CN105017406A - Novel polypeptide with nerve protection function - Google Patents

Abstract

The invention relates to novel polypeptide with a nerve protection function. The invention also relates to an application of the polypeptide and a medicinal composition containing the polypeptide. The polypeptide is small in molecular weight, can penetrate through various eye tissue barriers, is good in water solubility, and can keep a relatively high concentration in neutral tears, aqueous humor and vitreous humor.

Description

A new class of peptides with neuroprotective function

technical field

The invention relates to the field of biomedicine, in particular to a new class of polypeptides with neuroprotective function.

Background technique

Glaucoma (Glaucoma) is a group of optic nerve degenerative diseases caused by various factors such as increased pathological intraocular pressure (intraocular pressure, IOP) or decreased optic nerve blood perfusion pressure. the second leading cause of disease. At present, the main method of clinical treatment of glaucoma is to reduce the intraocular pressure through drugs, laser and surgery, so as to reduce the mechanical damage caused by pathological high intraocular pressure, improve the state of low blood perfusion, and thus slow down the apoptosis of retinal ganglion cells . However, clinical studies have found that a large proportion of patients whose intraocular pressure is controlled within the normal range are still developing, with typical optic disc changes and visual field defects. In addition, disease progression can occur in patients whose intraocular pressure remains within the normal range, known as normal tension glaucoma. Therefore, for the treatment of glaucoma, in addition to actively controlling intraocular pressure, attention should also be paid to the protection of the optic nerve.

There are many ways to protect the optic nerve, such as oral administration of drugs, topical eye drops or intravitreal injection to slow, prevent or even reverse the death of nerve cells; there is also gene therapy, that is, through recombinant adeno-associated virus (rAAV) or lentivirus (lentiviral) and other carrier-mediated expression of anti-apoptotic proteins or neurotrophic factors to slow down the apoptosis of RGCs; in addition, through stem cell transplantation, stem cells can be used to secrete neurotrophic factors and other regulatory factors to alleviate glaucoma-related neurotrophic Factor deprivation, inflammatory response, oxidative stress, excitotoxicity, etc. to protect RGCs. As far as drugs are concerned, traditional chemical drugs have severe side effects, high production costs and strong immunogenicity of recombinant protein drugs, all of which hinder their widespread clinical application.

When developing effective inhibitors of ocular inflammation, the particularity of ophthalmic drugs should be fully considered.

First, there are multiple anatomical and functional barriers in the eye. Systemic administration often fails to achieve sufficient drug concentration locally in ocular tissues due to the blood-aqueous humor barrier and blood-retinal barrier; local administration, such as intravitreal injection, is theoretically difficult for macromolecules larger than 76.5kDa to penetrate the retina Acts on retinal and choroidal neovascularization.

Second, the degree of drug dissolution in hydrophilic tears, aqueous humor, and vitreous humor is positively correlated with its effectiveness.

Third, based on the above-mentioned main reasons, the bioavailability of ophthalmic drugs is very low; in order to improve it, the concentration of administration needs to be increased. However, the toxic and side effects of high-concentration drugs are more obvious, and high-dose administration cannot be administered both systemically and locally.

Therefore, there is an urgent need in this field to develop a safe and effective small molecule neuroprotectant suitable for eyeball tissue.

Contents of the invention

The present invention provides a novel polypeptide with neuroprotective function, especially the polypeptide applicable to eyeball tissues.

The first aspect of the present invention provides a polypeptide represented by the following formula I, or a pharmaceutically acceptable salt thereof

[Xaa0]-[Xaa1]-[Xaa2]-[Xaa3]-[Xaa4]-[Xaa5]-[Xaa6]-[Xaa7]-[Xaa8]-[Xaa9]-[Xaa10]-[Xaa12]-[Xaa12 ]-[Xaa13]-[Xaa14]-[Xaa15]-[Xaa16]-[Xaa17]-[Xaa18]-[Xaa19] (I)

In the formula,

Xaa0 is none, or 1-5 amino acids constitute a peptide;

Xaa1 is an amino acid selected from the group consisting of Ile, Leu, Val, Met, Ala or Phe;

Xaa2 is an amino acid selected from the group consisting of Pen or Hcy;

Xaa3 is an amino acid selected from the group consisting of Lys, Arg, Gln or Asn;

Xaa4 is an amino acid selected from the group consisting of Gly, Pro, Ala or;

Xaa5 is an amino acid selected from the group consisting of Lys, Arg, Gln or Asn;

Xaa6 is an amino acid selected from the group consisting of Glu or Asp;

Xaa7 is an amino acid selected from the group consisting of Val, Ile, Leu, Met, Phe or Ala;

Xaa8 is an amino acid selected from the group consisting of Cys, Hcy or Pen;

Xaa9 is an amino acid selected from the group consisting of Thr or Ser;

Xaa10 is an amino acid selected from the group consisting of Acp or β-Ala;

Xaa11 is an amino acid selected from the group consisting of Asn, Gln, His, Lys or Arg;

Xaa12 is an amino acid selected from the group consisting of Ala, Val, Leu or Ile;

Xaa13 is an amino acid selected from the group consisting of Pro or Ala;

Xaa14 is an amino acid selected from the group consisting of Val, Ile, Leu, Met, Phe or Ala;

Xaa15 is an amino acid selected from the group consisting of Ser or Thr;

Xaa16 is an amino acid selected from the group consisting of Ile, Leu, Val, Met, Ala or Phe;

Xaa17 is an amino acid selected from the group consisting of Pro or Ala;

Xaa18 is an amino acid selected from the group consisting of Gln or Asn;

Xaa19 is none, or 1-5 amino acids constitute a peptide;

Wherein, a disulfide bond is formed between the Xaa2 and Xaa8, and the polypeptide has neuroprotective activity.

In another preferred example, the length of the polypeptide is ≤28 amino acids, preferably ≤25 amino acids, more preferably ≤20 amino acids.

In another preferred example, the polypeptide has at least 12 fixed amino acids, preferably 15, more preferably 16.

In another preferred example, the polypeptide is shown in SEQ ID NO.: 1-10.

In another preferred example, the polypeptide is:

Xaa0 is None;

Xaa1 is an amino acid selected from the group consisting of Ile or Leu;

Xaa2 is Pen;

Xaa3 is an amino acid selected from the group consisting of Lys or Arg;

Xaa4 is an amino acid selected from the group consisting of Gly or Ala;

Xaa5 is an amino acid selected from the group consisting of Lys or Arg;

Xaa6 is an amino acid selected from the group consisting of Glu or Asp;

Xaa7 is an amino acid selected from the group consisting of Val or Leu;

Xaa8 is an amino acid selected from the group consisting of Cys or Pen;

Xaa9 is an amino acid selected from the group consisting of Thr or Ser;

Xaa10 is an amino acid selected from the group consisting of Acp or β-Ala;

Xaa11 is an amino acid selected from the group consisting of Asn or Gln;

Xaa12 is an amino acid selected from the group consisting of Ala or Val;

Xaa13 is an amino acid selected from the group consisting of Pro or Ala;

Xaa14 is an amino acid selected from the group consisting of Val or Leu;

Xaa15 is an amino acid selected from the group consisting of Ser or Thr;

Xaa16 is an amino acid selected from the group consisting of Ile or Leu;

Xaa17 is an amino acid selected from the group consisting of Pro or Ala;

Xaa18 is an amino acid selected from the group consisting of Gln or Asn;

Xaa19 is None;

Wherein, a disulfide bond is formed between Xaa2 and Xaa8, and the polypeptide has neuroprotective activity, and the polypeptide undergoes at most 1-5, preferably 1-3, more preferably 1 - Substitution of 2 amino acids.

In another preferred example, the Xaa0 is a peptide segment consisting of 1-3 amino acids; and/or the Xaa19 is a peptide segment consisting of 1-3 amino acids.

In the second aspect of the present invention, a derivative polypeptide is provided, and the derivative polypeptide is a derivative polypeptide of the polypeptide shown in SEQ ID NO.: 1, and is selected from the following group:

(a) have the amino acid sequence shown in SEQ ID NO:1;

(b) The amino acid sequence shown in SEQ ID NO: 1 is formed by deletion, substitution or addition of 1-5 (preferably 1-3, more preferably 1-2) amino acid residues, and has neural Polypeptides derived from (a) that protect the function.

In another preferred example, (a) the length of the polypeptide is ≤28 amino acids, preferably ≤25 amino acids, more preferably ≤20 amino acids.

In another preferred embodiment, the derivative polypeptide is substituted by 1-5, preferably 1-3, more preferably 1-2 amino acid substitutions from the polypeptide shown in SEQ ID NO.:1; and/or

In another preferred example, the deletion is 1-3, preferably 1-2 deletions of amino acid residues at the C-terminal of the polypeptide; and/or

The two ends of the derivative polypeptide are respectively formed by adding 1-5, preferably 1-4 or 1-3, more preferably 1-2, amino acids.

In another preferred example, the polypeptide is substituted according to "Representative Substitutions" in Table 1.

In another preferred example, the polypeptide is substituted according to the "preferred substitution" in Table 1.

In another preferred example, the derivative polypeptide retains ≥70% of the neuroprotective activity of the polypeptide shown in SEQ ID NO:1.

In another preferred example, the homology between the derivative polypeptide and SEQ ID NO: 1 is ≥80%, preferably ≥90%; more preferably ≥95%.

The present invention also provides dimeric and multimeric forms of compounds of formula I that have neuroprotective functions.

In another preferred embodiment, the present invention also provides an isolated nucleic acid molecule encoding the above-mentioned polypeptide of the present invention.

In the third aspect of the present invention, a pharmaceutical composition is provided, which contains:

(a) a polypeptide according to any of the first aspects of the invention, a derivative polypeptide according to any of the second aspects of the invention, or a pharmaceutically acceptable salt thereof; and

(b) A pharmaceutically acceptable carrier or excipient.

In another preferred embodiment, the dosage form of the composition is eye drops, injections (such as periocular and intraocular injections, especially intravitreal injections), ophthalmic gels or ophthalmic ointments.

In another preferred example, the composition is a sustained-release dosage form.

The fourth aspect of the present invention provides a use of the polypeptide of the present invention or its derivative polypeptide or a pharmaceutically acceptable salt, which are used to prepare medicines for neuroprotection or prevention and treatment of diseases related to nerve cell damage.

In another preferred example, the subject is human.

In another preferred example, the nerve cell damage is the same as eye nerve cell damage.

In another preferred example, the diseases related to nerve cell damage are selected from the group consisting of diseases related to eye nerve cell damage, acute or chronic retinal optic nerve cell damage diseases.

In another preferred example, the diseases related to eye nerve cell damage include glaucoma, macular degeneration, diabetic retinopathy, and retinal artery and vein occlusion.

In the fifth aspect of the present invention, there is provided a method for enhancing the neuroprotection of mammals, comprising the step of: administering the polypeptide of the present invention or a pharmaceutically acceptable salt thereof to a subject in need.

In another preferred example, the administration includes ocular surface administration or intravitreal injection administration

In another preferred example, the subject is human.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows that NP-17 peptide has a pro-proliferative effect on RGC-5 cells in a dose-dependent manner. NP-17 is most active at the concentration of 1000ng/ml.

Figure 2 shows that the survival rate of RGC-5 cells is reduced under hypoxic conditions, and NP-17 can increase the survival rate of cells under hypoxic conditions and protect ganglion cells from damage caused by hypoxic conditions.

Figure 3 shows that NMDA has a toxic effect on RGC-5 cells in a dose-dependent manner. When NMDA is 100uM/L, the survival rate of RGC-5 cells is about 55% to 60%.

Figure 4 shows the protective effect of NP-17 on NMDA-induced RGC-5 cell injury: under NMDA injury, the survival rate of RGC-5 cells is reduced, and NP-17 can increase the cell survival rate under NMDA-induced injury and protect ganglion cells from immunity. Impaired by NMDA excitotoxicity.

Figure 5 shows that after NMDA injury, the apoptosis of RGC-5 cells increases, and NP-17 can reduce the apoptosis rate of cells after NMDA-induced injury and protect ganglion cells.

Figure 6 shows that NP-17 up-regulates the PI3K/Akt pathway: the phosphorylation level began to increase after NP-17 was treated for 5 minutes, reached a peak at 30 minutes, and then gradually decreased.

Figure 7 shows the effect of PI3K/Akt pathway inhibitors on the neuroprotective effect of NP-17: after adding pathway inhibitors, the cell death rate increased significantly; after adding signaling pathway inhibitors alone, the inhibitors had no significant effect on cell survival ; LY294002 can significantly reduce the level of p-Akt.

Figure 8 shows that NP-17 up-regulates the ERK pathway: the phosphorylation level of NP-17 began to increase after 2.5 minutes, and ERK1 reached a peak at 7.5 minutes, while ERK2 reached a peak at 10 minutes, and then gradually decreased.

Figure 9 shows the effect of ERK pathway inhibitors on the neuroprotective effect of NP-17: after adding the pathway inhibitor PD098059, the cell death rate increased significantly; after adding the signaling pathway inhibitor alone, the inhibitor had no significant effect on the cell survival rate; PD098059 can significantly reduce the level of p-Akt.

Figure 10 shows that NP-17 inhibits the expression of Caspase3: NP-17 can reduce the expression of Caspase3 in RGC-5 cells after NMDA injury, and its inhibitory ability is stronger than that of BDNF.

Figure 11 shows that Caspase3 inhibitors inhibit cell apoptosis: without adding NMDA group, after adding Caspase3 inhibitor Z-DEVD-FMK, there is no significant effect on cell death rate, and the inhibitor has no cytotoxicity on the surface; adding NMDA group, using inhibitory After treatment, the cell death rate was significantly reduced in a dose-dependent manner.

Figure 12 shows that NP-17 increases the expression of Bcl-2 and inhibits the expression of Bad and Bax: NP-17 can reduce the expression of Bad and Bax and increase the expression of Bcl-2 in RGC-5 cells after NMDA injury.

Figure 13 shows that NP-17 protects ganglion cells: the number of ganglion cells in the NMDA group decreased significantly, and after injection of NP-17, the number of residual ganglion cells was more than that in the NMDA group.

Figure 14 shows that NP-17 inhibits the apoptosis of ganglion cells: the number of apoptotic cells in the ganglion cell layer increased significantly in the NMDA group, and the number of apoptotic RGC cells decreased significantly after the addition of NP-17.

Figure 15 shows that NP-17 reduces the content of Caspase3 in retinal tissue: NMDA can significantly increase the content of Caspase3 in retinal tissue, and after intravitreal injection of NP-17, the content of Caspase3 in retinal tissue decreased significantly.

Figure 16 shows the effect of polypeptide N1 on the proliferation of RGC-5 cells: the polypeptide N1 in different concentration groups has no effect on the proliferation of RGC-5 cells, p>0.05.

Figure 17 shows the effect of polypeptide N2 on the proliferation of RGC-5 cells: polypeptide N1 in different concentration groups has no effect on the proliferation of RGC-5 cells, p>0.05.

Detailed ways

After extensive and in-depth research, the present inventors prepared for the first time a class of small molecule polypeptides derived from brain-derived neurotrophic factor, having neuroprotective activity, and a molecular weight of less than 5kD (eg, only about 2kD). Specifically, the inventors applied bioinformatics methods, based on homology analysis and biological characteristics analysis, designed several candidate sequences, synthesized and modified them by solid-phase method, separated and purified to obtain high-purity polypeptide NP -17, and was identified by HPLC and MS, and then screened by NMDA-induced apoptosis RGC cells in vivo and in vitro, and a new type of small molecule polypeptide with neuroprotective effect was obtained, and its activity level could reach that of BDNF. 80%, and the optimal effective concentration of the peptide was obtained. In addition, the inventors also found through experiments that the polypeptide NP-17 of the present invention is achieved by increasing the concentration of Bcl-2 protein and inhibiting the expression of Caspase3.

The polypeptide of the present invention has a small molecular weight and can penetrate various eye tissue barriers; has good water solubility and can maintain a relatively high concentration in neutral tears, aqueous humor and vitreous humor; has high safety and has little toxic and side effects on biological tissues; Ocular topical drugs have high bioavailability, which can reduce the dose, thereby reducing systemic side effects. The present invention has been accomplished on this basis.

Brain-derived neurotrophic factor (BDNF)

Brain-derived neurotrophic factor (BDNF) is a macromolecular protein with neurotrophic and neuroprotective effects, first discovered in 1982 by Barde et al. The BDNF molecular monomer is composed of 119 amino acids, the protein isoelectric point is 9199, and the molecular weight is 13.15kD. Multiple loop structures can be formed inside the molecule. BDNF is the most abundant neurotrophic factor in the body. It is widely distributed in the nervous system, endocrine system, and bone and cartilage tissues. It binds to tropomyosin receptor kinase (TrkB), activates downstream pathways, and regulates the behavior and function of nerve cells. , such as increasing synaptic plasticity, promoting neurogenesis, promoting neuronal differentiation, and repairing damaged neurons, etc., play a very important role in the growth and development of nerve tissue and damage repair.

BDNF has very strong biological activity, but due to its large molecular weight, it is difficult to pass through various physiological barriers, such as the blood-brain barrier, blood-retinal barrier, etc., and the half-life in the body is extremely short, so it is currently difficult to be used clinically. The research team used bioinformatics technology to screen out biological peptides with a hairpin-like LOOP structure similar to BDNF, which can bind to TrkB receptors and exert activities similar to BDNF. In the prior art, a number of biological peptides with LOOP structure have been synthesized, but the highest activity is only 60% of BDNF, and the activity is difficult to meet the clinical needs.

active peptide

In the present invention, the terms "polypeptide of the present invention", "NP-17 polypeptide", "NP-17 polypeptide", "short peptide NP-17" or "peptide NP-17" are used interchangeably, and all refer to neuroprotective The active peptide NP-17 amino acid sequence (I(Pen)KGKEVCT(Acp)NAPVSIPQ, as shown in SEQ ID NO: 1) is a protein or polypeptide, and the polypeptide of the present invention is a circular polypeptide containing an intrachain disulfide bond. In addition, the term also includes variant forms of the sequence of SEQ ID NO: 1 that have neuroprotective functions. These variations include (but are not limited to): 1-5 (usually 1-4, more preferably 1-3, and most preferably 1-2) amino acid deletions, insertions and/or substitutions, and One or several (usually within 5, preferably within 3-4, more preferably within 1-2) amino acids are added or deleted at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. Furthermore, the term also includes monomeric and multimeric forms of the polypeptides of the invention.

The present invention also includes active fragments, derivatives and analogs of NP-17 polypeptides. As used herein, the terms "fragment", "derivative" and "analogue" refer to polypeptides that substantially retain neuroprotective function or activity. The polypeptide fragments, derivatives or analogs of the present invention can be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more A polypeptide with substituent groups in amino acid residues, or (iii) a polypeptide formed by fusing the NP-17 polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional The polypeptide formed by fusing the amino acid sequence to the polypeptide sequence (subsequent protein formed by fusing with the leader sequence, secretory sequence or 6His and other tag sequences). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

A class of preferred active derivatives refers to that compared with the amino acid sequence of formula I, at most 5, preferably at most 3-4, more preferably at most 1-2 amino acids are replaced by amino acids with similar or similar properties. form peptides. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

Table 1

initial residue representative replacement preferred substitution Ala(A) Val;Leu;Ile Val

Arg(R) Lys;Gln;Asn Lys Asn(N) Gln;His;Lys;Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn;Gln;Lys;Arg Arg Ile (I) Leu;Val;Met;Ala;Phe Leu Leu(L) Ile;Val;Met;Ala;Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu;Phe;Ile Leu Phe(F) Leu;Val;Ile;Ala;Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr;Phe Tyr Tyr(Y) Trp;Phe;Thr;Ser Phe Val(V) Ile;Leu;Met;Phe;Ala Leu

The invention also provides analogs of NP-17 polypeptides. The difference between these analogs and the natural NP-17 polypeptide may be a difference in amino acid sequence, or a modification that does not affect the sequence, or both. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Some commonly used unnatural amino acids are listed in Table 1b below.

Table 1b

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

The polypeptides of the present invention can also be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include, but are not limited to, those formed with the following acids: hydrochloric, hydrobromic, sulfuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, succinic, oxalic, fumaric, maleic, acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as in the form of esters, carbamates or other conventional "prodrugs".

coding sequence

The present invention also relates to polynucleotides encoding NP-17 polypeptides. , which encodes the short peptide NP-17 shown in SEQ ID NO:1.

A polynucleotide of the invention may be in the form of DNA or RNA. DNA can be either the coding strand or the non-coding strand. The full-length NP-17 nucleotide sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the polypeptide (or its fragment, or its derivative) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.

The present invention also relates to vectors comprising the polynucleotides of the present invention, and host cells produced by genetic engineering using the vectors or NP-17 polypeptide coding sequences of the present invention.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific for NP-17 polypeptide.

Preparation

The polypeptides of the present invention may be recombinant polypeptides or synthetic polypeptides. The polypeptides of the invention may be chemically synthesized, or recombinant. Correspondingly, the polypeptide of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.

A preferred method is to use liquid-phase synthesis technology or solid-phase synthesis technology, such as Boc solid-phase method, Fmoc solid-phase method or a combination of the two methods. Solid-phase synthesis can quickly obtain samples, and the appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide. For example, the preferred solid phase carrier in the Fmoc system is Wang resin connected with the C-terminal amino acid in the peptide, the Wang resin structure is polystyrene, and the arm between the amino acid is 4-alkoxybenzyl alcohol; use 25% hexahydropyridine /dimethylformamide at room temperature for 20 minutes to remove the Fmoc protecting group, and extend from the C-terminal to the N-terminal one by one according to the given amino acid sequence. After the synthesis is completed, the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-cresol and the protective group is removed, and the resin can be filtered off and separated by ether precipitation to obtain the crude peptide. After lyophilization of the resulting product solution, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography. When using the Boc system for solid-phase synthesis, the preferred resin is a PAM resin connected to the C-terminal amino acid in the peptide. The structure of the PAM resin is polystyrene, and the arm between the amino acid is 4-hydroxymethylphenylacetamide; synthesized in Boc In the system, in a cycle of deprotection, neutralization, and coupling, the protecting group Boc is removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA/dichloromethane. Peptide chain condensation is completed Afterwards, use hydrogen fluoride (HF) containing p-cresol (5-10%), treat at 0°C for 1 hour, cut the peptide chain from the resin, and remove the protecting group at the same time. With 50-80% acetic acid (containing a small amount of mercaptoethanol) to extract the peptide, and after the solution is freeze-dried, further use molecular sieve SephadexG10 or Tsk-40f to separate and purify, and then obtain the desired peptide through high-pressure liquid phase purification. Various coupling agents known in the field of peptide chemistry can be used Each amino acid residue is coupled with a coupling method, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3,3-tetraurea hexafluorophosphate ( HBTU) for direct coupling. For the short peptides synthesized, their purity and structure can be confirmed by reversed-phase high performance liquid chromatography and mass spectrometry.

In a preferred example, the polypeptide NP-17 of the present invention is prepared by solid-phase synthesis according to its sequence, purified by high-performance liquid chromatography to obtain a high-purity lyophilized powder of the target peptide, and stored at -20°C.

Another approach is to use recombinant techniques to produce the polypeptides of the invention. The polynucleotides of the present invention can be used to express or produce recombinant NP-17 polypeptides by conventional recombinant DNA techniques. Generally speaking, there are the following steps:

(1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the NP-17 polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

Recombinant polypeptides can be expressed intracellularly, on the cell membrane, or secreted extracellularly. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Since the polypeptide of the present invention is relatively short, multiple polypeptides can be concatenated together to obtain a multimeric expression product after recombinant expression, and then the desired polypeptide can be formed by enzymatic cleavage or other methods.

Pharmaceutical compositions and methods of administration

In another aspect, the present invention also provides a pharmaceutical composition, which contains (a) a safe and effective amount of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient . The amount of the polypeptide of the present invention is usually 10 μg-100 mg/dose, preferably 100-1000 μg/dose.

For the purposes of the present invention, an effective dosage is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the present invention administered to an individual. In addition, the polypeptides of the present invention can be used alone or together with other therapeutic agents (eg formulated in the same pharmaceutical composition).

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. The term refers to pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon administration. These vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids, such as water, saline, glycerol and ethanol. In addition, there may also be auxiliary substances in these carriers, such as wetting agents or emulsifying agents, pH buffering substances and the like.

Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution, or suspension, in liquid carriers prior to injection can also be prepared.

Once formulated, the compositions of the present invention can be administered by conventional routes, including (but not limited to): ocular surface, periocular, intraocular (especially intravitreal), intramuscular, intravenous, subcutaneous , intradermal or topical administration. The subject to be prevented or treated can be an animal; especially a human.

When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the usage conditions. Preferably, there may be exemplified eye drops, injections (especially intravitreal injections), ophthalmic gels and ophthalmic ointments.

These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc. (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a conventional manner depending on the dosage form.

For example, the preparation of eye drops can be carried out like this: short peptide NP-17 or its pharmaceutically acceptable salt are dissolved in sterile water (surfactant is dissolved in sterile water) together with basic substance, adjust osmotic pressure and The pH is adjusted to the physiological state, and suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and viscosifiers can be added arbitrarily, and then completely dissolved.

The pharmaceutical compositions of the present invention can also be administered in the form of sustained release formulations. For example, the short peptide NP-17 or its salt can be incorporated into a pill or microcapsule supported by a slow-release polymer, and then the pill or microcapsule is surgically implanted into the tissue to be treated. In addition, the short peptide NP-17 or a salt thereof can also be applied by insertion into a pre-coated intraocular lens. Examples of sustained-release polymers include ethylene-vinyl acetate copolymers, polyhydroxymethacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.

When the pharmaceutical composition of the present invention is used for actual treatment, the dose of the short peptide NP-17 or its pharmaceutically acceptable salt as the active ingredient can be adjusted according to the body weight, age, sex, symptoms of each patient to be treated reasonably determined. For example, when topical eye drops, usually its concentration is about 0.1-10wt%, preferably 1-5wt%, can be administered 2-6 times a day, 1-2 drops each time.

experimental animal model

NMDA-induced apoptosis in RGC cells

The present invention uses NMDA-induced apoptosis RGC cells as a model for in vivo and in vitro experiments. Glutamate is the main excitatory neurotransmitter in the central nervous system, but the accumulation of high concentrations of glutamate in the extracellular fluid will produce toxic effects on cells through NMDA receptors. Excitotoxic effects are recognized as a common pathogenesis pathway in many neurological diseases, including acute ischemic diseases as well as chronic neurodegenerative diseases. Excess glutamate activates NMDA receptors, causing a large influx of Ca2+, which then initiates the intracellular apoptosis program and induces apoptosis. NMDA receptor-mediated cell death is also an important factor in retinal ganglion cell death in various eye diseases. Therefore, the experiment chose NMDA to induce retinal ganglion cell layer damage to mimic the pathological features of various ocular diseases.

Industrial Applicability

The pharmaceutical composition containing the peptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient has significant nerve cell protection function. It is confirmed by animal experiments that the polypeptide of the present invention can protect RGC cells from apoptosis induced by NMDA, improve the survival rate of RGC cells, and thus obtain better nerve cell protection.

The main advantages of the present invention include:

(a) The polypeptide NP-17 of the present invention has a small molecular weight and can penetrate the eye tissue barrier;

(b) It has good water solubility and can maintain a high concentration in neutral tear fluid, aqueous humor and vitreous humor;

(c) High safety, less toxic and side effects on biological tissues; and high bioavailability of topical ophthalmic drugs, with an activity of up to 80% of BDNF, which can reduce dosage and reduce systemic side effects;

(d) It can be prepared by solid-phase synthesis, with high purity, large yield and low cost;

(e) The stability of the polypeptide of the present invention is good.

Therefore, the polypeptide of the present invention is expected to be developed into a drug for treating eye diseases related to nerve cell damage and other related diseases.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.

Synthesis and identification of embodiment 1 polypeptide

The NP-17 (sequence: I(Pen)KGKEVCT(Acp)NAPVSIPQ) polypeptide was synthesized with a commercially available symphony peptide synthesizer, and purified by HPLC and MS methods. After synthesis and purification, the purity of the peptide is greater than 95%, and it is stored at -20°C for future use.

Effect of embodiment 2NP-17 on RGC-5 cells under hypoxic environment

1. Materials and Methods

1.1 Experimental cells and materials: Rat retinal ganglion cell-5 (RGC-5), purchased from Fudan University School of Medicine; DMEM high-glucose medium, purchased from Invitrogen, USA; Cell LIVE/DEAD Assay Kit, purchased from Invitrogen, USA ; MTS was purchased from Promega, USA.

1.2 Screening for the optimal concentration of NP-17 activity: RGC-5 cells were placed in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin, placed at 37°C, Expansion cultures were carried out in a 5% CO ₂ incubator. Cells in the logarithmic growth phase were taken, and the density was adjusted to 2×10 ⁴ /well and inoculated in a 96-well plate. When the cells adhered well and reached 60% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. The cells were randomly divided into blank control (Blank), NP-17 (1ng/ml), NP-17 (10ng/ml), NP-17 (100ng/ml), NP-17 (1000ng/ml) and NP-17 ( 5000ng/ml) group, with six replicate wells in each group, placed in a 37°C, 5% CO ₂ incubator for routine culture. After 24 hours, 20ul of MTS was added to each well, and after 2 hours, the OD value was measured with a spectrophotometer at a wavelength of 490nm.

1.3 Model establishment and intervention: RGC-5 cells were cultured in the same way as above. Cells in logarithmic growth phase were taken and seeded in two 96-well plates at a density adjusted to 2×10 ⁴ /well. When the cells adhered well and reached 60% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. The cells were randomly divided into control group (Control), hypoxia + blank group, hypoxia + NP-17 group, hypoxia + BDNF group, and each group had six replicate wells. The control group was cultured routinely in a 37°C, 5% CO ₂ incubator, and the other three groups were cultured in a 37°C, 5% CO ₂ , 5% O ₂ hypoxic incubator. After 24 hours, the CELL LIVE/DEAD Assay Kit was used. detection.

1.4 Statistical analysis: The experimental data were expressed as mean ± standard deviation, and statistical analysis was performed using SPSS17.0 statistical software package. Independent sample t-test was used, and P<0.05 was considered statistically significant.

2. Experimental results

2.1 The effect of NP-17 on the proliferation of RGC-5 cells: NP-17 peptide can promote the proliferation of RGC-5 cells in a dose-dependent manner. NP-17 is most active at the concentration of 1000ng/ml. See Figure 1, when NP-17 is at 1ug/ml, p<0.001; when NP-17 is at 100 and 5000ng/ml, p<0.01.

2.2 The effect of NP-17 on RGC-5 cells in hypoxic environment: in hypoxic environment, the survival rate of RGC-5 cells is reduced, and NP-17 can improve the survival rate of cells in hypoxic environment, and protect ganglion cells from hypoxia. Oxygen damage. See Figure 2 (* indicates p<0.05, **b indicates p<0.01).

3. Summary

Through cell proliferation experiments, the optimal active concentration of NP-17 was screened out. By further establishing the RGC-5 hypoxic injury model, the effect on the survival rate of RGC-5 cells was observed, and it was confirmed that NP-17 can improve RGC in hypoxic environment. -5 cell survival rate, protect nerve cells.

Example 3 Effect of NP-17 on NMDA-induced RGC-5 cell injury

1. Materials and Methods

1.1 Experimental cells and materials: Rat retinal ganglion cell-5 (RGC-5), purchased from Fudan University School of Medicine; DMEM high glucose medium, purchased from Invitrogen, USA; Cell LIVE/DEAD Assay Kit, purchased from Invitrogen, USA ; MTS was purchased from Promega, USA; Tunel apoptosis detection kit was purchased from Promega, USA; NMDA was purchased from Sigma, USA.

1.2 Model establishment and intervention: RGC-5 cells were placed in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin double antibodies, placed at 37°C, 5% CO ₂ Carry out expansion culture in the incubator, carry out the following experiments respectively:

1.2.1 Take the cells in the logarithmic growth phase, adjust the density to 2×10 ⁴ /well and inoculate them in a 96-well plate. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. Cells were randomly divided into control group (Control), NMDA (10uM/L) group, NMDA (25uM/L) group, NMDA (50uM/L) group, NMDA (100uM/L) group, NMDA (200uM/L) group, For NMDA (400uM/L) group, NMDA (800uM/L) group and NMDA (1600uM/L) group, each group had six replicate wells. Place them in a 37°C, 5% CO ₂ incubator for routine culture, and use the CELL LIVE/DEAD Assay Kit to detect after 24 hours.

1.2.2 Take the cells in the logarithmic growth phase, adjust the density to 2×10 ⁴ /well and inoculate them in a 96-well plate. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. Cells were randomly divided into control group (Control), NMDA+blank group, NMDA+NP-17 group, NMDA+BDNF group, with six replicate wells in each group. Place them in a 37°C, 5% CO ₂ incubator for routine culture, and use the CELL LIVE/DEAD Assay Kit to detect after 24 hours.

1.2.3 Take the cells in the logarithmic growth phase, adjust the density to 2×10 ⁵ /well and inoculate them in a 24-well plate. When the cells adhered well and reached 60% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. The cells were randomly divided into control group (Control), NMDA+blank group, NMDA+NP-17 group (the concentration of NP-17 was 1ug/ml), and NMDA+BDNF group, with 3 replicate wells in each group. Place them in a 37°C, 5% CO ₂ incubator for routine culture, and use the Tunel kit for detection after 24 hours.

1.3 Statistical analysis: The experimental data were expressed as mean ± standard deviation, and SPSS 17.0 statistical software package was used for statistical analysis. Independent sample t-test was used, and P<0.05 was considered statistically significant.

2. Experimental results

2.1 Injury effect of NMDA on RGC-5 cells: NMDA has a toxic effect on RGC-5 cells in a dose-dependent manner. When NMDA is 100uM/L, the survival rate of RGC-5 cells is about 55% to 60%, as shown in Figure 3. NMDA with a concentration of 100uM/L was selected as the experimental concentration.

2.2 The protective effect of NP-17 on NMDA-induced RGC-5 cell injury: Under NMDA injury, the survival rate of RGC-5 cells is reduced, and NP-17 can increase the survival rate of cells under NMDA-induced injury, and protect ganglion cells from NMDA excitation Sexual injury. See Figure 4 (* means p<0.05, **b means p<0.01).

2.3 The effect of NP-17 on NMDA-induced apoptosis of RGC-5 cells: After NMDA injury, the apoptosis of RGC-5 cells increased, and NP-17 could reduce the apoptosis rate of cells after NMDA-induced injury and protect ganglion cells. See Figure 5.

3. Summary

Through the NMDA injury experiment, the concentration of the NMDA injury model was screened out, and then NP-17 was used to intervene to observe the effect of NP-17 on the survival rate and apoptosis of RGC-5 cells. It was confirmed that NP-17 can improve the RGC- 5 cell survival rate, inhibit cell apoptosis, protect nerve cells.

Example 4 The role of PI3K/Akt pathway in NP-17 neuroprotection

1. Materials and Methods

1.1 Experimental cells and materials: Rat retinal ganglion cell-5 (RGC-5) was purchased from Fudan University School of Medicine; DMEM high-glucose medium was purchased from Invitrogen, USA; p-Akt antibody was purchased from CST Corporation, USA; beta- Actin antibody was purchased from Epitomic Company of the United States; LY294002 and Wortmannin were purchased from MERK Company of the United States; related instruments of Western blot were purchased from Bio-Rad Company of the United States; relevant reagents of Western Blot were purchased from Beyontian Company of China.

1.2 Experimental method: RGC-5 cells were placed in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 100U/ml penicillin and streptomycin double antibodies, placed at 37°C, 5% CO ₂ The expansion culture was carried out in the incubator, and the following experiments were carried out respectively:

1.2.1 The effect of NP-17 on the PI3K-Akt pathway: cells in the logarithmic growth phase were taken, adjusted to a density of 5×10 ⁵ and inoculated in a large dish, a total of 6 cells. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. Add the medium containing NP-17 biopeptide into the large dish respectively, start timing after adding, and extract cell protein at the following time points: 0min, 5min, 10min, 30min, 1h, 6h. After protein extraction, Western Blot experiments were performed.

1.2.2 Effect of PI3K-Akt pathway inhibitors on cell death rate: Cells in the logarithmic growth phase were harvested, adjusted to a density of 2×10 ⁴ /well and inoculated in a 96-well plate. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. Cells were randomly divided into Blank control group, NP-17 group, NP-17+LY294002 (30uM) group, NP-17+Wortmannin (100nM) group, LY294002 (30uM) group and Wortmannin (100nM) group, with six multiple holes. Place them in a 37°C, 5% CO ₂ incubator for routine culture, and use CELL LIVE/DEAD AssayKit to detect after 24 hours.

1.2.3 Effects of inhibitors on PI3K-Akt pathway: Cells in logarithmic growth phase were taken, adjusted to a density of 5×10 ⁵ and inoculated in a large dish, totaling 4 cells. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. They were randomly divided into the following 4 groups: Blank group, NP-17 group, LY294002 group and NP-17+LY294002 group. Inhibitor+NP-17 group was first added with inhibitor LY294002 for 15 minutes and then added NP-17 for 10 minutes.

1.3 Statistical analysis: the grayscale scanning software Bandscan scans the grayscale value of each group of strips, and performs quantitative analysis and statistics. Experimental data were expressed as mean ± standard deviation, and SPSS 17.0 statistical software package was used for statistical analysis. Independent sample t-test was used, and P<0.05 was considered statistically significant.

2. Experimental results

2.1 NP-17 up-regulates the PI3K/Akt pathway: The phosphorylation level of NP-17 starts to increase after 5 minutes of treatment, reaches a peak at 30 minutes, and then gradually decreases. See Figure 6 for details.

2.2 Effects of PI3K/Akt pathway inhibitors on the neuroprotective effect of NP-17: After adding pathway inhibitors, the cell death rate increased significantly; after simply adding signaling pathway inhibitors, the inhibitors had no significant effect on cell survival; LY294002 could Significantly decreased the level of p-Akt. See Figure 7 for details.

3. Summary

NP-17 can up-regulate the expression of PI3K/Akt pathway in a time-dependent manner; by using PI3K/Akt pathway inhibitors LY294002 and Wortmannin, it was found that the inhibitors can increase cell death after NMDA injury; inhibitors can effectively inhibit PI3K /Akt pathway, but itself had no significant effect on cell viability.

This experiment shows that NP-17 may play a neuroprotective role by activating PI3K/Akt.

Example 5 The role of ERK pathway in NP-17 neuroprotection

1. Materials and Methods

1.1 Experimental cells and materials: Rat retinal ganglion cell-5 (RGC-5) was purchased from Fudan University School of Medicine; DMEM high-glucose medium was purchased from Invitrogen, USA; p-ERK antibody was purchased from CST Corporation, USA; beta- Actin antibody was purchased from Epitomic Company of the United States; PD098059 was purchased from MERK Company of the United States; related instruments of Western blot were purchased from Bio-Rad Company of the United States; relevant reagents of Western Blot were purchased from Beyontian Company of China.

1.2 Experimental method: RGC-5 cells were placed in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 100U/ml penicillin and streptomycin double antibodies, placed at 37°C, 5% CO ₂ The expansion culture was carried out in the incubator, and the following experiments were carried out respectively:

1.2.1 The effect of NP-17 on the ERK pathway: Take cells in the logarithmic growth phase, adjust the density to 5×10 ⁵ and inoculate them in a large dish, a total of 7 cells. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. Add the medium containing NP-17 biopeptide into the large dish respectively, start timing after adding, and extract cell protein at the following time points: 0min, 2.5min, 5min, 7.5min, 10min, 30min, 1h. After protein extraction, Western Blot experiments were performed.

1.2.2 Effect of ERK pathway inhibitors on cell death rate: Cells in the logarithmic growth phase were seeded at a density of 2×10 ⁴ /well in a 96-well plate. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. Cells were randomly divided into Blank blank control group, NP-17 group, NP-17+PD098059 (20uM) group and PD098059 (20uM) group, with six replicate wells in each group. Place them in a 37°C, 5% CO ₂ incubator for routine culture, and use the CELLLIVE/DEAD Assay Kit to detect after 24 hours.

1.2.3 Effects of inhibitors on ERK pathway: Take cells in logarithmic growth phase, adjust the density to 5×10 ⁵ and inoculate them in a large dish, a total of 4 cells. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. They were randomly divided into the following 4 groups: Blank group, NP-17 group, PD098059 group and NP-17+PD098059 group. Inhibitor + NP-17 group was first added with inhibitor PD098059, acting for 15 minutes, and then adding NP-17 for 10 minutes.

1.3 Statistical analysis: the grayscale scanning software Bandscan scans the grayscale value of each group of strips, and performs quantitative analysis and statistics. Experimental data were expressed as mean ± standard deviation, and SPSS 17.0 statistical software package was used for statistical analysis. Independent sample t-test was used, and P<0.05 was considered statistically significant.

2. Experimental results

2.1 NP-17 up-regulates the ERK pathway: The phosphorylation level of NP-17 began to increase after 2.5 minutes, and ERK1 reached a peak at 7.5 minutes, while ERK2 reached a peak at 10 minutes, and then gradually decreased, as shown in Figure 8.

2.2 The effect of ERK pathway inhibitors on the neuroprotective effect of NP-17: After adding the pathway inhibitor PD098059, the cell death rate increased significantly; after adding the signaling pathway inhibitor alone, the inhibitor had no significant effect on the cell survival rate; PD098059 could significantly Reduced levels of p-Akt. See Figure 9 for details.

3. Summary

NP-17 can up-regulate the expression of the ERK pathway in a time-dependent manner; by using the ERK pathway inhibitor PD098059, it was found that the inhibitor can increase cell death after NMDA injury; the inhibitor can effectively inhibit the phosphorylation of the ERK pathway, while It has no significant effect on cell viability by itself.

This experiment shows that NP-17 may play a neuroprotective role by activating ERK.

Example 6 Effect of NP-17 on Caspase3 expression in RGC-5 cells

1. Materials and Methods

1.1 Experimental cells and materials: Rat retinal ganglion cell-5 (RGC-5) was purchased from Fudan University School of Medicine; DMEM high glucose medium was purchased from Invitrogen, USA; Caspase3/7Assay Kit was purchased from Ivitrogen, USA; Inhibitor Z - DEVD-FMK was purchased from Sigma.

1.2 Experimental method: RGC-5 cells were placed in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 100U/ml penicillin and streptomycin double antibodies, placed at 37°C, 5% CO ₂ The expansion culture was carried out in the incubator, and the following experiments were carried out respectively:

1.2.1 The effect of NP-17 on the expression of Caspase3: Take the cells in the logarithmic growth phase, adjust the density to 2×10 ⁴ and inoculate them in a 96-well plate. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. Replace the medium containing NP-17 biological peptide, incubate in the incubator for 24 hours, replace the medium containing NMDA for 15 minutes, then replace the previous medium, and continue to cultivate for 6 hours. Caspase3/7Assay Kit was used to detect the expression of Caspase3 in each group according to the kit instructions.

1.2.2 Effect of Caspase3 inhibitor Z-DEVD-FMK on cell death rate: Cells in the logarithmic growth phase were harvested, adjusted to a density of 2×10 ⁴ /well and inoculated in a 96-well plate. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. Cells were randomly divided into Blank control group, Z-DEVD-FMK (50uM) group, Z-DEVD-FMK (100uM) group, NMDA group, NMDA+Z-DEVD-FMK (50uM) group and NMDA+Z-DEVD- For the FMK (100uM) group, six replicate wells were set for each group. Place them in a 37°C, 5% CO ₂ incubator for routine culture, and use the CELL LIVE/DEAD Assay Kit to detect after 24 hours.

1.3 Statistical analysis: The experimental data were expressed as mean ± standard deviation, and SPSS 17.0 statistical software package was used for statistical analysis. Independent sample t-test was used, and P<0.05 was considered statistically significant.

2. Experimental results

2.1 NP-17 inhibits the expression of Caspase3: NP-17 can reduce the expression of Caspase3 in RGC-5 cells after NMDA injury, and its inhibitory ability is stronger than that of BDNF. See Figure 10 for details.

2.2 Caspase3 inhibitor inhibits cell apoptosis: No NMDA group was added, after adding the Caspase3 inhibitor Z-DEVD-FMK, there was no significant effect on cell death rate, and the inhibitor had no cytotoxicity on the surface; after adding the NMDA group, after using the inhibitor, The cell death rate was significantly reduced in a dose-dependent manner. See Figure 11 for details.

3. Summary

NP-17 can inhibit the expression of Caspase3; by using the Caspase3 inhibitor Z-DEVD-FMK, it was found that the use of the inhibitor can reduce the cell death rate after NMDA injury; the inhibitor itself has no cytotoxicity.

This experiment shows that Caspase3 plays an important role in the neuroprotective effect of NP-17.

Example 7 The effect of NP-17 on the expression of Bcl-2 family proteins in RGC-5 cells

1. Materials and Methods

1.1 Experimental cells and materials: Rat retinal ganglion cell-5 (RGC-5) was purchased from Fudan University School of Medicine; DMEM high-glucose medium was purchased from Invitrogen, USA; Bcl-2, Bad and Bax were purchased from CST, USA; Western blot related instruments were purchased from American Bio-Rad Company; Western Blot related reagents were purchased from China Beyontian Company.

1.2 Experimental method: RGC-5 cells were placed in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 100U/ml penicillin and streptomycin double antibodies, placed at 37°C, 5% CO ₂ The expansion culture was carried out in the incubator, and the cells in the logarithmic growth phase were taken, and the density was adjusted to 5×10 ⁶ and inoculated in a large dish. When the cells adhered well and reached 80% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. The experimental groups were as follows: Blank blank group, NP-17 group and BDNF group. Replace the medium containing NP-17 biological peptide, incubate in the incubator for 24 hours, replace the medium containing NMDA for 15 minutes, then replace the previous medium, and continue to cultivate for 6 hours. The histones were extracted and subjected to Western Blot experiments.

1.3 Statistical analysis: the grayscale scanning software Bandscan scans the grayscale value of each group of strips, and performs quantitative analysis and statistics. Experimental data were expressed as mean ± standard deviation, and SPSS 17.0 statistical software package was used for statistical analysis. Independent sample t-test was used, and P<0.05 was considered statistically significant.

2. Experimental results

NP-17 increases the expression of Bcl-2 and inhibits the expression of Bad and Bax: NP-17 can reduce the expression of Bad and Bax and increase the expression of Bcl-2 in RGC-5 cells after NMDA injury. See Figure 12 for details.

3. Summary

NP-17 can inhibit the expression of Bad and Bax and increase the expression of Bcl-2.

Example 8 The protective effect of NP-17 on NMDA-induced retinal damage

1. Materials and Methods

1.1 Experimental animals and materials: healthy male Wister rats, about 200g, were purchased from the Animal Center of the Chinese Academy of Medical Sciences; the Turnel kit was purchased from Promega Company of the United States; NMDA was purchased from Sigma Company of the United States; other related immunohistochemical reagents were purchased from Beyontian Company .

1.2 Model preparation and intervention: Rats were randomly divided into 5 groups: Control, Vehicle group, NMDA group, NMDA+NP-17 group and NMDA+BDNF group, with 10 rats in each group, and the right eye was selected as the experimental eye. The Vehicle group was injected with 4uL PBS buffer solution; the NMDA group was injected with 4uL NMDA with a concentration of 5mM in the vitreous cavity, 20nmol/eye; 10ug; NMDA+BDNF group was injected with 4uL NMDA and BDNF mixed solution, the content of NMDA in the solution was 20nmol, and the content of BDNF was 5ug. Seven days after the injection was completed, the animals were sacrificed, and the eyeballs or retinal tissues of the rats were removed for the following experiments:

1.2.1 Protective effect of NP-17 on retinal ganglion cells in rats: 7 days after intravitreal injection of the drug, the rat eyeballs were taken, fixed with fixative solution for 24 hours, and the eyeballs were sliced sagittal, including the optic nerve. Conventional HE staining. After the staining was completed, the samples were observed and photographed under a light microscope.

1.2.2 Effect of NP-17 on retinal ganglion cell apoptosis after NMDA-induced injury: 7 days after intravitreal injection of drugs, rat eyeballs were taken, fixed in fixative solution for 24 hours, and the eyeballs were sliced sagittal, including the optic nerve. Wax sections were made routinely, stained according to the instructions of the Turnel kit, observed and photographed under a confocal microscope.

1.3 Statistical analysis: Count the number of ganglion cells after HE staining and the number of apoptotic ganglion cells stained by Tunel, and perform quantitative analysis and statistics. Experimental data were expressed as mean ± standard deviation, and SPSS 17.0 statistical software package was used for statistical analysis. Independent sample t-test was used, and P<0.05 was considered statistically significant.

2. Experimental results

2.1 NP-17 protects ganglion cells: the number of ganglion cells in the NMDA group decreased significantly, and after injection of NP-17, the number of residual ganglion cells exceeded that of the NMDA group. See Figure 13 for details.

2.2 NP-17 inhibits the apoptosis of ganglion cells: the number of apoptotic cells in the ganglion cell layer increased significantly in the NMDA group, and the number of apoptotic RGC cells decreased significantly after the addition of NP-17. See Figure 14 for details.

3. Summary

NP-17 can protect the retina, alleviate the toxic effect of NMDA on the retina, and reduce the apoptosis of retinal RGC cells.

Example 9 Effect of NP-17 on Caspase3 expression in rat retina

1. Materials and Methods

1.1 Experimental animals and materials: healthy male Wister rats, about 200g, were purchased from the Animal Center of the Chinese Academy of Medical Sciences; Caspase3/7Assay Kit was purchased from Ivitrogen, USA; cell lysate was purchased from MERK, USA.

1.2 Take 3 eyeballs of each group of rats in "Example 7", separate the retinal tissue, add 200ul of cell lysate to each eyeball, ultrasonically disintegrate and lyse, centrifuge at 12000rpm for 5 minutes at high speed, and take the supernatant. According to the Caspase3/7 Assay Kit operation manual, the content of Caspase3 in each group was detected. Each group replicated 6 wells.

1.3 Statistical analysis: The experimental data were expressed as mean ± standard deviation, and SPSS 17.0 statistical software package was used for statistical analysis. Independent sample t-test was used, and P<0.05 was considered statistically significant.

2. Experimental results

NP-17 reduces the content of Caspase3 in retinal tissue: NMDA can significantly increase the content of Caspase3 in retinal tissue, and after intravitreal injection of NP-17, the content of Caspase3 in retinal tissue decreases significantly. See Figure 15 for details.

3. Summary

Intravitreal injection of NMDA can significantly increase the content of Caspase3 in retinal tissue, and NP-17 can reduce the content of Caspase3.

Preparation and Identification of Example 10 Derivative Polypeptides

According to the method of Example 1, and according to Table 1, some amino acids were substituted, added or deleted within a reasonable range, and the following polypeptides were prepared, and the polypeptides were identified:

Table 2

Embodiment 11 carries out activity test to the polypeptide in embodiment 10

According to the method shown in embodiment 3, measure each NP-17 each derivative polypeptide 1-9 (SEQ ID NO.:2-10) when the RGC-5 cell survival rate of NMDA-induced apoptosis at 1ug/ml concentration , and the results are shown in Table 3.

The results showed that, in the treatment groups of the above-mentioned derived polypeptides 1-9, the number of RGC cells with NMDA-induced apoptosis all increased to a certain extent. It can be seen that the derivative polypeptides of SEQ ID NO.: 1 can increase the activity of RGC-5 compared with the NMDA group, so they all have a certain degree of neuroprotective effect. It can be considered that, although the derivative polypeptides of the present invention are substituted, added or deleted within a reasonable range, almost all of them can have certain neuroprotective effects, although their activities vary.

Comparative example 1

Activity identification of two peptide pairs

2. Materials and Methods

1.1 Experimental cells and materials: Rat retinal ganglion cell-5 (RGC-5), a gift from Fudan University School of Medicine; DMEM high-glucose medium, purchased from Invitrogen, USA; MTS was purchased from Promega, USA.

1.2 Peptide activity identification: RGC-5 cells were placed in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin double antibodies, placed at 37°C, 5% CO ₂ Expansion culture in the incubator. Cells in the logarithmic growth phase were taken, and the density was adjusted to 2×10 ⁴ /well and inoculated in a 96-well plate. When the cells adhered well and reached 60% confluence, the DMEM medium without fetal bovine serum was replaced for serum starved culture for 24 hours. The cells were randomly divided into blank control (Blank), polypeptide (1ng/ml), polypeptide (10ng/ml), polypeptide (100ng/ml), polypeptide (1000ng/ml) and polypeptide (5000ng/ml) groups, and each group had six Each well was placed in a 37°C, 5% CO ₂ incubator for routine culture. After 24 hours, 20ul of MTS was added to each well, and after 2 hours, the OD value was measured with a spectrophotometer at a wavelength of 490nm.

1.3 Statistical analysis: The experimental data were expressed as mean ± standard deviation, and SPSS 17.0 statistical software package was used for statistical analysis. Independent sample t-test was used, and P<0.05 was considered statistically significant.

2. Experimental results

2.1 Effect of polypeptide N1 on the proliferation of RGC-5 cells: Polypeptide N1 in different concentration groups has no effect on the proliferation of RGC-5 cells, p>0.05. See Figure 16.

2.2 Effect of polypeptide N2 on the proliferation of RGC-5 cells: Polypeptide N1 in different concentration groups had no effect on the proliferation of RGC-5 cells, p>0.05. See Figure 17.

3. Summary

Through the cell proliferation experiment, polypeptide N1 and polypeptide N2 could not promote the proliferation of RGC-5 cells and were inactive.

Claims (10)

1. the polypeptide that represents of following formula I, or its pharmacy acceptable salt

[Xaa0]-[Xaa1]-[Xaa2]-[Xaa3]-[Xaa4]-[Xaa5]-[Xaa6]-[Xaa7]-[Xaa8]-[Xaa9]-[Xaa10]-[Xaa12]-[Xaa12]-[Xaa13]-[Xaa14]-[Xaa15]-[Xaa16]-[Xaa17]-[Xaa18]-[Xaa19](I)

In formula,

Xaa0 is nothing, or 1-5 Amino acid profile peptide section;

Xaa1 is selected from the amino acid of lower group: Ile, Leu, Val, Met, Ala or Phe;

Xaa2 is selected from the amino acid of lower group: Pen or Hcy;

Xaa3 is selected from the amino acid of lower group: Lys, Arg, Gln or Asn;

Xaa4 is selected from the amino acid of lower group: Gly, Pro, Ala or;

Xaa5 is selected from the amino acid of lower group: Lys, Arg, Gln or Asn;

Xaa6 is selected from the amino acid of lower group: Glu or Asp;

Xaa7 is selected from the amino acid of lower group: Val, Ile, Leu, Met, Phe or Ala;

Xaa8 is selected from the amino acid of lower group: Cys, Hcy or Pen;

Xaa9 is selected from the amino acid of lower group: Thr or Ser;

Xaa10 is selected from the amino acid of lower group: Acp or β-Ala;

Xaa11 is selected from the amino acid of lower group: Asn, Gln, His, Lys or Arg;

Xaa12 is selected from the amino acid of lower group: Ala, Val, Leu or Ile;

Xaa13 is selected from the amino acid of lower group: Pro or Ala;

Xaa14 is selected from the amino acid of lower group: Val, Ile, Leu, Met, Phe or Ala;

Xaa15 is selected from the amino acid of lower group: Ser or Thr;

Xaa16 is selected from the amino acid of lower group: Ile, Leu, Val, Met, Ala or Phe;

Xaa17 is selected from the amino acid of lower group: Pro or Ala;

Xaa18 is selected from the amino acid of lower group: Gln or Asn;

Xaa19 is nothing, or 1-5 Amino acid profile peptide section;

Wherein, between described Xaa2 and Xaa8, form disulfide linkage, and described polypeptide has neural activity of protecting.

2. polypeptide as claimed in claim 1, it is characterized in that, described polypeptide is:

Xaa0 is nothing;

Xaa1 is selected from the amino acid of lower group: Ile or Leu;

Xaa2 is Pen;

Xaa3 is selected from the amino acid of lower group: Lys or Arg;

Xaa4 is selected from the amino acid of lower group: Gly or Ala;

Xaa5 is selected from the amino acid of lower group: Lys or Arg;

Xaa6 is selected from the amino acid of lower group: Glu or Asp;

Xaa7 is selected from the amino acid of lower group: Val or Leu;

Xaa8 is selected from the amino acid of lower group: Cys or Pen;

Xaa9 is selected from the amino acid of lower group: Thr or Ser;

Xaa10 is selected from the amino acid of lower group: Acp or β-Ala;

Xaa11 is selected from the amino acid of lower group: Asn or Gln;

Xaa12 is selected from the amino acid of lower group: Ala or Val;

Xaa13 is selected from the amino acid of lower group: Pro or Ala;

Xaa14 is selected from the amino acid of lower group: Val or Leu;

Xaa15 is selected from the amino acid of lower group: Ser or Thr;

Xaa16 is selected from the amino acid of lower group: Ile or Leu;

Xaa17 is selected from the amino acid of lower group: Pro or Ala;

Xaa18 is selected from the amino acid of lower group: Gln or Asn;

Xaa19 is nothing;

Wherein, between described Xaa2 and Xaa8, form disulfide linkage, and described polypeptide has the activity of neuroprotective, and described polypeptide is through 1-5 at the most, preferably 1-3, more preferably 1-2 amino acid whose replacement.

3. polypeptide as claimed in claim 1, it is characterized in that, described Xaa0 is the peptide section of 1 ~ 3 Amino acid profile; And/or described Xaa19 is the peptide section of 1 ~ 3 Amino acid profile.

4. a derivative polypeptide, is characterized in that, described derivative polypeptide is the derivative polypeptide of polypeptide shown in SEQ ID NO.:1, and is selected from lower group:

A () has aminoacid sequence shown in SEQ ID NO:1;

B aminoacid sequence shown in SEQ ID NO:1 is formed through the replacement of 1-5 (preferably 1-3, more preferably 1-2) amino-acid residue or interpolation by (), and have the polypeptide derivative by (a) of neuroprotective function.

5. derivative polypeptide as claimed in claim 4, is characterized in that, described derivative polypeptide is by polypeptide shown in SEQID NO.:1 through 1-5, preferably 1-3, more preferably 1-2 aminoacid replacement; And/or

Through 1-3, the preferably aminoacid deletion at 1-2 polypeptide two ends; And/or

The two ends of described derivative polypeptide are respectively through 1-5, and preferably 1-4 or 1-3, more preferably 1-2, aminoacid addition is formed.

6. a pharmaceutical composition, it contains:

A polypeptide that () claim 1-3 is arbitrary or the arbitrary derivative polypeptide of claim 4-5, or its pharmacy acceptable salt; With

(b) pharmaceutically acceptable carrier or vehicle.

7. composition as claimed in claim 6, is characterized in that, the formulation of described composition be collyrium, injection (as near the eyes and intraocular injection, especially intravitreal), gel for eye use or spongaion.

8. a purposes for polypeptide of the present invention or pharmacy acceptable salt, they are used to the medicine preparing neuroprotective or control and neural cell injury disease.

9. purposes as claimed in claim 8, is characterized in that, described with group under being selected from of neural cell injury relative disease: ocular nerve cell injury relative disease, acute or chronic retinal optic cell injury disease.

10. purposes as claimed in claim 9, is characterized in that, described ocular nerve cell injury relative disease comprises glaucoma, maculopathy, diabetic retinopathy, retina arteriovenous obstruction.