CN103992376B - Small peptides inhibiting new vessels and application - Google Patents

Abstract

A new class of small peptides inhibiting angiogenesis and its application. The invention relates to a class of polypeptides for preventing and inhibiting angiogenesis and applications thereof. The present invention also relates to the preparation method and application of the polypeptide and the pharmaceutical composition containing the polypeptide. The polypeptide of the present invention has many advantages, such as small molecular weight, can pass through various eye tissue barriers; has good water solubility, and can maintain a relatively high concentration in neutral tear fluid, aqueous humor and vitreous humor, and the like.

Description

A small peptide for inhibiting neovascularization and its application

technical field

The present invention relates to the field of biomedicine, in particular to a new class of small peptides for inhibiting neovascularization, and also relates to the preparation method and application of the polypeptide and the pharmaceutical composition containing the polypeptide.

Background technique

The formation of neovascularization is an extremely complex process, which includes: dilation of existing blood vessels, increase of vascular permeability, degradation of perivascular matrix, activation of proliferation and migration of endothelial cells, and formation of new capillary-like lumens.

In the eye, about two-thirds of blinding diseases are associated with pathological neovascularization, such as corneal neovascularization in herpetic stromal keratitis, choroidal neovascularization in age-related macular degeneration, and diabetic retinopathy Or retinal neovascularization in retinopathy of prematurity, etc. Currently, laser photocoagulation, photodynamic therapy (PDT) and transpupillary thermotherapy (Thermal transpupillary therapy, TTT) are routinely used for the treatment of ocular pathological neovascularization. However, these treatment methods are not only easy to cause damage to local tissues, but also their long-term efficacy is not very satisfactory. Therefore, in recent years, people have been trying to develop more effective methods for treating ocular pathological neovascularization.

When developing effective angiogenesis inhibitors, 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. For ocular surface administration, the drug must penetrate the lipophilic tight junction of corneal epithelial cells and the hydrophilic corneal stroma successively. Therefore, only drugs with appropriate fat solubility, low molecular weight, or transporters in ocular surface tissues (such as: Amino acid transporter, oligopeptide transporter, etc.) can reach the anterior chamber to play a role.

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; to improve it, the concentration of administration can be increased. The compounds used to treat neovascularization of tumors have relatively obvious toxic and side effects, and cannot be administered in high doses both systemically and locally. In addition, exogenous proteins with large molecular weight are also sensitive sources of foreign matter, which can cause immune damage to ocular tissues (such as: uvea).

Fourth, although a series of relatively safe endogenous angiogenesis inhibitors have been confirmed successively, such as angiostatin, which consists of plasminogen Kringle domains 1-4 (plasminogen Kringle1-4), It can significantly inhibit the growth of blood vessel-dependent tumors, but due to its large molecular weight and complex spatial conformation, there are deficiencies in the preparation process such as cumbersome recombinant expression and purification processes and endotoxin residues. Due to the limitations of the above-mentioned conditions, the drugs currently used to treat ocular neovascularization are very limited, such as recombinant anti-human VEGF monoclonal antibody bevacizumab (Avastin), recombinant anti-human VEGF monoclonal antibody fragment ranibizumab (Lucentis), etc., but They are expensive and require repeated administration through the vitreous cavity, which may even cause risks such as vascular embolism.

It can be seen that looking for small molecule inhibitors with specific biological activity and biocompatibility can penetrate various ocular tissue barriers through non-invasive or minimally invasive drug delivery routes, improve the local bioavailability of the eye, and reduce the dosage , to reduce local and systemic side effects, which is of great significance to the clinical prevention and treatment of neovascular eye diseases. Therefore, there is an urgent need in this field to develop an effective and safe small molecule angiogenesis inhibitor suitable for eyeball tissue.

Contents of the invention

The object of the present invention is to provide a class of effective and safe small molecular polypeptides capable of inhibiting angiogenesis and fragments, analogs and derivatives thereof suitable for eyeball tissues.

Another object of the present invention is to provide the preparation method and application containing said polypeptide.

In the first aspect of the present invention, there is provided in the first aspect of the present invention, there is provided 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]-[Xaa11] (I)

In the formula,

Xaa0 is none, or 1-2 amino acids constitute a peptide segment;

Xaal 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: Ser; or Thr;

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 Glu; or Asp;

Xaa5 is an amino acid selected from the group consisting of: Thr; or Ser;

Xaa6 is an amino acid selected from the group consisting of: Lys; Arg; Gln; or Asn;

Xaa7 is an amino acid selected from the group consisting of Lys; Arg; Gln; or Asn;

Xaa8 is an amino acid selected from the group consisting of Lys; Arg; Gln; or Asn;

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

Xaa10 is an amino acid selected from the group consisting of Tyr; Trp; Phe; Thr; or Ser;

Xaa11 is none, or 1-2 amino acids constitute a peptide;

And the polypeptide has the activity of inhibiting angiogenesis, and the length of the polypeptide is 10-14 amino acids.

More preferably, in the polypeptide:

Xaa0 is None;

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

Xaa2 is an amino acid selected from the group consisting of: Ser; or or Thr;

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 Glu; or Asp;

Xaa5 is an amino acid selected from the group consisting of: Thr; or Ser;

Xaa6 is an amino acid selected from the group consisting of Lys; or Arg;

Xaa7 is an amino acid selected from the group consisting of: Lys; or Arg;

Xaa8 is an amino acid selected from the group consisting of: Lys; or Arg;

Xaa9 is an amino acid selected from the group consisting of: Tyr; or Phe;

Xaa10 is an amino acid selected from the group consisting of: Tyr; or Phe;

Xaa11 is None

In another preferred example, the length of the polypeptide is 10-14 amino acids;

In another preferred example, the length of the polypeptide is 9-14 amino acids.

In another preferred embodiment, the polypeptide is selected from the following group:

(a) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2;

(b) The amino acid sequence shown in SEQ ID NO: 2 is formed by substitution, deletion or addition of 1-4 (preferably 1-3, more preferably 1-2) amino acid residues, and has the ability to inhibit angiogenesis Functional polypeptides derived from (a).

In another preferred example, the derivative polypeptide retains ≥70% of the angiogenesis-inhibiting activity of the polypeptide shown in SEQ ID NO:2.

In another preferred example, the identity of the derived polypeptide to SEQ ID NO: 2 is ≥80%, preferably ≥90%; more preferably ≥95%.

The present invention also provides dimer and multimer forms of the compound of formula I having the function of inhibiting angiogenesis.

In a second aspect of the present invention, an isolated nucleic acid molecule encoding the above-mentioned polypeptide of the present invention is provided.

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

(a) the above-mentioned polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and

(b) A pharmaceutically acceptable carrier or excipient.

In another preferred example, the dosage form of the composition is eye drops, injections (such as periocular and intraocular injections), ophthalmic gel or ophthalmic ointment.

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

In the fourth aspect of the present invention, there is provided a use of the polypeptide or a pharmaceutically acceptable salt of the present invention, which are used in the preparation of drugs for inhibiting angiogenesis or preventing and treating diseases related to angiogenesis.

In the fifth aspect of the present invention, there is provided a method for inhibiting angiogenesis in 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 subject is human.

In another preferred example, the angiogenesis is angiogenesis associated with neovascular eye diseases.

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

The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention defined by the claims.

Figure 1 shows the effect of the small peptide ZY6 on the proliferation of human umbilical vein endothelial cells HUVECs, and the small peptide ZY6 has the effect of significantly inhibiting the proliferation of endothelial cells. Compared with the VEGF group, the VEGF+polypeptide ZY6 group (1 μM-100 μM) significantly inhibited the proliferation of HUVECs, *P<0.05, **P<0.01, the difference was statistically significant.

Figure 2 shows the effect of the small peptide ZY6 on the lumen formation of human umbilical vein endothelial cells HUVECs, and it can be seen that the small peptide ZY6 has the effect of significantly inhibiting the lumen formation of endothelial cells.

Figures 2a-2c show the inhibitory effect of the small peptide ZY6 on the lumen formation of HUVECs.

Figure 2a is the VEGF group; Figure 2b is the VEGF+ZY6 (100 μM) group; Figure 2c is the VEGF+ small peptide ZY6 group (1 μM-100 μM) significantly inhibited the lumen formation of HUVECs compared with the VEGF group, *P<0.05, **P<0.01, the difference is statistically significant.

Figure 3 shows that the small peptide ZY6 has a significant inhibitory effect on neovascularization on the allantoic membrane of chicken embryos.

Figures 3a-3c show the 3-5 microvessel counts within 2.5mm around the filter paper sheet.

Figure 3a is the PBS group; Figure 3b is the ZY6 (10 μl/tablet) group; Figure 3c is the ZY6 (50 μl/tablet) group; Figure 3d is that compared with the PBS group, the small peptide ZY6 group at various concentrations can significantly inhibit the chicken embryo allantois The number of membrane neovascularization, and the inhibitory effect was concentration-dependent, *P<0.05, **P<0.01, the difference was statistically significant.

Figure 4 shows that the small peptide ZY6 has a significant inhibitory effect on mouse retinal neovascularization.

Figure 4a is the PBS group; Figure 4b is the ZY6 (2 μg/μl) group; Figure 4c is the hyperoxia + ZY6 group (1 μg/μl～2 μg/μl) significantly inhibited the number of retinal neovascularization in mice compared with the hyperoxia + PBS group , and the inhibitory effect was concentration-dependent, *P<0.05, **P<0.01, the difference was statistically significant.

detailed description

After extensive and in-depth research, the present inventors have prepared a class of small molecule polypeptides with a molecular weight of less than 3kD that have the function of inhibiting angiogenesis for the first time. Specifically, the inventors applied the method of bioinformatics, based on the analysis of homology and biological characteristics, designed several candidate sequences, synthesized them by solid-phase method, separated and purified them to obtain the high-purity small peptide ZY6 , and identified by HPLC and MS, and then screened by chicken embryo allantoic membrane vascular model, oxygen-induced mouse retinal neovascularization model, VEGF-induced human umbilical vein endothelial cell proliferation model and human umbilical vein endothelial cell lumen model , obtained a new class of small molecular polypeptides that can prevent and treat angiogenesis-related diseases.

The small peptide 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 ; High bioavailability of ophthalmic topical drugs, can reduce the dose, thereby reducing systemic side effects. The present invention has been accomplished on this basis.

Human Neuropil in-1

Neuropilin is a transmembrane glycoprotein first reported by Satoda et al. in 1995. According to the selective splicing of the neuropilin gene, two isomers can be formed: Neuropilin-1 and Neuropilin-2, with 44% homology between them. Like other Neuropilin family members with highly conserved structures, Neuropilin-1 is a transmembrane protein with three different extracellular domains, called a1/a2, FV/FV III (b1/b2) and MAM (c) , its transmembrane region and intracytoplasmic region are included in the d domain. Neuropilin-1 gene is widely expressed in human tissues, such as nervous system, endothelial cells, certain tumor cells, heart, lung, placenta, etc. In the process of angiogenesis, VEGF-165, PlGF-2, VEGF-B, and VEGF-E can combine with Neuropilin-1 to activate downstream signal transduction pathways and exert important biological effects.

active peptide

In the present invention, the terms "polypeptide of the present invention", "ZY6 polypeptide", "ZY6 small peptide", "short peptide ZY6" or "peptide ZY6" are used interchangeably, and all refer to the amino acid sequence of peptide ZY6 with angiogenesis inhibitory activity (ISKETKKKYY, as shown in SEQ ID NO: 2) protein or polypeptide. In addition, the term also includes variant forms of the sequence of SEQ ID NO: 2 that have the function of inhibiting angiogenesis. These variations include (but are not limited to): 1-4 (preferably 1-3, more preferably 1-2, and most preferably 1) amino acid deletions, insertions and/or substitutions, and One or several (usually within 4, preferably within 3, more preferably within 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 term also includes linear as well as non-linear polypeptides (eg, cyclic peptides). A typical polypeptide has one amino acid missing from the C-terminal and/or N-terminal, that is, a polypeptide of 9 amino acids.

The present invention also includes active fragments, derivatives and analogs of the ZY6 polypeptide. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the function or activity of inhibiting angiogenesis. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) A polypeptide with substituent groups in amino acid residues, or (iii) a polypeptide formed by fusion of a ZY6 polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence The polypeptide fused to this polypeptide sequence (subsequent protein fused with 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.

One class of preferred active derivatives refers to that compared with the amino acid sequence of formula I, there are at most 4, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid is replaced by an amino acid with similar or similar properties. substitution to form a polypeptide. 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;Il e 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 Hi s(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 ZY6 polypeptides. The difference between these analogs and the natural ZY6 polypeptide may be the difference in amino acid sequence, or the difference in the modified form 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.

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 ZY6 polypeptides. A preferred coding sequence is (SEQ ID NO:1, coding sequence: ATTTCAAAAAAACCAAGAAGAAATATTAT), which encodes the short peptide ZY6 shown in SEQ ID NO:2.

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 coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, taking SEQ ID NO: 1 as an example, "degenerate variants" in the present invention refer to encoding polypeptides having the sequence of SEQ ID NO: 2, but having a sequence that is identical to the corresponding coding region in SEQ ID NO: 1 different nucleic acid sequences.

The full-length sequence of ZY6 nucleotides 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 a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector or the ZY polypeptide coding sequence of the present invention.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies specific to ZY6 polypeptide, especially monoclonal antibodies.

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 ZY6 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 polynucleotide of the present invention can be used to express or produce recombinant ZY6 polypeptide by conventional recombinant DNA technology. Generally speaking, there are the following steps:

(1). Use the polynucleotide (or variant) encoding the ZY6 polypeptide of the present invention, or transform or transduce a suitable host cell 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 small peptide can be formed by enzymatic digestion and other methods.

Angiogenesis-related diseases

In the present invention, angiogenesis-related diseases are not particularly limited, and include various angiogenesis-related diseases known in the art. Representative examples of diseases associated with angiogenesis include (but are not limited to): neovascular eye disease, neoplasm, ischemic heart disease, non-inflammatory cardiomyopathy, coronary atherosclerosis, arteriosclerosis obliterans, arterial embolism, Arterial thrombosis, Berger's disease, chronic inflammation, inflammatory bowel disease, ulcer, rheumatoid arthritis, scleroderma, psoriasis, infertility or sarcoid-like disease, etc.

Preferably, the neovascular eye diseases include (but are not limited to): involving the choroid, retina, cornea or iris, including age-related macular degeneration, proliferative diabetic retinopathy, retinal vascular occlusive disease, retinopathy of prematurity , corneal infection, neovascular glaucoma, etc.

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 may be administered by conventional routes including, but not limited to: ocular surface, periocular, intraocular, 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, eye drops, injections, ophthalmic gels and ophthalmic ointments can be exemplified.

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 as follows: the short peptide ZY6 or its pharmaceutically acceptable salt is dissolved in sterile water (surfactant is dissolved in sterile water) together with basic substances, and the osmotic pressure and pH are adjusted to Physiological state, and may optionally add suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and viscosifiers, and then make it 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 ZY6 or its salt can be incorporated into a pill or microcapsule with a slow-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. In addition, the short peptide ZY6 or its salts can also be applied by inserting 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 ZY6 or a pharmaceutically acceptable salt thereof as the active ingredient can be adjusted according to the body weight, age, sex, and degree of symptoms of each patient to be treated. be reasonably determined. For example, when topical eye drops, usually its concentration is about 0.1-10wt%, preferably 1-4wt%, can be administered 2-6 times a day, 1-2 drops each time.

Industrial applicability

The pharmaceutical composition containing the peptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient has significant inhibitory activity on angiogenesis. It is confirmed by animal experiments that the polypeptide of the present invention can not only inhibit angiogenesis of chicken embryo allantoic membrane and oxygen-induced retinal neovascularization of mice, but also inhibit the proliferation and lumen formation of human umbilical vein endothelial cells.

The main advantages of the present invention include:

(a) The polypeptide ZY6 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, which can reduce dosage, thereby reducing 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 the treatment of neovascular eye diseases and related neovascular diseases, such as tumor neovascularization.

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 methods not indicating specific conditions in the following examples are 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's instructions suggested conditions.

Preparation example

Synthesis of Small Peptide ZY6 and Derivative Peptides

Using a commercially available SYMPHONY 12-channel peptide synthesizer (Protein Technologies, USA), according to the operation manual of the synthesizer, the Fmoc solid-phase method was used to synthesize the ZY6 polypeptide with the sequence shown in SEQ ID NO: 2, and the sequence shown in SEQ ID NO: 2. NO.: Derivative polypeptides ZY6-1 to ZY6-6 shown in 3-6.

After the synthesis is complete, cut the polypeptide from the resin (cutting solution (10/g): TFA (J.T.Baker) 94.5%, water 2.5%, EDT (ALDRICH) 2.5%, TIS (ALDRICH) 1%; cutting time: 120min). The lysate was blown dry with nitrogen gas (Shanghai Bio Gas Industry Co., Ltd.), washed six times with ether (Shanghai Shiyi Chemical Reagent Co., Ltd.), and evaporated to dryness at room temperature.

Use HPLC (SHIMADZU high performance liquid chromatograph model: preparative type, analytical type, software: Class-VP.SevialSystem, manufacturer: SHIMADZU) to purify the peptide, dissolve the crude peptide in pure water or add a small amount of acetonitrile (Fisher), according to the following conditions The small peptide ZY6 and its derivative polypeptides were purified separately.

Pump A: 0.1% trifluoroacetic acid + ultrapure water

Pump B: 0.1% trifluoroacetic acid + acetonitrile

Flow rate: 1.0ml/min

Detection volume: 30ul

Wavelength: 220nm

Detection column: Column: Venusi MRC-ODS C18 column (30x250mm)

The detection process is shown in Table 2.

Table 2

Finally, the purified solution was lyophilized to obtain high-purity (>95%) small peptide ZY6 and derivative polypeptides ZY6-1 to ZY6-6.

A small amount of finished small peptide ZY6 was taken for purity identification by HPLC analysis and molecular weight identification by ESI-MS.

The results showed that the purity identification was greater than 99%. The small peptide ZY6 had a total of 10 amino acids and a molecular weight of about 1Kd, which was consistent with the predicted value.

Small peptides in white powder form are sealed and stored at -20°C for long-term storage.

Example 1

Effect of Small Peptide ZY6 on Proliferation of Human Umbilical Vein Endothelial Cells

Using the MTS method, the specific method is as follows:

Primary human umbilical vein endothelial cells HUVECs (purchased from ScienCell) were inoculated in 96-well plates at a concentration of 2×104/ml; after the cells adhered to the wall, serum-free culture medium ECM was added and cultured at 37°C for 24 hours; Serum-free culture agent ECM was added to the wells as a negative control, VEGF (100ng/ml) (purchased from Sigma) was used as a positive control, and VEGF (100ng/well) + small peptide ZY6 of different concentrations was used as a treatment group; continue to culture for 24 hours After that, 20 μl of MTS solution (purchased from Promega Company) was added to each well; after incubation for 4 hours at 37° C., the absorbance of each well at 490 nm was measured by a microplate reader (Bio-Rad Company), and the proliferation activity of the cells was judged according to OD490. Finally, SPSS11.0.1 is used for statistical analysis.

The results are shown in Figure 1. It can be seen that small peptide ZY6 can significantly inhibit the proliferation of HUVECs in human umbilical vein vascular endothelial cells in a concentration-dependent manner. Compared with the VEGF group, the VEGF + small peptide ZY6 group (1μM-100μM) can significantly inhibit the proliferation of HUVECs Effect, *P<0.05, **P<0.01, the difference was statistically significant.

Example 2

Effects of small peptide ZY6 on the lumen formation activity of human umbilical vein endothelial cells

Using the Matrigel method, the specific method is as follows:

50 μl/well of Matrigel (purchased from BD) was added to a 96-well plate, and incubated at 37° C. for 30 minutes. After they were solidified, primary human umbilical vein endothelial cells HUVECs were inoculated on the surface of Matrigel at a concentration of 8×10 ⁶ /ml; serum-free culture medium ECM was added to each well as a negative control, VEGF (100ng/ml) (purchased from Sigma) was used as a positive control, and VEGF (100ng/ml) + different concentrations of small peptide ZY6 was used as a treatment group, and cultured at 37°C. 6 hours after the treatment, the cells in the orifice plate were photographed at random under a 200-fold microscope in 3 fields of view, and the sum of the largest diameters of the lumen formed therein was calculated using the software Image-ProPlus Program5.1 (Media Cybernetics, Inc.), Finally, SPSS11.0.1 is used for statistical analysis.

The results are shown in Figure 2. The small peptide ZY6 has the effect of significantly inhibiting the lumen formation of human umbilical vein endothelial cells HUVECs within 6 hours in a concentration-dependent manner. Figures 2a-2c show the inhibitory effect of the small peptide ZY6 on the lumen formation of HUVECs. Figure 2a is VEGF group; Figure 2b is VEGF+ZY6 (100μM) group; Figure 2c is compared with VEGF group, VEGF+ small peptide ZY6 group (1μM～100μM) can significantly inhibit HUVECs lumen formation, *P<0.05 , **P<0.01, the difference is statistically significant.

Example 3

Determination of Anti-angiogenesis Effect of Small Peptide ZY6 on Chicken Embryo Allantoic Membrane

Using the chicken embryo allantoic membrane model, the specific method is as follows:

Put the breeding eggs 1-2 days after birth into a constant temperature and humidity box (T=37°C, humidity H=60-70%) after disinfection and incubate for 5 days (24 hours a day), and turn the eggs once a day in the morning and evening; Then add PBS (5 μl/piece) dropwise to filter paper pieces (Whatman quantitative filter papers, Sigma, ashless, Grade42, Cat No1442-042, 42.5 mmΦ×100 circles) containing cortisone acetate (5 μg/μl, 5 μl/piece) Or low-concentration (2μg/μl), high-concentration (10μg/μl) small peptide ZY6 (5μl/piece), air-dried the filter paper and place it between the allantoic membrane and large blood vessels of the breeding eggs and seal the breeding eggs; continue to put the breeding eggs Place in a constant temperature and humidity box (temperature T=37°C, humidity H=60-70%) and incubate for 2 days (24 hours count as one day), without turning the eggs; after that, the allantoic membrane of the chicken embryos is completely exposed, and photographed (the scope is a filter paper sheet) week 5mm) and count 3-5 microvessels (the range is 2.5mm around the filter paper), and use SPSS11.0.1 for statistical analysis.

The results are shown in Figure 3. It can be seen that compared with the PBS group, the small peptide ZY6 can significantly inhibit the neovascularization of the chicken embryo allantoic membrane at low concentrations (10 μg/tablet) and high concentrations (50 μg/tablet). Figures 3a-3c show microvessel counts of grades 3-5 within 2.5 mm of the filter paper disc. Figure 3a is the PBS group; Figure 3b is the ZY6 (10 μg/tablet) group; Figure 3c is the ZY6 (50 μg/tablet) group; Figure 3d is that compared with the PBS group, the small peptide ZY6 groups at various concentrations can significantly inhibit the chicken embryo allantois The number of membrane neovascularization, and the inhibitory effect was concentration-dependent, *P<0.05, **P<0.01, the difference was statistically significant.

Example 4

Determination of the Effect of Small Peptide ZY6 on Pathological Neovascularization of Mouse Retina

Using the oxygen-induced mouse retinopathy model, the specific method is as follows:

The 7-day-old C57BL/6 and lactating mother mice were raised in an environment with an oxygen concentration of about 75%±2%. After 5 days, the mice were taken out, and 0.5 μl of low concentration (0.5 μg/μl), medium concentration (1 μg/μl), and high concentration (2 μg/μl) were injected into the mouse vitreous on the day and the third day after taking out Small peptide ZY6 and control sample PBS, the mice were placed in normal air after operation and continued to be raised to establish an oxygen-induced mouse retinopathy model. After 5 days, the mouse eyeballs were taken out, fixed in 4% paraformaldehyde, and paraffin-embedded Sections were made and stained with HE. Under a light microscope of 200 times, the endothelial cell nuclei of retinal neovascularization were counted, and SPSS11.0.1 was used for statistical analysis.

The results are shown in Figure 4. The small peptide ZY6 can significantly inhibit pathological neovascularization of mouse retina at medium concentration (1 μg/μl) and high concentration (2 μg/μl), *P<0.05, **P<0.01, has statistical significane.

Example 5

Activity testing of derivatized peptides

According to the method shown in Example 3, the anti-angiogenesis effect of each ZY6-derived polypeptide was determined. The results are shown in Table 3:

table 3

sample sequence SEQ ID NO.: microvessel count Derivative Peptide 1 (ZY6-1) I SKETKKKY F 3 52 Derivative Peptide 2 (ZY6-2) IS Q ETKKKYY 4 49 Derivative Peptide 3 (ZY6-3) IS Q ETKKKY F 5 48 Derivative polypeptide 4 (ZY6-4) L SKETKK R YY 6 50 Derivative polypeptide 5 (ZY6-5) GA IS KETKKKYY VK 7 48 Derivative polypeptide 6 (ZY6-6) SKETKKKYY 8 53 Control (PBS) 64

The results showed that, compared with the control group, the derived polypeptides 1-6 had the effect of significantly inhibiting the neovascularization of the allantoic membrane of chicken embryos at low concentrations (10 μg/tablet).

Example 6

Preparation of eye drops

Using conventional techniques, the following ingredients are mixed to make 1% eye drops, the formula of which is as follows:

4 volunteers tried it for a week, 3 times a day, 1-2 drops/eye each time. The results showed that the eye drops can inhibit angiogenesis in the eye.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (6)

1. A polypeptide, or a pharmaceutically acceptable salt thereof, said polypeptide has the activity of inhibiting angiogenesis, and said polypeptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO.:2-8. 2. An isolated nucleic acid molecule, wherein the nucleic acid molecule encodes the polypeptide of claim 1. 3. A pharmaceutical composition, characterized in that, the pharmaceutical composition contains: (a) the polypeptide of claim 1, or a pharmaceutically acceptable salt thereof; and (b) A pharmaceutically acceptable carrier or excipient. 4. The pharmaceutical composition according to claim 3, wherein the dosage form of the pharmaceutical composition is injection, eye drops, or ophthalmic gel. 5. The use of the polypeptide according to claim 1, characterized in that it is used for preparing a drug for inhibiting angiogenesis or preventing and treating diseases related to angiogenesis. 6. The use according to claim 5, characterized in that the diseases related to angiogenesis are selected from the group consisting of neovascular eye diseases and tumors.