Peptide protein kinase c inhibitors and uses thereof

Abstract

The present invention relates to novel peptides, compositions and uses thereof in tissue penetration, in particular in the context of cancer prevention and / or treatment or induction of an immune response, in particular mucosal vaccination or anti-opioid treatment.

Description

Technical Field

[0001] This invention relates to novel inhibitors of the Cζ type of protein kinase and their use as tissue permeators, particularly in the context of cancer treatment. Background Technology

[0002] Tight junctions (TJs) are complex structures between adjacent epithelial or endothelial cells that regulate the passage of ions or molecules through the paracellular space. TJs also help distinguish cell differentiation by clearly differentiating the apical and basal sides. TJs are composed of various protein fragments, namely transmembrane proteins (closing proteins, interlocking proteins, junctional adhesion molecules (JAMs), etc.) and cytoplasmic scaffold proteins (ZO-1 (closing band-1), cingulates, afadin, MAGI1 (membrane-associated guanylate kinase), etc.). The cytoskeletal proteins of TJs are actin and microtubules (Van Itallie et al., 2014, Semin. Cell Dev. Biol. 36:157-165).

[0003] Protein kinase C (PKC) is a family of serine / threonine kinases containing regulatory and catalytic domains. PKC is involved in several cellular functions. PKC isotypes are classified into conventional isotypes (α, β1, β2, γ), novel isotypes (α, β1, β2, γ), and atypical isotypes (ζ, ι / λ). Unlike the conventional and novel isotypes, atypical PKC isotypes lack the C1 domain (phorbol ester / diacylglycerol binding domain), which is responsible for the membrane localization of other PKC isotypes. Peptide inhibitors of the protein kinase C isotype ζ have been developed and described as effective against a range of tumors, hyperproliferative diseases (such as psoriasis), and viral infections (such as HIV) (WO93 / 20101).

[0004] Several PKC isoforms are involved in the regulation of TJ. The atypical protein PKCζ is essential for TJ protein assembly, and atypical PKCs have been shown to be associated with cellular polarity (Steinberg, 2008, Physiol. Rev., 88:1341-1378; Hirai et al., 2003, J. Biochem., 133:1-7). PKCζ and PKCι share 72% homologous amino acid sequence identity. This includes a highly conserved pseudosubstrate region (Selbie et al., 1993, J. Biol. Chem., 268:24296-24302). The pseudosubstrate (PS) region, or PS prototope, is a sequence present in the regulatory domain that is responsible for keeping the protein kinase in an inactive cytoplasmic form by blocking the substrate binding site present in its kinase domain, and corresponds to amino acid sequence 113-126 of PKCζ (House et al., 1987, Science, 238:1726-1728).

[0005] Recent advances in understanding the molecular structure of epithelial junctions (TJs) have led to the development of tight junction modulators. Epithelial junction openers, which are tight junction modulators, can mitigate poor drug absorption, a major cause of failure in oral drug candidates during clinical development (Kennedy, 1997, Drug Discovery Today, 2:436-444; Lipinski, 2000, J. Pharmacol. Toxicol. Methods, 44:235-249). TJ modulators used as absorption enhancers are affected by narrow therapeutic windows and nonspecific modes of action. TJ toxicity and irreversible opening are major causes of these drug failures (Deli, 2009, Biochim. Biophys. Acta, 1788:892-910; Yamamoto et al., 1996, J. Pharm. Pharmacol., 48:1285-1289; Swenson et al., 1994, Pharm. Res., 11:1132-1142). For example, a small recombinant adenovirus serotype 3-derived protein, called linker-opener 1 (JO-1), has been developed that binds to the epithelial connexin desmosome core protein 2 (DSG2) and has been shown to improve drug penetration into tumors, particularly monoclonal antibodies (mAbs) used to treat solid tumors. Unfortunately, it has also been shown to induce immunogenicity (Beyer et al., 2011, Cancer Res., 71:7080-7090).

[0006] Cancer (including all subtypes) is a malignant transformation of epithelial cells, accounting for approximately 80% of cancer cases. Epithelial tumors are bound by tight junctions between cells, restricting penetration across the tumor, particularly the penetration of drugs larger than 500 Da (Lipinski et al., 2001, Adv. Drug Deliv. Rev., 46:3-26; Lavin et al., 2007, J. Exp. Biol., 210:2754-2764). Many receptors targeted by antitumor drugs have been found to be hidden / trapped within the tight junctions of tumor cells, inaccessible to antitumor drugs. This is considered a significant factor contributing to drug resistance and thus tumor recurrence (Beyer et al., 2011, Cancer Res., 71:7080-7090). Therefore, in most cases, antitumor drugs are ineffective due to target accessibility issues, rather than a lack of drug activity.

[0007] In addition, mucosal tissue, due to its approximately 400m 2The large surface area of ​​mucosa serves as an entry port for various pathogens. The immunological components of the mucosal surface, called mucosa-associated lymphoid tissue (MALT), can elicit an immune response to antigens, which then diffuse into the lamina propria region. Inducing mucosal immunity is a current research objective, and mucosal vaccination, which involves administering vaccines at one or more mucosal sites to induce immune responses at the site of administration, other mucosal sites, and / or systemically, has been extensively studied. Mucosal vaccination offers several advantages: a) ease of administration, and b) stimulation of immunoglobulin A expression, which inhibits microbial adhesion and invasion. However, mucosal vaccine delivery is hampered by the presence of intercellular TJs, thus limiting the passage of macromolecules (Borchard et al., 2012, Chitosan-Based Systems for Biopharmaceuticals: Delivery, Targeting and Polymer Therapeutics, John Wiley & Sons, Ltd, Chapter 12 (Chitosan-based delivery systems for mucosal vaccination), 211-224).

[0008] Therefore, there is a need to develop new TJ modulators that are safe, reversible, effective, and ideally have known modes of action. Summary of the Invention

[0009] This invention relates to the discovery of novel peptides that, once inside cells, act as inhibitors of protein kinase Cζ (PKCζ), which unexpectedly induce a transient decrease in tissue integrity, potentially indicating the opening of tight junctions within the tissue. This property of the peptides of the invention can be advantageously used to induce transient tissue penetration, particularly to enhance the penetration of large therapeutic molecules, such as antibodies or macromolecules for mucosal vaccination. The tissue penetration property can also be used for the transmucosal delivery of high molecular weight drugs (e.g., peptide and protein drugs such as insulin), avoiding parenteral administration of such drugs. Furthermore, transient induction of tissue penetration is beneficial in cases where the therapeutic agent is particularly toxic and requires entry into the basolateral aspect of the target cells, such as in the case of antitumor drugs. According to another aspect, the peptides of the invention can be used for ocular delivery of drugs (particularly biologics or macromolecules), for example as ophthalmic formulations for treating eye diseases or conditions.

[0010] When co-delivered, for example, as an adjuvant, the peptides of the present invention can enhance the efficacy of anticancer drugs by increasing drug penetration into tissues and significantly improve the immune response to mucosal vaccines.

[0011] One object of the present invention is to provide novel PKCζ inhibitors that have low toxicity, induce effective and transient tissue penetration, and are suitable for pharmaceutical use. In particular, an object of the present invention is to provide tissue penetration enhancers for therapeutic agents, especially mucosal penetration enhancers, and more particularly nasal penetration enhancers.

[0012] The first aspect of the invention provides compounds of formula (I) / SEQ ID NO.:1, and pharmaceutically acceptable salts and pharmaceutically active variants thereof.

[0013] Another aspect of the present invention relates to a pharmaceutical composition comprising at least one compound according to the present invention.

[0014] Another aspect of the invention is the use of compounds according to the invention for the prevention and / or treatment of cancer, particularly for the anticancer treatment of carcinoma.

[0015] Another aspect of the present invention is the use of the compound according to the invention for mucosal vaccination.

[0016] Another aspect of the invention is that the compounds according to the invention are used for the prevention and / or treatment of opioid use disorders, particularly opioid overdose or opioid dependence.

[0017] Another aspect of the invention is the use of the compounds according to the invention in the preparation of pharmaceutical compositions, particularly vaccine compositions.

[0018] Another aspect of the invention relates to a method for enhancing the therapeutic efficacy on a subject suffering from a disease or condition, the method comprising administering a compound or pharmaceutical preparation according to the invention, in combination with a therapeutic agent targeting the disease or condition of the subject, wherein the tissue penetration of the therapeutic agent is enhanced compared to when administered in the absence of the compound of the invention.

[0019] Another aspect of the invention is a method for preventing and / or treating a subject suffering from carcinoma cancer, the method comprising administering, in combination with anticancer therapy, a compound or pharmaceutical preparation according to the invention in the subject in need.

[0020] Another aspect of the present invention is a method for inducing immunity, the method comprising administering a mucosal vaccine in combination with a compound according to the present invention.

[0021] Another aspect of the invention is a method for preventing and / or treating subjects suffering from opioid use disorder, particularly opioid overdose or opioid dependence, said method comprising administering a compound or pharmaceutical preparation thereof according to the invention in combination with an antiopioid drug to the subject in need.

[0022] Another aspect of the invention is a method for preventing and / or treating a subject suffering from an eye condition, the method comprising administering, to a subject in need, a peptide or pharmaceutical preparation thereof according to the invention in combination with an ophthalmic agent.

[0023] Another aspect of the present invention is a transmucosal drug delivery system comprising an effective amount of at least one therapeutic agent and at least one mucosal penetration enhancer according to the present invention.

[0024] Another aspect of the invention is an ocular delivery system or formulation comprising an effective amount of at least one therapeutic agent and at least one mucosal penetration enhancer according to the invention.

[0025] Other objects and advantages of the invention will become apparent from the accompanying drawings, the claims, and / or from the following detailed description of embodiments of the invention. Attached Figure Description

[0026] Figure 1 The epithelial integrity of the nasal epithelial monolayer was shown in the presence of the peptide of the present invention (P4) (measured twice in detection 1 and detection 2), the comparative peptide (CP4), and the control solution (at t = 0, 20, 40, 60, and 80 min), as measured by TEER as described in Example 10.

[0027] Figure 2 The permeation of FITC-conjugated insulin was demonstrated, as described in Example 2, over a period of 300 minutes, measured by the release induced by peptides P3 and P4 of the present invention (final peptide concentration 50 μM), compared to the control (carrier) and comparative peptide CP4 that penetrated the nasal epithelial monolayer. Values ​​are mean ± SD (n = 3). Detailed Implementation

[0028] The term "cellular permeabilization fraction" refers to a peptide or non-peptide fraction that has the ability to be transported across a lipid bilayer (e.g., a cell membrane). When a cellular permeabilization fraction is conjugated to another molecule (cargo), it facilitates or enhances the efficient transport of said cargo molecule across the lipid bilayer (e.g., a cell membrane) into cells or tissues and across the blood-brain barrier; in other words, the cellular permeabilization fraction acts as a transmembrane carrier.

[0029] The cellular permeation portion can be a fatty acid portion, and it can be covalently linked to the peptide backbone, for example, by acylation, such as by N-myristoylation or palmitoylation. Examples of fatty acids that can be used as the cell permeation portion according to the present invention include octanoic acid (C8:0), decadecanoic acid (C10:0), dodecanoic acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), icosanoic acid (C20:0), docosanoic acid (C22:0), tetracosanoic acid (C24:0), and hexacosanoic acid.

[0030] Optionally, in the context of this invention, the cell-penetrating portion may be a lipid cell-penetrating portion conjugated to another cell-penetrating portion, such as in lipid cell-penetrating nanoparticles or cationic liposomes, for example in Lipofect. In formulations (Thermo Fisher Scientific, Waltham, MA, USA), myristoyl-Arg7, stearoyl-Arg8, cholesterol-Arg9, stearoyl-TP10 (named PepFect3), stearoyl-(Arg–Ahx–Arg)4, C12R9, C12dR9, C12dR9-1, C12dR9-2, C14R11, C14dR11 (Lee et al., 2013, ibid.; DiPisa et al., 2015, ibid.) or in carriers containing palmitoyl chains and arginine residues (Bonnet et al., 2001, J. Med. Chem., 44:468-471).

[0031] Optionally, the cell-penetrating moiety can be a peptide sequence derived from a natural protein, a chimeric peptide formed by the fusion of two natural sequences, or a rationally designed synthetic peptide. Examples of peptide cell-penetrating moieties include, but are not limited to, TAT (the transactivator of HIV transcription), Drosophila homologous protein antennal foot mutations (ANTp, the membrane-penetrating peptide), W / R, NLS (nuclear localization signal), and AlkCWK. 18 DiCWK 18 Transportan, Dipalytic, K16 RGD, Plae, Kplae, cKplae, MGP, HA2, LARL46, (LARL)n, Hel-11-7, KK, KWK, RWR, oligomers, herpesvirus VP22, SCWKn, RGD, 8-lysine, MPG, pVEC, ARF (1-22), BPrPp (1-28), VT5, MAP, SG3, Pep-7, FGF (fibroblast growth factor), stapling peptide, isopentenyl peptide, pepducins, Pep-1, polyarginine (9-arginine, 8-arginine, 6-arginine), R6W3, TP10, arginine-rich peptides such as (Arg–X–Arg). n Peptides (where X is a universal carbon chain spacer), proline-rich peptides (Schwartz et al., 2000, Curr. Opin. Mol. Ther., 2(2): 162-7; Lee et al., 2013, Methods MolBiol., 991: 281-92; Bechara et al., 2013, FEBS Lett., 587(12): 1693-1702; Di Pisa et al., 2015, J. Pept. Sci., 21(5): 356-369; Guo et al., 2016, Biomed. Rep., 4(5): 528-534).

[0032] Depending on the specific aspect, the cellular permeability is described in Svensen et al., 2012, Trends in Pharmacological Sciences, 33(4):186-192.

[0033] According to one aspect, the cell-permeable portion can be conjugated to the remainder of the peptide backbone of the present invention using known linking schemes via thiazolidinyl, thioether, disulfide, or hydrazone bonds (Bonnet et al., 2001, ibid.).

[0034] Optionally, in the context of this invention, the cell permeation portion may include a guide peptide (HP) sequence for specifically targeting tight junctions in certain cells (e.g., cancer cells in the case of a cancer guide peptide). For example, the cell permeation portion according to the invention may include a guide peptide (HP) sequence conjugated to the cell permeation portion or the cell permeation guide peptide (CPHP), as described, for example, in Svensen et al., 2012, Trends in Pharmacological Sciences, 33(4):186-192.

[0035] According to another aspect, the peptide according to the invention may, for example, be further conjugated to a guide peptide at its C-terminus.

[0036] "Directed peptide" or HP refers to a peptide that does not have inherent internalization properties and only delivers its cargo to specific cell surface receptors; other HPs themselves have cell permeability properties.

[0037] The term "myristylation" refers to the conjugation of a myristoyl group to the amino acid of the peptide of the present invention, particularly the α-amino group of the N-terminal residue, via an amide bond.

[0038] The term "therapeutic molecule" or "therapeutic active agent" refers to a molecule used to treat or prevent disease. In the context of this invention, examples of therapeutic molecules include, but are not limited to, molecules used in prophylactic vaccines (in the process of acquiring immunity against a specific disease or pathogen), molecules used in therapeutic vaccines (e.g., vaccines for cancer treatment or vaccines that induce tolerance to allergens), therapeutic antibodies (e.g., antibodies for cancer treatment), low molecular weight pharmaceutical products (e.g., cytotoxic drugs or enzyme inhibitors for cancer treatment), and anesthetics.

[0039] The term "protein kinase Cζ type" or "PKCζ" refers to the type of protein kinase C subtype.

[0040] The term "tight junction," abbreviated as "TJ," refers to a complex structure between adjacent epithelial or endothelial cells that regulates the passage of ions or molecules through the paracellular space. TJs are composed of various protein fragments, including transmembrane proteins such as closure proteins, latching proteins, and junctional adhesion molecules (JAMs), and cytoplasmic scaffold proteins such as ZO-1, cingulates, afadin, and membrane-associated guanylate kinase (MAGI1). The effect of the peptides of this invention on tight junctions can be monitored by: i) measurement of transepithelial electrical resistance (TEER); ii) determination of the apparent permeability (Papp) of bypass markers (e.g., fluorescein-glucan); iii) fluorescent immunostaining of TJ proteins followed by imaging; and iv) determination of TJ protein mRNA and protein expression.

[0041] The term "carcinoma" as defined in this article refers to diseases involving the malignant transformation of epithelial cells (including all subtypes of epithelial carcinoma). The term "carcinomas" refers to diseases such as, but not limited to, breast cancer, prostate cancer, lung cancer, pancreatic cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, colorectal cancer, and head and neck cancer. The term also covers gastric cancer and other solid tumors.

[0042] As defined herein, the term "mucosal vaccine" refers to a vaccine administered at one or more mucosal sites that induces an immune response at the site of administration, other mucosal sites, and / or systemically. Mucosal tissues include the tissues of the nose, mouth, intestines, lungs, eyes, rectum, and vagina. "Nasal vaccine" is defined as a vaccine administered at the mucosal site of the nose.

[0043] The term "eye or eye disease" as defined here refers to diseases affecting the eye, such as uveitis, scleritis, keratitis, snow blindness, Thygeson's superficial punctate keratopathy, corneal neovascularization, corneal endothelial dystrophy, keratoconjunctivitis sicca, iritis, Sjogren's syndrome, Wegener's granulomatosis, etc. Syndrome, uveitis, macular edema, choroidal neovascularization, retinal vasculitis, macular edema, age-related macular degeneration, diabetic retinopathy, diabetic macular edema, glaucoma, cataract, choroidal retinitis, choroidal retinal scarring, choroidal degeneration, choroidal dystrophy, choroidal hemorrhage, choroidal retinitis, hypertensive retinopathy, macular degeneration, anterior and posterior segment diseases.

[0044] The term "opioid use disorder" refers to clinically significant impairment or distress associated with the use of opioids, such as an intense craving for opioids, increased opioid tolerance, and withdrawal syndrome upon abrupt cessation of opioid use. In particular, addiction and dependence are the most severe components of opioid use disorder.

[0045] As defined herein, the term “neurological disease or condition” is a disease that affects the central nervous system (CNS) and / or the peripheral nervous system, and includes, for example, but not limited to, neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis, peripheral neuropathy, Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease; neuropsychiatric diseases such as depression, anxiety disorders, and psychosis; or diseases related to substance abuse such as opioid, alcohol, or nicotine abuse or addiction.

[0046] The term "efficacy" in the treatment according to the invention can be measured based on changes in the course of the disease in response to the use or method according to the invention. For example, the efficacy of a treatment according to the invention can be measured by its effect on disease signs or symptoms. A response is achieved when a subject experiences partial or complete remission, or a reduction in unexpected disease symptoms. According to specific embodiments, efficacy can be measured by assessing the increase in the effect of a therapeutic molecule used in combination with a compound of the invention compared to the effect of the same molecule used alone. For example, the efficacy of the anticancer treatment according to the invention can be monitored by following up on the effect on tumor size or by improving the survival of a group of patients so treated.

[0047] The term "efficacy" of a treatment according to the invention can be measured based on the reduction of treatment side effects compared to treatments administered without the use of the peptides of the invention.

[0048] The term "efficacy" in the vaccine according to the invention can be measured based on changes in the immune system response. For example, the efficacy of a vaccine according to the invention can be measured by its effect on acquired immunity against a specific disease / pathogen. For example, a response to a vaccine can be obtained when a subject acquires specialized systemic cells and treatments that eliminate or prevent the growth of pathogens.

[0049] The present invention can also be used to increase the efficacy (induction of tolerance) of allergic vaccines by increasing the penetration of allergens (e.g., recombinant allergens) into mucous membranes (e.g., mouth or nose).

[0050] As used in this article, "treatment" and "treating" generally refer to achieving the desired pharmacological and physiological effects. This effect can be preventative in preventing or partially preventing a disease, its symptoms, or symptom, and / or therapeutic in partially or completely curing a disease, symptom, or adverse reaction caused by the disease.

[0051] As used herein, the term "permeation" refers to the process of making a membrane permeable to a drug present on one side of the membrane. In the context of this invention, the permeation-enhancing molecules achieved by the compounds of this invention permeate across the epithelial cell layer. The ability of the compounds of this invention to increase tissue permeability to certain drugs can be detected in known assays, such as those described below.

[0052] As used herein, the term "subject" refers to a mammal. For example, mammals contemplated for this invention include humans, primates, domesticated animals such as cattle, sheep, pigs, horses, laboratory rodents, etc.

[0053] The term "D-amino acid" refers to the D-stereoisomer of an amino acid or the "right-hand" isomer of an amino acid.

[0054] In the context of this invention, the nonpolar amino acid may be selected from Gly, Ala, Val, Leu, Ile, Met, Trp, Phe and Pro or their conservative substitutions.

[0055] In the context of this invention, the positively charged amino acid is selected from Arg, Lys, or His or their conservative substitutions.

[0056] For example, “conservative amino acid substitution” can include replacing a natural amino acid residue with a non-natural residue such that the polarity or charge of the amino acid residue at that position has little or no effect. The desired amino acid substitution can be determined by someone skilled in the art when such substitution is required. The term “variant” also includes peptides or polypeptides that are substantially homologous to a reference peptide sequence but whose amino acid sequence differs from that of the reference sequence because one or more amino acids have been chemically modified or substituted by an amino acid analog. For example, non-natural residues can be introduced to enhance the pharmacological properties of peptide-based therapeutics (Geurink et al., 2013, J. Med. Chem., 56, 1262; Rand et al., 2012, Med. Chem. Commun., 3, 1282).

[0057] According to another specific embodiment, the peptides of the present invention may optionally undergo C-terminal amidation.

[0058] The term "pharmaceutical formulation" refers to a formulation in which the biological activity of the active ingredient is clearly proven and effective, and which does not contain any other components that would be toxic to a subject administering the formulation.

[0059] The compounds of the present invention

[0060] According to one aspect, peptides having a total of 5 to 10 amino acids of the following formula (I) are provided:

[0061] Z-Z1-Xaa4Xaa5RXaa7Xaa8-Z2

[0062] (I)

[0063] Where Z represents the cellular permeation component;

[0064] Z1 is an optional peptide moiety of 1 to 3 amino acids of formula (II):

[0065] Xaa1 Xaa2 Xaa3

[0066] (II)

[0067] Xaa1 and Xaa2 may or may not be present, and when present, Xaa1 and Xaa2 are independently positively charged amino acids, more specifically Arg, and Xaa3 is a nonpolar amino acid, especially Gly;

[0068] Xaa4 is an amino acid selected from Ala, Ser, and Val, especially Ala;

[0069] R stands for arginine;

[0070] Xaa5 and Xaa8 are independently positively charged amino acids, especially Arg;

[0071] Xaa7 is a nonpolar amino acid, and more particularly Trp;

[0072] Z2 is an optional peptide moiety of one or two amino acids of formula (III):

[0073] Xaa9 Xaa 10

[0074] (III)

[0075] Xaa9 is a positively charged amino acid, and more specifically, Lys, and Xaa 10 It may or may not exist, and when it exists, Xaa 10 These are nonpolar amino acids, especially Leu, in which at least one amino acid in formula (I) is a D-amino acid.

[0076] According to a particular embodiment, a peptide of formula (I) is provided, wherein the cell permeation portion Z is covalently attached to the N-terminus of the peptide.

[0077] According to another specific embodiment, a peptide of formula (I) comprising a total of 5 to 10 amino acids is provided, which may be represented by the common amino acid sequence of SEQ ID NO:1.

[0078] According to another specific aspect, a peptide of formula (I) is provided, wherein Z1 is absent.

[0079] According to another specific aspect, a peptide of formula (I) is provided, wherein Z2 is absent.

[0080] According to another specific aspect, a peptide of formula (I) is provided, wherein Z1 and Z2 are absent.

[0081] According to another specific aspect, a peptide of formula (I) is provided, wherein Xaa4 is Ala.

[0082] According to another specific aspect, a peptide of formula (I) is provided, wherein Xaa5 is Arg.

[0083] According to another specific aspect, a peptide of formula (I) is provided, wherein Xaa5 is Lys.

[0084] According to another specific aspect, a peptide of formula (I) is provided, wherein Xaa8 is Arg.

[0085] According to another specific aspect, a peptide of formula (I) is provided, wherein Xaa5 and Xaa8 are Arg.

[0086] According to another specific aspect, a peptide of formula (I) is provided, wherein Xaa5 is Lys and Xaa8 is Arg.

[0087] According to another specific aspect, a peptide of formula (I) is provided, wherein Xaa7 is Trp.

[0088] According to another specific aspect, a peptide of formula (I) is provided, wherein at least one, at least two, at least three, at least four, or at least five amino acids are D-amino acids.

[0089] According to another specific aspect, a peptide of formula (I) is provided, wherein five amino acids are D-amino acids.

[0090] According to another specific aspect, a peptide of formula (I) is provided, wherein all amino acids are D-amino acids.

[0091] According to another embodiment, the peptide of the present invention of formula (Ia) is provided:

[0092]

[0093] Wherein Z is as described herein, and R1 is selected from OH and amino groups, such as NH2, and where at least one, at least two, at least three, at least four, or at least five amino acids are D-amino acids.

[0094] According to another embodiment, the peptide of the present invention of formula (IIa) is provided:

[0095]

[0096] Wherein Z is as described herein, and R1 is selected from OH and amino groups, such as NH2, and where at least one, at least two, at least three, at least four, or at least five amino acids are D-amino acids.

[0097] Depending on other specific aspects, the cellular permeable portion Z is the fatty acid portion.

[0098] According to another specific aspect, the fatty acid portion is myristoyl.

[0099] In another specific embodiment, the peptide of the present invention is provided, said peptide being the peptide of SEQ ID NO:2 (peptide P4).

[0100] In another specific embodiment, the peptide of the present invention is provided, said peptide being the peptide of SEQ ID NO:4 (peptide P3).

[0101] According to one embodiment, the compounds of the present invention can be prepared by synthetic methods, particularly by solid-phase peptide synthesis. According to another embodiment, a non-commercial cell-permeable fraction can be prepared separately using standard methods prior to transplantation.

[0102] According to a specific embodiment, the compound of the present invention is an inhibitor of protein kinase Cζ (PKCζ).

[0103] According to a specific embodiment, the compound of the present invention is a transient tight-linkage opener.

[0104] Composition

[0105] The pharmaceutical compositions of the present invention may comprise one or more compounds according to the present invention and their pharmaceutically acceptable carriers, diluents or excipients.

[0106] Depending on a particular aspect, the composition may further comprise compounds that can be used to treat medical conditions or that can be used in vaccines.

[0107] According to certain aspects, the compositions of the present invention are anticancer compositions.

[0108] According to certain aspects, the compositions of the present invention are anti-opioid drug compositions.

[0109] According to certain aspects, the compositions of the present invention are ophthalmic compositions.

[0110] According to certain aspects, the compositions of the present invention are oral compositions.

[0111] According to another specific aspect, the compositions of the present invention are vaccine compositions, particularly mucosal vaccine compositions, such as vaccine compositions.

[0112] The compositions of the present invention may further comprise at least one agent that can be used to treat cancer, particularly epithelial cancer.

[0113] According to another specific aspect, the compositions of the present invention may further comprise at least one medicament that can be used to treat nervous system diseases or conditions, particularly neurodegenerative diseases or neuropsychiatric disorders.

[0114] According to another specific aspect, the compositions of the present invention may further comprise at least one agent that can be used to treat opioid use disorders, particularly opioid overdose or opioid dependence.

[0115] According to another specific aspect, the compositions of the present invention may further comprise at least one ophthalmic medication that can be used to treat ocular conditions such as uveitis.

[0116] According to another specific aspect, agents that can be used to treat cancer, particularly epithelial cancer, are selected from alkylating agents, angiogenesis inhibitors, antibodies (such as anti-tumor monoclonal antibodies selected from bevacizumab, dalizumab, etc.), antimetabolites, antimitotic drugs, antiproliferative drugs, aurora kinase inhibitors, apoptosis promoter (e.g., Bcl-xL, Bcl-w, and Bfl-1) inhibitors, death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell connective) antibodies, biological response modulators, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, demethylating agents, and histone deacetylases (HDACs). Inhibitors, hormone therapies, immunotherapies, apoptosis protein inhibitors (IAPs) of inserted antibiotics, kinase inhibitors, mammalian targets of rapamycin inhibitors, microRNA mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly(adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum-based chemotherapy drugs, polo-like kinase (Plk) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids / trigonelline alkaloids, small inhibitory ribonucleic acid (siRNA), topoisomerase inhibitors, agents used in dendritic cell therapy, or any other active substance suitable / approved for the treatment of cancer, such as those listed in WO 2011 / 156761. In particular, the drugs that can be used to treat cancer, especially cancer, are selected from trastuzumab, ramucirumab, docetaxel, doxorubicin hydrochloride, fluorouracil (5-FU), erlotinib tablets, afatinib, gefitinib, bevacizumab, crizotinib, ceritinib, cetuximab, nivolumab, pembrolizumab, methotrexate, and bleomycin.

[0117] According to a particular aspect, a pharmaceutical composition according to the invention is provided, wherein the agent for treating epithelial cancer is selected from proteins (e.g., antibodies), kinases, designed ankylosing spondylins (DARPins), small molecules, or any other active substance suitable / approved for cancer treatment.

[0118] According to other specific embodiments, a pharmaceutical composition according to the invention is provided, comprising at least one peptide of the invention and at least gefitinib.

[0119] According to another specific embodiment, a pharmaceutical composition according to the invention is provided, comprising at least one peptide of the invention and at least buserelin acetate.

[0120] According to another specific aspect, medications that can be used to treat opioid use disorder are anti-opioid drugs, such as opioid receptor antagonists or opioid receptor modulators.

[0121] According to another specific aspect, antiopioids are selected from naloxone and buprenorphine or combinations thereof.

[0122] According to another specific aspect, the agents that can be used to treat neurological diseases or conditions are selected from naltrexone, sumatriptan, zolmitriptan, nicotine, midazolam, lorazepam, fentanyl, ketamine, ketorazole, butorphanol, and hydromorphone.

[0123] According to a particular aspect, the compositions of the present invention may further comprise at least one agent selected from the group consisting of: drugs for the prevention and / or treatment of diseases or conditions related to alcohol use (e.g., naltrexone), anaphylactic shock (such as pinephrine, phentolamine, or entacapone), migraines (such as sumatriptan or zolmitriptan), perennial and seasonal allergic rhinitis (such as budesonide, beclomethasone dipropionate and its monohydrate (micronized), mometasone furoate, triamcinolone acetate, fluticasone propionate, fluticasone furoate, fluticasone with azelastine hydrochloride or sodium cromoglycate), nicotine withdrawal symptoms (such as nicotine), hypoglycemia (such as glucagon), epilepsy (such as midazolam or lorazepam), or drugs for the prevention and / or treatment of endometriosis (such as nafarelin acetate for ovarian stimulation), or drugs for the control of pain (such as fentanyl, ketamine, ketodrolic acid, butorphanol, or hydromorphone).

[0124] According to another aspect, the compositions of the present invention may further comprise at least one of the following therapeutic peptides suitable for intranasal delivery (such as desmopressin acetate, glucagon-like peptide-1 (GLP-1), interferon β, or those listed in Maggio et al., 2006, Expert Opinion on Drug Delivery, 3(4):529-539, or Lochhead et al., 2012, Advanced Drug Delivery Reviews, 64:614–628), particularly hormones and their analogues or derivatives (such as insulin, glucagon, angiotensin), interferons (such as interferon β), bioactive peptides (such as growth factors, interleukins, enzymes, etc.), compounds or molecules that regulate the function of neurotransmitters or neuroion channels in the central nervous system (such as antidepressants (bupropion), neurotransmitter receptor agonists / antagonists, antiepileptic drugs (topiramate, zonisamide), etc.) and any other active agents, for example, those listed in US 2008 / 0299079.

[0125] The compositions of the present invention may further comprise at least one agent that can be used for vaccination, particularly for mucosal vaccination, such as recombinant B subunit of cholera toxin and inactivated Vibrio cholerae O1 (Inaba and Ogawa serotypes), whole-cell killed Vibrio cholerae O1 and Vibrio cholerae O139, attenuated live rotavirus p1a(8), g1-g4 or rix 4414, attenuated live Salmonella Ty21a, attenuated live influenza virus, attenuated live monovalent or pentavalent rotavirus, attenuated live trivalent, bivalent and monovalent poliovirus, attenuated live Salmonella Tylerae, and inactivated Vibrio cholerae O1 classical and El Tor biotypes with or without cholera toxin B subunit (Mevyn et al., 2014, Human Vaccines & Immunotherapeutics, 10(8):2175–2187; Sae-Hae et al., 2014, Experimental & Molecular Medicine, 46:e85).

[0126] According to other specific aspects, a composition is provided comprising at least one therapeutic active agent and at least one peptide according to the invention, wherein the amount of the peptide according to the invention is from 0.01% to 20% w / v based on the weight of the active agent.

[0127] According to other specific aspects, a composition is provided comprising at least one therapeutic active agent and at least one peptide according to the invention, wherein the amount of the peptide according to the invention is from 0.01% to 80% w / v based on the weight of the active agent.

[0128] The compositions of the present invention may further comprise one or more pharmaceutically acceptable additional ingredients, such as alum, stabilizers, antimicrobial agents, buffers, colorants, flavorings, adjuvants, etc.

[0129] The compositions of the present invention can also be formulated for parenteral administration, including but not limited to injection or continuous infusion. Injectable formulations may be in the form of suspensions, solutions, or emulsions in oily or aqueous solvents, and may contain formulations, including but not limited to suspending agents, stabilizers, and dispersants. The compositions may also be provided in powder form, reconstituted with a suitable solvent, including but not limited to sterile, pyrogen-free water.

[0130] The compositions of the present invention can be formulated for inhalation and may be in the form of, but not limited to, solutions, suspensions or emulsions, and may be administered in dry powder form or as aerosols using propellants (such as dichlorodifluoromethane or trichlorofluoromethane).

[0131] According to a specific embodiment, the composition according to the invention is used for intratumoral injection.

[0132] According to specific embodiments, the compositions according to the invention are used for mucosal surface delivery. According to specific embodiments, the compositions according to the invention can be used for the delivery of biological agents or macromolecules across the intestinal epithelial barrier, particularly for the oral delivery of biological agents (such as peptides, hormones, and antibodies).

[0133] In another specific aspect, the compositions according to the invention are suitable for delivery by single or multiple administration.

[0134] Optionally, the compositions of the present invention can also be formulated into atomizable solutions or inhalable pharmaceutically acceptable compositions. In such formulations, the compounds according to the invention are prepared, for example, into inhalable dry powders or atomizable solutions. In particular, compositions suitable for nasal delivery can be formulated into drops (e.g., eye drops), sprays, gels, suspensions, emulsions, microemulsions, micelle formulations, liposome formulations, powders, microparticles, and nanoparticles.

[0135] According to a specific embodiment, the composition of the present invention is a veterinary composition.

[0136] Further details regarding materials and formulation processing techniques are described in Part 5 of Remington's "The Science and Practice of Pharmacy," 22nd edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, which is incorporated herein by reference.

[0137] The present invention provides peptides, compositions thereof, and methods of using thereof, which can be used to treat medical conditions, particularly as tissue penetration enhancers of therapeutically active substances, especially in combination with anticancer agents, antiopioid drugs, or as adjuvants in vaccine compositions.

[0138] This invention provides the peptides of the invention, compositions thereof, and methods of using thereof, which can be used to treat medical conditions, particularly cancer, or during vaccination.

[0139] Application method

[0140] The compositions of the present invention can be administered or delivered in any manner, including but not limited to oral, parenteral, sublingual, transdermal, transmucosal, topical, inhalation, buccal or nasal administration, or combinations thereof. Parenteral administration includes, but is not limited to, intratumoral, intravenous, intraarterial, intraperitoneal, subcutaneous, and intramuscular administration.

[0141] In another specific embodiment, the compound according to the invention is administered systemically by injection.

[0142] In another specific embodiment, the compound according to the invention is administered by inhalation.

[0143] In another specific embodiment, the compound according to the invention is applied across the mucous membrane.

[0144] In another specific embodiment, the compound according to the invention is administered intranasally.

[0145] In another specific embodiment, the compound according to the invention is administered intratumorally.

[0146] In another specific embodiment, the compound according to the invention is applied topically, particularly to the eyes.

[0147] In another specific embodiment, the compound according to the invention is administered orally.

[0148] In one specific embodiment, the method according to the invention is a method of administering a compound according to the invention to a lung tumor of a subject, including bronchoscopic-guided intratumoral injection of the compound or a combination thereof.

[0149] The dosage administered to an individual (in the form of a single or multiple doses) varies depending on a number of factors, including pharmacokinetic properties, the subject’s condition and characteristics (sex, age, weight, health status and body size), symptom severity, concurrent or combined therapy, treatment frequency and expected effect.

[0150] combination therapy

[0151] According to one aspect, the compounds of the present invention are to be administered in combination with at least one therapeutic molecule that can be used to prevent and / or treat diseases.

[0152] According to one aspect, the compounds of the present invention are administered in combination with at least one therapeutic molecule that can be used to prevent and / or treat cancer, particularly epithelial cancer.

[0153] According to one aspect, the compounds of the present invention are to be administered in combination with therapeutic molecules that can be used for vaccination, particularly for mucosal vaccination.

[0154] According to another aspect, the compounds of the present invention are to be administered in combination with at least one therapeutic molecule that can be used to prevent and / or treat nervous system diseases or conditions, particularly neurodegenerative diseases or neuropsychiatric disorders.

[0155] According to another aspect, the compounds of the present invention are to be administered in combination with at least one therapeutic molecule that can be used to prevent and / or treat opioid use disorder, particularly an antiopioid suitable for intranasal delivery.

[0156] According to another aspect, the compounds of the present invention are to be administered in combination with at least one medicament that can be used to treat neurological diseases or conditions, particularly neurodegenerative or neuropsychiatric diseases, and is suitable for intranasal delivery. For example, the compounds of the present invention are used to enhance the delivery of those medicaments that are delivered intranasally across the blood-brain barrier (BBB).

[0157] This invention covers the administration of the compounds of this invention, wherein the compounds are administered to a subject before, simultaneously with, or sequentially to a treatment regimen or at least one co-agent. Compounds of this invention administered simultaneously with said at least one co-agent may be administered in the same or different compositions and via the same or different routes of administration.

[0158] According to one aspect, the compounds of the present invention can be administered simultaneously, optionally simultaneously, in the same composition with at least one therapeutic molecule that can be used to treat lung cancer.

[0159] According to other specific aspects, the compounds of the present invention can be administered intratumorally under bronchoscopic guidance.

[0160] In other specific embodiments, the compounds according to the invention are applied topically to treat eye diseases or conditions, such as uveitis.

[0161] The compounds according to the invention can be administered simultaneously with at least one vaccine composition, optionally simultaneously in the same composition.

[0162] According to another aspect, the compounds of the present invention can be used in combination with at least one therapeutic molecule that can be used to prevent and / or treat opioid use disorder, particularly one or more antiopioids suitable for intranasal delivery, optionally simultaneously in the same composition.

[0163] patient

[0164] In this embodiment, the subject according to the invention has epithelial cancer or is at risk of developing epithelial cancer.

[0165] In other embodiments, the subject according to the invention has cancer or is at risk of having cancer, the cancer being selected from breast cancer, prostate cancer, lung cancer, pancreatic cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, colorectal cancer, head and neck cancer, and other solid tumors.

[0166] In other embodiments, the subject according to the invention has lung cancer or is at risk of developing lung cancer.

[0167] In other embodiments, the subject according to the invention has gastric cancer or is at risk of developing gastric cancer.

[0168] In other embodiments, the subject according to the invention suffers from a neurological disease or condition, particularly a neurodegenerative disease or neuropsychiatric disease, or is at risk of suffering from a neurological disease or condition, particularly a neurodegenerative disease or neuropsychiatric disease.

[0169] In other embodiments, the subject according to the invention has an opioid use disorder or is at risk of developing an opioid use disorder.

[0170] In other embodiments, the subject according to the invention suffers from an eye condition such as uveitis or is at risk of developing an eye condition such as uveitis.

[0171] In another embodiment, subjects according to the invention are vaccinated with mucosal vaccines, such as vaccines against influenza virus, rotavirus, Vibrio cholerae, Salmonella typhi, or poliovirus infection.

[0172] According to the uses of the invention

[0173] The compounds according to the invention can be used to enhance the effects of therapeutic molecules, particularly those used for the prevention and / or treatment of any disease, especially those used for the prevention and / or treatment of cancer (e.g., epithelial cancer) or opioid use disorder or vaccination.

[0174] According to another aspect, compounds according to the invention may be used in view of the agent crossing the blood-brain barrier (BBB), transdermal delivery, or in view of improving local anesthesia and improving the diffusion of anesthetic agents through tissues.

[0175] According to certain aspects, the peptides of the present invention have several advantages over known PKCζ pseudosubstrates, some of which have been used as tools for studying the role of PKCζ in tight junction regulation and have been described as capable of disrupting tight junctions in the mouse ileum (Jain et al., 2011, Biochem J., 437(2), 289-299), but are not recommended for use as permeation agents, or even for use in combination with therapeutic or vaccine macromolecules to enhance their efficacy. Another aspect of the invention relates to a method for enhancing the efficacy of treatment for a subject suffering from a disease or condition, the method comprising administering a compound according to the invention or a pharmaceutical formulation thereof in combination with a therapeutically effective agent targeting the disease or condition of the subject, wherein the tissue permeation of the therapeutically effective agent is enhanced compared to the tissue permeation of the therapeutically effective agent when administered in the absence of the compound of the invention.

[0176] The references cited herein are incorporated herein by reference in their entirety. This invention is not limited to the specific embodiments described herein, which are intended to serve as a single illustration of various aspects of the invention, and functionally equivalent methods and compositions are also within the scope of the invention. In fact, various modifications to the invention will become apparent to those skilled in the art, in addition to those shown and described herein, as previously stated. Such modifications are intended to fall within the scope of the appended claims. Having described the invention, the following embodiments are illustrative only and not limiting.

[0177] Example

[0178] The following abbreviations refer to the following definitions:

[0179] BSA (Bovine serum albumin); Caco-2 (human intestinal epithelial cells); CTRL (control); DIEA (N,N-diisopropylethylamine); DCM (dichloromethane); DMF (N,N-dimethylformamide); EDT (ethylene dithiol); EGFR (epidermal growth factor receptor); FD (fluorescein-conjugated dextran); Fmoc (9-fluorenylmethoxycarbonyl); HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate); HOBT (hydroxybenzotriazine); in (intranasal); myr (myristoyl); NSCLC (non-small cell lung cancer); OCA_EGFR19del (cell culture with tumors carrying EGFR ex19:del mutation); Ova (ovalbumin); P app (Apparent permeability); PBS (phosphate-buffered saline); PKCζ (protein kinase Cζ type); PS (pseudosubstrate); TEER (transepithelial resistance); TIS (triisopropylsilane); TFA (trifluoroacetic acid); TJ (tight junction); ZO-1 (closed band-1).

[0180] Example 1: Synthesis of the compound according to the present invention

[0181] The compounds of the present invention were prepared by solid-phase peptide synthesis. As an illustration, the synthesis steps of peptide P4 (SEQ ID NO:2) are provided as follows:

[0182] Step 1 - Wash the reaction vessel with dichloromethane (DCM), purge the bottom with nitrogen, and then drain it completely.

[0183] Step 2 - Resin swelling: Weigh the 2-chlorotriphenylmethyl chloride resin into the reaction vessel, and then swell the resin with dimethylformamide (DMF; 15 ml / g) for 30 min.

[0184] Step 3 - Coupling the first D-amino acid from the C-terminus of the peptide: Weigh 1.6 g of Fmoc-L-Arg(Pbf)-OH into a test tube, and dissolve the Fmoc (9-fluorenylmethoxycarbonyl)-amino acid in DMF / DCM (Sigma-Aldrich) (1:1) (15 ml / g). Transfer the solution to the above reaction vessel, add 10 times the amount of DIEA (N,N-diisopropylethylamine), and mix with nitrogen at room temperature for 30 min.

[0185] Step 4 - Blocking the active sites of the resin: Add 5 mL of methanol to the reaction vessel and purge the bottom for 10 min. Drain the reaction vessel and wash with DMF (3×), DCM (3×), and DMF (3×).

[0186] Step 5 - Deprotection: Drain the reaction vessel and add 20% piperidine (15 ml / g) to remove the Fmoc protecting group. Purge the bottom of the mixture for 10 min × 1 and 5 min × 1. Then wash the reaction vessel with DMF (3×), DCM (3×), and DMF (3×).

[0187] Step 6 - Coupling Monitoring: Take a resin sample, add 2 drops of 25% ninhydrin-alcohol solution and 1 drop of 20% phenol-alcohol solution, then add 1 drop of pyridine. Heat the sample at 105℃ for 5 minutes. A dark blue color indicates a positive reaction, and no color change indicates no reaction.

[0188] Step 7 - Condensation: Add 3 times excess of protected amino acids, 5g of HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate), HOBT (hydroxybenzotriazole) (1g), and DIEA (2ml) to DMF and dissolve them. Then add DCM (15ml / g) and allow the mixture to react for 1 hour.

[0189] Step 8 - Washing: Wash the reaction vessel three times alternately with DCM (15 ml / g) and DMF (15 ml / g).

[0190] Step 9 - Monitoring: Same as step 6.

[0191] Step 10 - Couple the remaining D-amino acids: Repeat steps 5-9 to couple the other amino acids.

[0192] Step 11 - Link the myr group to the N-terminus of the peptide.

[0193] Step 12 - Washing: After coupling with the last amino acid (the first amino acid at the N-terminus) and deprotecting it sequentially with the following reagents, wash the resin: twice with DMF (10 ml / g), twice with methanol (10 ml / g), twice with DMF (10 ml / g), twice with DCM (10 ml / g), and then dry it for 10 min.

[0194] Step 13 - Cleavage: Cleavage was performed using the following reagents: TFA 94.5% (trifluoroacetic acid), water 2.5%, EDT 2.5% (ethylene dithiol), and TIS 1% (triisopropylsilane). The lysis time was 2 hours.

[0195] Step 14 - Drying and Washing: Dry the lysis solution as much as possible with nitrogen gas, wash it 6 times with anhydrous ether, and dry it in air.

[0196] Step 15 - Purification by HPLC (High Performance Liquid Chromatography). The purified solution was dried by freeze-drying to obtain the product as a white powder.

[0197] HPLC purification

[0198] The crude peptide was dissolved in purified water and purified under the following conditions:

[0199] Pump A: 0.1% trifluoroacetic acid (TFA) in 100% water, 0.1% TFA in 100% aqueous solution.

[0200] Pump B: 0.1% trifluoroacetic acid, 0.1% TFA, and 100% ACN solution in 100% acetonitrile.

[0201] Preparation column: Venusi MRC-ODS C18 30 x 250mm

[0202] Preparation column: Venusi MRC-ODS C18 30 x 250mm

[0203] Total flow rate: 1.0 ml / min Flow rate: 1.0 ml / min

[0204] Loading volume: 3ml Sample volume: 3ml

[0205] Detection wavelength: 220nm

[0206]

[0207] Other peptides of the present invention can be synthesized in a similar manner to those for producing the peptide of SEQ ID NO:1, using different or additional amino acids. Grafting of the cell permeation portion can be achieved by standard methods known to those skilled in the art, such as solid-phase synthesis in the case of peptide cell permeation portions, or as described in this specification.

[0208] The compounds of the present invention can also be prepared by chemically selective linking synthesis (Bonnet et al., 2001, ibid.).

[0209] Example 2: The effect of increasing peptide lengths on the membrane permeability of macromolecules

[0210] To assess the potential role of the peptides of the present invention in penetrating therapeutic molecules, FITC insulin (Sigma-Aldrich, Buchs SG, Switzerland) was used as a model to evaluate transepithelial bypass drug transport (apex to basal lateral) across the epithelial monolayer.

[0211] To ensure the integrity of the monolayer during the experiments, transepithelial resistance (TEER) was measured before and after these studies, as described below.

[0212] Using Mucilair TM In human primary nasal and bronchial epithelial cells (Epithelixsarl, Geneva, Switzerland) and Caco-2 human intestinal epithelial cells (Huang et al., 2013, Toxicol. in Vitro 27:1151-1156).

[0213] Before each experiment, the culture medium was removed from each chamber, and the monolayer was washed once with 200 μl of physiological saline (0.9%) and once with warm Hank balanced salt solution (HBSS) (37°C). In the basolateral chamber, 600 μL of preheated HBSS was placed, and the cells were returned to an incubator at 37°C for 30 minutes to equilibrate. After equilibration, the peptides of the present invention and the cell bypass marker (FITC insulin) were applied to the apical side of the epithelial cell monolayer. The peptides P4 (SEQ ID NO:2) and P3 (SEQ ID NO:4) according to the present invention were detected compared with the comparative peptide CP4 (SEQ ID NO:3).

[0214] All peptides were used at a final concentration of 50 μM. The solvent was used as a control (CTRL). FD solution was added to the top chamber to make a final volume of 200 μl. Every 30 minutes over a 150-minute period, 100 μL of sample was taken from the base chamber of each well and replaced with an equal volume of fresh, warm buffer to maintain the leak. Fluorescence of FD was measured in black 96-well plates using a fluorescence microplate reader (BioTek Synergy Mx microplate reader, BioTek Instruments GmbH, Lucerne, Switzerland) at excitation and emission wavelengths of 485 and 520 nm, respectively. The cumulative release (ng) corresponds to the actual cumulative amount of drug released and is calculated as the amount in the suspension at any given time plus the amount of drug lost during each sampling.

[0215] like Figure 2 As shown, compared to the control, the comparative peptide CP4 slightly increased the penetration of the cellular bypass marker insulin FITC. Unexpectedly, the D-amino acid peptides of the invented P3&P4 increased the penetration of insulin FITC to a greater extent compared to the control peptide CP4.

[0216] Transepithelial resistance (TEER): After adding 200 μl of culture medium to the top chamber of the tissue culture, the resistance of the entire culture was measured three times at each time point using an EVOMX voltmeter (World Precision Instruments UK, Stevenage). TEER values ​​(Ω) were normalized using the following formula: TEER (Ωcm) 2 ) = (resistance value (Ω) - 100 (Ω)) × 0.33 (cm) 2 ), where 100Ω is the resistance of the membrane, and 0.33cm 2 It is the total surface area of ​​the epithelium.

[0217] Example 3: The role of membrane permeation groups

[0218] To evaluate the role of the membrane-permeable group (myristoyl group) in the peptides of the present invention in enhancing the permeability of macromolecules through the epithelial cell layer, the permeability of peptide P4 (SEQ ID NO:2) of the present invention was compared with that of a comparative peptide corresponding to the same peptide without the myristoyl group. Uptake experiments and TEER were performed as described in Example 2.

[0219] Example 4: Cell bypass permeability time frame

[0220] To assess the duration of the permeation effect of the peptides of the present invention, the apparent permeability of peptides of various lengths was evaluated as follows. Apparent permeability refers to the amount of FD released at the time point when the sample was collected. In this example, it indirectly represents the gradual closure of the bypass space of tightly connected cells, as the amount of FD released gradually decreases, as observed at the 30 or 60 minute time points.

[0221] The peptides detected, uptake assays, and TEER were as described in Example 2.

[0222] Apparent permeability of FD: The apparent permeability of FD (P) is calculated using Formula 1. app ):P app＝ (dQ / dt) / A*C0(1)

[0223] Assuming that since the peptide corresponds to a PKC pseudosubstrate, which may be a competitive substrate, the longer the pseudosubstrate sequence, the greater the inhibition of PKCζ, and thus the greater the increase in the cellular bypass permeability of the macromolecule. Therefore, the peptide of the present invention has an optimized length, which allows for the achievement of the desired cellular bypass permeability of the macromolecule while exhibiting greater reversibility of this effect, which is desirable for avoiding cytotoxicity (the longer the duration of PKCζ inhibition, the more likely it is to lead to increased degradation of TJ protein and affect cell proliferation) (Suzuki et al., 2002, J. Cell Sci., 115:3565-3573; Whyte et al., 2010, J. Cell Sci., 123:3316-3328).

[0224] Example 5: Effects of PKCζPS peptide on cell viability

[0225] The potential toxicity of the peptides of the present invention compared with comparative peptides was assessed by cell viability assays as follows.

[0226] The viability of Caco-2 cells (human intestinal epithelial cell line) was determined by a cell proliferation assay using a WST-1-based colorimetric method. Caco-2 cells (5*10⁻⁶) were... 3Cells were seeded at 1000 cells / well in 96-well multi-well plates and treated with peptide P4 and comparative peptide CP4 at different concentrations (i.e., 10, 50, and 100 μM) for 24 hours (long-term cell viability studies). WST-1 reagent (diluted 1:10 in cell culture medium) was added as described in the instruction manual (Roche Diagnostics GMBH, Mannheim, Germany). After incubation with the peptide or control at 37°C for 1–3 hours, absorbance was measured at 450 nm (690 nm as reference) using a BioTek Synergy Mx microplate reader. Cell viability percentage was calculated based on absorbance measurements relative to cells exposed only to culture medium (control group). 2% sodium dodecyl sulfate (SDS) was used as a positive control for cytotoxicity.

[0227] Example 6: Effect of the peptides of the present invention on the redistribution of tight junction proteins occlusion proteins and ZO-1

[0228] The effects of the peptides of the present invention on the tight junction structure of human primary nasal and bronchial epithelial cells were evaluated using confocal microscopy as follows.

[0229] Mucilair TM Human primary nasal and bronchial epithelial cells (Epithelix Sarl, Geneva, Switzerland) were incubated with 50 μM of the present invention peptide P4 or the comparative peptide CP4 for 2 hours, followed by incubation with phosphate-buffered saline (PBS) (Ca-free). 2+ / Mg 2+Wash twice at 37°C. Fix cells with methanol / acetone (50:50) at 20°C for 5 minutes, air dry, and wash with TBST (a mixture of Tris buffer (TBS) and polysorbate 20). Block cell monolayers with 3% bovine serum albumin (BSA) in PBS solution for 60 minutes at room temperature and incubate in dilution buffer with primary antibodies against occlusion protein (Cat: 331588, Invitrogen, Zug, Switzerland; dilution (1:200)) and ZO-1 (occlusion band-1 protein) (Cat: 339194, Invitrogen, Zug, Switzerland; dilution (1:200)). Samples were fixed (on Vectashield fixation medium containing DAPI (4',6-diamidinyl-2-phenylindole); Vector Laboratories) and evaluated over the next 24 hours using a laser scanning confocal microscope (CLSM; Plan-Apochromat 63 / 1.40 (CLSM; Plan-Apochromat 63 / 1.40 (oil) DIC objective, Zeiss Axiovert 100M-LSM 510; Carl Zeiss, Oberkochen, Germany). For each sample, at least five individual image capture locations were randomly selected in areas of uniform monolayer thickness. To establish comparable conditions between individual cell monolayers, equivalent images of the same number of horizontal sections (512 x 512 pixels) with the same vertical depth from the apex to the basement membrane were required between unstimulated and stimulated monolayers.

[0230] Example 7: The therapeutic effect of combination therapy containing the peptides of the present invention

[0231] The effects of the peptide of the present invention in combination with gefitinib (N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazoline-4-amine), a protein kinase inhibitor known as a selective inhibitor of the epidermal growth factor receptor tyrosine kinase domain (EGFR-TK), were investigated in a non-small cell lung cancer model as follows.

[0232] Gefitinib is a cytotoxic small molecule that was first approved by the U.S. Food and Drug Administration (FDA) in 2003 as a third-line treatment for non-small cell lung cancer (NSCLC), the most common type of lung cancer, and more recently as a first-line treatment (July 2015). Gefitinib has been shown to significantly improve progression-free survival (PFS = 7.7–12.9 months) compared to chemotherapy before resistance develops. Clinically, the standard dose of gefitinib is 250 mg / day, while in vitro, gefitinib exhibits micromolar (μM) inhibitory concentrations.

[0233] HCC827 cells (lung adenocarcinoma) with mutations in the tyrosine kinase domain of EGFR (epidermal growth factor receptor) (model OncoCilAir) were used. TM Non-small cell lung cancer (NSCLC) cells were labeled with green fluorescent protein (GFP). A total of 24 OncoCilAir tumors containing EGF receptor ex19:del mutations (denoted as OCA_EGFR19del) were included. TM Cultures (Mas et al., 2015, J. Biotechnol., 205:111-119) were treated for 14 days with gefitinib (or ZD-1839, “Iressa”) (Selleckchem (Luzern, Switzerland)) in combination with or without peptide P4. OCA_EGFR19del cultures were prepared from DMSO (dimethyl sulfoxide) stock solution diluted in medium at two final concentrations of 1 μM and 5 μM. Peptide P4 was administered top-side, and both administration methods were tested for gefitinib (either on the basal side or top-side of the insert). Starting with 200 μM stock solution, peptide P4 was used in medium at a final concentration of 1 μM. P4 was administered to the culture exactly 5 minutes before gefitinib administration.

[0234] Tumor morphometry was analyzed using fluorescence microscopy on the Zeiss Axiocam microscopy platform. Growth curves were based on various images acquired every 2 days, and green fluorescent protein positivity (GFP) was measured using Image-Pro Plus software (MediaCybernetics, Rockville, MD, USA). + The tumor area was calculated. For each time point, the ratio of the total area occupied by the tumor at the start of treatment to the insert size was calculated and expressed as a percentage on day 0 (tumor occupancy). The same analytical settings were used, i.e., a fixed fluorescence intensity threshold and a fixed size threshold (>900m). 2 This was applied to all processed images. The percentage of tumor growth inhibition was calculated using the following formula: (1 - [tumor occupancy in the treatment group / tumor occupancy in the control group] × 100).

[0235] Example 8: Adjuvant effect of the peptide of the present invention on antigen-specific serum IgG and IgG1 responses

[0236] The effects of the peptides of this invention on the efficacy of mucosal vaccines were studied by administering P4 in combination with soluble protein antigens, and the induced immunity has been investigated.

[0237] Six-week-old female C57-BL / 6 mice were purchased from Charles River Laboratories (Harlan, France) and housed under standard conditions in accordance with the relevant guidelines of the Animal Ethics Committee. Mice (n=5) were immunized intranasally under anesthesia using the following immunogens: physiological saline (PBS) as a negative control, a mixture of ovalbumin (Ova) (in 0.9% NaCl solution), or Ova+P4 Ova / dose (5 μg) (a mixture of Ova in 0.9% NaCl solution and P4 in 0.9% NaCl solution) at 12 μL (6 μL per nostril) for each group. P4 was used at a concentration of 5 μg / dose. Blood samples were collected one day before the first immunization and one week after the last immunization.

[0238] Antigen-specific serum antibodies (total IgG and IgG1) were measured by ELISA (enzyme-linked immunosorbent assay). In short, 96-well plates were coated overnight at 4°C with 100 ng of Ova antigen per well. The plates were blocked with 100 μl of DPBS (Dulbecco phosphate buffer) with 3% BSA (Sigma-Aldrich, Germany) at 37°C for 2 h, washed four times with wash buffer, and then incubated at 37°C for 1.5 h with 100 μl of serially diluted serum samples (1:50 to 1:819200 for IgG and IgG1). After four washes, the plates were incubated for 1 h with 100 μl of 1:8000 diluted HRP (horseradish peroxidase) conjugated anti-mouse IgG and IgG1 (Southern Biotech, France) antibody. The plate was washed four times, and HRP was quantified by adding 100 μl of TMB (3,3',5,5'-tetramethylbenzidine) substrate (Pierce Protein Research Products; Rockford, IL). After subtracting the original background, the antibody titer was determined at the midpoint of the optical density-log dilution curve. Unresponsive mice were given an arbitrary titer of 10.

[0239] Example 9: Intranasal delivery of the peptide combined with an antiopioid drug of the present invention

[0240] The following studies investigate the in vivo effects of intranasal delivery of the peptides of the present invention, alone or in combination with the antiopioid drug naloxone.

[0241] Female Wistar rats (weighing 225-250g) were anesthetized with a combination of ketamine and toluidine, and a cannula was inserted into the carotid artery. A three-way valve was inserted through the cannula to sample blood, which was then replaced with heparinized saline. Naloxone, alone or in combination with the peptide of this invention, was administered intranasally via the tip of a micropipette inserted 8 mm into the rat's nostril. Blood samples were collected before and at 5, 15, 30, 60, and 120 minutes after naloxone administration. Each blood sample (0.5 ml) was collected into a heparinized 1 ml syringe and then transferred to a frozen 1.5 ml polypropylene tube containing 10 μl of heparin (500 U / ml). The tubes were centrifuged at approximately 3,000 rpm for 20 minutes at 2-8°C, and the plasma supernatant was then transferred to a microcentrifuge tube stored at -200°C. The concentration and area under the curve of naloxone in the plasma were determined. C was determined by using HPLC (High Performance Liquid Chromatography) or LC-MS / MS (Liquid Chromatography-Mass Spectrometry). max (peak serum concentration of the drug) and T max (Observation C) max The value of the time) and the bioavailability value (compared to intravenous injection) are calculated from the area under the curve, which is obtained from the curve of plasma naloxone (or any other test drug) concentration over time.

[0242] Example 10: The effect of the peptide of the present invention on epithelial integrity

[0243] To assess the potential effect of the peptides of the present invention on epithelial integrity, transepithelial resistance was measured in the presence of the peptides of the present invention.

[0244] As in Example 2, the transepithelial resistance (TEER) was measured, except that the voltmeter was equipped with STX-2 chopstick electrodes (WPI, Sarasota, FL, USA). As in Example 2, a Mucilair was used... TM Human primary nasal epithelial cells (from a single donor; EP01-lot number MD069201). At the start of the experiment (t=0), peptide P4 (SEQ ID NO:2) and comparative peptide P4 (SEQ ID NO:3) were used in Mucilair. TM The 100 μM concentration in the culture medium and the control solution (i.e., 50 μl of physiological saline + 50 μl of Mucilair) TM Culture medium was applied to the top chamber of the tissue culture and measured after incubation for 20, 40, 60, and 80 minutes (t = 20 min, t = 40 min, t = 60 min, and t = 80 min). All TEER values ​​are expressed as a percentage relative to the control (baseline value was 242 Ω*cm). 2 All experiments were performed at least twice (tests 1 and 2).

[0245] Epithelial resistance is a measure of epithelial integrity and can indicate the regulation of epithelial tight junctions by a detected pharmacological agent (the peptide of the present invention), wherein a decrease in TEER value indicates an increase in cellular bypass space between tight junctions, which may therefore increase the transport of drugs and therapeutic macromolecules.

[0246] Compared to the control solution and comparative peptide P4 (CP4), peptide P4 decreased the TEER value at t = 20 min, and this effect persisted at t = 40 min and t = 60 min. Figure 1 Compared to values ​​measured at previous time points, the TEER value increased at t = 80 min (). Figure 1 ).

[0247] These results support the idea that the peptides of the present invention can increase the opening of epithelial tight junctions (an increase in the cellular bypass space between tight junctions), which in turn can allow the delivery of other molecules. The observed transient effects suggest that those peptides will be able to induce transient opening of tight junctions.

[0248] sequence list

[0249] SEQ ID NO:1 (common sequence) / Formula (I)

[0250] Z-[Xaa1 Xaa2 Xaa3] 0-1 -Xaa4Xaa5R Xaa7 Xaa8-[Xaa9 Xaa 10 ] 0-1

[0251] (I)

[0252] Where Z represents the cellular osmotic fraction; Xaa1 and Xaa2 may or may not be present, and when present, Xaa1 and Xaa2 are independently positively charged amino acids, and Xaa3 is a nonpolar amino acid; Xaa4 is an amino acid selected from Ala, Ser, and Val; R is arginine; Xaa5 and Xaa8 are independently positively charged amino acids; Xaa7 is a nonpolar amino acid; Xaa9 is a positively charged amino acid, and Xaa... 10 It may or may not exist, and when it exists, Xaa 10 It is a nonpolar amino acid, wherein at least one amino acid in formula (I) is a D-amino acid.

[0253] SEQ ID NO:2(P4)

[0254] Myr-D-Ala D-Lys D-Arg D-Trp D-Arg

[0255] SEQ ID NO:3 (Comparative peptide CP4)

[0256] Myr-AKRWR

[0257] SEQ ID NO:4(P3)

[0258] Myr-D-Ala D-Arg D-Arg D-Trp D-Arg sequence list <110> University of Geneva <120> Peptide kinase C inhibitors and their uses <130> P2249PC00 <150> EP18177281.5 <151> 2018-06-12 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> consensus sequence <220> <221> MISC_FEATURE <222> (1)..(1) <223> Cellular permeable portion covalently linked to groups of N-terminal amino acids <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is an optional peptide sequence Xaa1 Xaa2 Xaa3, where Xaa1 Xaa1 and Xaa2 may or may not exist, and when they exist, Xaa1 and Xaa2 are equal. Xaa2 is an amino acid with a positive charge; Xaa3 is... Nonpolar amino acids. <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is an amino acid selected from Ala, Ser, and Val. <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa is a positively charged amino acid. <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa is a nonpolar amino acid. <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa is a positively charged amino acid. <220> <221> MISC_FEATURE <222> (8)..(8) <223> Xaa is an optional peptide moiety [Xaa9 Xaa10], where Xaa9 is... Positively charged amino acids, and Xaa10 may or may not be present. Furthermore, when present, Xaa10 is a nonpolar amino acid, in which... At least one amino acid in SEQ ID NO: 1 is a D-amino acid. <400> 1 Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> P4 <220> <221> MISC_FEATURE <222> (1)..(1) <223> Myristoyl group covalently linked to the amino group of the N-terminal amino acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> D-Ala <220> <221> MISC_FEATURE <222> (2)..(2) <223> D-Lys <220> <221> MISC_FEATURE <222> (3)..(3) <223> D-Arg <220> <221> MISC_FEATURE <222> (4)..(4) <223> D-Trp <220> <221> MISC_FEATURE <222> (5)..(5) <223> D-Arg <400> 2 Xaa Xaa Xaa Xaa Xaa 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Comparison of peptide P4 <220> <221> MISC_FEATURE <222> (1)..(1) <223> Myristoyl group covalently linked to the amino group of the N-terminal amino acid <400> 3 Ala Lys Arg Trp Arg 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial sequence <220> <223> P3 <220> <221> MISC_FEATURE <222> (1)..(1) <223> Myristoyl group covalently linked to the amino group of the N-terminal amino acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> D‐No <220> <221> MISC_FEATURES <222> (2)…(2) <223> D‐Arg <220> <221> MISC_FEATURES <222> (3)…(3) <223> D‐Arg <220> <221> MISC_FEATURES <222> (4)..(4) <223> D‐Trp <220> <221> MISC_FEATURES <222> (5)…(5) <223> D‐Arg <400> 4 Blood Blood Blood Blood! 1 5

Claims

1. A peptide, wherein the peptide is a peptide of SEQ ID NO:2 or SEQ ID NO:

4.

2. The peptide according to claim 1, wherein the peptide is used as a drug or as a vaccine adjuvant.

3. A pharmaceutical composition comprising at least one peptide according to claim 1 and a pharmaceutically acceptable carrier, diluent or excipient thereof.

4. The pharmaceutical composition according to claim 3, wherein the composition is an ophthalmic or oral composition.

5. The pharmaceutical composition according to claim 3 or 4, wherein the pharmaceutical composition further comprises an anticancer agent.

6. The pharmaceutical composition according to claim 5, wherein the anticancer agent is gefitinib.

7. The pharmaceutical composition according to claim 3 or 4, wherein the pharmaceutical composition further comprises an antiopioid drug.

8. The pharmaceutical composition according to claim 7, wherein the opioid is naloxone.

9. The pharmaceutical composition according to claim 3 or 4, wherein the pharmaceutical composition further comprises a vaccine.

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