Designed and engineered skin-penetrating peptide and use thereof

Abstract

Provided are a designed and engineered skin-penetrating peptide with improved skin penetrability, a conjugate of the designed and engineered skin-penetrating peptide and a bioactive molecule, and a composition containing the designed and engineered skin-penetrating peptide and the bioactive molecule. Further provided are a method for determining the designed and engineered skin-penetrating peptide with improved skin penetrability; and the use of the designed and engineered skin-penetrating peptide for delivering the bioactive molecule through intact mammalian skin, thereby enhancing the efficacy and / or reducing the dosage of the bioactive molecule.

Description

Designed and engineered skin-penetrating peptides and uses thereof TECHNICAL FIELD

[0001] The present invention relates to a drug delivery vehicle for dermal drug delivery, a polypeptide, and drug delivery technology based on the polypeptide. In particular, the present invention relates to designed and engineered skin-penetrating peptides (e.g., Penetratin-derived peptides) that facilitate transdermal absorption for the delivery of biologically active molecules across intact mammalian skin. BACKGROUND

[0002] Transdermal drug delivery has many unique advantages, such as avoiding the first-pass effect, slow and sustained release of drugs, stable blood drug concentration in vivo, improved patient compliance, direct action on the lesion site near the skin, etc., and plays a very important role in various drug delivery routes.

[0003] Skin acts as a natural protective barrier to prevent pathogens and toxins from entering, but at the same time limits the delivery of drugs into the body. The low permeability of the skin hinders the administration of most drugs through the skin. The prior art has studied polypeptides with skin-penetrating potential, mainly including three categories, cell-penetrating peptides (CPPs), antimicrobial peptides (AMPs), and phage peptides.

[0004] CPP is a short peptide with positive charge under physiological conditions, usually composed of 5-30 amino acids, which can be used as a delivery carrier to facilitate the crossing of diagnostic or therapeutic drugs (such as small molecules, nucleic acids, proteins, viruses, etc.) through physiological barriers.

[0005] AMPs can form pores on bacterial cell membranes and increase skin permeability by disrupting the lipid structure of the stratum corneum.

[0006] Phage peptides are obtained through phage display technology, and these polypeptides are randomly displayed on each phage particle, forming a polypeptide library that can be used to screen skin penetration enhancers.

[0007] Using polypeptides to facilitate transdermal drug delivery as a simple and effective non-invasive drug delivery strategy has the advantages of high application value and wide application range. Compared with more complex active transdermal delivery methods (such as iontophoresis and ultrasound-mediated delivery), it significantly improves the possibility of clinical translation, and compared with invasive transdermal delivery strategies (such as injection and microneedles), it improves patient compliance.

[0008] Although the prior art reports the use of a cell-penetrating peptide to promote absorption of transdermal administration, there is still a need in the art for a more efficient and safe cell-penetrating peptide, and it is desirable that a drug delivery system based on the cell-penetrating peptide has higher delivery efficiency, while having excellent biodegradability and biocompatibility, and can avoid the safety problems associated with traditional chemical absorption promoters (such as oleic acid and lauric acid nitrogen

[0009] SUMMARY

[0010] The present inventors, through research, have developed new designed and engineered skin-penetrating peptides, such as Transportan-derived peptides and / or Penetratin-derived peptides, and provided a method for determining the skin-penetrating property of the designed and engineered skin-penetrating peptides, such as Transportan-derived peptides and / or Penetratin-derived peptides.

[0011] The present invention compares, analyzes and predicts the effect of polypeptide transdermal absorption by means of computer-aided design technology, determines the key factors that determine the efficiency of polypeptide transdermal delivery from the perspective of polypeptide structure and physicochemical properties, and finds the internal rules of polypeptide promotion of drug transdermal delivery. On this basis, new designed and engineered skin-penetrating peptides, such as Transportan-derived peptides and / or Penetratin-derived peptides, are obtained, and are verified by applying them to transdermal administration for treating melanoma.

[0012] Therefore, in a first aspect, the present invention provides a designed and engineered skin-penetrating peptide, which is a Transportan-derived peptide and / or a Penetratin-derived peptide, such as a Penetratin-derived peptide having a sequence as shown in any one of SEQ ID NO:21-SEQ ID NO:517.

[0013] In a preferred embodiment, the Penetratin-derived peptide of the present invention has a sequence as shown in SEQ ID NO:236 or SEQ ID NO:404.

[0014] In a second aspect, the present invention provides a method for determining a designed and engineered skin-penetrating peptide that improves skin-penetrating property, comprising

[0015] determining the cationic surface patch area (PSPA) and the hydrophobic surface patch area (HSPA) of the modified skin-penetrating peptide;

[0016] scoring the amphiphilicity of the polypeptide using the following equation:

[0017] Amphiphilicity score (SOA) = cationic surface patch area (PSPA) + 1 / 3 hydrophobic surface patch area (HSPA);

[0018] If the amphiphilicity score value is greater than the SOA value of a wild-type skin- penetrating peptide (e.g., a wild-type skin-penetrating peptide that has not been engineered), then the engineered skin-penetrating peptide is determined to have improved skin penetration.

[0019] In one embodiment, the present application provides a method of determining a Transportan-derived peptide having improved skin penetration, comprising

[0020] determining the cationic surface patch area (PSPA) and the hydrophobic surface patch area (HSPA) of the Transportan-derived peptide;

[0021] scoring the amphiphilicity of the polypeptide using the following equation:

[0022] Amphiphilicity score (SOA) = cationic surface patch area (PSPA) + 1 / 3 hydrophobic surface patch area (HSPA);

[0023] If the amphiphilicity score value is greater than the SOA value of a wild-type Transportan (e.g., a Transportan having the amino acid sequence set forth in SEQ ID NO: 20), then the Transportan-derived peptide is determined to have improved skin penetration.

[0024] In one embodiment, the present application provides a method of determining a Penetratin-derived peptide having improved skin penetration, comprising

[0025] determining the cationic surface patch area (PSPA) and the hydrophobic surface patch area (HSPA) of the Penetratin-derived peptide;

[0026] scoring the amphiphilicity of the polypeptide using the following equation:

[0027] Amphiphilicity score (SOA) = cationic surface patch area (PSPA) + 1 / 3 hydrophobic surface patch area (HSPA);

[0028] If the amphiphilicity score value is greater than the SOA value of a wild-type Penetratin (e.g., a Penetratin having the amino acid sequence set forth in SEQ ID NO: 19), then the Penetratin-derived peptide is determined to have improved skin penetration.

[0029] In some embodiments, the engineered skin-penetrating peptide in the methods of the present application is an engineered skin-penetrating peptide that has been altered, e.g., one or more amino acid residues, e.g., single, double, triple, four, five or more amino acid residue substitutions, deletions and / or additions, from the amino acid sequence of a wild-type skin-penetrating peptide.

[0030] In some embodiments, the Penetratin derivative peptide in the methods of the present application is a derivative peptide obtained by altering, e.g., replacing, deleting and / or adding one or more amino acid residues, e.g., a single, double, triple, four, five or more amino acid residue replacement, to the amino acid sequence of wild-type Penetratin.

[0031] In some embodiments, the Penetratin derivative peptide in the methods of the present application is a derivative peptide obtained by altering, e.g., replacing, deleting and / or adding one or more amino acid residues, e.g., a single, double, triple, four, five or more amino acid residue replacement, to the amino acid sequence of wild-type Penetratin.

[0032] In some embodiments, the Penetratin derivative peptide in the methods of the present application is a derivative peptide obtained by a triple amino acid residue mutation to wild-type Penetratin, e.g., the amino acid sequence of the wild-type Penetratin is set forth in SEQ ID NO: 19.

[0033] In some embodiments, the present application relates to a designed and engineered skin-penetrating peptide, e.g., a Transportan derivative peptide and / or a Penetratin derivative peptide, with improved skin penetration determined according to the methods of the present application.

[0034] In a third aspect, the present application provides a conjugate of a designed and engineered skin-penetrating peptide of the present application and a biologically active molecule, wherein the conjugate has improved skin penetration ability.

[0035] In some embodiments, the present application provides a conjugate of a Transportan derivative peptide of the present application and a biologically active molecule, wherein the conjugate has improved skin penetration ability.

[0036] In some embodiments, the present application provides a conjugate of a Penetratin derivative peptide of the present application and a biologically active molecule, wherein the conjugate has improved skin penetration ability. In some embodiments, the designed and engineered skin-penetrating peptide of the present application is directly conjugated or indirectly conjugated to the biologically active molecule via a linker.

[0037] In some embodiments, the Transportan derivative peptide of the present application is directly conjugated or indirectly conjugated to the biologically active molecule via a linker.

[0038] In some embodiments, the Penetratin-derived peptide of the present application is directly conjugated or indirectly conjugated via a linker to a biologically active molecule.

[0039] In some embodiments, the biologically active molecule in the conjugate of the present application is selected from at least one of a polypeptide, a protein, a nucleic acid, a lipid, a polysaccharide, a small molecule compound, for example, a chemotherapeutic agent.

[0040] In a fourth aspect, the present application provides a composition comprising component i) a designed and engineered skin-penetrating peptide of the present application; and component ii) a biologically active molecule, for example, the composition comprises the components i) and ii) in non-covalent combination.

[0041] In some embodiments, the present application provides a composition comprising component i) a Transportan-derived peptide of the present application; and component ii) a biologically active molecule, for example, the composition comprises the components i) and ii) in non-covalent combination.

[0042] In some embodiments, the present application provides a composition comprising component i) a Penetratin-derived peptide of the present application; and component ii) a biologically active molecule, for example, the composition comprises the components i) and ii) in non-covalent combination. In some embodiments, the biologically active molecule in the composition of the present application is selected from at least one of a polypeptide, a protein, a nucleic acid, a lipid, a polysaccharide, a small molecule compound, for example, a chemotherapeutic agent.

[0043] In a fifth aspect, the present application provides use of a designed and engineered skin-penetrating peptide of the present application for the manufacture of a skin-penetration enhancer for facilitating penetration of a biologically active molecule through intact skin of a mammal, preferably, increasing efficacy and / or reducing dosage of the biologically active molecule.

[0044] In some embodiments, the present application provides use of a Transportan-derived peptide of the present application for the manufacture of a skin-penetration enhancer for facilitating penetration of a biologically active molecule through intact skin of a mammal, preferably, increasing efficacy and / or reducing dosage of the biologically active molecule.

[0045] In some embodiments, the present application provides use of a Penetratin-derived peptide of the present application for the manufacture of a skin-penetration enhancer for facilitating penetration of a biologically active molecule through intact skin of a mammal, preferably, increasing efficacy and / or reducing dosage of the biologically active molecule.

[0046] In some embodiments, the designed and engineered skin-penetrating peptides of the present application (e.g., Transportan-derived peptides and / or Penetratin-derived peptides) are used to facilitate penetration of a chemotherapeutic agent, such as 5-fluoro-2'-deoxyuridine, through intact skin of a mammal, preferably, to increase efficacy and / or to reduce dosage of the chemotherapeutic agent, such as 5-fluoro-2'-deoxyuridine. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 shows the permeability of free FAM or FAM conjugates with each of the polypeptides shown in SEQ ID NOs: 1-20 to rat ex vivo skin and the corresponding apparent permeability coefficients (Papp). app Figure 1, panel a, shows the cumulative permeation amount-time curves of free FAM (abbreviated as "FAM" in the figure) and each of the FAM-polypeptide conjugates (abbreviated as the polypeptide name in Table 1, respectively) to rat dorsal skin. Figure 1, panel b, shows the apparent permeability coefficients of free FAM and each of the FAM-polypeptide conjugates to rat dorsal skin. All polypeptides were conjugated with FAM at their N-termini, and the cumulative permeation amount of the conjugates was measured by fluorescence intensity. The donor pool contained 50 μΜ of FAM or FAM-polypeptide conjugates.

[0048] Figure 2 shows the distribution and penetration depth of free FAM and FAM conjugates with each of the polypeptides in Table 1 in ex vivo skin. DAPI-stained skin frozen sections administered with free FAM (abbreviated as "FAM" in the figure) or each of the FAM-polypeptide conjugates (abbreviated as the polypeptide name in Table 1, respectively) were displayed using separate DAPI images and FAM-brightfield overlay images (scale bar = 500 μιη), and the overlay process was accomplished by ImageJ software. The average FAM fluorescence intensity along the white arrow was displayed on the fluorescence intensity- transdermal penetration depth (i.e., distance) curve on the right side of Figure 2 (maximum fluorescence intensity value: 255).

[0049] Figure 3 shows the skin distribution and penetration depth of free FAM (abbreviated as "FAM" in the figure) and FAM-polypeptide conjugates, where panel a is the distribution and penetration depth results of free FAM and the polypeptides of SEQ ID NO: 8 conjugated with FAM (abbreviated as "TAT" in the figure), the polypeptide of SEQ ID NO: 19 conjugated with FAM (abbreviated as "Penetratin" in the figure), and the polypeptide of SEQ ID NO: 20 conjugated with FAM (abbreviated as "Transportan" in the figure) in ex vivo skin; and panel b is the fluorescence distribution results of free FAM and FAM conjugates with each of the polypeptides in Table 1 within 200 μιη depth of skin.

[0050] Figure 4 shows the cytotoxicity of 50 μΜ free FAM (abbreviated as "FAM" in the figure) and conjugates of FAM and each polypeptide in Table 1 (abbreviated as each polypeptide name in Table 1 in the figure) to HeKa cells.

[0051] Figure 5 shows the polypeptide P app values of all polypeptides P app values of all polypeptides P app values of all polypeptides P app values of all polypeptides P app values of all polypeptides P app values of all polypeptides P app values of all polypeptides P app values of all polypeptides P. All correlation analyses were based on Pearson correlation coefficient, and p-test was performed using two-tailed test.

[0052] Figure 6 shows the prediction of transdermal efficiency of polypeptides based on single-factor fitting of PSPA, wherein, panel a: the effect of single amino acid mutation of wild-type Penetratin on PSPA, the abscissa is the amino acid residue after mutation of each site of wild-type Penetratin. Panel b: the effect of three amino acid mutations of wild-type Penetratin on PSPA, the abscissa is the amino acid residue after three mutations of wild-type Penetratin. Panel c: the measured P 2R8R9K values of wild-type Penetratin (abbreviated as "WILD" in the figure), polypeptide 3R5R9R Penetratin (abbreviated as "28R9K") and app Penetratin (abbreviated as "359R") in the figure). Panel d: the calculated surface patch area distribution of wild-type Penetratin and polypeptides 2R8R9K Penetratin, 3R5R9R Penetratin. The calculation of PSPA and surface patch area distribution was completed by Maestro software .

[0053] Figure 7 shows a two-factor fitting model based on amphiphilicity score optimization, wherein four derivatives of the wild-type polypeptide Penetratin, as set forth in SEQ ID NO: 19 (also referred to as “19” in the figure), were synthesized, namely derivative 1 (also referred to as derivative peptide “19*1” in the figure) with 2W8W two amino acid residue substitutions, derivative 2 (also referred to as derivative peptide “19*2” in the figure) with 2W9W two amino acid residue substitutions, derivative 3 (also referred to as derivative peptide “19*3” in the figure) with 8W9W two amino acid residue substitutions, and derivative 4 (also referred to as derivative peptide “19*4” in the figure) with 2W8W9W three amino acid residue substitutions, to verify the fitting model. Panel a: correlation of P app values of wild-type Penetratin and its derivatives with PSPA in rat skin and pig skin; panel b: correlation of P app values of wild-type Penetratin and its derivatives with SOA in rat skin and pig skin. Panel c: predicted folding structure of wild-type Penetratin and its derivative peptides. Panel d: surface patch distribution of wild-type polypeptide Penetratin and derivatives 1-4 from left to right (blue = cationic surface patch, green = hydrophobic surface patch, pink = anionic surface patch). Panel e: properties of wild-type Penetratin and its derivative peptides, including amphiphilicity score (SOA), Molinspiration partition coefficient (Milog P ), charge, rat skin and pig skin permeability (P app rat skin and P app pig skin). Panels c-d were generated using AlphaFold 2 algorithm and Maestro software, respectively.

[0054] Figure 8 shows polypeptide transdermal efficiency prediction based on two-factor fitting of SOA score, wherein panel a: effect of single amino acid mutation on PSPA, with the top five single amino acid mutations affecting PSPA labeled on the right. Panel b: effect of single amino acid mutation on HSPA, with the top five single amino acid mutations affecting HSPA labeled on the right. Panel c: effect of double amino acid mutation on SOA. Panel d: effect of triple amino acid mutation on SOA. Calculation of PSPA and HSPA was performed using Maestro software. Each point in panels c-d represents a polypeptide sequence.

[0055] Figure 9 shows the structure of wild-type Penetratin and its derivative peptides, wherein panel a: cumulative permeation amount-time curves of wild-type Penetratin and its derivative peptides in rat skin in vitro. Panel b: cumulative permeation amount-time curves of wild-type Penetratin and its derivative peptides in pig skin in vitro. Panel c: correlation of SOA values of wild-type Penetratin and its derivative peptides with P app values in rat skin and pig skin. Panel d: fluorescence intensity of Cy5 gel and Cy5-589WP gel at 0 hour, 4 hours or 8 hours after application to the back of a mouse; panel e: comparison bar chart of fluorescence intensity of Cy5 gel and Cy5-589WP gel at 4 hours and 8 hours after application to the back of a mouse; panel f: polypeptide molecular surface patch area distribution (blue = cationic surface patch, green = hydrophobic surface patch, pink = anionic surface patch).

[0056] Figure 10 shows the synthesis route, in vitro cytotoxicity and drug release of FUDR-589WP, wherein panel a: ① esterification reaction catalyzed by DCC / DMAP. ② click chemistry reaction between thiol group of 589WP (Cys-589WP) and maleimide of FUDR-Mal. Panel b: cell survival rate of B16F10 cells after 48 hours of co-incubation with 589WP, FUDR, FUDR-Mal and FUDR-589WP. Panel c: IC 50 values of B16F10 cells after 48 hours of co-incubation with 589WP, FUDR, FUDR-MAL and FUDR-589WP. Panel d: B16F10 cells calcein / propidium iodide double staining photos after 48 hours of treatment of B16F10 cells with 15 μΜ 589WP, FUDR, FUDR-MAL and FUDR-589WP. Panel e: cumulative release time curve of FUDR under different pH conditions. Panel f: cumulative release time curve of FUDR under different concentrations of porcine liver esterase (PLE) at pH = 7.4.

[0057] Figure 11 shows anti-melanoma activity and safety evaluation in mice. Panel a: Tumor volume-time curve of blank (vehicle) gel, 0.5% FUDR gel, 2.5% FUDR gel, 0.5% FUDR + 589WP gel, 0.05% FUDR-589WP gel, 0.5% FUDR-589WP gel. Panel b: Tumor volume-time curve comparison of blank (vehicle) gel, 0.5% FUDR gel, 2.5% FUDR gel, 0.5% FUDR + 589WP gel, 0.05% FUDR-589WP gel, 0.5% FUDR-589WP gel. Panel c: Isolated melanoma tissue. Panel d: Skin HE staining (Scale bar = 100 / 20 pm). From day 0 to day 21, C57BL / 6 mice bearing melanoma were topically treated with the gels once a day. Blank gel was used as negative control (n=8). The gel base consisted of 50% glycerol, 30% PEG 200 and 20% PEG 400 (mass ratio). The skin used to evaluate drug safety was selected from the area of skin above the melanoma. 3 The drug was applied topically on the skin above the melanoma of C57BL / 6 mice once a day starting from day 0. Blank gel was used as negative control (n=8). The gel base consisted of 50% glycerol, 30% PEG 200 and 20% PEG 400 (mass ratio). The skin used to evaluate drug safety was selected from the area of skin above the melanoma.

[0058] Figure 12 shows histopathology images of HE staining of skin overlying melanoma.

[0059] Figure 13 shows histopathology images of HE staining of major organs (heart, liver, spleen, lung, kidney).

[0060] DETAILED DESCRIPTION

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented. Further, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the application will be apparent from the description and drawings, and from the claims.

[0062] I. DEFINITIONS

[0063] As used herein, the term “subject” refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., swine, cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the subject is a human.

[0064] Various features of the drug delivery vehicle of the present application are discussed below.

[0065] As used herein, the term "about" is intended to designate an adjustment of ±10% of the specified value. For example, the term "about 5%" is intended to include a range of 4.5% to 5.5%.

[0066] As used herein, the term "comprising" or "including," means including the recited elements, integers or steps, but does not exclude any other elements, integers or steps.

[0067] The term "cell-penetrating peptide (CPP)" is a class of peptides with cell-penetrating ability, also known as cell transduction domain (CTD). CPPs can serve as delivery vehicles for bioactive molecules. Bioactive molecules can be classified as therapeutic drugs and diagnostic reagents according to their use. There is no particular limitation on the bioactive molecules that CPPs can deliver, including but not limited to peptides, DNA, siRNA, and small molecule drugs. The binding between CPPs and the delivered bioactive molecules can be non-covalent, such as electrostatic and hydrophobic interactions, or covalent.

[0068] The term "wild-type Penetratin" is a naturally occurring CPP derived from a fruit fly DNA binding protein with the ability to translocate across neuronal membranes. In some embodiments, the amino acid sequence of wild-type Penetratin is set forth in SEQ ID NO: 19.

[0069] The term "Penetratin-derived peptide" is a peptide obtained by amino acid modification or mutation of wild-type Penetratin. Wild-type Penetratin protein can be modified to include non-naturally occurring amino acid residues, such as α-aminobutyric acid, α-aminopentanoic acid, α-aminohexanoic acid, α-aminopentanoic acid. Techniques for adding naturally occurring amino acid residues and non-naturally occurring amino acid residues are well known to those of ordinary skill in the art.

[0070] Amino acid mutations can be amino acid substitutions, deletions, and additions. In some embodiments, the amino acid mutation is a substitution of one or more amino acids, for example, a single amino acid substitution or a combination of multiple amino acid substitutions. Amino acid deletions include deletions at the amino and / or carboxyl terminus of the wild-type Penetratin polypeptide sequence, as well as deletions within the wild-type Penetratin polypeptide sequence. Amino acid additions include additions at the amino and / or carboxyl terminus of the wild-type Penetratin polypeptide sequence, as well as additions within the wild-type Penetratin polypeptide sequence.

[0071] Amino acid substitutions referred to herein can be described in the following manner: single amino acid substitution: (position of original amino acid residue / replaced amino acid residue). For example, substitution of isoleucine (I) at position 5 of wild-type Penetratin with tryptophan (W) can be denoted as 5W. For cases of multiple amino acid substitutions, amino acid substitutions referred to herein can be denoted as a combination mutation at multiple given positions. For example, a Penetratin-derived peptide resulting from a 3M, 8W, and 9W combination mutation on wild-type Penetratin numbered according to SEQ ID NO: 19 can be denoted as: 3M8W9W; 3M89W; 3M89WP; or 3M8W9W Penetratin.

[0072] When referring to the amino acid residue positions of wild-type Penetratin, the amino acid sequence of wild-type Penetratin protein shown by reference to SEQ ID NO: 19 is used for determination. In the present application, unless otherwise specified, the amino acid positions of wild-type Penetratin polypeptide are the amino acid positions numbered according to SEQ ID NO: 19.

[0073] The term "amphiphilic" refers to a compound molecule containing both hydrophilic polar groups and hydrophobic non-polar groups. The increase in hydrophobicity of a polypeptide molecule can be achieved by introducing hydrophobic amino acid residues (e.g. leucine, isoleucine, alanine, valine, phenylalanine). It has been reported that the activity of a cell-penetrating peptide was increased by three-fold after replacing a methyl group with a more hydrophobic butyl group (Som, A. et al. Self-activation in de novo designed mimics of cell-penetrating peptides. Angew. Chem. Int., 2011, 50, 6147-6150).

[0074] The term "surface patch" is defined by a target residue and its spatially neighboring residues falling within a virtual sphere of diameter d centered on the target residue.

[0075] The term "cationic surface patch area (PSPA)" refers to the area of cationic patches exposed on the surface of a protein.

[0076] The term "hydrophobic surface patch area (HSPA)" refers to the area of hydrophobic patches exposed on the surface of a protein.

[0077] II. Methods of determining designed and engineered skin-penetrating peptides with improved skin-penetrating properties

[0078] Transdermal drug delivery systems (TDDS) have many advantages, including avoiding the first-pass effect, having sustained-release characteristics, being convenient and safe to use, and improving patient compliance, all of which are extremely important in medical practice. However, the skin, as the body's natural protective barrier, has low permeability, limiting the entry of exogenous substances and hindering the absorption of transdermal drug delivery. The main permeability barrier of the skin is the stratum corneum (SC), which is composed of tightly arranged and highly keratinized dead cells embedded in a lipid matrix. Therefore, drugs must pass through the complex lipid pathway between keratinocytes (intercellular pathway), or repeatedly distribute between cells and lipid matrix (intracellular pathway), or pass through skin appendages such as hair follicles to penetrate the stratum corneum. In general, methods to promote transdermal drug absorption can be divided into two categories: active or passive methods. Active delivery methods include iontophoresis, sonophoresis, electroporation, microneedles, etc., while passive methods include absorption enhancers, liposomes, micelles, nanoparticles, microemulsions, vesicles, etc.

[0079] In the field of transdermal drug delivery systems, the mechanisms of skin penetration of polypeptides have not been fully elucidated due to differences in the physicochemical properties of different polypeptides. These specific mechanisms include interactions between polypeptides and keratins, enhanced lipid fluidity, peptide transduction domains, amphiphilicity, and reversible regulation of intercellular junction proteins, etc. In these studies, polypeptides are used as absorption enhancers for biologically active molecules, mainly through covalent binding with drugs, while in some cases they are simply physically mixed with drugs (non-covalent binding). This method has been shown to increase the rate of transdermal drug absorption by several times, and in some cases even more than ten times. However, due to the lack of systematic comparison, optimization, and lack of theoretical guidance, the design of polypeptides as efficient transdermal absorption enhancers mainly relies on trial and error through in vitro skin penetration experiments, and there is a lack of reliable prediction methods. At the same time, there is still much room for improvement in the efficiency of transdermal drug delivery.

[0080] The present application provides a method for determining a designed and engineered skin-penetrating peptide with improved skin penetration, comprising the following steps:

[0081] - mutating or modifying one or more amino acid residues of a wild-type skin-penetrating peptide to obtain a designed and engineered skin-penetrating peptide;

[0082] - determining the positive ion surface patch area (PSPA) and the hydrophobic surface patch area (HSPA) of the designed and engineered skin-penetrating peptide;

[0083] - scoring the amphiphilicity of the designed and engineered skin-penetrating peptide using the following equation:

[0084] Amphiphilicity score (SOA) = cationic surface patch area (PSPA) + 1 / 3 hydrophobic surface patch area (HSPA);

[0085] - if the amphiphilicity score value is greater than the SOA value of the wild-type Penetratin, then the Penetratin derivative peptide is determined to have improved skin penetration;

[0086] - if the amphiphilicity score value is less than or equal to the SOA value of the wild-type Penetratin, then the Penetratin derivative peptide is determined to not have improved skin penetration.

[0087] In some embodiments, the present application provides a method of determining a Penetratin derivative peptide with improved skin penetration, comprising the steps of:

[0088] - mutating or modifying one or more amino acid residues of the wild-type Penetratin to obtain a Penetratin derivative peptide;

[0089] - determining the cationic surface patch area (PSPA) and the hydrophobic surface patch area (HSPA) of the Penetratin derivative peptide;

[0090] - scoring the amphiphilicity of the Penetratin derivative peptide using the following equation:

[0091] Amphiphilicity score (SOA) = cationic surface patch area (PSPA) + 1 / 3 hydrophobic surface patch area (HSPA);

[0092] - if the amphiphilicity score value is greater than the SOA value of the wild-type Penetratin, then the Penetratin derivative peptide is determined to have improved skin penetration;

[0093] - if the amphiphilicity score value is less than or equal to the SOA value of the wild-type Penetratin, then the Penetratin derivative peptide is determined to not have improved skin penetration.

[0094] In some embodiments, the present application provides a method of determining a Penetratin derivative peptide with improved skin penetration, comprising the steps of:

[0095] - mutating or modifying one or more amino acid residues of the wild-type Penetratin to obtain a Penetratin derivative peptide;

[0096] - determining the cationic surface patch area (PSPA) and the hydrophobic surface patch area (HSPA) of the Penetratin derivative peptide;

[0097] - amphipathicity of the Penetratin derivative peptide is scored using the following equation:

[0098] Amphipathicity Score (SOA) = Cationic Surface Patch Area (PSPA) + 1 / 3 Hydrophobic Surface Patch Area (HSPA);

[0099] - if the amphipathicity score value is greater than the SOA value of wild-type Penetratin, then the Penetratin derivative peptide is determined to have improved skin penetration;

[0100] if the amphipathicity score value is less than or equal to the SOA value of wild-type Penetratin, then the Penetratin derivative peptide is determined to not have improved skin penetration.

[0101] In some embodiments, the designing and engineering of the amino acid residues of the wild-type skin-penetrating peptide includes, but is not limited to, substitution, deletion, and / or addition of one or more amino acid residues to the wild-type skin-penetrating peptide.

[0102] In some embodiments, the designing and engineering of the amino acid residues of the wild-type Transportan includes, but is not limited to, substitution, deletion, and / or addition of one or more amino acid residues to the wild-type Transportan. In some embodiments, the amino acid sequence of the wild-type Transportan is set forth in SEQ ID NO: 20. In some embodiments, the amino acid sequence of the wild-type Transportan is substituted with single, double, triple, quadruple, quintuple, or more amino acid residues.

[0103] In some embodiments, the designing and engineering of the amino acid residues of the wild-type Penetratin includes, but is not limited to, substitution, deletion, and / or addition of one or more amino acid residues to the wild-type Penetratin. In one embodiment, the amino acid sequence of the wild-type Penetratin is set forth in SEQ ID NO: 19. In some embodiments, the amino acid sequence of the wild-type Penetratin is substituted with single, double, triple, quadruple, quintuple, or more amino acid residues.

[0104] In some embodiments, the PSPA and HSPA are obtained as follows: first, the folded conformation of the designed and engineered skin-penetrating peptide (e.g., a Transportan derivative peptide or a Penetratin derivative peptide) is obtained, and then the surface properties of the folded conformation of the designed and engineered skin-penetrating peptide (e.g., a Transportan derivative peptide or a Penetratin derivative peptide) are calculated, i.e., the surface patch area PSPA involved in the formation of the cationic residues and the surface patch area HSPA involved in the formation of the hydrophobic residues.

[0105] In some embodiments, the folded conformation of the designed and engineered skin- penetrating peptide (e.g., a Transportan-derived peptide or a Penetratin-derived peptide) is obtained by X-ray diffraction, nuclear magnetic resonance, and cryo-electron microscopy. However, obtaining the folded conformation of a protein by X-ray diffraction, nuclear magnetic resonance, and cryo-electron microscopy is time-consuming and costly.

[0106] In some embodiments, the folded conformation of the designed and engineered skin- penetrating peptide (e.g., a Transportan-derived peptide or a Penetratin-derived peptide) is predicted by computer, e.g., is predicted based on the AlphaFold2 algorithm or the AlphaFold3 algorithm.

[0107] In some embodiments, the surface properties of the folded conformation of the designed and engineered skin-penetrating peptide (e.g., a Transportan-derived peptide or a Penetratin-derived peptide) are calculated based on, e.g., Maestro software.

[0108] The method of the present application takes into account both the cationic and hydrophobic features of the designed and engineered skin-penetrating peptide (e.g., a Transportan-derived peptide or a Penetratin-derived peptide) and for the first time proposes to score the amphiphilicity of the designed and engineered skin-penetrating peptide (e.g., a Transportan-derived peptide or a Penetratin-derived peptide) using the following equation:

[0109] Amphiphilicity score (SOA) = cationic surface patch area (PSPA) + 1 / 3 hydrophobic surface patch area (HSPA)

[0110] If the amphiphilicity score value is greater than the SOA value of the wild-type skin-penetrating peptide (e.g., wild-type Transportan or wild-type Penetratin-derived peptide), then the designed and engineered skin-penetrating peptide (e.g., a Transportan-derived peptide or a Penetratin-derived peptide) is determined to have improved skin penetration.

[0111] III. Designed and engineered skin-penetrating peptides with improved skin penetration

[0112] By using the method of the present application for determining that a designed and engineered skin-penetrating peptide has improved skin penetration, the present application obtained the Penetratin-derived peptides with improved skin penetration shown in SEQ ID NO: 21-SEQ ID NO: 517 of Table 5.

[0113] In preferred embodiments, the Penetratin-derived peptide is a Penetratin-derived peptide set forth in SEQ ID NO: 236 or SEQ ID NO: 404.

[0114] The Penetratin-derived peptides of the present application having improved skin penetration are greater than the amphipathic score of wild-type Penetratin in terms of amphipathic score.

[0115] IV. Uses of the designed and engineered skin-penetrating peptides

[0116] The present application provides uses of the designed and engineered skin-penetrating peptides (e.g., Transportan-derived peptides or Penetratin-derived peptides) for the preparation of a conjugate or a composition of the designed and engineered skin-penetrating peptides (e.g., Transportan-derived peptides or Penetratin-derived peptides) and a biologically active molecule to penetrate the biologically active molecule through the intact skin of a mammal, preferably, to increase the efficacy of the biologically active molecule and / or to reduce the dosage of the biologically active molecule.

[0117] Biologically active molecules can be classified as therapeutic drugs and diagnostic reagents according to their uses. There is no particular limitation to the biologically active molecules that can be delivered by the designed and engineered skin-penetrating peptides (e.g., Transportan-derived peptides or Penetratin-derived peptides) of the present application, including but not limited to at least one of a polypeptide, a protein, a nucleic acid (e.g., DNA, siRNA), a lipid, a polysaccharide, and a small molecule compound. The binding between the designed and engineered skin-penetrating peptides (e.g., Transportan-derived peptides or Penetratin-derived peptides) of the present application and the delivered biologically active molecules can be non-covalent, such as electrostatic and hydrophobic interactions, or covalent.

[0118] In one embodiment, the biologically active molecule is 5-fluoro-2'-deoxyuridine (Floxuridine, 5-Fluoro-2'-deoxyuridine or FUDR, abbreviated as FUDR). 5-fluoro-2'-deoxyuridine (FUDR) is a metabolite of fluorouracil (5-FU). Human body has many tissues express thymidine phosphorylase (TP), while tumors express this enzyme at a higher concentration than the surrounding normal tissues. 5-FU can be converted to FUDR in vivo by the action of TP. FUDR exerts its anticancer effect by inhibiting thymidylate synthase (TS) as well as intercalating into RNA and DNA. The U.S. Food and Drug Administration (FDA) has approved 5% 5-FU cream for topical treatment of basal cell carcinoma (BCC). However, due to the limited skin cell penetration ability of hydrophilic 5-FU / FUDR, their topical application usually requires the use of high concentrations (usually 5% or 2.5%) of drug delivery, which increases the risk of side effects such as skin redness, itching and burning sensation.

[0119] After the designed and engineered skin-penetrating peptides (e.g., Transportan-derived peptides or Penetratin-derived peptides) of the present application are conjugated or physically mixed with FUDR, transdermal drug delivery can be used to treat melanoma. Melanoma is the most aggressive and deadly form of skin cancer. Systemic treatment of melanoma has limited anticancer potential and high toxicity. Transdermal drug delivery can directly deliver therapeutic molecules to the site of melanoma and is a promising treatment method.

[0120] The following examples are set forth to assist in understanding the application. No limitation on the scope of the application is intended or should be inferred from these examples. Examples

[0121] The present application generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting the scope of the application. These examples are not intended to represent that the experiments below are all or the only experiments performed.

[0122] Example 1 Permeability of polypeptides to ex vivo skin

[0123] In this example, conjugates of 5-Carboxyfluorescein (5-Carboxyfluorescein, also referred to herein as "FAM") and 20 different polypeptides in Table 1 were prepared, and the structures were identified by high performance liquid chromatography (HPLC) and mass spectrometry (MS). The permeability of each FAM-labeled polypeptide to ex vivo rat and pig skin was determined. Free FAM was used as a negative control.

[0124] Table 1. Name, source, sequence and classification of polypeptides

[0125] FAM was conjugated to the N-terminus of each polypeptide to obtain FAM-polypeptide conjugates. The permeation amount of each polypeptide through the ex vivo skin was calculated according to the fluorescence intensity. Briefly, 1 mM of FAM or each FAM-polypeptide conjugate was first dissolved in 100 μL of DMSO, and then further diluted into 20 mL of 0.9% NaCl solution (polypeptide = 50 μM, DMSO = 0.5% v / v). The transdermal experiment of free FAM or each FAM-polypeptide conjugate was performed using a Valia-Chienz diffusion cell, and the specific parameters of the experiment are listed in Table 2.

[0126] Table 2. Valia-Chienz diffusion cell and parameters

[0127] The permeation of free FAM or each FAM-polypeptide conjugate through the rat ex vivo skin was compared in 0.9% NaCl solution at 37°C. Specifically, the cumulative conjugate permeation amount was calculated as shown in Equation (1):

[0128] where C t is the conjugate concentration of the receiving solution at each sampling time point, C i is the conjugate concentration in the sample, V r and V s are the volume of the receiving solution and the volume of the sample, respectively. The obtained data is expressed as the cumulative conjugate permeation amount per unit skin surface area, i.e., Q t / S.

[0129] According to Equation (2), the apparent permeation coefficient (P app value) of each polypeptide was calculated by linear regression interpolation of the experimental data:

[0130] where C0is the conjugate concentration in the donor cell (50 μM), S is the skin surface area exposed to the permeation experiment, Δt refers to the length of the time change, and ΔQ refers to the change in the cumulative conjugate permeation amount per unit skin surface area within the length of the time change.

[0131] The cumulative conjugate permeation amount was plotted against time to obtain the cumulative conjugate permeation amount-time curve (i.e., Q-T curve) shown in Fig. 1, panel a, and the corresponding apparent permeation coefficient (P app ) is shown in Fig. 1, panel b.

[0132] As can be seen from FIG. 1, there was no significant difference in the permeability of ex vivo skin for the polypeptide conjugates of SEQ ID NO: 1-10 compared to free FAM (n=4, p>0.05). However, the polypeptide conjugates of SEQ ID NO: 11-20 exhibited significantly higher permeability of ex vivo skin than free FAM (n=4, *P<0.05, ***P<0.001, ****P<0.0001), and the polypeptides shown in SEQ ID NO: 11-20 enhanced the permeability of FAM coupled thereto to the ex vivo SD rat dorsal skin. In addition, the skin permeability of Penetratin of SEQ ID NO: 19 and Transportan of SEQ ID NO: 20 was significantly better than that of other polypeptides (***P<0.001).

[0133] The DAPI-stained skin frozen sections were examined under a fluorescence microscope, whereby the skin distribution and penetration depth of the FAM-polypeptide conjugates were directly observed. The images of the transdermal permeation of free FAM or each FAM-polypeptide conjugate are shown in FIG. 2. The enlarged images of the distribution and penetration depth of free FAM and the conjugates of FAM with the polypeptides shown in SEQ ID NO: 8, 19, and 20 in the ex vivo skin are shown in panel a of FIG. 3. Significant differences in the skin distribution and penetration depth between the FAM conjugates of Penetratin and Transportan and the other polypeptide FAM conjugates were observed.

[0134] Panel b of FIG. 3 shows the fluorescence distribution of FAM and the FAM-polypeptide conjugates within the depth of 200 μm of the skin. As can be seen from panel b of FIG. 3, Penetratin showed a sustained high level of distribution within the depth of 200 μm of the skin, i.e., the site of the stratum corneum and the epidermis, compared to the other polypeptides.

[0135] Example 2 In vitro toxicity of polypeptides to skin cells

[0136] In this example, the in vitro toxicity of each polypeptide shown in SEQ ID NO: 1-20 to HeKa cells (human keratinocytes) (purchased from Qingqi Shanghai Biotechnology Development Co., Ltd.) was determined.

[0137] Specifically, the cytotoxicity of the conjugates of each of the 20 polypeptides in Table 1 with FAM was detected using a CCK-8 kit (purchased from Dalian Milabio Company). The HeKa cells were pre-cultured in a 96-well plate for 24 hours, and then the original culture medium was replaced with DMEM medium containing 50 μM FAM or each FAM-polypeptide conjugate, and incubated for 48 or 72 hours. Then, the absorbance was detected at 450 nm according to the instructions of the CCK-8 kit. The results are shown in FIG. 4.

[0138] As shown in Figure 4, compared with Transportan, Penetratin did not exhibit significant cytotoxicity in HeKa cells and has a shorter peptide chain (sequence shown in Table 1). Considering both the safety and production cost of using peptides as absorption enhancers of bioactive molecules, Penetratin was chosen for further modification.

[0139] Example 3: Correlation analysis between the physicochemical properties of peptides and their permeability to isolated skin

[0140] To determine the key physicochemical properties affecting the transdermal permeability of peptides, the folding structures (PDB format) of the peptides shown in Table 1 were predicted using the AlphaFold 2 algorithm, and then Maestro software was used. The physicochemical properties of each peptide were calculated, and the correlation between the physicochemical properties of each peptide and its permeability to isolated skin was analyzed.

[0141] 3.1 AlphaFold 2 Algorithm

[0142] ColabFold v1.5.2 is used to predict peptide structures (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb#Instructions), and analytical steps are performed. ColabFold combines the rapid homology search of MMseqs2 with AlphaFold2 or RoseTTAFold to accelerate the prediction of peptide structures and complexes. ColabFold predicts peptide structures using AlphaFold2 and AlphaFolt2-multimer, and generates sequence alignment / templates using MMseqs2 and HHsearch.

[0143] 3.2 Maestro Software

[0144] use The Protein Surface Analyzer module in version 2020 was used to calculate and characterize the surface area of ​​all peptides. The OPLS3e force field was used in the computational method. All initial structures were prepared and processed using the Protein Preparation Wizard. Surface areas were analyzed and visualized using Maestro 2020-2. Additionally, energy minimization was intentionally omitted because the AlphaFold 2 algorithm already determined the 3D folded structure, and applying energy minimization could alter the peptide structure.

[0145] Figure 5 shows the apparent permeability coefficient P of the peptide.app The correlation of the values with various physicochemical properties. The physicochemical properties include: molecular weight (MW), charge, residue number, basic residue number, hydrophobic residue number, miLogP (lipid-water partition coefficient), alpha-helix %, hydrogen bond donor (HBD) area, hydrogen bond acceptor (HBA) area, hydrophobic moment, aggregate score sum, PSPA (cationic surface patch area), NSPA (anionic surface patch area), HSPA (hydrophobic surface patch area).

[0146] As shown in panel a of FIG. 5, when all polypeptides were analyzed comprehensively, no single physicochemical property of polypeptides was found to have a correlation coefficient (r value) with the P app values of all polypeptides exceeding 0.5, which can be attributed to the different biological membrane penetration mechanisms of different polypeptides. As shown in panels b, c, and d of FIG. 5, for specific types of polypeptides, such as cationic, amphiphilic, and hydrophobic polypeptides, the correlation coefficient between the P app values and some physicochemical properties exceeded 0.5, which can be because polypeptides in the same category exhibit similar secondary structure characteristics. For example, amphiphilic polypeptides usually have similar secondary structures, including alpha-helix or beta-turn. In addition, it was observed that the P app values of all cationic, amphiphilic, and hydrophobic polypeptides had a correlation coefficient exceeding 0.5 with PSPA (panel e of FIG. 5); the P app values of all cationic, amphiphilic, and hydrophobic polypeptides had a correlation coefficient exceeding 0.5 with charge (panel f of FIG. 5); the P app values of all cationic, amphiphilic, and hydrophobic polypeptides had a correlation coefficient exceeding 0.5 with HBD area (panel g of FIG. 5), but only the PSPA (cationic surface patch area) property passed the p test for correlation with in vitro skin permeability (P 疏水型多 肽 = 0.0411, P 两亲型多肽 = 0.0427, P 阳离子型多肽 = 0.0024). The results show that among the more than ten physicochemical parameters of polypeptides, PSPA is an important parameter that has a strong correlation with transdermal permeability.

[0147] Example 4: Establishment of a model for predicting the transdermal efficiency of polypeptides

[0148] In this example, one or more amino acid residues of wild-type Penetratin were replaced to obtain various Penetratin derivative peptides, and a model for predicting the transdermal efficiency of the various Penetratin derivative peptides was established.

[0149] 4.1 Polypeptide transdermal efficiency prediction model fitted by single factor

[0150] A polypeptide transdermal efficiency prediction model based on single factor fitting was developed. Briefly, the single factor prediction model is based on the single factor of PSPA, and considers that the higher the PSPA value, the higher the transdermal efficiency of the polypeptide, thereby predicting the transdermal efficiency of the polypeptide.

[0151] Single amino acid residue substitution was performed on wild-type Penetratin shown in SEQ ID NO: 19, each amino acid residue was replaced with other 19 natural amino acid residues, the PSPA values of the Penetratin derivative peptides after single amino acid residue substitution were predicted using the single factor fitting model, and compared with the PSPA value of wild-type Penetratin. Figure 6, panel a shows that mutation of single amino acid residue of wild-type Penetratin to acidic amino acid (D, E) significantly reduces the PSPA value of the Penetratin derivative peptide, while mutation to basic amino acid (K, R) significantly increases the PSPA value of the Penetratin derivative peptide.

[0152] Similarly, the wild-type Penetratin set forth in SEQ ID NO: 19 was subjected to two amino acid residue substitutions (i.e., 2R3R, 2R5R, 2R6R, 2R7R, 2R8R, 2R9R, 2R12R, 2R14R, 3R5R, 3R6R, 3R7R, 3R8R, 3R9R, 3R12R, 3R14R, 5R6R, 5R7R, 5R8R, 5R9R, 5R12R, 5R14R, 6R7R, 6R8R, 6R9R, 6R12R, 6R14R, 7R8R, 7R9R, 7R12R, 7R14R, 8R9R, 8R12R, 8R14R, 9R14R, 9R12R, 12R14R, 2K3K, 2K5K, 2K6K, 2K7K, 2K8K, 2K9K, 2K12K, 2K14K, 3K5K, 3K6K, 3K7K, 3K8K, 3K9K, 3K12K, 3K14K, 5K6K, 5K7K, 5K8K, 5K9K, 5K12K, 5K14K, 6K7K, 6K8K, 6K9K, 6K12K, 6K14K, 7K8K, 7K9K, 7K12K, 7K14K, 8K9K, 8K12K, 8K14K, 9K14K, 9K12K, 12K14K, 2R3K, 2R5K, 2R6K, 2R7K, 2R8K, 2R9K, 2R12K, 2R14K, 3R5K, 3R6K, 3R7K, 3R8K, 3R9K, 3R12K, 3R14K, 5R6K, 5R7K, 5R8K, 5R9K, 5R12K, 5R14K, 6R7K, 6R8K, 6R9K, 6R12K, 6R14K, 7R8K, 7R9K, 7R12K, 7R14K, 8R9K, 8R12K, 8R14K, 9R12K, 9R14K, 12R14K, 2K3R, 2K5R, 2K6R, 2K7R, 2K8R, 2K9R, 2K12R, 2K14R, 3K5R, 3K6R, 3K7R, 3K8R, 3K9R, 3K12R, 3K14R, 5K6R, 5K7R, 5K8R, 5K9R, 5K12R, 5K14R, 6K7R, 6K8R, 6K9R, 6K12R, 6K14R, 7K8R, 7K9R, 7K12R, 7K14R, 8K9R, 8K12R, 8K14R, 9K12R, 9K14R, 12K14R) and three amino acid residue substitutions, using the single-factor fitting model, the PSPA values of the resulting Penetratin derivative peptides were predicted. Fig. 6, panel b, presents the results of the Penetratin derivative peptides that exhibited a larger increase in PSPA value relative to the wild-type Penetratin. As can be seen from Fig. 6, panel b, relative to the KKK mutant form (i.e.,2K3K5K, 2K3K6K, 2K3K7K, 2K3K8K, 2K3K9K, 2K3K12K, 2K3K14K, 2K5K6K, 2K5K7K, 2K5K8K, 2K5K9K, 2K5K12K, 2K5K14K, 2K6K7K, 2K6K8K, 2K6K9K, 2K6K12K, 2K6K14K, 2K7K8K, 2K7K9K, 2K7K12K, 2K7K14K, 2K8K9K, 2K8K12K, 2K8K14K, 2K9K12K, 2K9K14K, 2K12K14K, 3K5K6K, 3K5K7K, 3K5K8K, 3K5K9K, 3K5K12K, 3K5K14K, 3K6K7K, 3K6K8K, 3K6K9K, 3K6K12K, 3K6K14K, 3K7K8K, 3K7K9K, 3K7K12K, 3K7K14K, 3K8K9K, 3K8K12K, 3K8K14K, 3K9K12K, 3K9K14K, 3K12K14K, 5K6K7K, 5K6K8K, 5K6K9K, 5K6K12K, 5K6K14K, 5K7K8K, 5K7K9K, 5K7K12K, 5K7K14K, 5K8K9K, 5K8K12K, 5K8K14K, 5K9K12K, 5K9K14K, 5K12K14K, 6K7K8K, 6K7K9K, 6K7K12K, 6K7K14K, 6K8K9K, 6K8K12K, 6K8K14K, 6K9K12K, 6K9K14K, 6K12K14K, 7K8K9K, 7K8K12K, 7K8K14K, 7K9K12K, 7K9K14K, 7K12K14K, 8K9K12K, 8K9K14K, 8K12K14K, 9K12K14K), RRK mutant forms (i.e.,2R3R5K, 2R3R6K, 2R3R7K, 2R3R8K, 2R3R9K, 2R3R12K, 2R3R14K, 2R5R6K, 2R5R7K, 2R5R8K, 2R5R9K, 2R5R12K, 2R5R14K, 2R6R7K, 2R6R8K, 2R6R9K, 2R6R12K, 2R6R14K, 2R7R8K, 2R7R9K, 2R7R12K, 2R7R14K, 2R8R9K, 2R8R12K, 2R8R14K, 2R9R12K, 2R9R14K, 2R12R14K, 3R5R6K, 3R5R7K, 3R5R8K, 3R5R9K, 3R5R12K, 3R5R14K, 3R6R7K, 3R6R8K, 3R6R9K, 3R6R12K, 3R6R14K, 3R7R8K, 3R7R9K, 3R7R12K, 3R7R14K, 3R8R9K, 3R8R12K, 3R8R14K, 3R9R12K, 3R9R14K, 3R12R14K, 5R6R7K, 5R6R8K, 5R6R9K, 5R6R12K, 5R6R14K, 5R7R8K, 5R7R9K, 5R7R12K, 5R7R14K, 5R8R9K, 5R8R12K, 5R8R14K, 5R9R12K, 5R9R14K, 5R12R14K, 6R7R8K, 6R7R9K, 6R7R12K, 6R7R14K, 6R8R9K, 6R8R12K, 6R8R14K, 6R9R12K, 6R9R14K, 6R12R14K, 7R8R9K, 7R8R12K, 7R8R14K, 7R9R12K, 7R9R14K, 7R12R14K, 8R9R12K, 8R9R14K, 8R12R14K, 9R12R14K), KKR mutant forms (i.e.,2K3K5R, 2K3K6R, 2K3K7R, 2K3K8R, 2K3K9R, 2K3K12R, 2K3K14R, 2K5K6R, 2K5K7R, 2K5K8R, 2K5K9R, 2K5K12R, 2K5K14R, 2K6K7R, 2K6K8R, 2K6K9R, 2K6K12R, 2K6K14R, 2K7K8R, 2K7K9R, 2K7K12R, 2K7K14R, 2K8K9R, 2K8K12R, 2K8K14R, 2K9K12R, 2K9K14R, 2K12K14R, 3K5K6R, 3K5K7R, 3K5K8R, 3K5K9R, 3K5K12R, 3K5K14R, 3K6K7R, 3K6K8R, 3K6K9R, 3K6K12R, 3K6K14R, 3K7K8R, 3K7K9R, 3K7K12R, 3K7K14R, 3K8K9R, 3K8K12R, 3K8K14R, 3K9K12R, 3K9K14R, 3K12K14R, 5K6K7R, 5K6K8R, 5K6K9R, 5K6K12R, 5K6K14R, 5K7K8R, 5K7K9R, 5K7K12R, 5K7K14R, 5K8K9R, 5K8K12R, 5K8K14R, 5K9K12R, 5K9K14R, 5K12K14R, 6K7K8R, 6K7K9R, 6K7K12R, 6K7K14R, 6K8K9R, 6K8K12R, 6K8K14R, 6K9K12R, 6K9K14R, 6K12K14R, 7K8K9R, 7K8K12R, 7K8K14R, 7K9K12R, 7K9K14R, 7K12K14R, 8K9K12R, 8K9K14R, 8K12K14R, 9K12K14R), RKK mutant forms (i.e.,2R3K5K, 2R3K6K, 2R3K7K, 2R3K8K, 2R3K9K, 2R3K12K, 2R3K14K, 2R5K6K, 2R5K7K, 2R5K8K, 2R5K9K, 2R5K12K, 2R5K14K, 2R6K7K, 2R6K8K, 2R6K9K, 2R6K12K, 2R6K14K, 2R7K8K, 2R7K9K, 2R7K12K, 2R7K14K, 2R8K9K, 2R8K12K, 2R8K14K, 2R9K12K, 2R9K14K, 2R12K14K, 3R5K6K, 3R5K7K, 3R5K8K, 3R5K9K, 3R5K12K, 3R5K14K, 3R6K7K, 3R6K8K, 3R6K9K, 3R6K12K, 3R6K14K, 3R7K8K, 3R7K9K, 3R7K12K, 3R7K14K, 3R8K9K, 3R8K12K, 3R8K14K, 3R9K12K, 3R9K14K, 3R12K14K, 5R6K7K, 5R6K8K, 5R6K9K, 5R6K12K, 5R6K14K, 5R7K8K, 5R7K9K, 5R7K12K, 5R7K14K, 5R8K9K, 5R8K12K, 5R8K14K, 5R9K12K, 5R9K14K, 5R12K14K, 6R7K8K, 6R7K9K, 6R7K12K, 6R7K14K, 6R8K9K, 6R8K12K, 6R8K14K, 6R9K12K, 6R9K14K, 6R12K14K, 7R8K9K, 7R8K12K, 7R8K14K, 7R9K12K, 7R9K14K, 7R12K14K, 8R9K12K, 8R9K14K, 8R12K14K, 9R12K14K), KRR mutant forms (i.e.,2K3R5R, 2K3R6R, 2K3R7R, 2K3R8R, 2K3R9R, 2K3R12R, 2K3R14R, 2K5R6R, 2K5R7R, 2K5R8R, 2K5R9R, 2K5R12R, 2K5R14R, 2K6R7R, 2K6R8R, 2K6R9R, 2K6R12R, 2K6R14R, 2K7R8R, 2K7R9R, 2K7R12R, 2K7R14R, 2K8R9R, 2K8R12R, 2K8R14R, 2K9R12R, 2K9R14R, 2K12R14R, 3K5R6R, 3K5R7R, 3K5R8R, 3K5R9R, 3K5R12R, 3K5R14R, 3K6R7R, 3K6R8R, 3K6R9R, 3K6R12R, 3K6R14R, 3K7R8R, 3K7R9R, 3K7R12R, 3K7R14R, 3K8R9R, 3K8R12R, 3K8R14R, 3K9R12R, 3K9R14R, 3K12R14R, 5K6R7R, 5K6R8R, 5K6R9R, 5K6R12R, 5K6R14R, 5K7R8R, 5K7R9R, 5K7R12R, 5K7R14R, 5K8R9R, 5K8R12R, 5K8R14R, 5K9R12R, 5K9R14R, 5K12R14R, 6K7R8R, 6K7R9R, 6K7R12R, 6K7R14R, 6K8R9R, 6K8R12R, 6K8R14R, 6K9R12R, 6K9R14R, 6K12R14R, 7K8R9R, 7K8R12R, 7K8R14R, 7K9R12R, 7K9R14R, 7K12R14R, 8K9R12R, 8K9R14R, 8K12R14R, 9K12R14R), RKR mutations (i.e.,2R3K5R, 2R3K6R, 2R3K7R, 2R3K8R, 2R3K9R, 2R3K12R, 2R3K14R, 2R5K6R, 2R5K7R, 2R5K8R, 2R5K9R, 2R5K12R, 2R5K14R, 2R6K7R, 2R6K8R, 2R6K9R, 2R6K12R, 2R6K14R, 2R7K8R, 2R7K9R, 2R7K12R, 2R7K14R, 2R8K9R, 2R8K12R, 2R8K14R, 2R9K12R, 2R9K14R, 2R12K14R, 3R5K6R, 3R5K7R, 3R5K8R, 3R5K9R, 3R5K12R, 3R5K14R, 3R6K7R, 3R6K8R, 3R6K9R, 3R6K12R, 3R6K14R, 3R7K8R, 3R7K9R, 3R7K12R, 3R7K14R, 3R8K9R, 3R8K12R, 3R8K14R, 3R9K12R, 3R9K14R, 3R12K14R, 5R6K7R, 5R6K8R, 5R6K9R, 5R6K12R, 5R6K14R, 5R7K8R, 5R7K9R, 5R7K12R, 5R7K14R, 5R8K9R, 5R8K12R, 5R8K14R, 5R9K12R, 5R9K14R, 5R12K14R, 6R7K8R, 6R7K9R, 6R7K12R, 6R7K14R, 6R8K9R, 6R8K12R, 6R8K14R, 6R9K12R, 6R9K14R, 6R12K14R, 7R8K9R, 7R8K12R, 7R8K14R, 7R9K12R, 7R9K14R, 7R12K14R, 8R9K12R, 8R9K14R, 8R12K14R, 9R12K14R), KRK mutant forms (i.e.,2K3R5K, 2K3R6K, 2K3R7K, 2K3R8K, 2K3R9K, 2K3R12K, 2K3R14K, 2K5R6K, 2K5R7K, 2K5R8K, 2K5R9K, 2K5R12K, 2K5R14K, 2K6R7K, 2K6R8K, 2K6R9K, 2K6R12K, 2K6R14K, 2K7R8K, 2K7R9K, 2K7R12K, 2K7R14K, 2K8R12K, 2K8R14K, 2K9R12K, 2K9R14K, 2K12R14K, 3K5R6K, 3K5R7K, 3K5R8K, 3K5R9K, 3K5R12K, 3K5R14K, 3K6R7K, 3K6R8K, 3K6R9K, 3K6R12K, 3K6R14K, 3K7R8K, 3K7R9K, 3K7R12K, 3K7R14K, 3K8R9K, 3K8R12K, 3K8R14K, 3K9R12K, 3K9R14K, 3K12R14K, 5K6R7K, 5K6R8K, 5K6R9K, 5K6R12K, 5K6R14K, 5K7R8K, 5K7R9K, 5K7R12K, 5K7R14K, 5K8R9K, 5K8R12K, 5K8R14K, 5K9R12K, 5K9R14K, 5K12R14K, 6K7R8K, 6K7R9K, 6K7R12K, 6K7R14K, 6K8R9K, 6K8R12K, 6K8R14K, 6K9R12K, 6K9R14K, 6K12R14K, 7K8R9K, 7K8R12K, 7K8R14K, 7K9R12K, 7K9R14K, 7K12R14K, 8K9R12K, 8K9R14K, 8K12R14K, 9K12R14K, 2K8R12K) compared to the RRR mutant form (i.e.,2R3R5R, 2R3R6R, 2R3R7R, 2R3R8R, 2R3R9R, 2R3R12R, 2R3R14R, 2R5R6R, 2R5R7R, 2R5R8R, 2R5R9R, 2R5R12R, 2R5R14R, 2R6R7R, 2R6R8R, 2R6R9R, 2R6R12R, 2R6R14R, 2R7R8R, 2R7R9R, 2R7R12R, 2R7R14R, 2R8R9R, 2R8R12R, 2R8R14R, 2R9R12R, 2R9R14R, 2R12R14R, 3R5R6R, 3R5R7R, 3R5R8R, 3R5R9R, 3R5R12R, 3R5R14R, 3R6R7R, 3R6R8R, 3R6R9R, 3R6R12R, 3R6R14R, 3R7R8R, 3R7R9R, 3R7R12R, 3R7R14R, 3R8R9R, 3R8R12R, 3R8R14R, 3R9R12R, 3R9R14R, 3R12R14R, 5R6R7R, 5R6R8R, 5R6R9R, 5R6R12R, 5R6R14R, 5R7R8R, 5R7R9R, 5R7R12R, 5R7R14R, 5R8R9R, 5R8R12R, 5R8R14R, 5R9R12R, 5R9R14R, 5R12R14R, 6R7R8R, 6R7R9R, 6R7R12R, 6R7R14R, 6R8R9R, 6R8R12R, 6R8R14R, 6R9R12R, 6R9R14R, 6R12R14R, 7R8R9R, 7R8R12R, 7R8R14R, 7R9R12R, 7R9R14R, 7R12R14R, 8R9R12R, 8R9R14R, 8R12R14R, 9R12R14R) showed the greatest increase in PSPA.

[0153] Table 3 shows the two Penetratin-derived peptides with the highest PSPA values obtained by the polypeptide transdermal efficiency prediction model by one-factor fitting after one, two or three amino acid residue substitutions of the wild-type Penetratin shown in SEQ ID NO: 19, i.e., 2R8R9K Penetratin and 3R5R9R Penetratin), determined by the method shown in Example 1 on the apparent permeation coefficient (P app value) on rat ex vivo skin.

[0154] Table 3. Sequences and properties of Penetratin wild-type and cationic derived peptides

[0155] Figure 6 panel c shows that, compared with wild-type Penetratin, 2R8R9K Penetratin and 3R5R9R Penetratin showed significantly reduced transdermal delivery efficiency. Figure 6 panel d shows that, 2R8R9K Penetratin exhibited more cationic surface patch area than wild-type Penetratin, while 3R1R5R9R Penetratin showed a looser structure.

[0156] From this single-factor fitting model, it can be inferred that the key property affecting transdermal permeation may not be only PSPA, and the reason for the decrease in transdermal efficiency may be the change in polypeptide structure caused by excessive positive charge.

[0157] 4.2 Two-factor fitting polypeptide transdermal efficiency prediction model

[0158] On the basis of Example 4.1, four derivatives of wild-type polypeptide Penetratin (SEQ ID NO: 19, also referred to as “19” in Figure 7) were synthesized, which were substitutions of two amino acid residues of 2W8W (i.e., derivative 1 of SEQ ID NO: 19, also referred to as derivative peptide “19*1” or 28W Penetratin), 2W9W (i.e., derivative 2 of SEQ ID NO: 19, also referred to as derivative peptide “19*2” or 29W Penetratin), 8W9W (i.e., derivative 3 of SEQ ID NO: 19, also referred to as derivative peptide “19*3” or 89W Penetratin), and 2W8W9W (i.e., derivative 4 of SEQ ID NO: 19, also referred to as derivative peptide “19*4” or 289W Penetratin). When single-factor fitting was performed based on PSPA as described in Example 4.1, the r value was less than 0.4 for both rat skin and pig skin (Figure 7 panel a). Therefore, a two-factor fitting polypeptide transdermal efficiency prediction model was further developed. When two-factor fitting was performed based on the amphiphilicity score (SOA), the r value was more than 0.9 for both rat skin and pig skin (Figure 7 panel b) when the apparent permeation coefficient (P app value) was determined on ex vivo skin by the method shown in Example 1, indicating that the correlation between SOA (amphiphilicity score) and P app value was significantly improved (P=<0.05).

[0159] Table 4: Sequences and properties of Penetratin-derived peptides used for the optimization of the predictive model

[0160] Therefore, in this two-factor fitting model, the transdermal permeability of a polypeptide can be predicted using the following equation:

[0161] SOA (score of amphiphilicity) = PSPA (cations surface patch area) + 1 / 3 HSPA (hydrophobic surface patch area) Equation (3)

[0162] The coefficient "1 / 3" is determined by the best linear fitting. The calculation of the polypeptide PSPA and HSPA is based on the polypeptide folding conformation calculated by the AlphaFold 2 algorithm (see Example 3.1 for details) and the polypeptide surface properties, i.e. PSPA and HSPA, calculated by the Maestro software (see Example 3.2 for details).

[0163] The comparative analysis of the wild-type polypeptide Penetratin and its derivatives showed that these derivatives maintained similar polypeptide folding structures (panel c in Figure 7) and had larger hydrophobic surface patch areas (see panel d in Figure 7). Since the hydrophobic surface patch area increased significantly, the hydrophobicity represented by miLogP was also monitored, in which the miLogP value was calculated using the miLogP algorithm disclosed on the website www.molinspiration.com.

[0164] In analyzing the correlation between the SOA and P app The change in the miLogP value while keeping the charge unchanged indicates that the increase in the P app value is not due to the increase in the hydrophobicity (miLogP), but the increase in the amphiphilicity (SOA) (panel e in Figure 7).

[0165] 4.3 Application of the two-factor fitting polypeptide transdermal efficiency prediction model

[0166] The optimized SOA-based two-factor fitting model was used to predict polypeptide sequences with better transdermal permeability.

[0167] Specifically, the polypeptide sequence shown in SEQ ID NO: 19 (wild-type Penetratin) was subjected to amino acid mutation to predict the physicochemical properties of the polypeptide, and thus the transdermal permeability of the polypeptide. The PSPA value and HSPA value of the mutant sequence were calculated using the method described in Example 3, and the SOA value (amphiphilicity score value) was calculated using Equation (3).

[0168] First, single amino acid mutations were made to wild-type Penetratin as shown in SEQ ID NO: 19 (the introduced amino acid mutations did not include acidic and basic amino acids), and the effects of the single amino acid mutations on PSPA and HSPA were calculated (Fig. 8, panel a and panel b). As can be seen from Fig. 8, panel a and panel b, after mutating the amino acid residue of wild-type Penetratin to P, the derived peptide ranked first in terms of ΔPSPA; after mutating the amino acid residue of wild-type Penetratin to W, the derived peptide ranked first in terms of ΔHSPA; after mutating the amino acid residue of wild-type Penetratin to I, the derived peptide ranked second in terms of both ΔPSPA and ΔHSPA; and after mutating the amino acid residue of wild-type Penetratin to M, the derived peptide ranked fifth in terms of both ΔPSPA and ΔHSPA.

[0169] Further, after selecting P, W, I, and M to make double amino acid residue mutations to wild-type Penetratin, the derived peptides were predicted for ΔSOA relative to wild-type Penetratin (Fig. 8, panel c). Among the various double amino acid mutation forms, the mutation combinations of WW and MM showed the top two and exhibited better ΔSOA distribution.

[0170] Therefore, the final ΔSOA prediction for triple amino acid mutations to wild-type Penetratin was based on the amino acid mutation combinations of M and W (Fig. 8, panel d). In order to prevent excessive disturbance to the polypeptide structure, the maximum number of allowed amino acid residue mutations was limited to three amino acid residue mutations.

[0171] By the above experiments, it was shown that the SOA value of the derived peptides was lower than that of the wild-type Penetratin after substitution of the amino acid residues selected from the group consisting of 7I, 3V, 5V, 7V, 14V, 3L, 6L, 7L, 8L, 12L, 14L, 3F, 5F, 6F, 8F, 14F, 2C, 3C, 5C, 7C, 8C, 12C, 14C, 3M, 5M, 7M, 14M, 5A, 6A, 7A, 8A, 9A, 12A, 14A, 2G, 3G, 5G, 6G, 7G, 8G, 9G, 12G, 14G, 2T, 3T, 5T, 6T, 7T, 12T, 14T, 2S, 3S, 5S, 6S, 7S, 8S, 9S, 12S, 14S, 7W, 9W, 12W, 3Y, 5Y, 6Y, 7Y, 8Y, 14Y, 9P, 12P, 14P, 3Q, 5Q, 6Q, 7Q, 9Q, 12Q, 14Q, 2N, 3N, 5N, 6N, 7N, 8N, 12N, 14N, 2D, 3D, 5D, 6D, 7D, 8D, 9D, 12D, 14D, 2E, 3E, 5E, 6E, 7E, 8E, 9E, 12E, 14E, 2P 3P, 2P 14P, 3P 5P, 3P 6P, 3P 9P, 5P 6P, 5P 12P, 5P 14P, 6P 7P, 6P 9P, 6P 12P, 6P 14P, 7P 9P, 9P 14P, 12P 14P, 2I 6I, 6I 7I, 6I 12I, 7I 8I, 7I 9I, 7I 12I, 7I 14I, 8I 14I, 12I 14I, 2M 14M, 3M 8M, 3M 14M, 5M 7M, 5M 8M, 5M 14M, 7M 8M, 7M 14M, 8M 14M, 6W 7W 14W, 6W 9W 14W, 6W 12W 14W, 6M 7W 14M, 3W 6M 14M, 2W 12W 14M, 2W 3W 7M to the wild-type Penetratin.

[0172] The following Table 5 shows the derived peptides having SOA values greater than that of the wild-type Penetratin obtained by the polypeptide transdermal efficiency prediction model fitted by two factors.

[0173] Table 5: Derived peptides having SOA values greater than that of the wild-type Penetratin and their SOA values

[0174] When the amphiphilicity score value is greater than the amphiphilicity score value 1195.3 of wild-type Penetratin, it is determined that the Penetratin derivative peptide has improved skin penetration.

[0175] Based on the above calculation results, three Penetratin derivative peptides with the largest SOA values were synthesized, which are 5W8W9W Penetratin (i.e., derivative peptide 5 of SEQ ID NO: 19, 589W Penetratin, also referred to as 589WP), 3M8W9W Penetratin (i.e., derivative peptide 6 of SEQ ID NO: 19, 3M89W Penetratin), 2W8M9M Penetratin (i.e., derivative peptide 7 of SEQ ID NO: 19, 2W89M Penetratin), and their cumulative permeation-time curves on rat and pig skin were obtained by the method shown in Example 1, as shown in Figure 9, panel a and panel b. In addition, they all conform to the SOA fitting model in terms of the apparent permeability coefficient (P app ) of rat skin (r=0.7824, *P<0.05) and the apparent permeability coefficient (P app ) of pig skin (r=0.8689, **P<0.01) (Figure 9, panel c).

[0176] Table 6: Sequences and properties of the top three Penetratin derivative peptides with SOA values

[0177] To visualize the transdermal penetration effect of polypeptide 589WP in vivo, Cy5 gel and Cy5-589WP gel were prepared. The gel matrix consists of 50% (w / w%) glycerol, 30% polyethylene glycol (PEG) 200 and 20% PEG 400.

[0178] To synthesize Cy5-589WP conjugate, 6.10 mg Cy5-NHS (Dalian Melin Biotech Co., Ltd.) and 10.30 mg 589WP were taken and dissolved in 1 mL PBS buffer (pH = 8.3), and allowed to react at 4°C for 3 hours under stirring. The preparation liquid was purified by liquid chromatography, and then freeze-dried. Subsequently, 1.75 mg of the freeze-dried powder obtained was dissolved with 3 milliliters of gel matrix (consisting of 50% (w / w%) glycerol, 30% polyethylene glycol (PEG) 200 and 20% PEG 400) (raw materials purchased from Dalian Melin Biotech Co., Ltd.) to obtain a Cy5-589WP gel with a final concentration of 0.2 mg / mL of Cy5. Similarly, 0.6 mg of Cy5 was dissolved in 3 mL of gel matrix to obtain a Cy5 gel.

[0179] At the initial zero time, the Cy5 gel and the Cy5-589WP gel showed the same fluorescence intensity; after 4 hours or 8 hours, the gels were carefully wiped with a wet cotton ball before being photographed. At 4 hours, the back of the mouse applied with the free Cy5 gel showed no fluorescence, indicating that the free Cy5 did not penetrate into the mouse back skin; while the Cy5-589WP group showed obvious fluorescence, indicating that the Cy5-589WP had penetrated into the mouse back skin; at 8 hours, the fluorescence intensity of the Cy5-589WP group was significantly higher than that of the free Cy5 group (Fig. 9, panel d). At the same time, the semi-quantitative results also showed that there was a significant difference in fluorescence intensity between the free Cy5 group and the Cy5-589WP group at 4 hours and 8 hours (p < 0.0001) (Fig. 9, panel e).

[0180] Compared with wild-type Penetratin, it was observed that the derived peptides 5W8W9W Penetratin, 3M8W9W Penetratin and 2W8M9M Penetratin maintained similar polypeptide folding structures and exhibited larger hydrophobic surface patch areas (Fig. 9, panel f).

[0181] After the above-mentioned in vitro screening, computer simulation, model optimization, transdermal efficiency prediction of polypeptides, and experimental verification, the wild-type Penetratin was structurally modified, and a number of optimized polypeptide derivatives were obtained. The results of Fig. 9 confirm that the skin permeability of these polypeptide derivatives designed according to the SOA model is improved if the SOA value of the polypeptide derivative is higher than that of the wild-type Penetratin. Experimental verification was carried out using the method of Example 1, and the results showed that compared with the wild-type Penetratin, the polypeptides 5W8W9W Penetratin in the P app value of the polypeptides increased by 41% in the Papp The values increased by 62% (Table 7). In addition, the P 5W8W9W P values of Penetratin conjugates in rat skin app The values increased by 72-fold in rat skin and 60-fold in pig skin (Table 7), showing that 5W8W9W Penetratin has significantly higher skin permeability.

[0182] Table 7: P values of Penetratin wild type and derived peptides in rat skin app P values of Penetratin conjugates in rat skin app Fold increase in P values compared to free FAM

[0183] Example 5 Synthesis and in vitro evaluation of FUDR-589WP

[0184] This example synthesized FUDR-589WP conjugate through a two-step process including esterification and click chemistry reaction (Fig. 10, panel a), and the product was verified using NMR and MS detection, respectively.

[0185] First, 250 mg of FUDR (5-fluoro-2'-deoxyuridine, 1.02 mmol) was weighed and dissolved in 25 mL of acetonitrile. Meanwhile, 186.8 mg of linker (4-maleimidobutyric acid, 1.02 mmol), 12.45 mg of DMAP (4-dimethylaminopyridine, 0.102 mmol) and 230.72 mg of DCC (N,N'-dicyclohexyl carbodiimide, 1.12 mmol) were weighed and dissolved in 3 mL of dichloromethane, respectively. The solution of FUDR and the solution of DCC were mixed and reacted at room temperature for 2 hours. Subsequently, the solution of DMAP and the solution of linker were added to the reaction mixture, which was then stirred overnight at room temperature. Then, the mixture was filtered and the filtrate was collected. The solvent was removed by rotary evaporation, and then the residue was resuspended in 7.2 mL of ultrapure water-methanol (1:1) using ultrasonic method. Purification was performed using preparative liquid chromatography, followed by lyophilization, to obtain FUDR-maleimide (hereinafter also referred to as "FUDR-MAL").

[0186] 10 mg of FUDR-MAL and 62.1 mg of Cys-589WP were dissolved in 10 mL of methanol, respectively. After mixing, 10 μL of triethylamine was added and reacted for 10 minutes on a shaking table at 37°C. Subsequently, methanol and triethylamine were removed by rotary evaporation, and the residue was resuspended in an appropriate solvent using ultrasonic method. Purification was performed using preparative liquid chromatography, followed by lyophilization, to obtain FUDR-589WP lyophilized powder. Finally, the products FUDR-MAL and FUDR-589WP were verified by mass spectrometry and nuclear magnetic resonance.

[0187] To investigate whether covalently coupled skin-penetrating peptides would affect the anti-tumor activity of FUDR, the in vitro activity of free 589WP, free FUDR, FUDR-MAL and FUDR-589WP was compared using B16F10 cells (mouse melanoma cells, purchased from Shanghai Institute of Cell Culture).

[0188] 5.1 In vitro cytotoxicity

[0189] The cytotoxicity of 589WP, FUDR, FUDR-MAL and FUDR-589WP on B16F10 cells was evaluated using CCK-8 solution in CCK-8 kit (purchased from Dalian Meilunbio Co., Ltd.). B16F10 cells were cultured in 96-well plates, after 24 hours of incubation, a series of 589WP, FUDR, FUDR-MAL and FUDR-589WP solutions (1, 5, 10, 20, 40, 60 μmol / L) were incubated with cells for 48 hours. Subsequently, CCK-8 solution was used to detect cytotoxicity. The absorbance value of each well was measured at 450 nm using a microplate reader. To visualize the difference in IC50 values, B16F10 cells were stained with Calcein-AM / PI double staining kit 48 hours after drug administration, and observed under an inverted fluorescence microscope.

[0190] 5.2 In vitro drug release

[0191] The ability of FUDR-589WP to release FUDR in esterase solution was studied. FUDR-589WP solutions were prepared using PBS buffer (pH 8.3, 7.4, 6.5) to make the final concentration of FUDR-589WP 0.5 mM, and the concentration of pig liver esterase (purchased from sigma-aldrich company) 0, 3 or 30 U / mL. The concentration of released FUDR was determined by HPLC at 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours and 96 hours at 37°C and 200 rpm.

[0192] Results:

[0193] In the cytotoxicity experiment of 589WP, FUDR, FUDR-MAL and FUDR-589WP on B16F10 cells, the IC50 of FUDR-MAL and FUDR-589WP was significantly increased compared with free FUDR (****P FUDR-MAL <0.0001, **P FUDR-589WP<0.01), indicating that covalent coupling affects the anti-tumor efficacy of FUDR. However, due to the cell penetration promotion of 589WP, FUDR-589WP still has growth inhibition effect on B16F10 cells at a relatively low concentration (IC50≈15 μM) (Fig. 10, panel b-c). This conclusion can be obtained by visual observation through Calcein-PI staining after 48 hours of treatment of B16F10 cells with 15 μM 589WP, FUDR, FUDR-MAL and FUDR-589WP, respectively (Fig. 10, panel d). Due to the selectivity of the dye to the cell membrane, Calcein can dye the living cells green, and PI can dye the dead cells red. In the negative control group (i.e., adding vehicle) and the free 589WP treatment group, similar living cell density and no dead cells were observed, indicating that 589WP itself has no anti-tumor effect.

[0194] Compared with the negative control group, the free FUDR treatment group showed a significant decrease in living cell density, while the FUDR-Mal treatment group showed a slight decrease in living cell density. At the same time, the FUDR-589WP treatment group showed nearly 50% of the living cell density (Fig. 10, panel d). Dead cells can be observed in all FUDR, FUDR-Mal and FUDR-589WP treatment groups. Notably, most of the dead cells floated in the culture dish due to the decrease in cell adhesion, which cannot be observed.

[0195] FUDR is slowly released from FUDR-589WP at physiological pH, almost not released at weakly acidic pH, and faster released at weakly basic pH (Fig. 10, panel e), which can prove that the release of FUDR is controlled by the ester bond between FUDR and the linker (i.e., 4-maleoyl butyric acid). In addition, porcine liver esterase (PLE) can accelerate the release of FUDR, and shows a dose-dependent manner (Fig. 10, panel f). Therefore, the in vivo process of FUDR-589WP can be predicted by the cumulative release time curve. When FUDR-589WP crosses the epidermis and dermis, in the tissues at physiological pH, free FUDR is slowly released by esterase hydrolysis of the ester bond, and remains stable in weakly acidic tissues, and the esterase concentration has little effect on this state. However, once it reaches the tumor tissue area, due to the rapid growth needs of melanoma, the esterase concentration increases significantly, and free FUDR can be rapidly released from FUDR-589WP, so as to simultaneously exert its anti-tumor effect in the form of FUDR-589WP and free FUDR.

[0196] Example 6 589WEffect of penetratin on drug efficacy in vivo

[0197] In this example, the effect of penetratin on drug delivery efficiency was investigated; and the therapeutic effect of the polypeptide drug conjugate (FUDR-589WP) was evaluated in a C57BL / 6 mouse model bearing melanoma. 589W Penetratin on drug delivery efficiency; and the therapeutic effect of the polypeptide drug conjugate (FUDR-589WP) was evaluated in a C57BL / 6 mouse model bearing melanoma.

[0198] Firstly, FUDR gel, FUDR+589WP gel, FUDR-589WP gel were prepared

[0199] The gel matrix was composed of 50% (mass ratio) glycerol, 30% (mass ratio) PEG 200 and 20% (mass ratio) PEG 400. The blank gel only contained the gel matrix. The FUDR gel contained 0.5% or 2.5% (mass ratio) free FUDR concentration. The FUDR+589WP gel was prepared by physically mixing FUDR and 589WP at a ratio of 1:1, resulting in a final FUDR concentration of 0.5% (mass ratio). The FUDR-589WP gel contained the covalent conjugate of FUDR and 589WP, FUDR-589WP, with a final concentration of 0.05% or 0.5% (mass ratio) calculated as FUDR.

[0200] To evaluate whether the FUDR-589WP gel could improve absorption and improve tumor treatment efficacy and safety in vivo, a subcutaneous tumor model was established by injecting 1 x 10 6 B16F10 cells into the left back of male C57BL / 6 mice. The mice were randomly divided into six groups: (i) blank gel; (ii) 0.5% FUDR gel; (iii) 2.5% FUDR gel; (iv) 0.5% FUDR+589WP gel; (v) 0.05% FUDR-589WP gel; (vi) 0.5% FUDR-589WP gel. Treatment was started when the tumor reached a volume of about 100 mm 3 , and 30 μL of gel was applied on the skin once a day. When the tumor grew to a volume of about 2000 mm 3 , the mice were euthanized considering the animal ethics, and the main organs (liver, kidney, lung, spleen, heart) and the skin covered by melanoma were collected for histopathological analysis by HE staining.

[0201] In this study, blank gel was used as negative control, 0.5% and 2.5% FUDR gels were used as low and high dose positive controls, respectively. In addition, 0.05% and 0.5% FUDR-589WP gel groups were introduced to investigate whether covalent conjugation could improve the skin penetration of FUDR at lower concentrations. Furthermore, 0.5% FUDR+589WP gel group was also set up to evaluate the effect of physical mixture (1:1) of FUDR and 589WP on therapeutic efficacy.

[0202] The results showed that 0.5% FUDR-589WP gel significantly inhibited tumor growth compared to all other groups (panel a in Fig. 11). This finding indicated that covalent conjugation with polypeptide effectively improved the skin penetration of FUDR even at lower concentration (1 / 5 concentration of 2.5% FUDR gel). In addition, 0.5% FUDR+589WP physical mixture gel showed similar tumor inhibition effect as 2.5% FUDR gel, indicating that non-covalent interaction of FUDR with 589WP was also able to significantly enhance the skin penetration of FUDR (P<0.0001). However, the non-covalent interaction of FUDR with 589WP was significantly weaker than the covalent interaction of FUDR with 589WP (P<0.0001). 0.5% FUDR gel and 0.05% FUDR-589WP gel did not significantly inhibit tumor growth compared to blank gel (P>0.05), indicating that the anti-tumor effect of FUDR and FUDR-589WP was concentration dependent (panel b in Fig. 11). These differences among all groups were also clearly visible in the dissected melanoma masses (panel c in Fig. 11).

[0203] Since FUDR can cause skin irritation, including symptoms such as skin erythema, itching, and burning sensation, at high doses in the clinic, it is important to ensure the safety of the gel formulation. To evaluate the skin irritation of the drug, healthy C57BL / 6 mice were used as a model, and the gel was applied to the skin once a day, 30 μL each time. After 7 days of administration, the skin at the administration site was taken for IL-1β antibody immunohistochemical (IHC) staining (panel d in FIG. 11). The results showed that both 0.5% FUDR and 2.5% FUDR caused more immunoreaction positive areas (indicated by black arrows) and deeper brownish yellow staining than the vehicle control group. In contrast, 0.5% 589WP or 0.5% FUDR-589WP did not have similar effects, indicating that 0.5% 589WP and FUDR-589WP did not cause significant skin irritation. This conclusion was further supported by the semi-quantitative average optical density (AOD) values of all groups (panel d in FIG. 11). The AOD values of the 0.5% 589WP and FUDR-589WP groups showed no statistically significant difference compared with the control group (P>0.05). However, their AOD values were significantly lower than those of the 2.5% FUDR group (P<0.01). This difference can be attributed to the reduction of the 0.5% FUDR-589WP dose, which is only 1 / 5 of the 2.5% FUDR. In addition, 589WP is safe as a polypeptide itself, which helps to improve the overall safety of the formulation.

[0204] Histopathological analysis of HE staining of skin covering melanoma showed that the structure of epidermis and dermis was intact, and no obvious irritation was observed (FIG. 12). In addition, HE staining of major organs (heart, liver, spleen, lung, kidney) also showed no obvious toxicity (FIG. 13).

[0205] The results of anti-tumor effect, IHC staining, and HE staining collectively indicate that 589WP is a novel, highly efficient, and safe transdermal absorption enhancer.

[0206] While certain representative embodiments and details have been shown for purposes of illustrating the subject application, it will be apparent to those skilled in the art that various changes in the embodiments and details can be made therein without departing from the scope of the subject application. In this regard, the scope of the present application is limited only by the claims that follow.

Claims

1. A designed and engineered skin-penetrating peptide that is a Transportan-derived peptide and / or a Penetratin-derived peptide, e.g., the Penetratin-derived peptide is a sequence set forth in any one of SEQ ID NO: 21-SEQ ID NO:

517.

2. The designed and engineered skin-penetrating peptide of claim 1, wherein the Penetratin-derived peptide is a sequence set forth in SEQ ID NO: 236 or SEQ ID NO:

404.

3. A method of determining a designed and engineered skin-penetrating peptide that has improved skin-penetrating properties, comprising determining the cationic surface patch area (PSPA) and the hydrophobic surface patch area (HSPA) of the designed and engineered skin-penetrating peptide; scoring the amphiphilicity of the polypeptide using the following equation: Amphiphilicity Score (SOA) = Cationic Surface Patch Area (PSPA) + 1 / 3 Hydrophobic Surface Patch Area (HSPA); determining that the designed and engineered skin-penetrating peptide has improved skin-penetrating properties if the SOA value is greater than the SOA value of the wild-type skin-penetrating peptide; for example, a method of determining a Transportan-derived peptide that has improved skin-penetrating properties, comprising determining the cationic surface patch area (PSPA) and the hydrophobic surface patch area (HSPA) of the Transportan-derived peptide; scoring the amphiphilicity of the polypeptide using the following equation: Amphiphilicity Score (SOA) = Cationic Surface Patch Area (PSPA) + 1 / 3 Hydrophobic Surface Patch Area (HSPA); determining that the Transportan-derived peptide has improved skin-penetrating properties if the SOA value is greater than the SOA value of the wild-type Transportan; for example, a method of determining a Penetratin-derived peptide that has improved skin-penetrating properties, comprising determining the cationic surface patch area (PSPA) and the hydrophobic surface patch area (HSPA) of the Penetratin-derived peptide; scoring the amphiphilicity of the polypeptide using the following equation: Amphiphilicity Score (SOA) = Cationic Surface Patch Area (PSPA) + 1 / 3 Hydrophobic Surface Patch Area (HSPA); determining that the Penetratin-derived peptide has improved skin-penetrating properties if the SOA value is greater than the SOA value of the wild-type Penetratin.

4. The method of claim 3, wherein the designed and engineered skin-penetrating peptide is a designed and engineered skin-penetrating peptide that has one or more amino acid residues, e.g., single, double, triple, quadruple, quintuple, or more amino acid residue substitutions, e.g., substitutions, deletions, and / or additions, to the amino acid sequence of a wild-type skin-penetrating peptide. For example, the Transportan-derived peptide is a derivative peptide obtained by altering, e.g., replacing, deleting and / or adding one or more amino acid residues, e.g., single, double, triple, quadruple, quintuple or more amino acid residue replacement, to the amino acid sequence of wild-type Transportan, preferably the Transportan-derived peptide is a derivative peptide obtained by single, double or triple amino acid residue mutation to wild-type Transportan, for example, the amino acid sequence of the wild-type Transportan is set forth in SEQ ID NO:

18. For example, the Penetratin-derived peptide is a derivative peptide obtained by altering, e.g., replacing, deleting and / or adding one or more amino acid residues, e.g., single, double, triple, quadruple, quintuple or more amino acid residue replacement, to the amino acid sequence of wild-type Penetratin, preferably the Penetratin-derived peptide is a derivative peptide obtained by triple amino acid residue mutation to wild-type Penetratin, for example, the amino acid sequence of the wild-type Penetratin is set forth in SEQ ID NO:

19.

5. A designed and engineered skin-penetrating peptide, e.g., a Transportan-derived peptide and / or a Penetratin-derived peptide, which is a designed and engineered skin-penetrating peptide with improved skin-penetrating property determined according to the method of claim 3 or 4.

6. The conjugate of a designed and engineered skin penetrating peptide and a biologically active molecule according to claim 1, 2 or 5, wherein, The conjugate has improved skin-penetrating ability, for example, the designed and engineered skin-penetrating peptide is directly conjugated to the biologically active molecule or indirectly conjugated through a linker.

7. The conjugate of claim 6, wherein the biologically active molecule is selected from at least one of a polypeptide, a protein, a nucleic acid, a lipid, a polysaccharide, a small molecule compound, for example, a chemotherapeutic agent.

8. A composition comprising component i) the designed and engineered skin-penetrating peptide of claim 1, 2 or 5; and component ii) a biologically active molecule, for example, the composition comprises the components i) and ii) in non-covalent combination.

9. The composition of claim 8, wherein the biologically active molecule is selected from at least one of a polypeptide, a protein, a nucleic acid, a lipid, a polysaccharide, a small molecule compound, for example, a chemotherapeutic agent.

10. Use of the designed and engineered skin-penetrating peptide of claim 1, 2 or 5 for the manufacture of a transdermal absorption enhancer to facilitate penetration of a biologically active molecule through intact skin of a mammal, preferably to increase efficacy and / or to reduce dosage of the biologically active molecule.

11. The use of claim 10 for facilitating penetration of a chemotherapeutic agent, such as 5-fluoro-2'-deoxyuridine, through intact skin of a mammal, preferably to increase efficacy and / or to reduce dosage of the chemotherapeutic agent, such as 5-fluoro-2'-deoxyuridine.

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