Disruption of the epithelial barrier for enhanced molecular delivery or extraction of extracellular fluid

Abstract

The present invention provides a method for delivering a payload molecule across an epithelial tissue barrier, the method comprising the steps of administering the payload molecule to the epithelial tissue barrier and further administering an agent to the epithelial tissue barrier. The agent is (i) a microbial quorum sensing signaling molecule (microbial QSSM) capable of disrupting epithelial tissue barrier function, or a derivative or variant thereof, or (ii) a carboxylic acid compound, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

Description

Technical Field

[0001] The present invention relates to methods of disrupting an epithelial tissue barrier for delivering payload molecules across the epithelial tissue barrier, reducing epithelial electrical resistance, or extracting extracellular fluid through an epithelial tissue barrier such as skin. The extracellular fluid can be used for assays such as glucose concentration or biomarker analysis. The reduced epithelial electrical resistance can be utilized to improve the sensitivity and signal-to-noise ratio of electrophysiological measurements. The present invention further relates to related compositions, uses, and processes.

Background Art

[0002] Treatment of serious diseases by modern precision medicine remains complicated because macromolecular therapeutics (non-limiting examples include immunotherapy, recombinant proteins, etc.) cannot cross tissue barriers (e.g., skin, lung, intestine, ocular surface, nose, oral, vagina) to reach their sites of action. Many of the most promising new therapeutics are large proteins such as monoclonal antibodies and recombinant proteins. The rapid growth of macromolecular protein-based drugs unfortunately does not coincide with the development of effective delivery systems for these new therapeutics. Most proteins are administered parenterally due to their high molecular weight that cannot permeate the epithelial barrier and their inability to withstand the acidic environment of the gastrointestinal tract. The drawbacks associated with the parenteral route, including patient discomfort and high cost, have stimulated the field to investigate non-invasive methods for administering macromolecular therapeutics.

[0003] The epithelium forms a barrier to protect tissues from ingested substances and pathogens. Those barrier properties result from a set of proteins (tight junctions, adherens junctions, and desmosomes) that limit the intercellular space between cells. The tight junction is the junctional complex that connects cells and provides a barrier to the free passage of molecules in the extracellular space. Tissue barriers are a formidable obstacle in drug delivery because drugs need to cross these barriers to reach their site of action and exert their therapeutic effects. The low permeability of polymeric therapeutics across tissue barriers inhibits the efficient treatment of costly prevalent diseases, including arthritis, macular degeneration, and cancer. The delivery of protein therapeutics across various epithelial barriers, including gastrointestinal, respiratory, nasal, and buccal epithelia, remains a challenge for both the pharmaceutical industry and clinical and academic communities.

[0004] Previous studies have shown that the integrity of epithelial cell junctions can be regulated by microbial quorum sensing signaling molecules (see Vikstrom, E. et al., Exp Cell Res, 2009. 315(2): 313-26; Vikstrom, E. et al., FEBS Lett, 2006. 580(30): 6921-8; Rejman, J. et al., Human Gene Therapy, 2007. 18(7): 642-652).

[0005] To communicate, bacteria secrete extracellular signaling molecules called autoinducers. This intercellular communication system, called "quorum sensing," helps bacteria estimate their population, monitor the environment, change gene expression, and as a result, change behaviors such as pathogenicity factor production and biofilm formation (see Whiteley, M. et al. Nature, 2017.551(7680):313 - 320; Papenfort, K. and B. L. Bassler, Nature Reviews Microbiology, 2016.14(9):576 - 588). The opportunistic human pathogen Pseudomonas aeruginosa uses acyl - homoserine lactone (AHL) quorum - sensing molecules to control and activate its gene expression.

[0006] To improve the permeability of polymeric therapeutics to and across tissue barriers, various strategies have been investigated. These include the use of absorption promoters, mucoadhesive excipients, and attempts to utilize epithelial transcytosis. Absorption (or permeation / permeability) promoters are a class of excipients that increase drug permeability across both epithelial and endothelial cell layers, leading to increased drug delivery to the systemic circulation.

[0007] Tight junctions can be disrupted by many agents, including toxins, cytokines, growth factors, surfactants, calcium chelators, polymeric vehicles such as chitosan, and some peptides. Disruption of tight junctions can enhance drug delivery, but permanent dysfunction of tissue barrier function often results from tissue disruption of tight junctions, which is not favorable for their general use in clinical medicine as drug delivery promoters. An ideal tight - junction modulator should transiently and reversibly disrupt the barrier properties of the epithelial layer.

[0008] There is a need to provide alternative, preferably improved, methods and compositions for disrupting the epithelial barrier, at least to overcome the above - mentioned problems and for delivering molecules across the epithelial barrier.

Summary of the Invention

[0009] The present invention has determined that a series of agents can enhance the paracellular permeability of payload molecules. This series of agents acts to translocate the ZO-1 protein and, without being bound by theory, may potentially act to impair the barrier function through induction of matrix metalloprotease (MMP) secretion.

[0010] The effectiveness of these agents has been demonstrated in relation to the paracellular permeability of 4 kDa FITC-dextran (FD4) across polarized Calu-3, ARPE-19, and Caco-2 cell layers, which are well-known in vitro models of the human airway, intestine, and retinal pigment epithelium. Surprisingly, macromolecular agents (aflibercept, bevacizumab, mepolizumab: approximately 150 kDa) have also been shown to be effectively delivered across the epithelial barrier by using the present invention. It would not have been expected that molecules of this size could be delivered. The present invention advantageously provides a general approach that can be used with a wide range of payload molecules. The ability to effectively transport drugs of various sizes by permeating the epithelial barrier is technically important and offers significant advantages over intravenous administration.

[0011] Advantageously, this series of agents according to the present invention has been found to reversibly cause modulation of tight junctions between barrier-forming cells and have a return to baseline TEER and barrier function against macromolecules. This is a beneficial and unexpected effect for the series of agents described herein. For being useful from a practical and therapeutic perspective, it is important to have a recovery phase, i.e., repair, since the disruption of the epithelial barrier needs to be transient rather than having permanent disruption or damage.

[0012] Thus, it is possible to "open" the epithelial barrier and allow payload molecules, such as therapeutic or prophylactic agents, to cross the epithelial barrier and be delivered to a desired target, after which the epithelial barrier returns to its normal configuration, i.e., "closes".

[0013] In addition to identifying this series of agents and demonstrating the ability to reversibly disrupt epithelial tissue barrier function using human cell lines, in order to facilitate the practical administration of these agents, we have successfully further tested this series of exemplary compounds both ex vivo and in vivo.

[0014] In this regard, N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL) and 2-n-heptyl-3-hydroxy-4(1H)-quinolone (C7 PQS), which are examples of two different classes of microbial quorum sensing molecules within the claimed series, have been confirmed to be effective in delivering drugs across the epithelial layer in in vitro and in vivo models, but experimental studies have also shown that they have a recovery phase. Experimental studies have shown that by including the agents according to the present invention, drugs that normally need to be injected can be successfully delivered using eye drops, and furthermore, no signs of toxicity are observed.

[0015] Thus, the agents according to the present invention provide a new and useful route for delivering payload molecules, such as therapeutic or prophylactic agents, to subjects such as human patients or companion or farm animals.

[0016] While being effective in enhancing the paracellular permeability of the payload molecule, the agent also has a recovery stage, that is, the destruction of the epithelial barrier is found by the inventors to be temporary. The agents have certain common structural features. They all have an alkane "tail" portion containing an R' group which is a C1-12 alkyl group, which may be unsubstituted or substituted, and any substituents present are independently selected from hydroxyl, halogen and NR''2, and each R'' is independently selected from hydrogen and methyl. Thus, this "tail" is saturated, contains only single bonds and is thus flexible. The agent also has either (i) a heterocyclic moiety or (ii) a carboxylic acid + alkene moiety; thus, this provides a less flexible / more rigid moiety containing at least one heteroatom.

[0017] In a first aspect, the present invention is a method for delivering a payload molecule across an epithelial tissue barrier, comprising: · administering the payload molecule to the epithelial tissue barrier; · further administering an agent to the epithelial tissue barrier; wherein the agent is (i) a compound capable of disrupting the epithelial tissue barrier function and comprising a heterocyclic moiety and an alkane moiety containing an R' group which is a C1-12 alkyl group which may be unsubstituted or substituted, and any substituents present are independently selected from hydroxyl, halogen, and NR''2, and each R'' is independently selected from hydrogen and methyl, a microbial quorum sensing signaling molecule (microbial QSSM) or a derivative or mutant thereof; or (ii) a carboxylic acid compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof: [Chemical formula] (wherein R 8 is H or R'; R 9 is H or R'; R 10is H or R’, provided that at least one R’ group is present; each R’ is independently selected from unsubstituted or optionally substituted C1-12 alkyl groups, and any substituents present are independently selected from hydroxyl, halogen, and NR’’2, where each R’’ is independently selected from hydrogen and methyl) selected from, provides a method.

[0018] The agent capable of disrupting the epithelial tissue barrier according to the present invention can be a microbial quorum sensing signaling molecule secreted by bacteria or other agents produced by bacteria. In this regard, the carboxylic acid compound of formula (I) may be produced by bacteria, for example, cis-2-decenoic acid is produced by bacteria. The agent may also be synthetic (i.e., non-natural). In particular, the agent may be synthetically / artificially manufactured, and the agent may or may not be produced or extracted from microorganisms.

[0019] The present invention further provides, in a second aspect, a pharmaceutically acceptable composition comprising (a) an agent capable of disrupting the epithelial tissue barrier and optionally (b) a payload molecule, wherein the agent capable of disrupting the epithelial tissue barrier is as defined in the first aspect.

[0020] In a third aspect, there is also provided a composition of the second aspect for use as a medicament, comprising a therapeutic or prophylactic payload molecule.

[0021] In a fourth aspect, there is provided a composition of the second aspect for use in a method of treating or preventing an ocular disorder, a respiratory disorder, a gastrointestinal disorder, a reproductive organ disorder, an autoimmune disorder, a mucosal disorder, a brain disorder, a microbial or parasitic infection, cancer, or a skin disorder in a subject.

[0022] Accordingly, this further provides a method of treating or preventing an ocular disorder, a respiratory disorder, an autoimmune disorder, a gastrointestinal disorder, a reproductive organ disorder, a mucosal disorder, a brain disorder, an infection, cancer, or a skin disorder, comprising Provided is a method comprising the step of administering to a subject a composition of a second aspect, the composition comprising a therapeutic or prophylactic payload molecule.

[0023] Treatment or prevention of a medical condition can involve direct delivery of a therapeutic or prophylactic payload molecule to a target tissue, or can involve indirect delivery of a therapeutic or prophylactic payload molecule to a target tissue. In particular, both local and systemic administrations are envisioned. Thus, in one embodiment, for example, a therapeutic or prophylactic payload molecule may be delivered directly to the eye to treat or prevent an eye disorder, or may be delivered directly to the skin to treat or prevent a skin disorder. In another embodiment, a therapeutic or prophylactic payload molecule may be delivered indirectly by being delivered to the bloodstream, or may be delivered indirectly by being delivered to a location adjacent to the target tissue or forming a broader portion of the body that includes the target tissue. For example, a therapeutic or prophylactic payload molecule may be administered to a position adjacent to the nail bed or nail so as to be subsequently delivered to a position under the fingernail or toenail, for example to treat a fungal infection, via the bloodstream. As another example, a therapeutic or prophylactic payload molecule may be administered to the nose so as to be subsequently delivered to the brain.

[0024] In a fifth aspect, a product comprising a composition of a second aspect, a) an eye drop dispenser, eye wash device or contact lens; and / or b) a suction device, inhaler, nebulizer, or vape device; and / or c) a controlled release tablet or capsule suitable for oral administration; and / or d) a transdermal patch or gel; and / or e) a vaccine further comprising an antigen or a nucleic acid suitable for expression of an antigen (such as a viral vector) is provided.

[0025] In a sixth aspect, (a) an agent capable of disrupting an epithelial tissue barrier, and (b) a payload molecule, A kit comprising a drug capable of disrupting the epithelial tissue barrier as defined in the first aspect is provided.

[0026] In a seventh aspect, the present invention also relates to the use of a drug capable of disrupting the epithelial tissue barrier for promoting the permeation of a payload molecule through the epithelial tissue barrier, wherein the drug capable of disrupting the epithelial tissue barrier is as defined in the first aspect.

[0027] In an eighth aspect, the present invention also relates to the use of a drug capable of disrupting the epithelial tissue barrier for facilitating the extraction of extracellular fluid from a subject, which includes the administration of the drug capable of disrupting the epithelial tissue barrier to the epithelial tissue barrier of the subject and the extraction of extracellular fluid through the epithelial tissue barrier,

[0028] Furthermore, in a ninth aspect, there is provided a method for the extraction of extracellular fluid from a subject, - administration of a drug capable of disrupting the epithelial tissue barrier to the epithelial tissue barrier of the subject, and - extraction of extracellular fluid through the epithelial tissue barrier, wherein the drug capable of disrupting the epithelial tissue barrier is as defined in the first aspect.

[0029] The invention of the present specification advantageously provides an agent that can be used to disrupt the epithelial barrier and enable the passage of payload molecules containing polymers. The payload molecules can be administered (i.e., topically) to the intended target epithelial tissue (e.g., respiratory tissue, skin surface, GI tract), but the payload molecules can also be applied to tissues that are not the intended target, and then the molecules are further transported to the intended target, for example, via the bloodstream (systemic administration). Thus, the payload molecules can be administered at a location adjacent to the target tissue or forming a wider part of the body including the target tissue. The payload molecules can be delivered to a distal location via the bloodstream from the point of introduction.

[0030] The agent according to the present invention has been shown to be effective in a Transwell polymer transport assay. The selected agent was then further tested by ex vivo assays, which supported the migration assay data showing the effectiveness of the agent that reversibly disrupts tight junctions, for example, with respect to the eye and skin. Still further tests confirmed the effectiveness of the agent according to the present invention in in vivo assays.

[0031] In this regard, it is advantageously shown herein that the use of agents according to the invention, such as 3OC12-HSL, can enhance the transport of macromolecular therapeutics such as bevacizumab (Avastin®), aflibercept (Eylea®), mepolizumab, and doxorubicin (a widely used first-line chemotherapy) across in vitro and ex vivo epithelial barrier tissue models. Bevacizumab (MW 149 kDa) is a recombinant humanized monoclonal IgG1 antibody that binds to human vascular endothelial growth factor (VEGF) to inhibit abnormal blood vessel growth in several cancers and is used in the treatment of age-related macular degeneration (AMD). Aflibercept (MW 115 kDa) is a recombinant fusion protein incorporating the VEGF-binding portions of the extracellular domains of human VEGF receptors 1 and 2, as well as the Fc portion of human IgG1. Bevacizumab and aflibercept are generally injected at significant expense to reach their sites of action in clinical practice. Doxorubicin (MW 543.52 Da) is an anthracycline antibiotic that binds to nucleic acids by specific intercalation of its planar anthracycline nucleus with DNA double helix and disrupts DNA synthesis in rapidly dividing cells.

[0032] Advantageously, the ability to transiently disrupt the epithelial barrier to deliver molecules can be used in a wide range of applications across a wide range of fields, such as delivery of vaccines, including nucleic acids and polypeptide-based vaccines for coronaviruses, through the skin; use in tattoo pigmentation; pulmonary or airway delivery of therapeutic agents; gastrointestinal or oral delivery of therapeutic agents; and ocular delivery.

[0033] Such delivery of molecules can be achieved via several routes of administration, such as gels, ointments, creams, patches, and aerosols (i.e., to the lungs).

[0034] It is beneficial that payload molecules, such as drugs, can be delivered without the need for injection. This improves the quality of life of patients, improves comfort, reduces the need for hospital visits, and reduces costs.

[0035] The present invention also recognizes that the ability to disrupt epithelial barriers for delivering molecules can be used conversely for extracting interstitial fluid between cells that can contain analytical biomarkers, vesicles or particles, or for draining body fluids in cases of peripheral edema.

[0036] The present invention also recognizes the use of disrupting epithelial barrier electrical resistance for improving the signal-to-noise ratio in electrophysiological recordings. Epithelial barrier electrical resistance can be recorded through the skin or other epithelial surfaces, for example, between an electrocardiogram, an electroencephalogram, an electroretinogram, or an electromyogram.

[0037] Further details regarding the molecules that can be delivered and the techniques that can benefit from the present invention are detailed below.

DETAILED DESCRIPTION OF THE INVENTION

[0038] Method The method can include the step of administering a payload molecule to an epithelial tissue barrier together with an agent capable of disrupting the epithelial tissue barrier. In another embodiment, the method can include the step of disrupting the epithelial tissue barrier function by contacting it with an agent capable of disrupting the epithelial tissue barrier, and then administering the payload molecule to the disrupted epithelial tissue barrier.

[0039] Generally, the payload molecule can be administered before, simultaneously with, or after the administration of the agent capable of disrupting the epithelial tissue barrier. The administration of the payload molecule and the agent can be simultaneous, sequential, or separate.

[0040] Disrupting the epithelial tissue barrier function can include, or consist of, reducing the transepithelial electrical resistance (TEER) of the epithelial tissue barrier. In one embodiment, disrupting the epithelial tissue barrier function includes, or consists of, a decrease in the diffusion resistance / limitation across the epithelial barrier. In another embodiment, disrupting the epithelial tissue barrier function includes, or consists of, reducing the electrical resistance (TEER) and / or the water permeability resistance of the epithelial tissue barrier.

[0041] The methods of the present invention can be performed in vivo, for example in a subject, or in vitro, for example in a tissue model or an extract. The methods of the present invention can be a treatment or prophylactic therapy for a disorder in a subject.

[0042] The method may involve topical delivery for direct delivery of the payload molecule to a local site, for example, there may be topical administration to the eye for delivering a drug to treat an eye condition.

[0043] Alternatively, the method may involve topical delivery for delivering the payload molecule into the body of the subject for forward delivery to a site different from the site of administration. Thus, for example, by delivering the payload molecule into the bloodstream of the subject, the payload molecule may be systemically distributed to downstream locations.

[0044] One example is the administration of the payload molecule to the skin adjacent to the subject's nail, such that the payload molecule can enter the blood supply extending under the nail, for example to target a fungal infection.

[0045] Generally, systemic or compartmental body distribution can be targeted by topical skin (or other barrier) administration that utilizes the subject's circulation to deliver the payload molecule downstream or systemically.

[0046] Agents capable of disrupting the epithelial tissue barrier Generally, an agent capable of disrupting an epithelial tissue barrier can be a carboxylic acid compound of formula (I) containing at least one R' group, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or can be a microbial quorum sensing signaling molecule (QSSM) or a derivative or variant thereof that is a compound containing a heterocyclic moiety and an alkane moiety containing an R' group. In each case, the R' group is an optionally substituted C1-12 alkyl group, and any substituents present are independently selected from hydroxyl, halogen and NR''2, and each R'' is independently selected from hydrogen and methyl.

[0047] The microbial QSSM can be a bacterial QSSM. A derivative or variant of the microbial QSSM can be synthetic (i.e., non-natural). In particular, a derivative or variant of the microbial QSSM can be produced synthetically / artificially. A derivative or variant of the microbial QSSM may or may not be produced or extracted from a microorganism.

[0048] In one embodiment, the agent capable of disrupting an epithelial tissue barrier is the Pseudomonas aeruginosa quorum sensing molecule N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL).

[0049] In another embodiment, the agent capable of disrupting an epithelial tissue barrier is 2-N-heptyl-3-hydroxy-4(1H)-quinolone (C7 PQS).

[0050] In another embodiment, the agent capable of disrupting an epithelial tissue barrier is any one of the agents shown in Table 1 of the examples as being effective in enhancing the translocation of 4 kDa FITC-dextran (i.e., indicated as "yes" in the last column of the table).

[0051] In another embodiment, the agent capable of disrupting an epithelial tissue barrier is any one of the agents shown in Table 2 of the examples as being an agent that causes MMP activation.

[0052] Agents that can disrupt epithelial tissue barriers, such as microbial QSSM, can be provided as solvates in solution.

[0053] Agents that can disrupt epithelial tissue barriers, such as microbial QSSM, may or may not be encapsulated or complexed within carrier particles such as nanostructured lipid particles (NLP).

[0054] In one embodiment, agents that can disrupt epithelial tissue barriers, such as microbial QSSM, can be encapsulated (trapped) or impregnated within a polymer, or adsorbed onto particles, polymeric carriers, or proteins.

[0055] Agents that can disrupt epithelial tissue barriers, such as microbial QSSM, are produced by crystallization, lyophilization, or other methods into nano-objects such as lipid micelles, droplets, or vesicles (i.e., particles in the range of 1 nm to 1000 nm). In another embodiment, agents that can disrupt epithelial tissue barriers, such as microbial QSSM, are bound or adsorbed onto the surface of nano-objects such as lipid micelles, droplets, or vesicles (i.e., particles in the range of 1 nm to 1000 nm).

[0056] In one embodiment, the agent can be used in an amount of 1 μM or more, such as 5 μM or more, or 10 μM or more, or 50 μM or more, for example, 100 μM or more, or 200 μM or more.

[0057] In one embodiment, the agent can be used in an amount of 1 - 5000 μM, such as 5 - 3000 μM, or 10 - 200 μM, or 50 - 1000 μM, for example, 100 - 750 μM or 200 - 500 μM.

[0058] The R' group R' is present in each of a series of agents according to the present invention and in each of formulas (I) - (V) and is independently a C1 - 12 alkyl group which may be unsubstituted or substituted.

[0059] Since the double bond is not present in the alkyl group and not present in any of the acceptable substituents, it will be understood that each R' does not contain a double bond. The R' group is saturated. Accordingly, the R' group is flexible.

[0060] It has been found that a flexible straight-chain alkyl chain is essential for the agent to have activity effective in disrupting the epithelial tissue barrier. The conformational restrictions (cis or trans) introduced through double bonds are significantly detrimental to activity.

[0061] The R' group is preferably a saturated hydrocarbon chain that does not contain an N heteroatom in the chain. In particular, conformational restrictions introduced through an amide functional group (which means the presence of N in the hydrocarbon chain) have been identified as being detrimental and reducing activity adversely.

[0062] In one embodiment, the R' group is preferably a saturated hydrocarbon chain that does not contain an N heteroatom or an S heteroatom in the chain. In one embodiment, the R' group is preferably a saturated hydrocarbon chain that does not contain an N heteroatom or an O heteroatom in the chain. In one particular embodiment, the R' group is a saturated hydrocarbon chain that does not contain an N heteroatom, an S heteroatom, or an O heteroatom in the chain. Accordingly, the backbone of the R' group chain can be based on carbon atoms only.

[0063] The hydrocarbon chain may optionally have substituents, but any substituents present must be independently selected from hydroxyl, halogen, and NR''2, and each R'' is independently selected from hydrogen and methyl. Accordingly, sterically bulky substituents are not tolerated, and the substituents present do not have a significant adverse effect on the flexibility of the chain. In one embodiment, 1 to 4 substituents, for example 1 or 2 substituents, are present.

[0064] In one embodiment, any substituents present are independently selected from hydroxyl and halogen.

[0065] In one preferred embodiment, any substituents present are independently selected from hydroxyl and F. The only substituent present may be -OH. The only substituent present may be F.

[0066] In one preferred embodiment, the C1-12 alkyl group is unsubstituted.

[0067] The unsubstituted or minimally substituted C1-12 alkyl group is flexible and thus contributes to having excellent activity to disrupt the epithelial tissue barrier.

[0068] When R' is a C3-12 alkyl group, this may be optionally branched, but preferably has no more than one C1 branch from the main chain (i.e., the main chain contains all or all but one of the carbon atoms), and more preferably the alkyl group is unbranched. Thus, in one embodiment, each R' is independently an unbranched C1-12 alkyl group. In another embodiment, each R' is independently a branched C3-12 alkyl group, and the branch is a C1 alkyl (methyl) group.

[0069] The conformational restriction introduced through branching is detrimental and unfavorably reduces the activity. Thus, only C1 branching is tolerated. As the degree of branching increases, the activity significantly decreases. For achieving optimal activity, no branching may be preferred.

[0070] It will be appreciated that R' can be C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 or C12 alkyl, optionally substituted, but preferably unsubstituted. In one embodiment, R' is C1-11 alkyl, optionally substituted, but preferably unsubstituted.

[0071] In one embodiment, R’ can be selected from C1 and C6-C12 alkyl, optionally substituted, but preferably unsubstituted. In one embodiment, R’ can be selected from C1 and C7-C12 alkyl, optionally substituted, but preferably unsubstituted. In one embodiment, R’ can be selected from C1, C7, C9 and C11, optionally substituted, but preferably unsubstituted.

[0072] In one embodiment, R’ can be unsubstituted C1-C12 alkyl, or unsubstituted C1-C11 alkyl, such as unsubstituted C6-C12 alkyl or unsubstituted C7-C11 alkyl.

[0073] In one embodiment, R’ may be an unsubstituted methyl group, or a methyl group substituted with one substituent, or an unsubstituted ethyl group.

[0074] When the R’ group is provided as a substituent on an aromatic or non-aromatic ring, it is preferred that there is no CH2NMe substituent at any position ortho to the R’ group.

[0075] In one embodiment, the substituent at any position ortho to the R’ group is selected from the group consisting of OH, CH3, H, R’, and O.

[0076] In one embodiment, the R’ group is provided as a substituent on an aromatic or non-aromatic heterocyclic ring containing N in the ring. N can be in the ortho position to the R’ substituent. N may be substituted with H, R’, or O. The other positions ortho to the R’ substituent may be a ring carbon optionally substituted with OH, CH3 or H. Formula (I)

Chemical formula

[0077] Formula (I) is a carboxylic acid compound. This can be characterized as an alkene having a carboxylic acid substituent and at least one R’ group substituent.

[0078] In this regard, R 8 is H or R'; R 9 is H or R'; R 10 is H or R', provided that at least one R' group is present. In one preferred embodiment, only one R' group is present.

[0079] Thus, formula (I) has an alkane "tail" portion containing a flexible R' group. It also has a carboxylic acid + alkene portion; thus, it provides a less flexible / more rigid portion containing at least one heteroatom.

[0080] The alkene can have E or Z stereochemistry. In some embodiments, the alkene can have Z stereochemistry, i.e., a cis double bond. This is typical of unsaturated fatty acids.

[0081] It is preferred that no other double bonds are present. In particular, the definition of the R' group substituent of the present invention does not permit the presence of double bonds. In this regard, the R' group is an unsubstituted or substituted C1-12 alkyl group, and any substituents present are independently selected from hydroxyl, halogen, and NR''2, and each R'' is independently selected from hydrogen and methyl.

[0082] As described above, it has been found that a flexible straight-chain alkyl chain is essential for the agent to have an activity effective in disrupting the epithelial tissue barrier. Conformational restrictions (cis or trans) introduced through double bonds other than those shown in formula (I) are significantly detrimental to the activity.

[0083] In some embodiments, at least one of R 8 and R 9 is H. In some preferred embodiments, R 8 is H and R 9 is H (thus, R 10 is R').

[0084] The compound can be cis-2-decenoic acid. In other embodiments, the compound is not cis-2-decenoic acid. Formula (II)

Chemical formula

[0085] Formula (II) contains two 6-membered rings that share two adjacent carbon atoms (known as bridging atoms), i.e., a fused bicyclic structure.

[0086] The first ring is an optionally substituted aromatic or alicyclic carbocyclic ring.

[0087] In this regard, each substituent R of the first ring x is independently selected from halo, C1-12 alkyl, C1-12 alkyl halo, OR y , and NR’’2, where R y is hydrogen or C1-12 alkyl, and each R’’ is independently selected from hydrogen and methyl.

[0088] The first ring may be substituted (n = 1-4) or unsubstituted (n = 0).

[0089] In one embodiment, when the first ring is substituted, it may have one or more substituents selected from C1-12 alkyl, C1-12 alkoxy, hydroxyl, and halogen. In one embodiment, when the first ring is substituted, it may have one or more substituents selected from C1-6 alkyl, C1-6 alkoxy, hydroxyl, and halogen (e.g., F).

[0090] In one preferred embodiment, when the first ring is substituted, it may have one or more substituents selected from hydroxyl and halogen (e.g., F), for example, one or two substituents selected from hydroxyl and halogen (e.g., F) may be present.

[0091] In one preferred embodiment, there is a halogen (e.g., F) substitution at the 7-position; this has been found to enhance the activity of the agent. In particular, this is the case when there is an OH group at the R 2 position. Without being bound by theory, this may be due to an increase in the polarizability of the OH at the R 2 position.

[0092] Effective results are achieved with a halogen (e.g., F) substitution at the 6-position. However, in one embodiment, when there is an OH group at the R 2 position, there is no halogen (e.g., F) substitution at the 6-position. Without being bound by theory, this is thought to possibly cause some decrease in the polarizability of the OH at the R 2 position.

[0093] In one embodiment, when there is an OH group at the R 2 position on the second ring, there is one or more substituents on the first ring. For example, there may be one or more substituents selected from hydroxyl and halogen (e.g., F) on the first ring.

[0094] The first ring may be aromatic, in one embodiment, as shown by the following formula (IIA).

Chemical formula

[0095] In formula (II), the second ring contains 5 carbon atoms and 1 nitrogen atom, and the nitrogen atom is located adjacent to the bridging atom.

[0096] The second ring is substituted. In this regard, R 1 is O or OH; R 2 is OH, CH3 or H; R 3 is R'; R 4 is H, R' or O.

[0097] Accordingly, the second ring is substituted with at least one R' group; in a preferred embodiment, this is substituted with only one R' group (i.e., R 4 is H or O).

[0098] Accordingly, formula (II) has an alkane "tail" portion containing a flexible R' group. It also has a heterocyclic portion; accordingly, this provides a less flexible / more rigid portion containing at least one heteroatom.

[0099] In formula (II), the R' group or each R' group is most preferably a C7 group from the perspective of optimal activity; however, other chain lengths are also effective. Thus, for example, the R' group or each R' group can be a C1-11 alkyl group, such as a C1-7 alkyl group, which is optionally substituted but preferably unsubstituted.

[0100] R 2 The small substituent (OH, CH3 or H) at the 2 position, i.e., adjacent to the R' group, is considered important in the structure-activity relationship. In this regard, the presence of basic functional groups such as F, -NH2 or -CH2NMe substituents at the 2 position has been shown to be extremely detrimental to the effectiveness of the agent and significantly reduce the activity of the agent. The acidic substituent OH at the

[0101] position is associated with excellent activity. 1 ~R 4 The second ring contains at least one double bond. Those skilled in the art will understand that the position of the double bond may depend on the substituents R

[0102] For example, the second ring can have one double bond (as in formulas (II-B) and (II-C)), or it can have two double bonds (as in formulas (II-D) and (II-E)):

Chemical formula

[0103] In some embodiments, R is such that the second ring contains a carbonyl group (C=O) adjacent to the bridging atom. 1 may be O (as in Formula (II-B) and Formula (II-C)). In some embodiments where R 1 is O, R 2 can be OH or H, and / or R 4 can be H or R'.

[0104] R 1 is O, R 2 is OH, and R 4 is H, the compounds may be usefully selected for use as drugs; these include C7 PQS, C1 PQS, C9 PQS, and C11 PQS shown below.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0105] R 1 is O, R 2 is H, and R 4 is H, the compounds may be usefully selected for use as drugs; these include HHQ, NHQ, and UHQ shown below.

Chemical formula

Chemical formula

Chemical formula

[0106] In one embodiment, R 1 can be OH (hydroxyl). In some embodiments where R 1 is OH, R 2 can be H and / or R 4 can be O. For example, the compound can have a structure such as formula (II-D) or formula (II-E) shown above.

[0107] In some embodiments, to provide an N-oxide (also known as an amine oxide), R 4 can be O. The N-oxide contains N + -O - and can be represented by an arrow as in formula (II-F). Although the first aromatic ring is shown, the first ring can be alicyclic.

Chemical formula

[0108] Specifically, to provide a compound having the structure shown in formula II-G, R 1 can be OH and R 4 can be O. Although the first aromatic ring is shown, the first ring can be alicyclic.

Chemical formula

[0109] R 4 In some embodiments where R 1 is O, R 2 can be OH and / or R 4 can be H or R'. Compounds where R 1 is O, R 2 is OH, and R 2 is H can be usefully selected for use as a drug; these include the C7 HHQ N-oxide and HNQ N-oxide shown below.

Chemical formula

[0110] Formula (III) contains a lactone moiety, i.e., a cyclic carboxylic acid ester.

[0111] The heterocyclic ring is optionally substituted. In this regard, each ring substituent R x is independently selected from halo, C1-12 alkyl, C1-12 alkyl halo, OR y , and NR’’2, R y is hydrogen or C1-12 alkyl, and each R’’ is independently selected from hydrogen and methyl.

[0112] The ring may be substituted (n = 1-4) or unsubstituted (n = 0).

[0113] In one embodiment, when the ring is substituted, it may have one or more substituents selected from C1-12 alkyl, C1-12 alkoxy, hydroxyl, and halogen. In one embodiment, when the ring is substituted, it may have one or more substituents selected from C1-6 alkyl, C1-6 alkoxy, hydroxyl, and halogen (e.g., F). In one embodiment, when the ring is substituted, it may have one or more substituents selected from hydroxyl and halogen (e.g., F).

[0114] Considering the rest of the molecule, R 5 is H or R’; R 6 is O, CH2, CHR’ or CR’2; R 7 is CH2C(O)R’ or R’. Thus, R 7Due to the base, there is at least one R' group present; in a preferred embodiment, there is only one R' group in the molecule.

[0115] Accordingly, formula (III) has an alkane "tail" portion containing a flexible R' group. This also has a heterocyclic moiety; thus, this provides a less flexible / more rigid portion containing at least one heteroatom.

[0116] In one embodiment, R is provided to provide an alkene that can have Z or E stereochemistry 6 can be CH2, CHR' or CR'2. R 6 In some embodiments where R is selected from CH2, CHR' or CR'2, R 5 can be H, and / or R 7 can be R'.

[0117] In one embodiment, R is provided to provide a carbonyl group (C=O) 6 can be O. R 5 is H, R 6 is O, and R 7 is R' can be usefully selected for use as a drug; these include C4 HSL, also known as N-butyl-homoserine-lactone (BHL):

Chemical formula

[0118] R 6 In some embodiments where R is O, R 5 can be H, and / or R 7 can be CH2C(O)R'. R 5 is H, R 6 is O, and R 7 is CH2C(O)R' can be usefully selected for use as a drug; these include HSL and 3-oxo-C12-HSL:

Chemical formula

Chem.

Chem.

[0119] Formula (IV) contains a thiazole ring. The thiazole ring is optionally substituted. In this regard, each ring substituent R x is independently selected from halo, C1-12 alkyl, C1-12 alkyl halo, OR y , and NR’’2, R y is hydrogen or C1-12 alkyl, and each R’’ is independently selected from hydrogen and methyl.

[0120] The ring may be substituted (m = 1 or 2) or unsubstituted (m = 0).

[0121] In one embodiment, when the ring is substituted, it may have one or more substituents selected from C1-12 alkyl, C1-12 alkoxy, hydroxyl, and halogen. In one embodiment, when the ring is substituted, it may have one or more substituents selected from C1-6 alkyl, C1-6 alkoxy, hydroxyl, and halogen (e.g., F). In one embodiment, when the ring is substituted, it may have one or more substituents selected from hydroxyl and halogen (e.g., F).

[0122] Considering the rest of the molecule, R 5 is H or R’; R 6 is O, CH2, CHR’ or CR’2; R 7 is CH2C(O)R’ or R’. Thus, due to the R 7 group, at least one R’ group is present; in a preferred embodiment, only one R’ group is present in the molecule.

[0123] Thus, formula (IV) has an alkane "tail" portion that includes a flexible R' group. This also has a heterocyclic moiety; thus, this provides a less flexible / more rigid portion that includes at least one heteroatom.

[0124] In one embodiment, to provide an alkene that can have Z or E stereochemistry, R 6 can be CH2, CHR', or CR'2. In some embodiments where R 6 is selected from CH2, CHR', or CR'2, R 5 can be H, and / or R 7 can be R'.

[0125] In one embodiment, to provide a carbonyl group (C=O), R 6 can be O. Compounds where R 5 is H, R 6 is O, and R 7 is R' can be usefully selected for use as a drug.

[0126] R 6 is O. In some embodiments where R 5 can be H, and / or R 7 can be CH2C(O)R'. Compounds where R 5 is H, R 6 is O, and R 7 is CH2C(O)R' can be usefully selected for use as a drug. Formula (V)

Chemical formula

[0127] Formula (V) includes a piperidine ring. The ring is optionally substituted. In this regard, each substituent R x of the ring is independently selected from halo, C1-12 alkyl, C1-12 alkyl halo, OR y and NR''2, and R yis hydrogen or C1-12 alkyl, and each R’’ is independently selected from hydrogen and methyl.

[0128] The ring may be substituted (m = 1 or 2) or unsubstituted (m = 0).

[0129] In one embodiment, when the ring is substituted, it may have one or more substituents selected from C1-12 alkyl, C1-12 alkoxy, hydroxyl, and halogen. In one embodiment, when the ring is substituted, it may have one or more substituents selected from C1-6 alkyl, C1-6 alkoxy, hydroxyl, and halogen (e.g., F). In one embodiment, when the ring is substituted, it may have one or more substituents selected from hydroxyl and halogen (e.g., F).

[0130] Considering the rest of the molecule, R 5 is H or R'; R 6 is O, CH2, CHR' or CR'2; R 7 is CH2C(O)R' or R'. Thus, due to the R 7 group, at least one R' group is present; in a preferred embodiment, only one R' group is present in the molecule.

[0131] Thus, formula (V) has an alkane "tail" portion containing a flexible R' group. It also has a heterocyclic portion; thus, it provides a less flexible / more rigid portion containing at least one heteroatom.

[0132] In one embodiment, to provide an alkene that may have Z or E stereochemistry, R 6 can be CH2, CHR' or CR'2. In some embodiments where R 6 is selected from CH2, CHR' or CR'2, R 5 can be H, and / or R 7 can be R'.

[0133] In one embodiment, to provide a carbonyl group (C=O), R 6 can be O. R 5 is H, R 6 is O, and R 7 is R'. The compound can be usefully selected for use as a drug.

[0134] R 6 In some embodiments where is O, R 5 can be H, and / or R 7 can be CH2C(O)R'. R 5 is H, R 6 is O, and R 7 is CH2C(O)R'. The compound can be usefully selected for use as a drug.

[0135] Payload molecule In one embodiment, the payload molecule is a therapeutic agent such as a polymeric therapeutic agent. It is unexpected that the present invention can effectively deliver payload molecules of various sizes, not just small molecules such as dextrin (4 kDa M w ). For example, molecules having M w of 10 kDa or more and 150 kDa or more can be effectively delivered by permeating the epithelial barrier in the presence of the drug according to the present invention. The payload molecule may not be able to naturally permeate the epithelial barrier in the absence of the drug according to the present invention.

[0136] The payload molecule may contain a protein. The payload molecule may contain a peptide or a protein. The payload molecule may contain a protein or peptide that is physiologically or metabolically relevant. The protein can be a glycoprotein. The protein or glycoprotein can be an enzyme. In another embodiment, the payload molecule may contain a polysaccharide. In another embodiment, the payload molecule may contain Botox.

[0137] The payload molecule is a non-small molecule (e.g., M w >900 Da) or a small molecule (e.g., Mw <900Da) may be included. The payload molecule is an M less than 200 kDa w of any molecule. In another embodiment, the payload molecule is an M less than 150 kDa w of any molecule. In another embodiment, the payload is an M between 1 kDa and 150 kDa w and may have.

[0138] The payload molecule may include a signaling protein that is a protein involved in a signaling pathway. The payload molecule may include a protein involved in regulating cell expression or metabolism. The payload molecule may include a protein involved in cell division. The payload molecule may include a marker such as a protein marker. The payload molecule may include a bacterium or a bacterium-derived protein. The payload molecule may include a mammal or a mammal-derived protein. The payload molecule may be any peptide, polypeptide, or protein. The payload molecule may include a research molecule, a diagnostic molecule, or a therapeutic molecule. The payload molecule may include an enzyme or its substrate, a protease, an enzyme activity modulator, a perturbimer, and a peptide aptamer, an antibody, a modulator of protein-protein interaction, a growth factor, or a differentiation factor. The payload molecule may be a preprotein or a prodrug. The payload molecule may include a virus particle or a virus-like particle. In one embodiment, the payload molecule is an antibody, or an antibody fragment or mimetic, such as a nanobody.

[0139] In one embodiment, the payload molecule is selected from any one of the group consisting of a therapeutic molecule; a drug; a prodrug; a functional protein or peptide such as an enzyme or a transcription factor; a microbial protein or peptide; a virus particle; a virus-like particle; and a toxin; or a nucleic acid encoding them.

[0140] In one embodiment, the payload molecule can include nucleic acids such as siRNA, messenger RNA (mRNA), microRNA, or DNA constructs. The payload molecule can include a nucleic acid complex. The nucleic acid can be recombinant.

[0141] The payload molecule can include non-covalent complexes such as protein-protein complexes, protein-mRNA, protein-non-coding RNA, protein-lipid, and protein-small molecule complexes.

[0142] The payload molecule can be a chemotherapeutic agent. In one embodiment, the payload molecule is an anti-VEGF antibody or a fragment thereof, or an inhibitor of VEGF. In one embodiment, the payload molecule is selected from the group consisting of bevacizumab (Avastin®), aflibercept (Eylea®), and doxorubicin. These are non-limiting examples, and those skilled in the art will understand that the present invention can be applied to a range of payload molecules having a certain range of molecular weights.

[0143] In another embodiment, the payload molecule is an anti-inflammatory agent. In one embodiment, the payload molecule includes a cytokine, such as interferon beta, preferably interferon beta 1a (20 kDa). Cytokines such as interferon beta can be cytokines of about 25 kDa or less. Those skilled in the art will recognize that interferon beta can be used to treat or prevent the effects of inflammation, such as inflammation in asthmatic disorders, or viral infections such as COVID-19 infection.

[0144] In one embodiment, the payload molecule is a therapeutic agent suitable for treating or preventing disorders of organs or tissues having an epithelial tissue barrier.

[0145] In one embodiment, the payload molecule is a therapeutic agent suitable for the treatment or prevention of eye disorders. The eye disorder can include any one of disorders selected from age-related macular degeneration, cancer, diabetic retinopathy, retinal vascular occlusion diseases, inflammatory eye diseases, and retinopathy of prematurity. In one embodiment, the eye disorder includes macular degeneration such as AMD (age-related macular degeneration). AMD can be exudative AMD or atrophic AMD. In another embodiment, the eye disorder includes cancers such as solid tumor cancer, melanoma, and retinoblastoma. In another embodiment, the eye disorder includes diabetic retinopathy. In another embodiment, the eye disorder includes retinal vascular occlusion diseases such as retinal vein stasis, central retinal vein occlusion, and branch retinal vein occlusion. In another embodiment, the eye disorder includes inflammatory eye diseases such as iritis, uveitis, scleritis, blepharitis, and orbital inflammatory diseases. In another embodiment, the eye disorder includes retinopathy of prematurity.

[0146] In another embodiment, the payload molecule is a therapeutic agent suitable for the treatment or prevention of respiratory disorders, such as mepolizumab. The respiratory disorder can include any one of disorders selected from microbial infections, cancer, asthma, interstitial lung disease, inflammatory lung disease, tumors, and COPD. In one embodiment, the respiratory disorder includes a microbial infection or an inflammatory effect associated with a microbial infection. In one embodiment, the microbial infection is a viral infection such as a coronavirus infection. The coronavirus can include SARS-CoV-2 (COVID-19). The viral infection can include influenza. In another embodiment, the microbial infection is a bacterial infection. The bacterial infection can be an infection with Bacillus anthracis. In another embodiment, the microbial infection is a fungal infection. The fungal infection can include an Aspergillus infection.

[0147] The respiratory disorder can include an inflammatory disease such as asthma. The respiratory disorder can include COPD (chronic obstructive pulmonary disease).

[0148] In another embodiment, the respiratory disorder includes cancer, such as lung cancer.

[0149] In another embodiment, the payload molecule is a therapeutic agent suitable for the treatment or prevention of gastrointestinal disorders. The gastrointestinal disorder may include any one of disorders selected from cancers such as inflammatory bowel disease, intestinal cancer, esophageal cancer, oral cancer, tongue cancer, or gastric cancer. In one embodiment, the gastrointestinal disorder includes inflammatory bowel disease such as Crohn's disease and ulcerative colitis. Therapeutic agents suitable for the treatment of inflammatory bowel disease include aminosalicylate preparations (such as sulfasalazine, mesalazine, olsalazine, and balsalazide), steroids (such as prednisolone, prednisone, hydrocortisone, methylprednisolone, beclometaone dipropionate, budesonide, budesonide-MMX, etc.), immunosuppressive agents (such as cyclosporine, azathioprine, mercaptopurine, and methotrexate), biopharmaceuticals or biosimilar pharmaceuticals (such as adalimumab, golimumab, vedolizumab, ustekinumab, and infliximab), or small molecules (such as tofacitinib, etc.).

[0150] In one embodiment, the payload molecule is a therapeutic agent suitable for the treatment or prevention of skin disorders. The skin disorder may include any one of disorders selected from microbial infections such as bacterial or fungal skin infections, skin neoplasms, and inflammatory skin disorders. In one embodiment, the skin disorder includes skin neoplasms that may be cancerous. The cancerous skin neoplasms may be cancers such as skin cancer (e.g., melanoma), or solid tumors present in the dermis or subcutaneous tissue. In another embodiment, the skin disorder includes inflammatory skin disorders such as eczema, acne, and psoriasis.

[0151] Therapeutic agents suitable for the treatment of skin disorders may include small molecules (such as lignocaine and triamcinolone), biological agents (such as adalimumab, etc.), or plasmid gene therapy.

[0152] In one embodiment, the payload molecule may be a prophylactic agent such as a vaccine.

[0153] Payload molecules such as therapeutic agents may be provided at therapeutically or prophylactically effective concentrations.

[0154] Combinations of payload molecules can be provided. Payload molecules such as therapeutic agents can be provided in combination with one or more other therapeutically active agents. For example, a second, third, or more therapeutic agents can be provided.

[0155] In one embodiment, the payload molecule can have a molecular weight (M w ) of at least 0.3 kDa, such as 0.4 kDa or more. In one embodiment, the payload molecule can have a molecular weight of at least 0.5 kDa. In another embodiment, the payload molecule can have a molecular weight of at least 1 kDa. The payload molecule can have a molecular weight of at least 4 kDa. Alternatively, the payload molecule can have a molecular weight of at least 5 kDa.

[0156] In one embodiment, the payload molecule can have a molecular weight (M w ) of at least 10 kDa. In one embodiment, the payload molecule can have a molecular weight of at least 15 kDa. In another embodiment, the payload molecule can have a molecular weight of at least 20 kDa.

[0157] In one embodiment, the payload molecule can have a molecular weight (M w ) of at least 25 kDa, or at least 50 kDa, or at least 100 kDa.

[0158] In some embodiments, the payload molecule can have a molecular weight (M w ) of 400 kDa or less. The payload molecule can have a molecular weight of 300 kDa or less.

[0159] In one non-limiting example, the payload molecule has a molecular weight (M w) may have. The payload molecule has a molecular weight (M) between about 50 kDa and about 200 kDa; or between about 100 kDa and about 150 kDa w ) may have.

[0160] If the payload molecule contains amino acids, the payload molecule can be between about 5 and about 30,000 amino acids in length, or more. The payload molecule can be between about 5 and about 10,000 amino acids in length. The payload molecule can be between about 5 and about 5,000 amino acids in length. The payload molecule can be between about 5 and about 1,000 amino acids in length. The payload molecule can be at least about 5 amino acids in length. The payload molecule can be at least about 100 amino acids in length.

[0161] One of ordinary skill in the art will recognize that the ability of a payload molecule to cross an epithelial barrier can depend on its size, shape, and / or charge.

[0162] The present invention can be suitable for enhancing the transport of any payload molecule. For example, the present invention can be particularly beneficial for molecules whose spontaneous rate of crossing an epithelial barrier is too low to be therapeutically effective without enhanced delivery.

[0163] Epithelial tissue barrier The epithelial tissue barrier can be in vivo or in vitro. The epithelial tissue barrier can be in situ in a subject.

[0164] The epithelial tissue barrier can be in the brain, eye, respiratory system, skin, gastrointestinal tract, urinary tract, male or female genital system, olfactory system, or any mucosa of a subject. In one embodiment, the epithelial tissue barrier is in the eye, for example, the epithelial tissue barrier can be the cornea and conjunctival epithelium. In another embodiment, the epithelial tissue barrier can be an internal barrier such as the blood-brain barrier, blood-testis barrier, or blood-retinal barrier.

[0165] In another embodiment, the epithelial tissue barrier is in the respiratory system, for example, the epithelial tissue barrier can include the lung epithelium.

[0166] In another embodiment, the epithelial tissue barrier is in the digestive tract. For example, the epithelial tissue barrier may include the epithelium of the mouth (buccal epithelium), esophagus, stomach, intestine (small intestine and / or large intestine), or rectum.

[0167] In another embodiment, the epithelial tissue barrier is in the urinary tract. For example, the epithelial tissue barrier may include the epithelium of the urethra or bladder. In one embodiment, the epithelial tissue barrier is in the kidney.

[0168] In another embodiment, the epithelial tissue barrier is in the female reproductive organs. For example, the epithelial tissue barrier may include the epithelium of the ovary, fallopian tube, uterus, or cervix.

[0169] In another embodiment, the epithelial tissue barrier may include the olfactory epithelium / nasal epithelium.

[0170] In another embodiment, the epithelial tissue barrier may include the epithelium of the tympanic membrane.

[0171] Subject The subject can be a mammal. In one embodiment, the subject is a human subject. The subject can be male or female. The subject can be a non-human animal such as a domesticated animal or livestock. In one embodiment, the use of the present invention can be veterinary or in animal research. The subject can be a rodent, rabbit, or monkey.

[0172] In one embodiment, the subject may require treatment for an eye disorder or may be at risk of developing an eye disorder. In another embodiment, the subject may require treatment for a respiratory disorder or may be at risk of developing a respiratory disorder. In another embodiment, the subject may require treatment for a gastrointestinal disorder or may be at risk of developing a gastrointestinal disorder. In another embodiment, the subject may require treatment for a urinary tract disorder or may be at risk of developing a urinary tract disorder. In another embodiment, the subject may require treatment for a disorder of the genital system or may be at risk of developing a disorder of the genital system. In another embodiment, the subject may require treatment for a skin disorder or may be at risk of developing a skin disorder. In another embodiment, the subject may require treatment for a brain disorder or may be at risk of developing a brain disorder. In another embodiment, the subject may require treatment for an autoimmune disorder.

[0173] It will be understood that the present invention may also be used to achieve a systemic concentration of a payload molecule for treating a systemic disease. The present invention may also be used to deliver a payload molecule to a particular compartment or tissue of the body, for example, via an epithelial barrier that may separate a particular compartment or structure of the body. For example, the payload molecule may be delivered to the brain via the nose.

[0174] The subject may require a delivery system that is more comfortable than invasive delivery methods such as injection. For example, the subject may benefit from payload molecule delivery via a topically applied patch or a topical composition such as a gel.

[0175] Composition The composition according to the present invention may comprise a carrier. Those skilled in the art will understand that the carrier may suitably be pharmaceutically acceptable.

[0176] In one embodiment, the composition is a pharmaceutically acceptable composition suitable for systemic delivery such as intravenous delivery.

[0177] In another embodiment, the composition is a pharmaceutically acceptable composition suitable for topical delivery such as application to the surface of the skin or the eye.

[0178] The composition can be an ophthalmically acceptable composition. Thus, any carrier present can be an ophthalmically acceptable carrier. The composition can be suitable for topical administration to the eye.

[0179] In one embodiment, the composition is an ophthalmic composition. An ophthalmic composition is understood to be a sterile liquid, semi-solid, or solid preparation containing one or more pharmaceutical active ingredients intended for administration to the eye or eyelid.

[0180] The composition can be in the form of a solution such as a gel, lotion, cream, ointment, or aqueous solution, suspended with an agent capable of disrupting the epithelial tissue barrier. In one embodiment, the composition is in the form of drops such as an eye drop formulation. In one embodiment, the composition is formulated using a gel such as a Pluronic® / poloxamer gel. The Pluronic® / poloxamer gel can contain poloxamer 407 (F127). The composition can be in the form of a spray. The composition can be formulated for oral delivery, for example, for sublingual delivery or as a buccal patch. The composition can be formulated for ocular delivery. In one embodiment, the composition can be provided in a contact lens, for example, to deliver a therapeutic agent to the eye.

[0181] In another embodiment, the composition is provided in the form of a patch, for example, for application to the skin or other epithelial tissue barriers. In another embodiment, the composition is provided in the form of a pessary or suppository.

[0182] In another embodiment, the composition contains a polymer such as a polymer matrix or film, for example, for controlled release. The agent of the present invention, and optionally the payload molecule, can be encapsulated by the polymer, for example, for controlled release. The agent of the present invention, and optionally the payload molecule, can be impregnated by the polymer, for example, for controlled release. The polymer can be a solid or a viscous liquid (i.e., having a viscosity substantially higher than water at 25°C). The polymer can be a gel or a paste.

[0183] The composition may contain one or more pharmaceutically acceptable excipients. The composition may contain water; physiological saline; salts; buffers; lubricants; wetting agents; thickeners; isotonic agents; cellulose derivatives such as sodium carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, or methyl cellulose; dextran 70; gelatin; polyols; glycerin; polyethylene glycols such as PEG300 or PEG400; polysorbate 80; propylene glycol; polyvinyl alcohol; and povidone (polyvinyl pyrrolidone); and may contain one or more ophthalmically acceptable components selected from the group consisting of these and combinations thereof. The composition may contain a poloxamer such as poloxamer 407 (F127 (trademark)).

[0184] The lubricant may contain or consist of a cellulose derivative, glycerin, polyvinyl alcohol, polyvinyl pyrrolidone, a cellulose derivative, polyethylene glycol, or a combination thereof.

[0185] The composition may be formulated as nanoparticles (e.g., lipid droplets / vesicles or polymer / sol-gel systems). Such formulations are known in the art. One of ordinary skill in the art will understand that targeted delivery can be achieved by then using such formulations that degranulate at a specific location and allow systemic delivery but enable local activity.

[0186] Medical Use The composition according to the present invention can be provided for use in medicine.

[0187] In one embodiment, this can be provided for use in treating or preventing an eye disorder of a subject.

[0188] Accordingly, provided is the use of the composition according to the present invention herein in the manufacture of a medicament for treating or preventing an eye disorder of a subject.

[0189] Also provided is a method of treating or preventing an eye disorder in a subject, the method comprising administering a composition according to the present invention to the eye of the subject.

[0190] The administration can be local to the surface of the eye or the eyelid.

[0191] The eye disorder can be selected from age-related macular degeneration, cancer, diabetic retinopathy, retinal vascular occlusion diseases, inflammatory eye diseases, and retinopathy of prematurity.

[0192] In one embodiment, the eye disorder includes macular degeneration such as AMD (age-related macular degeneration). AMD can be exudative AMD or atrophic AMD. The eye disorder can include diabetic retinopathy. In another embodiment, the eye disorder can include cancers such as solid tumor cancer, melanoma, and retinoblastoma. In another embodiment, the eye disorder includes diabetic retinopathy. In another embodiment, the eye disorder includes retinal vascular occlusion diseases such as central retinal vein occlusion, retinal vein branch occlusion, and retinal vein stasis retinopathy. In another embodiment, the eye disorder includes inflammatory eye diseases such as iritis, uveitis, scleritis, blepharitis, and orbital inflammatory diseases. In another embodiment, the eye disorder includes retinopathy of prematurity.

[0193] The eye disorder can be acute or chronic.

[0194] Also provided is a composition according to the present invention for use in treating or preventing a respiratory disorder in a subject.

[0195] Also provided is the use of a composition according to the present invention in the manufacture of a medicament for treating or preventing a respiratory disorder in a subject.

[0196] Also provided is a method of treating or preventing a respiratory disorder in a subject, the method comprising administering a composition according to the present invention to the lung of the subject.

[0197] Respiratory disorders can be selected from microbial infections (e.g., viruses or bacteria), cancer, asthma, interstitial lung disease, inflammatory lung disease, tumors, and COPD.

[0198] In one embodiment, the respiratory disorder includes cancer such as lung cancer. In another embodiment, the respiratory disorder includes a microbial infection or an inflammatory effect associated with a microbial infection. In another embodiment, the respiratory disorder includes a viral infection. The viral infection can be a coronavirus infection such as SARS-CoV-2 (COVID-19) or influenza. In another embodiment, the microbial infection is a bacterial infection such as Bacillus anthracis. In another embodiment, the microbial infection is a fungal infection. The fungal infection can include Aspergillus. In another embodiment, the respiratory disorder includes an inflammatory disorder such as asthma. In one embodiment, the respiratory disorder includes COPD.

[0199] The respiratory disorder can be acute or chronic.

[0200] Also provided is a composition according to the present invention herein for use in the treatment or prevention of a GI tract disorder in a subject.

[0201] Also provided is the use of a composition according to the present invention herein in the manufacture of a medicament for treating or preventing a GI tract disorder in a subject.

[0202] Also provided is a method for treating or preventing a GI tract disorder in a subject, the method comprising the step of administering a composition according to the present invention to the GI tract of the subject.

[0203] The GI tract disorder can be selected from inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, cancers such as intestinal cancer, esophageal cancer, oral cancer, tongue cancer, or stomach cancer.

[0204] The GI tract disorder can be acute or chronic.

[0205] The composition can be formulated for oral administration. The composition can include one or more orally acceptable excipients.

[0206] Also provided is a composition according to the present invention herein for use in treating or preventing a skin disorder of a subject.

[0207] Also provided is the use of a composition according to the present invention herein in the manufacture of a medicament for treating or preventing a skin disorder of a subject.

[0208] Also provided is a method of treating or preventing a skin disorder of a subject, the method comprising the step of administering a composition according to the present invention to the skin of the subject.

[0209] The skin disorder can be selected from skin neoplasms and inflammatory skin disorders. In one embodiment, the skin disorder includes a skin neoplasm that can be cancerous. The cancerous skin neoplasm can be a cancer such as skin cancer (e.g., melanoma), or a solid tumor present in the dermis or subcutaneous tissue. In another embodiment, the skin disorder includes inflammatory skin disorders such as eczema, acne, and psoriasis.

[0210] The administration can be topical to the surface of the skin. The administration can be via a transdermal patch or a gel.

[0211] Also provided is a composition according to the present invention herein for use in treating or preventing a brain disorder such as a brain cancer of a subject.

[0212] The use can be in a treatment method in which the composition is delivered systemically or nasally to the subject for transporting a payload molecule across the blood-brain barrier.

[0213] Also provided is a method of treating or preventing a brain disorder such as a brain cancer of a subject, the method comprising the step of administering a composition according to the present invention systemically or nasally to the subject.

[0214] Also provided is a composition according to the present invention herein for use as a vaccine.

[0215] There is also provided a method of vaccination, which includes the step of administering, as a vaccine, the composition according to the present invention herein to a subject.

[0216] Use as a vaccine can be for preventing or treating a disease of a subject. The disease can be an infectious disease such as a viral infection or a bacterial infection. In another embodiment, the disease can be cancer.

[0217] Generally, when considering the treatment or prevention of a medical condition, the administration of the composition can be a pharmaceutically effective amount of the composition. The treatment or prevention can include a single administration or repeated administrations. The administration can be once or more than once per 1 to 18 days. The administration can be once or more than once per 5 to 18 days. The administration can be once or more than once per 7 to 18 days. The administration can be once or more than once per 10 to 18 days. The administration can be once or more than once per 15 to 18 days. The administration can be once or more than once per about 7 days or once a month. The administration can be once per 1 to 30 days. The administration can be once or less per day. In one embodiment, the administration is once a week, once a month, or once every 2 to 3 weeks.

[0218] Products and Kits The present invention provides a product comprising the composition according to the present invention herein.

[0219] There is provided an eye drop dispenser or an eye wash device comprising the composition according to the present invention herein.

[0220] The eye drop dispenser may otherwise be known as an eye drop applicator. A typical eye drop dispenser comprises a reservoir for the composition and an outlet for the composition. The outlet may taper towards the distal end and the outlet orifice is at the tip / distal end. The dispenser can be configured to be sealed, for example, with a cap. Alternatively, the eye drop dispenser may comprise a syringe device. Alternatively, the eye drop dispenser may comprise a spray device.

[0221] There is provided a suction device comprising the composition according to the invention of the present specification. There is also provided an inhaler or nebulizer device comprising the composition according to the invention of the present specification.

[0222] The composition may be provided in a gel or liquid form suitable for administration through bronchoscopy.

[0223] There is also provided a controlled-release tablet comprising the composition according to the invention of the present specification.

[0224] There is also provided a transdermal patch or gel comprising the composition according to the invention of the present specification.

[0225] Thus, in one embodiment, there is provided a patch, such as a saline-soaked patch, containing the composition according to the invention. Such a patch can provide a recording portal into the body of a subject. The patch can be applied to an epithelial barrier such as the skin. The patch can be applied under a smartwatch or other personal electronic monitoring device. The monitoring can be for an acute or chronic condition. In one embodiment, the monitoring can be for electrophysiological signals, biomarkers, or analyte concentrations, either alone or in combination.

[0226] - a payload molecule; - an agent capable of disrupting the epithelial tissue barrier There is also provided a kit comprising.

[0227] The agent is as defined and described above. The payload molecule can be as defined and described above.

[0228] The payload molecule and the agent capable of disrupting the epithelial tissue barrier can be formulated into separate compositions or dispensers. The payload molecule and the agent capable of disrupting the epithelial tissue barrier can be formulated into separate containers and configured to be mixed before use. The payload molecule and the agent capable of disrupting the epithelial tissue barrier can be co-formulated into a single composition or dispenser. The separate compositions may be mixed before use / administration, or may be used / administered separately, such as by sequential or simultaneous administration.

[0229] The kit may further include one or more containers for the payload molecule and a composition containing an agent capable of disrupting the epithelial tissue barrier. The kit may further include an applicator such as an eye drop dispenser, a syringe, or a transdermal patch.

[0230] Other uses The agents defined and described above can be used to promote the permeation of the payload molecule through the epithelial tissue barrier.

[0231] The payload molecule can be co-formulated with an agent capable of disrupting the epithelial tissue barrier.

[0232] The agents defined and described above can be used to facilitate the extraction of extracellular fluid from a subject, and the use includes the administration of an agent capable of disrupting the epithelial tissue barrier to the epithelial tissue barrier and the extraction of extracellular fluid through the epithelial tissue barrier.

[0233] Also provided is a method for the extraction of extracellular fluid from a subject, the method including the administration of an agent capable of disrupting the epithelial tissue barrier to the epithelial tissue barrier of the subject and the extraction of extracellular fluid through the epithelial tissue barrier, wherein the agent capable of disrupting the epithelial tissue barrier is as defined and described above.

[0234] The extraction of extracellular fluid through the epithelial tissue barrier can be performed by evacuating the drug-treated surface to extract the extracellular fluid.

[0235] The extracted extracellular fluid can be used for analyses such as electrophysiological measurements and / or biomarker analysis. Biomarkers can include glucose (e.g., for diabetes management), proteins, volatile substances / gases, acidosis / pH / ion balance, and / or exosomes (such as up to about 30 nm).

[0236] The present invention can be used in applications such as needleless ink delivery (e.g., for the agricultural and / or tattoo industries); needleless delivery of therapeutic agents such as vaccines (e.g., COVID vaccines); targeted delivery of therapeutic agents to the lungs via aerosols; targeted delivery of therapeutic agents to the brain via the nasal epithelial barrier, etc.; long-term delivery of chemotherapeutic agents, etc.; or ocular delivery in contact lenses or contact lens solutions. In particular, the payload can be a molecule or therapeutic agent for use in such applications. In one embodiment, the composition according to the present invention is used, for example, for needleless delivery of therapeutic agents through the skin.

[0237] The composition can be formulated into a nebulizer / atomizer or vape for delivering the payload through the epithelial barrier of the airway.

[0238] In an embodiment where the present invention is used in tattoos, the present invention can provide painless subcutaneous delivery of pigments, particles, or polymers that can be patterned by painting, rolling, spraying, stamping, direct printing, or screen printing.

[0239] Fluorescent pigments and particles can be payloads delivered through or into the skin. The payload can include quantum dots, nanoparticles, non-fading Au nanoparticle-based colorants, or other polymers.

[0240] The present invention can be used for edema release or peripheral drainage. In such embodiments, one of ordinary skill in the art will recognize that the composition of the present invention may not include a payload for delivery.

[0241] The present invention can also be used in applications that require measurement of bioelectric signals that can be recorded from a subject. For example, epithelial barrier disruption can reduce transepithelial resistance and improve the signal-to-noise ratio of bioelectric signals such as electroencephalogram (EEG), electrocardiogram (ECG), and electroretinogram (ERG) signals.

[0242] Accordingly, according to another aspect of the present invention, there is provided a method for measuring a bioelectric signal from a subject, the method comprising administering a composition according to the present invention to an epithelial barrier surface such as the skin, and measuring an electric signal therefrom using an electrode. The composition according to the present invention can be applied to an epithelial barrier surface such as the skin in the form of a gel or a patch (e.g., an adhesive patch). The electrode capable of detecting an electric signal can be applied to an epithelial barrier surface such as the skin before, simultaneously with, or after the composition. In one embodiment, the electrode can be attached using the composition when provided in the form of a patch. In one embodiment, the composition is provided in the form of a gel and the electrode is applied with or after the gel.

[0243] Definitions The term "prevention" means the avoidance of a disorder or a protective treatment for a disorder. Prevention can include a reduction in the risk of a disorder, a reduction in the risk of infection, transmission, and / or progression, or a reduction in the severity of a disorder.

[0244] The term "treatment" means the cure of a condition or disease, the alleviation of symptoms, or a reduction in the severity of a disorder or the symptoms of a disorder.

[0245] References herein to "solid tumors" are intended to refer to an abnormal mass or growth of tissue within the body of a subject. Solid tumors can be either benign (non-cancerous) or malignant (cancerous).

[0246] "Antibody" includes substantially intact antibody molecules, as well as chimeric antibodies, human antibodies, humanized antibodies (where at least one amino acid is mutated relative to a naturally occurring human antibody), single-chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, single-domain antibodies, nanobodies, and antigen-binding fragments and derivatives thereof. The inventors also include antibody mimetic binding proteins such as SoloMER™ proteins within the meaning of the term "antibody". SoloMER™ is a small (11 kDa) stable protein that is similar to a single-domain antibody but has a fourth binding loop in a single-binding domain format. In particular, as used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen-binding sites that specifically bind to an antigen, whether made naturally or partially or fully synthetically. The term also encompasses any polypeptide or protein having a binding domain that is an antibody binding domain or is homologous to an antibody binding domain. These can be derived from natural sources or made partially or wholly synthetically. Examples of antibodies are immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, and IgA) and their isotype subclasses; fragments containing antigen-binding domains such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies can be polyclonal or monoclonal. Monoclonal antibodies can be referred to as "mAb".

[0247] It has been shown that fragments of the whole antibody can perform the function of binding to an antigen. Examples of the binding fragments of the present invention include: (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) an Fd fragment consisting of a VH domain and a CH1 domain; (iii) an Fv fragment consisting of a VL domain and a VH domain of a single antibody; (iv) a dAb fragment consisting of a VH domain; (v) an isolated CDR region; (vi) an F(ab’)2 fragment which is a bivalent fragment containing two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) in which the VH domain and the VL domain are linked by a peptide linker that enables the two domains to associate to form an antigen-binding site; (viii) a bispecific single-chain Fv dimer (PCT / US92 / 09965, incorporated herein by reference); and (ix) a “diabody” (WO 94 / 13804 pamphlet, incorporated herein by reference) which is a multivalent or multispecific fragment constructed by gene fusion.

[0248] Those skilled in the art will understand that any optional feature of an embodiment or aspect of the present invention may, where appropriate, be applicable to other embodiments or aspects of the present invention.

[0249] Embodiments of the present invention will be described in more detail by way of example only, with reference to the accompanying drawings and examples.

Brief Description of the Drawings

[0250]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11A

Figure 11B

Figure 12

Figure 13

[0251] Example A - In Vitro Tests Test Agents:

Chemical Formula

[0252] Methods Cell Culture Calu-3 cells obtained from the American Type Culture Collection in the United States were cultured using Dulbecco's Modified Eagle Medium (DMEM) supplemented with penicillin (100 U / mL), streptomycin (0.1 mg / mL), amphotericin (0.25 μg / mL), non-essential amino acids (NEM, 1%) and fetal bovine serum (FBS, 10% v / v, Sigma-Aldrich, UK). The human-derived RPE cell line ARPE-19 was obtained from the American Type Culture Collection. Cells were grown in Dulbecco's Modified Eagle Medium:F12 (1:1; Gibco, Invitrogen, Carlsbad, California) containing 10% inactivated fetal bovine serum, 100 units / mL penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine (Sigma-Aldrich). Caco-2 cells were grown in complete culture medium (DMEM supplemented with 17% FBS and 1% antibiotic-antifungal solution). All cells were maintained in a water-jacketed incubator (Model 3546, Forma Scientific, Inc., Marietta, Ohio, USA) with a humidified air atmosphere of 37 °C and 5% CO2. The medium was changed every other day until the cells reached 80 - 90% confluence, which is desirable for reseeding or culturing mature polarized barrier models.

[0253] Once confluent, the cells were seeded at 10 5 cells / cm 2 onto Transwell® filters (12 mm diameter, 0.4 μm pore size; Corning Life Sciences, Netherlands). Cell confluence and cell layer integrity were confirmed by TEER using an EVOM (World Precision Elements, USA) volt-ohm meter. The cells were used only when their TEER was stable and exceeded 600 Ω / cm 2 (for Calu-3 and Caco-2). The filter-cultured Calu-3 cell layer was used on days 13 - 14 after seeding, and the Caco-2 and ARPE-19 cell layers were used on day 21 after seeding.

[0254] Effect of QS molecules on Calu-3 metabolic activity: MTS assay To evaluate the cytotoxicity of 3OC12-HSL and C7 PQS molecules on the cell viability of Calu-3 cell monolayers and to monitor short-term cytotoxicity during the experiment, Calu-3 cells were treated with 3OC12-HSL and C7 PQS at concentrations of 5 nM, 1 μM, 50 μM, 100 μM and 200 μM for 6 hours. At the end of the treatment, the cell viability (by measurement of metabolic activity) of the cultures was determined by a colorimetric MTS assay. Assay:

[0255] The viability of Calu-3 cells was determined by a colorimetric MTS method based on the measurement of mitochondrial respiration evaluated by the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium to formazan. Calu-3 cells were cultured in 96-well plates (CellTiter 96® Aqueous, Promega) at a density of 10 4 cells / well and incubated at 37 °C, 5% CO2 for 24 hours. After 24 hours, the medium was replaced with 100 μl of the test compound dissolved in HBSS, and then the cells were incubated for 6 hours. HBSS and Triton X-100 (0.1% v / v in HBSS) were used as negative and positive controls, respectively. Then, the bath solution was aspirated and the cells were washed with PBS. Then, culture medium (100 μl at 37 °C) + MTS reagent (20 μl) was added to each well and the cells were incubated for 2 hours. The absorbance at 492 was measured using a Dynex absorbance microplate reader (Dynex Technologies, USA) and converted to percentage metabolic rate.

[0256] Fluorescein isothiocyanate-labeled dextran 4400 (FD4) permeability The culture medium was removed from the filter-cultured Calu-3 cells and replaced with Hank's balanced salt solution (HBSS), buffered with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, 20 mM). The cell layer was incubated with HBSS for 45 minutes. The quorum sensing molecules 3OC12-HSL and C7 PQS and dextran-FITC 4 kDa (FD4: 500 μg / ml [125 μM]) diluted in HBSS / HEPES were applied to the apical side chamber of each Transwell®.

[0257] The migration of FD4 from the apical side to the basolateral side was determined by sampling 100 μL every 30 minutes for 5 hours from the basolateral side. To maintain a constant basolateral volume, 100 μl of HBSS was added to the basolateral chamber after each sample was taken. Then, FD4 fluorescence (excitation 485 nm, emission 535 nm) was measured using an MFX microtiter plate fluorometer (Dynex Technologies, USA). FD4 in the basolateral solution was quantified by constructing a calibration curve and converting the fluorescence readings to FD4 concentration and amount.

[0258] To evaluate the tight junction integrity, the TEER of each cell layer was also measured at the same time points throughout the experimental period.

[0259] Immunostaining To visualize the effects of 3OC12-HSL and C7 PQS on tight junction integrity in the Calu-3 cell layer, ZO-1, immunostaining and confocal microscopy were performed on cell layers treated with 200 μM of 3OC12-HSL and C7 PQS and a control (0.15% DMSO in HBSS) for 1 hour.

[0260] The confluent cell monolayer was washed with PBS and fixed in 4% paraformaldehyde in PBS at room temperature for approximately 10 minutes. The cells were then washed three times with PBS and permeabilized by incubating with Triton X-100 (0.1% v / v in PBS) for 10 minutes. The cells were then washed with PBS and subsequently 1% BSA / PBS was applied for approximately 1 hour. Thereafter, the BSA / PBS solution was aspirated and replaced with mouse anti-human ZO-1 (primary) antibody, diluted with 1% BSA / PBS to a final concentration of 100 μg / ml. The cell samples were incubated with the primary antibody for 2 hours. The primary antibody solution was then removed and the cells were washed with PBS (5 times). Subsequently, FITC-labeled goat anti-mouse (secondary) antibody diluted with 1% BSA / PBS according to the manufacturer's instructions was applied to the cells for 1 hour. The secondary antibody solution was then aspirated and the cells were washed three times with PBS. The Transwell® filter membrane was excised, mounted on a glass slide (using ProLong® Gold antifade / mounting reagent containing DAPI), covered with a glass coverslip for confocal imaging, and confocal imaging was performed using a Zeiss LSM 880 confocal microscope.

[0261] Effect of 3OC12-HSL on in vitro drug permeability Using three polarized epithelial in vitro tissue models including lung epithelium (Calu-3), retinal pigment epithelium (ARPE-19) and intestinal epithelium (Caco-2), the efficiency of 200 μM 3OC12-HSL as a paracellular permeability enhancer for three different drugs: bevacizumab, aflibercept and doxorubicin (applied concentration 250 μg / mL) was tested. The amount of drug transported was determined by converting the fluorescence intensity to quantity (μg or ng) using a calibration curve constructed by serial standard dilution.

[0262] Transport assay Before the drug delivery experiment, the culture medium was removed, the cells were washed with PBS, and then HBSS / HEPES (warmed to 37°C; pH 7.4) was added to both sides (apical side and basolateral side) of the cell monolayer. Before the experiment, the cells were equilibrated in HBSS / HEPES for approximately 45 minutes (i.e., incubated at 37°C, 5% CO2).

[0263] The permeability experiment was initiated by applying 0.5 mL of the test solution to the apical side of the cells (donor compartment of Transwell®). The test solution contained the drug (250 μg / mL bevacizumab, aflibercept or doxorubicin diluted in HBSS / HEPES), with or without 3OC12-HSL (200 μM), and its permeation enhancing effect was investigated. The drug permeability was determined by sampling the basolateral solution at regular time intervals. Specifically, 100 μl volumes were removed from the basolateral chamber of the Transwell® system every 60 minutes for 5 hours. The sampled solution was immediately exchanged with an equal volume of the transport medium (HBSS).

[0264] Quantification of bevacizumab and aflibercept by ELISA after the transport experiment Solutions (100 μl volume) sampled during the bevacizumab and aflibercept transport experiments were placed into high-binding 96-well ELISA plates and incubated overnight at 4 °C to allow the IgG present in the sample solution to coat the surface of the multi-well plates. The next day, the sample solution was removed and the plates were washed three times with a wash buffer containing 0.05% v / v Tween 20 in PBS. The wells were then blocked for 1 hour at room temperature with 200 μl of 1% BSA (w / v) in PBS (blocking buffer). The blocking buffer was then removed and the wells were washed with the wash buffer. 100 μl of mouse anti-human IgG conjugated to horseradish peroxidase (HRP) diluted in the blocking buffer was added to each well. The plate was incubated for 2 hours at room temperature to label both bevacizumab and aflibercept in each well via the IgG Fc domain. The anti-human IgG-HRP solution was then removed and the plate wells were washed three times with the wash buffer. After adding the chromogenic TMB substrate (100 μl) to each well for 10 minutes, 20 μl of 2.5 M sulfuric acid was added to stop the reaction, and then the plate was transferred to a microplate reader to measure the absorbance (at 450 nm) of the HRP conversion product. The concentration of IgG, and thus the macromolecular therapeutic agent, was quantified by converting the absorbance readings to concentration through a complementary calibration curve created by serial dilution.

[0265] Quantification of Doxorubicin The apical-to-basolateral migration of endogenous fluorescent doxorubicin was determined using a fluorescence plate reader (λ ex 470 nm; λ em 585 nm) with an MFX microtiter plate fluorometer (Dynex Technologies, USA).

[0266] Statistical Analysis All experiments were performed using triplicate samples and repeated three times. One-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests were applied for comparisons between group means of three or more groups, and Student's t-tests were used for comparisons between two groups. Data are shown as the mean ± SEM of three different experiments. p-values less than 0.05 were considered statistically significant. *, **, and *** in the figures indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

[0267] Effect of 3OC12-HSL and C7 PQS on paracellular flux and transepithelial electrical resistance (TEER) of FITC-dextran (4 kDa) (FD4) across polarized Calu-3 cell monolayers To test the effects of 3OC12-HSL and C7 PQS on the barrier function of airway epithelium, human-derived Calu-3 cell monolayers were treated with 3OC12-HSL and C7 PQS, and the effects on the barrier function were determined by measuring TEER and monitoring the barrier-crossing transport of FD4 across mature Calu-3 cell monolayers.

[0268] Results Effect of 3OC12-HSL on drug permeability in vitro Figures 1 - 3 show the amount of drug present in the basolateral solution at different times after addition of the drug to the apical surface of the Transwell model tissue barrier.

[0269] The transport of all drugs across the epithelial cell model was significantly increased when administered with 3OC12-HSL compared to the same drugs administered in the absence of 3OC12-HSL.

[0270] Doxorubicin transport was enhanced 6-fold (62.33 μg vs. 9.85 μg) in the ARPE-19 cell monolayer (Figure 1), 17-fold (25.90 μg vs. 1.45 μg) in the Calu-3 cell monolayer (Figure 2), and 19-fold (59.33 μg vs. 3.07 μg) in the Caco-2 cell monolayer (Figure 3) by 3OC12-HSL after 5 hours. These enhancements in the delivered therapeutic agents were statistically significant at the α = 0.05 level.

[0271] In the presence of 3OC12-HSL, the transport of bevacizumab across the Calu-3 cell layer (10-fold: 663.43 ng vs. 61.03 ng) (Figure 2) and the Caco-2 cell layer (8-fold: 469.22 ng vs. 54.64 ng) (Figure 3) was also significantly enhanced. The permeability of bevacizumab across the ARPE-19 cell layer (Figure 1) was significantly increased 2.5-fold with 60-minute exposure (**p < 0.01).

[0272] The transport of aflibercept across the Calu-3 layer (18-fold: from 36.21 ng to 650.34 ng) (Figure 2) and the Caco-2 cell layer (11-fold: from 33.82 ng to 376.27 ng) (Figure 3) was significantly enhanced by 3OC12-HSL (***p < 0.001, n = 3), indicating that the opening of the paracellular pathway was efficient in these layers.

[0273] Cytotoxicity: MTS assay No significant decrease in cell metabolic activity was observed. Cell viability of more than 75% was maintained in all cases. Thus, 3OC12-HSL and C7 PQS do not have a cytotoxic effect detrimental to cell viability within the epithelial layer. Specifically, 200 μM of 3OC12-HSL did not change the metabolic activity in all three cell lines compared to the negative control, indicating that this high concentration of 3OC12-HSL was well tolerated by the cells.

[0274] Effects of 3OC12-HSL and C7PQS on paracellular flux of FITC-dextran (4 kDa) (FD4) and transepithelial electrical resistance (TEER) in polarized Calu-3 cell layers Treatment of the Calu-3 cell layer with 3OC12-HSL (concentrations of 200 μM or 1 μM in 0.15% DMSO in HBSS) resulted in a significant decrease in TEER (p < 0.001) compared to vehicle only (0.15% DMSO in HBSS). This decrease in TEER was correlated with an increase in paracellular passage of FD4 across the cell layer.

[0275] The flow of FD4 reached a peak after 30 minutes and slowly continued to increase over 4 hours (the duration of the test period).

[0276] Referring to Figure 4, the TEER began to increase after 4 hours and reached 520 + / - 20 Ω.cm after 7 hours 2 indicating that the effect on 3OC12-HSL cell layer integrity is reversible. At the end of the experiment, after removing 3OC12-HSL and replacing it with fresh cell culture medium, the TEER of the Calu-3 cell layer returned to baseline (about 940 + / - 40 Ωcm -2 ) after 24 hours.

[0277] Treatment of the Calu-3 cell layer with 200 μM C7 PQS also increased the flow of FD4 from the apical to the basolateral chamber in a similar manner. This also decreased the TEER from 1200 Ωcm-2 to 98 Ωcm -2 within the first hour of exposure. After 5 hours, the integrity of the cell layer began to recover and the TEER reached 510 Ωcm -2 (SEM + / - 20) after 7 hours.

[0278] Treatment with 200 μM C7 PQS also enhanced the trans-layer flow of FD4 across the Calu-3 cell layer.

[0279] Effect of 3OC12-HSL and C7 PQS on ZO-1 distribution Morphological changes within the tight junctions resulting from the application of 200 μM 3OC12-HSL and C7 PQS could be seen. In this regard, the continuous ring of fluorescence resulting from ZO-1 staining at cell-cell junctions was clearly visible in control cell layers exposed to vehicle (0.15% DMSO) only. In contrast, in cell layers treated with 200 μM 3OC12-HSL and PQS, a significant dislocation of the ZO-1 protein was evident.

[0280] These findings, combined with the impairment of the barrier function observed in the presence of both 3OC12-HSL and C7 PQS, strongly suggest that for some reason the fundamental mechanism disrupts tight junction organization.

[0281] Example B - Further In Vitro Tests Using an in vitro Transwell assay, several additional molecules were tested to evaluate whether they exhibited the same ability to enhance the translocation of drugs across an epithelial cell model.

[0282] The results were as follows: [Table 1 - 1] [Table 1 - 2] [Table 1 - 3] [Table 1 - 4]

[0283] Therefore, a series of agents according to the present invention have been shown to be effective in enhancing the paracellular permeability of payload molecules.

[0284] For all molecules indicated as "Yes" in the right - hand column, the results were comparable to 3OC12 - HSL and C7 PQS.

[0285] Looking at the molecules that were not effective (indicated as "No" in the right - hand column), these were unable to meet the structural requirements of the present invention for the following reasons: - The presence of a double bond or N - heteroatom within the R' chain; and / or - The presence of a - CH2NMe substituent at the 2 ortho position (with respect to the R' group).

[0286] Example C - MMP Activation Screening for Activity to the Extent Possible MMP Activity Assay - 3OC12 - HSL and C7 PQS To investigate the involvement of MMP in the disruption of tight junctions observed in the presence of 200 μM 3OC12-HSL and C7 PQS, a fluorimetric MMP activity assay was performed using the Amplite™ Universal Fluorimetric MMP Activity Assay Kit. This kit is designed to check the general activity of MMP enzymes.

[0287] After treatment with 3OC12-HSL and C7 PQS respectively, the conditioned medium was collected from the apical chamber every hour, and MMP activity was determined by using Amplite™ Universal Fluorimetric MMP Activity Assay Kit Green Fluorescence according to the manufacturer's instructions.

[0288] Review of samples taken at hourly intervals from the apical chamber during QS exposure revealed significant MMP activation (p < 0.01) in samples treated with 3OC12-HSL and samples treated with C7 PQS.

[0289] No fluorescence was observed in the control samples.

[0290] MMP activation was found to increase over the first 4 hours for both 3OC12-HSL and C7 PQS. Then, for samples taken after 4 hours, it was found that MMP activation began to decline.

[0291] This correlation between time and the observed decrease in TEER and increase in FD4 transport strongly implies that MMP activation by QS molecules underlies the disruption of the barrier function observed.

[0292] Further MMP screening The fluorimetric MMP activity assay was similarly performed for additional agents.

[0293] The following additional agents have been found to cause MMP activation and are thus considered useful as agents according to the present invention.

Table 2

[0294] Further tests: C4 HSL (BHL) was also tested and found to cause MMP activation and is thus similarly considered useful as an agent according to the present invention:

Chemical formula

[0295] Figure 5 shows agents that cause MMP activation together with negative and positive controls for reference.

[0296] Example D: Ex vivo test: An ex vivo test was conducted to confirm that the beneficial effects on the epithelial barrier shown in vitro are also reproduced ex vivo.

[0297] 3OC12-HSL was used as an exemplary agent for these tests.

[0298] Effect of 3OC12-HSL on drug permeability ex vivo To investigate the effectiveness of 3OC12-HSL as a drug delivery promoter in intact tissues, bevacizumab (250 μg / mL: 167 μM) and aflibercept (40 mg / mL: 348 μM) were administered to ex vivo rat eyes with and without 3OC12-HSL, and the distribution of the drugs was visualized in sagittal eye frozen sections by confocal microscopy. In a further series of experiments, the effectiveness of 3OC12-HSL through ex vivo porcine skin was tested.

[0299] Ex vivo test Six hooded rats weighing 280 - 300 g and having pigmented eyes at 7 - 8 weeks of age were provided by the Bio - Support Unit at the Queen’s Medical Centre in Nottingham. The experiments were designed to be carried out ex vivo immediately after sacrifice during experiments supporting other trials. The test therapeutic agents (bevacizumab (250 μg / mL) with / without 3OC12 - HSL (200 μM), aflibercept (250 μg / mL)) were applied (50 μL) to the corneal surface of each animal immediately after sacrifice for 1 hour and then washed away using ice - cold PBS. The eyeballs were enucleated, immersed in OCT, snap - frozen in liquid N2 - cooled isopentane and stored at - 80 °C for further processing.

[0300] Fresh full - thickness porcine skin was obtained immediately after sacrifice from the RB Elliot & Son Stud Farm in Carlow and held at 4 °C for 60 minutes. 5 cm squares were prepared with a scalpel and the central zone of the epidermis was demarcated with a lipophilic “Pap pen” (Abcam) to locally contain the applied liquid. The test therapeutic agents (bevacizumab (250 μg / mL) with / without 3OC12 - HSL (200 μM)) were applied to the zone demarcated by the Pap pen in PBS and the samples were held at 37 °C for 60 minutes. The skin surface was washed 3 times with room - temperature PBS and the samples were fixed overnight at 4 °C in 4% paraformaldehyde. The fixed samples were sliced in half with a scalpel to expose the central zone where the application was made, immersed in OCT and then plunge - frozen in liquid N2 - cooled isopentane and stored at - 80 °C for further processing.

[0301] Tissue section preparation The OCT - embedded eyes were sectioned into 15 - μm tissue sections using a cryostat (Leica 3050) and mounted onto gelatin - coated slides at a cutting temperature between - 15 and - 23 °C. The moisture trapped within the sections was sublimated on dry ice in an open box for 30 minutes to prevent the diffusion of the agents within the sections and the sections remained frozen throughout. The slides were then stored at - 80 °C.

[0302] OCT-embedded eye and skin samples were cut into 15 μm tissue sections using a cryostat (Leica 3050) and mounted on gelatin-coated slides at a cutting temperature between -15 and -23 °C. Trapped moisture within the sections was sublimated on dry ice for 30 min in an open box to prevent diffusion of drugs within the sections, which remained frozen throughout. Slides were then stored at -80 °C.

[0303] Tissue immunostaining Immediately after removal from the -80°C freezer, 50 μL of ice-cold methanol was added to each tissue section for 8 minutes to fix and precipitate all proteins. All sections were blocked by incubating with blocking buffer (1% horse serum in PBS) for 30 minutes at room temperature. DyLight 550-goat anti-human IgG Fc antibody (1:200) was applied to the sections for 1 hour, followed by thorough washing with PBS (3 times, 15 minutes) and mounting with antifade medium for confocal microscopy (Zeiss LSM 880 confocal microscope).

[0304] result Effect of 3OC12-HSL on ex vivo drug permeability Confocal laser scanning microscopy (CLSM) imaging demonstrated, via fluorescence, successful delivery of clinical formulations of aflibercept and bevacizumab across the cornea when 3OC12-HSL was applied, and thus into the diffusive reach of the anterior ciliary circulation, which directly perfuses the posterior retina.

[0305] No fluorescence was detected in eye sections where bevacizumab and aflibercept were applied without 3OC12-HSL, nor in eye sections treated with vehicle (HBSS / 0.1% DMSO) alone.

[0306] Example E - In vivo testing In vivo studies were performed to confirm that the effects on the epithelial barrier shown in vitro and ex vivo were reproduced in vivo.

[0307] 3OC12-HSL was used as an exemplary agent for these tests.

[0308] Hyperglycemia induction and glucose measurement The body weights of a total of 24 male Norway Brown rats (250 - 300 g) were measured, and a single intraperitoneal (i.p.) injection of streptozotocin (STZ) at 50 mg / kg (Sigma - Aldrich, Missouri, USA) was performed. For non - diabetic controls, an equal volume of physiological saline was injected. In addition to water, a 15% (w / v) sucrose solution was made available in a separate drinking bottle to mitigate the initial hypoglycemic spike after STZ induction. The sucrose intake was monitored over 72 hours. On the 4th day, the animals were anesthetized with isoflurane (2 - 5%), and 1 / 3 of a single Linplant insulin pellet (LinShin, Canada) was subcutaneously implanted into the neck muscle. Blood samples were collected from the tail vein on day 0, day 4, and before sacrifice (day 28), and blood glucose levels were measured using an Accu - Chek blood glucose monitor. Rats with blood glucose levels above 15 mmol / l were considered hyperglycemic. As outlined above, STZ - administered rats that did not become hyperglycemic on the 4th day were reinjected with STZ the next morning and then evaluated for diabetes.

[0309] Local eye treatment plan The animals were administered topical eye drops of 250 μg / ml bevacizumab with / without 3OC12 - HSL (200 μM) and control vehicle only (25 μL / eye) twice a day. The animals were gently restrained for approximately 30 seconds to convert the droplets into a gel upon thermal activation on the corneal surface. The animals were examined twice a day for signs of inflammation in the eyes and evidence of eye or nasal secretions.

[0310] Fluorescein fundus angiography (FFA) Fluorescein sodium salt (Na-Fl; M.W. 376.27) was prepared in sterile PBS to a final concentration of 10 mg / ml (w / v), filtered through a 0.2 μm filter, and stored away from direct light at room temperature until needed. Animals were anesthetized i.p. with a combination of ketamine hydrochloride, 37.5 mg / kg (Ketast (registered trademark), 100 mg / mL) and medetomidine hydrochloride, 0.25 mg / kg (Sedastart (registered trademark), 1 mg / mL), and transferred to an image cradle equipped with a heat mat. One drop each of phenylephrine hydrochloride, 5% and tropicamide, 0.8% was applied to the left eye to dilate the pupil. Viscotears (registered trademark) was applied to the corneas of both eyes to prevent dehydration, and a cotton tip was used to place the whiskers outside the visual field. A Micron IV ophthalmoscope (Phoenix Technology Group Inc.) was advanced towards the cornea, the optic nerve was centered in the visual field, and the brightness and focus were adjusted to achieve a clear image of the major retinal vessels. Once the eye was properly aligned, a bright-field fundus image of the retina was captured to confirm eye abnormalities. The FITC filter was selected, and a 3-minute video of the retina was recorded at 15 frames / second at maximum gain. Next, the rats were given a single 250 μl i.p. injection of Na-Fl (10 mg / ml, w / v). After imaging, the animals were recovered with atipamezole hydrochloride (Sedastop (registered trademark), 5 mg / mL). This was repeated on days 7, 14, 21, and 28, aligning the eye at the same position as captured on day 0.

[0311] FFA analysis The angiograms were imported as a virtual stack into Fiji software. The region of interest tool was used to define a first region within the major retinal vessels and a second region of the stroma (including unresolved capillaries) using the rectangular selection tool. The Na-Fl intensity in the retinal stroma and major retinal vessels was measured every 200 frames from 0 to 1800 frames (120 seconds) and plotted against time. The intensity values and time were processed through a Fiji macro to calculate retinal permeability.

[0312] Laser-induced choroidal neovascularization Female C57BL / 6J mice (16 - 20 g, 6 - 8 weeks old, Charles River, UK) were anesthetized by intraperitoneal (i.p.) injection of 50 mg / kg ketamine hydrochloride (Ketaset®, 100 mg / mL) and 0.5 mg / kg medetomidine hydrochloride (Sedastart®, 1 mg / mL). The pupils were dilated by instilling 5% phenylephrine hydrochloride and 0.8% tropicamide into each eye. In each eye, four photocoagulation lesions were obtained using a green Merilas laser (532α, 450 mW, 130 ms, 75 μm) in combination with a Micron IV ophthalmoscope at a distance of 1 - 2 disc diameters from the optic nerve and between retinal blood vessels in a peripapillary distribution. Only laser lesions with subretinal bubbles during treatment were included in the study. Immediately after laser photocoagulation, the animals were administered topical drops twice a day in both eyes (using 10 μL of 250 μg / mL bevacizumab with / without 3OC12 - HSL, 200 μM, and control vehicle only). On days 7 and 14, the animals were anesthetized, the pupils were dilated as described above, and an i.p. injection of 10 mg / mL Na - Fl in 100 μL of PBS was performed. Then, the retinas of both eyes were imaged using a Micron IV ophthalmoscope. Fundus angiography images were imported into Fiji software, and masked observers tracked the lesions, quantified the lesion area over the treatment time, and compared between treatment groups. Fourteen days after imaging, the animals were sacrificed, the eyeballs were enucleated, and fixed in 4% paraformaldehyde (w / v) for 1 hour.

[0313] Preparation of tissue sections The OCT - embedded eyes were sectioned into 15 - μm tissue sections using a cryostat (Leica 3050) and mounted on gelatin - coated slides at a cutting temperature between - 18 and - 23 °C. The moisture trapped within the sections was sublimated on dry ice in an open box for 30 minutes to prevent the diffusion of agents within the sections, and the sections remained frozen throughout. The slides were then stored at - 80 °C.

[0314] Tissue immunostaining 50 μL of ice-cold methanol was taken out from an -80 °C freezer and immediately added to each tissue section for 8 minutes to fix all proteins in place and precipitate them. All sections were blocked by incubating with blocking buffer (1% horse serum in PBS) at room temperature for 30 minutes. After applying DyLight 550-goat anti-human IgG Fc antibody (1:200) to the sections for 1 hour, they were washed thoroughly with PBS (3 times, 15 minutes each) and mounted with an anti-fading medium for confocal microscopy (Zeiss LSM 880 confocal microscope).

[0315] Statistical analysis Data were obtained from three independent replicate experiments. All data and graphs were formulated using Microsoft Excel (Microsoft Office software), GraphPad Prism v7 / 8 (GraphPad Software Inc., California, USA), Fiji, and Imaris. Data analysis was performed using one-way and two-way ANOVA, followed by Bonferroni post hoc correction.

[0316] Unless otherwise specified, all data are presented as mean ± SEM. All results were considered statistically significant at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

[0317] 3OC12-HSL enhances the delivery of bevacizumab locally administered to the posterior chamber of the eye in rodents.

[0318] Bevacizumab was formulated in Pluronic® F127 in the presence and absence of 3OC12-HSL (100 μM) and topically applied to the cornea. Two different rodent strains were selected to investigate the efficacy of 3OC12-HSL as a drug penetration enhancer in combination with bevacizumab. Bevacizumab is currently administered by multiple intravitreal injections to achieve therapeutic concentrations in the posterior segment of the eye and at the disease site. Local delivery of drug molecules to the back of the eye is a major challenge due to the many layers of the eye through which the drug must penetrate, the blood-aqueous barrier and the blood-retinal barrier, choroidal and conjunctival blood flow, lymphatic clearance, efflux pumps, and tear dilution. Drugs that have been successful in reaching the posterior segment do so by passive diffusion down a concentration gradient through the corneal, scleral, and conjunctival tissues. Thus, permeation aids such as 3OC12-HSL could revolutionize the way posterior segment diseases are currently treated.

[0319] Confocal imaging of sagittal frozen sections of the eye showed significantly enhanced permeability of bevacizumab when co-administered with 3OC12-HSL compared to administration of bevacizumab alone. The presence of 3OC12-HSL was found to significantly enhance the delivery of bevacizumab to the posterior chamber of the rat eye, as indicated by an increase in the expression of bevacizumab in confocal images.

[0320] To further confirm the above, in vivo data obtained from a diabetic rat model with increased retinal permeability showed a statistically significant decrease in vascular leakage (p = 0.023) after 28 days in rats topically treated twice daily with bevacizumab + 3OC12-HSL (0.083 ± 0.044 P フルオレセイン μm / s) (Figure 17D) or vehicle control (1.253 ± 0.253 P フルオレセイン μm / s) (Figure 6C) compared to bevacizumab alone (0.316 ± 0.078 P フルオレセイン μm / s). As previously published, vascular leakage increased significantly over time in the vehicle-only control group (Figures 6A-6D).

[0321] In addition to the diabetic retinopathy model, 3OC12-HSL was tested as a potential drug permeation enhancer in a laser-induced model of CNV. Treatment with bevacizumab (250 μg / mL) alone decreased the mean lesion area on day 7 (2.86 ± 0.47×10 4 ) compared to the vehicle control (3.75 ± 0.53). Interestingly, bevacizumab combined with 3OC12-HSL significantly decreased the lesion size on day 7 (0.19 ± 0.18×10 4 ) compared to the vehicle control (3.05 ± 0.45×10 4 , p = 0.002), confirming the effectiveness of 3OC12-HSL as a drug permeation enhancer in angiogenic eye diseases (Figure 7).

[0322] Example F: Further Tests The results of further Transwell tests are shown in Figures 8 and 9.

[0323] Figure 8 shows the results for 12 test molecules investigated in a rough dose response by FD4 delivery in Transwell cultures (Calu-3) to confirm the effect on FITC-dextran (4 kDa) transport. Each molecule was screened at 5 nM, 1 μM, and 200 μM levels. Figure 9 shows the results for 6 additional test molecules screened at the 200 μM level.

[0324] Discussion and Conclusion: Some agents have been shown to have advantages as excipient permeation enhancers that increase macromolecular therapeutic drug transport across epithelial layers in vitro and ex vivo through targeted reversible modulation of tissue barrier integrity.

[0325] For it to be useful from a practical and therapeutic perspective, it is important to have a recovery phase, i.e., repair, since disruption of the epithelial barrier needs to be transient rather than having permanent disruption or damage.

[0326] In summary, these results demonstrate that the agent according to the present invention is capable of inducing a reversible disruption of epithelial barrier integrity.

[0327] Enhanced paracellular delivery of both macromolecular (aflibercept, bevacizumab, mepolizumab) and small molecular (doxorubicin) therapeutic agents has been observed after administration of the agent according to the present invention, and this has been shown to be due to the opening of tight junctions rather than a result of toxicity.

[0328] It is surprising that macromolecular agents (aflibercept, bevacizumab, mepolizumab: approximately 150 kDa) as well as small molecules have been shown to be effectively delivered across the epithelial barrier by using the present invention. It would not have been expected that molecules of this size could be delivered. The present invention advantageously provides a versatile approach that can be used with a wide range of payload molecules. The ability to effectively transport drugs of various sizes by permeating the epithelial barrier is technically important and offers significant advantages over intravenous administration.

[0329] The in vitro experiments performed demonstrated that bevacizumab and aflibercept transport across mature ARPE-19 monolayers as well as across Calu-3 and Caco-2 model epithelial barriers was significantly enhanced in the presence of the agent according to the present invention. Co-administration of doxorubicin with the agent according to the present invention significantly enhanced transport in all model epithelial barriers tested.

[0330] Enhanced ex vivo delivery of aflibercept and bevacizumab across the cornea when co-administered with the agent according to the present invention was equivalent to that obtained using in vitro epithelial models. Enhanced delivery of aflibercept and bevacizumab across the corneal epithelium was confirmed by immunostaining of eye sections, and aflibercept and bevacizumab were not detected in the contralateral control eyes.

[0331] The topical co - administration of the agent and the polymeric therapeutic agent according to the present invention to the cornea and to the model epithelial barrier enhances the trans - epithelial delivery of the polymeric therapeutic agent. This is advantageous since very few drugs penetrate into eye tissues by topical administration and only drugs with a molecular weight of less than 500 Daltons can penetrate.

[0332] In conclusion, the agent according to the present invention can be used with polymeric therapeutic agents to increase the permeability of in vitro and ex vivo epithelial tissue models. By promoting the transport of these important modern therapeutic agents through the paracellular pathway in a non - toxic and reversible manner, a new beneficial pathway of excipient action is provided through the specific, non - toxic and reversible disruption of epithelial tight junctions.

[0333] Therefore, the claimed agent can be used as an efficient permeation enhancer of drugs to improve drug delivery across the epithelial tissue barrier, having beneficial implications for patient comfort and cost in clinical medicine.

Claims

Claim 1 A method for delivering a payload molecule across an epithelial tissue barrier, comprising: - applying the payload molecule to the epithelial tissue barrier; - further applying an agent to the epithelial tissue barrier; wherein the agent is (ii) a carboxylic acid compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof: (i)A compound that can disrupt the epithelial tissue barrier function and includes a heterocyclic moiety and an alkane moiety containing an R' group that is a C1-12 alkyl group which may be unsubstituted or substituted, wherein any substituents present are selected independently from hydroxyl, halogen, and NR'', ２ A microbial quorum sensing signaling molecule (microbial QSSM) or a derivative or variant thereof, wherein each R'' is independently selected from hydrogen and methyl; or (wherein 【Chemical 1】 selected from, a method. R ８ is H or R'; R ９ is H or R'; R １０ is H or R', provided that at least one R' group is present; Each R' is independently selected from unsubstituted or substituted C1-12 alkyl groups, and any substituents present are selected independently from hydroxyl, halogen, and NR'' ２ where each R'' is independently selected from hydrogen and methyl). Claim 2 The method according to claim 1, wherein the R' group is a saturated hydrocarbon chain containing no N heteroatom in the chain. Claim 3 The method according to claim 1 or 2, wherein the R' group is unbranched or the R' group is branched and contains one or less C1 branches from the main chain. Claim 4 Claim 5 When the R' group is provided as a substituent on an aromatic or non-aromatic ring, the substituent at any position ortho to the R' group is selected from the group consisting of OH, CH ３ , H, R', and O, the method according to any one of claims 1 to 3. The agent is a compound of formula (II), (III), (IV) or (V), or a pharmaceutically acceptable salt, hydrate or solvate thereof: (wherein 【Chemical 2】 is a microorganism QSSM, the method according to any one of claims 1 to 4. R １ is O or OH; R ２ is OH, CH ３ or H; R ３ is R'; R ４ is H, R', or O; R ５ is H or R'; R ６ is O, CH ２ , CHR' or CR' ２ and; R ７ is CH ２ C(O)R' or R'; Each R ｘ is independently selected from halo, C1-12 alkyl, C1-12 alkyl halo, OR ｙ , and NR'' ２ , and R ｙ is hydrogen or C1-12 alkyl, each R'' is independently selected from hydrogen and methyl; n is an integer of 0 or 1-4, and m is an integer of 0 or 1-2; Each R' is independently selected from unsubstituted or optionally substituted C1-12 alkyl groups, and any substituents present are selected independently from hydroxyl, halo, and NR'', ２ where each R'' is independently selected from hydrogen and methyl). Claim 6 One or more of the following are applied to the microorganism QSSM: The method according to claim 5, which is a compound of formula (II), or a pharmaceutically acceptable salt, hydrate or solvate thereof. ・R １ is O; and / or ・R ２ is OH or H; and / or ・R ４ wherein R is H or O Claim 7 The microorganism QSSM is a compound of formula (II), or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the condensed ring structure is or (i) halo, C1-12 alkyl, C1-12 alkyl halo, and OR ｙ (wherein R ｙ is hydrogen or C1-12 alkyl); or (ii) halo (e.g., F), C1-6 alkyl, C1-6 alkyl halo, and OR ｙ (wherein R ｙ is hydrogen or C1-6 alkyl); (iii) halo (e.g., F), methyl, and hydroxyl Claim 8 One to four (e.g., one or two) substituents R, each independently selected from ｘ The method according to claim 5 or 6, comprising a substituted aromatic group in which there is The microorganism QSSM is a compound of formula (II), or a pharmaceutically acceptable salt, hydrate or solvate thereof, optionally, the microorganism QSSM is the compound 7F-C7PQS or 6F,7F-C7PQS: The condensed ring structure contains a substituted aromatic group having 1 to 4 (e.g., 1 or 2) substituents R which are halo groups (e.g., F). ｘ and is present. The method according to claim 7. [Chemical Formula 3] Claim 9 The method according to claim 5 or 6, wherein the microorganism QSSM is a compound of formula (II) where n is equal to 0, or a pharmaceutically acceptable salt, hydrate or solvate thereof. Claim 10 The microorganism QSSM is a compound of formula (II-A), (II-B), (II-C), (II-D) or (II-E), or a pharmaceutically acceptable salt, hydrate or solvate thereof: The method according to claim 9. 【Chemical Formula 4】 Claim 11 Optionally, the microorganism QSSM is a compound selected from HHQ, NHQ, UHQ C7PQS, C1 PQS, C9 PQS, C11 PQS, and 7F-C7PQS, or a pharmaceutically acceptable salt, hydrate or solvate thereof: wherein the microorganism QSSM is R １ is O, and R ２ is OH or H, and R ４ is H or O, a compound of formula (II-B), or a pharmaceutically acceptable salt, hydrate or solvate thereof; ​ [Chemical Formula 5] The method according to claim 10, wherein.

12. wherein the microorganism QSSM is R ４ The method according to any one of claims 5 to 11, wherein is H or O, a compound of formula (II), or a pharmaceutically acceptable salt, hydrate or solvate thereof.

13. The microbial QSSM is a compound of formula (II-F) or (II-G), or a pharmaceutically acceptable salt, hydrate or solvate thereof: 【Chemical Formula 6】 being Optionally, the compound C7HHQ N-oxide or C9HNQ N-oxide, or a pharmaceutically acceptable salt, hydrate or solvate thereof: 【Chemical Formula 7】 The method according to claim 12, wherein.

14. One or both of the following are applied to the microbial QSSM: ・R ５ is H; and / or ・R ６ is O or CH ２ is, The method according to claim 5, wherein the compound is of formula (III), (IV) or (V), or a pharmaceutically acceptable salt, hydrate or solvate thereof.

15. The microbial QSSM is (i) halo, C1-12 alkyl, C1-12 alkyl halo, and OR ｙ (wherein R ｙ is hydrogen or C1-12 alkyl); or (ii) halo, C1-6 alkyl, C1-6 alkyl halo, and OR ｙ (wherein R ｙ is hydrogen or C1-6 alkyl); or (iii) halo (e.g., F), methyl, and hydroxyl One or two substituents R, each independently selected therefrom ｘ is present The method according to claim 5 or 14, wherein the compound is of formula (III), (IV) or (V), or a pharmaceutically acceptable salt, hydrate or solvate thereof.

16. The method according to claim 5 or 14, wherein the microbial QSSM is a compound of formula (III), (IV) or (V) in which m is equal to 0, or a pharmaceutically acceptable salt, hydrate or solvate thereof. wherein the microorganism QSSM is R ６ is O, a compound of formula (III), (IV) or (V), or a pharmaceutically acceptable salt, hydrate or solvate thereof,

17. 【Chemical 8】 Optionally, the microbial QSSM is a compound selected from OdDHL, C4HSL, OdDAT and OdDG, or a pharmaceutically acceptable salt, hydrate or solvate thereof: The method according to any one of claims 5 and 14 to 16, wherein.

18. The method according to any one of claims 1 to 17, wherein the microbial QSSM is (i) not C7PQS (2-heptyl-3-hydroxy-4(1H)-quinolone), and / or (ii) not 3-oxo-C12HSL, and / or (iii) not cis-2-decenoic acid. wherein the agent is R ８ is H or R'; R ９ is H or R'; R １０ is R', a carboxylic acid compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof, the method according to any one of claims 1 to 3.

19. wherein the agent is R ８ is H; R ９ is H; R １０ is R', a carboxylic acid compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof;

20. The method according to any one of claims 1 to 3, wherein optionally the agent is cis-2-decenoic acid.

21. The method according to any one of claims 1 to 20, wherein the payload molecule and the agent are administered simultaneously, sequentially, or separately to the epithelial tissue barrier.

22. The method according to any one of claims 1 to 21, wherein the payload molecule is a therapeutic or prophylactic agent.

23. The payload molecule is a) one or more of a peptide, a protein, a polysaccharide, a nucleic acid, a nanoparticle, or a small molecule having a molecular weight of less than 900 Da; and / or b) one or more of a virus particle, a virus-like particle, a viral protein, a nucleic acid encoding a viral protein, a nanoparticle, an antibody, an antibody fragment or mimetic, a protein or peptide complex, a cytokine, or a toxin; and / or c) an anti-VEGF antibody or fragment thereof, or an inhibitor of VEGF, and / or the payload molecule comprises an anti-inflammatory agent; and / or d) a therapeutic agent suitable for the treatment or prevention of an eye disorder, a respiratory disorder, a gastrointestinal disorder, a genital disorder, a mucosal disorder, a brain disorder, an infection, or a skin disorder; and / or e) a prophylactic agent such as a vaccine The method according to claim 22, comprising the above.

24. The epithelial tissue barrier is a) in the brain, eye, respiratory system, skin, digestive tract, urinary tract, male or female genital system, olfactory system, or any mucosa of the subject; and / or b) an internal barrier such as the blood-brain barrier, the blood-testis barrier, or the blood-retinal barrier; and / or c) in the respiratory system, digestive tract, urinary tract, or female genital organs The method according to any one of claims 1 to 23.

25. A pharmaceutically acceptable composition that can be a composition comprising (a) an agent capable of disrupting the epithelial tissue barrier and optionally (b) a payload molecule, wherein the agent capable of disrupting the epithelial tissue barrier is as defined in any one of claims 1 to 20; and the payload molecule is optionally as defined in claim 23.

26. a) the composition is in the form of a gel, lotion, cream, ointment, drops, spray, aerosol, or solution; and / or b) the composition further comprises one or more of water; physiological saline; salts; buffers; lubricants; wetting agents; thickeners; isotonic agents; cellulose derivatives such as sodium carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, or methyl cellulose; dextran 70; gelatin; polyols; glycerin; polyethylene glycols such as PEG 300 or PEG 400; polysorbate 80; propylene glycol; polyvinyl alcohol; and povidone (polyvinylpyrrolidone); poloxamers such as poloxamer 407; and combinations thereof; and / or c) the agent capable of disrupting the epithelial tissue barrier is encapsulated or impregnated within a polymer, or adsorbed onto a polymeric carrier or protein, or crystallized, lyophilized or produced into nanoparticles, The composition according to claim 25.

27. The composition according to claim 25 or 26 for use as a medicament, comprising a therapeutic or prophylactic payload molecule.

28. The composition for use according to claim 27, wherein the use is in a method of treating or preventing an eye disorder, a respiratory disorder, a gastrointestinal disorder, a reproductive organ disorder, a mucosal disorder, a brain disorder, a microbial or parasitic infection, cancer, or a skin disorder in a subject.

29. A method of treating or preventing an eye disorder, a respiratory disorder, a gastrointestinal disorder, a reproductive organ disorder, a mucosal disorder, a brain disorder, an infection, cancer, or a skin disorder, comprising the step of administering to a subject the composition as defined in claim 25 or 26, wherein the composition comprises a therapeutic or prophylactic payload molecule.

30. The composition for use in the treatment according to claim 28 or the treatment method according to claim 29, wherein the administration is topical administration to the epithelial barrier.

31. A product comprising the composition as defined in claim 25 or 26, a) an eye drop dispenser, an eye wash device or a contact lens; and / or b) a suction device, an inhaler, a nebulizer, or a vape device; and / or c) a controlled release tablet or capsule suitable for oral administration; and / or d) a transdermal patch or gel; and / or e) a vaccine further comprising an antigen or a nucleic acid suitable for antigen expression (such as a viral vector) thereof.

32. (a) an agent capable of disrupting the epithelial tissue barrier, and (b) a payload molecule, A kit comprising: The agent capable of disrupting the epithelial tissue barrier is as defined in any one of claims 1 to 20; The payload molecule is optionally as defined in claim 23, a kit.

33. Use of an agent capable of disrupting the epithelial tissue barrier for promoting the permeation of a payload molecule through the epithelial tissue barrier, The agent capable of disrupting the epithelial tissue barrier is as defined in any one of claims 1 to 20; The payload molecule is optionally as defined in claim 23, use.

34. Use of an agent capable of disrupting the epithelial tissue barrier for promoting the extraction of extracellular fluid from a subject, comprising the administration of the agent capable of disrupting the epithelial tissue barrier to the epithelial tissue barrier and the extraction of extracellular fluid through the epithelial tissue barrier, and the agent capable of disrupting the epithelial tissue barrier is as defined in any one of claims 1 to 20, use.

35. A method for the extraction of extracellular fluid from a subject, - administration of an agent capable of disrupting the epithelial tissue barrier to the epithelial tissue barrier of the subject, and - extraction of extracellular fluid through the epithelial tissue barrier, comprising, and the agent capable of disrupting the epithelial tissue barrier is as defined in any one of claims 1 to 20, a method.

36. The use according to claim 34 or the method according to claim 35, wherein the extraction of the extracellular fluid through the epithelial tissue barrier is performed by evacuating the surface treated with the agent to extract the extracellular fluid.

37. The use according to claim 34 or 36 or the method according to claim 35 or 36, wherein the extracted extracellular fluid is used for analysis such as electrophysiological measurement and / or biomarker analysis.

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