic effects by regulating mitochondrial metabolism and inducing abnormal mitochondrial autophagy. However, natural products are generally volatile, poorly soluble, physically and chemically unstable, and have extremely low bioavailability.

[0013] In one embodiment, the phosphoric acid-containing complex lipid compound is selected from one or more of L-α-glycerophosphocholine (L-α-GPC), phosphatidylserine (PS), phosphatidylcholine (PC), and glycerophosphatidylserine (GPS). Phosphate-containing complex lipid compounds enhance cellular protection by activating mitochondrial activity and maintaining mitochondrial structural integrity. However, phosphoric acid-containing complex lipid compounds are unstable, highly hygroscopic, and easily denatured.

[0014] In one embodiment, the vitamin or vitamin-like compound is selected from one or more of pyrroloquinoline quinone disodium salt (PQQ for short), coenzyme Q10, B vitamins, vitamin C, vitamin D, vitamin E, para-aminobenzoic acid (PABA for short) or taurine. The B vitamins include vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, folic acid, etc. Vitamins or vitamin-like compounds can participate in the formation and maintenance of nerve cells, promote nerve cell repair and energy metabolism, and scavenge free radicals. However, vitamins and vitamin-like compounds are highly sensitive to light, heat and humidity, and are easily degraded during processing or storage. They are also prone to interact with other ingredients, resulting in poor compatibility and stability, which affects the therapeutic effect.

[0015] In one embodiment, the phospholipid in the nanolipid carrier is selected from one or more of hydrogenated soybean lecithin, soybean lecithin, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine or dilauroylphosphatidylcholine; preferably hydrogenated soybean lecithin.

[0016] In one embodiment, the film-forming material is selected from one or more of pectin, gum, konjac glucan, oligofructose, sodium alginate, pullulan, sodium hyaluronate, collagen, β-glucan, hydroxypropyl methylcellulose (abbreviated as HPMC), hydroxypropyl cellulose (abbreviated as HPC), polyvinyl alcohol, povidone or polyethylene oxide.

[0017] In one embodiment, the excipients are selected from lyophilization excipients, plasticizers, disintegrants, flavoring agents, or surfactants. Among them, the lyophilization excipients can be selected from mannitol, glycine, sorbitol, lactose, glucose, or dextran; the plasticizer can be selected from glycerol, polyethylene glycol, diethyl phthalate, ethyl citrate, triacetin, or propylene glycol; the disintegrant can be selected from mannitol, lactose, or sorbitol; the flavoring agent can be sucralose, stevioside, aspartame, maltose, sucrose, starch sugar, or sodium cyclamate; and the surfactant can be Tween-80 or poloxamer 188.

[0018] The beneficial effects of the present invention are as follows: (1) the use of nanoparticle encapsulation technology solves a series of problems such as low solubility and poor stability of natural products, high hygroscopicity of phospholipid components, photothermal stability and poor compatibility of vitamin substances, thereby improving the stability and bioavailability of active ingredients; (2) the use of double-layer oral film technology overcomes the compatibility problems caused by differences in physical and chemical properties of the components in the compound preparation, thereby broadening the application range of the product; (3) through the synergistic effect of multiple targets, the protection and repair effects of nerve cells can be significantly enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The data are the effect data of the oral thin film of the present invention on promoting the protection of nerve cells. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0021] Example 1 Preparation method of lipid nanoparticles

[0022] The neuroprotective natural product or vitamin or vitamin-like compound is ultrasonically dissolved in a PBS buffer solution (pH = 7.4), which serves as the aqueous phase. Phospholipids and cholesterol are separately added to an organic solvent and ultrasonically dissolved, which serves as the organic phase. The aqueous phase is heated to 50-60°C, and the organic phase is slowly added under stirring to allow the organic phase to gradually evaporate, thereby forming crude liposomes. The crude liposomes are injected into a microfluidizer for homogenization, controlling the pressure of the microfluidizer at 10-30 Mbar, and the cycle is repeated 3-6 times to obtain uniform liposomes. The liposomes are purified through a dialysis bag, and then an appropriate amount of lyophilization excipient is added and freeze-dried to obtain neuroprotective natural product lipid nanoparticles or vitamin or vitamin-like compound lipid nanoparticles. The main components and dosages of the lipid nanoparticles are shown in Table 1 below.

[0023] Table 1

[0024]

[0025] Example 2 Preparation of Oral Thin Film

[0026] (1) Preparation of glue: According to the method of Example 1, the prepared neuroprotective natural product lipid nanoparticles, vitamin or vitamin-like compound lipid nanoparticles, film-forming materials and excipients are added to distilled water, heated to 50°C, and stirred until completely dissolved to obtain glue I; the phospholipid-containing complex lipid compound, film-forming materials and excipients are added to distilled water, heated to 50°C, and stirred until completely dissolved to obtain glue II.

[0027] (2) Defoaming and coating: Place the above-mentioned adhesive solution I and adhesive solution II in a vacuum degassing device to eliminate bubbles. Use a film coater to evenly coat the defoamed adhesive solution I and adhesive solution II on the release film, dry them at 60-80°C, and cut them into appropriate sizes to obtain oral thin films of prescriptions 1-5.

[0028] The amount (by weight) of each ingredient in prescriptions 1-5 is shown in Table 2 below.

[0029] Table 2

[0030]

[0031]

[0032] Example 3 Preparation of Comparative Prescriptions 1-10

[0033] Following a procedure similar to Example 2, the active ingredient, film-forming material, and excipients were added to distilled water, heated to 50°C, and stirred until completely dissolved to obtain a glue solution. The glue solution was placed in a vacuum degassing device to eliminate bubbles. The glue solution was evenly coated on a release film using a film coater, dried, and cut into appropriate sizes to obtain comparative formulations 1-10.

[0034] The amounts (by weight) of the ingredients in the comparative formulas 1-10 are shown in Table 3 below.

[0035] Table 3

[0036]

[0037] Example 4 In vitro cell experiment

[0038] An in vitro cell line model of rat pheochromocytoma cell line (PC-12 cells) was established. PC-12 cells (ATCC CRL-1721) were cultured in complete culture medium (RPMI-1640 + 10% FBS + 1% P / S) in poly-L-lysine-coated culture dishes in an incubator at 37°C and 5% CO2. The cells were proliferated for more than three passages and used in the following experiments.

[0039] A certain amount of oral thin films of prescriptions 1-5 and comparative prescriptions 1-10 were respectively weighed and dissolved in dimethyl sulfoxide (DMSO) to prepare a 5 μM solution for later use.

[0040] Cultured cells were harvested and plated. 24 hours after plating, complete medium supplemented with 5 ng / mL NGF (2.5S NGF, Invitrogen) was used to induce differentiation. The NGF-supplemented medium was replaced every three days. Blank, positive control, and experimental groups were established. Treatments were performed after the eighth day. The blank group received no treatment; the positive control group received 100 ng / mL NGF; and the experimental groups received 100 μl of each of Recipes 1–5 and Comparative Recipes 1–10. After 72 hours of culture, cells were washed with PBS and fixed in 1% paraformaldehyde for 20 minutes. They were washed three times with PBS and fluorescently labeled with a Texas Red maleimide probe, which reacts with the sulfhydryl groups of protein cysteine ​​residues, allowing for complete visualization of cell morphology. Immunofluorescence images were taken and analyzed under a confocal microscope. Images were acquired and the average axon length and number of synaptic branches in each group were measured using the measurement module in Metamorph software. The results are shown in Table 4 below.

[0041] Table 4

[0042]

[0043]

[0044] Example 5 PC-12 cell OGD experiment

[0045] An oxygen-glucose deprivation / reoxygenation (OGD / R) model was established to simulate an in vitro cell model of cerebral ischemia / reperfusion injury, and the protective effects of the test components of each formula on hypoxic injury in PC-12 cells were evaluated.

[0046] Experimental Procedure: PC-12 cells in the logarithmic growth phase were plated adherently in two 96-well culture plates. Culture plate 1 was used for normal culture conditions, while culture plate 2 was used for the oxygen-glucose deprivation (OGD) experiment. Culture plate 1 was assigned to the normal group; culture plate 2 was assigned to the model group (OGD) and the experimental group (OGD + test drug). The test drug was a DMSO solution of formulations 1-5 at a concentration of 5 μM, compared to formulations 1-10. Each group had six wells.

[0047] The culture plate 1 was placed in a common cell culture incubator at 37° C. and 5% CO 2 and cultured in a normal DMEM medium containing sugar and serum (concentration of 10% FBS).

[0048] In culture plate 2, the experimental group was pre-incubated with 5 μM DMSO solutions of formulations 1-5 and control formulations 1-10 for 2 hours in advance. The normal culture medium was then removed from all groups, washed twice with D-Hank solution, and the cells were then placed in low-glucose, serum-free DMEM medium. The culture plate was placed in a tri-gas culture flask (volume fraction N2: 95%, CO2: 5%, O2 <1%) at 37°C for 12 hours. The cells were then removed and replaced with high-glucose, serum-containing (10% FBS) DMEM medium in a standard cell culture incubator for another 4 hours. The culture was terminated, and cell survival was assessed using the MTT assay.

[0049] MTT, short for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, is a yellow dye. The MTT colorimetric assay is a method for measuring cell viability and growth. Its principle is that succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water-insoluble, blue-purple crystalline formazan, which then deposits within the cells. Dead cells, however, do not exhibit this behavior. Dimethyl sulfoxide (DMSO) dissolves the formazan in cells, and its absorbance at 490 nm, measured using an enzyme-linked immunosorbent assay (ELISA), indirectly reflects the number of viable cells. Within a certain cell population range, the amount of MTT crystals formed is proportional to the cell number.

[0050] Remove the culture medium in culture plate 1 and culture plate 2, add 10μl MTT solution (5mg / ml) to each well, and continue to culture for 4h. Terminate the culture and carefully remove the culture medium in the wells. Add 100μl DMSO to each well and shake at low speed on a shaker for 10min to fully dissolve the crystals. Measure the absorbance of each well at a wavelength of 490nm using an enzyme-linked immunosorbent assay (ELISA) and calculate the corresponding cell viability value. Using the cell viability value of the normal group as the benchmark, the calculation formula is: Cell viability (%) = OD value of the experimental group / OD value of the normal group * 100%. The results are as follows: Figure 1 shown.

[0051] The technical features of the above embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A compound oral thin film for protecting nerve cells, characterized in that: The invention comprises the following components: a neuroprotective natural product, a phosphoric acid-containing complex lipid compound, a vitamin or a vitamin-like compound, a nano-lipid carrier, a film-forming material and a pharmaceutically acceptable excipient, wherein the nano-lipid carrier comprises phospholipids and cholesterol, and the neuroprotective natural product, vitamin or vitamin-like compound is encapsulated in the nano-lipid carrier and exists in the form of lipid nanoparticles, and the weight ratio of phospholipid to cholesterol in the nano-lipid carrier is 1-6:

1.

2. The compound oral thin film according to claim 1, characterized in that The compound oral thin film consists of a double-layer structure of a basement membrane and a covering membrane, wherein the basement membrane contains neuroprotective natural product lipid nanoparticles, vitamin or vitamin-like compound lipid nanoparticles, film-forming materials and pharmaceutically acceptable excipients, and the covering membrane contains phosphoric acid-containing complex lipid compounds, film-forming materials and pharmaceutically acceptable excipients.

3. The compound oral film according to claim 2, characterized in that The weight ratio of the neuroprotective natural product to the nano lipid carrier in the neuroprotective natural product lipid nanoparticles is 1:1-20; the weight ratio of the vitamin or vitamin-like compound to the nano lipid carrier in the vitamin or vitamin-like compound lipid nanoparticles is 1:1-20.

4. The compound oral film according to claim 3, characterized in that The weight ratio of the neuroprotective natural product to the nano lipid carrier in the neuroprotective natural product lipid nanoparticles is 1:2-10; the weight ratio of the vitamin or vitamin-like compound to the nano lipid carrier in the vitamin or vitamin-like compound lipid nanoparticles is 1:2-10.

5. The compound oral film according to claim 2, characterized in that: The content of the neuroprotective natural product lipid nanoparticles is 1-15% by weight, the content of the phosphoric acid-containing complex lipid compound is 1-20% by weight, the content of the vitamin or vitamin-like compound lipid nanoparticles is 1-15% by weight, the content of the film-forming material is 20-70% by weight, the content of the excipient is 1-30% by weight, and the total content of each component is 100% by weight.

6. The compound oral film according to claim 5, characterized in that: The content of the neuroprotective natural product lipid nanoparticles is 5-15% by weight, the content of the phosphoric acid-containing complex lipid compound is 10-20% by weight, the content of the vitamin or vitamin-like compound lipid nanoparticles is 5-15% by weight, the content of the film-forming material is 30-70% by weight, the content of the excipient is 1-30% by weight, and the total content of each component is 100% by weight.

7. The compound oral thin film according to any one of claims 1 to 6, characterized in that: The neuroprotective natural product is selected from one or more of emodin, chrysophanol, tanshinone, shikonin, ginkgo lactone, ginsenoside, astragalus polysaccharide, huperzine A, ferulic acid, urolithin, gastrodin, gastrodilutein, eugenol, kaempferol, resveratrol, and curcumin; the phosphoric acid-containing complex lipid compound is selected from one or more of L-α-GPC, PS, PC, and GPS; and the vitamin or vitamin-like compound is selected from one or more of PQQ, coenzyme Q10, B vitamins, vitamin C, vitamin D, vitamin E, vitamin K3, PABA, or taurine.

8. The compound oral thin film according to any one of claims 1 to 6, characterized in that: The phospholipids in the nano lipid carrier are selected from one or more of hydrogenated soybean lecithin, soybean lecithin, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine or dilauroylphosphatidylcholine.

9. The compound oral thin film according to any one of claims 1 to 6, characterized in that: The film-forming material is selected from one or more of pectin, gum, konjac glucan, oligofructose, sodium alginate, pullulan, sodium hyaluronate, collagen, β-glucan, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl alcohol, povidone or polyethylene oxide.

10. The compound oral thin film according to any one of claims 1 to 6, characterized in that: The excipients include one or more of a lyophilizing excipient, a plasticizer, a disintegrant, a flavoring agent or a surfactant; the lyophilizing excipient is selected from mannitol, glycine, sorbitol, lactose, glucose or dextran; the plasticizer is selected from glycerol, polyethylene glycol, diethyl phthalate, ethyl citrate, triacetin or propylene glycol; the disintegrant is selected from mannitol, lactose or sorbitol; the flavoring agent is sucralose, stevioside, aspartame, maltose, sucrose, starch sugar or cyclamate; the surfactant is Tween-80 or poloxamer 188.

Images