CN103145799B - A group of polypeptides with the function of promoting transdermal and transmembrane penetration and their application - Google Patents

Abstract

A group of polypeptides with the function of promoting percutaneous and transmembrane permeation and application thereof are suitable for the fields of percutaneous and transmembrane drug delivery systems and cosmetics. The polypeptide contains 11 amino acid residues, 9 amino acid residues form a ring structure by disulfide bonds, and the structure is characterized in that N-5 and N-6 sites at the N-terminal of the polypeptide are basic amino acids or amidated at the C-terminal. Compared with the reported polypeptide with penetration promoting activity, the polypeptide synthesized by substituting alkaline amino acid at the N-5 or N-6 position of the N-terminal and the C-terminal amidated polypeptide can obviously improve the transdermal and transmembrane penetration capacity of insulin. The transdermal and transmembrane permeation-promoting capability of the polypeptide with N-5, N-6 positions simultaneously substituted by the basic amino acid is further enhanced, the blood sugar reducing effect on diabetic rats can be obviously improved along with the common transdermal administration of insulin, the in-vivo blood sugar level of the diabetic rats is reduced to 20-30% of the original level within 8 h after the transdermal administration, and the polypeptide group can be applied to solutions, gels and ointments to promote the transdermal and transmembrane absorption of biomacromolecule drugs such as insulin and vaccines.

Description

A group of polypeptides with the function of promoting transdermal and transmembrane penetration and their application

technical field

The invention relates to a group of polypeptides capable of promoting transdermal and transmembrane penetration and applications thereof, which are suitable for the fields of transdermal and transmembrane drug delivery systems and cosmetics.

Background technique

Transdermal administration means that the drug passes through the skin at a certain rate, and is absorbed into the body circulation through capillaries to produce drug effects. It also means that the drug is absorbed through the skin and enters the epidermis, dermis or subcutaneous tissue to exert local therapeutic effects. It means that the drug enters the circulatory system through the biomembrane to produce curative effect. Compared with traditional drug delivery methods, it has many advantages: transdermal and transmembrane drug delivery can reduce or avoid the first-pass effect of the liver, reduce toxic and side effects; avoid various factors that affect gastrointestinal absorption of drugs, such as pH value, enzymes, The combination of the drug and food has a slow-release effect on the effect of the drug; it can keep the blood drug level stable within the therapeutically effective concentration range; at the same time, the administration can be terminated at any time. Although these delivery methods are ideal delivery routes for enzymatically hydrolyzed hydrophilic macromolecules such as insulin, due to the low absorption of hydrophilic macromolecules through biomembranes, especially the stratum corneum, traditional drug delivery techniques cannot meet the treatment requirements. Insulin and other macromolecular drugs are mainly administered by injection, which has poor patient adaptability. Therefore, how to realize the effective transdermal administration of hydrophilic macromolecular drugs has become a major challenge in the field of preparations (Shaikh et al.2005, Cur.Pharma.Bio.6:387; Huang et al.2006, Jour.of control . Rel., 113:9-14; Li et al. 2008, Bio. & pharma. Bull. 31: 1574).

和

可以促进胰岛素类亲水性大分子经鼠皮吸收（Chen et al.2006,Nature bio.24:455；Hou etal.2007,Exp.Derma.16:999-1006；Hsu et al.2011,Proc.Natl.Acad.Sci.108:15816）。研究表明亲水性大分子药物主要是通过毛囊部位进入体内（Carmichael et al.2010,Pain,149:316.），而现有的促渗多肽存在着对胰岛素经皮促渗效能较低的问题，由于人体毛囊数量较少（Prausnitz,2006,Nature bio.24:416.），因此，研究开发对生物大分子药物具有更高促渗作用的多肽十分迫切和必要。At present, there are mainly the following methods reported to promote the passage of hydrophilic macromolecules through the skin: iontophoresis (Kanikkannan et al.1999, Jour.of control.Rel.59:99.), sonophoresis (Boucaud et al. 2002Jour.of control.Rel.81:113.), electroporation (Sen et al.2002, Bio.et bio.acta1564:5.) and microneedle introduction method (Zhou et al.2010, Inter.Jour. of pharma., 392:127.), etc. In addition, studies have reported that peptides

and

Can promote the absorption of insulin-like hydrophilic macromolecules through mouse skin (Chen et al.2006, Nature bio.24:455; Hou et al.2007, Exp.Derma.16:999-1006; Hsu et al.2011, Proc. Natl. Acad. Sci. 108:15816). Studies have shown that hydrophilic macromolecular drugs mainly enter the body through hair follicles (Carmichael et al.2010, Pain, 149:316.), while the existing penetration-enhancing polypeptides have a low effect on insulin transdermal penetration , due to the small number of human hair follicles (Prausnitz, 2006, Nature bio.24:416.), it is urgent and necessary to research and develop polypeptides with higher penetration-promoting effects on biomacromolecular drugs.

People's research on peptides in drug transport initially focused on cell-penetrating peptides rich in basic amino acids, such as Drosophila Antp (Derossi et al.1994, The Jour.of bio.chem.269:10444), Human immunodeficiency virus transactivator TAT (Brooks et al.2005, Adv.Dru.Del.Rev.57:559), PEP-1 (Hallbrink et al.2001, Bio.et bio.acta1515:101.) and Polyarginine (Futaki et al.2001, The Jour.of bio.chem.276:5836.), etc., these penetrating peptides can pass through human or animal skin, and some penetrating peptides can also pass through the protein Carry proteins through the skin or biomembrane by covalent linkage (Lopes et al. 2005, Pharma. Res. 22:750. Patel et al. 2009, Mol. Pharma. 6: 492.). In terms of the mechanism of promoting penetration, it has been found that the basic group of lysine or arginine has an important influence on the promoting activity of membrane-penetrating peptides (Beerens et al.2003, Curr.gene therapy3:486.), and found that The highly hydrophilic basic-group-rich membrane-penetrating peptide promotes the transmembrane transport of hydrophilic drugs through reversible loose intercellular tight junctions (Ohtake et al. 2002, J. controlled release, 82:263).

At present, the screening method for the transdermal penetration enhancing activity of polypeptides mainly adopts the in vivo animal experiment method, which is time-consuming, requires a large number of animal samples and has poor analysis stability due to individual animal differences. Caco-2 cells are widely used to evaluate the ability of drug transmembrane penetration and the in vitro evaluation of the effect of penetration enhancers on drug penetration (Torres-Lugo et al.2002, Biotechnol.Prog.18:612), reports have shown that epithelial tissue and Caco-2 cell monolayer membranes are similar, and there are tight junctions between cells (Morita et al. 2003, J. Dermatol. Sci. 31: 81).

Contents of the invention

结构改造，提供一组新型高效的促进经皮和跨膜渗透作用的多肽，提高胰岛素和疫苗等大分子药物经皮和跨膜渗透能力并提供相关技术手段。The present invention aims at the existing penetration-promoting activity polypeptide

Structural modification provides a group of new and efficient polypeptides that promote transdermal and transmembrane penetration, improves the transdermal and transmembrane penetration of macromolecular drugs such as insulin and vaccines, and provides related technical means.

基础上，通过碱性氨基酸扫描，明确碱性氨基酸种类、数量和位点对提高多肽经皮促渗活性的影响。通过对已有促渗活性多肽结构改造，合成更为高效的经皮和跨膜促渗多肽。该组多肽含有11个氨基酸残基，并由9个氨基酸残基以二硫键构成环状结构，N-端的N-5和N-6位点由碱性氨基酸残基构成，具体结构：

或C-端酰胺化，具体结构：

其他位点的碱性氨基酸取代的多肽与原有多肽

的经皮和跨膜促渗效果没有显著性差异。The technical scheme adopted in the present invention is: in the polypeptide

On the basis of basic amino acid scanning, the effect of basic amino acid type, quantity and position on improving the transdermal penetration-enhancing activity of polypeptides is clarified. By modifying the structure of existing permeation-promoting active polypeptides, more efficient transdermal and transmembrane permeation-promoting polypeptides are synthesized. This group of polypeptides contains 11 amino acid residues, and 9 amino acid residues form a ring structure with disulfide bonds. The N-5 and N-6 positions of the N-terminal are composed of basic amino acid residues. The specific structure:

Or C-terminal amidation, the specific structure:

Basic amino acid substituted polypeptides at other positions and original polypeptides

There was no significant difference in the effect of percutaneous and transmembrane permeation enhancement.

结构中的N-5，N-6位为结构改造活性位点，采用碱性氨基酸—赖氨酸（K）和精氨酸（R）取代或者C-端酰胺化的多肽可以显著提高对胰岛素经皮渗透的促渗活性，可以协助亲水性大分子药物穿过皮肤及生物膜屏障的各种制剂，包括胰岛素及各种疫苗等。多肽可以采用固相多肽合成法进行合成。The above-mentioned technical scheme adopts basic amino acid-arginine or lysine to scan, and studies the influence of basic amino acid type, quantity and position on percutaneous and transmembrane permeation-promoting activity. Peptide

The N-5 and N-6 positions in the structure are the active sites for structural modification, and the substitution of basic amino acids - lysine (K) and arginine (R) or the amidation of the C-terminus of the polypeptide can significantly improve the response to insulin The permeation-enhancing activity of percutaneous penetration can assist various preparations of hydrophilic macromolecular drugs to pass through the skin and biofilm barriers, including insulin and various vaccines. Polypeptides can be synthesized using solid-phase peptide synthesis.

中N-5，N-6位碱性氨基酸取代合成的多个多肽序列，与多肽

相比，亲水性大分子的经皮和跨膜渗透能力均有明显提高，对N-5，N-6位同时用K和/或R进行取代的多肽促渗活性得到进一步加强，提高了胰岛素经皮给药对糖尿病大鼠的降糖效果，经皮给药8h内糖尿病大鼠在体血糖水平到原有水平的20-30%，另外还可以显著提高胰岛素跨细胞膜的转运能力。The beneficial effects of the present invention are: the polypeptide having transdermal and transmembrane penetration-promoting effects contains 11 amino acid residues, and 9 amino acid residues form a ring structure with disulfide bonds.

N-5, N-6 basic amino acid substitutions synthesized multiple polypeptide sequences, and polypeptide

Compared with hydrophilic macromolecules, the transdermal and transmembrane penetration capabilities are significantly improved, and the penetration-promoting activity of polypeptides that are substituted with K and/or R at the N-5 and N-6 positions is further enhanced, improving the The hypoglycemic effect of transdermal insulin administration on diabetic rats, within 8 hours of transdermal administration, the blood glucose level in diabetic rats will be reduced to 20-30% of the original level, and it can also significantly improve the transport ability of insulin across the cell membrane.

Detailed ways

The present invention is further described in detail by non-limiting examples below:

Example 1

Diabetic rat model establishment: SD rats were weighed after fasting overnight, and the corresponding streptozotocin was injected according to 70 mg/kg fasting body weight, and the injection was completed within 30 minutes. After about 10 days of modeling, SD rats with a blood sugar level of 20-30 mmol/L were selected for polypeptide screening experiments. Moisten with saline and cut off the abdominal hair, so that the exposed area of the skin of the rat is within 4 cm ² and wash it with saline. After transdermal administration, the time-dependent changes in blood sugar in SD rats were observed in vivo and the ability of peptides to promote insulin transdermal penetration was studied, and the transdermal penetration enhancement ability of a series of synthetic peptides was screened.

Example 2

Caco-2 cell culture method: 40-60 generations of Caco-2 cells were planted in a 25cm ² cell culture flask. DMEM medium contains: 20% bovine serum, 1% (v/v) non-essential amino acids, 2mmol/L L-glutamine, 100IU/mL penicillin, 100μg/mL streptomycin (pH=7.4). The culture conditions were 37°C, 5% CO ₂ , 95% air and 90% relative humidity, and the medium was changed three times a week.

Example 3

Insulin transmembrane transport experiment: Caco-2 cells were seeded in 12-well Transwell culture plates at a seeding density of 2×105 cells/mL. Cell monolayers formed after 20-22 days of culture. Before the transport experiment, the Caco-2 cell monolayer was incubated with HBSS at 37°C for 10 min. At the same time, the resistance value of the cell monolayer membrane is detected to ensure the integrity of the cell membrane. Cell monolayer membranes with a resistance value greater than 400 Ω·cm ² were selected for transport experiments. Add 1.5mL 21IU/mL insulin solution, or 1.5mL 21IU/mL insulin and 5μmol/mL polypeptide (sequence 1-10) solution to the BL side, and add 0.5mL Hank's solution to the AP side. At different time points (0.5h, 1h, 1.5h and 2h), 100 μL of the receiving solution was drawn from the AP side and the transmembrane resistance of the cell monolayer was detected, and the insulin concentration in the receiving solution was detected by HPLC. Observe the changes of insulin concentration over time and study the ability of peptides to promote insulin transmembrane penetration, and screen the transmembrane penetration enhancement ability of a series of synthetic peptides. The change of insulin transmembrane permeability coefficient is shown in Table 1.

Example 4

Peptide sequence 1 As an example, the synthesis of peptides by solid phase synthesis is illustrated.

Microwave-assisted solid-phase synthesis of peptides was adopted: Wang resin was used as a solid-phase carrier, HBTU-HOBt was used as a condensation agent, and Fmoc was used as a synthetic strategy for the α-amino protecting group.

Weigh 0.1 mmol of Fmoc-glycine-Wang resin into a solid-phase reactor, add 10 mL of DCM to swell the resin for 30 min, wait until the resin is fully swollen, and vacuum the solvent to dry up. Wash once with 20% piperidine/DMF10mL, add 20% piperidine/DMF10mL to the resin and place it in a microwave-assisted solid-phase synthesizer, react at 60°C, 20W for 3min to remove the Fmoc protecting group, and sequentially use DCM , DMF washing, and the removal of the protecting group is detected by the Kaiser method. If the test is positive, the reaction can be continued. Dissolve 3eq of Fmoc-amino acid-OH, HBTU and HOBt in DMF, add dropwise 5eq of DIEA and let it stand for 2min to fully activate it, add it to a solid-phase reactor, react at 60°C, 20W for 5min, after the condensation is completed , wash the resin with DCM, DMF sequentially. Repeat the above steps to obtain the desired straight chain polypeptide, use TFA/TIS/H ₂ O 95:2.5:2.5 solution 10mL, react at room temperature for 2 hours to remove the N-terminal and side chain protecting groups and cut off the resin, wash with DCM three times, After the filtrate was concentrated to the minimum volume, 30 times the volume of glacial diethyl ether was added, centrifuged at 10,000 r/min for 15 min at 4°C, the supernatant was discarded, the crystallization was repeated twice, and the polypeptide linear product was obtained by vacuum drying. Dissolve the polypeptide in 25% methanol aqueous solution, add 2.5eq iodine 5 times at intervals of 30min for oxidation reaction, stir at room temperature for 2h and then concentrate to obtain the crude product. Purification was performed by gel chromatography Sephadex G-15, the eluent was 50% aqueous methanol, and the product was identified by HPLC and ESI-MS.

Example 5

的固相合成方法。Peptide sequence 9

solid-phase synthesis method.

Adopt 0.1mmol amide MBHA resin to carry out solid phase synthesis, other steps are the same as embodiment 2.

Example 6

0.5 μmol of polypeptide (sequence 1-9) was mixed with 2.1 IU insulin respectively, and then dissolved in 100 mg of normal saline, and evenly applied to the abdomen of 4 cm ² of diabetic rats in Example 1. Blood was taken from the tail vein at 0h, 2h, 5h, and 8h after administration, and the blood sugar level of SD rats was detected at the same time. Table 2 shows the blood sugar changes of diabetic rats.

Example 7

Mix PEG4000 and PEG400 at a ratio of 2:3, heat and melt to prepare an ointment base, mix 0.05~1.5 μmol polypeptide (sequence 1-9) with 0.21~2.1IU insulin respectively, and then mix them uniformly with 100mg ointment base to prepare a penetration-promoting Peptide insulin ointment.

Example 8

Prepare Carbopol941 gel matrix (containing 1%wt Carbopol941, 1.2%wt triethanolamine, pH=6~7), mix 0.05~1.5μmol polypeptide (sequence 1-9) with 0.21~2.1IU insulin respectively, and then mix with 100mg The gel matrix is evenly mixed to prepare the insulin gel containing the penetration-promoting polypeptide.

Comparative experiment 1

Mix 0.5 μmol of the comparison polypeptide (sequence 10) with 2.1 IU insulin, dissolve in 100 mg of normal saline, and apply evenly on 4 cm ² of the abdomen of diabetic rats in Example 1. Blood was taken from the tail vein at 0h, 2h, 5h, and 8h after administration, and the blood sugar level of SD rats was detected at the same time. Table 2 shows the blood sugar changes of diabetic rats.

Comparative experiment 2

2.1 IU of insulin was dissolved in 100 mg of normal saline as a control solution, and evenly applied to the abdomen of the diabetic rat in Example 1 (4 cm ^{2 )} . Blood was taken from tail vein at 0h, 2h, 5h, and 8h after administration, and the blood glucose level of SD rats was detected at the same time. Table 2 shows the changes in blood sugar of diabetic rats after transdermal administration of some embodiments to diabetic rats.

Table 1 Caco-2 cell membrane permeability coefficient of insulin

Table 2 Changes of blood sugar over time in diabetic rats

*P<0.05, **P<0.005 (n=6, compared with the comparison peptide).

The polypeptide sequence described in the present invention:

<110> Dalian University of Technology

<120> A group of peptides and their application to promote transdermal and transmembrane penetration

<130> Case reference number

<140> application number

<141>2013-02-17

<160>9

<210>1

<211>11

<212>PRT

<213> Synthetic sequence

<220>

<221> disulfide bond

<222>(2)...(10)

<223> The designed peptide is based on changing the position and charge of amino acid residues in the sequence, so as to increase the penetration-promoting effect on skin and biomembrane. (Designed peptide based on site and charge of amino acid residue to act as a enhancement activity on transdermal and membrane delivery)

<440>1

Ala Cys Ser Ser Lys Pro Ser Lys His Cys Gly

1 5 5 10

<210>2

<211>11

<212>PRT

<213> Synthetic sequence

<220>

<221> disulfide bond

<222>(2)...(10)

<223> The designed peptide is based on changing the position and charge of amino acid residues in the sequence, so as to increase the penetration-promoting effect on skin and biofilm. (Designed peptide based on site and charge of aminoacid residue to act as a enhancement activity on transdermal and membraneous delivery)

<440>2

Ala Cys Ser Ser Ser Lys Ser Lys His Cys Gly

1 5 5 10

<210>3

<211>11

<212>PRT

<213> Synthetic sequence

<220>

<221> disulfide bond

<222>(2)...(10)

<223> The designed peptide is based on changing the position and charge of amino acid residues in the sequence, so as to increase the penetration-promoting effect on skin and biofilm. (Designed peptide based on site and charge of aminoacid residue to act as a enhancement activity on transdermal and membraneous delivery)

<440>3

Ala Cys Ser Ser Arg Pro Ser Lys His Cys Gly

1 5 5 10

<210>4

<211>11

<212>PRT

<213> Synthetic sequence

<220>

<221> disulfide bond

<222>(2)...(10)

<223> The designed peptide is based on changing the position and charge of amino acid residues in the sequence, so as to increase the penetration-promoting effect on skin and biofilm. (Designed peptide based on site and charge of aminoacid residue to act as a enhancement activity on transdermal and membraneous delivery)

<440>4

Ala Cys Ser Ser Ser Arg Ser Lys His Cys Gly

1 5 5 10

<210>5

<211>11

<212>PRT

<213> Synthetic sequence

<220>

<221> disulfide bond

<222>(2)...(10)

<223> The designed peptide is based on changing the position and charge of amino acid residues in the sequence, so as to increase the penetration-promoting effect on skin and biofilm. (Designed peptide based on site and charge of aminoacid residue to act as a enhancement activity on transdermal and membraneous delivery)

<440>5

Ala Cys Ser Ser Lys Lys Ser Lys His Cys Gly

1 5 5 10

<210>6

<211>11

<212>PRT

<213> Synthetic sequence

<220>

<221> disulfide bond

<222>(2)...(10)

<223>Designed peptide based on site and charge of aminoacid residue to act as a enhancement activity on transdermal and membraneous delivery)

<440>6

Ala Cys Ser Ser Arg Arg Ser Lys His Cys Gly

1 5 5 10

<210>7

<211>11

<212>PRT

<213> Synthetic sequence

<220>

<221> disulfide bond

<222>(2)...(10)

<223> The designed peptide is based on changing the position and charge of amino acid residues in the sequence, so as to increase the penetration-promoting effect on skin and biofilm. (Designed peptide based on site and charge of aminoacid residue to act as a enhancement activity on transdermal and membraneous delivery)

<440>7

Ala Cys Ser Ser Arg Lys Ser Lys His Cys Gly

1 5 5 10

<210>8

<211>11

<212>PRT

<213> Synthetic sequence

<220>

<221> disulfide bond

<222>(2)...(10)

<223> The designed peptide is based on changing the position and charge of amino acid residues in the sequence, so as to increase the penetration-promoting effect on skin and biofilm. (Designed peptide based on site and charge of aminoacid residue to act as a enhancement activity on transdermal and membraneous delivery)

<440>8

Ala Cys Ser Ser Lys Arg Ser Lys His Cys Gly

1 5 5 10

<210>9

<211>11

<212>PRT

<213> Synthetic sequence

<220>

<221> disulfide bond

<222>(2)...(10)

<223> The designed peptide is based on changing the position and charge of amino acid residues in the sequence, so as to increase the penetration-promoting effect on skin and biofilm. (Designed peptide based on site and charge of aminoacid residue to act as a enhancement activity on transdermal and membraneous delivery)

<440>9

Ala Cys Ser Ser Ser Ser Pro Ser Lys His Cys Gly-NH2

1 5 5 10

Claims (1)

1. A polypeptide that promotes percutaneous and transmembrane penetration, the polypeptide contains 11 amino acid residues, and consists of 9 amino acid residues to form a ring structure with disulfide bonds, characterized in that: in the cyclic peptide structure, N The N -5 and N -6 positions at the -terminal are composed of arginine residues, and the specific structure is

. 2. The application of the polypeptide with transdermal and transmembrane penetration promoting effect according to claim 1, characterized in that: the polypeptide is used to prepare solutions, gels and ointments for transdermal and transmembrane administration.