CN115944713A - Neurotrophic factor and penetrating peptide composition and application thereof - Google Patents

Abstract

The present invention relates to a composition for treating optic nerve-related diseases, which comprises a neurotrophic factor, which exerts neurotrophic and protective effects, and a penetration peptide, which is a peptide fragment that significantly promotes the entry of the neurotrophic factor into the eyeball. On an SD rat model, the penetrating peptide can remarkably promote the recombinant human nerve growth factor to enter the eyeball; in an optic nerve clamp injury mouse model, the penetration peptide L-KR16 can promote the recombinant human nerve growth factor eye drops to generate the treatment effects of protecting retinal ganglion cells and improving visual evoked potential under the condition of lower concentration level.

Description

Neurotrophic factor and penetratin peptide composition and application thereof

Technical Field

The invention discloses a polypeptide composition, and belongs to the technical field of proteins or polypeptides.

Background

Optic neuropathy is often caused by trauma and by several pathological conditions, such as ischemic optic neuropathy, glaucoma, or diabetes, resulting in Retinal Ganglion Cell (RGC) axonal damage and irreversible RGC loss. Degeneration of RGCs and optic nerve atrophy are hallmarks of many ocular diseases and trauma, which can lead to permanent vision loss.

Neurotrophic factors are a class of protein molecules produced by tissues innervated by nerves and astrocytes that are essential for neuronal growth and survival. Nerve Growth Factor (NGF) is the most classical member of the neurotrophin family, and exogenous NGF has been widely reported to stimulate neuronal survival and restoration of visual function following optic Nerve injury. Topical application of NGF by intravitreal injection or eye drops can significantly reduce RGC loss following optic nerve injury. In clinical trials, recombinant Human Nerve Growth Factor (rhNGF) eye drops are safe and effective in improving symptoms and signs of glaucoma patients. In addition, topical application of rhNGF is beneficial for improving the structure and function of corneal nerves, and Cenegermin (20. Mu.g/mL rhNGF) eye drops have been approved by EMA and FDA for the treatment of neurotrophic keratitis.

However, ophthalmic drug delivery exhibits low bioavailability (< 5%) due to the presence of physiological barriers, and the corneal and conjunctival barriers, as well as the blood-ocular barrier, prevent drugs from entering the eye to reach the retina and optic nerve, and thus delivery of large and small molecule drugs into the eye by topical administration is a major challenge. Cell penetrating peptides are a diverse group of peptides, usually 5-30 amino acids, that can transport proteins, nucleic acid fragments, or small molecule drugs across cell membranes by non-covalent or covalent attachment. Cellular uptake based on penetrating peptides facilitates non-invasive local delivery of biomolecules, such as transdermal, nasal and ocular delivery, providing a new approach to drug delivery that breaches the physiological barriers of the human body. The invention aims to provide a composition for treating optic nerve related diseases, which can effectively break physiological barriers through effective component neurotrophic factors to reach pathological change parts to play a role in neuroprotection.

Disclosure of Invention

In view of the above, the present invention provides, in a first aspect, a composition comprising a neurotrophic factor and a polypeptide, wherein the polypeptide is selected from one or more of the following polypeptides:

(1) Polypeptide with sequence shown as SEQ ID NO. 1;

(2) Polypeptide with the sequence shown in SEQ ID NO. 2;

(3) Polypeptide with the sequence shown in SEQ ID NO. 3.

The polypeptide of the present invention, also referred to as a penetrating peptide, refers to a diverse group of peptides, usually having 5-30 amino acids, which can transport proteins, nucleic acid fragments or small molecule drugs across cell membranes by non-covalent or covalent binding. The polypeptide is rich in arginine and lysine with positive charges, and can promote electrostatic interaction with cell membranes with negative charges. The amino acid in the polypeptide can be D-type amino acid or L-type amino acid, the polypeptide which has L-type amino acid and has a sequence shown in SEQ ID NO.1 is named as 'L-RK 16', and the polypeptide which has D-type amino acid and has a sequence shown in SEQ ID NO.1 is named as 'D-RK 16'; the polypeptide with L-type amino acid and the sequence shown in SEQ ID NO.2 is named as 'L-KR 16', and the polypeptide with D-type amino acid and the sequence shown in SEQ ID NO.2 is named as 'D-KR 16'; the polypeptide having an L-type amino acid and having a sequence shown in SEQ ID NO.3 is named "L-DR16", and the polypeptide having a D-type amino acid and having a sequence shown in SEQ ID NO.3 is named "D-DR16".

In a preferred embodiment, the neurotrophic factor is selected from one or more of nerve growth factor, brain derived neurotrophic factor, neurotrophic factor 3, neurotrophic factor 4/5, neurotrophic factor 6, ciliary neurotrophic factor, glial cell derived neurotrophic factor.

In a more preferred embodiment, the neurotrophic factor is a nerve growth factor, and the weight ratio of the nerve growth factor to the polypeptide is 1 (2-50). In a specific embodiment of the present invention, when the final concentration of rhNGF is 50 μ g/mL and the final concentration of L-KR16 is 2.5 mg/mL, 1.0 mg/mL, 0.5 mg/mL, 0.25 mg/mL, or 0.1 mg/mL, the weight ratio of rhNGF to polypeptide is 1: (2-50), the content of rhNGF which passes through into rat retina has no significant difference, and the rhNGF has excellent passing efficiency.

Particularly preferably, the weight ratio of the nerve growth factor to the polypeptide is 1: (2-5). In a specific embodiment of the present invention, when the final concentration of rhNGF is 50. Mu.g/mL and the final concentration of L-KR16 is set to 250. Mu.g/mL, 200. Mu.g/mL, 150. Mu.g/mL or 100. Mu.g/mL, respectively, there is no significant difference in the content of rhNGF in the retina in the 4 concentration gradients.

In a specific embodiment of the invention, the sequence of the polypeptide is shown in SEQ ID No.1, and the weight ratio of the nerve growth factor to the polypeptide is 1:5. in a specific embodiment of the present invention, when the final concentration of rhNGF is 50 μ g/mL, L-RK16 sets 4 concentration gradients of 250 μ g/mL, 200 μ g/mL, 150 μ g/mL and 100 μ g/mL, the mean values of the contents of rhNGF in retina are 5.6 pg/mg, 4.4 pg/mg, 2.9 pg/mg and 2.3 pg/mg, respectively, the group of 250 μ g/mL is significantly higher than the group of 150 μ g/mL (P = 0.0065) and the group of 100 μ g/mL (P = 0.0013), i.e., the weight ratio of the nerve growth factor to the polypeptide for L-RK16 is 1: when 5, a more excellent crossing effect can be obtained.

In another specific embodiment of the invention, the sequence of the polypeptide is shown in SEQ ID No.2, and the weight ratio of the nerve growth factor to the polypeptide is 1:3. in a specific embodiment of the invention, under the condition of rhNGF 50 μ g/mL, L-KR16 is set to 4 concentration gradients of 250 μ g/mL, 200 μ g/mL, 150 μ g/mL and 100 μ g/mL, and the average values of the contents of rhNGF in the 4 concentration gradients in the retina are 22.0 pg/mg, 21.7 pg/mg, 35.3 pg/mg and 34.1 pg/mg respectively, without significant difference, but the content of rhNGF in the 150 μ g/mL group is the highest in the retina. Namely, for L-KR16, the weight ratio of the nerve growth factor to the polypeptide is 1: when 3, a more excellent crossing effect can be obtained.

In a preferred embodiment of the present invention, the amino acid in the polypeptide may be a D-form amino acid or an L-form amino acid, and in a more preferred embodiment, the polypeptide is an L-form amino acid.

The invention further provides an application of the composition in preparing a medicament for treating optic nerve related diseases.

In a preferred embodiment, the optic nerve-related disease comprises one or more of the following diseases: retinopathy, optic neuropathy-related diseases, glaucoma.

The retinopathy of the present invention includes acute retinal necrosis, retinitis pigmentosa, retinal vasculitis, retinal detachment, retinal artery/vein occlusion; the optic neuropathy related diseases comprise optic atrophy, retrobulbar optic neuritis; the glaucoma includes primary open-angle glaucoma, neovascular glaucoma and pigmentary glaucoma.

The treatment of optic nerve related diseases according to the present invention may include one or more of the following ways: anti-apoptosis, anti-inflammatory, promoting nerve cell survival, and promoting nerve regeneration.

In another preferred embodiment, the composition is prepared as a lyophilized formulation, an injection, an eye drop, a spray, a topical formulation. In one embodiment of the present invention, the composition is prepared as eye drops, and those skilled in the art will understand that the composition of the present invention can be prepared into corresponding suitable formulations to meet the specific needs of the implementation based on the specific needs of clinical administration.

The neurotrophic factor and penetrating peptide composition can obviously promote the efficiency of the neurotrophic factor entering eyes in the composition when being subjected to eye dropping, and the eye-entering efficiency of rhNGF in the composition is 2-80 times that of rhNGF in the composition when being singly dropped into eyes. The eye drop of the composition of the neurotrophic factor rhNGF and the penetration peptide L-KR16 can ensure that the rhNGF has the treatment effects of protecting retinal ganglion cells and improving visual evoked potentials on optic nerve injured mice under the condition of lower concentration level. The number of retinal ganglion cells of the L-KR16+ rhNGF 90 mu g/mL group is obviously higher than that of the rhNGF 90 mu g/mL group (the average value is 816 cells/mm) ² vs. 712 pieces/mm ² P = 0.0214) and model control (mean 816 pieces/mm) ² vs. 686/mm ² P = 0.0081); meanwhile, the P wave latency is also significantly lower than that of rhNGF 90 mug/mL group (average value of 64.0 ms vs.79.9 ms, P = 0.0305) and model control group (average value of 64.0 ms vs.85.9 ms, P = 0.0031), and is close to that of normal control group (average value of 64.0 ms vs.60.9 ms, P = 0.9492); in addition, the N-P wave amplitude was significantly higher than the model control group (mean 7.0 μ V vs. 5.3 μ V, P = 0.0346).

Drawings

FIG. 1 shows the results of evaluation of the effect of 10 penetrating peptides on rhNGF penetration into the eye;

FIG. 2 shows the results of further evaluation of the effect of 4 penetrating peptides on rhNGF penetration into the eye;

FIG. 3 shows the ocular effect of rhNGF and L-KR16+ rhNGF in eye drops, with different rhNGF concentrations, P <0.05; * P <0.01; * P <0.001;

FIG. 4 shows the results of evaluating the action concentration of L-KR16;

FIG. 5 shows the results of the evaluation of the concentration of L-RK16 action, where P <0.01;

FIG. 6 shows the results of the time-dependent dynamic changes in the content of rhNGF in the retina after eye-dropping of L-KR16+ rhNGF, P <0.0001;

FIG. 7 shows the therapeutic effect of L-KR16+ rhNGF eye drops on visual evoked potential of optic nerve clamp injury model mouse, P <0.05; * P <0.01; * P <0.001; * P <0.0001;

FIG. 8 shows the protection of retinal ganglion cells of mice model optic nerve clamp by eye drops of L-KR16+ rhNGF, where P is less than 0.05; * P <0.01.

Detailed Description

The invention is further described below in conjunction with specific embodiments, and the advantages and features of the invention will become more apparent as the description proceeds. These examples are only illustrative and do not limit the scope of protection defined by the claims of the present invention.

Example 1 penetration peptide enhances the ocular penetration efficiency of rhNGF

5 penetrating peptides based on L-amino acid and D-amino acid were synthesized, as shown in Table 1. Wherein RK-16 has been shown to be Effective in delivering antisense oligonucleotides (Tai et al, noninivative delivery of oligonucleotide by specificity-modi, 64257, polyplexes to inhibition protein expression of intracellular tumor, nanomedicine: nanotech-homology, biology, and Medicine (2017), doi: 10.1016/j.no. 2017.04.011) and the organic compounds methotrexate, vincristine (Kamei N et al, evaluation of Cell-pending Peptides Versations, effective Abstract Enhancers: translation Molecular Weight and endogenous expression Drug delivery, pharma 182/121: 11095/02874/0280); RK-16 and KR16 can increase Intestinal Absorption of oral Insulin in rats (Khafagy et al, region-Dependent Role of Cell-networking Peptides in Insulin Absorption Across The Rat Small Intestinal intracellular Membrane, the AAPS Journal,2015, published online 28 July 2015), and nasal atraumatic delivery of Insulin (Khafagy et al, one-single nuclear toxicity study of Cell-Dependent Peptides for Insulin delivery, european Journal of pharmaceuticals and pharmaceuticals 85 (2013) 736-743); DR-16 is derived from the Oct4 protein transduction domain and has a Cell penetrating function (Harreither et al, characteristics of a novel Cell penetrating peptide derived from human Oct4, cell Regeneration 2014, 3, http:// www.cellregenerationjoutlet. Com/content/3/1/2); GQ13 and acidic fibroblast growth factor fusion expression product eye drops can effectively infiltrate into rat retina (Wang et al, cell differentiation peptide TAT-mediated delivery of acidic FGF to recovery and protection against infection immunochemia reperfusion in rates, cell. Mol. Med. Vol 14, no 7, 2010 pp. 1998-2005); RR8 may be used for siRNA Delivery at the cellular level (Chu et al, rational modification of oligoarginine for high affinity siRNA Delivery: structure-activity relationship and mechanism of intracellular transduction of siRNA, nanomedicine: nanotechnology, biology, and Medicine 11 (2015) 435-446), as well as for promoting intravenous Insulin entry into the Brain across the Blood Brain barrier (Kamei N et al, non-molecular modification with Cell-peptide to surface of Brain 14. Biol. Shell. 41,546-554 (2018)). None of these penetrating peptides, except the GQ13 fusion expression product, was used for intraocular delivery of neurotrophic factors. rhNGF is designed based on a previous deletion mutant of the inventor (see CN 201110213670.8), is expressed in CHO cells, is purified by column chromatography, is detected by a UV method, and is verified to have biological activity not lower than that of a reference substance on TF-1 cells. And (3) dissolving and diluting the penetrating peptide to the required concentration by using PBS, mixing the peptide with the specific concentration with the rhNGF solution with the same volume and the specific concentration before the experiment, repeatedly sucking, fully and uniformly mixing, and placing in an ice bath for later use.

TABLE 1 sequence information of synthetic penetrating peptides

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The ocular efficacy of rhNGF was verified on SD rats (purchased from Tokyo Wintolidawa laboratory animal technologies, inc.). Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital based on body weight, the rats were sacrificed by adding a peptide-rhNGF composition or a control at a specific concentration to 30. Mu.L/eye with excessive anesthesia 30 minutes or 1, 2, 3, 6, 12 and 24 hours after eye dropping, the ocular surface was rinsed with PBS, the eyeball was removed, rinsed with PBS again, the eyeball was dissected under a stereomicroscope and the retina was removed, placed in an EP tube of a known weight, weighed and stored in a refrigerator at-70 ℃.

The rhNGF content in the retinas of the rats of each group was measured by ELISA. Taking out the sample to be detected from the low-temperature refrigerator, returning to the room temperature, adding 0.3 mL PBS into each tube, performing 60% power ultrasonic for 5 seconds, centrifuging at 4 ℃ and 12000 rpm for 5 minutes, transferring the supernatant into a new EP tube for detection, and storing the residual sample at the temperature of-70 ℃ or below after detection. According to the detection method of the attached instruction of the R & D Human beta-NGF DuoSet ELISA kit, the NGF Capture antibody Capture is diluted to 2.00 mu g/mL by PBS one day before detection, 100 mu L/hole is added into a 96-hole ELISA plate, and the plate is sealed and incubated overnight at room temperature; ELISA plates were washed 3 times with wash solution (PBS + 0.05% Tween-20), 300. Mu.L of sample dilution (PBS + 1% BSA) was added to each well and incubated for 1 hour at room temperature; preparing a series of concentration standards with concentration gradients of 2000 pg/mL, 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL and 31.2 pg/mL; pre-diluting a sample to be detected according to a pre-detection result; after the sealing is finished, washing the plate by the ELISA plate for 3 times, respectively adding a detection sample and a standard substance according to the plate layout, and incubating for 2 hours at room temperature; after washing for 3 times, adding 50.0 pg/mL biotin-labeled antibody to 100. Mu.L of each well, and incubating for 2 hours at room temperature; after washing the plate for 3 times, adding 40 times diluted Streptavidin-HRP into each 100 mu L of the well, and incubating for 20 minutes at room temperature in a dark place; after washing the plate for 3 times, adding 100 mu L of TMB color development liquid into each hole, developing for 6-10 minutes in a dark place at room temperature, and adding 50 mu L of stop solution into each hole to stop the reaction; and reading light absorption values at 450 nm and 570 nm by using a microplate reader, drawing a standard curve according to the concentration of the standard substance and the corresponding OD value (OD 450-OD 570), calculating the rhNGF concentration in the sample, and homogenizing according to the weight of the sample, wherein the unit of the rhNGF content is pg/mg.

36 male SD rats were divided into 12 groups of 3 animals (6 eyes), each of which was eye-dropped with PBS (Neg.), rhNGF alone, and rhNGF + respective penetration peptides at 30. Mu.L per eye, with a final concentration of rhNGF of 0.1 mg/mL and a final concentration of penetration peptides of 2.5 mg/mL. And (4) taking the retina to detect the rhNGF content 30 minutes after the eye drops are dropped. As shown in FIG. 1, rhNGF was not detected in the control group (Neg.), very low levels of rhNGF were detected in the rhNGF alone at an average concentration of 2.6 pg/mg, and the rhNGF content in the retina was increased in the rhNGF groups mixed with the penetration peptide, wherein the rhNGF content in the L-RK16+ rhNGF, D-RK16+ rhNGF, L-KR16+ rhNGF, D-KR16+ rhNGF and L-DR16+ rhNGF groups was highest in the retina at average values of 41.3 pg/mg, 14.6 pg/mg, 15.3 pg/mg and 12.0 pg/mg, respectively. The results showed that the penetration peptide could enhance the ocular penetration efficiency of rhNGF by eye drops, which was 4.7-16.1 times higher than that of rhNGF by eye drops alone under the condition that the concentration of rhNGF was 0.1 mg/mL and the concentration of penetration peptide was 2.5 mg/mL. In contrast, the rhNGF content in the L-GQ13+ rhNGF group and the D-GQ13+ rhNGF group was lower in retina, with average concentrations of 3.5 pg/mg and 4.6 pg/mg, respectively, which were only 1.3 times and 1.8 times that of rhNGF by eye drop alone, and were significantly lower than that in the L-RK16+ rhNGF, D-RK16+ rhNGF, L-KR16+ rhNGF, D-KR16+ rhNGF and L-DR16+ rhNGF groups.

Further examined the effect of the penetration peptides L-RK16, D-RK16, L-KR16 and D-KR16 on the ocular efficiency of rhNGF. 64 male SD rats were divided into 16 groups of 4 animals (8 eyes), each eye was dropped with PBS (Neg.), rhNGF alone and the peptide + rhNGF combination. rhNGF concentrations were set at three gradients of 100. Mu.g/mL, 50. Mu.g/mL and 25. Mu.g/mL, with a peptide concentration of 2.5 mg/mL. And (4) taking the retina to detect the rhNGF content 30 minutes after the eye drops are dropped. As shown in FIG. 2, in the case where the peptide concentration was 2.5 mg/mL, and the final rhNGF concentration was 100. Mu.g/mL, 50. Mu.g/mL or 25. Mu.g/mL, rhNGF was present in the highest concentration in the retinas in the L-KR16+ rhNGF groups, and the average values were 15.7 pg/mg, 12.2 pg/mg and 4.3 pg/mg, respectively, based on the results of FIG. 2, the lower graph shows that the eye-entry efficiency of rhNGF in the composition (100. Mu.g/eye) was 1.9 to 12.8 times that of rhNGF alone, and the upper graph shows that the eye-entry efficiency of NGF in the composition (50. Mu.g/eye) was 6.6 to 35.9 times that of ngf alone, and the upper graph shows that the eye-entry efficiency of rhNGF in the composition (25. Mu.g/eye) was 2.6 to 14.2 times that of ngf alone. As shown in fig. 3, the rhNGF content in the retina of both the 100 μ g/mL and 50 μ g/mL groups of rhNGF was significantly higher than that of the 25 μ g/mL group of rhNGF at the time of eye drop of L-KR16+ rhNGF (100 μ g/mL vs. 25 μ g/mL, P =0.0005, 50 μ g/mL vs. 25 μ g/mL, P = 0.0123), while there was no significant difference between the two groups of 100 μ g/mL and 50 μ g/mL (P = 0.3535); in contrast, the rhNGF eye drop alone was significantly higher in the group of 100. Mu.g/mL than in the blank control group (mean value of 1.2 pg/mg vs 0.17 pg/mg, P = 0.0006), and the content of rhNGF in the retina was 1.2 pg/mg, which was much lower than 4.3 pg/mg in the group of D-KR16+ rhNGF 25. Mu.g/mL. Based on the results of fig. 3, in the right panel, the ocular penetration efficiency of rhNGF in the composition was 49.2-80.2 times higher than that of rhNGF alone in the eye drops (left panel without rhNGF group alone).

The concentration of L-KR16 acting was examined under the condition that the final concentration of rhNGF was 50. Mu.g/mL. First, 5 SD rats (10 eyes) per group were set to have a final concentration of L-KR16 of 2.5 mg/mL, 1.0 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.1 mg/mL. And (4) taking the retina to detect the rhNGF content 30 minutes after the eye drops are dropped. As shown in FIG. 4, the average rhNGF content in rat retinas of the above 5 concentration gradients of L-KR16 was 12.9 pg/mg, 13.7 pg/mg, 13.3 pg/mg, 15.1 pg/mg and 10.5 pg/mg, respectively, and there was no significant difference between the groups, but the rhNGF content in retinas of the 0.1 mg/mL group was observed to be slightly lower than that of the other groups. Further examining the effect of L-KR16 from 0.25 mg/mL to 0.1 mg/mL, the final concentration of L-KR16 was set to 250. Mu.g/mL, 200. Mu.g/mL, 150. Mu.g/mL, 100. Mu.g/mL and 0. Mu.g/mL, respectively, and 4 rats (8 eyes) were each group. As shown in FIG. 4, the average values of the content of rhNGF in the 4 concentration gradients of L-KR16 in the retina were 22.0 pg/mg, 21.7 pg/mg, 35.3 pg/mg and 34.1 pg/mg, respectively, and there was no significant difference. Under the condition of rhNGF 50 mu g/mL, the final concentration of L-KR16 used was selected to be 150 mu g/mL.

The effect concentration of L-RK16 was investigated at a final rhNGF concentration of 50. Mu.g/mL. With reference to the results of L-KR16, 4 concentration gradients of 250. Mu.g/mL, 200. Mu.g/mL, 150. Mu.g/mL and 100. Mu.g/mL were set. As shown in FIG. 5, the average of the content of rhNGF in the retina of the above 4 concentration gradients of L-RK16 is 5.6 pg/mg, 4.4 pg/mg, 2.9 pg/mg and 2.3 pg/mg, respectively, and the content of rhNGF in the retina gradually decreases with the decrease of the concentration of L-RK16, wherein the 250 μ g/mL group is significantly higher than the 150 μ g/mL group (P = 0.0065) and the 100 μ g/mL group (P = 0.0013).

The retention time of rhNGF in rat eyes after entry into the eyes was examined under the conditions of rhNGF 50. Mu.g/mL and L-KR 16. Mu.g/mL. The experiment is totally designed to take the materials after dropping the eyes for 0.5 h, 1 h, 2 h, 3 h, 6 h, 12 h and 24 h to total 7 groups, and each group comprises 4 rats (8 eyes). As shown in fig. 6, the rhNGF content in the retina was the highest at 0.5 hour after eye drop, the average value was 21.7 pg/mg, and thereafter, a tendency was exhibited to gradually decrease, and a significant level of rhNGF could be detected by 3 hours after eye drop, the average value was 3.5 pg/mg, and the rhNGF content in the retina at 6 hours was significantly higher than that at 24 hours (average value 0.73 pg/mg vs. 0.083 pg/mg, P < 0.0001), and a low level of rhNGF could be detected at 12 hours, the average value was 0.23 pg/mg. The results suggest that L-KR16+ rhNGF was dropped twice daily to maintain the amount of rhNGF in the eyes.

In summary, L-RK16, D-RK16, L-KR16, D-KR16 and L-DR16 are penetration peptides which enhance rhNGF to have better eye penetration efficiency, wherein the L-type amino acid peptide has better effect than the D-type amino acid peptide, and the penetration peptide with the best effect is L-KR16; when L-KR16 is used, rhNGF is preferably used at a concentration of 50. Mu.g/mL, and at this concentration, L-KR16 is preferably used at a concentration of 150. Mu.g/mL; the content of the rhNGF in the eyes can be maintained by adding the L-KR16+ rhNGF to the eye twice a day.

Example 2 neuroprotective Effect of rhNGF in combination with L-KR16

The neuroprotective effect of rhNGF in combination with L-KR16 was evaluated in a mouse optic nerve clamp wound model. The experimental animals were adult male C57BL/6 mice, 6-8 weeks old, and 18-23 g in body weight, purchased from Schbefu (Beijing) Biotechnology Ltd. The experiment was divided into a normal control group (no treatment), a model control group (no administration of a clamp wound), a model single rhNGF 90. Mu.g/mL group, a model single rhNGF 180. Mu.g/mL group and a model L-KR16+ rhNGF 90. Mu.g/mL group, with a final concentration of L-KR16 of 270. Mu.g/mL.

And (3) establishing an optic nerve injury model by adopting a forceps clamping injury method. The mice were anesthetized with isoflurane gas, the bulbar conjunctiva was cut at the caudal side of the eye after the local sterilization of the left eye, the subconjunctival tissue was separated to expose the optic nerve, and the optic nerve was held vertically for 5 seconds with Dumont No. 5 forceps at a distance of 2mm from the eyeball for the pinching. And (5) resetting the bulbar conjunctiva after operation, smearing erythromycin eye ointment, and observing the condition of the eyeground under a microscope. The successful model is observed under the microscope on the 2 nd day, and the model shows that Marcus-gunn pupils, no obvious salient of eyeballs and no hemorrhage of eyeground appear.

The preparation is administered by eye drop 1 time in the morning and evening of mouse under anesthesia for 2 weeks. The blank control group was not treated at all. Visual evoked potentials were measured for each mouse on day 14, after which the mice were sacrificed under excess anesthesia and retinas were taken for visual ganglion cell staining and counting.

The visual evoked potential monitoring method is as follows. After dark adaptation for 2 hours, mice are anesthetized by intraperitoneal injection of 2% pentobarbital sodium with a certain volume according to body weight, compound tropicamide is added dropwise to fully mydriasis for 5 minutes, a recording electrode is arranged at the middle point of a double ear connecting line, a reference electrode is arranged at the outer canthus skin, a ground electrode is inserted into the tail of the mice, and the opposite side eyes are covered. The light intensity is 3 cd.s.m. by adopting full-visual field continuous white light stimulation ^-2 Frequency 1 Hz, recorder bandwidth 1-100 Hz, 50 times of superposition, P-wave latency (ms) and N-P-wave amplitude (μ V) of flash-visual Evoked Potential (F-VEP) recorded by the Retiport system. The test was repeated 3 times with healthy eyes as controls.

Retinal ganglion cell count methods were as follows. After mice were sacrificed by quantitative anesthesia and fixed by cardiac perfusion, the eyeballs were carefully removed and transferred to 4% paraformaldehyde for fixation overnight. The retinas were dissected and the upper and lower nasal-temporal 4 directions were marked, and dissected into 4 quadrants nasal, temporal, ventral, and dorsal. Retinas were soaked in Triton X-100 for 2 h and incubated overnight at 4 ℃ with rabbit anti-mouse RBPMS polyclonal antibody. After rinsing with PBS, alexa Fluor 594 labeled donkey anti-rabbit secondary antibody was added, and incubated for 2 hours at room temperature in the dark. After rinsing with PBS, omentum was spread on slides and mounted using DAPI-containing anti-fluorescence decay mounting medium. And (4) observing under a fluorescence microscope, photographing the positions 0.5 mm, 1 mm and 1.5 mm away from the concave position of the optic disc on the middle line of each quadrant respectively, drawing a counting area of 0.3 multiplied by 0.3 mm for each picture by using Photoshop software, and counting the cells of each counting area by using Image J software.

The visual evoked potential test results are shown in fig. 7. The average values of P-wave latencies of the normal control group, the model control group, the rhNGF 90. Mu.g/mL group, the rhNGF 180. Mu.g/mL group and the L-KR16+ rhNGF 90. Mu.g/mL group were 60.9 ms, 85.9 ms, 79.9 ms, 69.4 ms and 64.0 ms, respectively, 14 days after the operation. The P-wave latency time of the model control group mice was significantly prolonged compared to the normal control group mice (mean value 85.9 ms vs. 60.9 ms, P = 0.0008), indicating that optic nerve pinch caused a significant change in the P-wave latency of the mouse visual potential. rhNGF alone eye drops at 90 μ g/mL for 14 days failed to improve P-wave latency prolongation due to optic nerve pinch (mean value 79.9 ms vs. 85.9 ms, P =0.6889 compared to model control); after rhNGF was increased to 180 μ g/mL, the P-wave latency extension time was shortened, and was not statistically different from the normal control group (mean value 69.4 ms vs. 60.9 ms, P = 0.3665), significantly lower than the model result group (mean value 69.4 ms vs. 79.9 ms, P = 0.0315). The P-wave latency time of the L-KR16+ rhNGF 90 μ g/mL group was closest to that of the normal control group (mean value 64.0 ms vs. 60.9 ms, P = 0.9492) among the optic nerve pinch groups, while the P-wave latency time was significantly lower than that of the model control group (mean value 64.0 ms vs. 85.9 ms, P = 0.0031) and the rhNGF 90 μ g/mL group by eye drop alone (mean value 64.0 ms vs. 79.9 ms, P = 0.0305). These results indicate that eye drops of rhNGF at higher concentrations can improve the latency of P-wave in visual evoked potential in mice in optic nerve pinch models, while L-KR16 can promote rhNGF to achieve this effect at lower concentrations. In terms of N-P wave amplitude, the average values of N-P wave amplitudes of the normal control group, the model control group, the rhNGF 90. Mu.g/mL group, the rhNGF 180. Mu.g/mL group, and the L-KR16+ rhNGF 90. Mu.g/mL group were 16.0. Mu.V, 5.3. Mu.V, 5.9. Mu.V, 6.1. Mu.V, and 7.0. Mu.V, respectively, 14 days after the operation. The amplitude of the visual evoked potential N-P wave in the normal control group is obviously higher than that in each optic nerve clamping injury group. The N-P wave amplitude of the L-KR16+ rhNGF 90 μ g/mL group was significantly higher than that of the model control group (mean value 7.0 μ V vs. 5.3 μ V, P = 0.0346) between the respective optic nerve clamp groups, indicating that rhNGF in combination with L-KR16 could improve the N-P wave amplitude of the visual evoked potential of the optic nerve clamp model mouse.

The retinal ganglion cell count results are shown in figure 8. The mean values of retinal ganglion cells of the normal control group, the model control group, the rhNGF 90. Mu.g/mL group, the rhNGF 180. Mu.g/mL group and the L-KR16+ rhNGF 90. Mu.g/mL group were 3878 cells/mm, respectively, at 14 days after the operation ² 686/mm ² 712 pieces/mm ² 825 pieces/mm ² And 816 pieces/mm ² . The number of retinal ganglion cells of mice in each group of optic nerve clamp injury is obviously lower than that of the mice in a normal control group; between groups of optic nerve clamp injuryNo difference between rhNGF 90 mu g/mL group and model control group retinal ganglion cell number (average value 712/mm) ² vs. 686/mm ² P = 0.8361), the number of retinal ganglion cells in both the rhNGF 180 μ g/mL group and the L-KR16+ rhNGF 90 μ g/mL group were significantly higher than those in the model control group (rhNGF 180 μ g/mL group, mean 825 cells/mm ² vs. 686/mm ² P =0.0036; L-KR16+ rhNGF 90. Mu.g/mL group, mean 816 pieces/mm ² vs. 686/mm ² P = 0.0081) and rhNGF 90 μ g/mL group (rhNGF 180 μ g/mL group, mean 825/mm ² vs. 712 pieces/mm ² P =0.0093; L-KR16+ rhNGF 90. Mu.g/mL group, mean 816 pieces/mm ² vs. 712 pieces/mm ² P = 0.0214). The result shows that the lower concentration rhNGF eye drops can not protect retinal ganglion cells loss caused by optic nerve clamping injury, the increase of rhNGF can generate certain protection effect on the retinal ganglion cells, and L-KR16 can promote the rhNGF to achieve the protection effect under the lower concentration level.

In conclusion, eye drops with higher concentrations of rhNGF improved visual evoked potentials in optic nerve-pinched mice and protected retinal ganglion cells to some extent, while L-KR16 ensured that rhNGF was able to achieve these protective effects at lower concentration levels.

Claims (10)

1. A composition comprising a neurotrophic factor and a polypeptide, wherein the polypeptide is selected from one or more of the following:

(1) Polypeptide with sequence shown as SEQ ID NO. 1;

(2) Polypeptide with the sequence shown as SEQ ID NO. 2;

(3) Polypeptide with the sequence shown in SEQ ID NO. 3.

2. The composition of claim 1, wherein the neurotrophic factor is selected from one or more of nerve growth factor, brain-derived neurotrophic factor, neurotrophic factor 3, neurotrophic factor 4/5, neurotrophic factor 6, ciliary neurotrophic factor, and glial cell-derived neurotrophic factor.

3. The composition of claim 2, wherein the neurotrophic factor is nerve growth factor, and the weight ratio of the nerve growth factor to the polypeptide is 1: (2-50).

4. The composition of claim 3, wherein the weight ratio of the nerve growth factor to the polypeptide is 1: (3-5).

5. The composition of claim 4, wherein the polypeptide has a sequence shown in SEQ ID No.1, and the weight ratio of the nerve growth factor to the polypeptide is 1:5.

6. the composition of claim 4, wherein the polypeptide has a sequence shown in SEQ ID No.2, and the weight ratio of the nerve growth factor to the polypeptide is 1:3.

7. the composition of claim 3, wherein the amino acids in the polypeptide are L-amino acids.

8. Use of a composition according to any one of claims 1 to 7 for the manufacture of a medicament for the treatment of an optic nerve related disease.

9. The use of claim 8, wherein the optic nerve-related disease comprises one or more of the following: retinopathy, optic neuropathy-related diseases, glaucoma.

10. The use according to claim 8, wherein the composition is prepared as a lyophilizate, an injection, an eye drop, a nebulizer, a topical application.

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