WO2018224053A1 - 多肽类眼部吸收促进剂及其应用 - Google Patents

多肽类眼部吸收促进剂及其应用 Download PDF

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WO2018224053A1
WO2018224053A1 PCT/CN2018/095539 CN2018095539W WO2018224053A1 WO 2018224053 A1 WO2018224053 A1 WO 2018224053A1 CN 2018095539 W CN2018095539 W CN 2018095539W WO 2018224053 A1 WO2018224053 A1 WO 2018224053A1
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penetratin
drug
eye
derivative
intraocular
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PCT/CN2018/095539
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English (en)
French (fr)
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魏刚
江宽
陆伟跃
刘畅
太玲钰
高欣
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复旦大学
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Priority to US16/618,915 priority Critical patent/US11213591B2/en
Priority to JP2020517261A priority patent/JP2020526574A/ja
Publication of WO2018224053A1 publication Critical patent/WO2018224053A1/zh
Priority to US17/410,174 priority patent/US11826431B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
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Definitions

  • the invention belongs to the field of pharmaceutical preparations and relates to designing a series of lipophilic derivatives by using the wild type penetrating peptide penetropin.
  • These penetratin derivatives have strong ocular tissue penetration ability and do not produce ocular cytotoxicity.
  • intraocular drug delivery can be achieved through a non-invasive route, increasing the ocular bioavailability of the drug.
  • These penetratin derivatives and their constructed ocular delivery systems are used for eye drop administration, which can replace the patient's poorly administered intraocular injection, greatly enhancing the convenience and safety of treatment of intraocular and fundus diseases.
  • the eye is the most important sensory organ of the human body.
  • the unique physiological structure of the eye protects it from the intrusion of exogenous substances, but it is also detrimental to the intraocular delivery of the drug.
  • the physiological barriers of the eye include static barriers (such as the corneal epithelial barrier, blood-eye barrier, etc.) and dynamic barriers (such as tear wash, etc.), which are the main obstacles to drug absorption (Drug Discovery Today, 2008, 13 (3-4) :135-143; Adv. Drug Delivery Rev., 2006, 58(11): 1131-1135).
  • commercially available ophthalmic preparations are mainly in the form of eye drops, ophthalmic gels and eye ointments.
  • the drug is mainly transported through the cornea or conjunctiva to the eye. Due to the limited volume of conjunctival sac in the human eye, coupled with tear dilution and loss of nasolacrimal duct, the bioavailability of eye drops is usually less than 5%. Moreover, due to the long diffusion distance from the surface of the eye to the fundus and the convection of aqueous humor in the eye, very few drugs ( ⁇ 0.001%) can reach the posterior segment of the eye (J. Controlled Release, 2014, 193: 100-112). .
  • Systemic administration is another way to treat ocular diseases clinically, but practice has shown that due to the obstruction of the blood-eye barrier (such as the blood-retinal barrier), it is difficult to reach the retinal tissue and the vitreous cavity after systemic administration. In addition, large doses and frequent administration also present a risk of causing systemic side effects due to the large amount of drug entering the systemic circulation (Invest. Ophthalmol. Visual Sci., 2000, 41(5): 961-964).
  • the blood-eye barrier such as the blood-retinal barrier
  • Intraocular injections such as intravitreal injections
  • periocular injections such as subscleral injections
  • Intraocular injections are currently the most effective routes of administration for the treatment of intraocular and fundus diseases.
  • the drug can reach the eye directly, with quick onset and high bioavailability, but repeated injections can cause various complications (such as retinal detachment, endophthalmitis, etc.), which are difficult for patients to accept. Poor compliance (EYE, 2013, 27(7): 787-794).
  • eye drops are non-invasive to the eyes, and the preparation cost is low, the use is convenient, and the patient compliance is good. It is the most ideal ophthalmic dosage form in the clinic, but its main problem It is difficult to get the drug into the eye and it is more difficult to reach the bottom of the eye.
  • the use of pharmaceutics method to introduce an absorption enhancer into the prescription of eye drops can effectively improve the efficiency of intraocular delivery.
  • CPPs Cell-penetrating peptides
  • the penetrating peptide penetropin derived from the antennae of Drosophila has strong ocular tissue penetrating ability and does not produce ocular cytotoxicity (Mol. Pharm. 2014, 11(4): 1218-1227).
  • penetratin acts as an absorption enhancer that mediates the reporter gene reaching the posterior segment of the eye and is highly expressed in the retina (ACS Appl. Mater.Interfaces, 2016, 8(30) : 19256-19267; Nanomedicine: NBM 2017, DOI: http://dx.doi.org/10.1016/j.nano.2017.04.011; Chinese invention patent application: CN201610560173.8).
  • the ability of the wild-type penetratin to penetrate the membrane has room for further improvement.
  • the present application intends to provide a kind of artificially designed and modified penetratin derivative, which is used as an eye absorption enhancer and drops into the conjunctival sac.
  • Covalently linked or non-covalently linked drug molecules can be delivered more efficiently to the posterior segment of the eye, and the wild-type penetratin is retained for good eye safety.
  • this mode of topical administration also avoids drug distribution and side effects in non-target tissues.
  • the object of the present invention is to overcome the deficiencies of existing absorption enhancer ocular applications, and to provide a class of artificially engineered transmembrane peptides and methods for their design.
  • the engineered transmembrane peptide acts as an ocular absorption enhancer and can be administered to the eye by co-linked or non-covalently linked drugs by eye drop administration via a non-invasive route.
  • Ordinary eye drops have a short residence time in the conjunctival sac and poor absorption.
  • bio-molecular drugs such as genes, peptides and proteins
  • the eye bioavailability is extremely low and can hardly reach the back of the eye.
  • Segment; intraocular injections and intraocular implants have high bioavailability, but patients have poor compliance and are prone to serious complications.
  • the present invention is directed to the above problems, based on the natural source of the penetrating peptide penetropin, using amino acid mutation method, designed and prepared a series of peptide derivatives with good penetrating tissue and high biosafety, which can be adopted through a non-traumatic route.
  • Such artificially synthesized polypeptides act as eye absorption enhancers, which can mediate the effective absorption of the drug through the absorption barrier of the eye, promote the entry of the drug into the eye and reach the posterior segment of the eye, thereby improving the eye bioavailability of the drug.
  • the present invention provides a class of structurally modified penetratin derivatives.
  • the amino acid sequence of the wild type polypeptide penetratin is as follows:
  • the design principle of the penetratin derivative of the present invention is to introduce a hydrophobic amino acid into the molecule by using an amino acid mutation technique while maintaining the essential amino acid sequence of the wild type penetratin, thereby enhancing the obtained penetratin derivative. Eye tissue penetration ability.
  • the present invention maintains its original basic amino acid arginine (arginine, R), lysine (K) and the original hydrophobic amino acid isoleucine on the basis of wild-type penetratin ( Isoleucine, I), phenylalanine (F), tryptophan (W), methionine (M) sequence unchanged, using peptide solid phase synthesis technology, replaced by hydrophobic amino acids
  • the hydrophilic amino acids glutamine (Q) and asparagine (N) in the penetratin molecule yield a series of peptide derivatives.
  • X 1 , X 2 and X 3 represent a hydrophobic amino acid selected from the group consisting of amino acids alanine (A), valine (V), leucine (L), and isoluminescence.
  • the composition is substituted with different hydrophilic amino acids for different hydrophilic amino acid (glutamine and asparagine) sites of wild-type penetratin.
  • polypeptide derivative obtained by structural modification on the basis of wild type penetratin has the amino acid sequence shown in Table 1, wherein the mutated amino acid is underlined. Examples of non-natural amino acid mutations are not given in this table, and combined mutations of different hydrophobic amino acids give only representative examples.
  • the penetratin derivative described in the present invention may be linked to a drug having a diagnostic or therapeutic effect by an amide bond, a disulfide bond, a tetrahydrothiazole ring or other chemical bond.
  • One or more amino acids can also be used as a bridge, or other bifunctional groups can be used as a bridge to link to a drug having a diagnostic or therapeutic effect.
  • the above-mentioned diagnostic or therapeutic effect drug is selected from, but not limited to, one of the following drugs or a combination thereof:
  • cataract treatment drugs selected from vitamin C, vitamin E, pirenoxine, glutathione, benzal lysine, etc.;
  • Bacterial endophthalmitis treatment drugs selected from vancomycin, ceftazidime, isepamicin, neomycin, gentamicin, erythromycin, dexamethasone, trovafloxacin, cefuroxime sodium, Minocycline, etc.;
  • therapeutic drugs for fungal endophthalmitis selected from the group consisting of voriconazole, nystatin, amphotericin B, etc.;
  • glaucoma treatment selected from pilocarpine, carbachol, dipivoxil, timolol, betaxolol, metoprolol, levobromol, carteolol, dorzolamide, cloth Linzoline, brimonidine, latanoprost, travoprost, bimatoprost, bemelital, tafluprost, etc.;
  • antimetabolite selected from the group consisting of fluorouracil, mitomycin, etc.;
  • Treatment of uveal disease selected from glucocorticoids, acyclovir, penicillin, etc.;
  • Retinal disease treatment drugs selected from triamcinolone acetonide
  • Optic nerve disease treatment drugs selected from prednisolone, vitamin B1, vitamin B12, niacin and the like.
  • the penetratin derivative described in the present invention can also be modified on the surface of a delivery system such as liposome, micelle, nanoparticle by a bifunctional bridging molecule such as bifunctional polyethylene glycol (PEG).
  • a delivery system such as liposome, micelle, nanoparticle by a bifunctional bridging molecule such as bifunctional polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the penetratin derivative described in the present invention retains the positive charge characteristic of wild-type penetratin under physiological conditions, and can also self-assemble by biomolecular drugs such as negatively charged genes, polypeptides and proteins under electrostatic interactions under physiological conditions.
  • biomolecular drugs such as negatively charged genes, polypeptides and proteins under electrostatic interactions under physiological conditions.
  • Forming nanocomposites, or in the presence of cationic polymers such as polyethyleneimine (PEI), polylysine (DGLs), polyamide-amine dendrimers (PAMAM), etc.
  • Biomacromolecules such as negatively charged genes, polypeptides, and proteins self-assemble to form nanocomposites to achieve intraocular delivery of the above biomacromolecules.
  • biomacromolecular drug is selected from, but not limited to, one of the following drugs or a combination thereof:
  • Gene drug selected from the group consisting of plasmid DNA (pDNA), pegaptanib, Bevasiranib, antisense oligonucleotide, and the like;
  • Monoclonal antibody drugs selected from Bevacizumab, Ramucirumab, etc.;
  • VEGF anti-vascular endothelial growth factor
  • Conbercept Conbercept
  • EGF Epidermal growth factor
  • defensins interferon and cyclosporin.
  • the penetratin derivative described in the present invention forms a covalent complex with a drug having a diagnostic or therapeutic effect, or is modified on the surface of a drug delivery system such as a liposome, a micelle, a nanoparticle, or the like which contains a diagnostic or therapeutic drug, or Under the physiological conditions, biomacromolecules such as negatively charged genes, peptides and proteins self-assemble to form non-covalent nanocomposites, which can promote the passage of drugs through many eye absorption barriers after corneal administration in the conjunctival sac (the cornea, The conjunctiva, sclera, etc.
  • the concentration of the penetratin derivative in the covalent complex, the surface modified nano drug delivery system or the non-covalent nanocomplex constructed using the penetratin derivative is from 1 nM to 500 ⁇ M, preferably from 10 nM to 300 ⁇ M, further preferably from 100 nM to 100 ⁇ M.
  • the intraocular delivery system administered by the non-traumatic route helps to promote the absorption of the drug in the eye, improve the eye bioavailability of chemical drugs and biomacromolecules, and is clinically replaceable for patients such as intraocular injection. A mode of administration with low compliance.
  • the applicant uses a tryptophan (W) substituted penetratin derivative as an example, in color
  • W tryptophan substituted penetratin derivative
  • the C-terminus of the tyrosine-substituted penetratin derivative is additionally ligated with a lysine (K) and the fluorescent probe carboxyfluorescein (FAM) is attached to the amino side chain of lysine to form a covalent complex.
  • the present inventors examined the ocular cell uptake capacity of the penetratin derivative-modified fluorescent probe, the ocular detachment ability of the eye, and the eye of the living animal after administration through the conjunctival sac. Absorption and distribution. The results showed that the tryptophan-substituted penetratin derivative had significantly enhanced ocular absorption-promoting ability compared to wild-type penetratin.
  • the penetration ability of the penetratin derivative to the ocular tissue is related to its hydrophobicity (lipophilicity), and the stronger the hydrophobicity (lipophilicity), the stronger the penetration ability of the penetratin derivative to the ocular tissue. Therefore, a penetratin derivative prepared by using other hydrophobic amino acids will also obtain an enhanced ocular absorption promoting effect. Moreover, Applicants have demonstrated that the penetratin derivative is effective in delivering fluorescent probes to the eye, and that the same intraocular delivery effect will be obtained if other diagnostic or therapeutic agents are substituted for the fluorescent probe.
  • penetratin derivatives use antisense oligonucleotides as biomolecular drug models in the presence of polyamide-amine dendrimers (PAMAM) and hyaluronic acid (HA).
  • PAMAM polyamide-amine dendrimers
  • HA hyaluronic acid
  • the present inventors examined the ocular cell uptake capacity of the non-covalent complex modified by the penetratin derivative, the osmotic ability of the ocular tissue, and the in vivo administration of the intraocular lens through the conjunctival sac. Absorption and distribution of the eye. The results showed that the phenylalanine-substituted penetratin derivative had significantly enhanced ocular absorption-promoting ability compared to wild-type penetratin.
  • the advantage of the penetratin derivative described in the present invention is that the polypeptide-based absorption enhancer is easily degraded with respect to a small molecule absorption enhancer which is rarely applied to the eye due to safety problems, and thus is biosafety.
  • the polypeptide-based absorption enhancer is easy to modify and modify to achieve different application targets, and the penetratin derivative of the present invention has stronger ocular absorption-promoting ability than the wild-type penetratin of natural origin.
  • Figure 1 Qualitative evaluation of cell uptake by covalently linked complexes of penetratin derivatives and small molecules
  • human corneal epithelial cells and human conjunctival epithelial cells were incubated with pennetratin derivative-FAM covalent complex (100 nM) for 0.5 h, 1 h, 2 h and 4 h, respectively, and the scale was 100 ⁇ m.
  • Figure 2 Quantitative evaluation of cellular uptake of covalently linked complexes of penetratin derivatives and small molecules
  • human corneal epithelial cells and human conjunctival epithelial cells were incubated with pennetratin derivative-FAM covalent complex for 4 hours, and Fm was the average fluorescence intensity value of the cells.
  • human corneal epithelial cells and human conjunctival epithelial cells were incubated with penetratin derivatives for 12 h, and the survival rate of the experimental group relative to the negative control group was detected by MTT assay.
  • FIG. 4 Evaluation of permeation ability of excised eye tissue by covalently linking complexes of penetratin derivatives to small molecular substances
  • a is the permeation curve of the penetratin derivative-FAM covalent complex on the isolated rabbit eye
  • b is the penetration curve of the penetratin derivative-FAM covalent complex on the isolated rabbit eye sclera
  • c is a table
  • Fig. B is a fluorescent section of DAPI staining of the blank group and the experimental group
  • the scale is 100 ⁇ m.
  • Figure A is the corneal hydration value of the blank group and the penetratin derivative-FAM covalent complex group
  • Figure B is the scleral hydration value of the blank group and the penetratin derivative-FAM covalent complex group
  • Figure C is the blank group and The penetratin derivative-FAM covalent complex group was stained with HE stained in vitro, and the scale was 100 ⁇ m.
  • Figure 6 Distribution of covalently linked complexes of penetratin derivatives with small molecules in the eyes of living mice
  • Figure A shows the penetratin derivative-FAM covalent complex at the time of the conjunctival sac at different time points (10 min, 0.5 h, 1 h, 2 h, 4 h, 6 h) in the anterior segment of the mouse (corneal) and posterior segment of the eye ( Distribution of retina);
  • Figure B shows the distribution of penetratin derivative-FAM covalent complex in the anterior segment (corneal) and posterior segment (retinal) of mice 10 min after conjunctival sac administration;
  • Figure C is 10 min after administration. After 1h, 6h, semi-quantitative analysis results of intraocular distribution of penetratin derivative-FAM covalent complex.
  • Figure 7 shows the ocular absorption of a non-covalent complex of a Penetratin derivative and an antisense oligonucleotide drug
  • Figure A shows the non-covalent complexes (ASO/PG5, ASO/PG5/HA, ASO/PG5/HA/Pene) of different groups at different time points (1h, 2h, 6h, 8h) after administration of conjunctival sac Distribution of mouse retina;
  • Table B shows the distribution of green fluorescently labeled ASO in each region of the retina.
  • Figure 8 shows the cellular uptake evaluation of the non-covalent complex of the fluorouracil modified with the Penetratin derivative and the antisense oligonucleotide drug.
  • mouse fibroblasts were incubated with different prescription drug-loaded non-covalent complexes (dual/PG5, dual/PG5/HA, dual/PG5/HA/Pene) for 4 hours, and the average fluorescence intensity values of each group were determined.
  • the applicant also prepared alanine (A), valine (V), leucine (L), isoleucine (I), valine. (proline, P), phenylalanine (F), tryptophan (W), methionine (M), ⁇ -aminobutyric acid, ⁇ -amino a penetratin derivative substituted with natural and unnatural amino acids such as ⁇ -aminopentanoic acid, ⁇ -aminohexanoic acid, ⁇ -aminoheptanoic acid, and combinations thereof,
  • the amino acid sequence is shown in Table 1.
  • the applicant also separately treated cataract treatment drugs, bacterial endophthalmitis treatment drugs, fungal endophthalitis treatment drugs, glaucoma treatment drugs, antimetabolites, uveal disease treatment drugs, retinal diseases treatment drugs, and optic nerve diseases.
  • the therapeutic drug and the penetratin derivative are linked by a covalent bond to form a covalent complex of the penetratin derivative and the small molecule drug.
  • HCEC human corneal epithelial cells
  • NHS human conjunctival epithelial cells
  • the drug solution was discarded, and the positively-adsorbed substance was washed away with a PBS buffer solution containing 0.02 mg/mL of heparin sodium, and the nuclei were stained with dimethymidyl hydrazone (DAPI) and observed under an inverted fluorescence microscope.
  • DAPI dimethymidyl hydrazone
  • the fluorescence signal of FAM was still weak after incubation for 4 h, and the fluorescence signal of FAM was weaker for hydrophilic penetratin derivative 6A.
  • the lipophilic penetratin derivative has strong FAM fluorescence signal, especially the penetratin derivatives 9-W, 28-W, 89-W and 289-W.
  • the cells can see obvious FAM fluorescence signals.
  • the fluorescence signal of FAM is also enhanced. It is indicated that the use of hydrophobic amino acids to replace the hydrophilic amino acids in the penetratin molecule is beneficial to promote the uptake of small molecules by the polypeptide, while the increase in hydrophilicity leads to a decrease in cellular uptake.
  • HCEC and NHC cells with good growth status were inoculated into 12-well plates at 5 ⁇ 10 3 cells/cm 2 , and changed once a day after inoculation. The experiment was carried out after 2 to 3 days of culture. After discarding the culture solution, the cells were washed three times with sterile PBS, and a serum-free DMEM solution containing 3 ⁇ M of penetratin derivative and FAM complex was separately added, and incubated at 37 ° C, 5% CO 2 for 4 hours.
  • HCEC cells In HCEC cells, the cellular uptake of lipophilic penetratin derivatives and FAM complexes was significantly greater than that of wild-type penetratin and FAM (p ⁇ 0.001), and the mean fluorescence intensity was 1.7 (29-W) in the wild-type peneperatin group. ⁇ 7.7 (89-W) times the average fluorescence intensity of the hydrophilic penetratin derivative 6A was only 1/10 of the wild type peneperratin group (p ⁇ 0.001).
  • the average fluorescence intensity of the hydrophobic penetratin derivative is 2.8 (2-W) to 18.9 (29-W) times the wild-type penetratin (p ⁇ 0.01), and the average fluorescence intensity of the hydrophilic penetratin derivative. Only 1/4 of wild-type penetratin (p ⁇ 0.001). Taken together, the cellular uptake of lipophilic penetratin derivatives was significantly higher than that of wild-type penetratin in both HCEC and NHC cells, while the hydrophilic penetratin derivative was significantly lower than wild-type penepatin, consistent with qualitative uptake results.
  • HCEC and NHC cells with good growth in log phase were plated at 60 cells/well in 60 wells of 96-well plates, and the edges were filled with sterile PBS buffer solution at 37 °C. Incubate under 5% CO 2 until the cell monolayer is covered with the bottom of the plate, discard the culture solution, wash 3 times in sterile PBS buffer, add 200 ⁇ L of the culture solution containing different concentrations of penetratin derivative, incubate for 12 hours in the cell culture incubator. After that, the drug solution was discarded, washed 3 times in sterile PBS buffer, and the culture medium was added for further 12 hours.
  • thiazole basket (MTT) solution (5 mg/mL) to each well. After 4 hours of incubation, carefully discard the liquid. After washing three times with PBS buffer, add 150 ⁇ L of dimethyl sulfoxide (DMSO) per well to the shaker at low speed. After shaking for 20 min, the absorbance values of the wells were measured at OD490nm. Blank wells (medium, MTT, DMSO) and negative control wells (cell, medium, MTT, DMSO) were set at the same time.
  • MTT thiazole basket
  • the freshly isolated cornea and sclera were carefully sandwiched between the two diffusion cells, and the epithelial surface was oriented toward the left diffusion cell (supply cell) with a diffusion window diameter of 10.25 mm and a diffusion area of 0.825 cm 2 .
  • Add 3.5 mL Ringer solution to the supply tank and the right receiving tank respectively, and pass the circulating water. The water temperature is maintained at 34 °C ⁇ 0.5 °C. After the system is equilibrated for 10 min, the solution in the supply tank is removed, and the Penetratin derivative and the FAM complex are added. The solution was at a concentration of 7.5 ⁇ M.
  • samples were taken at a predetermined time point, each time 500 ⁇ L was sampled in the receiving pool, and 500 ⁇ L of fresh Ringer's solution was immediately added.
  • the sample fluorescence intensity value was measured.
  • the cornea and sclera were removed, the surface was carefully washed three times with Ringer's solution, and the part other than the diffusion surface was removed. The remaining tissue was fixed with Davidson's solution and subjected to DAPI-stained frozen sections for observation of the tissue distribution of the peptide-linked fluorescent probe. .
  • Figure 4A shows that the process by which a fluorescent probe attached to a polypeptide penetrates the excised cornea and sclera is a time-dependent linear process.
  • the P app values were 1.1, 1.5, and 2.1 fold (p ⁇ 0.001) for wild-type penetratin, respectively, while 6A was only 2/3 (p ⁇ 0.001) for wild-type penetratin.
  • the excised corneal and scleral fluorescence sections of DAPI staining in Figure 4B showed no green fluorescence in the blank tissue sections, indicating no fluorescence background interference in the tissue.
  • the lipophilic penetropin derivative treated corneal fluorescence intensity was stronger than that of the wild-type peneperratin group, and the stronger the lipophilicity, the more the corneal volume entered, while the hydrophilic penetratin derivative 6A group did not.
  • Significant fluorescence signal; in the isolated sclera the fluorescent signal of the lipophilic derivative group was stronger than that of the wild type penepatin group, while the 6A was significantly weaker than the penetratin group.
  • Fig. 6A show that there is no green fluorescence signal in the anterior segment (corneal) and posterior segment (retinal) of the eye group of the blank group, indicating that the ocular tissue has no fluorescence background interference.
  • the lipophilic penetratin derivative group had stronger green FAM fluorescence signals in the anterior and posterior segments of the mouse 10 min after the instillation, indicating that the lipophilic penetratin derivative had better eye penetration ability.
  • Figure 6B shows that after administration of the conjunctival sac, the lipophilic penetratin derivative can be effectively distributed in the corneal stromal layer and the intraretinal layer of the mouse, while the distribution of 6A in the cornea is significantly less than that of the wild type penetratin, and there is almost no FAM.
  • Figure 6C shows the results of semi-quantitative analysis.
  • the lipophilic penetratin derivative enters the cornea and retina more (p ⁇ 0.001), and the intraocular retention time is longer, for the lipophilic 28- W, 29-W, 89-W and 289-W, the retention time was as long as 6h, while for 6A, the amount entering the eye was significantly less than that of wild-type penetratin (p ⁇ 0.05), indicating that it penetrated the eye barrier. Poor sex.
  • ASO/PG5 complex 500 ⁇ L of the experimental concentration of PAMAM solution was added, and an equivalent volume (500 ⁇ L) of ASO solution was added dropwise under vortex conditions. After the addition, the mixture was vortexed for 30 s, and allowed to stand at room temperature for 30 min to obtain an ASO/PG5 complex. Take 1 mL of stabilized ASO/PG5 complex, add 500 ⁇ L of experimental concentration of HA solution dropwise under vortex conditions, continue to vortex for 30 s after completion of the addition, and stabilize at room temperature for 30 min to obtain ASO/PG5/HA complex. .
  • ASO/PG5 ASO/PG5/HA and ASO/PG5/HA/Pene complexes were prepared using fluorescently labeled ASO, respectively.
  • the groups all contained 30 ⁇ g/mL ASO.
  • the mice were randomly divided into 3 groups: ASO/PG5, ASO/PG5/HA and ASO/PG5/HA/Pene, 12 rats in each group, and 10 ⁇ L of the experimental group solution was administered to the right eye. The drug was administered once every 10 minutes, and each group was administered a total of 3 times. Three mice were randomly selected from each group for 1h, 2h, 6h and 8h after the last administration.
  • the eyeballs of the mice were removed and dehydrated in 30% sucrose solution.
  • the slices were sectioned perpendicular to the cornea and the middle section of the tissue was taken. Frozen sections were prepared and stained with DAPI working solution for nuclear staining. The elimination of complexes in the posterior segment of the eye was observed under a confocal microscope.
  • the ASOs delivered by the three complexes were distributed in the posterior segment of the mouse eye 1 h after the eye drops, and the distribution increased with time, but only ASO/PG5/HA/Pene delivery.
  • the ASO showed a significant distribution in the posterior segment of the eye, and was still distributed in the outer mesh layer (OPL) and retinal pigment epithelial cells (RPE) after 8 hours.
  • OPL outer mesh layer
  • RPE retinal pigment epithelial cells
  • the results suggest that ASO can be effectively delivered to the posterior segment of the eye and distributed in the retinal pigment epithelial cell layer (RPE) by intraocular administration using the penetratin derivative 2-M, 8-W, 9-Y modified oligonucleotide complex. ), and the retention time in the back of the eye exceeds 8h (Fig. 7A).
  • Fig. 7B A comparison of the relative fluorescence intensities of each major ASO distribution layer is shown (Fig. 7B).
  • antisense oligonucleotide (anti-TGF ⁇ 2-ASO) antagonizing TGF ⁇ 2 was added, and 3 mL of a buffer solution was added thereto, and the mixture was vortexed for 30 s to be sufficiently dissolved to obtain an ASO solution having a concentration of 10 ⁇ g/mL.
  • 100 ⁇ L of ASO solution was added dropwise to the same volume of Fu/PG5 solution under vortex conditions. After the addition was completed, vortexing was continued for 30 s, and the mixture was allowed to stand at room temperature for 30 min to obtain a dual/PG5 complex.

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Abstract

属药物制剂领域，涉及利用野生型穿膜肽penetratin设计一系列亲脂性衍生物。这些penetratin衍生物具有很强的穿透眼组织能力，而且不会产生眼部组织毒性，作为眼部吸收促进剂可通过无创途径实现眼内药物递送，增加药物的眼部生物利用度。这些penetratin衍生物及其构建的眼部给药系统用于滴眼给药，可替代患者顺应性差的眼内注射给药，增强了眼内和眼底疾病治疗的便利性和安全性。

Description

多肽类眼部吸收促进剂及其应用 技术领域
本发明属药物制剂领域，涉及利用野生型穿膜肽penetratin设计一系列亲脂性衍生物。这些penetratin衍生物具有很强的眼组织穿透能力，而且不会产生眼部细胞毒性，作为眼部吸收促进剂可通过无创途径实现眼内药物递送，增加药物的眼部生物利用度。这些penetratin衍生物及其构建的眼部给药系统用于滴眼给药，可替代患者顺应性差的眼内注射给药，极大地增强眼内和眼底疾病治疗的便利性和安全性。
背景技术
眼睛是人体最重要的感觉器官。眼睛独特的生理结构保护其免受外源性物质侵入，但也不利于药物的眼内递送。眼部的生理屏障包括静态屏障(如角膜上皮屏障、血-眼屏障等)和动态屏障(如泪液冲刷等)，是阻碍药物吸收的主要原因(Drug Discovery Today，2008，13(3-4)：135-143；Adv.Drug Delivery Rev.，2006，58(11)：1131-1135)。目前市售的眼用制剂以滴眼剂、眼用凝胶剂和眼膏剂等剂型为主。临床实践中，滴眼剂滴入结膜囊后，药物主要透过角膜或结膜向眼内转运。由于人眼结膜囊容积有限，再加上泪液稀释和鼻泪管流失等原因，通常滴眼剂的生物利用度小于5％。而且，由于从眼表面到眼底较长的扩散距离和房水在眼内的对流，极少药物(＜0.001％)可以到达眼后段组织(J.Controlled Release，2014，193：100-112)。
全身给药是临床上治疗眼部疾病的另一途径，但实践表明，由于血-眼屏障(如血-视网膜屏障)的阻碍，药物经全身给药后很难到达视网膜组织和玻璃体腔内。另外，由于大量药物进入体循环，大剂量和频繁给药还存在引起全身性副作用的风险(Invest.Ophthalmol.Visual Sci.，2000，41(5)：961-964)。
眼内注射(如玻璃体内注射)和眼周注射(如巩膜下注射)是目前临床上治疗眼内疾病和眼底疾病最为有效的给药途径。借助这些创伤性的给药方式，药物 可直接到达眼内，起效快、生物利用度较高，但反复注射会引起多种并发症(如视网膜脱落、眼内炎等)，患者难以接受，顺应性差(EYE，2013，27(7)：787-794)。
综合考虑各种眼内和眼底药物递送方法，滴眼剂对眼部无创伤性，并且制备成本低、使用方便、患者顺应性好，是临床上最理想的眼用剂型，但其主要的问题在于药物难以吸收到眼内，更难以到达眼底部位。采用药剂学方法，在滴眼剂处方中引入吸收促进剂，可以有效提高其眼内递药效率。
由于眼睛极其敏感，有研究报道小分子吸收促进剂(如乙醇、二甲基亚砜等)的眼部刺激性强，并且不易被代谢，不适用于眼部应用(Toxicol.Lett.，2001，122(1)：1-8)。因此，新型眼部吸收促进剂的开发十分必要。
穿膜肽(cell-penetrating peptides，CPPs)是一种生理条件下荷正电的短肽，可以介导共价或非共价连接的分子或给药系统(如双链DNA、脂质体等)进入细胞(J.Controlled Release，2011，155(1SI)：26-33；Biomaterials，2013，34(32)：7980-7993)。来源于果蝇触角的穿膜肽penetratin具有很强的眼组织穿透能力，而且不会产生眼部细胞毒性(Mol.Pharm.2014，11(4)：1218-1227)。有报道在几种非创伤性的眼部递药系统中，penetratin作为吸收促进剂，可以介导报告基因到达眼后段并在视网膜高效表达(ACS Appl.Mater.Interfaces，2016，8(30)：19256-19267；Nanomedicine：NBM 2017，DOI：http：//dx.doi.org/10.1016/j.nano.2017.04.011；中国发明专利申请：CN201610560173.8)。但野生型penetratin的穿膜能力还有进一步提升的空间。
基于已公开文献报道对眼组织穿透能力较强的天然来源多肽penetratin，本申请拟提供一类人工设计和改造的penetratin衍生物，此类多肽作为眼部吸收促进剂，滴入结膜囊后，可以更有效地递送共价连接或非共价连接的药物分子到达眼后段组织，并且保留野生型penetratin良好的眼部安全性。此外，这种局部给药的方式也可以避免药物在非靶组织中分布和产生副作用。
发明内容
本发明的目的是克服现有吸收促进剂眼部应用的缺陷，提供一类人工改造的穿膜肽及其设计方法。经人工改造的穿膜肽作为眼部吸收促进剂，可通过非创伤 途径滴眼给药，将共价连接或非共价连接的药物递送至眼内。
普通滴眼剂在结膜囊内滞留时间短，吸收效果差，特别是基因、多肽和蛋白质等生物大分子药物经局部滴眼给药后，眼部生物利用度极低，并且几乎不能到达眼后段；眼内注射剂和眼内植入剂生物利用度虽高，但患者顺应性差，且容易导致严重的并发症。本发明针对上述问题，基于天然来源的穿膜肽penetratin，采用氨基酸突变的方法，设计并制备了一系列对眼组织穿透性好、生物安全性高的多肽衍生物，可通过非创伤途径将与其共价连接的分子甚至非共价连接的分子递送至眼内。此类人工合成的多肽作为眼部吸收促进剂，能够介导药物高效地透过眼部的吸收屏障，促进药物进入眼内并到达眼后段，进而提高药物的眼部生物利用度。
本发明提供了一类经结构改造的penetratin衍生物。野生型多肽penetratin的氨基酸序列如下：
RQIKIWFQNRRMKWKK
申请人通过总结大量的实验数据意外地发现，野生型penetratin对眼组织的穿透能力随着分子的疏水性增强而得到改善。因此，本发明所述penetratin衍生物的设计原理，是在保持野生型penetratin基本氨基酸序列不变的前提下，利用氨基酸突变技术，在其分子中引入疏水性氨基酸，进而增强所得到的penetratin衍生物的眼组织穿透能力。
具体地说，本发明是在野生型penetratin的基础之上，保持其原有碱性氨基酸精氨酸(arginine，R)、赖氨酸(lysine，K)以及原有疏水氨基酸异亮氨酸(isoleucine，I)、苯丙氨酸(phenylalanine，F)、色氨酸(tryptophan，W)、甲硫氨酸(methionine，M)的序列不变，利用多肽固相合成技术，以疏水性氨基酸取代penetratin分子中的亲水性氨基酸谷氨酰胺(glutamine，Q)和天冬酰胺(asparagine，N)，进而得到一系列多肽衍生物。
所述penetratin衍生物的特征在于具有下述氨基酸序列：
R X 1 IKIWF X 2X 3 RRMKWKK
其中，X 1、X 2和X 3代表疏水性氨基酸，选自天然来源的氨基酸丙氨酸(alanine， A)、缬氨酸(valine，V)、亮氨酸(leucine，L)、异亮氨酸(isoleucine，I)、脯氨酸(proline，P)、苯丙氨酸(phenylalanine，F)、色氨酸(tryptophan，W)、甲硫氨酸(methionine，M)和非天然来源的氨基酸α-氨基丁酸(α-aminobutyric acid)、α-氨基戊酸(α-aminopentanoic acid)、α-氨基己酸(α-aminohexanoic acid)、α-氨基庚酸(α-aminoheptanoic acid)等，以及它们的组合物。所述组合物为对野生型penetratin不同亲水性氨基酸(谷氨酰胺和天冬酰胺)位点用不同的上述疏水性氨基酸进行取代。
在野生型penetratin基础之上进行结构改造得到的多肽衍生物，其氨基酸序列如表1所示，其中，突变的氨基酸用下划线标示。该表格中未给出非天然氨基酸突变的实例，不同疏水氨基酸的组合突变仅给出代表性实例。
表1 野生型penetratin经结构改造后得到的penetratin衍生物
Figure PCTCN2018095539-appb-000001
Figure PCTCN2018095539-appb-000002
本发明中所述的penetratin衍生物可通过酰胺键、二硫键、四氢噻唑环或其他化学键与具有诊断或治疗作用的药物连接在一起。也可以利用一个或多个氨基酸作为桥连，或利用其他双功能基团作为桥连，与具有诊断或治疗作用的药物连接在一起。
上述起诊断或治疗作用的药物选自但不局限于下列药物中的一种或它们的复方：
1)白内障治疗药物：选自维生素C、维生素E、吡诺克辛、谷胱甘肽、苄达赖氨酸等；
2)细菌性眼内炎治疗药物：选自万古霉素、头孢他啶、异帕米星、新霉素、庆大霉素、红霉素、地塞米松、曲伐沙星、头孢呋辛钠、米诺环素等；
3)真菌性眼内炎治疗药物：选自伏立康唑、制霉菌素、两性霉素B等；
4)青光眼治疗药物：选自毛果芸香碱、卡巴胆碱、地匹福林、噻吗洛尔、倍他洛尔、美替洛尔、左布诺洛尔、卡替洛尔、多佐胺、布林佐胺、溴莫尼定、拉坦前列素、曲伏前列素、比马前列素、贝美前列素、他氟前列素等；
5)抗代谢药物：选自氟尿嘧啶、丝裂霉素等；
6)葡萄膜疾病治疗药物：选自糖皮质激素类药物、阿昔洛韦、青霉素等；
7)视网膜疾病治疗药物：选自曲安奈德等；
8)视神经疾病治疗药物：选自泼尼松龙、维生素B1、维生素B12、烟酸等。
本发明中所述的penetratin衍生物还可以通过双功能桥连分子，如双功能聚乙二醇(PEG)，修饰在脂质体、胶束、纳米粒等给药系统表面，利用这些给药系统包载上述药物，实现上述药物的眼内递送。
本发明中所述的penetratin衍生物保留了野生型penetratin在生理条件下带正电荷的特性，还可以通过静电相互作用与生理条件下带负电荷的基因、多肽和蛋白质等生物大分子药物自组装形成纳米复合物，或者与在阳离子聚合物如聚乙烯亚胺(PEI)、多聚赖氨酸(DGLs)、聚酰胺-胺树枝状大分子(PAMAM)等存在的情况下与生理条件下带负电荷的基因、多肽和蛋白质等生物大分子药物自组装形成纳米复合物，实现上述生物大分子药物的眼内递送。
上述生物大分子药物选自但不局限于下列药物中的一种或它们的复方：
1)基因药物：选自质粒DNA(pDNA)、哌加他尼(Pegaptanib)、贝伐西尼(Bevasiranib)、反义寡核苷酸(Antisense oligonucleotide)等；
2)单克隆抗体药物：选自贝伐单抗(Bevacizumab)、雷莫芦单抗(Ramucirumab)等；
3)其他多肽蛋白类药物：选自抗血管内皮生长因子(VEGF)融合蛋白康柏西普(Conbercept)、表皮生长因子(Epidermal growth factor，EGF)、防御素、干扰素和环孢菌素等。
本发明中所述的penetratin衍生物与具有诊断或治疗作用的药物形成共价复合物，或修饰在包载诊断或治疗药物的脂质体、胶束、纳米粒等给药系统表面，或与生理条件下带负电荷的基因、多肽和蛋白质等生物大分子药物自组装形成非共价纳米复合物，经结膜囊内滴眼给药后，可促进药物穿过诸多眼部吸收屏障(角膜、结膜、巩膜等)进入眼内，甚至将其携带的化学药物或基因、多肽、蛋白质类生物大分子药物递送至眼后段的视网膜部位。所述利用penetratin衍生物构建的共价复合物、表面修饰的纳米给药系统或非共价纳米复合物中，penetratin衍生物的浓度为1nM-500μM，优选10nM-300μM，进一步优选100nM-100μM。这种通过非创伤途径给药的眼内递药系统有助于促进药物在眼部的吸收，提高化学药物和生物大分子药物的眼部生物利用度，在临床上可替代眼内注射等患者顺应度低的给药方式。
为了直观地展示本发明中所述的penetratin衍生物对与其共价连接的化学药物的眼部吸收促进效果，申请人以单纯色氨酸(tryptophan，W)取代的penetratin衍生物为例，在色氨酸取代的penetratin衍生物的C端额外接上一个赖氨酸(lysine，K)并将荧光探针羧基荧光素(FAM)连接在赖氨酸的氨基侧链上，形成共价复合物。通过一系列体外和体内实验，本发明考察了penetratin衍生物修饰的荧光探针的眼部细胞摄取能力、眼部离体组织透过能力以及经结膜囊内滴眼给药后在活体动物眼部的吸收与分布。结果表明，与野生型penetratin相比，色氨酸取代的penetratin衍生物具有显著增强的眼部吸收促进能力。
更重要的是，申请人发现penetratin衍生物对眼组织的穿透能力与其疏水性(亲脂性)相关，疏水性(亲脂性)越强，penetratin衍生物对眼组织的穿透能力也越强。因此，利用其它疏水性氨基酸制备的penetratin衍生物也将获得增强的眼部吸收促进效果。而且，申请人证实penetratin衍生物能够有效地将荧光探针递送至眼内，如果用其他具有诊断或治疗作用的药物替代荧光探针，也将获得同样的眼内递送效果。
为了直观地展示本发明中所述的penetratin衍生物对纳米药物载体以及与其非共价结合的生物大分子药物的眼部吸收促进效果，申请人以单纯苯丙氨酸(phenylalanine，F)取代的penetratin衍生物为例，以反义寡核苷酸(Antisense oligonucleotide)为生物大分子药物模型，在聚酰胺-胺树枝状大分子(PAMAM)和透明质酸(hyaluronic acid，HA)存在的条件下，构建了包载反义寡核苷酸的非共价复合物。通过一系列体外和体内实验，本发明考察了penetratin衍生物修饰的非共价复合物的眼部细胞摄取能力、眼部离体组织透过能力以及经结膜囊内滴眼给药后在活体动物眼部的吸收与分布。结果表明，与野生型penetratin相比，苯丙氨酸取代的penetratin衍生物具有显著增强的眼部吸收促进能力。
本发明中所述penetratin衍生物的优势在于，相对于因安全性问题极少在眼部应用的小分子吸收促进剂，这种多肽类吸收促进剂易于降解，因而生物安全性更好。另一方面，多肽类吸收促进剂易于修饰和改造，以实现不同的应用目标，而且本发明所述的penetratin衍生物较天然来源的野生型penetratin具有更强的眼部吸收促进能力。
附图说明
图1：实施penetratin衍生物与小分子物质共价连接复合物细胞摄取定性评价
其中，人角膜上皮细胞和人结膜上皮细胞与penetratin衍生物-FAM共价复合物(100nM)分别孵育0.5h，1h，2h和4h，标尺为100μm。
图2：实施penetratin衍生物与小分子物质共价连接复合物细胞摄取定量评价
其中，人角膜上皮细胞和人结膜上皮细胞与penetratin衍生物-FAM共价复合物分别孵育4h后测定，Fm为细胞平均荧光强度值。
图3：实施penetratin衍生物细胞毒性评价
其中，人角膜上皮细胞和人结膜上皮细胞与penetratin衍生物分别孵育12h，采用MTT法检测实验组相对于阴性对照组细胞存活率。
图4：实施penetratin衍生物与小分子物质共价连接复合物离体眼组织透过能力评价
其中，图A中a为penetratin衍生物-FAM共价复合物对离体兔眼角膜透过曲线，b为penetratin衍生物-FAM共价复合物对离体兔眼巩膜透过曲线，c为表观透过 系数；图B为空白组与实验组离体组织DAPI染色的荧光切片，标尺为100μm。
图5：实施penetratin衍生物与小分子物质共价连接复合物离体组织毒性评价
其中，图A为空白组与penetratin衍生物-FAM共价复合物组角膜水化值；图B为空白组与penetratin衍生物-FAM共价复合物组巩膜水化值；图C为空白组与penetratin衍生物-FAM共价复合物组离体组织HE染色切片，标尺为100μm。
图6：实施penetratin衍生物与小分子物质共价连接复合物在活体小鼠眼内的分布
其中，图A为结膜囊给药后，不同时间点(10min，0.5h，1h，2h，4h，6h)penetratin衍生物-FAM共价复合物在小鼠眼前段(角膜)和眼后段(视网膜)分布情况；图B为结膜囊给药后10min，penetratin衍生物-FAM共价复合物在小鼠眼前段(角膜)和眼后段(视网膜)分布情况；图C为给药后10min，1h，6h后，penetratin衍生物-FAM共价复合物眼内分布半定量分析结果。
图7实施Penetratin衍生物与反义寡核苷酸药物非共价复合物的眼部吸收
其中，图A为结膜囊给药后，不同时间点(1h，2h，6h，8h)各组非共价复合物(ASO/PG5，ASO/PG5/HA，ASO/PG5/HA/Pene)在小鼠视网膜分布情况；表B为各组绿色荧光标记的ASO在视网膜各区域分布情况。
图8实施Penetratin衍生物修饰的氟尿嘧啶与反义寡核苷酸药物双载药非共价复合物的细胞摄取评价：
其中，小鼠成纤维细胞与不同处方双载药非共价复合物(dual/PG5，dual/PG5/HA，dual/PG5/HA/Pene)孵育4h后，测定各组细胞平均荧光强度值。
具体实施方式
以下结合本发明的具体实施例进一步阐明本发明，但并不限制其保护范围。
实施例1
Penetratin衍生物与小分子物质共价连接复合物的制备：以野生型penetratin的结构为基础，保持其原有的碱性氨基酸和疏水氨基酸的序列不变，利用多肽固 相合成技术，以疏水性的色氨酸(tryptophan，W)分别取代penetratin分子中亲水性的谷氨酰胺(glutamine，Q)和天冬酰胺(asparagine，N)，进而得到一系列多肽衍生物。在上述色氨酸取代的penetratin衍生物的C端额外接上一个赖氨酸(lysine，K)并将荧光探针羧基荧光素(FAM)连接在赖氨酸的氨基侧链上，形成共价复合物，其氨基酸序列见表2。
同时，保持野生型penetratin原有的碱性氨基酸和亲水氨基酸的序列不变，利用多肽固相合成技术，以丙氨酸(alanine，A)取代penetratin分子中疏水性更强的异亮氨酸(isoleucine，I)、苯丙氨酸(phenylalanine，F)、色氨酸(tryptophan，W)和甲硫氨酸(methionine，M)，得到一条亲水性的penetratin衍生物6A，并进一步利用FAM对其进行荧光标记，作为亲水性对照多肽。
表2 Penetratin衍生物与荧光探针共价复合物的氨基酸序列
Figure PCTCN2018095539-appb-000003
利用上述方法，申请人还分别制备了丙氨酸(alanine，A)、缬氨酸(valine，V)、亮氨酸(leucine，L)、异亮氨酸(isoleucine，I)、脯氨酸(proline，P)、苯丙氨酸(phenylalanine，F)、色氨酸(tryptophan，W)、甲硫氨酸(methionine，M)、α-氨基丁酸(α-aminobutyric acid)、α-氨基戊酸(α-aminopentanoic acid)、α-氨基己酸(α-aminohexanoic acid)、α-氨基庚酸(α-aminoheptanoic acid)等天然与非天然氨基酸以及它们的组合物取代的penetratin衍生物，其氨基酸序列见表1。
利用上述方法，申请人还分别将白内障治疗药物、细菌性眼内炎治疗药物、真菌性眼内炎治疗药物、青光眼治疗药物、抗代谢药物、葡萄膜疾病治疗药物、视网膜疾病治疗药物以及视神经疾病治疗药物与penetratin衍生物通过共价键连接在一起，形成penetratin衍生物与小分子药物共价复合物。
实施例2
Penetratin衍生物与小分子物质共价连接复合物的细胞摄取定性评价：取生长状态良好的人角膜上皮细胞(HCEC)和人结膜上皮细胞(NHC)按5×10 3个细胞/cm 2分别接种到24孔板中，接种后每天换液一次，培养至2～3天后进行实验。弃去培液后，无菌PBS洗三次，分别加入含100nMpenetratin衍生物与FAM复合物的无血清DMEM溶液，37℃、5％CO 2条件下孵育一定时间(0.5h、1h、2h和4h)。结束后弃去药液，用含0.02mg/mL肝素钠的PBS缓冲溶液洗去正电性吸附物质，二咪基苯基吲哚(DAPI)染细胞核后在倒置荧光显微镜下观察。
结果表明，对于野生型penetratin，孵育4h后，细胞中FAM的荧光信号仍然较弱，对于亲水性penetratin衍生物6A，FAM的荧光信号更弱。显而易见，亲脂性penetratin衍生物有较强的FAM荧光信号，特别是penetratin衍生物9-W、28-W、89-W以及289-W，给药1h后即可见细胞发出明显的FAM荧光信号，并且随孵育时间延长，FAM的荧光信号也随之增强。表明利用疏水性氨基酸取代penetratin分子中原有的亲水性氨基酸，有利于促进多肽携载小分子物质被细胞摄取，而亲水性增强则使细胞摄取减少。
实施例3
Penetratin衍生物与小分子物质共价连接复合物的细胞摄取定量评价：取生长状态良好的HCEC和NHC细胞按5×10 3细胞/cm 2分别接种到12孔板中，接种后每天换液一次，培养2～3天后进行实验。弃去培液后，无菌PBS洗三次，分别加入含3μMpenetratin衍生物与FAM复合物的无血清DMEM溶液，37℃、5％CO 2条件下孵育4h。结束后弃去药液，用含0.02mg/mL肝素钠的PBS缓冲溶液洗去正电性吸附物质，将细胞消化下来，重悬于200μL无菌PBS缓冲溶液中，吹打均匀后进行流式检测，每个样品细胞计数在10 4，未给药细胞作为阴性对照组。
在HCEC细胞中，亲脂性penetratin衍生物与FAM复合物的细胞摄取量明显多于野生型penetratin与FAM的复合物(p＜0.001)，平均荧光强度是野生型penetratin组的1.7(29-W)～7.7(89-W)倍不等，而亲水性penetratin衍生物6A的平均荧光强度仅为野生型penetratin组的1/10(p＜0.001)。对于NHC细胞，疏水性penetratin衍生物的平均荧光强度是野生型penetratin的2.8(2-W)～18.9(29-W)倍不等(p＜0.01)，亲水性penetratin衍生物的平均荧光强度则仅为野生型penetratin的1/4(p＜0.001)。综合上述结果，无论对于HCEC还是NHC细胞，亲脂性penetratin衍生物的细胞摄取量均明显多于野生型penetratin，而亲水性penetratin衍生物则显著低于野生型penetratin，与定性摄取结果一致。
实施例4
Penetratin衍生物的细胞毒性评价：取对数期生长状态良好的HCEC和NHC细胞按3000细胞/孔浓度分别铺于96孔板的60内孔中，边缘用无菌PBS缓冲溶液填充，在37℃、5％CO 2条件下培养至细胞单层铺满板底，弃去培液，无菌PBS缓冲液洗3次后，加入200μL含不同浓度penetratin衍生物的培液，细胞培养箱中孵育12h后，弃去药液，无菌PBS缓冲液洗3次后，加入完全培养基继续培养12h。之后每孔加入20μL噻唑篮(MTT)溶液(5mg/mL)，继续培养4h后，小心弃去液体，PBS缓冲液洗三次后每孔加入150μL二甲基亚砜(DMSO)，在摇床上低速振荡20min后，于酶标仪OD490nm处测定各孔吸光度值。同时设置空白调零孔(培养基、MTT、DMSO)和阴性对照孔(细胞、培养基、MTT、DMSO)。
结果表明，在考察浓度范围条件下(≤30μM)，细胞生长状况未见显著改变，penetratin及其衍生物对HCEC和NHC细胞未见毒性作用。
实施例5
Penetratin衍生物与小分子物质共价连接复合物的离体组织透过性评价：采用耳缘静脉注射戊巴比妥钠(30mg/kg)麻醉实验兔，并注射过量水合氯醛(150mg/kg)处死后，分离结膜后小心取出整个眼球，在距角膜缘约2mm处环切，取下角膜，并去掉虹膜等组织，得到实验用角膜，剩余眼球部分除去视网膜等组织，得到实验用巩膜，角膜与巩膜均用林格溶液小心冲洗3次后备用。动物处死 后30min内开始实验。将新鲜离体的角膜与巩膜小心的夹在两扩散池中间，上皮面均朝向左侧扩散池(供给池)，扩散窗直径10.25mm，扩散面积0.825cm 2。向供给池和右侧接收池分别加入3.5mL林格溶液，通入循环水，水温保持在34℃±0.5℃，系统平衡10min后，撤去供给池中溶液，加入Penetratin衍生物与FAM复合物的溶液，浓度为7.5μM。整个扩散装置置于扩散仪上，保持磁力搅拌，恒温水浴保持在34℃±0.5℃，并向扩散介质中通入混合气体(O 2∶CO 2＝95∶5，体积比)。实验开始后，在预先设定的时间点取样，每次在接收池取样500μL，并立即补加500μL新鲜林格溶液。实验结束后，立即测定样品荧光强度值。角膜和巩膜取下后，用林格溶液小心冲洗表面3次，去掉扩散面以外部分，剩余组织用Davidson’s溶液固定后，做DAPI染色的冰冻切片，用于观察多肽连接的荧光探针的组织分布。
图4A结果表明，与多肽连接的荧光探针透过离体角膜和巩膜的过程是一个时间依赖的线性过程。根据各曲线得到的表观透过系数(P app)值可知，对于离体角膜，亲脂性衍生物2-W、28-W、289-W的P app值分别是野生型penetratin的1.2、1.3和1.4倍(p＜0.05)，而亲水性衍生物6A则仅为野生型penetratin的1/5(p＜0.001)，而对离体巩膜，2-W、28-W、289-W的P app值分别是野生型penetratin的1.1、1.5和2.1倍(p＜0.001)，而6A则仅为野生型penetratin的2/3(p＜0.001)。图4B中DAPI染色后的离体角膜和巩膜荧光切片结果显示，空白组织切片未见绿色荧光，表示组织无荧光本底干扰。在离体角膜中，与野生型penetratin组相比，亲脂性penetratin衍生物处理过的角膜荧光强度更强，并且亲脂性越强，进入角膜量越多，而亲水性penetratin衍生物6A组无明显荧光信号；在离体巩膜中，亲脂性衍生物组荧光信号强于野生型penetratin组，而6A则明显弱于penetratin组。
实施例6
Penetratin衍生物与小分子物质共价连接复合物的离体组织毒性评价：Davidson’s溶液固定后的组织取部分做苏木素-伊红(HE)染色的石蜡切片，用于观察透过实验后组织的完整性；另取部分组织用滤纸吸干表面水分后称重记为m 0，然后置于60℃烘箱中干燥48h，称重后记为m t，按如下公式计算组织水化值：
ΔH＝(m 0-m t)/m 0×100％
图5A结果显示，penetratin衍生物处理过的离体角膜和巩膜的水化值与未经处理的新鲜角膜和巩膜的水化值相比，没有显著差异，并且与文献报道的正常值相符，表明在7.5μM浓度条件下，penetratin衍生物对离体眼组织未见毒性作用。
图5B中HE染色切片结果显示，所有经penetratin衍生物处理过的角膜均保持了完整的角膜上皮结构，无空泡化或损伤的情况。所有经penetratin衍生物处理过的巩膜也保持了完整结构，无纤维断裂情况。表明penetratin衍生物在7.5μM浓度条件下对离体角膜和巩膜未见毒性作用。
实施例7
Penetratin衍生物与小分子物质共价连接复合物的眼内分布评价：向雄性小鼠结膜囊滴入penetratin衍生物与FAM复合物的溶液5μL(浓度30μM)，轻轻闭合眼睑使溶液均匀分布，每10min给药一次，共3次，将最后一次给药时间作为零时刻，在预先设定的时间点腹腔注射过量水合氯醛致死小鼠，取出小鼠眼球，用Davidson’s溶液固定0.5h后包埋，制备眼球冰冻纵向切片并用DAPI染细胞核，在倒置荧光显微镜下观察多肽在眼部分布情况。
图6A结果显示，空白组小鼠眼球切片中眼前段(角膜)和眼后段(视网膜)均无绿色荧光信号，表明眼组织无荧光本底干扰。而相比于野生型penetratin组，亲脂性penetratin衍生物组在滴眼10min后小鼠眼前段和眼后段均有更强的绿色FAM荧光信号，表明亲脂性penetratin衍生物眼部透过能力更强，而亲水性penetratin衍生物6A组FAM荧光信号减弱，表明其眼部透过能力弱于野生型penetratin，与离体实验结果相一致。此外，图6B显示，经结膜囊给药后，亲脂性penetratin衍生物可以有效分布于小鼠角膜基质层和视网膜内网层，而6A在角膜中分布较野生型penetratin明显较少，几乎没有FAM的绿色荧光信号，而且在视网膜中6A也只有少量分布。
图6C半定量分析结果表明，与野生型penetratin相比，亲脂性penetratin衍生物进入角膜和视网膜的量更多(p＜0.001)，并且眼内滞留时间更长，对于亲脂性较强的28-W，29-W，89-W和289-W，滞留时间长达6h，而对于6A，进入眼内的量明显少于野生型penetratin(p＜0.05)，表明其对眼部屏障的透过性较差。
实施例8
Penetratin衍生物与反义寡核苷酸药物非共价复合物的制备：取60μg拮抗转化生长因子(TGFβ2)基因的反义寡核苷酸(anti-TGFβ2-ASO)，加入2mL缓冲液，涡旋30s使其充分溶解，得到浓度为30μg/mL的ASO溶液。取5代氨基端的聚酰胺-胺树状大分子(PAMAM，缩写为PG5)甲醇溶液10μL(PAMAM浓度为0.1mg/μL)，于40℃水浴中进行氮气吹干，后将PAMAM复溶于1mL蒸馏水中并用蒸馏水进行稀释，得到实验浓度的PAMAM溶液。取透明质酸(HA)1mg溶于1mL蒸馏水中并用蒸馏水进行稀释，得到实验浓度的HA溶液。取2-M，8-W，9-Ypenetratin衍生物(R MIKIWF WYRRMKWKK，缩写为Pene)溶于缓冲液中，得到浓度为500μM的penetratin衍生物溶液。
取500μL实验浓度的PAMAM溶液，在涡旋条件下逐滴加入同等体积(500μL)的ASO溶液，滴加完毕后继续涡旋30s，室温静置下稳定30min，得到ASO/PG5复合物。取稳定后的ASO/PG5复合物1mL，在涡旋条件下逐滴加入500μL实验浓度的HA溶液，滴加完毕后继续涡旋30s，室温静置下稳定30min，得到ASO/PG5/HA复合物。取稳定后的ASO/PG5/HA复合物1.5mL，在涡旋条件下逐滴加入500μL浓度为500μM的penetratin衍生物溶液，滴加完毕后继续涡旋30s，室温静置下稳定30min，得到ASO/PG5/HA/Pene复合物。
实施例9
Penetratin衍生物与反义寡核苷酸药物非共价复合物的眼部吸收：应用荧光标记的ASO分别制备ASO/PG5，ASO/PG5/HA和ASO/PG5/HA/Pene复合物，所有实验组均含30μg/mL ASO。将小鼠随机分为3组，分别为ASO/PG5，ASO/PG5/HA和ASO/PG5/HA/Pene组，每组12只，分别用10μL实验组溶液进行右眼滴眼给药，每隔10min给药一次，每组每只共给药3次。每组分别自最后一次给药1h，2h，6h和8h后随机取3只小鼠进行处死，摘除小鼠眼球，并在30％蔗糖溶液中脱水过夜，垂直于角膜进行切片，取组织中段切片制备冰冻切片并用DAPI工作液进行细胞核染色，共聚焦显微镜下观察眼后段中复合物的消除情况。
通过冰冻切片的观察结果可知，3种复合物递送的ASO均在滴眼1h后在小 鼠眼后段有所分布，且分布随着时间延长而增多，但只有ASO/PG5/HA/Pene递送的ASO在眼后段呈现明显分布，且8h后在外网层(OPL)和视网膜色素上皮细胞(RPE)仍有分布。结果提示，利用penetratin衍生物2-M，8-W，9-Y修饰的寡核苷酸复合物可通过滴眼给药将ASO有效送达眼后段并分布于视网膜色素上皮细胞层(RPE)，且在眼后段存留时间超过8h(图7A)。各主要ASO分布层的荧光相对强度对比见(图7B)。
实施例10
Penetratin衍生物修饰的氟尿嘧啶与反义寡核苷酸药物双载药非共价复合物的制备：取5代氨基端的聚酰胺-胺树状大分子(PAMAM，缩写为PG5)甲醇溶液10μL(PAMAM浓度为0.1mg/μL)，于40℃水浴中进行氮气吹干，后将PAMAM复溶于1mL蒸馏水中并用蒸馏水进行稀释，得到浓度为6.23μM的PG5溶液。取氟尿嘧啶(Fu)1mg溶于1mL蒸馏水中，40℃水浴5min后涡旋溶解，后将母液用蒸馏水稀释至所需浓度得到Fu溶液。将Fu溶液滴加入PG5溶液并搅拌一定时间，得到混合溶液。停止搅拌，将混合溶液移至截留分子量为3000Da的超滤离心管中，3000rpm/min的速度下离心去除游离Fu，得到Fu/PG5复合物溶液。
取30μg拮抗TGFβ2的反义寡核苷酸(anti-TGFβ2-ASO)，加入3mL缓冲液，涡旋30s使其充分溶解，得到浓度为10μg/mL的ASO溶液。将100μL ASO溶液在涡旋条件下滴加至同体积Fu/PG5溶液中，滴加完毕后继续涡旋30s，室温静置下稳定30min，得到dual/PG5复合物。取稳定后的dual/PG5复合物200μL，在涡旋条件下逐滴加入100μLHA溶液(含HA 20μg/mL)，滴加完毕后继续涡旋30s，室温静置下稳定30min，得到dual/PG5/HA复合物。取300μLdual/PG5/HA溶液，在涡旋条件下逐滴加入100μL浓度为300μM的289-Fpenetratin衍生物(R FIKIWF FFRRMKWKK，缩写为Pene)溶液，滴加完毕后继续涡旋30s，室温静置下稳定30min，得到dual/PG5/HA/Pene复合物。
实施例11
Penetratin衍生物修饰的氟尿嘧啶与反义寡核苷酸药物双载药非共价复合物的细胞摄取评价：采用荧光标记的PAMAM制备dual/PG5、dual/PG5/HA及 dual/PG5/HA/Pene复合物，以游离荧光素FAM作为阴性对照，在共聚焦显微镜下观察Penetratin衍生物修饰的双载药非共价复合物在L929细胞中的摄取情况，并用流式细胞仪定量检测3种复合物的摄取比例。
Penetratin衍生物289-F修饰的双载药非共价复合物的胞内平均荧光强度相对于其他实验组有显著性提高，统计结果显示，dual/PG5/HA/Pene相对于dual/PG5及dual/PG5/HA，在L929细胞中的摄取效率分别提高了近3倍及1倍(图8)。

Claims (8)

  1. 一类野生型穿膜肽penetratin的衍生物，作为眼部吸收促进剂可通过无创途径实现眼内药物递送，其特征在于，所述penetratin衍生物具有下述氨基酸序列：
    R X 1 IKIWF X 2X 3 RRMKWKK
    其中，X 1、X 2和X 3代表疏水性氨基酸，选自天然来源的氨基酸丙氨酸(alanine，A)、缬氨酸(valine，V)、亮氨酸(leucine，L)、异亮氨酸(isoleucine，I)、脯氨酸(proline，P)、苯丙氨酸(phenylalanine，F)、色氨酸(tryptophan，W)、甲硫氨酸(methionine，M)和非天然来源的氨基酸α-氨基丁酸(α-aminobutyric acid)、α-氨基戊酸(α-aminopentanoic acid)、α-氨基己酸(α-aminohexanoic acid)、α-氨基庚酸(α-aminoheptanoic acid)，以及它们的组合物。
  2. 根据权利要求1所述的penetratin衍生物，其特征在于，X 1、X 2和X 3为色氨酸(tryptophan，W)，所述penetratin衍生物的氨基酸序列为：
    R WIKIWFQNRRMKWKK
    RQIKIWF WNRRMKWKK
    RQIKIWFQ WRRMKWKK
    R WIKIWF WNRRMKWKK
    R WIKIWFQ WRRMKWKK
    RQIKIWF WWRRMKWKK
    R WIKIWF WWRRMKWKK
  3. 根据权利要求1所述的penetratin衍生物，其特征在于，与具有诊断或治疗作用的药物通过化学键连接形成共价复合物，或修饰在包载诊断或治疗药物的给药系统表面，经结膜囊内滴眼给药可促进药物吸收进入眼内。
  4. 根据权利要求3所述的具有诊断或治疗作用的药物，其特征在于选自白内障治疗药物维生素C、维生素E、吡诺克辛、谷胱甘肽、苄达赖氨酸，细菌性眼内炎治疗药物万古霉素、头孢他啶、异帕米星、新霉素、庆大霉素、红霉素、 地塞米松、曲伐沙星、头孢呋辛钠、米诺环素，真菌性眼内炎治疗药物伏立康唑、制霉菌素、两性霉素B，青光眼治疗药物毛果芸香碱、卡巴胆碱、地匹福林、噻吗洛尔、倍他洛尔、美替洛尔、左布诺洛尔、卡替洛尔、多佐胺、布林佐胺、溴莫尼定、拉坦前列素、曲伏前列素、比马前列素、贝美前列素、他氟前列素等；抗代谢药物氟尿嘧啶、丝裂霉素，葡萄膜疾病治疗药物糖皮质激素类药物、阿昔洛韦、青霉素，视网膜疾病治疗药物曲安奈德，视神经疾病治疗药物泼尼松龙、维生素B1、维生素B12、烟酸，以及它们的组合物。
  5. 根据权利要求3所述的包载诊断或治疗药物的给药系统，其特征在于选自脂质体、胶束和纳米粒。
  6. 根据权利要求1所述的penetratin衍生物，其特征在于，与生物大分子药物自组装形成非共价复合物，经结膜囊内滴眼给药可促进药物吸收进入眼内。
  7. 根据权利要求6所述的生物大分子药物，其特征在于选自基因药物质粒DNA(pDNA)、哌加他尼(Pegaptanib)、贝伐西尼(Bevasiranib)、反义寡核苷酸(Antisense oligonucleotide)，单克隆抗体药物贝伐单抗(Bevacizumab)、雷莫芦单抗(Ramucirumab)，其他多肽蛋白类药物抗血管内皮生长因子(VEGF)融合蛋白康柏西普(Conbercept)、表皮生长因子(Epidermal growth factor，EGF)、防御素、干扰素和环孢菌素，以及它们的组合物。
  8. 按权利要求1所述的penetratin衍生物，其特征在于，眼部应用的浓度为1nM-500μM，优选10nM-300μM，进一步优选100nM-100μM。
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