Brain targeting peptide-nervonic acid conjugate crossing blood brain barrier, preparation method and application

Abstract

The invention belongs to the technical field of biological medicine, and discloses a brain targeting peptide-nervonic acid conjugate crossing a blood brain barrier and a preparation method and application thereof.The brain targeting peptide and nervonic acid are covalently coupled, so that the targeting property of nervonic acid is improved, the blood brain barrier crossing capacity of nervonic acid is enhanced, and by increasing the accumulation amount of nervonic acid in brain parenchyma, the brain targeting peptide-nervonic acid conjugate crossing the blood brain barrier is obtained. Precise delivery of nervonic acid is achieved, and the brain-targeted peptide-nervonic acid conjugate can be applied to treatment of brain-related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a brain-targeting peptide - nervonic acid conjugate that crosses the blood-brain barrier, a preparation method thereof, and an application thereof. Background Art

[0002] The incidence of neurodegenerative diseases has been increasing year by year. In the treatment of these diseases, the blood-brain barrier is considered to be the main reason for preventing drugs from entering the brain tissue and thus affecting the curative effect. The blood-brain barrier is one of the internal barriers participating in innate immunity of the body, which is composed of the pia mater between the blood circulation and the brain parenchyma, the cerebral capillary wall of the choroid plexus, and the glial membrane outside the wall, and can prevent pathogenic organisms and other macromolecular substances from entering the brain tissue and cerebral ventricles from the blood circulation. Basically, 100% of macromolecular drugs, including polypeptides, recombinant proteins, monoclonal antibodies, drugs based on RNA interference technology, gene therapy-related drugs, etc. cannot cross the blood-brain barrier, and more than 98% of small-molecule drugs also cannot cross the blood-brain barrier. Therefore, the present invention is dedicated to studying to make existing drugs better penetrate the blood-brain barrier.

[0003] In order to overcome the limitation of the blood-brain barrier on drug delivery to the brain, researchers have discovered a non-invasive drug delivery method. Using a brain-targeting peptide as a carrier, the drug is conjugated to the brain-targeting peptide by a chemical bond. The brain-targeting peptide can cross the blood-brain barrier and bring the drug into the brain parenchyma, thereby achieving the therapeutic purpose. Compared with antibody-drug conjugates, brain-targeting peptide-drug conjugates have gradually become one of the hotspots in new drug research and development due to the characteristics of polypeptides themselves, such as lower relative molecular weight, better ability to penetrate the blood-brain barrier, lower immunogenicity, and easy large-scale synthesis. For example: the polypeptide Penetratin is covalently coupled with doxorubicin for the treatment of breast cancer; the polypeptide TAT is covalently coupled with the drug P53 for the treatment of eye cancer metastasis; the polypeptide YTA4 is covalently coupled with methotrexate for the treatment of breast cancer.

[0004] Nervonic acid, also known as selacholeic acid, with the scientific name cis-15-tetracosenic acid, is a type of ω-9 monounsaturated fatty acid. Nervonic acid is currently recognized as the only dual-effect substance that can repair and dredge damaged brain nerve pathways - nerve fibers and promote the regeneration of nerve cells. Nervonic acid is an important component of biological membranes, mainly present in nerve tissues and brain tissues, and has functions such as promoting the proliferation and differentiation of nerve cells, delaying brain aging, enhancing learning and memory ability, regulating blood lipid and blood sugar, and improving immunity. It plays a crucial role in the development and maintenance of the brain as well as in the biosynthesis and improvement of nerve cells. Nervonic acid is a natural component present in breast milk and can promote the myelin formation and brain development in infants in the early stage. Intaking nervonic acid is an effective treatment method for various nervous system diseases, such as demyelinating diseases, sequelae of stroke, Alzheimer's disease, cerebral palsy, brain atrophy, memory loss, insomnia and forgetfulness and other brain diseases. However, nervonic acid also has problems such as weak targeting and low bioavailability, which limit its further application and become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] The first object of the present invention is to provide a brain-targeting peptide-nervonic acid conjugate that crosses the blood-brain barrier to solve the problems of weak targeting and low bioavailability of nervonic acid. The brain-targeting peptide-nervonic acid conjugate is NA-[TGNYKALHPHNG]-NH2.

[0006] Preferably, the brain-targeting peptide and nervonic acid are formed through an amide bond.

[0007] Preferably, the structure of the brain-targeting peptide-nervonic acid conjugate is.

[0008]

[0009] Preferably, the brain-targeting peptide consists of 12 amino acids, and the amino acid sequence is: TGNYKALHPHNG. This polypeptide itself does not have physiological activity, but has the function of crossing the blood-brain barrier. The brain-targeting peptide is [TGNYKALHPHNG]-NH2.

[0010] Preferably, the structure of the brain-targeting peptide is:

[0011]

[0012] The second object of the present invention is to provide a coupling method for a brain-targeting peptide and nervonic acid that crosses the blood-brain barrier for preparing the above-mentioned brain-targeting peptide-nervonic acid conjugate, including the following steps:

[0013] (1) Add 0.5 mmol of polypeptide resin containing nervonic acid (2 eq) into a solid-phase synthesis tube for polypeptides.

[0014] (2) Add O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate / 1-hydroxybenzotriazole (3 eq) and N,N-diisopropylethylamine (6 eq), dissolve in DMF, and protect with nitrogen. After stirring at 27 °C for 2 h, terminate the reaction and cleave with TFA / TIS / H2O 95 / 2.5 / 2.5.

[0015] (3) Use a high-resolution liquid chromatography-mass spectrometry instrument to determine the molecular weight, and purify the crude product by RP-HPLC.

[0016] Preferably, the reaction formula is:

[0017]

[0018] The third object of the present invention is to provide an application of a brain-targeting peptide-nervonic acid conjugate. Utilizing the characteristic that the above-mentioned brain-targeting peptide-nervonic acid conjugate is easy to cross the blood-brain barrier, it is applied to the treatment of neurological diseases.

[0019] The covalent coupling of the brain-targeting peptide and nervonic acid in the present invention can improve the targeting of nervonic acid, enhance its ability to cross the blood-brain barrier, increase the accumulation of nervonic acid in the brain parenchyma, and achieve the precise delivery of nervonic acid.

[0020] The fourth object of the present invention is to provide a preparation method of a brain-targeting peptide that crosses the blood-brain barrier for preparing the above-mentioned brain-targeting peptide. The required polypeptide is prepared by solid-phase synthesis. Solid-phase synthesis is a process of gradually adding amino acids from the carboxyl terminus to the amino terminus with a resin as the solid-phase carrier, and specifically includes the following steps:

[0021] (1) Add 0.5 mmol of Rink Amide MBHA resin into a polypeptide synthesis tube, swell with dichloromethane and wash with DMF.

[0022] (2) After removing the Fmoc protecting group with a 20% piperidine / DMF solution, add Fmoc-amino acid (3 eq), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate / 1-hydroxybenzotriazole (3 eq) and DIEA (6 eq), protect with nitrogen, and react at room temperature for 2 hours.

[0023] (3) Repeat step (2) until the coupling of the last amino acid is completed, remove the Fmoc protecting group, wash, and cleave the polypeptide with TFA / TIS / H2O 95 / 2.5 / 2.5.

[0024] (4) Use a high-resolution liquid chromatography-mass spectrometry analyzer to determine the molecular weight of the polypeptide, and purify the crude peptide by RP-HPLC.

[0025] The present invention has the following beneficial effects:

[0026] In the present invention, a brain-targeting peptide-neural acid conjugate is obtained by coupling a brain-targeting peptide with neural acid, which can cross the blood-brain barrier, achieving the purpose of improving the therapeutic effect of brain diseases. In Example 2 of the present invention, the brain-targeting peptide TGNYKALHPHN (TGN) is covalently coupled with neural acid. The TGN peptide can be recognized by receptors on the BBB and has great potential for brain transport. To confirm the targeted delivery of neural acid to the brain, an in vitro model of co-culturing immortalized mouse brain microvascular endothelial cells and mouse brain astrocytes (bEnd.3 and C8-D1A) was established in Example 6. The results show that the BBB transport ratio of TGN@NA–Cy5 obtained in Example 3 was 20.95% at 12 hours, which was approximately 4.8 times that of NA-Cy5 obtained in Example 4. In the in vivo fluorescence imaging shown in Example 5, the CY5-labeled brain-targeting peptide-neural acid conjugate obtained in Example 3 aggregated in the brains of mice and the fluorescence signal was stronger than that of the mice labeled in Example 4. This indicates that the targeting of the conjugated drug is improved and the level of neural acid crossing the blood-brain barrier is enhanced. Description of the Drawings

[0027] Figure 1 Shows the HPLC test result diagram of the brain-targeting peptide.

[0028] Figure 2 Shows the mass spectrometry test result diagram of the brain-targeting peptide.

[0029] Figure 3 Shows the mass spectrometry test result diagram of the brain-targeting peptide-neural acid conjugate.

[0030] Figure 4 Shows the concentration distribution of the brain-targeting peptide-neural acid conjugate in the mouse brain.

[0031] Figure 5 Shows the distribution concentrations of the brain-targeting peptide-neural acid conjugate in the mouse brain, heart, liver, spleen, lung, and kidney at 4 hours.

[0032] Figure 6 Shows the TEER value of the established bEnd.3 / C8-D1A cell co-culture in the Transwell BBB model.

[0033] Figure 7 Shows the in vitro blood-brain barrier transport rate of the brain-targeting peptide-neural acid conjugate. Detailed Embodiments

[0034] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Example 1: Solid-phase synthesis of brain-targeting peptide TGN

[0036] A solid-phase polypeptide synthesis method using the Fmoc strategy (the amino acids used were purchased from Shanghai Gil Co., Ltd.).

[0037] (1) Take 3.15 g of Rink Amide MBHA resin with a substitution degree of 0.317 mmol / g as a solid-phase carrier and add it to a solid-phase reaction column. Add 10 ml of DMF to swell for 1 h, then drain DCM and wash 3 times with DMF; add 15 mL of 20% piperidine / DMF (volume ratio) deprotection solution, react for 3 min, then drain the liquid, add the deprotection solution again and react for 5 min. After the reaction, add 20 ml of DMF to wash, wash a total of 6 times, and take the resin to be detected as positive by ninhydrin; then weigh 0.892 g of Fmoc-Gly-OH (3 eq), 1.138 g of O-benzotriazole-tetramethylurea hexafluorophosphate (3 eq), and 0.459 g of 1-hydroxybenzotriazole (3 eq) and add them to 15 mL of DMF solution and stir to dissolve. While stirring, add 0.992 g of DIEA (6 eq) as a condensing agent to obtain an amino acid mixture; finally, pour the above amino acid mixture into the reaction column and react at room temperature for 2 h. After the reaction, filter by suction, add 20 mL of DMF to wash 2 times to obtain Fmoc-Gly-MBHA, and use ninhydrin to detect the above resin as negative.

[0038] (2) Repeat step (1) to remove the Fmoc protecting group with 20% piperidine / DMF and use DIC / HOBT as a condensing agent to sequentially couple the amino acids Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Lys(mtt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH to obtain the brain-targeting peptide resin peptide TGNYKALHPHNG-MBHA.

[0039] (3) Add 20 mL of cleavage solution to the obtained brain-targeting peptide resin, where 1 g of brain-targeting peptide resin corresponds to 8 mL of cleavage solution, and the ratio is TFA: EDT: H2O (95: 2.5: 2.5). React at 20 - 30 °C for 2 h, then filter, wash the resin with an appropriate amount of trifluoroacetic acid, combine the filtrates, then add 5 times the volume of methyl tert-butyl ether to precipitate the crude peptide, filter to remove the liquid, and wash the filter cake 5 times with methyl tert-butyl ether to obtain the crude peptide.

[0040] (4) The obtained crude peptide was purified using an RP-HPLC C18 semi-preparative column (purchased from Agilent Technologies). The detection conditions were as follows: detection wavelength: 220 nm, mobile phase A: acetonitrile (containing 0.1% trifluoroacetic acid), mobile phase B: water (containing 0.1% trifluoroacetic acid), elution conditions: from 35% A to 50% A within 30 minutes. The molecular weight of the obtained pure product was determined by MS, and the sample purity was determined by HPLC. The powder was obtained after desalting and freeze-drying when the purity was greater than 95%. The HPLC test results are shown in Figure 1 , Table 1, and the spectrum detection results are as shown in Figure 2 . The theoretical molecular weight is 1308.4, and the measured molecular weight is 1308.7. The mass spectrometry confirmed it was correct.

[0041] Table 1 HPLC test results of brain-targeting peptide

[0042]

[0043] Example 2: Preparation of brain-targeting peptide - nervonic acid conjugate

[0044] Take 1 g of the resin peptide (1 eq) obtained in Example 1, swell it with 12 ml of methanol, remove the Fmoc protecting group with 20% piperidine / DMF, use 0.361 g of O-benzotriazole-tetramethylurea hexafluorophosphate (3 eq) / 0.1289 g of 1-hydroxybenzotriazole (3 eq) as the condensing agent, use 0.315 g of DIEA (6 eq) as the acid-binding agent, add 1.08 g (2 eq) of nervonic acid and dissolve it, and oscillate the reaction overnight. After the reaction, filter it by suction, add 20 mL of DMF and wash it 3 times. Use ninhydrin to detect that the above resin is negative; according to steps (3) and (4) in Example 1, cut and purify to obtain the brain-targeting peptide - nervonic acid conjugate.

[0045] Example 3: Preparation of CY5-labeled brain-targeting peptide - nervonic acid conjugate

[0046] The brain-targeting peptide resin obtained in Example 2 was added with 20 mL of mtt protecting group removing agent, and the ratio was TFA:TIS:DMF = 3:3:94. After reacting at room temperature for 15 min, it was washed 3 times with DMF. The above operation was repeated 3 times. After the washing was completed, the DMF was dried by suction, and the resin turned blue when detected by ninhydrin. Then, 0.494 g of Cy5-COOH (3 eq) fluorescent dye, 0.361 g of O-benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate (3 eq) / 0.1289 g of 1-hydroxybenzotriazole (3 eq) were weighed and added to 15 mL of DMF solution and stirred until dissolved. While stirring, 0.315 g of DIEA (6 eq) was added and mixed evenly; finally, the above amino acid mixture was poured into the reaction column and reacted at room temperature for 12 h. After the reaction was completed, suction filtration was carried out, 20 mL of DMF was added and washed 3 times, and the above resin was detected to be negative by ninhydrin to obtain CY5-resin peptide; according to the steps (3) and (4) of Example 1, cleavage and purification were carried out to obtain CY5-labeled brain-targeting peptide-nervonic acid conjugate.

[0047] Example 4: Preparation of CY5-labeled nervonic acid

[0048] Weigh 0.469 g (1.0 mmol, 1.0 eq) of Fmoc-Lys(Mtt)-OH, dissolve it in 50 mL round-bottom flask with 10 ml of tetrahydrofuran. Weigh 0.341 g (4 mmol, 4.0 eq) of hexahydropyridine to remove Fmoc, protect with nitrogen, react at room temperature for 2 h, and detect the reaction completion by TLC. After the reaction is completed, concentrate under reduced pressure to obtain a white solid. Dissolve the product with 150 m of acetonitrile, filter by suction and detect the pH of the filtrate. If the pH is greater than 7, continue to wash with acetonitrile until the filtrate is washed to neutral. Dry and weigh.

[0049] Add 0.469 g (1.0 mmol, 1.0 eq) of the product NH2-Lys(Mtt)-OH from the above step to a 50 ml round-bottom flask, and stir and dissolve it with 10 ml of tetrahydrofuran. Measure 20 ml of tetrahydrofuran to dissolve 0.439 g (1.2 mmol, 1.2 eq) of nervonic acid, then add 0.162 g (1.2 mmol, 1.2 eq) of HoBt and 0.151 g (1.2 mmol, 1.2 eq) of DIC in sequence, stir and activate for 3 min, and add to the reaction solution. Protect with nitrogen and react at 25 °C for 4 h, and detect the reaction completion by TLC. After the reaction is completed, concentrate under reduced pressure to obtain a white solid, wash with 150 ml of acetonitrile, make a slurry, filter by suction to obtain an off-white solid, dry and weigh

[0050] Add 0.469 g (1.0 mmol, 1.0 eq) of the product NA-Lys(Mtt)-OH from the above step to a 50 ml round-bottom flask. Weigh 0.456 g (4 mmol, 4.0 eq) of TFA to remove Fmoc. Under nitrogen protection, react at room temperature for 2 h, and detect the completion of the reaction by TLC. After the reaction is completed, concentrate under reduced pressure to obtain a white solid. Dissolve the product in 150 ml of acetonitrile, filter by suction and detect the pH of the filtrate. If the pH is less than 7, continue to wash with acetonitrile until the filtrate is washed to neutral. Dry and weigh.

[0051] Add the product from the above step to a 50 ml round-bottom flask. Measure 20 ml of tetrahydrofuran to dissolve 0.637 g (1.2 mmol, 1.2 eq) of CY5-COOH, then add 0.162 g (1.2 mmol, 1.2 eq) of HoBt and 0.151 g (1.2 mmol, 1.2 eq) of DIC in sequence, stir and activate for 3 min, and add to the reaction solution. Under nitrogen protection, react under light protection at 25 °C for 4 h, and detect the completion of the reaction by TLC. After the reaction is completed, concentrate under reduced pressure, wash with 150 ml of acetonitrile, slurry, filter by suction to obtain an off-white solid, dry and weigh.

[0052] Example 5: In vivo evaluation of the conjugate's ability to cross the blood-brain barrier

[0053] Examine the blood-brain barrier crossing ability of the CY5-labeled brain-targeting peptide-nervonic acid conjugate obtained in Example 3 and the CY5-labeled nervonic acid obtained in Example 4 through a small animal in vivo imaging system. Anesthetize (purchased from the Experimental Animal Center of Lanzhou Institute of Animal Science, Chinese Academy of Sciences, Kunming mice) with isoflurane, and intravenously inject through the tail vein (200 μL / animal, 6 animals / group) the brain-targeting peptide-nervonic acid conjugate of Example 2 calculated and prepared according to the dose of 10 mg / kg of the cyanine dye Cy5-COOH, and the injection method is tail vein injection. Record the time as the 0th hour (0 h) after the administration is completed. Perform in vivo imaging at different time points (2 h, 4 h, 6 h, 8 h, 12 h) after the tail vein injection, and dissect and photograph the mouse brain to count the fluorescence content when the imaging is completed at 6 h.

[0054] It can be seen from Figure 4 that the CY5-labeled brain-targeting peptide-nervonic acid conjugate obtained in Example 3 shows a characteristic of systemic distribution after being injected into the mouse blood circulation through the tail vein. As the administration time prolongs, the fluorescence intensity of the CY5-labeled brain-targeting peptide-nervonic acid conjugate in the mouse brain gradually increases; at 6 hours after administration, the fluorescence signal of the CY5-labeled brain-targeting peptide-nervonic acid conjugate in the mouse brain is strong, but the fluorescence signal of the CY5-labeled nervonic acid obtained in Example 4 in the mouse brain is weak; at 12 hours after administration, a large amount of fluorescence signal still remains in the mouse brain of Example 3, and there is almost no fluorescence in the mouse brain labeled in Example 4.

[0055] Six hours after the CY5-labeled brain-targeting peptide - nervonic acid conjugate obtained in Example 3 and the CY5-labeled nervonic acid obtained in Example 4 were administered via the tail vein, the mice were humanely sacrificed. The brain, heart, liver, spleen, lung, and kidney were dissected and imaged in vitro as Figure 5 shown. Consistent with the results of in vivo imaging, 12 hours after drug metabolism, the fluorescence intensity in the mouse brain was Example 3 > Example 4. The above results indicate that the brain-targeting peptide - nervonic acid conjugate has the best blood-brain barrier crossing ability.

[0056] Example 6: In vitro evaluation of the conjugate's penetration through the blood-brain barrier

[0057] In this example, the cells used were bEnd.3 and C8-D1A (purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). The C8-D1A cells were seeded on the outer bottom surface of the Transwell insert at a concentration of 1×104 cells / well, and the bEnd.3 cells were seeded on the inner bottom surface of the Transwell insert at a concentration of 5×104 cells / well. The cells were cultured until the electrical resistance was greater than 200 Ω. The Cy5-labeled brain-targeting peptide - nervonic acid conjugate was dissolved in PBS and incubated with the cells to a final concentration of 10 μmol / L. After 1 h, the fluorescence intensities in the upper and lower chambers of the Transwell insert were measured.

[0058] In this example, an in vitro BBB model was constructed using Transwell. As can be seen from Figure 6 it, when the bEnd.3 cells and C8-D1A cells were continuously cultured for about 7 days, the trans-epithelial electrical resistance of the Transwell insert reached TEER > 200 Ω·cm2 and was relatively stable, indicating that the model was successfully established and was a dense cell layer. Further, Figure 7 shows the permeability of the CY5-labeled brain-targeting peptide - nervonic acid conjugate obtained in Example 3 and the CY5-labeled nervonic acid obtained in Example 4 in the in vitro BBB model. The compounds obtained in Example 3 and Example 4 both increased with the increase of the penetration time. However, 24 hours after cell treatment, the compound obtained in Example 3 was 4.8 times that of the compound obtained in Example 4. When the compounds obtained in Example 3 and Example 4 were used to treat the cells for 4 hours, the CY5-labeled brain-targeting peptide - nervonic acid conjugate obtained in Example 3 was 15 times that of the CY5-labeled nervonic acid obtained in Example 4. This indicates that the CY5-labeled brain-targeting peptide - nervonic acid conjugate obtained in Example 3 can effectively improve the ability of nervonic acid to cross the blood-brain barrier.

[0059] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A brain-targeting peptide-nervonic acid conjugate across the blood-brain barrier, characterized in that, The brain-targeting peptide-nervonic acid conjugate is NA-[TGNYKALHPHNG]-NH2.

2. The brain-targeting peptide-nervonic acid conjugate crossing the blood-brain barrier according to claim 1, characterized in that, The brain-targeting peptide-nervonic acid conjugate is formed by the brain-targeting peptide and nervonic acid through an amide bond.

3. The brain-targeting peptide-nervonic acid conjugate crossing the blood-brain barrier according to claim 1, characterized in that, The structure of the brain-targeting peptide-nervonic acid conjugate is:

4. A coupling method for a brain-targeting peptide-nervonic acid conjugate across the blood-brain barrier, which is used to prepare the brain-targeting peptide-nervonic acid conjugate according to any one of claims 1-3, characterized in that, It includes the following steps: (1) Add polypeptide resin containing nervonic acid into the polypeptide solid-phase synthesis tube. (2) Add O-benzotriazole-tetramethylurea hexafluorophosphate / 1-hydroxybenzotriazole dissolved in DMF, protect with nitrogen, stir and react at a certain temperature and then cleave. (3) Use a high-resolution liquid chromatography-mass spectrometry analyzer to determine the molecular weight and purify the crude product using RP-HPLC.

5. Application of a brain-targeting peptide-nervonic acid conjugate for crossing the blood-brain barrier. Use the brain-targeting peptide-nervonic acid conjugate as described in claim 1 and apply it to the treatment of neurological diseases.

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