Application of fat cell extract in repairing nerve damage

Abstract

An extract is a water-soluble substance obtained from isolated fat tissue and has the function of repairing nerve damage. It is used as an active ingredient to make medicines or medical devices for repairing and treating nerve damage.

Description

Technical Field

[0001] This invention relates to a tissue extract, and more particularly to a mixture extracted from isolated adipose tissue that has a repairing effect on damaged nerves. Background Technology

[0002] Trauma, tumors, cerebrovascular diseases, and metabolic abnormalities are just some of the various causes that can lead to nerve damage. Nerve damage is a general and unified description of diseases affecting the nervous system. Acute and chronic nerve injuries are common clinical conditions that seriously endanger human health. Based on the location of the injury, they can be divided into central and peripheral nerve injuries. Central nerve injuries mainly include spinal cord injury, stroke, cerebral hemorrhage, cerebral infarction, cerebral palsy, and their sequelae, which have high morbidity, disability, and mortality rates. Peripheral nerve injuries mainly include injuries to the optic nerve, facial nerve, sciatic nerve, and brachial plexus. Current treatment methods for nerve injuries mainly include surgical treatment, anti-inflammatory treatment, and free radical scavenging therapy. However, current treatment methods still have problems such as high surgical risks, complex procedures, and uncertain drug efficacy; effective therapeutic drugs are still lacking. Summary of the Invention

[0003] One object of the present invention is to provide an extract obtained from isolated adipose tissue for use in the preparation of drugs for repairing nerve damage.

[0004] Another object of the present invention is to provide a composition having an extract obtained from isolated adipose tissue as an active ingredient for repairing nerve damage.

[0005] Another object of the present invention is to provide a medical device that uses an extract obtained from isolated adipose tissue as an active ingredient for repairing nerve damage.

[0006] The extract referred to in this invention should be understood as an extract obtained through separation and purification methods, such as, but not limited to, extraction, static separation, centrifugation, chromatography, and crystallization, and is a water-soluble substance.

[0007] The extract of this invention is a water-soluble substance obtained from adipose tissue by first breaking it down through physical means such as emulsification, and then separating it (Stem Cell Res Ther. 2018, 8; 9, 294).

[0008] The extract of this invention is a cell-free fat extract (CEFFE).

[0009] The extract of the present invention is obtained by emulsifying isolated fat and then centrifuging it, and the liquid layer is taken from it.

[0010] The extract of the present invention is obtained by first emulsifying isolated fat, then subjecting it to one or more freeze-thaw cycles (e.g., freezing at -80°C and then thawing at 37°C), and finally centrifuging to obtain the liquid layer.

[0011] The extracted extract can be used clinically to repair nerve damage. Excipients can be added to the extract to prepare a liquid formulation. Alternatively, freeze-drying can be used to effectively preserve the extract's bioactivity, and excipients can be added before use to form a composition (e.g., a formulation). It can also be combined with other materials, such as biocompatible or biodegradable materials, to create medical devices, such as dressings or stents.

[0012] The compositions of this invention use extracts obtained from isolated adipose tissue as active ingredients and also include various pharmaceutical excipients adapted to the contained compounds or compositions to formulate dosage forms that facilitate drug delivery, such as, but not limited to, aqueous injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder mists, sustained-release agents, and controlled-release agents. These pharmaceutical excipients can be those conventionally used in various formulations, such as, but not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants; or they can be selected for compatibility with the substances, such as emulsifiers, solubilizers, antibacterial agents, analgesics, and antioxidants. These excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release and absorption rates of the compounds, thereby improving the metabolism of various compounds in vivo and enhancing the drug delivery effect of the composition. In addition, excipients may be used to achieve specific drug delivery purposes or methods, such as sustained-release drug delivery, controlled-release drug delivery, and pulsatile drug delivery. These excipients include, but are not limited to, gelatin, albumin, chitosan, polyethers, and polyester polymers, such as, but not limited to, polyethylene glycol, polyurethane, polycarbonate, and their copolymers. The main manifestations of what is referred to as "facilitating drug delivery" include, but are not limited to, improved therapeutic efficacy, increased bioavailability, reduced toxicity and side effects, and improved patient compliance.

[0013] In aqueous injections, excipients generally include isotonic agents and buffer solutions, as well as necessary emulsifiers (such as Tweeen-80, Pluronic, and Poloxamer), solubilizers, and antibacterial agents. In addition, they may include other pharmaceutically acceptable excipients, such as antioxidants, pH adjusters, and analgesics.

[0014] Excipients used in the preparation of oral liquid formulations generally include solvents, as well as necessary flavoring agents, antibacterial agents, emulsifiers, and coloring agents.

[0015] Excipients used in tablet manufacturing generally include fillers (such as starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, and mannitol), binders (such as ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution, and aqueous or alcoholic solutions of polyvinylpyrrolidone), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose, and croscarmellose sodium), and lubricants (such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, and magnesium lauryl sulfate).

[0016] The excipients used in the preparation of emulsions are generally water, oil (such as fatty acids), emulsifiers, and necessary preservatives and flavoring agents.

[0017] The excipients used to produce granules are similar to those used for tablets, but the granulation process is different. Depending on the requirements, the produced granules are mixed with a gliding agent and then encapsulated to obtain capsules.

[0018] When the extract of this invention was administered to test animals, it was confirmed that CEFFE treatment could effectively reduce brain tissue damage after cerebral ischemia in mice, reduce neurological function damage and improve behavioral recovery, indicating that CEFFE can repair nerve damage and has a nerve regeneration effect. This suggests that CEFFE has a therapeutic effect on central nervous system injury diseases (such as, but not limited to, spinal cord injury, stroke, cerebral hemorrhage, cerebral infarction, cerebral palsy and its sequelae) or peripheral nerve injury diseases (such as, but not limited to, optic nerve, facial nerve, sciatic nerve and brachial plexus injury). Attached Figure Description

[0019] Figure 1 This is a statistical graph showing the volume of cerebral infarction in each group of mice. Detailed Implementation

[0020] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0021] The specific experimental methods used in the following embodiments of the present invention are described below:

[0022] 1) Ex vivo adipose tissue

[0023] Fat tissue is obtained from healthy women undergoing routine liposuction. The specific method is as follows: After local anesthesia with tumescent fluid, a 3mm liposuction cannula with a large side hole (2mm × 7mm) is connected to a 20mL syringe. Radial aspiration is performed under artificial negative pressure. The obtained fat is held upright and stationary. After removing the tumescent fluid, the fat is rinsed 2-3 times with saline solution and set aside. The rinsed fat tissue is then centrifuged at 1200g at 4℃ for 3 minutes.

[0024] 2) Neurological function score

[0025] To evaluate the balance, movement, and reflexes of mice, the Modified Neurological Severity Scores (mNSS) method (DOI: 10.1212 / wnl.59.4.514) was used to assess mice on days 1, 3, 7, and 14. The severity score ranged from 0 to 14, where 0 represented normal and higher scores indicated more severe impairment.

[0026] 3) Calculation of cerebral infarction volume

[0027] ImageJ software was used to draw the boundary between infarcted and non-infarcted tissues in the cerebral hemisphere. The volume of the contralateral cerebral hemisphere minus the volume of the ipsilateral non-infarcted hemisphere equals the infarct volume.

[0028] 4) Statistical analysis

[0029] Data were analyzed using SPSS software for independent group t-tests to assess the significance of differences. Results are expressed as mean ± standard deviation.

[0030] Example 1

[0031] After centrifugation of isolated adipose tissue in a container, the upper oil layer and lower water layer were removed. The central fat layer was mechanically emulsified by repeatedly and uniformly pushing it 30 times using two 10mL syringes connected to three-way stopcocks. After emulsification, the fat was frozen at -80℃ and then thawed at 37℃. After a single freeze-thaw cycle, 1200g of fat was centrifuged at 4℃ for 5 minutes. After centrifugation, the fat separated into four layers: the first layer was the oil layer, the second layer was the residual adipose tissue, the third layer was the liquid layer, and the fourth layer was cell / tissue debris. The first and second layers were discarded. The liquid layer was carefully aspirated, avoiding contamination from the bottom sediment. The liquid was filtered through a 0.22μm filter for sterilization to remove cell debris, aliquoted, and frozen at -20℃. It was thawed at 4℃ before use.

[0032] Example 2

[0033] Six-week-old male C57 mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The common carotid artery, external carotid artery, internal carotid artery, and their branches were isolated. A small incision was made in the wall of the external carotid artery, and a 6-0 nylon suture was inserted through the external carotid artery. The suture plug was then advanced to the internal carotid artery and then upward to the middle cerebral artery. The success of the occlusion was determined by monitoring the surface cerebral blood flow using a laser Doppler flowmeter (Moor Instruments, Devon, UK) (reduction to 10% of baseline cerebral blood flow). One hour after occlusion, the suture plug was slowly removed from the artery, and the suture entry point was ligated. The cerebral blood flow was observed to have returned to 100%. The experimental group was treated immediately after modeling (Day 0) with CEFFE injection (5 μl / g body weight) via the tail vein, once every 3 days for a total of 5 injections. The blank control group was injected with an equal volume of phosphate buffered saline (PBS, pH 7.4). The sample size for each group was 8 mice.

[0034] Example 3

[0035] This experiment used the mNSS scoring system to evaluate neuromotor dysfunction in mice after CEF treatment, and the results are shown in Table 1. Compared with PBS, the neurological function scores of mice in the CEF treatment group were significantly lower on days 7 and 14 (p < 0.01, p < 0.05), indicating that CEF treatment can reduce the neurological function scores of MCAO mice and effectively alleviate neurological damage.

[0036] Table 1. mNSS scores of mice in each group

[0037]

[0038] * p < 0.05 ** p < 0.01

[0039] Example 4

[0040] To measure the recovery of motor function in mice, a rotation test was performed on days 3, 7, and 14. This test required the mice to maintain balance on a fatigue rotundus. The animals first adapted to the bar for 1 minute, then the bar began to rotate and steadily accelerated to 40 revolutions per minute within 2 minutes. The time the mice maintained their balance on the rotundus was recorded. The test was repeated three times, and the results are shown in Table 2.

[0041] Mice in the CEFFE treatment group showed significantly longer exercise time above the fatigue rotundus on days 3, 7, and 14 compared to the PBS group (P < 0.0001, P < 0.0001, P < 0.01). This indicates that CEFFE can promote the recovery of behavioral function in MCAO mice.

[0042] Table 2. Duration of mice on the rotarod in each group

[0043]

[0044]

[0045] * p < 0.05 *** p < 0.0001

[0046] Example 5

[0047] Fourteen days after surgery, the model mice underwent neurobehavioral testing. Brain tissue was then extracted after perfusion with 0.9% saline and 4% paraformaldehyde solution, immersed overnight in 4% paraformaldehyde solution, thoroughly dehydrated with 30% sucrose, and embedded using OCR. The brain tissue was then serially sectioned in the coronal plane at a thickness of 20 mm, stained with cresol purple, and the infarct volume of the model mice was calculated. Results are shown below. Figure 1 The infarct volume in mice treated with CEFFE was significantly lower than that in the control group (p < 0.05), indicating that CEFFE treatment can effectively reduce brain tissue damage.

Claims

1. Use of an extract in the preparation of a drug or medical device for repairing nerve damage, wherein the extract is a water-soluble substance obtained from ex vivo adipose tissue, and the extract is acellular adipose active protein; The extract is obtained by first emulsifying the in vitro fat, then subjecting it to a single or multiple freeze-thaw cycles, and then centrifuging to obtain the liquid layer; The nerve damage is brain tissue damage after cerebral ischemia.

2. The use according to claim 1, characterized in that: Application in the preparation of drugs or medical devices for alleviating neurological damage.

3. The use according to claim 1, characterized in that: Used to prepare drugs or medical devices that promote nerve regeneration.

4. The use according to claim 1, characterized in that: The method for preparing the water-soluble substance is as follows: After the ex vivo adipose tissue was centrifuged in the container, the upper oil layer and the lower water layer were removed, and the central layer of fat was mechanically emulsified by repeatedly pushing and beating 30 times at a uniform speed through a syringe connected to a three-way tube. After emulsification, the fat was frozen at -80°C and then thawed at 37°C. After a single freeze-thaw cycle, the fat was centrifuged at 1200g and 4°C for 5 minutes; after centrifugation, the fat was divided into 4 layers, the first layer was the oil layer, the second layer was the residual adipose tissue, the third layer was the liquid layer, and the fourth layer was the cell / tissue fragments; The first and second layers were discarded, the liquid layer was aspirated to avoid contamination by sedimentation at the bottom, and the liquid was sterilized by filtering through a 0.22 μm filter to remove cell debris.

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