KR101327555B1 - Transdermal peptides traversing across hair follicles in the skin - Google Patents

Abstract

The present invention relates to a transdermal delivery peptide that penetrates the skin through pores, wherein the peptide ligand for perforated transdermal delivery identified by the present invention can be usefully used to promote the delivery of large drug molecules through the transdermal route. have.

Description

Transdermal peptides traversing across hair follicles in the skin}

The present invention relates to a transdermal delivery peptide that penetrates the skin through pores, and more specifically to SSHTISF, FPQPPQR, AFTPAKT and SPWIPAV peptides that can promote the delivery of large drug particles through the transdermal delivery mechanism through pores. It is about.

Transdermal Drug Delivery System (TDS) is one of the recent delivery methods of Drug Delivery System (DDS) due to local application, hepatic first-pass avoidance, continuous release of drug, and patient convenience. Prausnitz and Langer, 2008. Recently, many studies on enhancers such as liposomes or nanoparticles and peptide ligands for effectively delivering macromolecular drugs have been reported [Lademann et al. , 2007, Chen et al., 2006], several researchers believe that these promoters effectively deliver drugs through the skin appendages of skin tissue such as pores and sebaceous glands [Amoh et al., 2007, Toll et al., 2004, Vogt et al., 2006].

Phage display is one of the effective methods of identifying peptide ligands as a delivery medium for various DDS strategies. In particular, phage display is widely used in targeting and convenient delivery strategies. Chen et al., 2006, Kang et al., 2008, Wan et al., 2009.

In relation to transdermal delivery of drugs, Korean Patent No. 10-0062547 'transdermal administration composition of protein and peptide drugs' includes a solvent storage layer containing a solvent and a polyelectrolyte and peptide binding units of amino acids and three or more proteins and While the peptide drug is separated into a semipermeable membrane, the drug is ionized by an ionizing agent composed of a solvent, a polymer electrolyte, and an accelerator that exits the semipermeable membrane, and the ionized drug is discharged out of the administration device by an electrical force so that the skin The transdermal composition of protein and peptide drugs is characterized by transdermal administration of proteins and peptide drugs by electrical force using a patch-type administration device so that the ionized drug is percutaneously administered through the passage formed by saliva. Listed,

Korean Patent Application No. 10-2006-7022824 'transdermal delivery device and method of the influenza vaccine' includes a microprojection member having a plurality of microprojections for stratum corneum-piercing the biocompatible coating containing an immunological active agent disposed on top A system for transdermal delivery of an immunologically active agent is described,

Korean Patent Application No. 10-2006-7010339 'Delivery through the coated microprojection of the polymer conjugate of the therapeutic peptide and protein' is a microprojection member having a plurality of microprojections for piercing the stratum corneum with a biocompatible coating disposed thereon Wherein the biocompatible coating comprises a biologically active agent selected from the group consisting of peptide and protein conjugates, a device for transdermal delivery of a biologically active agent to a patient.

In addition, the Republic of Korea Patent Registration No. 10-0461458 'Multi-layer liposomes and its preparation method' has a particle size of 200 to 5000nm, it describes a transdermal absorption multi-layer liposomes and a method for preparing the same that can collect the bioactive active ingredient therein. It is.

However, none of the prior art has ever been suggested or described for transdermal delivery peptides that deliver drugs through pores as described herein.

Until recently, the transdermal drug delivery method has been largely an approach related to delivery through the stratum corneum, but this means that the deliverable drug must meet characteristics such as size (less than 1 kDa) or high hydrophobicity. Because of the problem, it was difficult to deliver large molecular weight drugs (protein, oligo-DNA / RNA, etc.) to the transdermal route. Recently, studies to improve the delivery efficiency through the skin tissue peripheral organs other than the stratum corneum by using the permeation accelerator has been actively reported, but there is a problem that there is a limitation in use depending on the size, nature, passive permeation of the accelerator.

Therefore, an object of the present invention is to identify and provide peptide ligands having high skin permeation efficiency through phage display.

The present invention consists in identifying efficient skin permeable peptide ligands through in vivo phage display screening and verifying the efficacy of the identified skin permeable peptide ligands.

More specifically, the specific steps of transdermal recombinant phage screening using the phage display technique of the present invention are as follows:

Random phage-peptide libraries are transdermally administered to rats. Preferably a Ph.D.-M13 phage-peptide library is used.

To collect the permeated phage, the heart blood is drawn after a certain time after skin smearing. In the case of abdominal smearing, the largest amount of phage is recovered from the heart blood after 30 to 60 minutes.

The harvested phage mixture is amplified through the infected host. Preferably, E. coli ER 2738 is used as the infecting host.

Repeated rounds (biopanning) are performed using the amplified phage. The preferred number of biopannings is three times.

The DNA and peptide sequences of the skin permeation phage recovered in the last round are identified. Four peptides identified in the present invention are ST1 peptide (SSHTISF: Ser-Ser-His-Thr-Ile-Ser-Phe), ST2 peptide (FPQPPQR: Phe-Pro-Gln-Pro-Pro-Gln-Arg), ST3 Peptides (AFTPAKT: Ala-Phe-Thr-Pro-Ala-Lys- Thr) and ST4 peptides (SPWIPAV: Ser-Pro-Trp-Ile-Pro-Ala-Val).

The transdermal efficacy of the peptide is confirmed by smearing the recombinant phage containing the peptide onto the skin, and using the real-time PCR quantitative method for transdermal phage recovered from blood collection. Phage specific primers available in real time PCR quantification are as follows:

In addition, the transdermal efficacy of the peptide is also confirmed in comparison with the transdermal efficacy of control phage. Preferably, the preferred control phage clones in the present invention are randomly selected from the Ph.D.-7 phage-peptide library, preferably 5'-TCTATTCTCACTCTGCGCCT-3 'as the specific primer.

In addition, the phage transmission pathway in the present invention is confirmed through immunofluorescence staining method.

The pore-permeable transdermal delivery peptide ligands identified by the present invention can be usefully used to facilitate the delivery of large drug molecules through the transdermal route.

1 is a schematic diagram showing a method for screening transdermal permeate phage using phage display technique.
In FIG. 2, A shows the amount of phage-peptide library and wild type M13 collected through blood over time, and B shows the amount of phage-peptide library and wild type M13 collected through blood per biopanning round.
Figure 3 shows the transdermal delivery peptides ST1, ST2, ST3 and ST4 selected through phage display and their respective phage identification primer sequences.
Figure 4 schematically shows a method for assaying the transdermal permeation efficiency of individual phage using real-time PCR quantification method after treatment of the mixed solution of phage.
5 is a graph showing the specificity and sensitivity of individual phage primer sets used in real time PCR quantification.
In Figure 6 A is a graph showing the transdermal permeation efficiency of each of the phage mixtures ST1, ST2, ST3 and ST4 phage per B, B is the percutaneous permeation efficiency of the phage mixture solution 30 minutes, 60 minutes, 90 minutes and 120 minutes reference C is a graph showing phage values counted through real-time PCR quantification and floc quantification.
In Figure 7 A is a graph comparing the transdermal penetration efficiency of ST3 phage and control C2 phage, B is the transdermal penetration of ST3 phage, control C2 phage and wild-type phage 30 minutes, 60 minutes, 120 minutes and 240 minutes after skin It is a graph showing the measurement results, C to F is an image showing that the phage was moved through the pores by using immunofluorescence staining method.

The present invention will be described in detail through the following examples. These examples are only for illustrating the invention, and the scope of the present invention is not limited thereto.

Example 1 Screening of Transdermal Delivery Peptides Using Phage Display

Rats (Sprague Dawley, 250 g, Samtako, South Korea) are anesthetized with tiletamine (25 mg / kg, Ip) and zolazepam (25 mg / kg, Ip). The abdomen was then depilated and the phage library was plated to collect recombinant phages labeled with transdermal delivery peptides by recovering phages that penetrated the skin through blood sampling from the heart (FIG. 1).

Phage library used for phage screening was used Ph.D-7 M13 phage library of New England Biolab (NEB), USA, the abdomen with 2x10 ¹¹ pfu / 200 ul PBS (pH 7.9) was removed by an electric hair removal machine Plates were applied to the skin (2 cm x 2 cm).

After smearing, 500 ul of blood was collected at 0, 15, 30, and 60 minutes through the heart, treated with the same amount of acidic buffer (0.1M glycine-HCl, pH 2.0) and centrifuged at 14,000 × g for 2 minutes. The supernatant was recovered and neutralized by treatment with 20 ul of 2M Tris-base, and the recovered samples were plated on LB / tetracyclin / IPTG / X-gal agar plates to quantify the number of phages per mL of blood (pfu). .

First, the optimal recovery time for recovering skin-permeated phages from the blood for optimal biopanning was investigated, and the recovered phages were most recovered at settling time between 30 and 60 minutes after abdominal smearing of the phage library ( 2A).

After 30 minutes and 60 minutes were amplified through the mix all of the recovered phage infected host is E. coli ER2738 (NEB, USA), the amplified phage is a total of three times, by treatment by the same amount each round by (2x10 ¹¹ pfu) repeated rounds (Biopanning) was performed. Skin permeation phage recovered in the final round of third screening was confirmed to be about 400-fold greater than recovered phage in experiments using the previous round and control (wild-type M13 phage; New England Biolab, USA) (FIG. 2B).

In order to identify peptide sequences of the recombinant phage in the recovered third round sample, DNAs of randomly selected individual phages were extracted and sequenced to confirm DNA sequences. As a sequencing primer, a 5'-CCCTCATAGTTAGCGTAACG-3 'primer (-96gIII reverse primer; Bioneer, Korea) for the -96th sequence from the peptide coding region-gIII of the recombinant phage was used. The DNA sequences encoding the identified peptides were translated into 7 amino acid sequences to finally identify 182 peptide sequence populations.

Example 2: Selection of Four Candidate Peptide Ligands by Primary Screening

The homology of amino acid sequences was confirmed using the alignment program Clustal X (Thomson et al., 1997) for 182 sequences identified after in vivo screening, among which the ST1 peptide appeared 16 times in the same manner. (SSHTISF), 9 appeared ST2 peptide (FPQPPQR) and ST3 peptide (AFTPAKT), and 8 appeared ST4 peptide (SPWIPAV) were selected according to the appearance frequency advantage (FIG. 3). In addition, phage specific primers were designed and synthesized (Bioneer, Republic of Korea) to effectively evaluate and evaluate the skin permeation efficacy by real-time PCR quantitation using recombinant phages that label each candidate peptide (FIG. 3). ). Each of the primers (forwards) has a specificity by including the DNA sequence of each peptide, the reverse primer was analyzed in common using -96gIII primer (Fig. 3).

Example 3: Comparative evaluation between efficacy evaluation and quantification method of recombinant phage through real-time PCR quantification

To investigate the sensitivity and selectivity of the designed phage specific primers, each of the four phage (1x10 ⁶ pfu / ul) DNA prepared after independent amplification, each 5 pmol forward / reverse primer, 1 U Taq polymerase / PCR buffer / 10mM dNTP (NEB, USA), mixed with ddH ₂ O to a final volume of 25 ul 95 ° C 5 minutes (one cycle) / 95 ° C 30 seconds-50 ° C 30 seconds-72 ° C 60 seconds (35 cycles) PCR analysis was performed using a temperature condition of / 72 ° C for 7 minutes (one cycle). Through agarose gel electrophoresis after analysis, as a result, it was confirmed that the primer specific for one phage has no amplification effect on the other three candidate phage (Fig. 4A).

Next, in real-time PCR (i-cycler; Bio-rad, USA) analysis, it was tested whether each primer set can specifically amplify the phage. Total genomic DNA, including phage DNA, was extracted from the blood using the DNeasy kit (Qiagen, USA), 5ul of extracted DNA sample, 10ul of Bio-Rad SYBR pre-mixture (Bio-Rad, USA), and ddH ₂ O. The analysis was performed with the final 20 ul sample volume included. As a result, as shown in Figure 4B, the primer set of phage was confirmed by melting curve analysis (melting curve) that can specifically amplify each phage DNA. Also derived a correlation graph for gripping can values (copy number) and the C _T (threshold cycle) accordingly in order to quantify the amount of phage by real-time PCR (Figure 4C). This result was obtained by preparing each phage in 10 ² ~ 10 ⁶ copy numbers / 5ul and performing a real-time PCR under the above conditions to measure the C _T value for each diluent. Correlation graphs were used as a standard curve for the quantification of phage DNA in real-time PCR quantitation using samples containing four different phages.

Thereafter, as shown in FIG. 5, the recombinant phage candidates selected through primary screening were mixed together in equal amounts (1 × ^{10 11} pfu), and smeared onto the abdominal hair of the epilated rats, and the phages that penetrated the skin were collected through hourly blood collection. Hourly phage recovery, DNA extraction and real-time PCR analysis methods are the same as described in Examples 1 and 3. When the permeation efficiency was compared using the real-time PCR quantification method to evaluate the skin permeation efficiency of each recovered phage, the ST3 phage labeling the AFTPAKT peptide ligand (ST3) among the four recombinant phages had the highest skin permeation efficiency. (* p <0.05) was confirmed (FIG. 6A). Phage counts in blood samples were quantified by conventional plaque titration methods to compare the efficiency of candidate and control phages with quantitative analysis of each phage through real-time PCR as in FIG. 6A. As a result, the skin permeation of the mixed candidate phage was significantly lower than that of the control phage per hour when the control phage equivalent (C2 phage; 4x10 ¹¹ pfu) was performed (* p <0.05, **). p <0.01, *** p <0.001) were confirmed (FIG. 6B). In addition, to compare the accuracy of real-time PCR quantification, total phages per ml of blood performed by real-time PCR quantification and plaque titration were compared. By counting it was possible to compare and confirm the accuracy of the quantification through the method (Fig. 6C).

Example 4: Reassessment of Percutaneous Delivery Effect of Phage by ST3 Peptide and Confirmation of Permeation Pathway

The final selected ST3 phage was found to be significantly higher than the control phage (** p <0.01) even when mixed with control C2 phage (1x10 ¹¹ pfu) (FIG. 7A), ST3 phage, control C2 phage , Or when wild type M13 phage was treated with different experimental animals individually, the skin permeation efficiency of ST3 phage was also the highest (* p <0.05, *** p <0.001). . Experiments with separate treatments were performed by quantifying each phage (1 × ^{10 11} pfu) on blood anesthetized and epilated rat abdomen, then recovering phage from blood at 30/60/120/240 min and then quantifying by floc quantification. This confirmed that the identified ST3 peptide has the ability to permeate the phage particles continuously as macromolecules for up to 4 hours.

In addition, immunofluorescence staining was performed to confirm the permeation pathway of phage. ST3 phage and C2 control phage were plated separately and skin tissue of the plated area was taken from the anesthetized rats after 1 hour. The tissues were washed three times with 4 ° C. PBS (pH 7.4) and then fixed in 4% paraformaldehyde solution at 4 ° C. for 2 hours. After fixing, the cells were washed three times with cold PBS, and then the tissues were placed in 30% sucrose solution and incubated overnight at 4 ° C. The tissue was then choked to an appropriate size and placed in an Optical Cutting Temperature (OCT) solution and rapidly frozen at -70 ° C. The frozen tissue block was then frozen using a cryographer (Cryotome HM 505E; Microm, Germany). Section to a thickness of 10 um. Tissue sections were blocked in PBS solution containing 10% FBS, 20 mM HEPES for 30 minutes and then treated with mouse anti-M13 g8p antibody (Abcam, UK) for 1 hour. After washing the PBS three times for 5 minutes after the first antibody treatment, the goat anti-mouse IgG antibody (Molecular Probes, USA) labeled with alexa-488 was treated as a secondary antibody for 1 hour, and the phage was stained with fluorescent green color. Finally, the nuclei were stained blue using DAPI reagent. As a result, it was visually confirmed that the ST3 peptide ligand could deliver its phage to the blood through the hair follicle (ST3 phage; Figures 7C and 7D, control C2 phage; Figures 7E and 7F).

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Claims (2)

Peptide sequence Peptides for pore penetration or transdermal delivery characterized by an amino acid sequence selected from the group consisting of FPQPPQR, AFTPAKT and SPWIPAV. delete

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